Botanical Research: Series Editors

Advances in
BOTANICAL RESEARCH
Series Editors
JEAN-CLAUDE KADER
Laboratoire Physiologie Cellulaire

et Moleculaire des Plantes, CNRS,
Universite de Paris, Paris, France
MICHEL DELSENY
Laboratoire Genome et
Developpement des Plantes,
CNRS IRD UP, Universite de
Perpignan, Perpignan, France
Academic Press is an imprint of Elsevier

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ISBN: 978-0-12-386479-6
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CONTRIBUTORS TO VOLUME 58
ADELBERT BACHER Institute of Food Chemistry, University of

Hamburg, Hamburg, Germany; Ikosatec GmbH, Garching, Germany
CHRISTOPHER I. CAZZONELLI ARC Centre of Excellence in Plant
Energy Biology, Research School of Biology, Australian National
University, Canberra, ACT 0200, Australia
ABBY J. CUTTRISS Molecular Biosciences and Bioengineering, University
of Hawaii at Manoa, Honolulu, HI, USA; Department of Biological
Sciences, Lehman College, The City University of New York, Bronx,
New York, USA
MARKUS FISCHER Institute of Food Chemistry, University of
Hamburg, Hamburg, Germany; Ikosatec GmbH, Garching, Germany
JUTTA HAGER Institut of Biologie des Plantes, UMR8618 CNRS/
Universite de Paris sud 11, Batiment 630, Universite de Paris sud 11,
91405 Orsay CEDEX, France
SHENGCHUN LI Institut of Biologie des Plantes, UMR8618 CNRS/
GRAHAM NOCTOR Institut of Biologie des Plantes, UMR8618 CNRS/
BARRY J. POGSON ARC Centre of Excellence in Plant Energy Biology,
Research School of Biology, Australian National University, Canberra,
ACT 0200, Australia
MARIA RAPALA-KOZIK Department of Analytical Biochemistry,
Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian
University, Krakow, Poland
ALISON G. SMITH Department of Plant Sciences, University of
Cambridge, Cambridge, United Kingdom
MICHAEL E. WEBB School of Chemistry and Astbury Centre
for Structural Molecular Biology, University of Leeds, Leeds, United
Kingdom
ELEANORE T. WURTZEL Department of Biological Sciences, Lehman
College, The City University of New York, Bronx, New York, USA
PREFACE
VITAMINS: A PLANT AFFAIR

All organisms need to synthesize, transform and interconvert a myriad of
molecules to enable them to grow and reproduce. All these reactions are
catalysed by enzymes (the living tools) which facilitate chemical modifications of substrates owing to their specific binding properties. In many cases,
suitable coenzymes (nicotinamide adenine dinucleotide [NAD], nicotinamide adenine dinucleotide phosphate [NADP], flavin adenine dinucleotide
[FAD], flavin mononucleotide [FMN], pyridoxal 50 -phosphate, biotin, coenzyme A, etc.) may assist in biochemical transformations. Some of these
coenzymes may be more or less tightly bound to enzymes as part of prosthetic groups (biotin, FMN, etc.). Coenzymes may also be loosely bound to
enzymes as detachable molecules. In that case, they are acting as substrates,
being often recycled through other set of reactions (NAD(P), folates,
ascorbate, etc.).
Vitamin (a combination word from vita and amine) are by definition
dietary substances required for good health and normal development that
are only synthesized by microorganisms and plants. During the course of
animal evolution, the ability to biosynthesize these compounds has been lost
and, instead, elaborate uptake mechanisms have been developed. There are
13 recognized vitamins, involved in various catalytic functions. The largest
number of vitamins serve as precursors to coenzymes (vitamins B1 [thiamine],
B2 [riboflavin], B3 [niacin], B5 [pantothenic acid], B6 [pyridoxine], B9 [folic
acid]) or as coenzymes themselves (vitamins B8 [biotin], B12 [cobalamin], C
[ascorbic acid], K [phylloquinone, menaquinone]). Some of these vitamins,
especially the hydrophobic (vitamins A [retinol, pro-vitamin A carotenoids],
E [tocopherols, tocotrienols] and D [ergocalciferol, cholecalciferol]), cannot
be truly considered as coenzymes: vitamins A and D display hormonal effects
in the human body, and vitamin E has a protective role in membranes by
scavenging free radicals. Vitamins are involved in almost all important
cellular functions, displaying protective (antioxidant) functions or participating to numerous metabolisms, including the energetic metabolism (respiration, photosynthesis) and the metabolisms of sugars, amino acids, fatty acids
and nucleic acids. The daily amount of vitamins required for a good health
depends on the considered vitamin and fluctuates widely, from a few micrograms (B12, D, K) to several milligrams (B3, B5, C). Vitamin deficiencies are
PREFACE
quite common in low-resource countries but also occur in developed

countries due to bad food habits. Well-known vitamin-related diseases
include, among others, blindness (vitamin A), beriberi (vitamin B1), pellagra
(vitamin B3), anaemia (vitamins B6 and B9), scurvy (vitamin C), rickets
(vitamin D) or neural tube defects (vitamin B9). In addition, antioxidant
vitamins (such as A, C, E and B6) have protective roles as efficient quenchers
of reactive oxygen species.
Plants synthesize an impressive diverse array of natural products including
vitamins, and plants are considered as a major nutritional source for these
essential molecules. Plants are able to synthesize 12 out of the 13 vitamins.
Indeed, plants have no cobalamin-dependent proteins and use for methionine synthesis an alternate catalytic mechanism that does not need vitamin
B12. Vitamin B12 is only synthesized in prokaryotes, and humans primarily
obtained it from animal food, thanks to the intestine flora of herbivores. Two
of the vitamins (vitamins A and D) have hormonal functions in animals,
which functions do not exist in plants. Plants do not synthesize vitamin A,
but carotenoids. Some of these carotenoids are pro-vitamin A, which are
transformed in retinol once assimilated by animals. Vitamin D (D2 and D3) is
formed from the precursors ergosterol (mainly present in fungal cells) and
cholesterol (mainly present in mammalian cells) following sun exposure (UV
radiation). Although vitamins D2 and D3 can be found in low amounts in the
membranes of some Solanaceous plants, higher plants are not considered as a
source of vitamin D and plant food cannot compensate insufficient synthesis
in the human body. Thus, the plant kingdom is a recognized dietary source
for 11 out of the 13 vitamins.
As many vitamins are only required in trace quantities, their biosynthesis is
normally strictly controlled and the involved enzymes are generally produced
in very small amounts. This is why it has been extremely difficult to elucidate
their complete biosynthetic pathways, and it still remains the case that several
steps within the biosynthesis of vitamins are poorly understood (e.g. thiazole
ring scaffolding). However, the advent of modern recombinant DNA techniques, coupled with the completion of many genome projects, made possible
to decipher pathways in plants, thus allowing now a more complete understanding of how these molecules are made. The general picture emerging
from these recent data indicates that the metabolic web represented by these
molecules is of a rare complexity. Indeed, not only may the synthesis of
vitamins require some 10 enzymatic steps but also several of these metabolic
routes are split between various compartments of the plant cell, adding a
further level of complexity when compared to prokaryotes. Since all cell
compartments need their vitamins, this situation implies transport and
trafficking of intermediates and end products of the pathways. Today,
there is no explanation for such compartmentalization.
The actual understanding of how these biosynthetic pathways operate can
be exploited for health and wealth creation. Vitamin synthesis is largely
PREFACE
xi
restricted to plants and microorganisms, a biochemical feature that can be

harnessed for the development of specific pesticides (bactericides, herbicides,
fungicides, etc.). Taking into account the health problems related to vitamin
deficiencies, together with an increase in the use of vitamin supplements for
human and animal nutrition, there is also a requirement, from a nutritional
and commercial standpoint, to enhance the production of many of these
vitamins. Overproduction of the vitamins can be achieved in a number of
ways, by removing transcriptional controls, overproduction of key enzymes
that represent bottlenecks in the pathways of biosynthesis, suppression of
metabolic feedbacks, limitation of the catabolism and increase of the storage.
It is clear that the optimization of these systems requires a complete understanding of (i) their endogenous regulation and (ii) their integration within
the metabolism as a whole.
This book includes comprehensive and authoritative reviews from leading
experts on vitamins in plants, and we are thankful for their time and effort.
The aim of this book is to collect and interpret the rapid growing experimental information on vitamins in plants, especially in the challenging area of
their biosynthesis. We also hope that this book may be useful as a starting
point for those graduates and undergraduate students and researchers
wishing to pursue special studies in this field.
FABRICE REBEILLE AND ROLAND DOUCE
CONTENTS OF VOLUMES 3557

Series Editor (Volumes 3544)
J.A. CALLOW
School of Biosciences, University of Birmingham,
Birmingham, United Kingdom
Contents of Volume 35
Recent Advances in the Cell Biology of Chlorophyll Catabolism
H. THOMAS, H. OUGHAM and S. HORTENSTEINER
The Microspore: A Haploid Multipurpose Cell
A. TOURAEV, M. PFOSSER and E. HEBERLE-BORS
The Seed Oleosins: Structure Properties and Biological Role
J. NAPIER, F. BEAUDOIN, A. TATHAM and P. SHEWRY
Compartmentation of Proteins in the Protein Storage Vacuole:
A Compound Organelle in Plant Cells
L. JIANG and J. ROGERS
Intraspecific Variation in Seaweeds: The Application of New Tools
and Approaches
C. MAGGS and R. WATTIER
Glucosinolates and Their Degradation Products
R. F. MITHEN
xiv
PLANT VIRUS VECTOR INTERACTIONS
Edited by R. Plumb
Aphids: Non-Persistent Transmission
T. P. PIRONE and K. L. PERRY
Persistent Transmission of Luteoviruses by Aphids
B. REAVY and M. A. MAYO
Fungi
M. J. ADAMS
Whitefly Transmission of Plant Viruses
J. K. BROWN and H. CZOSNEK
Beetles
R. C. GERGERICH
Thrips as Vectors of Tospoviruses
D. E. ULLMAN, R. MEIDEROS, L. R. CAMPBELL,
A. E. WHITFIELD, J. L. SHERWOOD and T. L. GERMAN
Virus Transmission by Leafhoppers, Planthoppers and Treehoppers
(Auchenorrhyncha, Homoptera)
E. AMMAR and L. R. NAULT
Nematodes
S. A. MacFARLANE, R. NEILSON and D. J. F. BROWN
Other Vectors
R. T. PLUMB
ANTHOCYANINS IN LEAVES
Edited by K. S. Gould and D. W. Lee
Anthocyanins in Leaves and Other Vegetative Organs: An Introduction
D. W. LEE and K. S. GOULD
Le Rouge et le Noir: Are Anthocyanins Plant Melanins?
G. S. TIMMINS, N. M. HOLBROOK and T. S. FEILD
Anthocyanins in Leaves: History, Phylogeny and Development
D. W. LEE
The Final Steps in Anthocyanin Formation: A Story of
Modification and Sequestration
C. S. WINEFIELD
Molecular Genetics and Control of Anthocyanin Expression
B. WINKEL-SHIRLEY
Differential Expression and Functional Significance of
Anthocyanins in Relation to Phasic Development in
Hedera helix L.
W. P. HACKETT
Do Anthocyanins Function as Osmoregulators in Leaf Tissues?
L. CHALKER-SCOTT
The Role of Anthocyanins for Photosynthesis of Alaskan Arctic
Evergreens During Snowmelt
S. F. OBERBAUER and G. STARR
Anthocyanins in Autumn Leaf Senescence
D. W. LEE
A Unified Explanation for Anthocyanins in Leaves?
K. S. GOULD, S. O. NEILL and T. C. VOGELMANN
xv
xvi
An Epidemiological Framework for Disease Management
C. A. GILLIGAN
Golgi-independent Trafficking of Macromolecules to the Plant Vacuole
D. C. BASSHAM
Phosphoenolpyruvate Carboxykinase: Structure,
Function and Regulation
R. P. WALKER and Z.-H. CHEN
Developmental Genetics of the Angiosperm Leaf
C. A. KIDNER, M. C. P. TIMMERMANS, M. E. BYRNE
and R. A. MARTIENSSEN
A Model for the Evolution and Genesis of the Pseudotetraploid
Arabidopsis thaliana Genome
Y. HENRY, A. CHAMPION, I. GY, A. PICAUD,
A. LECHARNY and M. KREIS
Cumulative Subject Index Volumes 138
Starch Synthesis in Cereal Grains
K. TOMLINSON and K. DENYER
The Hyperaccumulation of Metals by Plants
M. R. MACNAIR
Plant Chromatin Learning from Similarities and Differences
J. BRZESKI, J. DYCZKOWSKI, S. KACZANOWSKI,
P. ZIELENKIEWICZ and A. JERZMANOWSKI
xvii
The Interface Between the Cell Cycle and Programmed Cell Death in
Higher Plants: From Division unto Death
D. FRANCIS
The Importance of Extracellular Carbohydrate Production by Marine
Epipelic Diatoms
G. J. C. UNDERWOOD and D. M. PATERSON
Fungal Pathogens of Insects: Cuticle Degrading Enzymes and Toxins
A. K. CHARNLEY
Multiple Responses of Rhizobia to Flavonoids
During Legume Root Infection
JAMES E. COOPER
Investigating and Manipulating Lignin Biosynthesis
in the Postgenomic Era
CLAIRE HALPIN
Application of Thermal Imaging and Infrared Sensing in Plant
Physiology and Ecophysiology
HAMLYN G. JONES
Sequences and Phylogenies of Plant Pararetroviruses, Viruses, and
Transposable Elements
CELIA HANSEN and J. S. HESLOP-HARRISON
Role of Plasmodesmata Regulation in Plant Development

ARNAUD COMPLAINVILLE and MARTIN CRESPI
xviii
Chemical Manipulation of Antioxidant Defences in Plants
ROBERT EDWARDS, MELISSA BRAZIER-HICKS,
DAVID P. DIXON and IAN CUMMINS
The Impact of Molecular Data in Fungal Systematics
P. D. BRIDGE, B. M. SPOONER and P. J. ROBERTS
Cytoskeletal Regulation of the Plane of Cell Division: An Essential
Component of Plant Development and Reproduction
HILARY J. ROGERS
Nitrogen and Carbon Metabolism in Plastids: Evolution, Integration,
and Coordination with Reactions in the Cytosol
ALYSON K. TOBIN and CAROLINE G. BOWSHER
Defensive and Sensory Chemical Ecology of Brown Algae
CHARLES D. AMSLER and VICTORIA A. FAIRHEAD
Regulation of Carbon and Amino Acid Metabolism: Roles of Sucrose
Nonfermenting-1-Related Protein Kinase-1 and General Control
Nonderepressible-2-Related Protein Kinase
NIGEL G. HALFORD
Opportunities for the Control of Brassicaceous Weeds of Cropping
Systems Using Mycoherbicides
AARON MAXWELL and JOHN K. SCOTT
Stress Resistance and Disease Resistance in Seaweeds: The Role of
Reactive Oxygen Metabolism
MATTHEW J. DRING
Nutrient Sensing and Signalling in Plants: Potassium and Phosphorus
ANNA AMTMANN, JOHN P. HAMMOND,
PATRICK ARMENGAUD and PHILIP J. WHITE
xix
Angiosperm Floral Evolution: Morphological
Developmental Framework
PETER K. ENDRESS
Recent Developments Regarding the Evolutionary
Origin of Flowers
MICHAEL W. FROHLICH
Duplication, Diversification, and Comparative Genetics of Angiosperm
MADS-Box Genes
VIVIAN F. IRISH
Beyond the ABC-Model: Regulation of Floral Homeotic Genes
LAURA M. ZAHN, BAOMIN FENG and HONG MA
Missing Links: DNA-Binding and Target Gene Specificity of Floral
Homeotic Proteins
RAINER MELZER, KERSTIN KAUFMANN
NTER THEIEN
and GU
Genetics of Floral Development in Petunia
ANNEKE RIJPKEMA, TOM GERATS and
MICHIEL VANDENBUSSCHE
Flower Development: The Antirrhinum Perspective
BRENDAN DAVIES, MARIA CARTOLANO and
ZSUZSANNA SCHWARZ-SOMMER
Floral Developmental Genetics of Gerbera (Asteraceae)
TEEMU H. TEERI, MIKA KOTILAINEN, ANNE UIMARI,
SATU RUOKOLAINEN, YAN PENG NG, URSULA MALM,
NEN, SUVI BROHOLM, ROOSA LAITINEN,
LLA
EIJA PO
PAULA ELOMAA and VICTOR A. ALBERT
Gene Duplication and Floral Developmental Genetics of Basal Eudicots
ELENA M. KRAMER and ELIZABETH A. ZIMMER
xx
Genetics of Grass Flower Development

CLINTON J. WHIPPLE and ROBERT J. SCHMIDT
Developmental Gene Evolution and the Origin of Grass
Inflorescence Diversity
SIMON T. MALCOMBER, JILL C. PRESTON, RENATA
REINHEIMER, JESSIE KOSSUTH and ELIZABETH A. KELLOGG
Expression of Floral Regulators in Basal Angiosperms and the Origin and
Evolution of ABC-Function
PAMELA S. SOLTIS, DOUGLAS E. SOLTIS, SANGTAE KIM,
ANDRE CHANDERBALI and MATYAS BUZGO
The Molecular Evolutionary Ecology of Plant Development: Flowering
Time in Arabidopsis thaliana
KATHLEEN ENGELMANN and MICHAEL PURUGGANAN
A Genomics Approach to the Study of Ancient Polyploidy and
Floral Developmental Genetics
JAMES H. LEEBENS-MACK, KERR WALL, JILL DUARTE,
ZHENGUI ZHENG, DAVID OPPENHEIMER and
CLAUDE DEPAMPHILIS
Series Editors (Volume 45 )
JEAN-CLAUDE KADER
Laboratoire Physiologie Cellulaire et Moleculaire des Plantes, CNRS,
Universite de Paris, Paris, France
MICHEL DELSENY
Laboratoire Genome et Developpement des Plantes,
CNRS IRD UP, Universite de Perpignan,
Perpignan, France
RAPESEED BREEDING
History, Origin and Evolution
S. K. GUPTA and ADITYA PRATAP
xxi
Breeding Methods
B. RAI, S. K. GUPTA and ADITYA PRATAP
The Chronicles of Oil and Meal Quality Improvement in Oilseed Rape
ABHA AGNIHOTRI, DEEPAK PREM and KADAMBARI GUPTA
Development and Practical Use of DNA Markers
KATARZYNA MIKOLAJCZYK
Self-Incompatibility
RYO FUJIMOTO and TAKESHI NISHIO
Fingerprinting of Oilseed Rape Cultivars
URN and JANA ZALUDOVA

VLADISLAV C
Haploid and Doubled Haploid Technology
L. XU, U. NAJEEB, G. X. TANG, H. H. GU, G. Q. ZHANG,
Y. HE and W. J. ZHOU
Breeding for Apetalous Rape: Inheritance and Yield Physiology
LIXI JIANG
Breeding Herbicide-Tolerant Oilseed Rape Cultivars
PETER B. E. MCVETTY and CARLA D. ZELMER
Breeding for Blackleg Resistance: The Biology and Epidemiology
W. G. DILANTHA FERNANDO, YU CHEN and
KAVEH GHANBARNIA
Development of Alloplasmic Rape
MICHAL STARZYCKI, ELIGIA STARZYCKI and JAN PSZCZOLA
Honeybees and Rapeseed: A PollinatorPlant Interaction
D. P. ABROL
xxii
Genetic Variation and Metabolism of Glucosinolates

NATALIA BELLOSTAS, ANNE DORTHE SRENSEN,
JENS CHRISTIAN SRENSEN and HILMER SRENSEN
Mutagenesis: Generation and Evaluation of Induced Mutations
SANJAY J. JAMBHULKAR
Rapeseed Biotechnology
VINITHA CARDOZA and C. NEAL STEWART, JR.
Oilseed Rape: Co-existence and Gene Flow from Wild Species
RIKKE BAGGER JRGENSEN
Evaluation, Maintenance, and Conservation of Germplasm
RANBIR SINGH and S. K. SHARMA
Oil Technology
US
BERTRAND MATTHA
INCORPORATING ADVANCES IN PLANT PATHOLOGY
Nitric Oxide and Plant Growth Promoting Rhizobacteria: Common Features
Influencing Root Growth and Development
NICA CREUS, MARIA
CELESTE MOLINA-FAVERO, CECILIA MO
LUCIANA LANTERI, NATALIA CORREA-ARAGUNDE, MARIA
CRISTINA LOMBARDO, CARLOS ALBERTO BARASSI
and LORENZO LAMATTINA
How the Environment Regulates Root Architecture in Dicots
RIE LEFEBVRE, PHILIPPE
MARIANA JOVANOVIC, VALE
LAPORTE, SILVINA GONZALEZ-RIZZO, CHRISTINE
LELANDAIS-BRIE`RE, FLORIAN FRUGIER, CAROLINE
HARTMANN and MARTIN CRESPI
xxiii
Aquaporins in Plants: From Molecular Structure to Integrated Functions

OLIVIER POSTAIRE, LIONEL VERDOUCQ and
CHRISTOPHE MAUREL
Iron Dynamics in Plants
JEAN-FRANC
OIS BRIAT
Plants and Arbuscular Mycorrhizal Fungi: Cues and Communication in the
Early Steps of Symbiotic Interactions
VIVIENNE GIANINAZZI-PEARSON, NATHALIE
SEJALON-DELMAS, ANDREA GENRE, SYLVAIN
JEANDROZ and PAOLA BONFANTE
Dynamic Defense of Marine Macroalgae Against Pathogens: From Early
Activated to Gene-Regulated Responses
AUDREY COSSE, CATHERINE LEBLANC and
PHILIPPE POTIN
INCORPORATING ADVANCES IN PLANT PATHOLOGY
The Plant Nucleolus
EZ-VA
SQUEZ AND FRANCISCO JAVIER MEDINA
JULIO SA
Expansins in Plant Development
DONGSU CHOI, JEONG HOE KIM AND YI LEE
Molecular Biology of Orchid Flowers: With Emphasis on Phalaenopsis
WEN-CHIEH TSAI, YU-YUN HSIAO, ZHAO-JUN PAN, CHIACHI HSU, YA-PING YANG, WEN-HUEI CHEN AND
HONG-HWA CHEN
xxiv
Molecular Physiology of Development and Quality of Citrus

S, JOSE M.
FRANCISCO R. TADEO, MANUEL CERCO
COLMENERO-FLORES, DOMINGO J. IGLESIAS, MIGUEL A.
NARANJO, GABINO RIOS, ESTHER CARRERA, OMAR
RUIZ-RIVERO, IGNACIO LLISO, RAPHAE L MORILLON,
PATRICK OLLITRAULT AND MANUEL TALON
Bamboo Taxonomy and Diversity in the Era of Molecular Markers
MALAY DAS, SAMIK BHATTACHARYA, PARAMJIT SINGH,
TARCISO S. FILGUEIRAS AND AMITA PAL
Molecular Mechanisms Underlying Vascular Development
JAE-HOON JUNG, SANG-GYU KIM, PIL JOON SEO
AND CHUNG-MO PARK
Clock Control Over Plant Gene Expression
ANTOINE BAUDRY AND STEVE KAY
Plant Lectins
ELS J. M. VAN DAMME, NAUSICAA LANNOO
AND WILLY J. PEUMANS
Late Embryogenesis Abundant Proteins
MING-DER SHIH, FOLKERT A. HOEKSTRA
AND YUE-IE C. HSING
Phototropism and Gravitropism in Plants
MARIA LIA MOLAS AND JOHN Z. KISS
xxv
Cold Signalling and Cold Acclimation in Plants

ERIC RUELLAND, MARIE-NOELLE VAULTIER,
ALAIN ZACHOWSKI AND VAUGHAN HURRY
Genome Evolution in Plant Pathogenic and Symbiotic Fungi
GABRIELA AGUILETA, MICHAEL E. HOOD,
GUISLAINE REFREGIER AND TATIANA GIRAUD
Aroma Volatiles: Biosynthesis and Mechanisms
of Modulation During Fruit Ripening
BRUNO G. DEFILIPPI, DANIEL MANRIQUEZ,
LEZ-AGU
ERO
KIETSUDA LUENGWILAI AND MAURICIO GONZA
Jatropha curcas: A Review
NICOLAS CARELS
You are What You Eat: Interactions Between Root Parasitic
Plants and Their Hosts
LOUIS J. IRVING AND DUNCAN D. CAMERON
Low Oxygen Signaling and Tolerance in Plants
FRANCESCO LICAUSI AND PIERDOMENICO PERATA
Roles of Circadian Clock and Histone Methylation in
the Control of Floral Repressors
RYM FEKIH, RIM NEFISSI, KANA MIYATA,
HIROSHI EZURA AND TSUYOSHI MIZOGUCHI
xxvi
PAMP-Triggered Basal Immunity in Plants
RNBERGER AND BIRGIT KEMMERLING
THORSTEN NU
Plant Pathogens as Suppressors of Host Defense
TRAUX, ROBERT WILSON JACKSON,
JEAN-PIERRE ME
ESTHER SCHNETTLER AND ROB W. GOLDBACH
From Nonhost Resistance to Lesion-Mimic Mutants:
Useful for Studies of Defense Signaling
ANDREA LENK AND HANS THORDAL-CHRISTENSEN
Action at a Distance: Long-Distance Signals in Induced Resistance
MARC J. CHAMPIGNY AND ROBIN K. CAMERON
Systemic Acquired Resistance
R. HAMMERSCHMIDT
Rhizobacteria-Induced Systemic Resistance
FTE
DAVID DE VLEESSCHAUWER AND MONICA HO
Plant Growth-Promoting Actions of Rhizobacteria
STIJN SPAEPEN, JOS VANDERLEYDEN AND YAACOV OKON
Interactions Between Nonpathogenic Fungi and Plants
M. I. TRILLAS AND G. SEGARRA
Priming of Induced Plant Defense Responses
UWE CONRATH
Transcriptional Regulation of Plant Defense Responses
MARCEL C. VAN VERK, CHRISTIANE GATZ
AND HUUB J. M. LINTHORST
xxvii
Unexpected Turns and Twists in Structure/Function of PR-Proteins

that Connect Energy Metabolism and Immunity
MEENA L. NARASIMHAN, RAY A. BRESSAN,
MATILDE PAINO DURZO, MATTHEW A. JENKS
AND TESFAYE MENGISTE
Role of Iron in PlantMicrobe Interactions
P. LEMANCEAU, D. EXPERT, F. GAYMARD,
P. A. H. M. BAKKER AND J.-F. BRIAT
Adaptive Defense Responses to Pathogens and Insects
LINDA L. WALLING
Plant Volatiles in Defence
MERIJN R. KANT, PETRA M. BLEEKER, MICHIEL VAN WIJK,
ROBERT C. SCHUURINK AND MICHEL A. HARING
Ecological Consequences of Plant Defence Signalling
MARTIN HEIL AND DALE R. WALTERS
Oxidation of Proteins in PlantsMechanisms and Consequences
LEE J. SWEETLOVE AND IAN M. MLLER
Reactive Oxygen Species: Regulation of Plant Growth and Development
HYUN-SOON KIM, YOON-SIK KIM, KYU-WOONG HAHN,
HYOUK JOUNG AND JAE-HEUNG JEON
Ultraviolet-B Induced Changes in Gene Expression and Antioxidants in Plants
S. B. AGRAWAL, SURUCHI SINGH
AND MADHOOLIKA AGRAWAL
xxviii
Roles of -Glutamyl Transpeptidase and -Glutamyl Cyclotransferase in

Glutathione and Glutathione-Conjugate Metabolism in Plants
NAOKO OHKAMA-OHTSU, KEIICHI FUKUYAMA
AND DAVID J. OLIVER
The Redox State, a Referee of the LegumeRhizobia Symbiotic Game
DANIEL MARINO, CHIARA PUCCIARIELLO, ALAIN PUPPO
AND PIERRE FRENDO
Arabidopsis Histone Lysine Methyltransferases
FREDE RIC PONTVIANNE, TODD BLEVINS,
AND CRAIG S. PIKAARD
Advances in Coffea Genomics
ALEXANDRE DE KOCHKO, SELASTIQUE AKAFFOU, ALAN
ANDRADE, CLAUDINE CAMPA, DOMINIQUE CROUZILLAT,
ROMAIN GUYOT, PERLA HAMON, RAY MING,
LUKAS A. MUELLER, VALERIE PONCET,
CHRISTINE TRANCHANTDUBREUIL, AND SERGE HAMON
Regulatory Components of Shade Avoidance Syndrome
JAIME F. MARTINEZ-GARCIA, ANAHIT GALSTYAN,
S CIFUENTES-ESQUIVEL,
MERCE`SALLA-MARTRET, NICOLA
MARC AL GALLEMI, AND JORDI BOU-TORRENT

Responses of Halophytes to Environmental Stresses with Special
Emphasis to Salinity
KSOURI RIADH, MEGDICHE WIDED, KOYRO HANS-WERNER,
AND ABDELLY CHEDLY
Plant Nematode Interaction: A Sophisticated Dialogue
PIERRE ABAD AND VALERIE M. WILLIAMSON
xxix
Optimization of Nutrition in Soilless Systems: A Review

NGELES CALATAYUD
ELISA GORBE AND A
Pollen Germination and Tube Growth
HUEI-JING WANG, JONG-CHIN HUANG,
AND GUANG-YUH JAUH
Molecular Mechanisms of Sex Determination in Monoecious
and Dioecious Plants
GEORGE CHUCK
The Evolution of Floral Symmetry
HELE`NE CITERNE, FLORIAN JABBOUR, SOPHIE NADOT,
AND CATHERINE DAMERVAL
Protein Turnover in Grass Leaves
LOUIS JOHN IRVING, YUJI SUZUKI, HIROYUKI ISHIDA,
AND AMANE MAKINO
Carpel Development
NDIZ, CHLOE FOURQUIN,
CRISTINA FERRA
NATHANAEL PRUNET, CHARLIE P. SCUTT, EVA SUNDBERG,
CHRISTOPHE TREHIN, AND AURELIE C. M.
VIALETTE-GUIRAUD
Root System Architecture
PAUL A. INGRAM AND JOCELYN E. MALAMY
xxx
Functional Genomics of Cacao

FABIENNE MICHELI, MARK GUILTINAN, KARINA PERES
GRAMACHO, MIKE J. WILKINSON, ANTONIO VARGAS DE
LIO CEZAR DE MATTOS CASCARDO,
OLIVEIRA FIGUEIRA, JU
SIELA MAXIMOVA, AND CLAIRE LANAUD
The Ecological Water-Use Strategies of Succulent Plants
R. MATTHEW OGBURN AND ERIKA J. EDWARDS
Nodule Physiology and Proteomics of Stressed Legumes
M. I. QURESHI, S. MUNEER, H. BASHIR, J. AHMAD,
AND M. IQBAL
Molecular Aspects of Fragrance and Aroma in Rice
APICHART VANAVICHIT AND TADACHI YOSHIHASHI
Miscanthus: A Promising Biomass Crop
EMILY A. HEATON, FRANK G. DOHLEMAN, A. FERNANDO
MIGUEZ, JOHN A. JUVIK, VERA LOZOVAYA, JACK WIDHOLM,
OLGA A. ZABOTINA, GREGORY F. MCISAAC, MARK B. DAVID,
THOMAS B. VOIGT, NICHOLAS N. BOERSMA,
AND STEPHEN P. LONG
Plant Adaptations to Salt and Water Stress: Differences and Commonalities
RANA MUNNS
Recent Advances in Understanding the Regulation of Whole-Plant Growth
Inhibition by Salinity, Drought and Colloid Stress
PETER M. NEUMANN
xxxi
Recent Advances in Photosynthesis Under Drought and Salinity

MARIA M. CHAVES, J. MIGUEL COSTA AND
NELSON J. MADEIRA SAIBO
Plants in Extreme Environments: Importance of Protective Compounds
in Stress Tolerance
SZLO
CS, AVIAH ZILBERSTEIN
SZABADOS, HAJNALKA KOVA
LA
AND ALAIN BOUCHEREAU
Ion Transport in Halophytes
SERGEY SHABALA AND ALEX MACKAY
The Regulatory Networks of Plant Responses to Abscisic Acid
TAISHI UMEZAWA, TAKASHI HIRAYAMA, TAKASHI
KUROMORI AND KAZUO SHINOZAKI
Molecular Mechanisms of Abscisic Acid Action in Plants and Its Potential
Applications to Human Health
ARCHANA JOSHI-SAHA, CHRISTIANE VALON
AND JEFFREY LEUNG
Signalling Strategies During Drought and Salinity, Recent News
TIJEN DEMIRAL, ISMAIL TURKAN AND A. HEDIYE SEKMEN
An Overview of the Current Understanding of Desiccation Tolerance in the
Vegetative Tissues of Higher Plants
MONIQUE MORSE, MOHAMED S. RAFUDEEN AND
JILL M. FARRANT
Root Tropism: Its Mechanism and Possible Functions in Drought Avoidance
YUTAKA MIYAZAWA, TOMOKAZU YAMAZAKI, TEPPEI
MORIWAKI AND HIDEYUKI TAKAHASHI
xxxii
Roles of Circadian Clock in Developmental Controls and Stress Responses in

Arabidopsis: Exploring a Link for Three Components of Clock Function in
Arabidopsis
RIM NEFISSI, YU NATSUI, KANA MIYATA, ABDELWAHED
GHORBEL AND TSUYOSHI MIZOGUCHI
Engineering Salinity and Water-Stress Tolerance in Crop Plants: Getting
Closer to the Field
ZVI PELEG, MARIS P. APSE AND EDUARDO BLUMWALD
Drought Stress: Molecular Genetics and Genomics Approaches
MELDA KANTAR, STUART J. LUCAS AND HIKMET BUDAK
Carotenoids
ABBY J. CUTTRISS,*,{ CHRISTOPHER I. CAZZONELLI,{

ELEANORE T. WURTZEL{ AND BARRY J. POGSON{,1
*Molecular Biosciences and Bioengineering,

University of Hawaii at Manoa, Honolulu, HI, USA
{
Department of Biological Sciences, Lehman College,
The City University of New York, Bronx, New York, USA
{
ARC Centre of Excellence in Plant Energy Biology,
Research School of Biology, Australian National University,
Canberra, ACT 0200, Australia
I. Biological Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Dietary Carotenoids............................................................
B. Carotenoids in Photosynthetic Organisms..................................
II. Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
III. Carotenoid Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Isoprenoid Precursors..........................................................
B. Carotene Synthesis .............................................................
C. Xanthophyll Synthesis .........................................................
D. Cleavage Products ..............................................................
IV. Regulation of Carotenoid Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Transcriptional Regulation ...................................................
B. Metabolite Feedback...........................................................
C. Catabolism.......................................................................
D. Storage Capacity................................................................
V. Nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Rice ...............................................................................
B. Maize .............................................................................
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Corresponding author: E-mail: [email protected]
Advances in Botanical Research, Vol. 58

Copyright 2011, Elsevier Ltd. All rights reserved.
0065-2296/11 $35.00
DOI: 10.1016/B978-0-12-386479-6.00005-6
A. J. CUTTRISS ET AL.
C. Wheat ............................................................................
D. Cassava...........................................................................
E. Sorghum .........................................................................
F. Banana and Plantain ...........................................................
G. Sweet Potato ....................................................................
H. Potato ............................................................................
VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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ABSTRACT
Carotenoid pigments provide fruits and flowers with distinctive red, orange and
yellow colours as well as a number of aromas, which make them commercially
important in agriculture, food, health and the cosmetic industries. Carotenoids
comprise a large family of C40 polyenes that are critical for the survival of plants
and animals alike. -carotene and its derivatives contain unmodified -ionone
groups, which serve as precursors for vitamin A and are therefore essential dietary
components for mammals. Significant progress has been made towards producing
staple food crops with elevated provitamin A carotenoids, an important first step in
alleviating worldwide vitamin A deficiency. Recent insights into the regulatory
processes that control carotenoid composition and content may further advance
biofortification projects.
ABBREVIATIONS
LCY
eLCY
OH
eOH
ABA
CCD
CRTISO
CsZCD
DMAPP
DXP
DXS
GGPP
IPP
MEP
MVA
NCED
NPQ
NXS
lycopene -cyclase
lycopene e-cyclase
-hydroxylase
e-hydroxylase
abscisic acid
carotenoid cleavage dioxygenases
carotenoid isomerase
crocus zeaxanthin 7,8(70 ,80 )-cleavage dioxygenase
dimethylallyl diphosphate
deoxy-D-xylulose 5-phosphate
deoxy-D-xylulose 5-phosphate synthase
geranylgeranyl diphosphate
isopentenyl diphosphate
methylerythritol 4-phosphate
mevalonic acid
9-cis-epoxycarotenoid dioxygenase
non-photochemical quenching
neoxanthin synthase
CAROTENOIDS
PDS
PSY
VDE
ZDS
ZEP
Z-ISO
phytoene desaturase
phytoene synthase
violaxanthin de-epoxidase
-carotene desaturase
zeaxanthin epoxidase
15-cis--carotene isomerase
I. BIOLOGICAL FUNCTION
A. DIETARY CAROTENOIDS
Carotenoids are a vital component of mammalian diets, providing precursors

for vitamin A biosynthesis. Antioxidants and their dietary uptake can pigment the tissues of animals such as fish, crustaceans and birds. Vitamin A
(all-trans-retinol) is generated from unmodified -ring containing provitamin
A carotenoids in the diet (von Lintig, 2010), of which -carotene (two
nonhydroxylated -ionone rings), is the most efficient, because it can generate up to two retinol molecules. -carotene and -cryptoxanthin also contain
provitamin A potential, but only have a single nonhydroxylated -ring
(Davis et al., 2008).
Vitamin A deficiency is responsible for a number of disorders that range
from impaired iron mobilization, growth retardation and blindness to a
depressed immune response, as well as increased susceptibility to infectious
disease (Sommer and Davidson, 2002). Between 140 and 250 million children
are at risk of vitamin A deficiency (Underwood, 2004); 250,000500,000
become blind every year and half will die within 12 months after losing
their sight (http://www.who.int/nut/vad.htm). Simply improving the vitamin
A status of children, by increasing the uptake of provitamin A (e.g. - and
-carotene), can reduce overall child mortality by 25% (http://www.unicef.
org/immunization/facts_vitamina.html).
Low serum levels of vitamin A (less than 0.7 mol L 1) can be used as a
population-based indicator of health risks (Underwood, 2004). Recommended daily allowances for vitamin A range from 300600 g for children
to 9001300 g for adults of retinol activity equivalents (retinol and provitamin A carotenoids; Fig. 1). There is no recommended daily allowance for
provitamin A carotenoids, as the conversion efficiency remains imprecise;
however, between 3 and 6 mg of -carotene daily is sufficient to maintain
healthy serum carotenoid levels, as would five or more servings of fruits and
vegetables per day (Panel on Micronutrients, 2001).
HO
b-Cryptoxanthin
a-Carotene
b-Carotene
O
All-trans-retinal
OH
All-trans-retinol
O
OH
Retinoic acid
11-cisRetinal
O
Fig. 1. Vitamin A and carotenoid precursor structures. Common dietary provitamin A carotenoids with unmodified -ionone rings (highlighted in orange/dark grey)
are processed to form C20 retinoids, including all-trans-retinol (vitamin A, highlighted
in yellow/light grey), all-trans-retinal, retinoic acid and 11-cis-retinal, a photoreceptor
chromophore.
B. CAROTENOIDS IN PHOTOSYNTHETIC ORGANISMS
Carotenoids play a variety of crucial roles in photosynthetic organisms.

Carotenoids are involved in photosystem assembly where they contribute
to harvesting light in a broader range of wavelengths in the blue region of the
visible light spectrum and subsequently transfer the energy to chlorophyll
(Fig. 2). The distinctive yellow colours of light-harvesting carotenoids
become visible during autumn when chlorophyll degrades. The colour of
carotenoids, typically ranging from pale yellow to red is defined by the
number of conjugated double bonds along the C40 backbone as well as
other structural and oxygenic modifications that impart different spectral
properties. Carotenoids also provide protection from excessive light via
CAROTENOIDS
Chloroplast
PSII
PsbS
LHC
Xanthophyll
cycle
Thylakoid
membrane
Fig. 2. Photoprotective carotenoids in chloroplast membranes and proteins. Carotenoids accumulate in chloroplast thylakoid membranes, as indicated by this simplified schematic. Xanthophylls, such as lutein, zeaxanthin, violaxanthin and
neoxanthin, accumulate in light-harvesting complex proteins (LHC) where they
have a structural role and contribute to light harvesting. -carotene molecules in
the photosystem II reaction centre (PSII) could quench singlet oxygen or possibly
have a role in electron transfer. In high light, zeaxanthin is formed from violaxanthin
via the xanthophyll cycle. Zeaxanthin, lutein, PsbS and specific antenna proteins all
contribute to non-photochemical quenching of chlorophyll fluorescence; note, the
exact locations of each are not depicted in this cartoon.
energy dissipation and free radical detoxification, which limits damage to

membranes and proteins (DellaPenna and Pogson, 2006).
Plants need to maintain a balance between absorbing sufficient light for
photosynthetic processes and avoiding oxidative damage caused by high
light. Complementary photoprotective mechanisms are employed to minimize photodamage induced by exposure to high light and these include (1)
the harmless dissipation of excess energy via non-photochemical quenching
(NPQ) that is mediated by certain xanthophylls (zeaxanthin, antheraxanthin
and lutein), (2) quenching of triplet chlorophylls by carotenoids, (3) accumulation of antioxidants (ascorbate, tocopherols and carotenoids) and (4)
activation of antioxidant enzymes such as ascorbate peroxidase that
de-toxify free radicals, as well as repair damaged proteins (Bailey and
Grossman, 2008; Niyogi, 1999).
The physiological relevance of xanthophylls is exemplified by the bleaching, delayed greening, viviparous and semi-lethal phenotypes observed in
several carotenoid- and NPQ-deficient mutants (Neill et al., 1986; Niyogi
et al., 1997; Pogson et al., 1998; Robertson et al., 1966; Treharne et al., 1966;
Wurtzel, 2004). Alterations in the carotenoid pool size make the xanthophyll
cycle affect plant fitness. Increasing the xanthophyll cycle pool by overexpressing the bacterial OH gene (chyB) enhances stress tolerance in
Arabidopsis (Johnson et al., 2008). Zeaxanthin prevents oxidative damage
of the thylakoid membranes and plants with reduced zeaxanthin exhibit
increased sensitivity to light stress (Havaux and Niyogi, 1999; Verhoeven
et al., 2001). Conversely, a lycopene -cyclase (LCY) mutant that lacks
zeaxanthin but accumulates additional lutein and -carotene (suppressor of
zeaxanthin-less1, szl1) exhibits a partially restored quenching efficiency,
suggesting that lutein may substitute for zeaxanthin (Li et al., 2009).
II. DISTRIBUTION
Carotenoids are synthesized by all photosynthetic organisms, some bacteria
and fungi. Other organisms, such as humans, must acquire carotenoids
through dietary intake. For instance, the commercially significant pigment
astaxanthin is primarily synthesized by microorganisms, such as the green
alga Haematococcus pluvialis and is accumulated by fish such as salmon, thus
colouring their flesh red. In the case of lobster and other crustaceans, astaxanthins spectral properties are modified by the protein, crustacyanin, which
results in blue pigmentation that shifts to red upon cooking, which causes
protein-pigment denaturation (Britton et al., 1997). Flamingos can also
make use of carotenoids cosmetically and when the birds applied canthaxanthin-rich secretions onto their feathers, their courting behaviour became
more frequent during mating seasons due to a visually more attractive
breeding partner (Amat et al., 2010). Humans have been using carotenoids
and their derivatives, such as bixin, as food additives, as well as for cosmetic
purposes (Bouvier et al., 2003a).
Curious exceptions to the lack of synthesis of carotenoids by animals include
the synthesis of carotenoids in the human protist parasites, Plasmodium and
Toxoplasma (Tonhosolo et al., 2009), which is explained by the existence of a
remnant plastid, known as an apicoplast. An aphid genome was found to
encode enzymes for carotenoid biosynthesis, which was the result of lateral
gene transfer from a fungus, thus making aphids the only known animal to date
capable of synthesizing their own carotenoids (Moran and Jarvik, 2010).
Carotenoid accumulation relies on the presence of structures capable of
storing and retaining carotenoids. During the transformation of a chloroplast
into a chromoplast, carotenoids become localized in plastoglobuli before incorporation into the chromoplast (Tevini and Steinmuller, 1985). Carotenoids
within plastoglobuli exhibit much higher light stability than carotenoids within
CAROTENOIDS
chloroplast membranes, suggesting that pigments are better protected from

light destruction in these structures (Merzlyak and Solovchenko, 2002).
Cyanobacterial mutants with inactivated plastoglobulin-like genes are especially sensitive to altered light regimes, and the plastoglobulin-like peptides
accumulate to a greater extent in wild-type cultures that are exposed to high
light (Cunningham et al., 2010). Chromoplasts also accumulate carotenoids in
lipoprotein structures (Bartley and Scolnik, 1995; Vishnevetsky et al., 1999) that
are sequestered as crystals. For example, in a novel cauliflower mutant with
orange curd, Or, -carotene accumulates in the plastids of the pith and curd as
sheets, ribbons and crystals (Li et al., 2001; Lu et al., 2006).
There are other plastid organelles capable of storing carotenoids. These
include the colourless amyloplasts, which store starch granules (Kirk and
Tiliney-Bassett, 1978). Lutein is the predominant carotenoid present in many
seed amyloplasts such as wheat (Hentschel et al., 2002; Howitt et al., 2009),
whereas maize exhibits great diversity in terms of pigment composition
(Harjes et al., 2008). Leucoplasts are characteristic of mature root cells and
accumulate trace levels of neoxanthin and violaxanthin, which amount to
only 0.030.07% of the levels in light-grown leaves (Parry and Horgan, 1992).
Elaioplasts are specialized lipid-storing plastids and provide an ideal hydrophobic sink for accumulation of carotenoids. The dark-grown etioplast is
distinguished by the prolamellar body, a uniformly curved lattice of tubular
membranes, which contains several of the biochemical building blocks
required for the chloroplast (Gunning and Jagoe, 1967) including the xanthophylls, lutein and violaxanthin (Joyard et al., 1998). The Arabidopsis crtiso
(ccr2) mutant accumulates tetra-cis-lycopene and lacks a prolamellar body.
Thus, a mutation in carotenoid biosynthesis apparently disrupts membrane
curvature and stabilization of the prolamellar body (Park et al., 2002).
The absence of this structure in CRTISO mutants suggests that different
carotenoids either directly or indirectly impede formation of the membrane
lattices, which results in a delay in plastid development and greening upon
exposure to light. These data demonstrate an important role for carotenoids
in plastid differentiation (Park et al., 2002).
III. CAROTENOID BIOSYNTHESIS

A. ISOPRENOID PRECURSORS
Isoprenoids (or terpenoids) are a large and diverse class of naturally occurring organic chemicals derived from five-carbon isoprene units. Carotenoids
are derived from two isoprene isomers, isopentenyl diphosphate (IPP) and
dimethylallyl diphosphate (DMAPP). The same precursors are used to make

a diverse range of compounds that include tocopherols, chlorophylls,
phylloquinone, gibberellins, abscisic acid (ABA), monoterpenes and plastoquinone. The biosynthesis of isoprenoid precursors has been covered in detail
elsewhere (Rodriguez-Concepcion, 2010).
Two distinct pathways exist for IPP production: the mevalonic acid
(MVA) pathway and the mevalonate-independent, methylerythritol 4-phosphate (MEP) pathway (Lange et al., 2000). The plastid-localized MEP
pathway combines glyceraldehyde-3-phosphate and pyruvate to form
deoxy-D-xylulose 5-phosphate (DXP), a reaction catalysed by DXP synthase
(DXS). A number of steps are then required to form geranylgeranyl diphosphate (GGPP), the precursor to carotenoid biosynthesis. The Arabidopsis
Cla1 mutant, in which the DXS gene of the MEP pathway is disrupted, is
photobleached because of the absence of protective carotenoids (Araki et al.,
2000; Estevez et al., 2000). Conversely, overexpression of PSY (phytoene
synthase) resulted in increased carotenoid accumulation and a concomitant
accumulation of the DXS enzyme (Rodriguez-Villalon et al., 2009).
B. CAROTENE SYNTHESIS
1. Phytoene synthase
The first committed step is the condensation of two molecules of GGPP to
produce phytoene (Fig. 3). This reaction is catalysed by PSY in higher plants
and bacteria (CrtB; Armstrong, 1994). PSY is a single-copy gene in Arabidopsis but present in multiple copies in other plants such as rice, maize and
cassava, all of which have three copies that are expressed in different tissues
and show differential responses to environmental stimuli (Arango et al.,
2010; Li et al., 2008a,b; Welsch et al., 2008). PSY is a rate-limiting step and
a dosage effect of the maize Y1 allele was noted as early as 1940 (Randolph
and Hand, 1940). Overexpression of an exogenous daffodil PSY in rice
endosperm leads to phytoene accumulation, the first instance of carotenoid
engineering in rice (Burkhardt et al., 1997).
2. Desaturases (PDS and ZDS)

Phytoene is produced as a 15-cis isomer, which is subsequently converted to
all-trans isomer derivatives (Beyer et al., 1989; Chen et al., 2010). Two
desaturases, phytoene desaturase (PDS) and -carotene desaturase (ZDS),
catalyse a series of dehydrogenation reactions by introducing four double
bonds to form lycopene. Desaturation is linked to a plastidic respiratory
CAROTENOIDS
GGPP
OPP
PSY
15-cis-Phytoene
PDS
9,15,9-Tri-cis--carotene
Z-ISO
9,9-Di-cis--carotene
ZDS
7,9,9,7-Tetra-cis-lycopene
CRTISO
All-trans-lycopene
bLCY
eLCY
bLCY
aCarotene
bCarotene
bOH
bOH
Zeinoxanthin
OH
eOH
Zeaxanthin
OH
HO
Lutein
OH
VDE
ZEP
HO
Violaxanthin
OH
O
O
HO
NCED
ABA
HO
NXS
9-cis-Neoxanthin
C
OH
OH
Fig. 3. Carotenoid biosynthetic pathway in higher plants. The pathway shows the
primary reactions found in nearly all plant species. Grey shaded areas on carotenoid
structures indicate site of activity for each biosynthetic enzyme. ABA, abscisic acid;
LCY, lycopene -cyclase; OH, -hydroxylase; CRTISO, carotenoid isomerase;
eLCY, lycopene e-cyclase; eOH, e-hydroxylase; NCED, 9-cis-epoxycarotenoid dioxygenase; NXS, neoxanthin synthase; PDS, phytoene desaturase; PSY, phytoene
synthase; VDE, violaxanthin de-epoxidase; ZDS, -carotene desaturase; ZEP,
zeaxanthin epoxidase; Z-ISO, 15-cis--carotene isomerase.
10
redox chain (Nievelstein et al., 1995) and evidence for a quinone requirement
was demonstrated in daffodil and Arabidopsis (Beyer, 1989; Norris et al., 1995).
3. Isomerases (Z-ISO and CRTISO)
Recent biochemical evidence confirms that the desaturation reactions in
plants proceed via various cis intermediates, including 9,15,90 -tri-cis--carotene, 9,90 -di-cis--carotene and 7,9,90 -tri-cis-neurosporene (Chen et al., 2010;
Isaacson et al., 2004). Thus, all-trans-lycopene, the preferred substrate for the
cyclases, is produced by the desaturases in concert with two isomerases.
The first isomerase was identified in Arabidopsis and tomato (Isaacson
et al., 2002; Park et al., 2002). Lesions in CRTISO result in accumulation
of cis-carotene isomers in dark-grown plants (Park et al., 2002). Characterization of the maize recessive y9 mutant demonstrated that, like crtiso
mutants, the phenotype could be rescued by light exposure, to form 9,90 -dicis-zeta-carotene, the substrate for ZDS (Li et al., 2007). The Z-ISO gene was
identified in both maize and Arabidopsis and found to be similar to NnrU
(for nitrite and nitric oxide reductase U), which is required for bacterial
denitrification, a pathway that produces nitrogen oxides as alternate electron
acceptors for anaerobic growth. An Escherichia coli assay proved that Z-ISO
was capable of 15-cis bond isomerization in 9,15,90 -tri-cis--carotene
(Chen et al., 2010).
In the Arabidopsis CRTISO (ccr2) and Z-ISO mutants, cis intermediates
are photoisomerized in the light, which raises questions about the necessity of
carotenoid isomerases in plants and why there are four genes required for the
synthesis of lycopene in plants but only one in bacteria. In chromoplasts,
CRTISO activity is required for all-trans-lycopene accumulation, regardless
of the light regime, because the tangerine mutant accumulates tetra-cislycopene in the light (Isaacson et al., 2002). Carotenoids are deposited in a
crystalline form in tomato chromoplasts and these may be more resistant to
photoisomerization. Further, although the biosynthetic pathway proceeds in
chloroplasts, a delayed greening and substantial reduction in lutein occurs
in mutants defective in CRTISO in Arabidopsis and some chlorosis occurs in
rice and tomato leaves (Fang et al., 2008; Isaacson et al., 2002; Park et al.,
2002). Thus, carotenoid synthesis in dark-grown tissues absolutely requires
isomerase activity. Such tissues include the endosperm, a target for provitamin A carotenoid biofortification.
4. Cyclases
After lycopene, the carotenoid biosynthetic pathway divides into two
branches, distinguished by different cyclic end groups, namely beta or epsilon. Two -rings form the , branch (-carotene and its derivatives) with
CAROTENOIDS
11
one - and one e- forming the ,e branch (-carotene and its derivatives).
LCY introduces a -ionone ring to either end of all-trans-lycopene to
produce -carotene, whereas both the -cyclase and e-cyclase enzymes are
required to form -carotene (Cunningham and Gantt, 2001). Curiously,
mutated maize endosperm tissue lacking LCY activity was also found to
accumulate lactucaxanthin (e,e-ring) and other unusual carotenes, including
-carotene, and e-carotene. The ratio of LCY:eLCY transcripts correlated
with the accumulation of different cyclization products in embryo and endosperm tissues (Bai et al., 2009). eLCY expression is important in controlling
pathway flux to carotenes with higher provitamin A value and the breeding
alleles that have been developed for breeding high-provitamin A maize
(Harjes et al., 2008).
Other cyclase activities include the capsanthincapsorubin synthase (CCS)
(Lefebvre et al., 1998) in capsicum that cyclizes lycopene to produce the
-cyclic carotenoids, capsanthin and capsorubin. CCS was found to contain
a noncovalently bound flavin adenine dinucleotide (FAD), though it was
only required for activity in the presence of NADPH, which functions as the
FAD reductant. The CCS flavoproteins catalyse reactions with no net redox
change as the reaction did not transfer hydrogen from the dinucleotide
cofactors to -carotene or capsanthin. Thus, FAD in its reduced form
could be implicated in the stabilization of the carbocation intermediate
(Mialoundama et al., 2010).
C. XANTHOPHYLL SYNTHESIS
Xanthophylls are oxygenated derivatives of carotenes and play important

roles in photoprotection and light-harvesting antennae formation (Niyogi,
1999).
1. Hydroxylases
Nearly all xanthophylls in higher plants have hydroxyl moieties on the
3-carbon in the - or -carotene rings to form zeaxanthin and lutein, respectively. There are two distinct hydroxylation reactions of the e- and -rings,
confirmed by the identification of the e-hydroxylase (eOH) locus, lut1
(Pogson et al., 1996), and the -hydroxylase (OH) genes in higher plants
(Sun et al., 1996). OH enzymes are ferredoxin dependent and contain an
iron-coordinating histidine cluster that is required for activity (Bouvier et al.,
1998). In contrast, eOH is a plastid-targeted cytochrome P450-type monooxygenase with a distinctly different enzymatic mechanism from the OHs
(Tian et al., 2004).
12
OH activity is an important provitamin A biofortification target, as

hydroxylation or any other modification of -ionone rings depletes vitamin
A potential. Thus, reduced hydroxylase activity will result in fewer -rings
modifications, thereby maintaining -carotene pool and maximum vitamin
A potential. Of the six loci encoding this enzyme, one locus, HYD3, was
found to be critical for maize endosperm -carotene levels and alleles were
identified in a population of 51 maize lines (Vallabhaneni et al., 2009) and
further association and linkage population studies in maize found that
this gene was indeed responsible for a QTL associated with -carotene
accumulation (Yan et al., 2010), and in combination with LCY alleles
(Harjes et al., 2008), it is now possible to use molecular markers to select
for high-provitamin A carotenoid maize seeds.
2. Zeaxanthin epoxidase and violaxanthin de-epoxidase
An epoxide group is introduced into both rings of zeaxanthin by zeaxanthin
epoxidase (ZEP) to form violaxanthin. Under high light stress, the reverse
reaction is rapidly undertaken by the violaxanthin de-epoxidase (VDE;
Yamamoto, 1979). Light is critical in modulating the interconversion of
zeaxanthin and violaxanthin. Under normal light conditions, when the incident light can be safely utilized for photosynthetic electron transport, ZEP
converts zeaxanthin to violaxanthin by introducing 5,6-epoxy groups to the
3-hydroxy--rings. However, when incident light is in excess, VDE converts
a substantial pool of violaxanthin to zeaxanthin (Pfundel et al., 1994).
VDE is soluble and inactive at neutral pH, but following acidification
(below pH 6.5) it attaches to the thylakoid membrane and its violaxanthin
substrate (Hager and Holocher, 1994). The thylakoid membrane lipid monogalactosyldiacylglycerol is needed for optimal VDE activity when assayed
in vitro and it requires ascorbate as a reductant (Schaller et al., 2010).
Structural analyses revealed that at neutral pH, VDE is monomeric and its
active site occluded within a lipocalin barrel, but acidification causes the
barrel to open and the enzyme dimerizes. The carotenoid substrate could fit
in a channel linking the two active sites of the dimer enabling de-epoxidation
of both violaxanthin -rings, thus forming zeaxanthin (Arnoux et al., 2009).
Site-directed mutagenesis of amino acid residues lying in close contact with
the two substrates supported the proposed substrate-binding sites and identified two residues, Asp-177 and Tyr-198, that are required for catalytic
activity (Saga et al., 2010).
ZEP mutants, aba1, are deficient in ABA and display a partially
de-etiolated phenotype, including reduced hypocotyl growth, cotyledon
expansion and the development of true leaves during late skotomorphogenic
growth. However, other ABA-deficient mutants lack this phenotype and
CAROTENOIDS
13
ABA application did not rescue the skotomorphogenesis, though it could be

phenocopied by the addition of fluridone, a carotenoid inhibitor that blocks
PDS activity. Thus, ZEP appears to have a role in skotomorphogenic growth
(Barrero et al., 2008).
3. Neoxanthin synthase
Conversion of violaxanthin to neoxanthin is performed by the enzyme
neoxanthin synthase (NXS), which was unequivocally identified in a novel
ABA-deficient Arabidopsis mutant, aba4. The predicted gene product is a
novel chloroplast membrane protein, and constitutive expression of ABA4 in
Arabidopsis led to increased accumulation of trans-neoxanthin. Significantly
reduced levels of ABA were synthesized in dehydrated aba4 mutants, demonstrating that ABA biosynthesis in response to stress must occur mainly via
neoxanthin isomer precursors (North et al., 2007). Detached aba4.1 leaves
were more sensitive to oxidative stress than the wild type and aba4.1 npq1
double mutants, lacking both zeaxanthin and neoxanthin, underwent stronger PSII photoinhibition (DallOsto et al., 2007).
D. CLEAVAGE PRODUCTS
Characterization of the carotenoid-cleavage gene family has yielded some

interesting results in recent years. The enzyme products are varyingly referred
to as carotenoid-cleavage dioxygenases (CCD) or 9-cis-epoxycarotenoid dioxygenases (NCED), reflecting the first characterized member of this gene family
(Schwartz et al., 1997; Tan, 1997). The nine members of the gene family in
Arabidopsis show different substrate specificity and tissue distribution
(Schwartz et al., 2001, 2003; Tan et al., 2003). The CCD gene family is
responsible for the formation of vitamin A, phytohormones (e.g. ABA and
strigolactones), coloured spices (e.g. saffron and bixin) and novel signalling
molecules as well as plant volatiles used in the perfume industry (Fig. 4).
1. Vitamin A
Vitamin A is a C20 cleavage product of carotenoids, which, in addition to its
retinoid derivatives, is essential for animal survival and vitamin A biosynthesis has recently been reviewed in detail (von Lintig, 2010). Cleavage of
-carotene was postulated as an important step in the formation on vitamin
A, but it was not until 2000 that a -carotene 15,150 -dioxygenase was cloned
from Drosophila melanogaster (von Lintig and Vogt, 2000) and chicken
(Wyss et al., 2000). The deduced amino acid sequence showed homology to
the maize carotenoid dioxygenase, VP14, involved in the synthesis of ABA.
14
Carotenoids
NCED/CCD Cleavage
OH
COOH
O
Abscisic acid
O
Strigol
b -Ionone
O
O
COOR
O
Geranyl acetone
Volatile apocarotenoids
Hormones
ROOC
Mycorradicin
External signaling
compounds
Fig. 4. Carotenoid cleavage products have diverse roles. Carotenoids are cleaved
by 9-cis-epoxycarotenoid dioxygenase (NCED) or carotenoid cleavage dioxygenase
(CCD) enzymes and further modified to form apocarotenoids with diverse functions.
Geranyl acetone and -ionone are volatile apocarotenoids that are commonly used in
fragrance manufacture. Mycorradicin is involved in recruiting beneficial fungi. Strigolactones such as strigol enhance the germination of harmful parasitic plant seeds
and modulate shoot branching as well as stimulate beneficial mycorrhizal fungi
symbiosis. Abscisic acid mediates plant stress responses, playing an important role
in controlling stomatal aperture and transpiration as well as promoting seed development and dormancy.
Any carotenoid containing an unmodified -ionone ring has provitamin A

activity; thus, -carotene is one of the most active because a single -carotene
molecule is cleaved to form two all-trans-retinal molecules, which are
reduced to form all-trans-retinol (vitamin A). All retinoids are derived from
this compound and maintain the characteristic -ionone ring. Different end
groups or -ionone ring modifications characterize the various retinoids. For
example, retinoic acid (or 11-cis-retinal), which is required for reproduction,
embryonic development, cell differentiation, immunity and other biological
processes, binds to opsin to provide a chromophore for the visual pigments
that mediate phototransduction (von Lintig, 2010).
2. Phytohormones
The plant hormone ABA is primarily involved in plant stress responses, seed
development and dormancy (Seo and Koshiba, 2002). ABA is a cleavage
product of 9-cis-violaxanthin and/or 90 -cis-neoxanthin, an idea that was first
proposed by Taylor and Smith (1967). Cleavage of 90 -cis-neoxanthin by
CAROTENOIDS
15
NCED produces xanthoxin and was first identified in the maize viviparous14
(vp14) mutant (Schwartz et al., 1997; Tan, 1997). Xanthoxin is followed in
the pathway by a number of further modified products that are required to
produce ABA (Seo and Koshiba, 2002). For the ABA signal to be transmitted, it must first bind a receptor molecule. The putative identification of such
receptors has been the topic of recent controversy, though the recent crystal
structure of a PYR/PYL (pyrabactin resistance/pyrabactin resistance-like) or
RCAR (regulatory component of ABA receptor) protein appears to resolve
this question (Park et al., 2009). ABA-bound PYR/PYL/RCAR protein
inhibits a phosphatase 2C that is known to participate in ABA signalling
(Ma et al., 2009).
Strigolactones are carotenoid-derived terpenoid lactones that inhibit shoot
branching and can be exuded from plant roots to recruit beneficial mycorrhizal fungi. This apocarotenoid signal has been hijacked by parasitic plant
seeds to encourage germination (Dun et al., 2009; Matusova et al., 2005).
Such a signal was initially proposed after novel CCD mutants were found to
exhibit increased shoot branching in Arabidopsis max4 and pea rms1
mutants (Sorefan et al., 2003). MAX3 (CCD7) (Booker et al., 2004) and
MAX4 (CCD8) can sequentially cleave -carotene to form the C18 compound 13-apo-carotenone (Schwartz et al., 2004). The recent discovery that
both rice and pea branching mutants were deficient in strigolactones resolved
years of speculation about the nature of the branching signal. It has been
shown that strigolactone application restores the wild-type branching phenotype in pea CCD8 mutants, confirming that strigolactones are necessary
and sufficient to inhibit shoot branching in plants. Further, the CCD8
mutants exhibited additional typical strigolactone-deficient phenotypes
including alterations to mycorrhizal symbiosis and parasitic weed interaction
(Gomez-Roldan et al., 2008). Concurrent studies confirmed that synthetic
strigolactone application inhibits tillering in rice D10 (CCD8) and D17
(CCD7) mutants as well as rescuing the equivalent Arabidopsis mutants.
An elegant indirect assay confirmed that these mutants were deficient in
strigolactone synthesis, as root exudates did not stimulate germination of
parasitic Striga seeds to the same extent as wild-type exudates (Umehara
et al., 2008). The CCD7 knockdown in tomato exhibited increased branching, but a metabolic screen did not identify any significant changes in root
carotenoid substrate. However, C13 cyclohexenone and C14 mycorradicin
apocarotenoids were reduced in response to mycorrhizal colonization, indicating that CCD7 is required for arbuscular mycorrhiza-induced apocarotenoid synthesis (Vogel et al., 2010).
Other components of the strigolactone biosynthetic pathway have been
identified, including MAX1, which encodes a cytochrome p450 that modifies
16
an apocarotenoid product of the CCD7 and CCD8 cleavage reactions to

produce another mobile intermediate (Booker et al., 2005). MAX2/RMS4/
D3 encode F-box proteins and the mutants are not rescued by exogenous
strigolactones and are thus predicted to have a role in signalling via ubiquitin-mediated protein degradation (Beveridge et al., 1996; Stirnberg et al.,
2002). Additional steps have been identified in rice, including another hightillering rice mutant, d27, which does not exude strigolactones. D27 is chloroplast localized, though its enzymatic activity has not been described.
Crosses with d10 (CCD8) are not additive and the d27 mutant can be rescued
by strigolactone application, thus is thought to be required for the biosynthesis of strigolactones (Lin et al., 2009). The D14 gene encodes a /-fold
hydrolase, and the d14 mutant is strigolactone insensitive, but exhibits
increased tillering and does not show an additive phenotype when crossed
with d10 (Arite et al., 2009). Characterization of this curious mutant could
provide insights into strigolactone signalling or have a role in producing a
bioactive strigolactone-derived hormone.
Strigolactone and ABA composition were analysed in plants treated with
inhibitors of specific carotenoid-cleavage enzymes. Strigolactone content was
reduced in plants treated with the CCD inhibitor, D2, but root ABA levels
were maintained. Conversely, plants with genetically or chemically inhibited
ABA biosynthesis also had reduced strigolactones and a concomitant reduction in CCD7 and CCD8 transcript abundance, implying a potential crosstalk role for ABA in the regulation of strigolactone biosynthesis (Lopez-Raez
et al., 2010). Finally, strigolactone biosynthesis and the concomitant branching phenotype are responsive to phosphate deficiency in Arabidopsis
(Kohlen et al., 2010). The role of strigolactones in controlling plant morphology and response to the environment has become an exciting area of active
research.
3. Bixin, saffron and plant volatiles
Carotenoid cleavage metabolites are vital for plants and animals. They are
also highly prized in the food and cosmetic industries. Bixin (annatto) is a
red-coloured, di-carboxylic monomethyl ester apocarotenoid, traditionally
derived from the plant Bixa orellana. Bouvier and colleagues identified a
lycopene cleavage dioxygenase, bixin aldehyde dehydrogenase and norbixin
carboxyl methyltransferase that are required to produce bixin from lycopene.
Co-transforming the appropriate constructs into E. coli, engineered to produce lycopene, resulted in bixin production at a level of 5 mg g 1 dry weight
(Bouvier et al., 2003a).
Saffron, another commercially important coloured compound, can attribute the majority of its characteristic colour, flavour and aroma to the
CAROTENOIDS
17
accumulation of carotenoid derivatives. A crocus (Crocus sativus) zeaxanthin

7,8(70 ,80 )-cleavage dioxygenase (CsZCD) was cloned and found to be targeted to the chromoplast and initiated the production of the cleavage products. Another enzyme, 9,10(90 ,100 )-cleavage dioxygenase was also cloned
and found to be a less specific cleavage enzyme (Bouvier et al., 2003b).
Beta-ionone is the predominant norisoprenoid volatile in the mature stigma tissue. Four CCD genes were isolated from crocus that were capable of
cleaving -carotene at the 9,10(90 ,100 ) positions to yield -ionone, though
with different expression patterns indicative of sub-functionalization (Rubio
et al., 2008). Differential expression was also observed for LCY genes,
CstLcyB1 and CstLcyB2a. The CstLcyB2a is chromoplast specific and conspicuously absent in crocus species with low apocarotenoid content, suggesting that it encodes an important step in determining the accumulation of
-carotene substrate that is required to produce the distinctive saffron apocarotenoids (Ahrazem et al., 2010).
4. Novel-signalling molecules
A putative novel signal has been observed in Arabidopsis bps1 mutants,
which are developmentally defective but the shoot can be rescued if the
roots are removed or carotenoid biosynthesis is chemically blocked with
norflurazon. It is hypothesized that an unknown substance moves constitutively from the root to the shoot to arrest growth, and this is supported by
experiments demonstrating that mutant roots are sufficient to arrest wildtype shoot development (Van Norman et al., 2004). BYPASS1 encodes a
novel protein of unknown function that is widespread in plant genomes
(Sieburth and Lee, 2010), though the tobacco homologue contains a transmembrane domain and GFP fusion proteins were endoplasmic reticulum
associated (Kang et al., 2008). It is likely that more novel carotenoid-derived
signalling molecules remain to be identified.
IV. REGULATION OF CAROTENOID BIOSYNTHESIS

A. TRANSCRIPTIONAL REGULATION
Carotenoid composition is responsive to environmental stimuli, oxidative

stress, redox poise and metabolite feedback regulation. In general, increases
in carotenoid accumulation, be it during fruit ripening, flower development
or production of stress-induced carotenoids in algae, coincide with increased
transcript abundance of some key (but not all) steps in the pathway.
18
Phytoene biosynthesis is a rate-limiting step in carotenogenesis and transcript abundance can dramatically alter carotenoid pool size, thus making
PSY a logical target in the study of carotenoid regulation. Changes in
transcript abundance are particularly evident during morphogenic changes
from etioplast to chloroplast or chloroplast to chromoplast. PSY transcript
abundance is upregulated during photomorphogenesis via a phytochromemediated (red-light) pathway, a response that is abolished in the phyA
mutant (Welsch et al., 2000, 2008). Phytochrome-mediated light signals
regulate carotenoid biosynthesis in plants by way of phytochrome-interacting
factor 1 (PIF1), which directly binds to the PSY promoter, thus repressing
PSY expression. Light-triggered degradation of PIFs by photoactivated
phytochromes during deetiolation permits PSY expression, which enables
rapid production of carotenoids (Toledo-Ortiz et al., 2010).
Further evidence that PSY controls metabolic flux was obtained by paclobutrazol treatment, which inhibits gibberellin synthesis and enables deetiolation despite the absence of light. PSY activity and carotenoid levels increased
in the dark following treatment with paclobutrazol, and this increase was
supported by feedback regulation of DXS protein abundance. Overexpression of DXS alone in etiolated tissue did not increase carotenoid accumulation; however, PSY overexpression resulted in increased carotenoid
accumulation and a concomitant post-transcriptional accumulation of
DXS (Rodriguez-Villalon et al., 2009).
PSY is present as a single copy in Arabidopsis, but additional homologues
have been identified in tomato, poplar and cereal crops such as rice, wheat
and maize (Chaudhary et al., 2010; Howitt et al., 2009; Li et al., 2008a,b;
Welsch et al., 2008). PSY homologues respond differently to abiotic stimuli
and have unique tissue specificities though their function remains redundant.
For example, salt and drought induce PSY3 transcript abundance in maize
roots, which correlated with increased carotenoid flux and ABA in maize
roots (Li et al., 2008a). Rapid disappearance of PSY2 and PSY3 mRNA
after rewatering suggests mRNA instability or strict control of transcription
(Li et al., 2008a). Similar responses were observed in rice PSY homologues
(Welsch et al., 2008). Cassava also has three sub-functionalized PSY genes;
however, it was not PSY3, but a PSY1 paralogue that responded to abiotic
stress (Arango et al., 2010). Perhaps the most dramatic enhancement of
carotenoid accumulation has been achieved in the oil seeds of canola (Brassica napus) and Arabidopsis, where overexpression of PSY in seeds resulted
in a 43- to 50-fold increase in total carotenoid content (Lindgren et al., 2003;
Shewmaker et al., 1999). PSY overexpression in Arabidopsis seedlings did
not alter carotenoid content. However, non-photosynthetic calli and roots
overexpressing PSY accumulated 10- to 100-fold more carotenoids than
CAROTENOIDS
19
corresponding wild-type tissues, predominantly -carotene and its derivatives, which were deposited as crystals. Similarly, overexpression of the
bacterial PSY, crtB, in white carrot roots also initiated carotenoid crystal
formation (Maass et al., 2009).
The complexity of carotenoid regulation is further demonstrated by the
analysis of the PSY promoter where a cis-acting motif (ATCTA) was identified to be involved in mediating the transcriptional regulation of photosynthetic genes, including PSY (Welsch et al., 2003). Manipulation of RAP2.2,
APETALA2 transcription factors that bind to the PSY promoter, resulted in
only minor carotenoid alterations in root calli (Welsch et al., 2007).
The relative activities of the eLCY and LCY at the branch point of the
pathway have a major regulatory role in modulating the ratio of lutein to that
of the -branch carotenoids (Cuttriss et al., 2007). CRTISO is a major
regulatory node at the branch point of the biosynthetic pathway
(Cazzonelli et al., 2009; Isaacson et al., 2004). A chromatin-modifying histone methyltransferase enzyme (SET DOMAIN GROUP 8, SDG8) has been
shown to be necessary for maintaining CRTISO gene expression (Cazzonelli
et al., 2009). The CRTISO and SDG8 promoters show overlapping patterns
of expression specifically in the shoot apical meristem and pollen, which are
active sites of cell division and epigenetic programming (Cazzonelli and
Pogson, 2010). The absence of SDG8 reduces CRTISO transcript abundance, thereby altering carotenoid flux through the pathway, which might
potentially impair strigolactone biosynthesis. This was the first report implicating epigenetic regulatory mechanisms in the control of carotenoid composition (Cazzonelli et al., 2009).
Allelic variation is another important source of carotenoid regulation.
For example, alternative splicing of the PSY-A1 allele altered the relative
abundance of functional PSY transcript and appeared to be a major QTL
determinant of flour colour in bread wheat (Howitt et al., 2009). This was
reiterated by a detailed analysis of natural genetic variation in maize.
Association analysis, linkage mapping, expression analysis and mutagenesis
confirmed that variation at the eLCY locus altered flux partitioning.
Four polymorphisms controlled 58% of the variation between - and
-branch accumulation, thus enabling the selection of alleles that confer
high-provitamin A status for improved maize varieties (Harjes et al., 2008).
Natural variation in OH activity also has a significant impact on carotenoid composition (Vallabhaneni et al., 2009; Yan et al., 2010). Multiple
control points both within the carotenoid pathway and MEP precursor
pathway were identified in maize, and the timing of gene expression was
found to be critical in determining carotenoid composition (Vallabhaneni
and Wurtzel, 2009).
20
B. METABOLITE FEEDBACK
Feedback regulation by ABA increases PSY3 gene expression in rice and

plays a critical role in the formation of a positive feedback loop that mediates
abiotic stress-induced ABA formation (Welsch et al., 2008). The LCY gene
from the eubacterium Erwinia herbicola and daffodil (Narcissus pseudonarcissus) flowers were introduced into the tomato plastid genome resulting in
increased accumulation of xanthophyll cycle pigments in leaves and -carotene in fruits. Surprisingly, transplastomic tomatoes showed > 50% increase
in total carotenoid accumulation (Apel and Bock, 2009), which may be due
to a carotenoid product or intermediate feedback.
Lutein levels are altered when the higher plant desaturases and isomerases
are bypassed, and thus cis-carotene intermediates are not produced (Misawa
et al., 1994). Similarly, the absence of CRTISO or specific carotene isomers
results in less lutein (Isaacson et al., 2002; Park et al., 2002). The mechanism
of this flux partitioning is unclear, though flux through the two branches can
be determined by eLCY mRNA levels (Cuttriss et al., 2007; Harjes et al.,
2008; Pogson et al., 1996; Pogson and Rissler, 2000) and recent experiments
indicate that both CRTISO (ccr2) and SDG8 (ccr1) mutants have
aberrant eLCY transcript levels. It is thus possible that feedback may
account for at least part of the reduction in lutein (Cazzonelli et al., 2009;
Cuttriss et al., 2007).
C. CATABOLISM
Accumulation of carotenoids in photosynthetic tissue requires a balance

between their rate of synthesis and catabolism. Recent 14CO2 uptake data
demonstrates that synthesis, and by inference, turnover, is much more rapid
than expected (Beisel et al., 2010). The incorporation of 14C into different
carotenoids was not uniform and varied between mutants and under high
light (Beisel et al., 2010), implying active degradation both enzymatically and
by oxidative damage.
Studies in Arabidopsis, strawberry (Fragaria ananassa) and chrysanthemum (Chrysanthemum morifolium) petals have all demonstrated that the
pool of carotenoids is determined in part by CCD catalysed degradation
(Auldridge et al., 2006; Garcia-Limones et al., 2008; Ohmiya et al., 2006). In
Arabidopsis seeds, loss of CCD function leads to significantly higher carotenoid levels (Auldridge et al., 2006).
CCD1 expression levels in strawberry correlate with ripening and a decrease in lutein content, which suggests that lutein could constitute the main
natural substrate of FaCCD1 activity (Garcia-Limones et al., 2008). High
CAROTENOIDS
21
expression of CCD1 associated with certain maize alleles was correlated with
low carotenoid levels in maize endosperm (Vallabhaneni et al., 2010). Petal
colour in chrysanthemums is also regulated by CCD activity; white petals
contain elevated transcript levels of CmCCD4a, which catabolizes the yellow
carotenoid pigments (Ohmiya et al., 2006). Curiously, when CCD1 was
overexpressed in high carotenoid golden rice lines (GR2), there appeared to
be little impact on carotenoid levels in the endosperm. In fact, a similar
carotenoid content was observed in both GR2 and antisense lines. Surprisingly, in vitro analyses suggested that apocarotenoids were the primary
substrates of OsCCD1 (Ilg et al., 2010).
D. STORAGE CAPACITY
Carotenoid biosynthesis appears to take place largely at the chloroplast

envelope and, in some cases, the thylakoid membrane (Joyard et al., 2009).
Storage capacity is a major determinant of carotenoid pool size; the high
pigment2 (hp2) tomato mutant (DEETIOLATED1, a negative regulator of
light signalling) has a larger plastid and thus increased pigmentation
(Kolotilin et al., 2007). Similarly, the hp3 tomato mutant (ZE) revealed an
ABA deficiency, an enlarged plastid compartment and 30% more carotenoids
in mature fruit (Galpaz et al., 2008). Plastid differentiation is an important
mechanism in determining storage capacity, as demonstrated by the cauliflower (Brassica oleracea) Orange (Or) gene that creates a metabolic sink to
accumulate -carotene in the chromoplast (Li et al., 2001; Li and Van Eck,
2007; Lu et al., 2006). During the chloroplast to chromoplast transformation
process, carotenoids become localized in plastoglobuli (Steinmuller and
Tevini, 1985). Carotenoids within plastoglobuli exhibit much higher light
stability than carotenoids within chloroplast membranes (Merzlyak and
Solovchenko, 2002).
V. NUTRITION
A. RICE
Golden rice (Oryza sativa) was developed to alleviate vitamin A deficiency as

this important staple crop does not typically accumulate any carotenoids in
edible endosperm tissue. Daffodil PSY and bacterial desaturases (crtI, Erwinia uredovora) were targeted to endosperm tissue, where they produced up to
1.6 g g 1 carotenoids, predominantly -carotene due to endogenous cyclase
activity (Ye et al., 2000). A second generation line Golden Rice 2 overcame
22
a metabolic bottleneck by incorporating a more active PSY gene from maize,

which substantially improved carotenoid biosynthesis, with some lines
accumulating up to 37 g g 1 (Paine et al., 2005). More recent work has
focused on transgene stability and the transformation of high-yielding
cultivars (Datta et al., 2006, 2007). A dietary study of Golden Rice confirmed
that deuterium-labelled [2H]a-carotene produced by these plants could
be converted to retinol and is thus an effective biofortification strategy
(Tang et al., 2009).
B. MAIZE
Zea mays is an essential staple cereal crop that naturally accumulates

provitamin A carotenoids in the endosperm of the seed. There are vast
diverse collections from which to source favourable alleles for plant breeding
programmes. Such collections have been extensively utilized to identify
important regulatory points in determining provitamin A potential. A significant QTL analysis determined that PSY1 was responsible for 6.627.2% of
phenotypic variation in carotenoid content (Chander et al., 2008). Genetic
variation in eLCY was responsible for 58% of the variation in flux between
the two branches of the pathway and is critical for driving provitamin A
levels (Harjes et al., 2008). Two recent studies identified different OH alleles
of one locus that were important in determining the extent of -ionone ring
hydroxylation, and thus loss of provitamin A activity (Vallabhaneni et al.,
2009; Yan et al., 2010). The most favourable alleles were found in temperate
varieties and will be bred into tropical maize germplasm to help alleviate
vitamin A deficiency in third world countries (Yan et al., 2010). Recent
studies also identified additional control points that offer future possibilities
for further enhancing carotenoid levels in maize (Vallabhaneni et al., 2010;
Vallabhaneni and Wurtzel, 2009). Transgenic approaches to maize biofortification have also played a significant role in modifying -carotene content
(Aluru et al., 2008; Zhu et al., 2008) and laid the foundation for targeting
alternative approaches. Analyses of tropical varieties (Menkir et al., 2008)
and sweet corn (Fanning et al., 2010) have identified further diversity for
carotenoid enhancement projects.
C. WHEAT
Triticum spp. endosperm colour is an important agronomic trait and thus has
been the focus of several QTL studies. Lutein is the predominant carotenoid
in wheat endosperm tissue and is frequently heavily esterified (Atienza et al.,
2007; Howitt et al., 2009). A targeted molecular marker was developed for
CAROTENOIDS
23
the PSY1 gene on wheat chromosome 7A, and found to co-segregate with
yellow pigmentation in a collection of Chinese wheat cultivars (He et al.,
2008). Further, the total carotenoid pool size was found to be modulated by
eLCY alleles and/or PSY-A1 spice variants (Howitt et al., 2009). Transgenic
wheat expressing endosperm-specific PSY1 from maize and bacterial CRTI
(desaturases) produced a 10.8-fold increase (up to 4.96 g g 1 dry weight) in
total seed carotenoid content (Cong et al., 2009). Thus, both targeted breeding and transgenic approaches are likely to improve wheat lutein content,
which is correlated with protection against age-related macular degeneration
(AMD) of the eyethe leading cause of blindness in the developed world.
Whether such strategies can increase provitamin A levels in wheat has not
been reported thus far.
D. CASSAVA
Manihot esculenta is an important staple crop, especially in arid regions such

as sub-Saharan Africa, though it is nutrient poor and typically accumulates
very little provitamin A. Analysis of diversity collections identified landraces
that accumulate lycopene (5 mg kg 1) or -carotene (4 mg kg 1) (Nassar
et al., 2007) and such variation was harnessed to identify natural PSY alleles
that altered metabolic flux (Welsch et al., 2010). Cassava has three PSY
genes, one of which (PSY1) responded strongly to abiotic stress (Arango
et al., 2010). A single nucleotide polymorphism in PSY2 was found to
co-segregate with yellow-rooted cultivars in a breeding population that
accumulated between 6.0 and 11.5 g g 1 carotenoids in fresh tissue. This
genetic variant was used to successfully produce transgenic cassava with
increased carotenoid accumulation in the roots (Welsch et al., 2010). Bioavailability of -carotene in cassava was analysed and found to be as efficacious as -carotene supplementation; thus, biofortification of cassava is a
valid approach to alleviating vitamin A deficiencies (Howe et al., 2009)
E. SORGHUM
Sorghum bicolor is a major staple crop grown in semiarid regions due to its
drought tolerance, which makes it a good candidate for biofortification.
Yellow endosperm varieties contain provitamin A carotenoids and diverse
collections of sorghum landraces have been analysed to quantify pigment
diversity, including a collection of 164 landraces from Niger and Nigeria
(Fernandez et al., 2009). Several QTL were identified that correlated with
total carotenoids or individual pigments, such as -carotene. A strong QTL
that accounted for between 11% and 15% of phenotypic variation was
24
associated with PSY3, thus pinpointing a focal point for breeding highprovitamin A sorghums (Fernandez et al., 2008).
F. BANANA AND PLANTAIN
Banana and plantain (Musa spp.) are tropical crops and some of the most highly
consumed fruits in the world. They have a high genetic diversity, as exemplified
by the Embrapa international germplasm collection of more than 400 accessions, including wild diploids, triploids and tetraploids; however, they are not
readily bred. Analysis of pigment composition identified 42 high pigment lines
that accumulate between 1.06 and 19.24 g g 1 of total carotenoids. Genetic
variability was estimated using Diversity Arrays Technology molecular markers to establish a biofortification programme (Amorim et al., 2009). A similar
study identified broad pigment diversity but limited accumulation of mineral
micronutrients in a 171 genotype collection (Davey et al., 2009).
G. SWEET POTATO
Proof of the biofortification principle was established in Kenya where consumption of the orange-fleshed sweet potato (Ipomoea batatas) increased the
vitamin A status of women and children (Hagenimana et al., 1999). A similar
study in South Africa demonstrated a reduction in vitamin A deficiency of
children (van Jaarsveld et al., 2005). However, analysis of carotenoid degradation in stored sweet potato, which is typically dried and stored for months,
indicated losses of around 70% of the total carotenoid pool after 4 months
storage in Uganda. This demonstrates the necessity for establishing diversity
in carotenoid-rich agricultural products and underlines the difficulty in
maintaining provitamin A intake outside of the growing season (Bechoff
et al., 2010).
H. POTATO
Another staple food crop with limited micronutrient content is potato (Solanum tuberosum). Potato has been successfully fortified to produce provitamin
A carotenoids. Overexpression of three bacterial genes for -carotene synthesis (CrtB, CrtI and CrtY, encoding PSY, PDS and LCY, respectively)
from Erwinia were targeted to the tuber. The transgenic lines accumulated up
to 47 g g 1 -carotene (Diretto et al., 2007). Detailed transcript analyses of
lines carrying various combinations of transgenes found that -cyclase had
the greatest impact on regulating the amount of carotenoid accumulation
(Diretto et al., 2010).
CAROTENOIDS
25
VI. CONCLUSIONS
The essential roles that carotenoids play in human health, as well as plant
photosynthesis, photoprotection and reproduction, make them obvious candidates for enhancement and manipulation. To this end, molecular genetics,
in concert with classical biochemistry, has facilitated an advanced understanding of the biosynthetic pathway. Breakthroughs in understanding the
regulation of carotenoid accumulation are paving the way for improving the
provitamin A content of staple food crops that would otherwise be of low
nutritional value. This is of utmost importance for developing countries,
where food storage is a problem and effective agriculture practices are still
being developed. Further characterisation of regulatory processes that determine carotenoid accumulation, composition and storage capacity, as well as
developing new transgenic technologies and breeding varieties, will all continue to strengthen biofortification projects in diverse crop species.
ACKNOWLEDGEMENTS
The authors acknowledge the support of Professor David Christopher, the
New Zealand Foundation for Research Science and Technology to A. J. C.;
funding from the United States National Institutes of Health (GM081160)
and New York State to E. T. W. and A. J. C; and the Australian Research
Council Centre of Excellence in Plant Energy Biology (CE0561495) to C. I.
C. and B. J. P.
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Vitamin B1 (Thiamine): A Cofactor for Enzymes Involved

in the Main Metabolic Pathways and an Environmental
Stress Protectant
MARIA RAPALA-KOZIK
Department of Analytical Biochemistry, Faculty of Biochemistry,

Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Structure and Biological Functions of Phosphorylated
Thiamine Analogues ...........................................................
B. Thiamine Deficiency Symptoms in Mammals..............................
II. Thiamine Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Thiamine Biosynthesis in Bacteria and Yeast ..............................
B. Genes and Proteins Involved in Plant Thiamine Biosynthesis and the
Cellular Distribution of the Biosynthetic Pathways.......................
C. Regulation of Plant Thiamine Biosynthesis ................................
III. TDP-dependent Enzymes in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Catalytic Mechanisms of TDP-Dependent Enzymes .....................
B. Classification of TDP-Dependent Enzymes and their Localization
within the Plant Cell ...........................................................
IV. Thiamine Transport, Distribution and Storage in Plant Tissues . . . . . . . . . . .
V. Role of Thiamine in the Sensing, Response and Adaptation to Plant Stress
A. Abiotic Stress Responses ......................................................
B. Thiamine Function in Biotic Stress ..........................................
C. Rescue of Stressed Plants by Thiamine SupplementationIs Thiamine
a Real Antioxidant? ............................................................
VI. Practical Aspects and Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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DOI: 10.1016/B978-0-12-386479-6.00004-4
38
M. RAPALA-KOZIK
ABSTRACT
Thiamine (vitamin B1) is essential for human metabolism and is particularly important
for proper brain functioning. Plants, which are the best source of this vitamin for human
nutrition, synthesize thiamine in three stages. The first of these involves the independent
formation of thiazole and pyrimidine moieties. In the next phase, these are coupled
together to form thiamine monophosphate. The final step results in the formation of the
active form of vitamin B1, thiamine diphosphate, which functions as a major enzymatic
cofactor. The biosynthesis of thiamine is regulated through feedback inhibition by the
end product of the pathway, that is, thiamine diphosphate. This regulatory mechanism
involves the binding of thiamine diphosphate by mRNA elements, riboswitches (THIBOXes). The transport of thiamine and thiamine diphosphate between plant tissues and
into cell compartments determines the proper functioning of major metabolic pathways
such as the acetyl-CoA synthesis, the tricarboxylic acid cycle, the pentose phosphate
pathway, CalvinBenson cycle and isoprenoid biosynthesis pathway. The recently
reported activation of thiamine production in plant cells under biotic or abiotic stress
conditions also suggests a non-cofactor role of this vitamin as a stress alarmone or stress
protectant to enable plants to survive in unfavourable environments.
I. INTRODUCTION
The discovery of vitamin B1 was made from studies of plants, with the finding
by Umetaro Suzuki in 1910 that unpolished rice could cure patients with a
nutritional deficiency-based disease, beriberi. Two years later, Casimir Funk
isolated the compound from rice bran (Funk, 1912) and its biosynthesis was
accomplished in 1935 by Robert R. Williams who first coined the name
thiamine for this vitamin (Williams and Cline, 1936). Thiamine is essential
for the normal growth and development of all living organisms. It plays a
crucial role in carbohydrate metabolism, NADPH and ATP biosynthesis and
in the production of nucleic acid pentoses. In mammals, thiamine is also
essential for the proper functioning of the heart, muscles and nervous system.
The biologically active form of vitamin B1 is thiamine diphosphate (TDP),
which in most organisms is formed from free thiamine in a one-step process
catalysed by thiamine pyrophosphokinase (TPK). Thiamine can be synthesized de novo, that is, from simple precursors, in bacteria, yeast and plants.
However, humans and other mammals are dependent on its dietary uptake.
A. STRUCTURE AND BIOLOGICAL FUNCTIONS OF PHOSPHORYLATED
THIAMINE ANALOGUES
The thiamine molecule is composed of two heterocyclic moieties, a substituted pyrimidine (4-amino-2-methyl-5-pyrimidyl) and substituted thiazole
(4-methyl-5-(2-hydroxyethyl)-thiazolium) rings which are linked by a
methylene bridge (Fig. 1).
39
VITAMIN B1 (THIAMINE)
NH2
CH3
+
N
OH
CH3
Thiamine
NH2
CH3
OH
+
N
O
CH3
OH
O
OH
O
OH
TMP
TDP
TTP
NH2
OH
OH
+
N
N
CH3
NH2
CH3
O
S
P
O
P
O
OH
O
P
O
O
O
ATTP
OH
OH
Fig. 1. Chemical structure of thiamine (vitamin B1) and its biologically occurring
phosphorylated derivatives.
In living organisms, thiamine is present in its free form and also as four
phosphorylated derivatives, thiamine monophosphate (TMP), TDP, thiamine triphosphate (TTP) and adenosine thiamine triphosphate (ATTP).
TMP is a product of thiamine biosynthetic pathways in bacteria, plants
and yeast and is a reservoir for further transformations to thiamine or
TDP. However, no other physiological function has been proposed for this
compound. TDP is the main thiamine compound that functions as a cofactor
for a number of enzymes involved in major metabolic pathways. These
critical TDP-dependent enzymes include pyruvate dehydrogenase (PDH),
-ketoglutarate dehydrogenase (KGDH), branched-chain -ketoacid dehydrogenase (BCKDH), transketolase (TK) and pyruvate decarboxylase
(PDC; Frank et al., 2007). TTP represents the smallest fraction of the total
thiamine pool in humans, but it has been proven to play an important role in
the physiology of the nervous system (Gangolf et al., 2010b) owing to its
involvement in the phosphorylation of key regulatory proteins (Nghiem
et al., 2000) and in the activation of high-conductance anion channels in
nerve cells (Bettendorff et al., 1993). Recent reports of the common
40
M. RAPALA-KOZIK
occurrence of this compound in bacteria, fungi and plants under specific

metabolic conditions (e.g. amino acid starvation) suggest its more general
cellular function as an alarmone (Bettendorff and Wins, 2009;
Makarchikov et al., 2003). ATTP has only recently been identified
(Bettendorff et al., 2007) and has been since detected at a minimal level in
mammalian tissues and in some cell lines (Frederich et al., 2009; Gangolf
et al., 2010a). However, the levels of ATTP are dramatically increased in
Escherichia coli in response to carbon starvation (Gigliobianco et al., 2010).
In human, the main fraction of total thiamine contains TDP (7280%)
which exists mostly in a form that is bound to TDP-dependent enzymes. Free
thiamine and TMP constitute about 2026% of the total thiamine content
and appear to be a flexible fraction of this vitamin pool that is easily
transferable and transformable into TDP or TTP, depending on the requirements at the time. TTP constitutes only 12% of the total thiamine (Gangolf
et al., 2010a; Lonsdale, 2006). The best sources of vitamin B1 for human
consumption are cereals, whole grains (especially wheat germ), fortified
bread, beans, peas, soybeans, nuts, fish, eggs and lean meats (especially
pork). The average physiologic requirement for thiamine is about 1.5 mg
per day for humans but this value may vary with age, gender and living
conditions (e.g. physical activity, stress or pregnancy; Linus Pauling Institute
Recommendation; Rakel, 2007). A content of thiamine in various plants of
nutritional interest is presented in Table I.
B. THIAMINE DEFICIENCY SYMPTOMS IN MAMMALS
The clinical symptoms of thiamine deficiency in humans manifest in the

cardiovascular system (such as wet beriberi, which is associated with
vasodilatation, myocardial failure, edema and fulminant cardiovascular collapse) and the nervous system (dry beriberi which is related to mental
confusion, a disordered ocular motility and ataxia; also WernickeKorsakoff
syndrome, a neuropathy). However, a thiamine deficiency may not only be
the result of a low-vitamin diet problem but may also arise due to metabolic
dysfunction. The available thiamine levels in cells depend on (i) the absorption of thiamine from gastrointestinal tract, (ii) the effective phosphorylation
to TDP, (iii) active TDP transport to organelles and (iv) the incorporation of
TDP into properly functioning TDP-dependent enzymes. For each of these
events, some disturbances may evoke thiamine deficiency symptoms. Additionally, in some countries, seasonal ataxia is observed due to the consumption of local special meals (shellfish, raw fermented fish or pupae of
the African silkworm) that contain heat labile enzyme, thiaminase,
which effectively degrades thiamine molecules (Bos and Kozik, 2000;
41
TABLE I
Average Thiamine Content in Plant Foods
Thiamine (mg/100 g)
Thiamine (mg/100 g)
Cereal:
Cornmeal
Oatmeal
Rice (brown)
Sorghum
Wheat (whole grain)
0.18
0.62
0.33
0.15
0.41
Pulses, nuts and seeds:

Beans
Chickpeas
Groundnuts
Lentils
Peas
Soybeans (dry whole seeds)
0.460.63
0.40
0.84
0.50
0.72
1.03
Tubers/starchy roots:
Cassava
Potato
Yam
0.06
0.10
0.09
Vegetables:
Broccoli
Cabbage
Carrots
Cassava leaves
Cauliflower
Spinach
Tomatoes
0.10
0.06
0.06
0.16
0.11
0.11
0.06
Fruits:
Bananas
Breadfruit
Grapes
Mangoes
Oranges
Pineapples
0.05
0.09
0.06
0.05
0.08
0.08
Source: WHO (1999). Thiamine deficiency and its prevention and control in major emergencies.
Jenkins et al., 2007). In addition, heat-stable polyphenolic compounds which

are produced in plants (ferns, tea, coffee, betel nuts) may react with thiamine
to yield non-absorbable forms of this molecule (Hilker and Somogyi, 1982).
1. Damage to the uptake or transport of thiamine and TDP
Thiamine is distributed between tissues via the bloodstream and the blood
thiamine level is critically dependent on the intestinal thiamine absorption
which requires thiamine to cross the brush border and basolateral membranes of the enterocytes (Ricci and Rindi, 1992; Rindi and Laforenza,
2000). To be available for uptake by neuronal cells, thiamine must additionally cross the bloodbrain barrier to reach the cerebrospinal fluid (Tallaksen
et al., 1993). The active intestinal transport of thiamine at its physiological
concentrations involves two types of membrane transporters, THTR1 (the
product of SLC19A2 gene in humans) and THTR2 (SLC19A3 gene; Fig. 2)
which probably function through a thiamine/H antiport mechanism.
THTR1 operates at the brush border membrane and undergoes saturation
at micromolar thiamine concentrations, whilst THTR2 becomes saturated at
nanomolar levels (Said et al., 2004). The entry of thiamine into the
42
M. RAPALA-KOZIK
Diabetes
Alcohol
TRMA
TA
THTR1
TPK
TA
TA
TK
TDP
THTR2
DVC
PH
TMP
TDP
RFC1
TMP
RFC1
TDP
RFC1
RFC1
TDP PDC
KGDH
MITOCHONDRION
WKS
OUT
LA
IN
Fig. 2. Diseases caused by damages to thiamine transport or functionality of

TDP-dependent enzymes in mammalian cells. Thiamine (TA) enters the cell via two
types of specific thiamine/H antiporters, THTR1 and THTR2. A mutation in
slc19a2 gene which codes for THTR1 causes a thiamine-responsive megaloblastic
anaemia (TRMA). In the cytosol, thiamine is converted to TDP by the action of
thiamine pyrophosphokinase (TPK). The cytosolic TDP pool can be enriched by the
uptake of external TMP via a cell membrane-bound reduced folate carrier (RFC1);
this relatively non-specific anion transport is coupled with H symport into the cell
and OH antiport out of the cell. Intracellular TMP must be dephosphorylated by
unspecific phosphatase (PH) to become the TPK substrate. TDP can be (i) exported
from the cell via plasma membrane-bound RFC1, (ii) bound by cytosolic TDPdependent enzymes such as transketolase (TK) or (iii) cross the inner mitochondrial
membrane via the same anion transporter RFC1 to be used by intramitochondrial
TDP-dependent enzymes such as pyruvate dehydrogenase (PDH) or ketoglutarate
dehydrogenase (KGDH). The thiamine transporters as well as TPK can be damaged
by alcohol to cause the thiamine deficiency and WernickeKorsakoff syndrome
(WKS). Neurological effects of thiamine deficiency are associated with the impairment of PDH or KGDH which ensure the biosynthesis of several neurotransmitters such as acetylcholine, glutamate and GABA. Reduced activity of these enzymes
can also lead to lactic acid accumulation within the brain (lactic acidosis, LA).
In diabetics, a low plasma thiamine level is accompanied with low transketolase
activity, decreasing the utilization of high carbohydrate levels and resulting in a raised
level of advanced glycation end products which promotes a diabetic vascular complication (DVC).
bloodstream across the basolateral membrane is dependent on the Na

concentration and upon ATP hydrolysis by the universal Na/K ATP-ase
(Rindi and Laforenza, 2000). Most cell types actively take up thiamine from
43
the blood via THTR1 or THTR2 located on their plasma membranes.

A reduced folate transporter RFC1 (SCL19A1) seems also to be involved
in cellular TMP import (Zhao et al., 2002) and TDP export (Zhao et al.,
2001). Mutations in the SLC19A2 gene that encodes THTR1 cause thiamine
deficiency and thiamine-responsive megaloblastic anaemia syndrome
(Fleming et al., 1999).
In the cytosol, thiamine is rapidly phosphorylated to TDP by TPK, and
TDP is taken up by mitochondria to be bound by the main TDP-dependent
dehydrogenases. TDP transfer across the inner mitochondrial membrane
probably occurs via a TDP/TMP antiport mechanism with the engagement
of the RFC1 transporter (Barile et al., 1990; Song and Singleton, 2002).
A large body of evidence also suggests that intestinal thiamine absorption
and further thiamine phosphorylation in the peripheral tissues and brain are
impaired by alcohol (Martin et al., 2003).
2. Functional disorders in mitochondria

Three dehydrogenase complexes involved in mitochondrial energy production, PDH, KGDH and BCKDH, utilize TDP as their cofactor. One of the
best recognized thiamine deficiency-based disorders is the WernickeKorsakoff syndrome in which the selective damage of mammillary bodies, the
thalamus and pons has been commonly observed. Analyses at the cellular
level have shown that this disorder is associated with neuronal loss, microglial activation and astrocyte proliferation (Hazell, 2009; Hazell et al., 1998;
Wang and Hazell, 2010). It has been demonstrated also in WernickeKorsakoff syndrome that the activity of all TDP-dependent enzymes is reduced,
but KGDH is principally affected. The treatment of experimental animals
with pyrithiamine, a known thiamine antagonist (Fig. 3), has confirmed that
KGDH depletion leads to a decrease in glutamate, aspartate and -aminobutyric acid (GABA) production (Heroux and Butterworth, 1995), as well
as mitochondria disintegration and chromatin clumping (Zhang et al., 1995).
This impairment of cerebral energy metabolism causes lactate accumulation and
acidosis, which results in neuronal cell loss (Hakim, 1984; Navarro et al., 2005).
Recent brain studies in rats treated with pyrithiamine have provided
evidence that in thiamine deficiency, it is oxidative stress that causes
cellular energy depletion and neuronal damage. The increase in hemooxygenase and ICAM-1 levels, as well as microglial activation and the induction
of neuronal peroxidase, have also been observed during a thiamine
deficiency (Gibson and Zhang, 2002). A high NOS expression level, NO
production as well as nitrotyrosine immunodetection have indicated that
the formation of peroxynitrites is likely to be responsible for KGDH
44
M. RAPALA-KOZIK
NH2
CH3
+
N
N
CH3
OH
Pyrithiamine
OH
CH3
NH2
O
N
N
N
OH
O
S
CH3
Benfothiamine
Fig. 3. Chemical structure of pyrithiamine and benfothiamine. Pyrithiamine is a

thiamine antagonist commonly used in model studies of thiamine deficiency in animals. Benfothiamine, a lipophilic thiamine analogue which easily crosses biological
membranes, can be used for treatment of thiamine deficiency-related diseases.
deactivation. These observations have led to the hypothesis that thiamine

may act as an antioxidant (Gibson and Blass, 2007; Huang et al., 2010) but
the chemical mechanism underlying this putative activity remains unknown.
Analogical changes have been detected also in Alzheimers disease and
Parkinsons disease (Hazell and Butterworth, 2009).
3. Diabetes and diabetic complications
The high glucose cytosolic concentration associated with hyperglycemia
leads to triosephosphate accumulation and the development of diabetic
complications such as diabetic nephropathy, neuropathy and retinopathy.
Decreased thiamine concentrations and TK activity in whole blood samples
are often observed in diabetic patients. Supplementation with thiamine or its
lipophilic analogue, benfothiamine (Fig. 3), applied in cell culture or diabetic
rat models restore the disposal of excess triosephosphate by the pentose
phosphate pathway (Thornalley, 2005). Benfothiamine, a lipid-soluble compound from the allithiamine family, was originally described in onions and
leeks (Fujiwara, 1976). Its high cellular bioavailability depends on a thiazole
ring-open structure that facilitates cell membrane crossing more readily.
After oral administration, benfothiamine is dephosphorylated by alkaline
phosphatase in the brush border of intestinal mucosal cells. The product of
this reaction, S-benzoylthiamine, enters the cells by passive diffusion and is
45
further converted to thiamine, mostly in erythrocytes. Equivalent doses of

thiamine have a fivefold lower bioavailability (Balakumar et al., 2010).
II. THIAMINE BIOSYNTHESIS

Thiamine biosynthesis pathways in bacteria, yeast and plants (Figs. 4 and 5)
consist of three general stages: (i) the independent formation of phosphorylated pyrimidine (4-amino-2-methyl-5-hydroxmethylypyrimidine diphosphate, HMP-PP) and thiazole (4-methyl-5-(2-hydroxyethyl)thiazole
phosphate, HET-P) precursors, (ii) their condensation into TMP molecules
and (iii) the formation of biologically active TDP (Goyer, 2010; Jurgenson
et al., 2009; Kowalska and Kozik, 2008).
In spite of this common general scheme, however, the biosynthetic pathways in the main groups of thiamine-synthesizing organisms differ in many
details. These differences are highest between prokaryotic and eukaryotic
organisms and, among the latter, plants seem to have a combination of the
synthetic systems of bacteria and yeast (Begley et al., 2008; Nosaka, 2006;
Rapala-Kozik et al., 2009). The reaction rate of the entire pathway is tightly
regulated by the final product, TDP, albeit via different mechanisms in
bacteria, yeast and plants (Bocobza and Aharoni, 2008; Miranda-Ros,
2007; Nosaka, 2006).
NH2
O
OH
OH
P
OH
O
N
CH3
4-Amino-2-methyl-5-hydroxymethylpyrimidine diphosphate
(HMP-PP)
CH3
OH
N
O
S
OH
4-Methyl-5-(2-hydroxymethyl)-thiazole phosphate
(HMT-P)
Fig. 4.
Biosynthetic precursors of pyrimidine and thiazole moieties of thiamine.
46
M. RAPALA-KOZIK
HMP
AIR
thiD
THI20/21
At-th1, Zm-thi3
HMP-P
DXP
+
cysteine
+
tyrosine or glycine
thiC
THI5/11/12/13
Histidine
+
pyridoxal-5-P
thiC
AIR
HMP-PP
dxs, thiF, thiS

thiG, tenl
glycine
+
NAD+
+
S-donor
thiD
THI20/21
At-th1, Zm-thi3
HET
glycine
+
NAD+
+
S-donor
THI4
thiM
THI6
?
HET-P
At-thi1,
Zm-thi1,
Zm-thi2
thiE
THI6
At-th1, Zm-thi3
TMP
??
TA
THI80
At-TPK1, At-TPK2
thiL
TDP
Fig. 5. A comparative scheme of thiamine biosynthesis in bacteria, yeast and

plants. Thiamine biosynthesis pathways use different sets of substrates in bacteria
(red), bakers yeast Saccharomyces cerevisiae (blue) and plants (green), but the late
steps are common to all thiamine-synthesizing organisms and include the independent
formation of pyrimidine (HMP-PP) and thiazole (HET-P) precursors, followed by
their condensation into TMP. The symbols of genes coding for the proteins involved
in these late steps of thiamine synthesis are specified on the scheme with correspondingly coloured fonts (AtArabidopsis thaliana, ZmZea mays). These enzymes
include HMP-P synthase, HMP-P kinase, HET-P synthase and TMP synthase.
Common to bacteria, yeast and plants utilize also the salvage pathways that engage
HMP kinase and HET kinase. Only in bacteria, the metabolically active coenzyme,
TDP, can be formed through a direct phosphorylation of TMP by TMP kinase. In
yeast and plants, TMP is first dephosphorylated by non-specific phosphatases to give
free thiamine which is used as a substrate by TPK.
A. THIAMINE BIOSYNTHESIS IN BACTERIA AND YEAST
1. Pyrimidine component synthesis

Extensive genetic and biochemical studies in E. coli and Bacillus subtilis have
revealed that 4-amino-2-methyl-5-hydroxymethylpyrimidine phosphate
(HMP-P) is formed by a rearrangement of 5-aminoimidazole ribonucleotide
(AIR), an intermediate in the purine nucleotide biosynthesis pathway.
47
This process is catalysed by the product of the thiC gene (Begley et al., 1999;
Zhang et al., 1997). HMP-P synthase (ThiC) activity is dependent on
S-adenosyl methionine (SAM) and a functional ironsulphur cluster localized on the ThiC C-terminal domain. It has been suggested that this FeS
cluster is available to bind SAM and that a reductive cleavage may generate
the 50 -deoxyadenosyl radical (Martinez-Gomez et al., 2009). This step may
enable the further rearrangement of AIR to HMP-P. The presence of the
FeS cluster within the structure of ThiC and possible free radical reaction
chemistry has confirmed this enzyme as a member of the radical SAM
superfamily (Chatterjee et al., 2008a).
The next HMP-P phosphorylation event, resulting in HMP-PP formation,
is performed by a thiD gene product. This kinase is bifunctional as it also can
take part in a salvage pathway through which external 4-amino-2-methyl-5hydroxymethylpyrimidine (HMP) may be phosphorylated to HMP-P
(Mizote et al., 1999).
In Saccharomyces cerevisiae, the pyrimidine moiety of thiamine is derived
from histidine and pyridoxal 50 -phosphate, with the involvement of the THI5
gene family (THI5/THI11/THI13) but the exact mechanism underlying
HMP-P formation remains insufficiently understood (Nosaka, 2006;
Zeidler et al., 2003). In the final HMP-P phosphorylation step, another
multigene family (THI20/THI21) is engaged. Again, the latter kinases perform the salvage HMP phosphorylation reactions (Kawasaki et al., 2005).
2. Thiazole component synthesis
For the biosynthesis of the thiazole moiety, bacteria utilize 1-deoxy-D-xylulose-5-phosphate (DXP), glycine or tyrosine and a sulphur carrier protein
(ThiS). This reaction is initiated by thiazole phosphate synthase (ThiG), an
enzyme that performs DXP tautomerization and further oxidative condensation with glycine and cysteine, in cooperation with a Ten1 protein
(Dorrestein et al., 2004; Kriek et al., 2007). Early isotopic labelling studies
have identified cysteine, glycine and D-pentulose-5-phosphate (D-ribulose-5phosphate or D-xylulose-5-phosphate) as primary precursors of the thiazole
moiety in S. cerevisiae. However, a recent study of the structure and mechanism underlying enzymatic thiazole formation by thiazole synthase (THI4)
and analysis of a thiazole derivative tightly bound to this protein has revealed
that NAD is the most likely source of the carbohydrate (ribose) required for
thiazole synthesis (Chatterjee et al., 2007).
The final product of these pathways, HET-P, may be also regenerated
from HET by a salvage enzyme, HET kinase, encoded by the thiM gene in
E. coli (Mizote and Nakayama, 1989) and THI6 gene in S. cerevisiae (Nosaka
et al., 1994).
48
M. RAPALA-KOZIK
3. Condensation of pyrimidine and thiazole components into TMP

A TMP synthase (also known as thiamine-phosphate diphosphorylase),
encoded by the thiE gene in bacteria, couples the diphosphorylated pyrimidine compound and the phosphorylated thiazole compound to form TMP.
The structure of this enzyme and its catalytic mechanism suggests a dissociative mechanism of TMP formation (Chiu et al. 1999; Peapus et al., 2001;
Reddick et al., 2001), with a pyrimidine carbocation as an intermediate
(Hanes et al., 2007). The same condensation reaction in S. cerevisiae is
performed by the bifunctional TMP synthase/HET kinase encoded by the
THI6 gene (Nosaka et al., 1994).
4. Formation of TDP, a biologically relevant cofactor
The last step in the TDP biosynthesis pathway differs between bacteria and
yeast. In most bacteria, TMP is simply further phosphorylated to TDP by a
kinase encoded by the thiL gene (Webb and Downs, 1997). The active site
structure of ThiL suggests a direct, inline transfer of the -phosphate of ATP
to TMP (McCulloch et al., 2008). Yeast utilize a more complex mechanism to
produce TDP. First, TMP is dephosphorylated to thiamine, probably by an
unspecific but not yet identified phosphatase. The free thiamine is then
diphosphorylated by TPK, encoded by a single THI80 gene (Nosaka et al.,
1993). The structure of this enzyme is well documented but its mechanism of
catalysis is still under debate (Baker et al., 2001; Voskoboyev and Ostrovsky,
1982).
5. Synthesis of TTP and ATTP
The biosynthetic pathways for the TTP and ATTP in bacteria and yeast have
not yet been identified. However, in the rat brain, TTP synthesis was recently
suggested to occur in mitochondria and to be coupled to the respiratory
chain (Gangolf et al., 2010a,b).
B. GENES AND PROTEINS INVOLVED IN PLANT THIAMINE BIOSYNTHESIS AND
THE CELLULAR DISTRIBUTION OF THE BIOSYNTHETIC PATHWAYS
A large number of auxotrophic mutants have been used to elucidate the

specific steps involved in thiamine biosynthesis in plants. Thiamine auxotrophs identified in the model plant, Arabidopsis thaliana, manifest seedling
lethal phenotypes that can be complemented by exogenous thiamine. The
first identified group of auxotrophs, th1 (chromosome I), th2 (chromosome
V) and th3 (chromosome IV) were rescued only using thiamine supplementation, indicating that the respective genes are involved in the latest steps of
TDP formation. The second group of auxotrophs, py (chromosome II) and tz
49
(chromosome V), require thiamine or HMP (py) and thiamine or HET (tz)
for growth, suggesting the involvement of these genes in the biosynthesis of
the pyrimidine and thiazole moiety, respectively (Koornneef and Hanhart,
1981).
1. Pyrimidine component biosynthesis
Plants appear to take advantage of both bacterial and yeast thiamine biosynthetic processes (Fig. 5). A search for a HMP-P synthase candidate in
A. thaliana revealed only one homologue of the thiC gene, which encodes a
protein with a 60% sequence identity to ThiC from B. subtilis and E. coli.
A partial confirmation of the involvement of plant THIC in thiamine biosynthesis came from a previous finding that seedlings of THIC knockdown
mutants possess a significantly decreased thiamine level, present a chlorotic
phenotype and are unable to develop beyond the cotyledon stage. However,
supplementation with an external dose of thiamine was found to rescue this
phenotype (Kong et al., 2008; Raschke et al., 2007). Further analyses of the
THIC mutant for metabolites originating from the reactions which engage
TDP as a cofactor (the tricarboxylic acid cycle, the CalvinBenson cycle and
the oxidative pentose phosphate pathway) have suggested that THIC is
essential for plant viability (Raschke et al., 2007). This hypothesis was
further supported by findings that THIC-overexpressing plants possess a
higher thiamine content in their tissues and that the A. thaliana THIC gene
can complement a bacterial thiC mutant (Kong et al., 2008). Similarly to
bacteria, plant THIC requires a reducing agent and a FeS cluster for the
catalytic conversion of AIR into HMP-P. Raschke et al. (2007) have demonstrated in A. thaliana that a cysteine desulfurylase (NifS) may be the sulphur
source for the FeS cluster and speculated that the thioredoxin system could
be involved in the activation of this enzyme. However, the mechanism of this
reaction has not yet been elucidated.
YFP-fusion protein analysis has indicated that THIC is localized in chloroplasts. This finding confirmed early suggestions that plant thiamine synthesis occurs in plastids (Faith et al., 1995; Julliard and Douce, 1991). The THIC
transcript was found to be expressed in leaves, flowers and siliques, and at
small amounts in roots. The expression levels are dependent on the stage of
seedling development (commencing on the fifth day after imbibition) and
also the thiamine levels in the medium. The THIC transcript levels increase
also under light exposure (Kong et al., 2008; Raschke et al., 2007). The THIC
gene is tightly regulated by a riboswitch-dependent mechanism (see Section II.C) in which TDP plays a role of a feedback inhibitor whilst thiamine,
available in the medium, may be easily converted to TDP inside the cells
(Fig. 6).
50
M. RAPALA-KOZIK
HET- P
2
TDP-dependent
enzymes
TMP
4
HMP- P
TDP
TA
5
6
TA
TDP
TDP
TDP-dependent
enzymes
TDP-dependent
enzymes
Fig. 6. Compartment localization of biosynthetic pathways and intracellular

traffic of thiamine diphosphate (TDP) in the plant cell. The plant biosynthesis of
thiamine monophosphate (TMP) is localized in chloroplasts where the thiazole precursor (HET-P) and pyrimidine precursor (HMP-P) are formed on independent
pathways catalysed by HET-P synthase (1) and HMP-P synthase (2), respectively.
Coupling of these two moieties (after additional phosphorylation of HMP-P) is
performed by TMP synthase (3). Since thiamine (TA) is present in the cytosol, the
product of its biosynthesis (TMP) must first be dephosphorylated by yet unidentified,
probably non-specifc phosphatases (4). Hence, this process is proposed to proceed in
the chloroplast but its occurrence in the cytosol is also possible. After dephosphorylation, TA is transported to cytosol by a yet unidentified transporter (5) and is further
converted to TDP by cytosolic thiamine pyrophosphokinase (6). TDP plays the
cofactor function for the cytosol-, mitochondrion- or chloroplast-localized enzymes.
TDP must be transported into mitochondria and chloroplasts by high effective
specific transporter(s) (7, 70 ) that have not yet been identified.
2. Thiazole component biosynthesis

For the building of the thiazole component, plants use the same pathway
developed in yeast (Fig. 5), in which NAD and glycine are converted to
HET-P by thiazole synthase (THI4) in cooperation with a protein sulphur
donor (Chatterjee et al., 2006, 2008b). Numerous genes with high sequence
similarity to THI4 have been identified in the genomes of Zea mays (thi1 and
thi2; Belanger et al., 1995), Alnus glutinosa (agthi1; Ribeiro et al., 1996), A.
thaliana (thi1; Machado et al., 1996) and Oryza sativa (OsDR8; Wang et al.,
2006). Complementation studies using Arabidopsis THI1 in E. coli mutant
strains defective in DNA repair pathways or THI4-defective yeast mutant
51
strains further supported a role for THI1 in thiamine biosynthesis and its
possible involvement also in plant tolerance to mitochondrial DNA damage
(Machado et al., 1996, 1997).
Sequence analysis of the THI1 protein encoded by a single gene in Arabidopsis (tz locus) has identified an N-terminal chloroplast transit peptide and
a mitochondria targeting-like presequence just downstream, suggesting the
dual targeting of this gene product to both plastids and mitochondria. The
resolved crystal structure of Arabidopsis THI1, heterologously expressed
and overproduced in E. coli (Godoi et al., 2006) revealed that the protein
(244 kDa) is an octamer containing dinucleotide binding domains adapted to
NAD binding. To date, this is the only plant thiamine biosynthetic enzyme
whose three-dimensional structure has been elucidated at an atomic resolution (Fig. 7).
Similarly to the yeast THI4 protein, the tightly bound 2-carboxylate-4methyl-5-(-ethyl adenosine 50 -diphosphate) thiazole was identified within
the THI1 structure and was suggested to be a late intermediate on the
thiazole biosynthetic route, additionally supporting the hypothesis that
yeast-like biosynthetic pathways are utilized by plants, with NAD as the
substrate. The dual function of this gene was confirmed by the observation
that some site-directed mutations of THI1 prevent thiazole biosynthetic
Fig. 7. Structure of thiazole-synthesizing protein THI1 from Arabidopsis thaliana.

(A) The structure of THI1 monomer with a visualized molecule of tightly bound
2-carboxylate-4-methyl-5-(-ethyl adenosine 50 -diphosphate) thiazole (ADT), an apparent product of the catalysed reaction. (B) Amino acid residues which surround the
ADT molecule bound in the active centre of THI1 protein. The structure was
imported from UniProt KB (access No Q38814) and drawn with PyMol program
(ExPASy server).
52
M. RAPALA-KOZIK
activity but do not affect mitochondrial DNA stability (Godoi et al., 2006),
the latter being controlled by the same gene through a yet unidentified
mechanism.
Thiazole synthesis was found to be localized to chloroplasts in spinach
(Julliard and Douce, 1991) and maize (Belanger et al., 1995). The dominant
accumulation of transcripts of thi2, the maize paralog of thi1, was observed
in young, rapidly dividing tissues, whilst thi1 is detectable in mature green
leaves. This may reflect a subfunctionalization of both encoded proteins. The
thi2-blk1 mutant is a thiamine auxotroph which shows defects in shoot
meristem maintenance and a novel leaf blade reduction phenotype
(Woodward et al., 2010).
In Arabidopsis, an analysis of the organelle localization of a -glucuronidase-fused THI1 protein (GUS-THI1) confirmed chloroplasts and mitochondria as the targets of THI1 localization and provided evidence that
two isoforms of THI1 are produced from a single nuclear transcript.
Hence, this targeting occurs through a post-transcriptional mechanism
(Chabregas et al., 2001, 2003; Ribeiro et al., 2005). The intensive expression
of THI1 was observed in all organs at different plant development stages, for
example, during nodule differentiation (Ribeiro et al., 1996) and ethyleneinduced fruit maturation (Jacob-Wilk et al., 1997). THI1 expression was also
found to predominate in shoot tissues as compared with roots (Ribeiro et al.,
2005) and is twofold higher in plants grown under light (Papini-Terzi et al.,
2003). The presence of thiamine in the medium did not affect the THI1
expression level, in sharp contrast to the strong repression of the yeast
orthologous gene (THI4) by external thiamine.
3. Coupling the pyrimidine and thiazole compounds
The condensation of pyrimidine and thiazole components to form TMP is
the common step in thiamine biosynthesis in all autotrophic organisms.
Similarly to S. cerevisiae, plants use a bifunctional enzyme for this reaction
(Fig. 5), although the additional activity (HMP/HMP-P kinase) combined
with TMP synthase activity in one molecule is different from that in the yeast
THI6 protein (HET kinase). The occurrence of TMP synthase in plants was
demonstrated in studies on the functional complementation of thiaminerequiring mutants in bacteria (Ajjawi et al., 2007b; Kim et al., 1998).
The protein identified in Z. mays (THI3) shows a 39% sequence similarity
to B. subtilis ThiD and a 6080% similarity to several plant orthologues in
O. sativa, Medicago truncatula, A. thaliana and Brassica napus (RapalaKozik et al., 2007). This analysis also indicated that THI3, similarly to all
of its plant orthologues, possesses two putative conserved domains, an
N-terminal domain with a high sequence similarity to bacterial HMP-P
53
kinases, and a C-terminal domain highly similar in sequence to the bacterial

TMP synthases. In contrast, the yeast bifunctional TMP synthase (THI6) is
associated with HET kinase activity localized in a C-terminal domain, that is,
downstream from the TMP synthase domain (Kawasaki, 1993). On the basis
of sequence similarity of THI3 to structurally characterized bacterial TMP
synthase (B. subtilis) and HMP-P kinase (Salmonella typhimurium) (Cheng
et al., 2002; Chiu et al., 1999), the overall structures of the THI3 domains as
well as the arrangements of conserved amino acid residues within the active
centres have been modelled (Rapala-Kozik et al., 2007). The kinase domain
reveals a ribokinase-like fold, whilst the synthase domain harbours a triose
phosphate isomerase fold.
THI3 was heterologously expressed in E. coli and yielded as a soluble
dimer of 55 kDa subunits which possessed the expected enzymatic activities
(Rapala-Kozik et al., 2007). These included TMP synthesis and two successive steps of HMP phosphorylation, with the production of HMP-P and
HMP-PP, the latter serving as the substrate for TMP synthase. HMP phosphorylation to HMP-P also appears to be a salvage pathway, as in bacteria.
The predicted arrangements of the active centre amino acid residues were
confirmed by site-directed mutagenesis experiments. Detailed kinetic analysis
showed that TMP formation was strongly inhibited by an excess of one of
TMP synthase substrates (HMP-PP) and uncompetitively inhibited by ATP.
Both compounds are involved in the reaction catalysed by the HMP-P kinase
domain of THI3, one as a substrate (ATP) and the other as a product (HMPPP). It was suggested that this unique fusion of both enzyme activities in one
protein molecule may provide a regulatory mechanism for TMP biosynthesis
in plants.
All members of the plant TMP synthase family contain the N-terminal
signal sequence responsible for chloroplast targeting (Rapala-Kozik et al.,
2007). The detection of fluorescent protein-fused TMP synthase in Arabidopsis mesophyll protoplasts also indicated the chloroplasts as the location
of TMP biosynthesis (Ajjawi et al., 2007b). During seedling development,
most plants on the early stage of growth utilize thiamine reserves accumulated in the seeds and de novo thiamine biosynthesis, manifested by the
induction of TMP synthase activity, which starts between days 3 and 6
after imbibition (Golda et al., 2004).
4. TDP synthesis
In the final step of thiamine coenzyme formation, plants, unlike bacteria but
similar to yeast, do not perform a direct phosphorylation of de novo synthesized TMP. Instead, TMP is first dephosphorylated to free thiamine which
is then pyrophosphorylated to TDP, in a reaction catalysed by TPK (Fig. 5).
54
M. RAPALA-KOZIK
It is generally assumed that the production of free thiamine from TMP in

plants involves numerous broad-specificity phosphatases. However, this does
not exclude the possibility that some phosphatases may be more important
than others in this process. Recently, a homogeneous acid phosphatase (a
dimer of 24 kDa subunits) with a broad specificity was isolated from Z. mays
seedlings on the basis of its ability to dephosphorylate TDP and TMP
(Rapala-Kozik et al., 2009). The purified enzyme showed some preference
for thiamine phosphates (TDP > TMP) over other organic phosphate esters.
Purified TPK preparations have been obtained from parsley leaves (Mitsuda
et al., 1979), soybean seedlings (Molin and Fites, 1980) and maize seedlings
(Rapala-Kozik et al., 2009). They differed slightly in terms of subunit size,
subunit association states and basic kinetic parameters. For example, the
maize TPK is a 29-kDa monomeric protein. In Arabidopsis, this enzyme is
encoded by two genes, At-TPK1 and At-TPK2, and the predicted amino acid
sequence of their protein products show a significant similarity with the
structurally characterized fungal and animal TPKs (Ajjawi et al., 2007a).
Both genes are expressed at comparable levels, predominantly in leaves but
also in the stems, siliques and flowers. However, in the roots, their expression
levels differ, with a clear preference for At-TPK1. An analysis of a TPK
double knockout mutant in Arabidopsis further showed that the seedling had
a lethal phenotype and survived only in the presence of external doses of
TDP (Ajjawi et al., 2007a).
Negative regulation by light was suggested for TPK activity in Z. mays
seedlings, whilst a presence of thiamine in the culture medium exerted only
minor effects upon TPK expression (Rapala-Kozik et al., 2009). TPK is
involved in TDP biosynthesis from the very early stages of seed germination
when thiamine reserves stored in seeds serve as the substrate for TPKcatalysed pyrophosphorylation (Molin et al., 1980; Golda et al., 2004). The
de novo formation of TDP was found to be localized to the plant cell cytosol,
as in yeast and mammals (Barile et al., 1990; Bettendorff, 1995; Hohmann
and Meacock, 1998). As TDP is necessary for many biochemical processes in
different cell compartments, effective systems for its transport must exist in
plants, but the underlying mechanisms have not yet been characterized.
5. Thiamine triphosphate and adenosine thiamine triphosphate
The presence of the highly phosphorylated thiamine compound, TTP, in the
germ axes of higher plants was reported many years ago (Kochibe et al.,
1963; Yusa, 1961) and recently, its presence in withering plants was also
confirmed (Makarchikov et al., 2003). Whereas TTP accumulation was
detected in E. coli in response to amino acid starvation in carbon containing
medium, leading to the hypothesis of a general alarmone function of this
55
compound (Bettendorff and Wins, 2009; Lakaye et al., 2004), its actual
significance and functions in plants remain unknown. The newly discovered
thiamine compound, ATTP, first detected in bacteria upon carbon starvation, has also been found in the roots of higher plants (Bettendorff et al.,
2007). It has been further proposed that ATTP may serve as a TTP precursor
deposited in a less reactive storage form (Jordan, 2007) or as a source of
TDP. To date, however, nothing is known about the biosynthetic routes for
TTP and ATTP in plants.
C. REGULATION OF PLANT THIAMINE BIOSYNTHESIS
For half a century, it has been well established that thiamine synthesis and
transport in bacteria and yeast are strongly repressed by the presence of
exogenous thiamine in the culture media (Begley et al., 2008; Kowalska and
Kozik, 2008; Nosaka, 2006). It is actually the intracellular TDP concentration that provides this regulatory signal. Although less frequent, a similar
system of feedback regulation has been reported in plants (Kim et al., 1998;
Rapala-Kozik et al., 2009). Only recently, however, have the molecular
mechanisms underlying the regulation of plant thiamine biosynthesis been
characterized at the molecular level with the discovery of plant TDP-dependent riboswitches that regulate the expression of the THIC pyrimidinesynthesizing gene and, albeit not in the entire plant kingdom, of the THI1
thiazole-synthesizing gene (Bocobza et al., 2007; Sudarsan et al., 2003). Other
reports have also suggested that the expression of thiamine-synthetic
enzymes may depend on some tissue-specific transcription factors (Ribeiro
et al., 2005), light (Kong et al., 2008; Rapala-Kozik et al., 2009; Raschke
et al., 2007; Ribeiro et al., 1996, 2005), the thioredoxin system (Balmer et al.,
2003; Lemaire et al., 2004; Raschke et al., 2007) and elements responding to
abiotic and biotic stress signalling (see Section VI). Additionally, the allosteric inhibition of plant TMP synthase activity by ATP and HMP-PP has been
reported (Rapala-Kozik et al., 2007).
1. Riboswitch-dependent regulation of HMP-P synthase (THIC) and HET-P
synthase (THI1)
The precise mechanism of plant THIC gene regulation by accessible TDP has
recently been identified and shown to engage a TDP-binding riboswitch
(THI-BOX) (Bocobza et al., 2007; Sudarsan et al., 2003; Wachter et al.,
2007). Riboswitches are non-coding mRNA domains that can selectively
bind some metabolites and subsequently affect the expression of adjacent
coding sequences (Breaker, 2010; Serganov, 2010; Smith et al., 2010;
Wachter, 2010). They are believed to be the modern descendents of an
56
M. RAPALA-KOZIK
ancient sensory and regulatory system which may have functioned in the
RNA world (Breaker, 2010). The THI-BOXes represent the most abundant class of riboswitches, and are found in prokaryotes, archea and eukaryotes (Bocobza and Aharoni, 2008). Like other riboswitches, they are
composed of a highly conserved TDP-binding domain (aptamer) responsible for coenzyme sensing and a more variable expression platform which,
when forced to rearrange by the ligand-induced conformational change in
the aptamer, affects gene expression (Rodionov et al., 2002; Winkler et al.,
2002). The high-resolution crystal structure of a complex between TDP and
the Arabidopsis THI-BOX that controls THIC gene expression has been
reported (Thore et al., 2006) and is schematically presented in Fig. 8.
Two helical domains are involved in TDP binding. The first of these forms
a deep pocket for the pyrimidine moiety of TDP and the other is responsible
for the binding of the diphosphate tail, bridged by an Mg2 ion (MirandaRos, 2007; Serganov et al., 2006; Thore et al., 2006). The TDP molecule lies
in a perpendicular orientation against the two parallel helices and adopts an
extended conformation, in contrast to its V-conformation observed in
Pre-mRNA
5
UAA
INT1
Poly(A) signal
3
EX1 INT2
EX2
TDP riboswitch
Diphosphate
binding helix
Low TDP level
Pyrimidine
binding helix
TDP
EX2
UAA
EX1 INT2
UAA
EX1 INT2 AAA
Intron retention
Short, stable transcript

Switching
helix
3
High TDP level

UAA
UAA
EX1
EX2
EX1
AAA
EX2 AAA
AA
Long, unstable transcript
A
Intron splicing
Fig. 8. Structure and action mechanism of TDP-binding riboswitch (THI-BOX)

which regulates the expression of the thiC gene in Arabidopsis thaliana. (Left panel)
The three-dimensional structure of THI-BOX (Thore et al., 2006; Protein Data Bank
access No 3D2G, drawn with PyMol program). (Right panel) A suggested mechanism
of THI-BOX-dependent regulation of plant thiC expression (Bocobza and Aharoni,
2008). Different modes of thiC pre-mRNA processing are dependent on the intracellular TDP level. At low TDP concentration, the riboswitch folding enables its interaction with 50 splice site and prevents splicing. The major processing site is retained
(the intron retention variant), resulting in the formation of short transcript that
permits a high THIC expression. At high TDP level, it binds to riboswitch and induces
its conformational changes that prevent the riboswitch interaction with 50 splice site.
The splicing takes place (the intron splaced variant), leading to the removal of poly(A)
signal and the formation of long unstable transcripts.
57
TDP-dependent enzymes. The apparent dissociation constant of the complex

formed (500 nM) is consistent with the TDP level detected in plants after they
initiate its biosynthesis (0.255 M; Winkler et al., 2002; Bocobza and
Aharoni, 2008). The accommodation of a pyrithiamine diphosphate by the
THI-BOX aptamer suggests that the thiazole ring is not essential for fixing
TDP in the binding pocket. However, the binding of TMP by a bacterial
THI-BOX is weaker by 3 orders of magnitude, suggesting incomplete stabilization of both helical domains and presenting additional evidence for the
preferential regulation of THIC expression by TDP (Agyei-Owusu and
Leeper, 2009; Winkler et al., 2002).
The binding of TDP generates a parallel localization of sensor helices and
alters the expression platform (Bocobza and Aharoni, 2008; Thore et al., 2006;
Winkler et al., 2002). This ancient-origin mechanism is widespread in both
prokaryotes and eukaryotes, although differs in terms of gene expression
alteration (Bocobza and Aharoni, 2008; Sudarsan et al., 2003). In Grampositive bacteria, TDP binding by THI-BOX, such as that involved in the
tenA regulation in B. subtilis, causes structural rearrangements that lead to the
formation of a transcription termination hairpin. In Gram-negative bacteria
(e.g. thiM in E. coli), the presence of TDP leads to translation repression via
the sequestration of the ShineDelgarno sequence and the prevention of
ribosome binding (Mironov et al., 2002; Ontiveros-Palacios et al., 2008;
Winkler et al., 2002). In some non-yeast fungi (e.g. Aspergillus oryzae or
Neurospora crassa), THI-BOX is located within an intron in the 50 -untranslated region (50 -UTR) of THI4 (an orthologue of plant THI1) mRNA and TDP
binding alters the mRNA splicing so that it does not occur at the 50 splice site
(distal) as normal but at a more proximal site. This alternative splicing leads to
upstream open-reading frame (ORF) expression and premature termination
(Cheah et al., 2007; Sudarsan et al., 2003).
In THIC transcripts in flowering plants, the TDP riboswitch element is
located in the 30 -UTR and controls the splicing toward an alternative 30 end
processing of precursor THIC mRNA (Bocobza et al., 2007, Sudarsan et al.,
2003; Wachter et al., 2007). The pre-mRNA 30 -UTR consists of a constitutively spliced intron just after the ORF stop codon, followed by a sequence
which contains a polyadenylation signal and a potential splice site (Fig. 7B).
The TDP riboswitch is located 70 bp downstream of the polyadenylation
signal and is followed by the last variable-length exon. The 30 splice site of the
second intron is located within the riboswitch (P2 box). At a low TDP
concentration, the riboswitch interacts with 50 splice site and splicing of the
second intron is prevented. In this situation, mRNA processing leads to
variant transcripts with the second intron retained and harbouring the
major processing site that permits transcript cleavage and polyadenylation.
58
M. RAPALA-KOZIK
This variant is short and can be translated. At a high TDP concentration, the
conformational change of the riboswitch exposes the 50 splice site of the
second intron and an alternative splicing reaction proceeds and removes
the normal processing site to form an intron-spliced variant which is both
long and unstable (Bocobza and Aharoni, 2008). Analysis of the effects of
TDP supplementation upon the THIC transcript levels in Arabidopsis and
tomato auxotrophic mutants with low endogenous TDP contents directly
supports the hypothesis of TDP involvement in thiamine biosynthesis regulation (Bocobza et al., 2007; Wachter et al., 2007).
The THI-BOX-dependent regulation of pyrimidine-synthesizing THIC gene
expression has been confirmed in all major plant taxa, from species of moss
(bryophytes) to flowering plants (angiosperms). A similar type of riboswitchdependent regulation of the expression of the thiazole-synthesizing THI1 gene
was lost during gymnosperm evolution. Cycas revoluta is the plant of the highest
evolutionary order for which a TDP-dependent riboswitch in the 30 -UTR of
THI1 mRNA can be detected (Bocobza and Aharoni, 2008).
III. TDP-DEPENDENT ENZYMES IN PLANTS

TDP functions as the cofactor for enzymes involved in key metabolic pathways such as ethanolic fermentation, acetyl-CoA formation, the tricarboxylic acid cycle, the oxidative pentose phosphate pathway, the CalvinBenson
cycle, the mevalonate-independent isoprenoid synthesis pathway and
branched-chain amino acid biosynthesis. In all of these processes, the first
step depends on the special structure and charge of the TDP molecule in the
enzyme active centres. A comparison of the crystal structures of the major
TDP-dependent enzymes reveals that this cofactor is accommodated by
the protein in the V-conformation in which the amino group of the pyrimidine ring is closely positioned with the C2 atom of the thiazole ring.
This orientation influences the mechanism of enzyme catalysis (Frank
et al., 2007).
Although the sequence similarity between TDP-dependent enzymes is low
(less than 20% amino acid identity), the tertiary structures of these proteins
show high similarities in terms of the TDP-binding folds, particularly at the
geometric positions of the conserved residues which are involved in TDP
binding. It has been demonstrated that TDP-dependent enzymes contain at
least two conserved domains: (i) a phosphate-binding domain in which the
TDP cofactor is bound primarily through its diphosphate group coordinated
by a divalent cation and (ii) a pyrimidine-binding domain containing the
conserved glutamic acid residue which plays a crucial role in the catalytic
59
mechanism (Duggleby, 2006; Widmann et al., 2010). TDP-dependent

enzymes form either dimers or tetramers, in which TDP is bound at the
dimer interface, with the pyrimidine moiety associated with one monomer
and the diphosphate residue bound to the other monomer (Duggleby, 2006;
Lindqvist et al., 1992).
A. CATALYTIC MECHANISMS OF TDP-DEPENDENT ENZYMES
The close proximity of the 40 -amino group to the C2 atom of thiazolium ring
in the enzyme-bound TDP molecule permits an intramolecular proton abstraction which leads to the formation of a nucleophilic C2-ylide. This is
the initiating step of all reactions catalysed by TDP-dependent enzymes
(Fig. 9).
It has been demonstrated in many earlier reports that TDP first undergoes
tautomerization into an imino-form and that the nitrogen atom of the imine
is responsible for abstracting the C2 proton in the thiazolium ring of TDP
(Jordan et al., 2003; Nemeria et al., 2007; Tittmann et al., 2003). This process
is rendered possible by the charge of the N10 atom, which forms a hydrogen
bond with the conserved glutamate residue located in the catalytic centres of
many TDP-dependent enzymes (Shaanan and Chipman, 2009). Ylide stabilization and further catalytic steps are also favoured by the effective polarity
of the binding site (Zhang et al., 2005).
NH2
N
H3C
N
(N1) protonation
N1
+
NH2
R1
N
+
H3C
N
CH3
R1
CH3
H
4-Aminopyrimidinium
4-Aminopyrimidine
H+
NH2
N
H3C
N
+
H
NH
N
+
H3C
N
C2carbanion (ylide)
N
R1
CH3
N
+
R1
CH3
1 ,4-Iminopyrimidine
Fig. 9. Generation of active ylide-like carbanion as an initiating step of all

reactions catalysed by TDP-dependent enzymes.
60
M. RAPALA-KOZIK
OH
S
+
N
+
H3C
N
+
H
PY
H3C
R1
H3C
NH2
S
PY
N
+
CH3
CO2
R1
CH3
Ylide
OH
OH
H 3C
H3C
S
PY
N
+
R1
PY
CH3
R1
CH3
H+
PY
N
+
R1
H3C
CH3
Activated aldehyde
Fig. 10. Generation of activated aldehyde intermediate in the pyruvate decarboxylase reaction. The activated aldehyde, also known as the enamine-carbanion intermediate, is a common early stage in catalytic mechanisms of all TDP-dependent
enzymes, in spite of very different first substrate and downstream reaction steps.
The reactions catalysed by TDP-dependent enzymes can be generally

divided into decarboxylation and transferase-type reactions. All share certain mechanistic similarities, that is, the first part of the reaction involves the
breaking of a CC or CH bond adjacent to a carbonyl group of the
substrate and the formation of a metastable enamine intermediate (Fig. 10).
In the next step of the catalysis reaction, the second substrate is bound and
the final product is eventually released with ylide regeneration.
B. CLASSIFICATION OF TDP-DEPENDENT ENZYMES AND THEIR
LOCALIZATION WITHIN THE PLANT CELL
The TDP-dependent enzymes involved in plant vital functions belong to

three of the main enzyme classes, namely oxidoreductases, transferases and
lyases. Each enzyme has an important function in major metabolic pathways
and their localizations within the plant cell are presented in Fig.11.
1. Oxidoreductases
The plant TDP-dependent oxidoreductases are -ketoacid dehydrogenase
complexes that have crucial functions in all aerobic organisms. These
enzymes link glycolysis with the tricarboxylic acid cycle, drive the further
61
Pentose phosphate
pathway
Isoprenoid phosphate
pathway
PDH
DXPS
TK
CalvinBenson
pathway
AHAS
TDP
Tricarboxylic acid
cycle
BCKDH
PDH
KGDH
PDC
TK
Pentose phosphate
pathway
Fig. 11. Localization of TDP-dependent pathways in the plant cell. The major
mitochondrial TDP-dependent enzymes include pyruvate dehydrogenase (PDH)
involved in the acetyl-CoA synthesis, -ketoglutarate dehydrogenase (KGDH) of
the tricarboxylic acid cycle and branched-chain -ketoacid dehydrogenase
(BCKDH). The chloroplast contains PDH, acetohydroxyacid synthase (AHAS),
2-deoxy-D-xylulose-5- phosphate synthase (DXPS) of non-mevalonate isoprenoid
synthesis pathway and transketolase (TK), involved in the pentose phosphate pathway and the CalvinBenson cycle. Pyruvate decarboxylase (PDC) and, at least in
some species, an additional pool of TK are present in the cytosol.
oxidation of glucose in this cycle and are involved in substrate production for
fatty acid biosynthesis (Randall et al., 1996; Money et al., 2002). In general,
these dehydrogenase complexes consist of multiple copies of three components: (E1) a specific -ketoacid dehydrogenase that also decarboxylates
-ketoacid with the participation of TDP (E2) dihydrolipoyl acyltransferase
that transfers the acyl group to CoA and (E3) dihydrolipoyl dehydrogenase
that regenerates the E2 prosthetic group and produces NADH. The E2
component forms the core of the complex to which E1 and E3 are noncovalently attached. Plants possess three types of -ketoacid dehydrogenase
complex as described in further detail below.
a. Mitochondrial pyruvate dehydrogenase complex. Mitochondrial pyruvate dehydrogenase complex (mtPDH) converts pyruvate to acetyl-CoA.
Localized in the mitochondrial matrix, mtPDH is a linker between cytoplasmic glycolysis and the mitochondrial tricarboxylic acid cycle (Fig. 10).
62
M. RAPALA-KOZIK
The precise regulation of mtPDH activity is based on product inhibition,

metabolite effectors (Tovar-Mendez et al., 2003), compartmentalization and
the plant developmental stage (Luethy et al., 2001; Thompson et al., 1998).
Additionally, a light-dependent, reversible inactivation of this complex has
been observed during its phosphorylation by a bound E2 kinase, suggesting
that E2 phosphatase may play a regulatory function (Roche et al., 2001;
Thelen et al., 1998).
It is interesting to note that mammalian mitochondrial enzymes that use
TDP as the cofactor are usually isolated from tissues as holoenzymes in
which TDP is tightly bound to the apoenzyme forms. In contrast, the plant
mitochondria easily loose TDP during the isolation process but the purified
enzymes (mtPDH, KGDH) rapidly recapture the coenzyme after its external
supply. This suggests a weaker binding of TDP by these enzymes in plants
with possible benefits of a more effective transport which could be important
for a effective regulation of enzyme activity or for a more sensitive detection
of TDP biosynthetic needs (Douce and Neuburger, 1989).
b. Plastidial pyruvate dehydrogenase complex. The plastidial pyruvate dehydrogenase complex (ptPDH) supplies the acetyl-CoA and NADH for de
novo fatty acid biosynthesis in the stroma (Camp and Randall, 1985; Ke
et al., 2000). Unlike mtPDH, ptPDH is upregulated under photosynthetic
conditions by an increase in the stromal pH and Mg2 concentrations
(Miernyk et al., 1985; Williams and Randall, 1979), and is not regulated by
reversible phosphorylation (Miernyk et al., 1998).
c. -Ketoglutarate dehydrogenase. As a component of the tricarboxylic
acid cycle, KGDH catalyses the oxidative decarboxylation of -ketoglutarate to succinyl-CoA and NADH and is localized at the inner mitochondrial
membrane (Millar et al., 1999). Analyses of KGDH activity in the presence
of some inhibitors (Araujo et al., 2008; Bunik and Fernie, 2009) have shown
that it is the limiting enzyme for cellular respiration and plays a role in
nitrogen assimilation and amino acid (glutamate, glutamine and GABA)
metabolism. It has also been proposed that at low levels of NAD, KGDH
may be involved in a side reaction of reactive oxygen species (ROS) production, thus being a signal of a metabolic disorder (Bunik and Fernie, 2009).
d. Branched-chain -ketoacid dehydrogenase. BCKDH is a mitochondrial
enzyme (Anderson et al., 1998) which catalyses the irreversible oxidative
decarboxylation of the branched-chain -ketoacids derived from valine, leucine and isoleucine (Paxton et al., 1986; Wynn et al., 1996; Yeaman, 1989).
63
Its regulation by light is probably dependent on a mechanism similar to that of

mtPDH (Fujiki et al., 2000).
2. Transferases
The transketolases (TKs) belong to the class of transferases and catalyse the
reversible transfer of a keto group from a ketose to an aldose via a nonoxidative mechanism (Schenk et al., 1998).
a. Transketolase. Plant TKs operate mostly in chloroplasts (Debnam and

Emes, 1999; Schnarrenberger et al., 1995). The spinach TK gene harbours a
plastid-targeting sequence (Flechner et al., 1996; Teige et al., 1995) and is
expressed in both photosynthesizing and non-photosynthesizing tissues
(Bernacchia et al., 1995; Teige et al., 1998). In chloroplast stroma, TK
takes part in the photosynthesis-associated carbon fixation that occurs in
the CalvinBenson cycle (Raines, 2003). Its activity is a limiting factor for the
maximum rate of photosynthesis. In the CalvinBenson cycle, TK catalyses
the conversion of glyceraldehyde-3-P and fructose-6-P to xylulose-5-P and
erythrose-4-P, as well as that of glyceraldehyde-3-P and sedoheptulose-7-P to
ribose-5-P and xylulose-5-P. Although TK is a non-regulated enzyme, its
decreased level can suppress sucrose production and the photosynthesis rate
(Henkes et al., 2001). TK is also universally required for the pentose phosphate pathway. Most of the enzymes involved in NADPH generation in the
oxidative part of this pathway are present in both plastids and the cytosol.
However, the plant cell localization of the non-oxidative part of pentose
phosphate pathway, where TK is responsible for the carbon skeleton production for nucleotide biosynthesis, is still under debate (Bernacchia et al.,
1995). Previous TK activity analyses (Hong and Copeland, 1990; Journet
and Douce, 1985; Nishimura and Beevers, 1979) and isotopic carbohydrate
labelling studies (Krook et al., 1998; Rontein et al., 2002) have indicated that
TK catalysis can vary between species, tissues and different stages of plant
development, and may also depend on the environmental conditions (Kruger
and von Schaewen, 2003).
3. Lyases
Among the well-characterized plant TDP-dependent lyases are (i) PDC,
the key enzyme in ethanolic fermentation; (ii) acetolactate synthase
(AHAS) which is involved in branched-chain amino acid synthesis; and
(iii) 1-deoxy-D-xylulose-5-phosphate synthase (DXPS), the enzyme for
isoprenoid formation.
64
M. RAPALA-KOZIK
a. Pyruvate decarboxylase. PDC catalyses the irreversible, non-oxidative

decarboxylation of pyruvate to acetaldehyde with CO2 liberation (Fig. 9).
This enzyme is predominant in seeds and has been detected in O. sativa
(Hossain et al., 1994; Rivoal et al., 1990), Z. mays (Forlani et al., 1999) and
Pisum sativum (Mucke et al., 1995). During ethanolic fermentation, acetaldehyde is reduced to ethanol by alcohol dehydrogenase. The activation of
PDC, resulting in ethanol production, has mostly been observed under stress
conditions, for example, in the adaptation of rice plants to low temperature,
probably owing to alterations in the physical properties of membrane lipids
(Kato-Noguchi and Yasuda, 2007) or in changes in plant growth under
anoxia and hypoxia (Ismail et al., 2009; Ismond et al., 2003; Kursteiner
et al., 2003). The induction of fermentative metabolism was also observed
previously under aerobic conditions in the roots of pea plants as a result of
the inhibition of branched-chain amino acid biosynthesis (Zabalza et al.,
2005). PDC was also shown to be critically involved in the growth of pollen
tubes in Petunia hybrida (Gass et al., 2005).
b. Acetohydroxyacid synthase. AHAS catalyses the first step in the biosynthesis of branched-chain amino acids (Duggleby et al., 2008), the condensation
of two pyruvate molecules during the synthesis of Val and Leu, or that of
pyruvate and -ketobutyrate for the synthesis of Ile. This enzyme is unstable
during purification, but its activities have been demonstrated in maize
(Muhitch et al., 1987), barley (Durner and Boger, 1988) and wheat (Southan
and Copeland, 1996) and, using a heterologous expression system in bacteria,
also in cocklebur (Bernasconi et al., 1995), Arabidopsis (Chang and Duggleby,
1997; Dumas et al., 1997; Ott et al., 1996) and tobacco (Chang et al. 1997). Plant
AHASs are composed of a catalytic subunit with a TDP-binding site and a
regulatory subunit necessary for feedback inhibition by branched-chain amino
acids (Lee and Duggleby, 2001; McCourt and Duggleby, 2006). The identified
N-terminal signal sequences suggest the translocation of this protein to chloroplasts (Ott et al., 1996). The AHAS enzymes are also involved in the binding of
several herbicide classes (McCourt et al., 2006). However, some herbicideresistant mutations in the AHAS gene have been reported in rice, tobacco
and Arabidopsis (Chang and Duggleby, 1998; Kawai et al., 2007; Okuzaki
et al., 2007; Shimizu et al., 2002; Tan et al., 2005). These observations have
prompted a number of attempts to produce transgenic, herbicide-resistant crop
plants (Ott et al., 1996).
c. 1-Deoxy-D-xylulose-5-phosphate synthase. DXPS catalyses the first reaction in an alternative, non-mevalonate pathway of isoprenoid biosynthesis, in
which glyceraldehyde 3-phosphate is condensed with pyruvate
65
(Lichtenthaler, 1999; Sprenger et al., 1997). The product, DXP, was also
identified as a precursor in the thiamine and pyridoxol (a form of vitamin
B6) biosynthesis pathways in plants and in E. coli (Begley et al., 1999; Hill
et al., 1996). Multiple DXPS genes have been found in several plant species
that encode isoforms involved in the biosynthesis of different classes of
isoprenoids (Cordoba et al., 2009). DXPS expression has also been detected
in all photosynthetic tissues, with an unequivocal plastidial localization of this
enzyme (Zhang et al., 2009). DXPS overexpression in tomato, Arabidopsis
and tobacco correlates with the accumulation of chlorophyll, carotenoids,
tocopherols and abscisic acid (ABA), indicating that this enzyme catalyses the
rate-limiting reaction in the isoprenoid phosphate pathway (Estevez et al.,
2001; Lois et al., 2000, Zhang et al., 2009). Some growth conditions, for
example, light exposure (Kim et al., 2005), mechanical wounding or fungal
elicitors (Phillips et al., 2007), also modulate DXPS transcript accumulation.
IV. THIAMINE TRANSPORT, DISTRIBUTION AND

STORAGE IN PLANT TISSUES
Depending on the development stage, plants use different sources for thiamine acquisition. These include seed storage tissues, biosynthetic processes
and soils. During seed maturation, thiamine accumulates in the germ in
parallel with the increase in the total soluble protein content (Shimizu et al.,
1990). Thiamine is stored in the unphosphorylated form and even in mature
seeds, the phosphate esters represent only 5% of the total thiamine content.
The long-term thiamine storage in seeds depends on specific thiamine-binding
proteins (TBPs) which are present in many plant species (AdamekSwierczynska and Kozik, 2002; Adamek-Swierczynska et al., 2000; Kozik
and Rapala-Kozik, 1995; Mitsunaga et al., 1986a,b, 1987; Nishimura et al.,
1984; Nishino et al., 1983, Rapala-Kozik and Kozik, 1998; Shimizu et al.,
1995). The chemical mechanism of thiamine binding by these proteins has
been extensively studied (Kozik and Rapala-Kozik, 1996; Rapala-Kozik and
Kozik, 1992, 1996; Rapala-Kozik et al., 1999). TBPs are suggested to represent specific variants of the major seed storage globulins (AdamekSwierczynska and Kozik, 2002; Rapala-Kozik et al., 2003).
Developing seedlings first utilize the thiamine that is stored in seeds, as
demonstrated from previous analyses of the total seed thiamine content
which does not change (Kylen and McCready, 1975; Mitsunaga et al.,
1987) or decrease (Golda et al., 2004) during seed germination. Depending
on the species, this takes 24 days after imbibition, before the seedlings
commence thiamine biosynthesis (Golda et al., 2004). At least in cereal
66
M. RAPALA-KOZIK
seeds, TPK activity progressively increases during seed germination and

seedling growth (Golda et al., 2004; Mitsunaga et al., 1987).
As discussed in the preceding sections, the biosynthesis of thiamine takes
place in chloroplasts but TDP is formed in the cytosol (Fig. 6). As different
compartments utilize TDP as an enzyme cofactor, it is highly probable that
plants possess thiamine-, TMP- or TDP-specific cellular transporters, but
none have yet been identified. It has been shown that TMP, synthesized de
novo in chloroplasts, readily undergoes dephosphorylation by relatively nonspecific phosphatases (Rapala-Kozik et al., 2009), but the actual subcellular
localization of this process remains unknown. Free thiamine is pyrophosphorylated in the cytosol but the TDP produced is also important for
fundamental mitochondrial functions. This suggests that a TDP transporter
should exist in the inner mitochondrial membrane. Mitochondrial TDP
transporters were previously identified in human, yeast and Drosophila melanogaster (Iacopetta et al., 2010; Lindhurst et al., 2006; Marobbio et al.,
2002) and belong to a broad mitochondrial carrier family, the members of
which have also been detected in Arabidopsis (Millar and Heazlewood,
2003). Similar hypothetical transporters may also be useful for thiamine
uptake from seed storage tissues or soil.
Owing to the chloroplastic localization of the entire pathway of TMP de
novo synthesis, green tissues are the primary location where thiamine is
formed and from which it is transported to thiamine-requiring tissues such
as the roots. Accordingly, the genes encoding HMP-P synthase, HET-P
synthase and HMP-P kinase/TMP synthase are predominantly or sometimes
exclusively detected in leaves (Belanger et al., 1995; Kim et al., 1998; Kong
et al., 2008; Papini-Terzi et al., 2003; Raschke et al., 2007; Ribeiro et al.,
1996). In contrast, TPK is expressed in all plant tissues, albeit at variable
levels (Ajjawi et al., 2007a,b), to ensure that both endogenous and exogenous
thiamine sources will be equally useful for the synthesis of TDP.
An alternative way to acquire thiamine is via absorption from the soil by
the roots (Mozafar and Oertli, 1992, 1993), which in most plant species have
no thiamine-synthetic capacity. The transport of external thiamine appears
to be independent of the level of metabolic energy and probably represents a
passive transpiration-mediated process. Root-absorbed thiamine flows to
other plant parts via the xylem (Mozafar and Oertli, 1992, 1993). Thiamine
and its phosphate esters can also be introduced into plant seedlings through
the leaves (Mozafar and Oertli, 1992, 1993). After a sufficient period of time
from its application, thiamine appears to be uniformly distributed between
different parts of the plant. This transport probably occurs via the phloem
and may be strictly polarized (basopetal), as seen in the tomato petiole
(Kruszewski and Jakobs, 1974) or may proceed in both the acropetal and
67
basopetal directions (Mozafar and Oertli, 1992, 1993). More recently, it has
been reported that the foliar application of TMP and TDP can trigger plant
disease resistance (Ahn et al., 2005, 2007) and complement the Arabidopsis
TPK double mutant (Ajjawi et al., 2007a,b). These findings provide good
evidence for thiamine-phosphate transport by the plant vascular system via
an apoplastic route. Leaves which develop after thiamine application can
concentrate this vitamin, suggesting its possible re-mobilization from older
parts of the plant (Mozafar and Oertli, 1993). Thiamine transport via the
phloem from leaves to the kernels in maize, wheat and rice was reported
many years ago (Kondo et al., 1951; Shimamoto and Nelson, 1981). The
thiamine levels decrease in the glumes, leaves and stem and increase in the
kernels towards the end of kernel-filling process (Geddes and Levine, 1942).
In maize, the concentration of thiamine in the embryo is more than 10-fold
greater than that in the endosperm (Shimamoto and Nelson, 1981).
In summary, the current knowledge of thiamine transport in plant tissues
and cells is not well advanced and further research, paying particular attention
to the identification of the TDP- and/or thiamine transporters, is necessary.
V. ROLE OF THIAMINE IN THE SENSING,

RESPONSE AND ADAPTATION TO PLANT STRESS
The environmental conditions which exert abiotic stress in plants (drought,
high salinity, heavy metals, drastic changes in temperature or light intensity)
can significantly alter plant metabolism, growth and development. However,
the mechanisms underlying the responses or even perception of these environmental stresses by plants are not well understood. The current evidence with
regards to the pathways by which plants sense or adapt to stress is based on
transcript changes (genetic analyses), protein induction or suppression (proteomics) or protein activity determination. However, an increase in the mRNA
levels could be interpreted in terms of increased requirement for the translated
protein product during stress conditions but it may also indicate that this
protein is susceptible to damage during stress and its resulting degradation
requires an increase in transcription to maintain its normal cellular levels. These
possibilities must be taken into account in future data analysis.
A. ABIOTIC STRESS RESPONSES
As plants are unable to avoid exposure to extreme environmental conditions,

they have developed many types of specific responses in order to survive.
Most metabolic analyses in this regard have been concerned with changes in
68
M. RAPALA-KOZIK
various pathways of carbon metabolism including glycolysis, the tricarboxylic acid cycle and photosynthesis, which probably represent the primary
responses of plants to stress, mediated by chemical reactions and enzymatic
components. However, the changes observed in thiamine biosynthesis processes should be considered as a second line of defence, once the stress
stimulus has been sensed by the plant and transcriptional, translational or
post-translational responses have been initiated.
Previous studies that have focused on the activity of the main metabolic
pathways that operate during abiotic stress conditions have shown that
primary anabolic metabolism is largely downregulated in favour of catabolic
and antioxidant metabolism. For example, in Arabidopsis roots or in the
cells of other organs subjected to oxidative stress, an impairment of the
tricarboxylic acid cycle and of amino acid metabolism has been observed
and this was followed by the initiation of a backup system for glycolysis
comprising a redirection of carbon metabolism to the oxidative pentose
phosphate pathway for NADPH production (Baxter et al., 2007; Lehmann
et al., 2009).
As many enzymes which operate in the sensing, response activation and
adaptation to plant stress require TDP as a cofactor (Fig. 11), it is not
surprising that the de novo biosynthesis of this compound is upregulated in
plants under stress conditions. The upregulation in the transcript levels
(three- to fourfold) of two initial thiamine biosynthetic genes, THI1 and
THIC, was observed during the adaptation of Arabidopsis seedlings to
growth under paraquat-induced oxidative stress (Tunc-Ozdemir et al.,
2009). Additionally, a twofold increase in -GUS activity was observed
under salt stress or flooding conditions in transgenic plants carrying the
GUS promoter gene fused to THI1 promoter fragments (Ribeiro et al.,
2005). These results confirmed earlier findings from proteomic and DNA
microarray studies of plant responses to cold, heat and drought (Ferreira
et al., 2006; Wong et al., 2006). The THI1 gene may be precisely regulated
under stress conditions since its promoter possesses an ABA-responsive
element (Ribeiro et al., 2005). It has also been suggested that the THIC
promoter possesses a stressresponse element (Tunc-Ozdemir et al., 2009).
However, in both cases, there is no evidence of the actual functioning of these
putative regulatory elements. A three- to sixfold increase of the levels of TMP
synthase and TPK transcripts was also observed in Arabidopsis seedlings
under oxidative stress conditions and these results correlated with a detectable
increase of the levels of thiamine and its phosphate esters (Tunc-Ozdemir et al.,
2009). Analogical responses were observed in Z. mays seedlings under salt,
water and oxidative stress conditions under which the activities of both TMP
synthase and TPK, as well as total thiamine levels, significantly increased
69
(Rapala-Kozik et al., 2008). Interestingly, the latter effect in stressed Z. mays

seedlings was predominantly due to an increase of free thiamine, whilst in
Arabidopsis, a TDP increase was more predominantly detected under similar
stress conditions. This could be explained by the different plant response phases
analysed in these two studies. In the study of Z. mays seedlings, an overproduction of thiamine, ready to be transported into the appropriate organelles, was
detected and in the Arabidopsis model, the response may be shifted to the
production of the functional coenzyme form of thiamine. A drop in the steadystate TDP levels may be important as TDP is the major regulatory factor for
thiamine biosynthesis (Nosaka, 2006), and is known to operate via a riboswitch
which is present in the 30 -UTR of the THIC gene (Bocobza et al., 2007; Raschke
et al., 2007; Wachter et al., 2007).
After the regeneration of a significant source of TDP, damaged pathways
can be restarted, probably at a higher rate to compensate for any stressinduced deficiencies and to support adaptive responses (Fig. 12).
Stress sensing and response

Adaptation
CBC
TK
or
PPP
PDH
Abiotic
stress
KGDH
DXPS
Thiamine
biosynthesis
pathways
(THI1, THIC,
THI3, TPK)
NADPH, ribose-5P,
glutatione,
nucleic acids,
coenzymes
PDH
TCAC
Glutamate,
proline,
GABA
IPBP
Izoprenoids,
gibberellins,
ABA
Fig. 12. Thiamine biosynthesis and TDP-dependent pathways in the sensing,

response and adaptation to plant stress. A sensing of environment stress factors by
the plant involves damages to the main TDP-dependent enzymes (TK, PDH, KGDH,
DXPS). In a response, the activities of thiamine biosynthetic enzymes (THI1, THIC,
THI3,TPK) increase and subsequently a regeneration of the main metabolic pathways occurs. In an adaptation phase, some of the TDP-dependent pathways such as
the CalvinBenson cycle (CBC), the pentose phosphate pathway (PPP), the tricarboxylic acid cycle (TCAC) and isoprenoid phosphate biosynthesis pathway (IPBC)
can be upregulated to compensate for the previous damages and to provide important
defence molecules (e.g. antioxidants) and stress protectants.
70
M. RAPALA-KOZIK
It has been reported that the oxidative pentose phosphate pathway (Baxter
et al., 2007; Couee et al., 2006), isoprenoid biosynthesis pathway (Paterami
and Kanellis, 2010; Schroeder and Nambara, 2006), the tricarboxylic acid
cycle (Lehmann et al., 2009) and ethanolic fermentation (Conley et al., 1999;
Drew, 1997; Kursteiner et al., 2003) are accelerated or induced under different abiotic stress conditions, in which an intensive increase in ROS production was observed in all plant cell compartments in most cases (Zhu, 2002).
The cytosolic enzymes involved in the early stages of glycolysis, triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase, may be
partly inhibited by excessive ROS, causing a rerouting of the main carbohydrate-metabolic flux from the glycolytic to the pentose phosphate pathway
(Ralser et al., 2007). This pathway is activated by the upregulation of
regulatory enzymes involved in the oxidative steps (Couee et al., 2006;
Debnam et al., 2004; Valderrama et al., 2006) and produces more
NADPH, which is recycled via numerous antioxidant systems, such as the
ascorbate-glutathione cycle, to quickly restore the cytoplasmic redox equilibrium (Valderrama et al., 2006).
TK is one of the major TDP-dependent enzymes for which the increased
transcript and protein levels, as well as a higher enzymatic activity, has been
shown in several plant species under different stress conditions (Bernacchia
et al., 1995; Ferreira et al., 2006; Rapala-Kozik et al., 2008; Wolak et al.,
2010). TK operates in chloroplasts and probably, at least in some species,
also in the cytoplasm, and is involved in the CalvinBenson cycle and pentose
phosphate pathway. These two processes produce NADPH which feeds a
variety of ROS-scavenging systems such as the plastidial AsadaHalliwell
pathway that engages two powerful antioxidants, reduced glutathione and
ascorbate (Arora et al., 2002). Although TK is not a regulatory enzyme, its
levels need to be suitably adjusted during the response to environmental
stresses to assure a balanced flow of all intermediates of the NADPH producing pathways (Henkes et al., 2001).
Another stress defence system which operates in chloroplast is dependent
on the non-mevalonate isoprenoid synthesis pathway which engages another
TDP-dependent enzyme, DXPS (Lange et al., 1998). This pathway provides
precursors for the synthesis of carotenoids, terpenes, tocopherols and is also a
source of chlorophyll, plastoquinone, gibberellins and ABA (Lichtenthaler
et al., 1997). Carotenoids are powerful antioxidants (Hix et al., 2004;
Vallabhaneni and Wurtzel, 2010) and ABA participates in the signal transduction pathways required for plant adaptation to several types of abiotic
stress. DXPS transcript accumulation is induced in Cistus creticus in response
to heat, drought, wounding and elicitors including salicylic acid and methyl
jasmonate (Paterami and Kanellis, 2010). These results are consistent with
71
previous finding that isoprenoids participate also in thermotolerance-related

activities involved in plant adaptation (Penuelas and Munne-Bosch, 2005).
The activation of the ethanolic fermentation pathway in plants which grow
at low temperatures, or under water deficiency or hypoxia, is well documented (Ismond et al., 2003; Kato-Noguchi and Yasuda, 2007). A cytosolic
TDP-dependent enzyme, PDC, is the main regulatory enzyme in this pathway (Kursteiner et al., 2003) and its overexpression in Arabidopsis improves
the plant tolerance to hypoxia (Ismond et al., 2003). This finding suggests
that mitochondrial dysfunction and the inhibition of pyruvate conversion to
acetyl-CoA cause a redirection of the main glycolytic pathway to cytoplasmic
ethanolic fermentation. Ethanol production prevents lipid degradation in the
plant membrane and enables the maintenance of energy production until the
more effective aerobic respiration processes are recovered (Kursteiner et al.,
2003; Tadege et al., 1999).
The major stress sensing pathway in plants seems to be the tricarboxylic
acid cycle and mitochondrial production of acetyl-CoA (Baxter et al., 2007;
Sweetlove et al., 2002; Taylor et al., 2004a). Both pathways engage the TDPactivated complex enzymes PDH and KGDH which are readily inactivated
by oxidative damage of their lipoic acid-dependent components (Taylor
et al., 2004b). After antioxidant stress responses are activated, these pathways are restored during the adaptation phase (Taylor et al., 2004a).
B. THIAMINE FUNCTION IN BIOTIC STRESS
The improved growth of plants in the presence of thiamine was observed

some years ago, but it has only been recently that a better understanding of
this effect of thiamine has come to light, particularly under biotic stress
conditions. The thiamine supplementation of plants undergoing bacterial,
fungal or viral infection triggers systemic acquired resistance (SAR) to these
pathogens (Ahn et al., 2005; Malamy et al., 1996). It was further found that
in the presence of thiamine, the expression of defence-related (PDF1.2) and
SAR-related (PR1) genes is induced more rapidly compared with pathogen
inoculation. The expression of these genes was found to be even higher when
pathogen inoculation was followed by thiamine treatment (Ahn et al., 2005).
Interestingly, thiamine-phosphate esters were also found to rescue infected
plants, and at even lower concentrations than thiamine. This could be due to
either a higher effectiveness of TDP/TMP or a slower effect of thiamine due
to restrictions in its transport.
The signalling processes that are affected by thiamine during pathogen
infections involve the salicylic acid-dependent and mobilized calcium-related
signalling pathways and also the priming of plant defence responses that
72
M. RAPALA-KOZIK
suppress pathogen propagation (Ahn et al., 2005). SAR activation by thiamine is accompanied by hydrogen peroxide accumulation which can trigger a
stress response (Ahn et al., 2007). This suggestion is supported by the
observation that rice plants with a repressed disease resistance-responsive
gene (OsDR8) produce a lower level of thiamine (Wang et al., 2006). This
effect can be related to the high sequence similarity between the OsDR8
protein and maize thiazole synthase (THI1, THI2). Additionally, OsDR8silenced plants express lower levels of several defence-responsive genes suggesting the involvement of OsDR8 in the regulation of signal transduction
pathways that function in the defence response.
C. RESCUE OF STRESSED PLANTS BY THIAMINE SUPPLEMENTATIONIS
THIAMINE A REAL ANTIOXIDANT?
In many types of plant stress, ROS production in the cells is considered to be a

secondary stress event and the prime activator of antioxidative response pathways. Some reports have suggested that thiamine can function directly as an
antioxidant. The products of thiamine oxidation in vitro are thiochrome or
thiamine disulphide analogues (Lukienko et al., 2000; Stepuro et al., 2005).
Thiochrome can be easily detected owing to its strong fluorescence but for its
formation, a non-physiological highly basic environment is necessary. Thiamine
disulphide-related compounds are difficult to analyse in cell extracts. A similar
hypothesis for the antioxidant activity of thiamine has come from analyses of
human nerve cells with a thiamine deficiency (Hazell and Butterworth, 2009). It
has been documented that thiamine can normalize the lipid peroxidation levels
and elevate the activity of glutathione reductase, and that thiamine deficiency
leads to peroxynitrite accumulation. However, thiamine was found not to exert
a phytotoxic effect at any concentration tested. In addition, the participation of
thiamine in DNA repair in bacteria, yeast and plants has been proposed
(Machado et al., 1996, 1997). However, the mechanism underlying the role of
thiamine action as an antioxidant defence trigger remains obscure.
VI. PRACTICAL ASPECTS AND FUTURE

PERSPECTIVES
The current knowledge of the physiological functions of thiamine compounds in human, including the crucial role of TDP as cofactor in cellular
metabolism and the non-cofactor neurophysiological role of TTP, is well
advanced. Modern medicine has taken advantage of this knowledge in the
development of treatments for numerous pathophysiological conditions
73
which are the results of a low vitamin B1 content in the diet, inefficient
intestinal thiamine absorption, an impaired uptake of thiamine by individual
tissues and cells or thiamine-dependent metabolic malfunctions. At least
some of these pathogenic influences can be prevented or eliminated nutritionally through the enrichment of foods with vitamin B1, supplementation
with more easily absorbable derivatives (e.g. benfothiamine) and the elimination of antithiamine factors (thiaminases, polyphenolic compounds)
among others. In developed countries, thiamine imbalances in the diet are
usually overcome by industrial fortification of foods such as bread with this
vitamin. However, in developing countries, crops are the major source of
thiamine. Unfortunately, the limited advances thus far in thiamine-focused
plant science seriously hinder the potential for improving plant constituents
as a strategy to lower the rate of thiamine deficiency-related diseases.
As noted in this review, considerable progress has been made in our
understanding of thiamine biosynthesis and metabolism in higher plants in
recent years. One of the highlights in this regard has been the discovery of the
riboswitch-dependent feedback inhibition of thiamine synthesis. This and
other regulatory mechanisms must be further elucidated to the point where it
is possible to engineer plant cultivars with a higher thiamine content in the
consumable tissues. However, thiamine produced by microorganisms in the
soil can be absorbed by roots, transported to plant cells and converted to
TDP, but our current understanding of these transport processes is still in its
infancy. It is possible that cytoplasmic TPK, which is probably less tightly
regulated than chloroplastic enzymes of the main thiamine biosynthetic
route, may be a viable target for genetic manipulation (overexpression) to
increase the production of TDP and, after its quick dephosphorylation,
augment the total thiamine content in plant tissues. It is likely, however,
that the best material to increase the nutrition value with respect to vitamin
B1 will prove to be the seeds in which specific globulins are deposited together
with captured thiamine, to provide the necessary reserves for the growing
seedling. An increase in the expression of these TBPs by genetic engineering
should be possible and thereby provide an enriched store of this vitamin.
The recent unequivocal establishment of the critical role of thiamine in the
plant response and adaptation to biotic and abiotic stresses should have a
practical impact, for example, in developing plant cultivars with higher stress
resistance. Once our general understanding of the mechanism of thiamine
transport in plants is improved, methods for a more effective supplementation of plants may be developed to increase plant resistance to stress factors
such as high temperature, drought or environmental pollution. The development of plant cultivars with high stress tolerance should improve global plant
production levels, which would represent another approach in contemporary
74
M. RAPALA-KOZIK
agriculture to overcoming thiamine deficiency problems. Recently, a strategy

for engineering herbicide-tolerant crops has been proposed which utilizes the
known properties of a specific TDP-dependent enzyme, AHAS. This enzyme,
which is critical for the biosynthesis of branched-chain amino acids in plants,
is a potential target for herbicide action (Duggleby et al., 2008; McCourt
et al., 2006). It was found that the imidazolinone herbicides bind to the AHAS
regulatory subunit, blocking the active centre of this enzyme (Trucco et al.,
2006). In mutagenesis analysis of the herbicide-binding pocket in AHAS,
some amino acids were selected whose substitution resulted in the resistance
of this enzyme to these herbicides (Jung et al., 2004; Kolkman et al., 2004).
Mutagenesis or selection approaches, that utilize conventional plant breeding
techniques, have created many imidazolinone-resistant crops including
maize, rice, wheat, sunflower and oilseed rape (Tan et al., 2005). The application of imidazolinone herbicides in the cultivation of resistant crops has
facilitated the control of a broad spectrum of grasses and broadleaf weeds.
However, effectiveness at low doses, low mammalian toxicity as well as a
favourable environmental profile have made imidazolinone herbicides attractive agents for efficient crop production. In addition, DXPS, the key enzyme
in mevalonate-independent isoprenoid biosynthesis, has been suggested to be
a promising target for new herbicide development as well as for improving the
nutritional value of crop plants (Cordoba et al., 2009; Muller et al., 2000).
In summary, the recent strong progress in the biochemical and physiological
study of thiamine in plants, albeit less advanced than analogous research in
animals and microorganisms, is expected to continue in the near future and to
have an important impact in modern agriculture for improving the nutritional
value of plant crops, thereby reducing the rate of chronic disease states that are
dependent on the impaired uptake and metabolism of vitamin B1.
ACKNOWLEDGEMENTS
The author thanks prof. Andrzej Kozik for helpful discussion and critical
chapter reading. This work was supported in part by the Ministry of Science
and Higher Education, Poland (the grant No. NN303 320937) and the
Jagiellonian University (statutory funds No. DS/15/WBBiB).
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Biosynthesis of Vitamin B2 and Flavocoenzymes in Plants
MARKUS FISCHER*,{,1 AND ADELBERT BACHER*,{
*Institute of Food Chemistry, University of Hamburg,

Hamburg, Germany
{
Ikosatec GmbH, Garching, Germany
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
II. A Single Plant Enzyme, RibAB Generates Both Committed
Precursors for the Riboflavin Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
III. Deaminase/Reductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
IV. Lumazine Synthase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
V. Riboflavin Synthase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
VI. Riboflavin Kinase and FAD Synthetase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
VII. Cellular Topology of Flavocoenzyme Biosynthesis in Plants . . . . . . . . . . . . .
VIII. Regulation of Riboflavin Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
IX. Excretion and Enhanced Formation of Riboflavin by Iron-Deficient
Roots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
X. Evolution of Flavocoenzyme Biosynthesis Enzymes in Plants. . . . . . . . . . . .
XI. Riboflavin Biosynthetic Enzymes as Potential Herbicide Targets . . . . . . . .
XII. Riboflavin and Plant Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
XIII. Biosynthesis of 5-deaza-7,8-didemethyl-8-hydroxy-riboflavin in Algae . .
XIV. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
94
100
105
108
116
126
127
128
129
131
132
132
133
137
138
ABSTRACT
Riboflavin (vitamin B2) derivatives serve as cofactors for a very wide variety of redox
enzymes but are now also known to participate in the catalysis of certain non-redox
reactions and as cofactors of blue-light photoreceptors. In parallel with the unique
1

0065-2296/11 $35.00
DOI: 10.1016/B978-0-12-386479-6.00003-2
94
M. FISCHER AND A. BACHER
features of its chemical reactivity, the vitamin is biosynthesised from one molecule of
GTP and two molecules of ribulose phosphate by a mechanistically unique series of
enzyme-catalysed reactions. Although the work on its biosynthesis has predominantly
involved microorganisms, a reasonably detailed picture is now also emerging for
plants. A central topic of this review is the emerging role of riboflavin biosynthesis
enzymes in connection with plants iron acquisition and pathogen resistance.
I. INTRODUCTION
Vitamin B2 (riboflavin) is the obligatory precursor of flavocoenzymes, which
are involved in a wide variety of redox reactions as well as certain reactions
without net exchange of redox equivalents. Flavocoenzymes have been estimated to serve as cofactors for up to 2% of all enzymes and are essential in all
organisms.
Riboflavin, the committed precursor of flavocoenzymes, is biosynthesised
in plants and in numerous, although by no means all, microorganisms.
Animals, as well as microorganisms that are not endowed with the capacity
to obtain riboflavin by biosynthesis, are absolutely dependent on extrinsic
sources. For animals, plants are the most important source of the vitamin.
The potential contribution to the vitamin supply by the gastrointestinal flora,
notably in mammals, is incompletely understood.
Riboflavin is widely distributed as an essential constituent of all living cells.
The most relevant dietary sources are milk and milk products, meat, eggs, fish
and green leafy vegetables (Souci et al., 2008) (Table I). The absorption of
animal-derived riboflavin is better than riboflavin from vegetables.
TABLE I
Vitamin B2 Content of Foods (Souci et al., 2008)
Food
Vitamin B2 (mg/100 g)
Pork liver
Beef liver
Chicken breast
Wheat germ
Camembert/Parmesan
Sardine
White mushrooms
Egg
Spinach
Pork
Beef
Milk/Yoghurt
Avocado
Maracuja
3.2
3.0
0.9
0.7
0.6
0.4
0.4
0.3
0.23
0.2
0.2
0.18
0.1
0.1
BIOSYNTHESIS OF RIBOFLAVIN IN PLANTS
95
The daily requirement for an adult human is reported to be in the range of

around 1.3 mg (Yates et al., 1998). Recommendations for a daily riboflavin
intake are to be increased during pregnancy and lactation (to 1.4 and 1.6 mg)
(Yates et al., 1998) (Table II). The symptoms of human riboflavin deficiency
are poorly characterised, as riboflavin is widely distributed in human nutrients and any deficiency tends to be overshadowed by deficiencies for other
essential food components. Riboflavin deficiency may be difficult to recognise because it is normally accompanied by other vitamin deficiencies and is
most commonly found among people whose diets are inadequate. The first
deficiency symptoms are sores at the corners of the mouth and a sore throat.
These may be followed by a magenta coloration of the tongue (glossitis), red
and raw lips and skin sores.
Urinary excretion of less than 30 mg of riboflavin/g creatinine is associated
with clinical signs of riboflavin deficiency. The degree of stimulation of the
activity of erythrocyte glutathione reductase activity by the addition of FAD
is used to estimate the sufficiency of riboflavin in humans. The use of oral
TABLE II
Riboflavin: Dietary Reference Intake (Yates et al., 1998)
Age
Infants
06 months
712 months
Children
13 years
48 years
mg/day
0.3a
0.4a
0.5
0.6
Males
913 years
1418 years
18 years
0.9
1.3
1.3
Females
913 years
1418 years
18 years
0.9
1.0
1.1
Pregnancy
1930 years
3150 years
1.4
1.4
Lactation
18 years
1930 years
3150 years
1.6
1.6
1.6
a
Values are Adequate Intakes (AI), others are Recommended Daily
Allowance (RDA). (Adapted from www.vitaminherbuniversity.com).
96
contraceptives has been reported to be conducive to reduced riboflavin

saturation.
Despite its presence as riboflavin and/or its coenzyme forms in most typical
human food components (Table I), vitamin B2 is produced on an estimated
global scale of about 3000 metric tons per year by technically advanced
fermentation processes using Bacillus subtilis, Ashbya gossypii and Candida
famata strains, which have replaced the chemical synthesis (Stahmann et al.,
2000). Commercial riboflavin is predominantly used as feed supplement for
animal husbandry and as food colourant. Only minor amounts of the global
production become part of vitamin supplements for direct human
consumption.
Although plants are the ultimate source of most of the riboflavin that is
consumed by animals, the biosynthesis of the vitamin has been predominantly studied in microorganisms (yeasts and bacteria). This may have been due
to the relative ease of experimentation with microorganisms as compared to
plants. Moreover, the prevailing opinion seems to have been that, with the
exception of photosynthesis and directly related aspects, much of primary
metabolism in plants is basically a boilerplate variant of microbial and
mammalian metabolism. In any case, apart from some minor studies in
the 1960s, most work on riboflavin biosynthesis in plants is of relatively
recent origin.
Work on the biosynthesis of riboflavin (Fig. 1) in microorganisms started
with the seminal observation that riboflavin production by the naturally
flavinogenic ascomycete, Eremothecium ashbyi, could be increased by the
addition of purines to the culture medium (MacLaren, 1952). Subsequent
work with radioisotopes by several research groups established that all the
nitrogen atoms and the carbon atoms of the vitamins pyrimidine ring are
entirely derived from a purine precursor (for review, see Plaut et al., 1974).
Another pioneering discovery was the isolation of a green fluorescent compound (6,7-dimethyl-8-ribityllumazine, 10, Fig. 1) from the culture fluid of E.
ashbyii (Masuda, 1957a,b), which was established as the direct biosynthetic
precursor of riboflavin around 1960 (Katagiri et al., 1958a,b,c,d,e; Maley and
Plaut, 1959; Plaut, 1960, 1963).
For an extended period, the preferred objects of research on riboflavin
biosynthesis were naturally occurring flavinogenic microorganisms and, later
on, flavinogenic mutants of non-flavinogenic organisms. The reason for
that was twofold. (i) Flavinogenic strains were expected to provide favourable conditions for analysis because they were correctly expected to have
enhanced levels of flavin biosynthetic enzymes. (ii) Microbial flavinogenesis
was of practical interest as a potential technology for the production of
97
Fig. 1. Biosynthesis of riboflavin and flavocoenzymes. (A) GTP cyclohydrolase

III; (B) GTP cyclohydrolase II; (C) 2-amino-5-formylamino-6-ribosylamino-4(3H)pyrimidinone 50 -phosphate hydrolase; (D) 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 50 -phosphate deaminase; (E) 5-amino-6-ribosylamino-2,4
(1H,3H)-pyrimidinedione 50 -phosphate reductase; (F) 2,5-diamino-6-ribosylamino-4
(3H)-pyrimidinone 50 -phosphate reductase; (G) 2,5-diamino-6-ribitylamino-4(3H)pyrimidinedione 50 -phosphate deaminase; (H) hypothetical phosphatase; (I) 3,4-dihydroxy-2-butanone 4-phosphate synthase; (J) 6,7-dimethyl-8-ribityllumazine synthase;
(K) riboflavin synthase; (L) riboflavin kinase; (M) FAD synthetase; 1, GTP; 2, 2,5diamino-6-ribosylamino-4(3H)-pyrimidinone 50 -phosphate; 3, 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 50 -phosphate; 4, 5-amino-6-ribosylamino2,4(1H,3H)-pyrimidinedione 50 -phosphate; 5, 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinedione 50 -phosphate; 6, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione
50 -phosphate; 7, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione; 8, ribulose
5-phosphate; 9, 3,4-dihydroxy-2-butanone 4-phosphate; 10, 6,7-dimethyl-8-ribityllumazine; 11, riboflavin; 12, FMN; 13, FAD. Green arrows mark the plant pathway;
red, fate of the 4-carbon precursor 9 derived from ribulose 5-phosphate.
98
the vitamin; in fact, fermentation has by now completely superseded

the chemical synthesis as a method for the technical production of the
vitamin.
Ever since the early studies, there has been an uninterrupted stream of
additional data on flavin biosynthesis and function. The inclusion of Archaea
and plants began in the 1990s. The early work on riboflavin biosynthesis has
been reviewed repeatedly, and readers are directed to these articles (Bacher,
1991; Bacher et al., 1993, 2000, 2001; Brown and Neims, 1982; Brown and
Reynolds, 1963; Brown and Williamson, 1987; Fischer and Bacher, 2005,
2006, 2008, 2011; Plaut et al., 1974; Young, 1986).
There are significant differences in the riboflavin pathways of different
taxonomic groups. The early steps constitute a moderately complex maze as
shown in Fig. 1. Invariably, all pathway variants start with GTP. In eubacteria, fungi and plants, GTP is converted into 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone monophosphate by GTP cyclohydrolase II, which catalyses the release of C-8 of the purine moiety by cleavage of two different
carbon nitrogen bonds, resulting in the formation of formate as well as the
cleavage of a phosphoanhydride bond resulting in the formation of inorganic
pyrophosphate (Foor and Brown, 1975). Archaea, however, use two enzymes
to achieve the same end result, with the formamide 3 as an intermediate
(Graham et al., 2002; Grochowski et al., 2009; Spoonamore et al., 2006).
The reaction product of GTP cyclohydrolase II can be converted to
5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 50 -phosphate (6) via
two different reaction sequences. In bacteria and in plants, the pyrimidine
ring of 2 undergoes deamination resulting in 4, and the subsequent reduction
of the ribosyl side chain affords 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 50 -phosphate (6) (Burrows and Brown, 1978). In yeasts and Archaea, however, the ribosyl residue of 2 is first converted into a ribityl moiety
by a reductase affording 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 50 phosphate (5) (Bacher and Lingens, 1970; Hollander and Brown, 1979). The
subsequent deamination of the pyrimidine ring of 5 affords 6. Notably, the
yeast and fungal pathways converge at the level of the 50 -phosphate 6.
However, that compound is not used by the next known enzyme in the
pathway, lumazine synthase, which accepts only the dephosphorylated
form of 6 as substrate. It follows that 6 must be dephosphorylated in order
to enter the final stretch of the biosynthetic pathway, but it is still unknown
how the required dephosphorylation occurs.
The final two reactions involve the assembly of the xylene ring of the
vitamin from two identical 4-carbon molecules. Work around 1990 established that ribulose 5-phosphate (8) is transformed into 3,4-dihydroxy-2butanone 4-phosphate (9) by a mechanistically complex rearrangement
99
resulting in the extrusion of C-4 of the substrate as formate (Richter et al.,

1992; Volk et al., 1990; Volk and Bacher, 1988, 1990, 1991) (notably, GTP
cyclohydrolase II also affords formate as a second product).
The condensation of the pyrimidine derivative 7 with the carbohydrate 9,
catalysed by lumazine synthase, was shown in the 1990s to afford the green
fluorescent pteridine derivative, 6,7-dimethyl-8-ribityllumazine (10) (Kis and
Bacher, 1995; Volk and Bacher, 1988), several decades after the compound
had been established to serve as the sole substrate for the terminal intermediate in the pathway, riboflavin synthase. The latter enzyme catalyses the
transfer of a 4-carbon unit between two identical substrate molecules resulting in the formation of equivalent amounts of riboflavin and the pyrimidine
7. That second product of riboflavin synthase is also the substrate of lumazine synthase and can be recycled; hence, on average, all molecules generated
by the early stage of the biosynthetic pathway must be processed twice by
lumazine synthase and by riboflavin synthase.
In summary, the biosynthesis of one molecule of riboflavin requires one
molecule of GTP and two molecules of ribulose phosphate. No cofactors are
required except divalent metal ions and NADPH. The reactions catalysed by
GTP cyclohydrolase II, pyrimidine deaminase, 3,4-dihydroxy-2-butanone
4-phosphate synthase, lumazine synthase and riboflavin synthase are all
exergonic, and the reverse reaction defies experimental detection. Clearly,
the vitamin arises by a unique sequence of chemical reactions. Despite their
extraordinary complexity that will be described below in more detail, some of
these reactions can proceed at appreciable rates in the absence of enzyme
catalysis. That said, the riboflavin pathway is extraordinary in many respects.
Around the turn of the century, when many aspects of flavin biosynthesis
had been worked out with microorganisms, the newly available and rapidly
growing body of available DNA sequences from a variety of plants provided
a basis for rapid identification of candidate genes for riboflavin biosynthesis
in plants by way of database mining. The role of candidate genes could then
be verified by heterologous expression. In that way, most riboflavin biosynthesis genes and proteins were recovered during a short period. As shown in
more detail below, plants, green algae and cyanobacteria all use the bacterial
pathway, as opposed to the yeast/archaeal pathways shown in Fig. 1. Specifically, however, the plant mRNAs invariably specify N-terminal extensions
which enable the targeting of the enzymes to chloroplasts.
All organisms need to convert riboflavin into the coenzyme forms, FMN
(12) and FAD (13). Remarkably, in species endowed with de novo riboflavin
biosynthesis, a phosphate residue is removed from the 50 -position of the
ribityl side chain of the precursor 6 and needs to be replaced after the
formation of the alloxazine ring.
100
In Fig. 1, the reactions catalysed by the enzymes of Gram-positive bacteria

and plants are shown in colour for ease of orientation. The following section is
predominantly focused on plant aspects. However, it should be noted that indepth studies of the respective plant enzymes are few in number. Notably, an
X-ray structure has only been reported for lumazine synthase of plant origin,
and detailed kinetic and mechanistic studies of the plant enzymes are mostly
elusive. However, based on homology aspects, realistic conjectures are possible on the basis of the more extensive knowledge of the bacterial orthologues.
II. A SINGLE PLANT ENZYME, RibAB

GENERATES BOTH COMMITTED
PRECURSORS FOR THE RIBOFLAVIN PATHWAY
The first committed substrates of the convergent riboflavin synthase are
produced by GTP cyclohydrolase II and 3,4-dihydroxy-2-butanone 4-phosphate synthase. The cognate genes ribA (Richter et al., 1993) and ribB
(Richter et al., 1992) of Escherichia coli were both cloned by marker rescue
in the early 1990s. With that information in hand, it became obvious that the
ribA gene at the 50 -end of the rib operon of B. subtilis (Mironov et al., 1989)
specifies a bifunctional enzyme with an N-terminal 3,4-dihydroxy-2-butanone 4-phosphate synthase domain and a C-terminal GTP cyclohydrolase II
domain (it should be noted that, for historical reasons, the monofunctional
GTP cyclohydrolase II gene of E. coli and the bifunctional B. subtilis gene are
both designated ribA).
A cDNA segment of Arabidopsis thaliana was cloned in 1998 by
marker rescue of a ribA- mutant of E. coli. Its sequence was similar to the
ribA gene of E. coli (Kobayashi et al., 1998), but no attempt was made to
characterise the translation product. Later work showed the reported sequence to be a fragment of an open reading frame specifying a bifunctional
3,4-dihydroxybutanone 4- phosphate synthase/GTP cyclohydrolase II (Herz
et al., 2000). The sequence of the two catalytic domains was similar to the
bifunctional RibA protein of B. subtilis. A homologous gene was also cloned
from tomato. The 3,4-dihydroxybutanone 4-phosphate synthase domains of
the plant genes are preceded by N-terminal segment of about 120 amino acid
residues which fulfil the criteria for chloroplast targeting sequences. The
recombinant full-length protein expressed in E. coli and a pseudomature
form were both enzymatically active.
The ribA gene of A. thaliana comprises seven exons. The gene fusion has
been proposed to guarantee a balanced substrate input from the convergent
pathways (Moore, 2004). However, detailed kinetics studies or informations
101
on substrate concentrations inside the chloroplast compartment, which

would enable simulations, are not available.
Whereas the bifunctional RibA proteins that are characteristic for plants
and Gram-positive bacteria have not been studied yet in closer detail, important insights on the structure of their catalytic domains and their reaction
mechanisms can be gleaned from sequence comparison and by extrapolation
from the detailed study of the monofunctional RibA and RibB proteins of
bacteria and fungi which have been studied in considerable detail.
NMR studies with 13C-labelled substrates showed that the 3,4-dihydroxy2-butanone 4-phosphate synthase of E. coli extracts C-4 of the substrate,
ribulose phosphate (8), as formate by way of a strictly intramolecular rearrangement (Fischer et al., 2002; Volk and Bacher, 1991) (Fig. 2). Based on
these findings, it has been proposed that the reaction is initiated by the
formation of the endiol structure 14. Following the formation of the
3-ketulose 15 and the elimination of water, a Lobry de Bryn van Ekenstein
isomerisation is believed to afford the branched aldose intermediate 18 which
can subsequently eliminate formate. The reaction is terminated by isomerisation of the resulting endiol 19. Reprotonation is stereospecific; only the L
enantiomer of the product is formed. Mg2 is required for catalysis.
Crystal structures (Fig. 3) have been reported for the (monofunctional)
3,4-dihydroxy-2-butanone 4-phosphate synthases from E. coli (Liao et al.,
2001a), Salmonella typhimurium (Kumar et al., 2010), Candida albicans (Echt
et al., 2004), Methanococcus jannaschii (Steinbacher et al., 2004) and from the
Fig. 2. Hypothetical reaction mechanism of 3,4-dihydroxy-2-butanone 4-phosphate synthase (Fig. 1, step I) (Fischer et al., 2002; Steinbacher et al., 2003; Volk and
Bacher, 1990).
102
Fig. 3. Overlay of single subunits of 3,4-dihydroxy-2-butanone 4-phosphate

synthases of C. albicans (green), E. coli (blue), M. jannaschii (red) and M. grisea
(olive green). Only the substrate ribulose 5-phosphate (8) of the C. albicans complex is
shown (yellow) (Echt et al., 2004; Liao et al., 2001a, 2002; Steinbacher et al., 2003,
2004).
rice-pathogenic fungus, Magnaporthe grisea (Liao et al., 2002; Steinbacher

et al., 2003). The solution structure of the E. coli enzyme has also been
determined by NMR (Kelly et al., 2001). The structure of the 3,4-dihydroxy-2-butanone 4-phosphate synthase domain of the bifunctional RibA
protein of Mycobacterium tuberculosis has also been reported (Singh et al.,
2011). More than 20 structures have been reported, some of them with very
range. The monofunctional proteins are homohigh resolution in the 1 A
dimers with subunits comprising about 200 amino acid residues (depending
on pH, the recombinant M. tuberculosis domain can be monomeric or
dimeric, but the quaternary structure of the naturally occurring, bifunctional
full-length protein has not been reported).
All available three-dimensional structures of 3,4-dihydroxy-2-butanone
4-phosphate synthases from different organisms show similar folds with
complex connectivity (Fig. 3). The dimers interact predominantly by hydrophobic interactions. The two topologically equivalent active sites are located
at the subunit interface, and residues from both subunits are involved in each
active site. A segment with the strictly conserved polar residues (arginine 25,
glutamates 26 and 28 and aspartates 21 and 30 (E. coli numbering)), which
103
are all essential for catalysis, is located in a flexible loop that becomes ordered
upon binding of substrate and divalent metal ions.
In 3,4-dihydroxy-2-butanone 4-phosphate synthase from M. jannaschii,
three of the four hydroxy groups of ribulose 5-phosphate are coordinated
by the metal ions. Based on crystallographic refinement, the metals were
assigned as zinc and calcium, which were present in the crystallisation buffer
(but neither of these metal ions supports catalysis) (Steinbacher et al., 2004).
The reaction mechanism of GTP cyclohydrolase II is characterised by
extraordinary complexity (Fig. 4). Specifically, the enzyme catalyses the hydrolytic cleavage of no less than three different bonds: two in the imidazole
ring of the purine moiety and one in the triphosphate motif. The available data
suggest that the first reaction step involves the formation of a covalent adduct
(20) between the protein and a GMP moiety under release of inorganic
pyrophosphate (Ritz et al., 2001). The X-ray structure of the E. coli enzyme
(Ren et al., 2005) suggests specifically that a phosphoamide bond is formed
between Asp128 and the -phosphate of the substrate, GTP. The formation of
the phosphoamide derivative is followed by the hydrolytic cleavage of the
bonds connecting C-8 and N-9 of the purine moiety, which results in
the formation of the formamide-type intermediate 21. In a subsequent step,
the formamide motif is hydrolysed under formation of formate. A Zn2 ion is
essential for both hydrolysis steps, which are believed to be further supported
by Tyr 105 serving as a proton donor (Kaiser et al., 2002). Finally, the
phosphoamide bond is cleaved hydrolytically. Very well in line with this
scenario is the observation that a fraction of substrate GMP (about 10% in
case of the E. coli enzyme) is converted to GMP (22) by the enzyme, rather than
to the riboflavin precursor 2 (Ritz et al., 2001). This implicates that, occasionally, the phosphoamide bond can be hydrolysed even before the opening of
the imidazole ring. Also in line with the hypothesis is the observation that
2-amino-5-formamido-6-ribosylamino-4(3H)-pyrimidinone
50 -pyrophosphate (21) can serve as a non-natural substrate for GTP cyclohydrolase II
(Schramek et al., 2001). Most surprisingly, the initial formation of the phosphoamide bond between protein and a GMP moiety at the beginning of the
reaction is the rate-determining step (Schramek et al., 2001).
The crystal structure of GTP cyclohydrolase II from E. coli has been
determined at near-atomic resolution in complex with a non-hydrolysable
GTP analogue (Fig. 5) (Ren et al., 2005).
104
Fig. 4. Hypothetical mechanism for release of formate by GTP cyclohydrolase II

(Fig. 1, step I). Arg128 refers to GTP cyclohydrolase II from E. coli. (Kaiser et al.,
2007; Ren et al., 2005).
Fig. 5.
105
Structure of GTP cyclohydrolase II from E. coli (Ren et al., 2005).
III. DEAMINASE/REDUCTASE
The transformation of the GTP cyclohydrolase II product 2 into lumazine
synthase substrate 8 requires the hydrolytic removal of the amino group in
position 2 of the pyrimidine ring, the reduction the ribosyl side chain affording a ribityl side chain, and dephosphorylation. In yeasts and Archaea, side
chain reduction precedes deamination and the pathway proceeds via 2,5diamino-6-ribitylamino-4(3H)-pyrimidinone 50 -phosphate (5) as intermediate (Bacher and Lingens, 1970; Hollander and Brown, 1979). In eubacteria,
however, deamination precedes side chain reduction and the reaction sequence proceeds via 5-amino-6-ribosylamino-4(3H)-pyrimidinone 50 -phosphate (4) as intermediate (Burrows and Brown, 1978). Numerous
eubacteria have genes that specify fusion proteins with an N-terminal deaminase domain and a C-terminal reductase domain (Richter et al., 1997).
Recent evidence indicates that plants and blue-green algae use the bacterial
pathway via 5-amino-6-ribosylamino-4(3H)-pyrimidinone 50 -phosphate (4)
(Fischer et al., 2004a) (Fig. 1). Specifically, a pyrimidine deaminase from
A. thaliana (GenBank Accession Code: At4g20960) has been expressed in
pseudomature form and is described in more detail below.
106
The A. thaliana open reading frame At4g20960 specifies a putative terminal

plastid-targeting sequence followed by a segment with relatively close similarity to eubacterial pyrimidine deaminase, and a C-terminal domain of about
170 amino acid residues whose function is currently unknown (Fig. 6).
Marker rescue experiments using a ribD mutant of E. coli showed that the
plant gene product serves as deaminase but has no reductase activity. The
putative deaminase domain (residues 64250), when expressed in a recombinant E. coli strain, catalysed the deamination of 2 at a similar rate as the
homologous RibD-protein of E. coli. In analogy to bacterial homologues,
the plant deaminase domain binds a Zn2 ion which is believed to be essential
for catalysis.
The structure of the plant deaminase can be anticipated in significant detail
on basis of the X-ray structures of the bifunctional deaminase/reductase
orthologues of E. coli (RibD-protein) (Stenmark et al., 2007) and of B.
subtilis (RibG protein) (Chen et al., 2006, 2009) (Fig. 7). The deaminases
are members of the pyrimidine deaminase superfamily.
Structures of cytidine deaminases of E. coli and the yeast Saccharomyces
) and can
cerevisiae have been determined to very high resolution (< 1.2 A
also serve as models for the deaminase domain from A. thaliana. The essential Zn2 ion of the cytosine deaminases and paralogs is bound by two
cysteines and one histidine. The highly conserved zinc coordination site is
indicated by asterisks in Fig. 6.
The eubacterial and archaeal reductases, although catalysing different
reactions, are all structurally related to dihydrofolate reductase (Chatwell
et al., 2006; Chen et al., 2006; Romisch-Margl et al., 2008). An in vivo study
on the mechanism of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidine 50 -phosphate reductase using the ascomycete A. gossypii showed that the incoming
hydrogen atom ends up in the pro-S position at C-10 of the ribityl side chain
of 5 (Keller et al., 1988) (Fig. 8). Thus, a hypothetical reaction mechanism via
an Amadori-type intermediate could be ruled out. Instead, it was proposed
that the reaction proceeds via the Schiff base intermediate 23. This is well in
line with a recent crystallographic study on RibG-protein from B. subtilis
(Chen et al., 2009).
The bifunctional deaminase/reductase of E. coli has been shown to transfer
the C4-pro-R hydrogen of NADPH to C-1 of 4 (Magalhaes et al., 2008); the
same stereochemistry is supported for the pyrimidine reductase of M. jannaschii
(using 2 as substrate) by crystallographic evidence (Chatwell et al., 2006).
Putative genes of A. thaliana (Accession No. AX546684) and Oryza sativa
(Accession No. Q6K6I4) have been suggested to specify pyrimidine reductase
on basis of sequence similarity Chatwell et al., 2006 but direct experimental
confirmation is as yet not available (Fig. 9).
Fig. 6. Sequence alignment of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 50 -phosphate deaminase domains from eubacteria and
plants with cytosine deaminase from S. cerevisiae. AthDeam_At4g20960, A. thaliana (At4g20960); BsuRibG, B. subtilis ribG (P17618);
OsaDEam_B9FAG7, Oryza sativa (B9FAG7); and SceFcy1 (cytosine deaminase of S. cerevisiae) (Q12178). The highly conserved zinc
coordination site of yeast cytosine deaminase is indicated by asterisks (Fischer et al., 2004a).
108
Fig. 7.
Structure of RibG-protein from B. subtilis (Chen et al., 2006).
Fig. 8. The proposed deamination and reduction mechanisms catalysed by the

deaminase and reductase domains of eubacterial RibG, respectively (Chen et al., 2009).
IV. LUMAZINE SYNTHASE

In the field of riboflavin biosynthesis research, lumazine synthase occupies a
commanding position. At least 38 X-ray structures from a variety of pathogenic and non-pathogenic microorganisms have been reported, and the
bibliography comprises at least 14 major protein structure papers. Moreover,
Fig. 9. Sequence alignment of putative 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 50 -phosphate reductase domains from Archaea,
plants and eubacteria. MjaRED, M. jannaschii (Accession No. Q58085); Ath_AX546684, Arabidopsis thaliana (Accession No. Ax546684);
Osa_Q6K6I4, Oryza sativa (Accession No. Q6K6I4); BsuRibG, B. subtilis (Accession No. P17618). Conserved residues are shown in black,
homologous in grey (Chatwell et al., 2006).
110
lumazine synthase is the only riboflavin biosynthesis enzyme with the

structure of a plant orthologue published (Jordan et al., 1999).
The lumazine synthase nomenclature is unfortunately confusing for historical reasons. The protein subunit catalysing the condensation of 7 with 9 had
been discovered in the 1970s as a component of a large protein complex from
B. subtilis, which had been designated as heavy riboflavin synthase (Bacher
et al., 1980; Bacher and Mailander, 1978). Whereas it was clear that the riboflavin synthase activity was exclusively due to a 25-kDa peptide designated
subunit, the enzymatic role of the 15-kDa peptide (designated subunit)
was assigned only much later as lumazine synthase (Neuberger and Bacher,
1986). The 1-MDa enzyme complex from B. subtilis and other Bacillaceae is
now known to be a bifunctional lumazine synthase/riboflavin synthase.
On the surface, the reaction now known to be catalysed by lumazine
synthase looks simple enough (Fig. 10). The diaminopyrimidine derivative
7 condenses with the carbohydrate 9 to form one equivalent each of the
lumazine derivative 10 and inorganic phosphate as well as two equivalents of
water. In line with that, the reaction can proceed uncatalysed in dilute
aqueous solutions of the reactants at room temperature at neutral pH (Kis
et al., 2001). So fast is the spontaneous process that blanks without enzyme
are really mandatory in order to monitor the relatively modest rate acceleration brought about by the enzyme.
On closer inspection, the reaction mechanism of lumazine synthase is
anything but simple. The well-known regiochemistry of the reaction (C-2
Fig. 10.
Hypothetical reaction mechanism of lumazine synthase (Kis et al., 1995).
111
of 9 becomes directly linked to N-5 of 7) suggests that the formation of a

Schiff base is the first reaction step (Keller et al., 1988; Kis et al., 1995;
Nielsen et al., 1986). Stopped flow experiments have identified a major
transient with absorption maxima at 455 and 408, and that suggests one or
several intermediates with extended conjugated double bond systems, possibly 25 and/or 26 (Haase et al., 2003; Schramek et al., 2003). The elimination
of inorganic phosphate could then generate a new carbonyl group that could
now react with the position 6 ribitylamino group of the pyrimidine 27.
Ultimately, the elimination of water could generate the final product, either
as the neutral molecule 10 or as the exomethylene anion of 33 (cf. Fig. 14)
which could be converted to the neutral form by protonation. As already
mentioned, the reaction can proceed without enzyme catalysis, but the
uncatalysed reaction is only partially regiospecific and is believed to involve
partitioning via two different reaction trajectories (Kis et al., 2001).
Studies on the bifunctional lumazine synthase/riboflavin synthase of
B. subtilis were initiated in the early 1970s. The 1-MDa protein was shown
to consist of 3 subunits, which accounted for its riboflavin synthase activity
and of 60 subunits, which were only later assigned as lumazine synthase.
The 60 subunits were shown to form a spherical shell with icosahedral 532
symmetry, which had some similarity to small virus capsids (Bacher et al.,
1986; Ladenstein et al., 1986, 1988) (Fig. 11). The three subunits were
shown to be enclosed in the central core. Following the induced dissociation
of the large, hetero-oligomeric particles, it was possible to reassemble the
subunits as hollow, icosahedral capsids of 60 monomers. The assembly of
much larger hollow capsids comprising more than 100 monomers was also
possible under in vitro conditions.
The icosahedral, 60-meric lumazine synthase capsid is best described as a
dodecamer of pentamers, although free pentamers have not been observed
(Fig. 11). The pentameric modules of the icosahedral/dodecahedral capsids
have channels running along their c5 axes. The central part of the pentamer
module can be described as a superhelical assembly of the 3 helices of the
five subunits which constitute the pentamer. The subunits are characterised
by a flavodoxin-like fold. The active sites, which will be described in closer
detail below, are all located at the interfaces of adjacent subunits in the
pentamer modules. Notably, each 60-mer has 60 topologically equivalent
active sites.
Over the past three decades, the X-ray structures of lumazine synthases
have been reported for a range of widely different species including fungi,
eubacteria, Archaea and one plant (spinach) (Braden et al., 2000; Gerhardt
et al., 2002a; Klinke et al., 2005, 2007; Koch et al., 2004; Meining et al., 2000;
Morgunova et al., 2005, 2006, 2007, 2011; Persson et al., 1999; Zylberman
112
Fig. 11. Space filling model of the lumazine synthase capsid from B. subtilis.
Subunits in one pentamer are shown in colour. Selected amino acid residues are
also shown in colour to indicate areas where the fivefold (red), threefold (violet) and
twofold (red) symmetry axes penetrate the molecular surface.
et al., 2004, 2006) (Fig. 12). The microbial species include several pathogens
(e.g. Bacillus anthracis, Brucella abortus, M. tuberculosis, S. typhimurium,
C. albicans). The X-ray structure of lumazine synthase from the important
rice pathogen, M. grisea, has also been reported (Persson et al., 1999).
Despite the large molecular size (15 nm), the X-ray structure of the enzyme
from the hyperthermophilic eubacterium Aquifex aeolicus could be deter ) (Zhang et al., 2001).
mined to near-atomic resolution (1.4 A
The preferred coverage of human- and plant-pathogenic microorganisms
is due to the fact that the enzymes of the riboflavin biosynthesis pathway are
under evaluation as potential targets for the design of inhibitors with antiinfective or fungicidal properties. The unifying tenet of these different quests
is the fact that the riboflavin biosynthetic enzymes are essential in plants and
in many animal and plant pathogens but are absent in humans and animals.
Hence, inhibitors used in medicine or plant crop protection (herbicides,
fungicides) would be exempt from target-related human toxicity (although
that would of course not rule out off-target toxicity).
The extensive comparative studies have revealed the existence of four
different quaternary structures for lumazine synthase, namely (i) c5-pentamers (yeasts and fungi, B. abortus, M. tuberculosis) (Braden et al., 2000;
113
Fig. 12. (Left) Structural superposition of monomers of the pentameric lumazine

synthase from Schizosaccharomyces pombe (yellow) and the icosahedral lumazine
synthases from Aquifex aeolicus (green) and spinach (red). A bound substrate analogue inhibitor (5-nitro-6-(D-ribitylamino)-2,4(1H,3H)-pyrimidinedione, 29) to mark
the substrate binding site is shown (based on the spinach structure, shown in yellow)
(Gerhardt et al., 2002a, Persson et al., 1999, Zhang et al., 2001). (Right) Pentameric
assembly of spinach lumazine synthase viewed along the fivefold non-crystallographic
symmetry axis as seen from the inner capsid wall. The active sites are all located at
interfaces of adjacent monomers (Persson et al., 1999; Zhang et al., 2003).
Gerhardt et al., 2002a; Meining et al., 2000; Morgunova et al., 2005); (ii) d5symmetric decamers (B. abortus) (Zylberman et al., 2004); (iii) hollow icosahedral capsids (certain eubacteria) (Kumar et al., 2011); and (iv) heterooligomers consisting of 3 cores inside 60 capsids (Bacillaceae) (Bacher
et al., 1986). Recombinant lumazine synthase of spinach is a hollow icosahedral capsid, but it is unknown whether 360 complexes can be assembled
inside plant cells (Persson et al., 1999).
Sequence comparison has been explored as a method to predict the association state of different lumazine synthase orthologues (Fornasari et al.,
2004). However, in practice, the quaternary structure outcome of sequence
modification has been incompletely predictable. The assembly process of the
icosahedral capsids appears to be very complex; notably, the hypothetical
pentamer building blocks of the icosahedral ensembles are conjectural, on
basis of the symmetry properties and intersubunit interfacing in the capsid,
and have not been directly observed as an isolated molecular species.
114
Pseudomaturated lumazine synthase from spinach has been crystallised as

a recombinant, homo-oligomer consisting of subunits with 156 amino acid
residues (Persson et al., 1999). The mRNA predicts a plastid-targeting
sequence comprising 66 amino acid residues. The structure was solved at a
. The subunit fold and the general topology of the 532resolution of 3.1 A
symmetric capsid closely resemble those of the icosahedral lumazine
synthases of bacterial origin. Notably, the N-terminus of each subunit participates as a fifth strand in the central four-stranded sheet of the respective,
adjacent subunit in the pentamer ensemble.
The active site of lumazine synthase has been subject to intense studies
employing substrate, intermediate and product analogues; cocrystal structures implying at least 17 different analogues have been reported. The spinach enzyme has been cocrystallised with the substrate analogue, 5-nitro-6ribitylamino-2,4(1H,3H)-pyrimidinedione (29, Fig. 13); this relatively strong
inhibitor (Otto and Bacher, 1981) differs from the natural substrate 7 only by
the replacement of the position-5 amino group by a nitro group. However, in
contrast to the strongly oxygen-sensitive substrate 7, the inhibitor 29 is a
stable compound. The pyrimidine ring of the inhibitor is located in a hydrophobic pocket involving the residues Phe22, Ala56, Val81 and Val92 and
forms an essentially coplanar complex with the side chain of Phe22 that is
believed to play a major role for binding. The carbonyls of the inhibitor
interact with the side chain of Asn23 and the backbone NH group of Ala56.
The ribityl side chain is involved in hydrogen bonds with a variety of mainand side-chain elements.
Analogues emulating the structural features of the putative intermediate
24 believed to result from the reaction of the pyrimidine substrate 7 with the
carbonyl group of the second substrate 9 have played an important role in
order to gain an understanding of the substrate and intermediate topology.
Fig. 13.
Inhibitors for lumazine synthase (Zhang et al., 2007a).
115
Specifically, derivatives of ribitylaminopyrimidines, 9-ribitylpurines and

8-ribityllumazines with aliphatic side chains carrying terminal phosphate or
phosphonate residues were investigated by X-ray crystallography of cocrystals with various lumazine synthases of microbial or yeast origin. These data
leave no doubt that the hypothetical Schiff base intermediate 24 of the
lumazine synthase trajectory is bound in an extended conformation, with
the negatively charged phosphate residue located in the close proximity of
Arg127 and Lys131 and Lys135 residues (B. subtilis) (Fischer et al., 2003a;
Meining et al., 2000). These results confirmed a hypothesis that had been
proposed much earlier on basis of structural work with lumazine synthase
from B. subtilis that had located an inorganic phosphate residue in that
position (Ritsert et al., 1995). It is also noteworthy in this connection that
inorganic phosphate is a potent stabiliser of the icosahedral capsid.
The finding that the reaction intermediate 24 must be bound in an extended conformation adds yet another complex twist to the complex reaction
mechanism. If indeed the Schiff base 24 is generated with the proposed,
extended conformation, a cistrans isomerisation of the imine motif becomes
necessary in order to obtain an acceptable reactant topology for the formation of the pyrazine ring of the product.
The extensive work, by several research groups, on substrate and intermediate analogues has afforded potent lumazine synthase inhibitors with Ki
values in the nanomolar range (Cushman et al., 1986, 1990, 1991, 1992, 1997,
1998, 1999a,b,c, 2001, 2002a,b, 2004, 2005; Talukdar et al., 2007, 2009;
Zhang et al., 2007a,b, 2008; Zhao et al., 2009). Regretfully, none of these
compounds caused inhibition of microbial growth, most probably due to
difficulties of entry into bacterial or yeast cells.
Screening of compound libraries has also been employed in the search for
lumazine synthase inhibitors (Chen et al., 2005). That work has resulted in
the discovery of the pentacyclic inhibitor 30 (Zhang et al., 2007a) (Fig. 13).
It is as yet unknown whether plant lumazine synthases can encapsidate
riboflavin synthase, in parallel to the 3 60 lumazine synthase/riboflavin
synthase complexes of bacteria (however, complex formation has been
ruled out experimentally for the icosahedral lumazine synthases of certain
Gram-negative bacteria) (Bacher et al., 1986). The bifunctional enzyme
complex of Bacillaceae does, however, pose numerous unanswered structural
and mechanistic problems. Thus, as the homotrimeric 3 riboflavin synthase
is devoid of trimeric symmetry, it is unknown how it could be accommodated
inside the 532-symmetric cavity inside the icosahedral capsid. Moreover, at
least for the B. subtilis enzyme, it remains enigmatic how riboflavin (being the
product of the riboflavin synthase moiety in the core of the enzyme complex)
can penetrate the capsid wall in order to be unloaded into the bulk solvent
116
compartment. Interestingly, a modelling study has shown that this problem

would be non-existent in the case of the spinach enzyme where the channels
along the fivefold axis are wide enough to allow the passage of riboflavin into
and out of the core of the capsid (Persson et al., 1999).
The detailed structure elucidation of lumazine synthase served as the basis
for an extensive mutagenesis study conducted with the B. subtilis enzyme
(Fischer et al., 2003a). Every residue of the first amino acid shell around the
spacious reactive site cavity was subjected to conservative as well as less
conservative replacement. Whereas the removal of positively charged residues from the site accommodating the phosphate moiety of the substrate 9
and the early intermediate 24 resulted in a significant decline of enzyme
activity, the attempt to nail down any residues that might be involved in
covalent catalysis failed. Although the reaction mechanism clearly has a
requirement for several proton exchange steps, the search for residues that
might facilitate these reactions by acting as acids and/or bases also turned up
essentially empty. Based on these data, it must be assumed that lumazine
synthase exerts its catalytic effect mainly by establishing a favourable topological relation between the reactants. In other words, the catalytic power of
the enzyme appears predominantly based on modulating the activation
entropy (Fischer et al., 2003a).
It is in order at this point to remind the readers that the rate acceleration by
lumazine synthase is rather modest (Kis et al., 1995). The reaction can run at
easily detectable rates without any enzyme catalysis in neutral aqueous
solution with concentrations of 7 and 9 in the low millimolar range. In fact,
it is mandatory to run blanks without enzyme in order to measure the
velocity of the enzyme-catalysed reaction with sufficient accuracy. It is
tempting to speculate that there has been significant selective pressure that
has locked the evolution of lumazine synthase at the level of a fairly limited
rate enhancement.
V. RIBOFLAVIN SYNTHASE
Riboflavin synthase catalyses one of the most puzzling and mechanistically
complex reaction trajectories (Fischer and Bacher, 2011). Without any requirement for cofactors, the enzyme engineers the transfer of a 4-carbon unit
between two identical substrate molecules. To fully appreciate that catalytic
achievement, it must be noted that the 4-carbon segment that must be carved
out of the donor substrate is linked to the diaminopyrimidine moiety by two
carbon nitrogen bonds which are both part of a heteroaromatic ring system
with a significant level of resonance stabilisation. Last but not least, the
117
4-carbon segment that is cut out of the lumazine donor has been
incorporated into the heterocyclic moiety precisely by the preceding reaction
step which is catalysed by lumazine synthase; notably, the formation of the
lumazine is exergonic and is not detectably reversible. One of the products of
riboflavin synthase, the pyrimidine 7, is structurally identical with the substrate of lumazine synthase; in other words, whereas the lumazine synthase
reaction is not per se reversible (under regeneration of both substrates,
namely 7 and 9) at a measurable rate, the pyrimidine substrate 7 of lumazine
synthase can be regenerated by riboflavin synthase in a reaction that goes to
completion; this quasi-reversal of the lumazine formation is enabled, with
regard to its thermodynamic aspects, by the resonance stabilisation of the
newly formed xylene ring of riboflavin synthases other product, namely
riboflavin. The quantitative conversion of the pyrimidine 7 into riboflavin
requires two equivalents of the carbohydrate 9. That, in conjunction with the
unique reaction topology summarised in Fig. 1, has the consequence that, on
average, every pyrimidine molecule must be handled twice by both lumazine
synthase and riboflavin synthase (note that the emphasis here is on on
average; stochastically, certain pyrimidine molecules do end up as riboflavin after being handled exactly once by each of the enzymes).
The discovery of riboflavin synthase followed rapidly on the serendipitous
discovery of its substrate as a green fluorescent substance (originally
named G compound to connotate the colour of its fluorescence) of the
naturally flavinogenic ascomycete E. ashbyii (Kuwada et al., 1958; Maley
and Plaut, 1959; Masuda, 1957b). The structure was obviously similar to
riboflavin, and an enzyme activity converting G compound to riboflavin
was discovered in cell extracts of A. gossypii and also of E. coli around
1960. It then came as a major surprise that the enzyme required nothing
besides the lumazine (no second substrate and no cofactors) for its conversion into the vitamin (Plaut, 1960, 1963). Classical studies conducted by
the group of Plaut then unravelled the 4-carbon transfer described in the
previous paragraph.
Also in the 1960s, riboflavin activity was partially purified from spinach
(Mitsuda et al., 1965, 1970). However, that early work on riboflavin biosynthesis remained without significant follow-up for more than two decades.
The cloning and expression of the genes for lumazine synthase and riboflavin
synthase from plants (spinach, tobacco and A. thaliana) were reported in
patents around 2000 (Bacher and Eberhardt, 2001; Viitanen et al., 2000,
2002). The same patents also revealed the cloning of the genes for both
enzymes from the rice pathogen, M. grisea. This work was focused on the
discovery of potential agents with herbicide or fungicide activity for use
in crop protection. Only a single research paper has been published so
118
far on the cloning and expression of riboflavin synthase genes of plant origin
(A. thaliana) (Fischer et al., 2005).
In contrast to the limited work on plant riboflavin synthase, the enzyme of
E. coli has been studied in close detail, and the sequence similarity between
riboflavin synthases from plants and bacteria suggests that the bacterial data
can be extrapolated to the plant enzyme to a significant extent. The basic
details of the riboflavin synthase reaction were established in the 1960
(Harvey and Plaut, 1966; Plaut and Harvey, 1971; Plaut et al., 1970a). The
early work showed that all carbon atoms of the xylene ring of riboflavin are
exclusively derived from the substrate 9. Specifically, it was shown that the
carbon atoms 6, 6, 7 and 7 of one substrate molecule are sacrificed and
serve as building blocks for the extension of the chromophore system of a
second substrate molecule that results in the formation of riboflavin (Beach
and Plaut, 1970a; Paterson and Wood, 1969, 1972; Sedlmaier et al., 1987).
The donor substrate is converted to the pyrimidine 7 in this process. The data
strongly suggested that the exchange of the 4-carbon unit between the two
substrate molecules was a concerted, intermolecular process that did not
proceed via a free 4-carbon unit.
If the enzymatic transformation of a single substrate into a mixture of
riboflavin and 7 was puzzling, it was even more surprising that the reaction
could proceed without any enzyme catalysis, and under relatively mild conditions (Beach and Plaut, 1970b; Paterson and Wood, 1969; Plaut et al.,
1970b). Specifically, riboflavin was formed in high yield when a neutral
aqueous solution of the lumazine derivative 10 was refluxed under an inert
atmosphere. Later, it was shown that the uncatalysed reaction could also
proceed under acidic conditions (Beach and Plaut, 1969).
Based on these observations, initial hypotheses for the reaction mechanisms were proposed by the research groups of Plaut and Wood. These
hypotheses set out from the exceptional CH acidity of 8-substituted
6,7-dimethyllumazines. In case of 6,7,8-trimethyllumazine, the abstraction
of a proton yields the structurally unusual exomethylene anion 33 (Fig. 14).
Deprotonation affords a complex mixture comprising small amounts of the
exomethylene anion 33 in equilibrium with at least four molecular species
(3437) arising by nucleophilic attack of the position 7 carbon atom of 33 by
one of the hydroxy groups of the ribityl side chain, whereas the model
compound 6,7,8-trimethyllumazine (31) has a pK of 9.8 and forms a single
anion species (32) with a 7-exomethylene group (Beach and Plaut, 1970b,
1971; Bown et al., 1986; Pfleiderer et al., 1971). The riboflavin synthase
substrate 10 is even more acidic, with a pK of 8.4 (Pfleiderer and
Hutzenlaub, 1973), but the deprotonation affords a complex mixture that
is dominated by a series of tricyclic molecular species 3437, whereas the
119
Fig. 14. Structures of lumazine derivatives. 10, 6,7-dimethyl-8-ribityllumazine;

33, 7-exomethylene anion of 10; 3437, tricyclic adduct anions of 10; 31, 6,7,8trimethyllumazine; 32, 6,7,8-trimethyllumazine anion.
exomethylene anion is only present in small amounts. The tricyclic species

are believed to arise by nucleophilic attacks of the position 20 - or 30 -hydroxy
groups of the ribityl side chain at the C-7 carbon of the exomethylene anion
33, lumazine derivatives devoid of hydroxy groups in the position 8 substituent can only form the exomethylene anion species (Fig. 15).
Up to date, all mechanistic hypotheses for riboflavin formation use the
unique exomethylene anion as a starting point. The initial mechanistic
120
Fig. 15. Hypothetical reaction mechanisms of riboflavin synthase. (A) Mechanism proposed by Plaut, Wood and co-workers (Beach and Plaut, 1970b; Paterson
and Wood, 1972; Plaut and Beach, 1976; Plaut et al., 1970b) and modified after
discovery of the pentacylic reaction intermediate 40 (Illarionov et al., 2001a); (B)
hydride transfer/cycloaddition mechanism (Kim et al., 2010). 10a, donor lumazine;
10b, acceptor lumazine; R, ribityl chain.
121
suggestions of the Plaut and Wood group assumed that the exomethylene
anion could execute a nucleophilic attack on a second substrate molecule
(Beach and Plaut, 1970b; Plaut and Beach, 1976; Plaut et al., 1970b).
Around 1970, studies with deuterium-labelled substrate established
that both the enzyme-catalysed and -uncatalysed reactions are regiospecific
in the sense that the incoming and the pre-existing 4-carbon unit of the
acceptor substrate are linked in a head-to-tail fashion (Beach and
Plaut, 1970a; Fischer et al., 2004b; Paterson and Wood, 1969, 1972;
Sedlmaier et al., 1987). That unexpected result necessitated a modification
of the mechanism. Later on, Beach and Plaut suggested a mechanism that
involves tricyclic intermediates (Rowan and Wood, 1968); that mechanism
could be ruled out by later work and is not discussed in detail in this
review. Readers are directed to earlier reviews for a detailed discussion of
these early mechanistic concepts (Plaut, 1971; Plaut and Beach, 1976; Plaut
et al., 1974).
Not surprisingly, as flavocoenzymes are trace metabolites, the enzymes for
their biosynthesis have low expression levels. Whereas riboflavin synthase
had been purified 4000-fold from yeast (Harvey and Plaut, 1966; Plaut et al.,
1970b) and 700-fold from spinach (Mitsuda et al., 1970), the accessibility of
reasonably pure protein for biophysical studies was limited. In the 1990s, the
accessibility of pure protein in significant amount was improved by recombinant homologous or heterologous expression, and detailed structural and
biophysical studies became possible. A major breakthrough was the determination of X-ray structures of riboflavin synthase from E. coli (Liao et al.,
2001b) and Schizosaccharomyces pombe (Gerhardt et al., 2002a) (to be discussed in detail below). Moreover, sufficient amounts of protein were now
available for single turnover studies which resulted in the photometric discovery of a transient which could be isolated by preparative rapid quench
experiments (Illarionov et al., 2001a). Although transient species was only
obtained in substoichiometric amounts (based on the amount of enzyme), its
structure could be determined by NMR spectroscopy (the structure determination depended critically on the use of 13C-labelled substrate in order to
enhance the sensitivity of the 13C NMR measurements) (Illarionov et al.,
2001a, 2003).
The structure of the transient species is shown in Fig. 15; the pentacyclic
(40) molecule is a covalent dimer of the substrate. Notably, the reaction
generates two novel chiral centres. The covalent adduct can serve as a
substrate for E. coli riboflavin synthase and fulfils the criteria for a kinetically
competent intermediate (i.e. it is consumed more rapidly than the substrate
10) (Illarionov et al., 2001a). The treatment of the adduct with riboflavin
synthase was shown to afford three products, namely riboflavin, the
122
pyrimidine 7 and 6,7-dimethyl-8-ribityllumazine (10) (upon longer incubation, the latter is consumed under formation of 7 and 11) (Fig. 16).
The cleavage of the pentacyclic intermediate affording riboflavin and the
pyrimidine 7 is easily explained. The tetrahydropyrazine ring of 40b can be
opened by a vinylogous -elimination, and a second elimination step affords
riboflavin and the pyrimidine 7. However, the formation of the pentacyclic
intermediate 40 from the lumazine derivative 10 is anything but easy to
explain. An attempt to reconcile the novel information with the Plaut/
Wood mechanism is shown in Fig. 15A. However, whereas that hypothesis
does not seem to violate established chemical facts, the path from the
substrate to the intermediate is long and tortuous.
Figure 15B shows a recent hypothesis that represents a break with established ideas in so far as it introduces a cryptic redox process into the
formation of the pentacyclic intermediate (Kim et al., 2010). That hypothesis, which requires further study, will be best discussed after a description of
the structures of the bacterial/fungal/plant riboflavin synthase and the independently evolved pentameric riboflavin synthase from Archaea.
Fig. 16. Stereochemistry of 6,7-dimethyl-8-ribityllumazine conversion into riboflavin catalysed by trimeric eubacterial and pentameric archaeal riboflavin synthase.
Q and Q0 , pentacyclic reaction intermediates; R, ribityl.
123
The amino acid sequence of riboflavin synthase from E. coli revealed two
segments with marked sequence similarity and a C-terminal segment that
was devoid of similarity to the other two segments (Schott et al., 1990).
This suggested that each subunit could form two domains which would
interact to form a pseudo-c2-symmetric ensemble that would be naturally
conducive to the experimentally determined regiospecificity of the riboflavin
synthase reaction. The N-terminal domain could be expressed as a stable,
recombinant protein which retained the capacity to bind riboflavin with high
affinity (Eberhardt et al., 2001), whose three-dimensional structure was
determined by X-ray crystallography (Meining et al., 2003) and NMR spectroscopy (Gerhardt et al., 2002b; Liao et al., 2001b; Truffault et al., 2001).
Riboflavin synthase of B. subtilis had been shown to be a homotrimer by
hydrodynamic studies (Fig. 16). A breakthrough came with the determination of the crystal structure of riboflavin synthase from E. coli (Liao et al.,
2001b). That structure confirmed that each subunit folds into two domains
with closely similar structure. Each domain consists of a six-stranded
-barrel. The two domains of each subunit are related by pseudo-c2 symmetry. Moreover, the N-terminal domain of one (and only one) subunit forms a
dimer contact with the C-terminal domain of one adjacent subunit, and that
ensemble is also characterised by pseudo-c2 symmetry. Last but not least, the
homotrimer is kept together by a short triple-helix motif formed by the
helical C-termini. Notably, however, the subunits of the homotrimer are
not related by trigonal symmetry (the fact that, nevertheless, the Patterson
analysis of the refraction data had appeared to indicate some threefold
symmetry relations does not in conflict with that, (cf. Liao et al., 2001b)).
Additional information was gleaned from the X-ray structure of riboflavin
synthase from the yeast, S. pombe (Gerhardt et al., 2002b). Whereas that
protein is a trimer in solution, the crystal structure shows only monomers.
That is probably due to the breakdown of hydrophobic contacts between the
C-terminal helices, which may have been caused by the methylpentanediol
(MPD) that had been used as precipitant. Most important, however, each
monomeric subunit of the S. pombe crystal form carried two molecules of
a substrate analogue, with one respective ligand molecule bound to each
-barrel domain. At this point, it was logical to try to model the experimentally
observed ligand of the yeast protein into the trimeric structure of the E. coli
protein (which had been crystallised without ligand) (Gerhardt et al., 2002b).
The model studies leave no doubt that the active site is located between
the special pair comprising the N-terminal domain of one subunit and the
C-terminal domain of the adjacent subunit. Two ligand molecules are bound
at the domain interface, each located in a surface depression of one of the
adjacent subunits. Due to the pseudo-c2 symmetric arrangement of the domain
124
pair, the two bound ligands are also related by pseudo-twofold symmetry.
This is perfectly in line with the known regiochemistry of the catalytic reaction
and had already been predicted on basis of sequence arguments.
Although only just one homotrimer structure is as yet available, one may
speculate that the protein has dynamic properties that could allow different
domains, at different times, to come close in order to form an active site,
although at any one time only a single active site is believed to exist. The
kissing active site domains could come apart to allow the unloading of
products and the loading of new substrate. 19F NMR studies are well in line
with the hypothesis that all six domains can bind a given ligand at the same
time, but that the ligands loaded to different domains are not in topologically
equivalent environments (Fischer et al., 2003b; Scheuring et al., 1994, 1996).
The model study allowed to assign the absolute stereochemistry of two
novel stereocentres of the pentameric reaction intermediate 40 that are
generated by the dimerisation of 10 (cf. Fig. 15) (Gerhardt et al., 2002b).
The X-ray structure of S. pombe riboflavin synthase and the X-ray and
NMR structures of the artificial, homodimeric N-terminal domain (obtained
by recombinant expression) provide a detailed description of the environment of the bound substrate at the active site (Eberhardt et al., 2001; Meining
et al., 2003; Truffault et al., 2001). At both domains, the lumazine chromophore is involved in hydrogen bonding with the backbone amide motifs and
in hydrophobic interactions. The ribityl side chain is involved in hydrogen
bond contacts with backbone and side chain elements.
Virtually all highly conserved residues of E. coli riboflavin synthase have
been probed by mutagenesis (Illarionov et al., 2001b). Surprisingly, the
replacement or deletion of some residues of the N-terminal MFTG motif
caused dramatic reductions of the catalytic activity, although this segment is
located at a considerable distance from both substrate binding sites. Replacement of Ser41 or His 102 also has a massive impact on activity, although the
amino acids are relatively far remote from the substrate molecules bound at
the active site.
The pentacyclic intermediate 40 can be cleaved by the E. coli riboflavin
synthase in two different directions affording either one equivalent each of
riboflavin and the pyrimidine 7 (forward reaction) or two equivalents of 6,7dimethyl-8-ribityllumazine (10) (reverse reaction). The velocities of the forward and backward reaction are similar for the wild-type enzyme but are
differentially affected by mutations (Illarionov et al., 2001b). The novel
stereocentres of 40 (marked by asterisks in Fig. 15) are quenched by both
fragmentation reactions.
Recent 13C NMR studies have shown that the artificial dimer of the
N-terminal domain selectively binds and stabilises the exomethylene anion
125
form 33 of the substrate 10 (Kim et al., 2010). None of the tricyclic molecular
species can be bound. In full agreement with the crystallographic data, the
hypothetical mechanism implying tricyclic lumazine adduct structures such as
3437 is thereby ruled out. The affinity for the exomethylene anion is modulated by replacement of threonine residues 50 and 67 whose side chains are in
direct hydrogen bonding contact with N-5 and C-30 of the bound substrate.
These findings have recently prompted yet another mechanistic hypothesis
which introduces the concept of a hidden redox step in order to explain the
enigmatic riboflavin synthase reaction trajectory. More specifically, it has
been proposed that an exomethylene anion 33 located at the N-terminal
domain inside the active site donates a hydride ion to an electroneutral
substrate molecule located at the C-terminal domains binding site. The
hydride donor will thereby be converted to the quasi-quinoid dehydrolumazine structure 38, and the hydride acceptor is converted to the dihydrolumazine 39a, which is known to be in equilibrium with the tautomer 39b. The
dehydrolumazine 38 and the dihydrolumazine 39b could then undergo a
4 2 cycloaddition which is directly conducive to the pentacyclic enantiomer
in the tautomer form 40a. Following conversion to the tautomer 40b, the
pentacyclic intermediate could then be fragmented under formation of riboflavin and 7. Alternatively, the tautomer 40a could react backward under
regeneration of 6,7-dimethyl-8-riboflavin, a reaction that has indeed been
directly observed (see above). In fact, the two tautomers 40a and 40b of the
pentacyclic intermediate have different, predetermined breaking points
which enable the two different exit pathways in the forward and backward
direction, respectively (Fischer and Bacher, 2011; Kim et al., 2010) (Fig. 15).
The proposed hydride transfer has precedent in the Cannizzaro reaction
which converts aromatic and heterocyclic aldehydes into equimolar mixtures
of the cognate alcohol and carboxylic acid. The Cannizzaro reaction proceeds via a hydroxide adduct that is formed under alkaline conditions and
donates a hydride to a free aldehyde molecule. Cannizzaro reactions can
proceed at room temperature in aqueous solution, literally in the twinkling of
an eye, and with excellent yields. It should also be noted that hydride
transfers are one of the most common elementary reactions in biochemistry.
Last but not least, it should be noted that the riboflavin synthase domain
has significant similarity to certain flavocoenzyme binding domains. Moreover, the topology of the bound riboflavin synthase substrates is essentially
the same as that of the flavin cofactor in the respective cofactor binding
domains. Obviously, many flavocoenzymes act as professional hydride transponders. By analogy, a hydride transfer as part of the riboflavin synthase is
less strange than it may appear at first sight.
126
Whereas the reaction catalysed by riboflavin synthase shows massive

isotope effects (Plaut et al., 1970a), these are unfortunately not suitable to
distinguish in a simple way between hypotheses A and B (Fig. 15), as carbon
hydrogen bonds need to be broken in both cases. Thus, further data will be
required in order to rule out either hypothesis.
Studies in the 1990s showed that Archaea use riboflavin synthases without
similarity to the trimeric enzymes from eubacteria, fungi and plants
(Eberhardt et al., 1997). The archaeal enzymes are c5-symmetric homopentamers whose sequence and structure have significant similarity with lumazine synthase (Fischer et al., 2004b; Ramsperger et al., 2006). The reaction
proceeds via a pentameric adduct which differs from the intermediate of the
homotrimeric enzymes with regard to the stereochemistry. More specifically,
the intermediates of trimeric and pentameric riboflavin synthases are diastereomers as shown in Fig. 16. Hence, the pentacyclic intermediate generated by
archaeal riboflavin synthase cannot be processed by trimeric riboflavin
synthase and vice versa (for review, see Fischer and Bacher, 2008).
VI. RIBOFLAVIN KINASE AND FAD SYNTHETASE

Despite the involvement of early 50 -phosphorylated precursors (compounds
1 and 6), the de novo biosynthetic pathway affords unphosphorylated riboflavin, as 50 -phosphates are unable to serve as substrates for lumazine
synthase and riboflavin synthase and must be dephosphorylated prior to
being converted into riboflavin by these enzymes. Riboflavin kinase and
FAD synthase are therefore required in all organisms to obtain the coenzyme
forms, FMN (riboflavin 50 -phosphate) and FAD (flavin adenine dinucleotide), either from biosynthetic of nutritional riboflavin.
Riboflavin kinase (flavokinase, E.C. 2.7.1.26) converts riboflavin into
riboflavin 50 -phosphate (FMN) using ATP as phosphate donor (recent
work has shown that riboflavin kinase from Archaea uses CTP instead
(Mashhadi et al., 2008)). FAD synthetase (EC 2.7.7.2) converts FMN to
flavin adenine dinucleotide by the transfer of an adenylate unit under formation of inorganic pyrophosphate as second product. Research on riboflavin
kinase and FAD synthetase spans a period of more than five decades. The
early work has been reviewed elsewhere (Bacher, 1991).
A bifunctional flavokinase/FAD synthetase was first demonstrated in
Brevibacterium ammoniagenes (Manstein and Pai, 1986); the N-terminal
domain catalyses the formation of FAD, and the C-terminal domain serves
as riboflavin kinase. Similar bifunctional proteins are now known to exist in
127
numerous eubacteria. However, riboflavin kinase and FAD synthetase are

separate proteins in animals, fungi, plants and certain eubacteria including E.
coli and B. subtilis.
A bifunctional enzyme whose C-terminal domain resembles the monofunctional riboflavin kinase of yeast has been cloned from A. thaliana. The
N-terminal domain of the plant protein is a member of the haloacid dehydrogenase superfamily which acts as a phosphatase that accepts FMN as a
substrate (Sandoval and Roje, 2005). Genes specifying similar riboflavin
kinase/FMN hydrolase fusion proteins have been noted in other plants;
gymnosperm but not angiosperm orthologues specify N-terminal extensions
that are believed to act as organelle-targeting sequences. It has been suggested that the fusion of the riboflavin kinase and FMN phosphatase domain
has occurred at an early time in plant evolution.
The A. thaliana genome also comprises two genes with similarity to bacterial riboflavin kinase/FAD synthetase. However, whereas the N-terminal
domain is clearly a functional FAD synthetase, sequence arguments suggest
that the C-terminal domain is unable to function as riboflavin kinase but may
have acquired a different, as yet unknown function. Both A. thaliana orthologues comprise N-terminal organelle-targeting segments. Orthologous
genes are present in other plant genomes. Sequence arguments suggest that
the plant FAD synthetases are of cyanobacterial origin (Sandoval et al.,
2008; Yruela et al., 2010). The relatively sparse information concerning the
cellular topology of riboflavin kinases and FAD synthetases in plants is
reported below (see Section VII).
X-ray structures have been reported for monofunctional riboflavin kinases
of human (Karthikeyan et al., 2003) and yeast (S. pombe) (Bauer et al., 2003)
origin, the monofunctional FAD synthetases of yeast (S. cerevisiae) (Leulliot
et al., 2010) and the thermophilic archaeon Thermotoga maritima (Wang
et al., 2005), for the bifunctional riboflavin kinase/FAD synthetase of Corynebacterium ammoniagenes (Herguedas et al., 2010), and a CTP-dependent
archaeal riboflavin kinase of M. jannaschii (Ammelburg et al., 2007). Structures of plant orthologues have not been reported as yet.
VII. CELLULAR TOPOLOGY OF

FLAVOCOENZYME BIOSYNTHESIS IN PLANTS
The genes that code for the four currently known plant enzymes involved in
the biosynthesis of riboflavin (GTP cyclohydrolase II/3,4-dihydroxy-2-butanone 4-phosphate synthase, pyrimidine reductase, lumazine synthase and
riboflavin synthase) specify N-terminal segments that fulfil the criteria for
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plastid-targeting sequences. By comparison with the catalytic domains, the

putative targeting segments show a low degree of conservation.
Chloroplasts have been shown to import full-length lumazine synthase
in vitro (Jordan et al., 1999). More recently, lumazine synthase has been
shown to localise to chloroplasts in vivo (Wu et al., 2010). Hence, it appears
likely that the biosynthesis of riboflavin proceeds entirely inside chloroplasts.
As already mentioned above, the spatial pattern is more complex for
riboflavin kinase and FAD synthase which convert riboflavin into the coenzyme forms. Recent data suggest that the conversion of riboflavin into the
coenzyme forms can proceed in the cytoplasm as well as inside mitochondria
and plastids (Giancaspero et al., 2009; Sandoval et al., 2008; Torchetti et al.,
2010; Yruela et al., 2010). Each compartment appears to individually generate its own flavocoenzymes from riboflavin that is excreted into the cytoplasm by chloroplasts and can be extracted from the cytoplasm by
mitochondria.
More specifically, the plant-type FAD synthetases carry putative organelle-targeting sequences. However, putative organelle-targeting motifs precede the riboflavin kinase/FMN hydrolase from gymnosperms but not from
angiosperms. The present picture is probably incomplete. Notably, the transporters enabling the exchange of riboflavin (and possibly also FAD) between
the different compartments are as yet unknown.
VIII. REGULATION OF RIBOFLAVIN BIOSYNTHESIS

The regulation of riboflavin biosynthesis has been studied in considerable
detail in Bacillaceae and other Gram-positive bacteria where all riboflavin
biosynthesis genes form part of a single operon. The coding region of the
operon is preceded by a regulatory region comprising more than 100 bp that
had been identified early on by genetic high-resolution mapping (Mironov
et al., 1990). Certain mutations in that region are conducive to riboflavin
overproduction.
Transcription of the rib operon begins with this regulatory region, and the
50 -end of the resulting mRNA folds into a highly specific three-dimensional
motif that binds FMN with a Kd of about 10 nM and with remarkably high
selectivity. FMN binding is accompanied by an allosteric transition of this
so-called riboswitch (Gelfand et al., 1999).
When the rib operon (preceding the coding region) is transcribed in the
absence of FMN, the riboswitch structure acts as an antiterminator which
allows the transcription of the operon to go to completion. However, if FMN
binds to the riboswitch at the 50 -end of the nascent mRNA, transcription of
129
the operon is aborted (Vitreschak et al., 2002, 2004; Winkler et al., 2002).
Certain missense mutations in the ribC gene specifying riboflavin kinase are
conducive to riboflavin overproduction by lowering the intracellular
concentration of FMN.
The riboswitch mechanism has been initially discovered by studies on the
rib operon of B. subtilis but is now known to be involved in the control of
numerous metabolic pathways in bacteria. A detailed description of this
regulatory principle exceeds the limits of this chapter, and readers are directed to excellent reviews of the topic (Mandal and Breaker, 2004; Sutak et al.,
2008; Vitreschak et al., 2004).
Studies on the regulation of flavin biosynthesis in plants have been mostly
centred around aspects of iron availability as summarised below. At a molecular level, these impressive phenomena are still incompletely understood.
IX. EXCRETION AND ENHANCED FORMATION OF

RIBOFLAVIN BY IRON-DEFICIENT ROOTS
Whereas iron is one of the most abundant elements in the earth crust, it is not
readily available to plants. Ferric iron (Fe3) is poorly soluble, especially at
relatively high pH, and the situation is further aggravated in soil containing
calcium carbonate. Ferrous iron (Fe2) has substantially higher solubility
but is subject to oxidation under aerobic conditions. Hence, a substantial
fraction of the global flora, and notably crop plants for human nutrition,
grow under conditions of iron deficiency.
Two strategies have evolved in the plant community in order to deal with iron
deprivation: (i) the excretion of protons, organic acids and flavins in conjunction with upregulation of Fe(III) chelate reductase (Vorwieger et al., 2007) and
(ii) the excretion of chelators (Vorwieger et al., 2007).
In plants using strategy I, iron deficiency is conducive to massive enhancements of riboflavin biosynthesis in root tips at the transcriptomic, proteomic
and metabolic level.
Evidence for increased production and for excretion of flavins by the hair
roots of certain plants under iron deficiency stress has been building over a
period of several decades (Higa et al., 2008, 2010; Lopez-Millan et al., 2000;
Susin et al., 1994 and references cited therein). Early reports noted that irondeficient hair roots developed yellow autofluorescence, whose spectral character was well in line with the optical properties of flavins. Sugar beets were
shown to accumulate hitherto unknown 30 - and 50 -sulphates of riboflavin
under conditions of iron deficiency (Susin et al., 1993). Their concentration in
extracts of iron-deficient roots reached values above 1 mM, well above of the
solubility limit of unsubstituted riboflavin.
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More recent studies have shown dramatic increases in the root concentrations of riboflavin biosynthetic enzymes under iron deficiency conditions.
Thus, Medicago trunculata roots grown under conditions of iron deficiency
and calcium excess showed 99-fold increase of lumazine synthase and
36-fold increase of GTP cyclohydrolase II/3,4-dihydroxy-2-butanone
4-phosphate synthase; the increase of deaminase and riboflavin synthase
was 24- and 6-fold, respectively (Rodriguez-Celma et al., 2011). A massive
increase of lumazine synthase and the cognate mRNA was also observed
in roots of sugar beets (Beta vulgaris) (Rellan-Alvarez et al., 2010).
The modulation of biosynthesis and excretion of flavins by roots of irondeficient plants must be viewed in the context of a complex network including,
but not limited to Fe(III) chelate reductase, a member of the flavochrome
superfamily which shuttles electrons across membranes, and a transporter for
Fe(II) (Li et al., 2004; Mori, 1999; Robinson et al., 1999; Waters et al., 2002).
These membrane proteins have been characterised in some detail but cannot be
addressed in detail in the present review. Various hypotheses trying to integrate
the modules of this network into a coherent model have been proposed. Thus, it
has been argued that the enhanced activity of the Fe(III) chelate reductase
causes an increased requirement for flavocoenzymes to serve as cofactors.
More direct roles for flavins have also been proposed, such as complexation
of iron ions. However, the proposed models fail to describe the interactions in
the complex system in significant detail and remain speculative.
In A. thaliana seedlings, the mRNA level for two transcription factors
(AtbHLH38 (At3g56970) and AtbHLH39 (At3g56980)) showed a massive
increase within days after initiation of iron deficiency conditions. The expression of these genes in tobacco was conducive to riboflavin synthesis and
excretion in transgenic tobacco plants even under conditions of abundant
iron supply (Vorwieger et al., 2007).
Despite the abundance of iron in the earth crust, not only plants but also
many millions of humans suffer from insufficient iron supplies. Notably, for
populations whose nutrition is predominantly plant based, plants are the
main dietary iron source. Thus, iron deficiency of crop plants affects human
nutrition by reduced crop size on one hand and poor iron supply on the other
hand (Mori, 1999).
Iron supply and riboflavin biosynthesis are also known to be connected in
certain yeasts and bacteria, but the research extending over many decades
cannot be addressed in this chapter (Almeida et al., 2009; Crossley et al.,
2007; Hsu et al., 2011; Sutak et al., 2008).
131
X. EVOLUTION OF FLAVOCOENZYME
BIOSYNTHESIS ENZYMES IN PLANTS
In contrast to the shikimate pathway which appears to have been maintained
constant throughout evolution, the riboflavin pathway is a theme with variations. For the first time, this has become evident in the 1970s with the discovery
that the pathway proceeds via different, although structurally similar intermediates (compounds 4 and 5, respectively) in eubacteria and fungi. Work in recent
years has revealed the riboflavin pathway of plants as a patchwork that appears
to have been assembled from pieces acquired from different taxa.
Several features of the plant pathway suggest a bacterial origin. (i) The first
committed intermediates of both branches of the converging riboflavin pathway, that is, 2 and 9, are both generated by a single fusion protein whose
general architecture and sequence shows strong resemblance with bacterial
orthologues (Herz et al., 2000). (ii) Pyrimidine deamination precedes the
reduction of the ribityl side chain; hence, the plant pathway proceeds via the
bacterial intermediate 4 (Fischer et al., 2004a). (iii) In line with that, the
sequence of the plant deaminase resembles the bacterial orthologues. Notably,
however, the bacterial prototypes are fusion proteins comprising a deaminase
and a reductase domain which jointly catalyse two consecutive reactions of the
riboflavin pathway. (iv) The plant-type FAD synthetase resembles the bifunctional riboflavin kinase/FAD synthetase that is present in numerous eubacteria; notably, however, the C-terminal domain of the plant protein, despite its
general sequence similarity to the bacterial riboflavin kinase domain, may now
catalyse a different reaction (if any). All of these features may have been
brought to plants via the genome of a cyanobacterial endosymbiont.
Several features of the plant pathway are shared with eubacteria and fungi,
but not with Archaea. (i) The formation of the pyrimidine type intermediate
2 from GTP is catalysed by a single enzyme, GTP cyclohydrolase II, in plants,
eubacteria and fungi, whereas Archaea require the consecutive action of two
enzymes, neither of which has similarity to GTP cyclohydrolase II; notably,
however, the product of the first-acting archaeal enzyme, the formamide 3, has
been shown to serve as a (non-released) intermediate of GTP cyclohydrolase II.
(ii) Plants, eubacteria and fungi use heterotrimeric riboflavin synthases whose
subunits fold into two closely similar domains, whereas Archaea use homopentameric riboflavin synthases which are paralogs of lumazine synthase.
Plants, many eubacteria and Archaea use lumazine synthases consisting of
60 identical subunits; the quasispherical proteins have icosahedral 532 symmetry (see Section IV for details). Fungi and certain eubacteria use pentameric lumazine synthases.
132
XI. RIBOFLAVIN BIOSYNTHETIC ENZYMES AS

POTENTIAL HERBICIDE TARGETS
As flavocoenzymes are essential in all organisms, the enzymes of riboflavin
biosynthesis are potential targets for herbicides and fungicides for crop protection. However, they are also potential targets for novel anti-infective drugs.
In each case, it is advantageous that animals do not synthesise riboflavin;
hence, there would be no risk of target-related toxicity.
Assays for high-throughput screening (HTS) with riboflavin biosynthesis
enzymes have been reported (Kaiser et al., 2007). Specifically, a HTS assay
for 3,4-dihydroxy-2-butanone 4-phosphate synthase can be monitored using
riboflavin synthase and/or lumazine synthase as auxiliary enzyme(s) in order
to generate a signal that can be recorded photometrically in the visible range.
The reactions catalysed by lumazine synthase and riboflavin synthase can be
monitored photometrically or flurometrically, respectively. It is also possible
to include riboflavin synthase as auxiliary enzyme in order to screen for
inhibitors of lumazine synthase (Kaiser et al., 2007).
XII. RIBOFLAVIN AND PLANT RESISTANCE

Over the past decade, a number of papers from different research groups
have reported that riboflavin can enhance the resistance of various plant
species including rice against certain viral, bacterial and fungal pathogens.
The mechanisms of riboflavin-mediated resistance involved enhanced formation of reactive oxygen species (ROS) and lignin. Specifically, riboflavin
treatment was found to prime the expression of lipoxygenase and upregulation of phenylalanine ammonia lyase (Averyanov et al., 2000; Dong
and Beer, 2000; Taheri and Hofte, 2007; Taheri and Tarighi, 2010; Zhang
et al., 2009).
Recent studies have shown that the expression of rice lumazine synthase in
transgenic tobacco results in elevated levels of jasmonic acid and ethylene
production. Moreover, the expression of defence-related genes and resistance
to viral, bacterial and oomycete pathogens were increased (Wu et al., 2010).
Earlier, lumazine synthase had already been implicated in the regulation of
jasmonate-mediated defence by genetic studies on the coronatine insensitive1
(Coi1) gene of A. thaliana (Xiao et al., 2004). The failure of the recessive coi1
mutant to react to jasmonate results in a pleiotrophic phenotype involving
male sterility as well as sensitivity to insect attack and pathogen infection.
COI1 protein interacts with CULLIN1, RBX1 and the Skp1-like proteins
ASK1 and ASK2 to form a multi-protein E3 ubiquitin ligase complex
133
designated SCFCOI1, which catalyses the ubiquitination of proteins destined

for proteasomal degradation. The cos1 (coi1 supressor1) mutation restores
the defects of the coi1 mutation. cos1 coi1-double mutants have wild-typelike phenotypes with regard to senescence and defence response. The cos1
gene was isolated by a map-based cloning strategy and was found to specify
lumazine synthase. The cos1 mutation involves a single nucleotide change
that results in the destruction of the splice site at the start of the second exon
and is believed to result in the formation of a lumazine synthase with a
deletion of six amino acids and with reduced catalytic activity. The authors
conclude that the riboflavin biosynthesis pathway is relevant for signal
transmission downstream from jasmonate.
Virus-induced silencing of the ribA gene specifying the bifunctional GTP
cyclohydrolase II/3,4-dihydroxy-2-butanone 4-phosphate synthase of Nicotiana benthamiana caused the loss of hypersensitivity response via cell death
and the synthesis of NO (nitric oxide) and ROS (reactive oxygen species)
(Asai et al., 2010). As expected, silencing of the ribA gene also reduced the
levels of endogenous riboflavin, FMN and FAD in the plants. RibA-silenced
leaves showed high sensitivity for infection by the oomycete Phytophtora
infestans. The compromised phenotypes could be rescued by treatment with
riboflavin, FMN or FAD.
Whereas animals do not biosynthesise riboflavin, it is noteworthy that
human riboflavin kinase couples TNF receptor 1 (tumour necrosis factor
receptor 1) to NADPH oxidase, thus playing an important role in the
generation of ROS that function as defence and signalling molecules
related to innate immunity and various cellular responses (Yazdanpanah
et al., 2009).
XIII. BIOSYNTHESIS OF 5-DEAZA-7,8DIDEMETHYL-8-HYDROXY-RIBOFLAVIN IN ALGAE

It has been known for some time that cyanobacteria employ DNA photolyases that use 7,8-dimethyl-8-hydroxy-5-deazaflavin (FO, 46, Fig. 17) as
light-harvesting chromophore (Carell et al., 2001). Recent studies indicate
that the deazaflavin also serves as the second chromophore of DNA photolyase of green algae (Eker et al., 1988; Glas et al., 2009; Maul et al., 2008;
Muller and Carell, 2009; Petersen and Ronan, 2011), although not of higher
plants. This implicates that the deazaflavin, which was initially discovered in
Archaea, is considerably more widespread than had been originally assumed
(Eirich et al., 1978, 1979). As the biosynthesis of FO is a side branch of the
134
Fig. 17. Hypothetical mechanisms for biosynthesis of 5-deaza-7,8-didemethyl-8hydroxy-riboflavin (FO, 46). 7, 5-Amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione; 41, 4-hydroxyphenylpyruvate. (A) Via the quinoid pyrimidine intermediate
42 (Eisenreich, 1991; Reuke et al., 1992); (B) by free radical recombination of 43 and
44 (Graham et al., 2003).
riboflavin biosynthetic pathway, the state of knowledge on its presence and

utilisation in algae and its biosynthesis is summarised below.
DNA photolyases catalyse the light-driven repair of light-induced damage
in the DNA of light-exposed cells (for review, see Sancar, 2008). Specifically,
two different types of DNA photolyases carry out the repair of cyclobutane
pyrimidine dimers and of (6-4) thymidine dimers. DNA photolyases are
widespread in nature but are absent in mammals where thymidine dimers
are exclusively processed by excision repair.
The reaction catalysed by DNA photolyases starts with the absorption of a
photon (for review, see Sancar, 2008). A fully reduced FADH2 chromophore
in the optically excited S1 state transfers an electron to the pyrimidine dimer
lesion under generation of a radical pair. The free-radical-type intermediate
135
then undergoes fragmentation, and an electron is transferred back to the

flavin; thus, there is no net transfer of redox equivalents. All known DNA
photolyases use FADH2 as the chromophore for catalysis. Besides that, a
second chromophore is used for efficient light harvesting. Energy is then
transferred from the antenna chromophore to the catalytic FADH2 centre by
Forster transfer.
As light-harvesting chromophore, DNA photolyases can use a tetrahydrofolate derivative (5,10-methenyltetrahydrofolate), a second flavin cofactor (FAD in the fully oxidised state), or factor FO. Higher plants use folate
derivatives, and so do many eubacteria.
The field of DNA photolyases has been progressing vigorously, with an
average of at least 10 papers per year, and exceeds the scope of this review (cf.
Sancar, 2008). However, we will briefly review the evidence for deazaflavin
biosynthesis and use in algae and the literature on deazaflavin biosynthesis in
Archaea and certain eubacteria.
FO (46) was initially identified as the chromophoric moiety of coenzyme
F420 of methanogenic bacteria, which plays a central role in methanogenesis.
FO was also shown to serve as antenna for DNA photolyases of nonmethanogenic Archaea and of mycobacteria. Moreover, recent evidence
suggests the occurrence of numerous coenzyme F420-dependent enzymes in
M. tuberculosis (Selengut and Haft, 2010).
Early studies showed a biosynthetic relationship between the deazaflavin
cofactor and purine nucleotides (Eisenreich et al., 1991; Jaenchen et al., 1984;
Le Van et al., 1985; Schwarzkopf et al., 1990). Moreover, the phenolic ring
was shown to be derived from the shikimate pathway. The riboflavin precursor 7 and 4-hydroxyphenylpyruvate (41) were subsequently identified as
specific biosynthetic precursors (Reuke et al., 1992) (interestingly, Methanobacterium thermoautotrophicum was found to convert the radiolabelled 7 into
FO (46) without noticeable dilution).
More recently, the cofG and cofH gene products were shown to be sufficient for the formation of FO from 7 and 41 in M. jannaschii (Graham et al.,
2003). Both peptides comprise CXXXCXXC motifs which are believed to
enable the binding of [4Fe4S] clusters essential for catalysis. Putative orthologues of cofG and cofH have been found in blue-green algae. The recombinant coexpression of CofG and CofH in E. coli enables the formation of
small amounts of of FO in vivo, and cell extracts catalyse FO formation
in vitro, albeit at low levels in the range of 100 pmol mg 1 min 1.
In Mycobacterium smegmatis, the FbiC (EC 2.5.1.77) fusion protein comprising segments with homology to CofG and CofH has been reported to be
sufficient for the formation of FO from 7 and 41 (Graham et al., 2003;
Guerra-Lopez et al., 2007). Specifically, the N-terminal half of the
136
bifunctional FO synthases resembles CofG, and the C-terminal half is most

similar to CofH of M. jannaschii.
The phr1 mutant of the green alga, Chlamydomonas reinhardtii, has a
severe defect in photoreactivation after ultraviolet irradiation. Recently, it
was found that the PHR1 gene is similar to the fbiC gene of mycobacteria
(Petersen and Ronan, 2011). The gene specifies a putative, N-terminal plastid-targeting sequence which precedes the catalytic domains. A recombinant
E. coli strain expressing the phr1 gene (without the plastid-targeting sequence) generated detectable amounts of FO. This is well in line with recent
work indicating that the green alga Ostreococcus tauri uses FO as antenna for
DNA photolyase (Glas et al., 2009).
Whereas the reaction catalysed by FbiC protein has been as yet incompletely characterised, a plausible scenario is shown in Fig. 17 (Eisenreich
et al., 1991; Reuke et al., 1992). The primary reaction step, to be discussed in
more detail below, could yield a covalent adduct of the pyrimidine 7 and 4hydroxyphenylpyruvate (41), which could subsequently lose ammonia and
oxalate by a coordinated elimination reaction. Tautomerisation could prepare the reaction product for a nucleophilic attack of the phenolate moiety
(in its quinoid tautomer form, 42) by the ribityl-substituted amino group,
thus affording the tricyclic intermediate 45 which could be converted to FO
(46) by a two-electron oxidation step; as this reaction is accompanied by
aromatisation of the carbocyclic ring, it would be highly exergonic.
An early proposal for the formation of the covalent adduct involved the
oxidative conversion of the pyrimidine 7 into a quinoid form (42) which
could then serve as an electrophile attacking the enolate form of 4-hydroxyphenylpyruvate (41). More recently, it has been proposed that the FO
synthases (FbiC, Phr1 and CofF/CofG, respectively) could strip one electron
each from the pyrimidine 7 and from 4-hydroxyphenylpyruvate (41); the two
radicals (43, 44) could then combine to form a further intermediate 45. This
proposal is based on the presence of amino acid motifs that are characteristic
binding sites for iron sulphur clusters and on reaction accelerations in crude
cell extracts upon addition of dithionite, iron ions and S-adenosylmethionine. However, the involvement of iron sulphur clusters and S-adenosylmethionine would per se not rule out a quinoid intermediate whose
formation they might in fact enable.
137
XIV. CONCLUSIONS
Whereas plants are the most important producers of flavins for human and
animal nutrition, the investigation of the biochemical background of flavin
metabolism in plants had a tardive start. Due to the emerging technology of
data base mining, however, the knowledge gap could be substantially narrowed during the past decade.
Flavins are essential for all organisms. As enzymes for riboflavin biosynthesis are essential in plants and in many plant pathogens and human
pathogens, but absent in humans and in animals, they represent potential
targets for novel herbicides, fungicides and anti-infective drugs which should
all be exempt from target-related toxicity. Specifically, the silencing of the
ribA gene of N. benthamiana is conducive to a light-sensitive phenotype; thus,
inhibitors of riboflavin biosynthesis might have an attractive mode of action
as herbicides. Notably, no novel mode of action has been introduced for
commercial herbicides since three decades, and the emergence of herbicide
resistance in conjunction with the growth of the human population is cause
for concern. The knowledge basis for target-oriented search for agents directed against plant enzymes for riboflavin biosynthesis is now available,
although there is still a dearth of structural biology work; only a single
riboflavin biosynthesis enzyme of plant origin has been studied by X-ray
crystallography.
The topology of flavocoenzyme biosynthesis and metabolism in plant cells
and the trafficking of proteins and metabolites between different cell compartments are still incompletely known, despite considerable recent progress.
Connections between the regulation of riboflavin biosynthesis, iron acquisition and resistance mechanisms involving the generation of nitrous oxide
and ROS are now firmly documented, but their mechanisms are poorly
understood. A better understanding of these phenomena at the molecular
and cellular level could have important ramifications for plant breeding and
biotechnology. Notably, crop plants grow under conditions of iron limitation in large geographic areas; moreover, plants are important providers of
iron for human and animal nutrition, and insufficient iron supply is the cause
of iron deficiency anaemia affecting 20% of women, 50% of pregnant women
and 3% of men.
138
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Biosynthesis of NAD and Its Manipulation in Plants
GRAHAM NOCTOR,1 JUTTA HAGER AND SHENGCHUN LI
Institut of Biologie des Plantes, UMR8618 CNRS/Universite

de Paris sud 11, Batiment 630, Universite de Paris sud 11, 91405
Orsay CEDEX, France
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
II. NAD in Human Nutrition and Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Niacin (vitamin B3)............................................................
B. Biosynthesis of NAD in Humans ...........................................
C. Measuring Niacin, NAD, and Related Compounds .....................
III. NAD in Plant Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Interconversion of NAD and NADP.......................................
B. Redox Links Between the NAD and NADP Pools ......................
C. Non-Redox Roles of NAD ..................................................
IV. Compartmentation and Transport of NAD in Plants . . . . . . . . . . . . . . . . . . . . .
A. Intracellular Distribution.....................................................
B. Subcellular Transport .........................................................
C. Plasticity of Tissue NAD(H) Contents .....................................
V. Plant Pathways of NAD Synthesis and Recycling . . . . . . . . . . . . . . . . . . . . . . . . .
A. De novo Production of NaMN in Bacteria and Plants...................
B. Conversion of NaMN to NAD .............................................
C. Recycling Pathways ...........................................................
D. Regulation of NAD Synthesis ...............................................
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In this chapter, NAD and NADP are used to refer to oxidized forms while NADH and
NADPH denote reduced forms. Where no distinction is drawn between the oxidized and reduced
forms, or where both forms may be concerned, only NAD is used.

0065-2296/11 $35.00
DOI: 10.1016/B978-0-12-386479-6.00002-0
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VI. Manipulating NAD Contents in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A. NAD Enrichment in Bacteria and Human Cells .........................
B. Enriching Tissue NAD in Plants ............................................
C. Inducible Increases in NAD(H) are Associated with Activation
of Pathogenesis-Related Pathways..........................................
D. Links Between NAD, ROS, and Thiol Status? ...........................
VII. Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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ABSTRACT
NAD and NADP are key brokers in cell redox and energy metabolism, but they also
play important roles in signalling pathways. Several of these latter roles involve NAD
cleavage, requiring continuous resynthesis of the molecule to maintain cellular pools.
Mammals synthesize their cellular NAD from pyridine precursors that are obtained
as niacin (vitamin B3) or produced from tryptophan. In many foods, the major source
of niacin can be the pyridine ring of NAD and NADP. Plant NAD contents show
considerable plasticity in response to environmental conditions, and recent advances
have opened up new perspectives for rational manipulation of tissue NAD contents.
In the model plant Arabidopsis, the de novo pathway of plant NAD synthesis has been
identified, as have proteins involved in the subcellular transport of NAD and in NAD
cleavage and recycling. Here, we discuss emerging concepts on the regulation of NAD
contents in plants, and we report the use of transformation technology to enrich NAD
several-fold in plant tissues through overexpression of the de novo synthesis pathway.
Microarray analyses reveal that these increases trigger a characteristic transcriptomic
signature, notably involving specific genes in defence phytohormone signalling. Thus,
manipulation of plant NAD biosynthesis may offer interesting perspectives to (1)
dissect redox-related signalling in stress responses, (2) enhance the nutritional value of
plants, and (3) modify plant resistance to environmental stress.
I. INTRODUCTION
Life on earth is driven by reactions in which energy is extracted from
thermodynamically favourable respiratory electron flow from relatively reducing compounds to more oxidizing ones. Photoautotrophic organisms
such as plants continually replenish the biospheres supply of reducing
compounds through the process of photosynthesis. Both respiration and
photosynthesis involve membrane-bound electron transfer chains coupled
to metabolic reactions in the aqueous phase. Chief among the compounds
that link the soluble and membrane-bound components are the pyridine
nucleotides, NAD(H) and NADP(H). During aerobic respiration, NADH
formed by mitochondrial matrix enzymes is reoxidized by dehydrogenases
bound to the inner mitochondrial membrane (Douce and Neuburger, 1989;
Rasmusson et al., 2004). In photosynthesis, NADPH generated by
BIOSYNTHESIS OF NAD AND ITS MANIPULATION IN PLANTS
155
ferredoxin-NADP reductase (FNR) is reconverted to NADP by the

photosynthetic isoform of glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) in the single reductive reaction of the CalvinBenson cycle, or
reductive pentose phosphate pathway (Harbinson et al., 1990; Scheibe et al.,
2005). As well as these essential core processes, both pyridine nucleotides are
involved in a host of other reactions. It is estimated that hundreds of enzymes
depend on NAD or NADP in a redox-carrying role (Waller et al., 2010 and
references therein). As a very general rule, NAD is more closely linked to
catabolic reactions whose physiological role is to generate energy while
NADP is the preferred cofactor of enzymes involved in biosyntheses and
cellular redox homeostasis. However, this distinction is far from absolute.
For example, NADPH as well as NADH can be oxidized by the plant
mitochondrial electron transport chain (Douce and Neuburger, 1989;
Rasmusson et al., 2004), NADH is required for assimilatory nitrate reductase (NR) activity (Kaiser et al., 2000, 2002), and the antioxidative enzyme
monodehydroascorbate reductase can use either NADH or NADPH
(Hossain and Asada, 1985).
In addition to its cofactor function for oxidoreductases, NAD has been
known for some time to be required by other enzymes, such as ADP-ribose
transferases (Hayaishi and Ueda, 1977; Henderson, 1983). As these reactions
do not involve oxidoreduction, they can be termed non-redox. From a
physiological standpoint, the redox versus non-redox distinction may be
somewhat simplistic. Even if the reaction itself does not involve redox
exchange, it may still be important in redox metabolism, homeostasis, or
signalling. Nevertheless, a key difference is that the non-redox roles involve
consumption (cleavage) of the nucleotide, and therefore, resynthesis or new
synthesis is required to avoid NAD depletion.
Recent years have seen the appearance of several reviews on NAD synthesis
and metabolism in plants (Hashida et al., 2009; Hunt et al., 2004; Katoh and
Hashimoto, 2004; Noctor et al., 2006; Roje, 2007) and interest in the direct,
targeted manipulation of intracellular NAD concentrations has grown.
Among the reasons for this burgeoning interest are (1) the plant biosynthetic
pathway has been clarified; (2) it has become evident that NAD contents can
be quite plastic in plants; (3) ever-growing attention is being paid to the roles of
redox-related factors not only in cellular energy metabolism but also in
signalling; and (4) the non-redox reactions mentioned above have continued
to receive increasing attention. The aim of the present chapter is to discuss
some recent advances in understanding NAD metabolism and turnover within
the context of the perspectives for manipulation of these pathways. With this
aim in mind, we present previously unpublished data from an ongoing study
seeking to engineer increased NAD contents in plant tissues.
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II. NAD IN HUMAN NUTRITION AND HEALTH

The fundamental redox exchange function of pyridine nucleotides involves
the nicotinamide moiety, which is identical in NAD and NADP (Fig. 1).
Thus, the two nucleotides undergo the same redox reactions with Em
320 mV at pH 7. The difference between the two compounds resides in
the phosphate group attached to the 20 of the ribose ring of the adenosine
moiety of NADP (Fig. 1), a group which is absent in NAD, explaining the
preference that enzymes may have for one form rather than the other. The
only known pathway for the synthesis of NADP is from NAD, a reaction
catalysed by NAD kinases (NADKs). Thus, in order to maintain these
nucleotide pools or to produce new nucleotides for growth, synthesis of
NAD is required. This requires either a dietary source of the pyridine ring
or its production from other molecules. Humans can obtain the NAD
pyridine precursors either from niacin present in food or from degradation
of the indole ring of tryptophan.
A. NIACIN (VITAMIN B3)
The best known pyridine precursors are nicotinic acid and nicotinamide,
collectively known as vitamin B3 or niacin (or sometimes niacinamide in
the case of nicotinamide). Nicotinic acid was identified as the substance able
Fig. 1. Basic structures of NAD(P) and NAD(P)H. For simplicity, not all C and
H atoms are shown. The redox-active pyridine ring is highlighted within the blue
square. The difference between NAD and NADP, at C-2 of the AMP ribose ring, is
highlighted red. In NAD, R H. In NADP, R phosphate group.
157
to cure canine black-tongue disease (Elvehjem et al., 1938). This advance led
to the recognition that niacin deficiency was responsible for the related
human disease, pellagra, outbreaks of which were prevalent in the early
part of the twentieth century. Although it is still found in undeveloped
regions during certain seasons, pellagra has largely disappeared from the
developed world, except in certain conditions such as chronic alcoholism
(Bogan and Brenner, 2008).
The U.S. recommended daily amount (RDA) of niacin (nicotinic acid
nicotinamide) is 1416 mg (Bogan and Brenner, 2008), while the EU recommends between 9 and 18 mg (SCF, 1993). Like many other organisms,
however, humans can make the NAD pyridine ring from tryptophan.
Thus, provided tryptophan intake is sufficient, niacin may not be required
at all in many conditions (SCF, 1993). Tryptophan has been assigned a
niacin equivalent value of 1/60 (60 mg tryptophan 1 mg niacin), though
this value is necessarily approximate (Horwitt et al., 1981). In many food
sources (including milk, meat, and cereals), tryptophan is much more abundant than niacin, but the capacity to convert tryptophan is variable between
individuals (Horwitt et al., 1981).
Especially good sources of niacin are tuna, chicken, and liver (Table I).
Espresso coffee is rich in niacin, though instant coffee contains much less.
Milk and eggs are also niacin-poor (Horwitt et al., 1981). Enrichment with
niacin means that one or two modest servings of many breakfast cereal
products can be sufficient to meet the above RDAs (Table I; Rose-Sallin
et al., 2001; Windahl et al., 1998). Cereal grains naturally contain quite
high amounts of niacin, but much of it is complexed, mainly with polysaccharides, leaving only a minor part available for uptake (Lahely et al.,
1999). The unavailability of niacin in corn (maize) is thought to explain
outbreaks of pellagra in the first decades of the twentieth century in
regions where much of the diet was corn-based. This problem may have
been conditional on food preparation (Henderson, 1983). Pellagra is uncommon in Central America, even though corn makes up a large part of
the calorie intake. However, corn in Central America is traditionally
prepared by soaking in limewater (nixtamalization), a procedure that
releases much of the bound niacin. An additional factor that could favour
the development of pellagra is removal of the embryo (germ) from cereal
grains, causing depletion of both niacin and tryptophan, a low-abundance
amino acid in corn endosperm (Henderson, 1983). Plant foods that are
quite good sources of niacin include potatoes and peas (Table I). Cooking
does not generally diminish niacin contents in a wide range of meats and
vegetables, and may increase the amount of the vitamin in some cases
(Windahl et al., 1998).
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TABLE I
Typical Amounts of Niacin in Some Foods
Foodstuff
Fruits and vegetables
Apple
Banana
Tomato (raw)
Lettuce
Potato (boiled)
Peas (boiled)
Mushrooms (raw)
Cereals and derived foods
Rice (white, boiled)
Wheat flour
French bread
Multigrain bread
Maize (sweetcorn)
Cornflakes (enriched)
Fish
Cod (dry-cooked)
Haddock
Tuna (canned)
Meats
Chicken (breast, roasted)
Beef (sirloin steak, grilled)
Pork (chops, grilled)
Lamb (roasted)
Beef liver
Beverages
Milk
Orange juice
Beer
Wine (red)
Tea
Coffee (espresso)
Weight (g)
Niacin (mg)
% RDA
140
120
120
100
150
160
100
0.13
0.79
0.73
0.36
2.0
2.4
3.5
0.9
5.3
4.9
2.3
13.0
16.0
23.9
160
120
25
25
80
30
2.3
5.9
1.2
1.1
1.3
7.0
15.3
39.3
7.9
7.3
8.7
46.7
100
100
100
1.3
4.1
13.3
8.6
27.5
88.7
100
100
100
100
100
13.7
8.2
8.1
7.0
17.4
91.2
54.9
54.1
46.8
115.8
250 ml
250 ml
250 ml
250 ml
250 ml
60 ml
0.2
0.7
1.0
0.6
0
3.1
1.3
4.7
6.7
3.9
0
20.7
Source: the USDA National Nutrient Database. A more detailed list can be found at
http://www.ars.usda.gov/SP2UserFiles/Place/12354500/Data/SR23/nutrlist/sr23a406.pdf. The
recommended daily amount (RDA) is taken as 15 mg. Apart from breakfast cereals, values are
for unenriched foodstuffs.
Nicotinic acid, nicotinamide, NAD, and NADP are all recognized niacin
vitamers. At least in relatively unprocessed foods, particularly meats, NAD
and NADP are considered to provide the largest part of the available niacin
(Bogan and Brenner, 2008; Henderson, 1983; Lahely et al., 1999). Although
the forms taken up across the gut mucosa for intracellular conversion to
NAD are predominantly nicotinic acid and nicotinamide, these are produced
from the nucleotides by digestive enzymes in the gut (Henderson, 1983; SCF,
159
1993). In other foodstuffs, free nicotinic acid or nicotinamide may make a

more substantial contribution to the total intake. In most cases, however,
identifying the chemical nature of the different vitamers is not a primary aim
of niacin assays in foodstuffs (see Section II.C), and so uncertainty remains in
this area.
Vitamin B3 present in supplements can consist of either nicotinic acid or
nicotinamide. High doses in the range of 100 mg to several grams per day of
nicotinic acid have been used for decades to treat hyperlipidaemias (Bogan
and Brenner, 2008; SCF, 1993). However, the mechanisms responsible for the
effect of nicotinic acid are not clearly established. The mechanisms may be
independent of NAD production, because nicotinamide is much less effective
as a treatment, though it is possible that the different efficacies rather reflect
preferential use of nicotinic acid for NAD synthesis in some tissues (Bogan
and Brenner, 2008). In high doses, nicotinic acid has clear side effects, including painful facial flushing linked to hypotension, though this is often temporary and tolerance develops within a few days (Henderson, 1983; SCF, 1993).
B. BIOSYNTHESIS OF NAD IN HUMANS
Nicotinic acid and nicotinamide acquired from the gut are converted to
pyridine mononucleotides (Fig. 2). These are then adenylated to produce
the dinucleotide. In the case of NAD synthesis from nicotinic acid, a terminal
amination step is required and so the conversion involves three enzymes that
constitute the PreissHandler pathway (Preiss and Handler, 1958). Conversion of nicotinamide to NAD was thought for some years to involve
nicotinic acid production by nicotinamidase, and therefore also to require
the same three intracellular enzymes. However, it was established in the mid1970s that conversion of nicotinamide to NAD requires only two enzymes, a
phosphoribosyltransferase and an adenylyltransferase (Henderson, 1983).
The second of these enzymes shows dual substrate specificity. It either converts nicotinate mononucleotide (NaMN) to nicotinate adenine dinucleotide
(NaAD), which is then aminated by NAD synthetase (NADS) using glutamine or ammonia, or produces NAD directly from nicotinamide mononucleotide (NMN; Fig. 2).
Another dietary source of the pyridine ring has been described in foodstuffs, such as milk, which are low in other recognized forms of niacin
(Bieganowski and Brenner, 2004). This is nicotinamide riboside. As well as
being present in some foodstuffs, it may be a product of NAD(P) digestion
via NMN in the gut (Bogan and Brenner, 2008). Whatever its source, it can
be either phosphorylated to produce NMN directly (Fig. 2) or, alternatively,
cleaved to nicotinamide and ribose with nicotinamide being subsequently
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G. NOCTOR ET AL.
Fig. 2. NAD synthesis pathways in humans. Niacin obtained in the diet as

nicotinic acid, nicotinamide, NAD, or NADP can be converted to pyridine nucleotides, which are then converted to NAD. Alternatively, the pyridine group can be
synthesized de novo by degradation of the indole group of tryptophan. NADS, NAD
synthetase; NaMN/NMNAT, nicotinic acid mononucleotide/nicotinamide mononucleotide adenylyltransferase; NaPRT, nicotinic acid phosphoribosyltransferase;
NPRT, nicotinamide phosphoribosyltransferase; NRK, nicotinamide riboside kinase; QPRT, quinolinate phosphoribosyltransferase. As well as being obtained in
food, nicotinamide is also produced from intracellular NAD by several enzymes
(see Fig. 3) and can be recycled into NAD by the two-step pathway shown on the
right.
converted to NMN through phosphoribosyltransferase activity (Belenky

et al., 2009).
Like other animals, humans can synthesize the pyridine ring of NAD
through a de novo route. In the so-called eukaryotic pathway of de novo NAD
synthesis, quinolinate is obtained by degradation of the indole ring of tryptophan and then converted to NaMN by quinolinate phosphoribosyltransferase (QPRT). NaMN can then be adenylated and aminated through the
final two enzymes of the PreissHandler pathway (Fig. 2). Thus, the NAD
synthesis pathways from quinolinate and nicotinate converge at NaMN and
thereafter involve the same enzymes. While still a subject of ongoing research, human tissues have different capacities for the different pathways
of NAD synthesis and turnover. For further details, the reader is referred to
the recent review by Bogan and Brenner (2008).
161
It should be noted that the term eukaryotic pathway of NAD synthesis

is misleading. First, some prokaryotes can use this pathway (Gazzaniga et al.,
2009; Kurnasov et al., 2003). Second, as we discuss later in this chapter, the
best characterized pathway in plants is not this sequence but rather the socalled prokaryotic pathway (Katoh et al., 2006).
C. MEASURING NIACIN, NAD, AND RELATED COMPOUNDS
In determining the vitamin B3 content of foodstuffs, the contribution of the

different vitamers is often not considered. Common extraction treatments
such as alkaline or acid hydrolysis mean that any NAD, NADP, and nicotinamide present in the sample are converted to nicotinic acid, which is the
form that is then measured as niacin (Lahely et al., 1999; Rose-Sallin et al.,
2001; Windahl et al., 1998). Acid extraction is considered to yield the total
bioavailable niacin in a sample (i.e. that which can be absorbed from the gut)
while alkaline hydrolysis can also release bound forms of niacin (Lahely
et al., 1999; Windahl et al., 1998).
Widely used methods for niacin determination in foods are microbial
bioassay and a chemical colorimetric method based on the reaction of
pyridine with cyanogen bromide (Fuller, 1980). More recent developments
include high-performance liquid chromatography (HPLC) and capillary
electrophoresis (CE) methods. Nicotinic acid can be readily detected by CE
or by HPLC with UV absorbance detection (Windahl et al., 1998). A more
sensitive method using HPLC-fluorescence measured nicotinic acid in various foodstuffs (Lahely et al., 1999; Rose-Sallin et al., 2001). Although
nicotinic acid is not volatile, derivatization allows ready detection by gas
chromatographymass spectrometry (GCMS). While this method only
offers relative quantification, it can be useful for large-scale metabolite
profiling of plant tissues (e.g. Hager et al., 2010).
While HPLC techniques exist for separating and quantifying NAD and
NADP, both can readily be quantified through well-established and sensitive
enzymatic cycling assays. It is a simple matter to distinguish between NAD
and NADP through the use of specific enzymes (or by separation on HPLC
columns). More problematic is discrimination between the reduced and
oxidized forms. Usually, this is achieved by exploiting the differential lability
of the two forms in acid and alkaline extraction buffers. High-throughput
methods have been developed that can measure the two nucleotides at the
same time as other redox compounds (Queval and Noctor, 2007).
A full understanding of NAD(P)-related biological questions is likely to
benefit from techniques that are able to report on the status of specific pools
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in different compartments. The fluorescent properties of reduced pyridine

nucleotides have been exploited in the analysis of plant mitochondrial metabolism (Kasimova et al., 2006; Neuburger et al., 1984). However, these
measurements do not separate the NADH and NADPH signals from each
other, or give unambiguous information on redox states. The limitations of
the information generated by the different methods of measuring pyridine
nucleotides have been nicely summarized by Hagedorn et al. (2007). A recent
interesting technique coupled CE separation to an enzymatic recycling method with fluorescence detection to measure NAD and NADH concentrations in a single rat cell (Xie et al., 2009).
As plant biologists have focused on measuring NAD and NADP rather
than their metabolites or precursors, and food nutritionists are mainly
interested in total niacin, quantitative information on the different compounds involved in NAD synthesis and turnover in plants is relatively
scarce. This is likely to change in the coming years, as metabolite profiling
techniques continue to become increasingly powerful and widespread. As
well as nicotinate, GCMS can be used to measure quinolinate and other
similar compounds (Smythe et al., 2002), though not large, phosphorylated
compounds such as nucleotides. A recent report described an LCMS
method able to profile NAD alongside most of its precursors and metabolites in mouse erythrocytes (Yamada et al., 2006). This analysis showed
that NaMN and NMN could be detected but that they were at least 10fold less abundant than NAD itself, whereas nicotinate, nicotinamide, and
NaAD were below the threshold of detection (Yamada et al., 2006). In
Arabidopsis rosette leaves, CEMS quantification of NaMN and NaAD
revealed that both were about 20-fold less abundant than NAD
(Takahashi et al., 2009). Using the same technique in rice leaves, the
contents of quinolinate, nicotinate, nicotinamide, NaMN, and NaAD
were quantified (Takahara et al., 2010). While nicotinamide, NaMN, and
NaAD were present at about 1 nmol g 1 leaf fresh weight (20-fold less
abundant than NAD and NADP), quinolinate and nicotinate were considerably more abundant, at about one-third of NAD and NADP contents
(Takahara et al., 2010). In terms of niacin, the summed amounts of the
measured pyridine compounds would represent about 0.8 mg/100 g fresh
leaf weight, a fairly similar value to those recorded for edible leaf vegetables such as lettuce (Table I). Based on this value, and discounting any
contribution from tryptophan, it would require almost 2 kg of rice leaves
to supply the U.S. and European RDAs of niacin, with NAD supplying
44%, free nicotinate 12%, and free nicotinamide 1.5% of the total. However, a modest serving of rice grains satisfies about one-sixth of the
recommended daily niacin intake (Table I).
163
III. NAD IN PLANT BIOLOGY

Given the plethora of NAD-dependent enzymes, it is not possible to cover
the redox roles of NAD in this chapter. As a prelude to the subsequent
sections on NAD synthesis, the following discussion focuses on reactions
that consume NAD, including NADP synthesis. Redox aspects are largely
confined to a brief overview of the relationship between NAD and NADP,
because this issue is relevant to any attempt to evaluate the functional
interaction between the two nucleotides.
A. INTERCONVERSION OF NAD AND NADP
NAD is converted to NADP through ATP-dependent phosphorylation catalysed by NADK. A single gene has been described encoding NADK in
humans, whereas three genes are found in Arabidopsis (Waller et al., 2010).
Chloroplast NADK activity has long been known to be influenced by light,
which may in some cases be reflected in light-dependent changes in chloroplast NAD or NADP contents (Heineke et al., 1991; Takahama et al.,
1981). Based on effects on NADPH, H2O2, and oxidative stress sensitivities
in human cell lines in which the cytosolic NADK was knocked down or
overexpressed, it has been proposed that this enzyme works together with
NADP-linked dehydrogenases in ensuring NADPH production. Perhaps
unsurprisingly, however, the dehydrogenases were found to play the major
role in producing NADPH during oxidative stress (Pollak et al., 2007).
As well as the two NAD-specific kinases found in the chloroplast
(NADK2) and cytosol (NADK1), a third enzyme shows preference for
NADH and is thus able to produce NADPH directly (Turner et al., 2005).
Of the different Arabidopsis knockout mutants, only nadk2 shows a clear
phenotype in optimal growth conditions (Chai et al., 2005; Takahashi et al.,
2009). However, this mutation is not lethal, suggesting either that the other
NADKs can partly replace NADK2 and that the chloroplast envelope can
take up NADP produced by these enzymes, or that other chloroplast
enzymes competent in NADP synthesis await discovery. Mutants for nadk3
lacking the NADH-preferring kinase are hypersensitive to several types of
stress (Chai et al., 2006). Interestingly, NADK3, previously considered to be
cytosolic, has recently been shown to be targeted to the peroxisome (Waller
et al., 2010). A thorough study showed that targeting required a previously
undescribed peroxisomal targeting sequence 1 (PTS1) composed of the
C-terminal tripeptide SRY and accessory targeting amino acids immediately
upstream of SRY (Waller et al., 2010). Some NADKs interact with calmodulin (Delumeau et al., 2000; Harding et al., 1997; Turner et al., 2004)
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G. NOCTOR ET AL.
although the in vivo significance of some of the interactions has been questioned (Waller et al., 2010).
Overexpression of NADK in Arabidopsis or rice produced marked effects
on photosynthesis and carbon and nitrogen metabolism (Takahara et al.,
2010; Takahashi et al., 2009). These observations add to others that point to
close links between NAD(P) status, and the coordination of plant C and N
metabolism (Dutilleul et al., 2005; Hager et al., 2010; Pellny et al., 2008).
Despite the effects of altering NADK capacity, neither mutants nor overexpressors have been shown to exhibit the changes in either NAD(H) or
NADP(H) that might be predicted, though decreased NADPH was reported
in nadk3 mutants (Chai et al., 2006).
NADP can be converted to NAD by a phosphatase activity. This activity
has been studied in plants, notably in seeds (Gallais et al., 2000a,b). A recent
study revealed an inverse correlation between NADK and NADP phosphatase activities that was associated with the differing dormancy of Arabidopsis
ecotypes (Hunt and Gray, 2009). Enzymes and genes responsible for NADP
phosphatase activity remain to be fully characterized.
How do NADKs and NADP-dependent dehydrogenases interact in supplying NADPH? NADK has been implicated in NADPH oxidase function
linked to pathogenesis and related responses (Harding et al., 1997), while
other studies have underlined the importance of enhanced respiratory flux
through enzymes such as glucose-6-phosphate dehydrogenase (G6PDH;
Pugin et al., 1997). However, G6PDH is only one of several relatively
abundant NADP-linked cytosolic dehydrogenases in plants (Foyer and
Noctor, 2009; Hayashi et al., 2005). Recent data suggest that the cytosolic
NADP-isocitrate dehydrogenase may also play some role during oxidative
stress and pathogenesis responses (Mhamdi et al., 2010a). Despite the attention drawn to the roles of NADK in stress responses, our own studies on
plants showing obvious oxidative stress do not point to marked reciprocal
changes in NAD and NADP contents in these conditions relative to controls
(Mhamdi et al., 2010a,b). Whether NADNADP cycling is stimulated under
stress conditions remains to be established.
B. REDOX LINKS BETWEEN THE NAD AND NADP POOLS
Metabolite pools in mitochondria, chloroplast, and cytosol link NAD and

NADP pools through redox transfer. Plants have been known for many
years to contain an irreversible cytosolic NADP-linked GAPDH, alongside
the better known reversible cytosolic NADGAPDH (Kelly and Gibbs,
1973). The NADP-linked enzyme is called non-phosphorylating, because
it does not involve the incorporation of phosphate during glyceraldehyde-
165
3-phosphate oxidation, and so produces 3-phosphoglycerate (3-PGA) directly without ATP formation by PGA kinase. Several roles for this enzyme have
been proposed, including export of NADPH from the chloroplast, production of NADPH for biosyntheses or, more recently, generation of reducing
power in oxidative stress responses (Kelly and Gibbs, 1973; Rius et al., 2006).
The last function is of particular interest, given that the NAD-dependent
GAPDH undergoes numerous stress-related post-translational modifications that tend to inhibit its activity (Holtgrefe et al., 2007). Other major
soluble dehydrogenases such as isocitrate and malate dehydrogenases are
also found as isoforms that can use NAD, NADP, or both (Lancien et al.,
2000; Rasmusson and Mller, 1990).
Direct redox transfer between NAD and NADP pools is possible via
transhydrogenase activity through the following reversible reaction:
NADH NADP ! NAD NADPH
This enzyme, which is found in bacteria and the inner mitochondrial membrane, is usually but not always coupled to transmembrane proton transfer.
In the presence of an electron transport chain-dependent proton gradient, the
reaction is driven in the direction shown above, and this is thought to
contribute to the high NADP reduction state compared to NAD in bacteria
(Jackson, 2003). In mitochondria, the enzyme capacity is lower than in
bacteria, and the main role of transhydrogenase may be in fine-tuning the
TCA cycle (Jackson, 2003). Biochemical studies in plants have reported
two independent transhydrogenase activities associated with the mitochondrial inner membrane, one of which was associated with complex I (Bykova
et al., 1999).
C. NON-REDOX ROLES OF NAD
The NAD molecule can be considered either as a dinucleotide or as an ADPribose group joined to nicotinamide (Fig. 3). Cleavage of the pyrophosphate
bond that joins the two mononucleotides or the b-N-glycosidic linkage
between the ADP-ribose and the nicotinamide group is energetically favourable. Several types of enzyme are able to cleave the glycosidic bond. This
liberates the pyridine ring as nicotinamide and produces several different
compounds as partner products, including cyclic ADP-ribose, free ADPribose, acetylated ADP-ribose, or protein-bound ADP-ribose (Fig. 3). Cyclic
ADP-ribose synthase has been implicated in abscisic acid (ABA) signalling in
plants (Sanchez et al., 2004). Like cADP-ribose, NaADP, which is produced
by deamidation of NADP, is involved in calcium signalling in a variety of
organisms, including plants (Guse and Lee, 2008; Navazio et al., 2000).
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G. NOCTOR ET AL.
Fig. 3. Enzymes involved in NAD turnover. Different NUDIX hydrolases have

different substrate specificities. NUDIX hydrolases so far described that cleave NAD
preferentially use NADH rather than NAD.
NAD-dependent deacetylases (named SIRTUINS in humans and yeast)

have been linked to cell death and oxidative stress responses in plants
(Huang et al., 2007). In other organisms, SIRTUINS have been implicated
in the regulation of various biological processes, notably ageing (Imai et al.,
2000; Lin et al., 2000). Recently, enzymes of NAD recycling and NAD itself
have been implicated in the regulation of mammalian circadian clock function (Ramsey et al., 2009).
A major demand on NAD (and potentially, therefore, NAD synthesis)
may come from poly(ADP-ribose) polymerase (PARP). This protein transfers ADP-ribose groups from NAD to acceptor proteins, a modification that
has been implicated in regulation of gene expression and DNA repair. PARP
has been implicated in stress responses in different types of organisms,
including plants (Adams-Phillips et al., 2010; De Block et al., 2005;
Graziani et al., 2005; Pellny et al., 2009; Vanderauwera et al., 2007; Ying
et al., 2003). The RADICALCELLDEATH1 (RCD1) protein has a PARPtype domain and has been implicated in oxidative stress responses in Arabidopsis (Ahlfors et al., 2004).
Other potential NAD-cleaving enzymes that have recently received attention in plants are NUDIX hydrolases. The original member of this family
(MutT) was discovered in Escherichia coli as a suppressor of mutations
linked to oxidized dGTP: all members of the family cleave a nucleoside
disphophate bond linked to some other group, X, with variable specificity
for nucleoside triphosphates, nucleotide sugars, and NADH (Bessman et al.,
167
1996). NUDIX hydrolases that cleave NADH produce the two mononucleotides (with the pyridine ring of NMN in the reduced form) while others can
metabolize ADP-ribose (Fig. 3). The physiological functions of these reactions may be to prevent excessive accumulation of NADH, which would
inhibit oxidative metabolism, or ADP-ribose, which could affect a number of
processes (Bessman et al., 1996; McLennan, 2006). Yeast deficient in the
yeast NUDIX hydrolase Ysa1 had decreased reactive oxygen species (ROS)
levels and oxidative stress sensitivity linked to increased ADP-ribose. It was
suggested that these effects were caused by ADP-ribose inhibition of
GAPDH and mitochondrial complex I, thus decreasing ROS production
and supporting NADPH generation through the oxidative pentose phosphate pathway (Tong et al., 2009). In Arabidopsis, there are more than 20
genes predicted to encode NUDIX hydrolases (AtNUDT or AtNUDX).
Analysis of nine of the corresponding recombinant proteins showed that
NUDT2, NUDT6, NUDT7, and NUDT10 were able to use ADP-ribose,
with the first three also able to use NADH (Ogawa et al., 2005). Knockout
mutants for NUDT7 show pleiotropic phenotypes, most notably increased
salicylic acid (SA) and pathogen resistance (Bartsch et al., 2006; Ge et al.,
2007; Jambunathan and Mahalingam, 2006). Differences in phenotypes have
been reported between some of the studies and may be explained by different
growth nutrition regimes (Jambunathan et al., 2010). A large part of the total
hydrolase activity against ADP-ribose and NADH may be due to NUDT7
(AtNUDX7) activity and modified expression of this enzyme affected in vivo
contents of both compounds (Ishikawa et al., 2009). Other data point to a
role for NUDT7 in metabolizing PARP-derived ADP-ribose during oxidative stress (Ishikawa et al., 2009). In a subsequent study, NUDT6/NUDX6
was implicated in regulation of NADH contents during SA signalling
through the redox-regulated NONEXPRESSOROFPATHOGENESISRELATEDGENES1 (NPR1) pathway (Ishikawa et al., 2010). A third member
of this family with activity against ADP-ribose, AtNUDX2, has been implicated in oxidative stress tolerance (Ogawa et al., 2009).
The above discussion underlines that the non-redox roles of NAD may be
important in stress, of which redox changes are a key part. Emerging evidence from work on other organisms suggests that in some circumstances,
NAD concentrations may be limiting for these enzymes (Belenky et al.,
2009 and references cited therein). A key issue here could be that nucleotide
concentrations determined by direct assay are unlikely to reflect the biochemically relevant free concentration, which is probably significantly lower
than the measured concentration. This is because the high abundance of
redox enzymes that use NAD(H) means that a large fraction of the measurable nucleotide pool within any compartment is bound at any one time to
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G. NOCTOR ET AL.
protein. While this issue has been discussed in relation to redox functions of
NAD in plants (Heineke et al., 1991; Igamberdiev and Gardestrom, 2003;
Rasmusson and Mller, 1990), it may also be important in evaluating the
regulation of enzymes that use NAD or NADH as a cleavable substrate.
IV. COMPARTMENTATION AND TRANSPORT

OF NAD IN PLANTS
The distribution of NAD between compartments is a key issue in understanding NAD function and regulation of NAD synthesis and turnover. To
date, most studies in plants have focused on NAD and NADP themselves,
and this is reflected in the discussion below. These nucleotides are generally
considered unlikely to move at significant rates between cells, though their
precursors could be transported between tissues. The discussion below focuses on the relatively limited information currently available on their distribution and transport within plant cells.
A. INTRACELLULAR DISTRIBUTION
While the fluorescence of the reduced pyridine ring can be used to measure
relative changes in NAD(P)H, determination of nucleotide concentrations
and redox states (reduced:oxidized ratios) has thus far used techniques that
require extraction. A major challenge in such measurements is avoiding
alterations in metabolite status during sample preparation, a problem that
becomes particularly acute when fractionation of cells is required prior to
assay. Artefacts can arise from exchange between compartments during fractionation and/or changes in redox state as cellular integrity and functions are
perturbed. Approaches that have been used to circumvent these problems in
plants include non-aqueous fractionation and rapid filtration of protoplasts
into acid or alkaline buffers. While the former technique is relatively slow, it
avoids leakage and inhibits metabolism. The latter is much more rapid as it
can arrest metabolism in a fraction of a second, but it requires prior preparation of protoplasts that can be easily ruptured by passage though a nylon mesh
with an appropriate pore size (Gardestrom, 1987; Stitt et al., 1982).
A summary of some of the data available using these techniques is presented in Table II. NAD and NADP concentrations in the chloroplasts,
mitochondria, and cytosol are between 0.1 and 2 mM (Heineke et al., 1991;
Igamberdiev and Gardestrom, 2003; Szal et al., 2008). These values are in the
range of intracellular concentrations measured in other organisms such as yeast
(Bogan and Brenner, 2008). Concentrations measured in isolated intact chloroplasts are also similar (Takahama et al., 1981). Based on data obtained for
TABLE II
Pyridine Nucleotide Concentrations (mM) in Three Compartments of Plant Cells in the Light and the Dark (n.m., not measured; n.d., not detected)
Compartment
Chloroplast (stroma)
Mitochondria
Cytosol
Condition
Light
Light
Dark
Light
Light
Dark
Light
Light
Dark
NAD
0.21
0.19
0.92
0.68
1.55
1.52
0.72
0.52
0.57
NADH
0.05
n.d.
n.d.
1.76
0.46
0.08
0.09
0.06
0.02
Total NAD
0.26
0.19
0.92
2.44
2.01
1.60
0.81
0.58
0.59
NADP
n.m.
0.59
0.51
n.m.
0.08
0.27
n.m.
0.14
0.15
NADPH
n.m.
0.29
0.12
n.m.
0.24
0.05
n.m.
0.18
0.17
Total NADP
Reference
n.m.
0.88
0.63
n.m.
0.32
0.32
n.m.
0.32
0.32
c
a
a
c
b
b
c
b
b
Data from Heineke et al. (1991); chloroplasts obtained by non-aqueous fractionation of spinach leaves.
Data from Igamberdiev and Gardestrom (2003); rapid fractionation of pea protoplasts by filtration. Light values are for protoplasts incubated in limiting
CO2.
c
Data from Szal et al. (2008); rapid fractionation of cucumber protoplasts by filtration. The concentrations shown were calculated from values given in nmol mg 1
chlorophyll assuming subcellular volumes reported for spinach leaves (Winter et al., 1994).
b
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G. NOCTOR ET AL.
three compartments in leaves or leaf cell protoplasts, NAD concentrations are

highest in the mitochondria and lowest in the chloroplast (Table II). However,
given the relative contributions of the different compartments to the intracellular volume (in leaf mesophyll cells, cytosol volume > 6 total mitochondrial
volume: Queval et al., 2011; Winter et al., 1994), the cytosolic pool would make
the major contribution to overall leaf mesophyll cell NAD contents.
NADP concentrations are about twice as high in the chloroplast as in the
other two compartments (Table II). Interestingly, the mitochondrial NADP
redox state is quite strongly affected by light (Igamberdiev and Gardestrom,
2003). Mitochondrial NAD pools are more reduced than either cytosolic or
chloroplastic NAD pools. However, this is less apparent when photorespiration is not active, for example, in the dark or at high CO2 (Igamberdiev and
Gardestrom, 2003). A highly oxidized cytosolic NAD pool is also observed in
some animal cells and is probably important to drive the GAPDH reaction in
the glycolytic direction (Heineke et al., 1991). A large cytosolic NAD pool
explains the redox state of global pools extracted from whole tissues, only 5
20% of which is NADH even in samples taken in the light (see Fig. 4). Light
dark changes in chloroplast NAD may reflect the activity of light-activated
stromal NADK (Heineke et al., 1991).
B. SUBCELLULAR TRANSPORT
Reduced and oxidized forms of pyridine nucleotides do not cross most

biological membranes at high rates. Instead, shuttle systems allow redox
equivalents to be transferred between subcellular compartments through
fast exchange of metabolites such as malate and oxaloacetate or triose
phosphate and 3-PGA (Day and Wiskich, 1981; Heineke et al., 1991;
Journet et al., 1981; Reumann et al., 1994). These systems allow the chloroplast stromal and mitochondrial matrix to be redox-coupled to the cytosol
and, potentially therefore, to each other (Foyer et al., 2009; Raghavendra
and Padmasree, 2003; Scheibe et al., 2005). In animal and yeast mitochondria, a glycerol-3-phosphate dehydrogenase (G3PDH) located in the inner
mitochondrial membrane has long been known to participate in the oxidation of cytosolic redox equivalents. More recently, the enzymes involved in
this shuttle have been studied in Arabidopsis (Shen et al., 2003, 2006). As in
other organisms, the catalytic site of the mitochondrial G3PDH is oriented
towards the outer surface. Since most small molecules traverse the outer
mitochondrial membrane quite freely, the enzyme would act to oxidize
cytosolic NADH in a similar though more indirect manner than the externally oriented inner membrane NADH dehydrogenase activity (Douce and
Neuburger, 1989; Rasmusson et al., 2004). The mitochondrial G3PDH could
be particularly important in oxidizing glycerol produced from lipid reserves
171
in germinating seeds and young seedlings (Shen et al., 2003). Observations in

knockout mutants for cytosolic G3PDH (which is likely to produce some
of the G3P for mitochondrial oxidation) suggest that a shuttle similar to
those occurring in other organisms is required for redox homeostasis in
Arabidopsis (Shen et al., 2006).
Despite the existence of these high-flux redox exchangers, current knowledge of NAD(P) synthesis implies that lower capacity transporters must
operate to allow entry of nucleotides themselves into organelles. Indeed,
slow uptake of NAD has been observed in purified mitochondria
(Neuburger and Douce, 1983; Neuburger et al., 1985). Recently, the first
specific proteins responsible for NAD transport across plastid and mitochondrial membranes have been identified and studied. Two types of proteins that are competent in transmembrane nucleotide transport via exchange
mechanisms have been identified. First, nucleotide transporter-type (NTT)
carriers are found in plastids, as well as in bacteria that obtain their nucleotides from the extracellular milieu such as a host cell. They catalyse phosphate-neutral reactions involving ATP versus ADP plus phosphate or, in
some cases, NAD versus ADP (Haferkamp et al., 2004; Trentmann et al.,
2008). However, the three Arabidopsis NTTs do not transport NAD
(Palmieri et al., 2009). The second type belongs to the mitochondrial carrier
family (MCF). Despite the nomenclature, it is now clear that these transporters are also found in other intracellular membranes such as those bounding
the plastids and peroxisomes (Palmieri et al., 2009). Based on homology to an
MCF yeast gene encoding the first identified mitochondrial NAD carrier
(Todisco et al., 2006), the Arabidopsis proteins AtNDT1 and AtNDT2 were
shown to be competent in NAD transport and to localize, respectively, to
chloroplasts and mitochondria (Palmieri et al., 2009). Their KM values for
NAD were 0.150.24 mM, suggesting that they would work effectively at
typical intracellular substrate concentrations even if, as discussed above, the
free concentrations were somewhat lower than those shown in Table II.
Less information is available on transport of pyridine mononucleotides in
plants. Both AtNDT1 and AtNDT2 were shown to exchange NAD against
NaMN, NMN, or NaAD as well as other nucleotides such as AMP or ADP
but not nicotinamide or nicotinate (Palmieri et al., 2009). Indeed, evidence
has been presented that NMN and (at lower rates) NaMN can be taken up
and converted to NAD by mitochondria isolated from Jerusalem artichoke
tubers (Di Martino and Pallotta, 2011). Although yeast contain an NADHpreferring NADK that is localized in the mitochondria (Strand et al., 2003),
the Arabidopsis NADHK (NADK3) has been localized to peroxisomes
(Waller et al., 2010). Uptake of NAD for the peroxisomal NAD(H) kinase
(and other NAD-dependent functions) are still to be described. A mitochondrial (and chloroplast) NADP transporter may await discovery: neither of
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G. NOCTOR ET AL.
the two recently described NDTs showed significant exchange of NADP or

NADPH against NAD (Palmieri et al., 2009). Comparison of NAD turnover rates in nucleated and enucleated mammalian cells suggests that a
substantial proportion of breakdown and recycling of NAD may take
place in the nucleus (Imai, 2009). Whether this is the case in plants remains
unclear. Likewise, the extent to which the nuclear and cytosolic NAD pools
are in equilibrium remains to be established.
C. PLASTICITY OF TISSUE NAD(H) CONTENTS
As described above, there are significant differences in both NAD concentrations and redox states between different cellular compartments. However,
perhaps because the key roles of pyridine nucleotides imply careful control of
NAD(H) (nmol.g-1FW)
Tobacco leaves
Nitrogen nutrition
40
B
100
30
75
20
50
10
25
Complex I mutant
0
Low N
High N
WT
CMSII
Arabidopsis leaves
Flowering/senescence
NAD(H) (nmol.g-1FW)
C
60
D
60
45
45
30
30
15
15
Intracellular H2O2
0
41 d
62 d
Col-0
cat2
Fig. 4. Response of NAD(H) pools to environmental and developmental factors.

(A) Effect of N nutrition on NAD contents. N. sylvestris plants grown at low N
(0.1 mM NH4NO3) or high N (5 mM NH4NO3). Data are redrawn from Hager et al.
(2010). (B) Increases in NAD(H) in tobacco mitochondrial mutants lacking complex I
(CMSII; Dutilleul et al., 2005). (C) Changes in leaf NAD(H) during Arabidopsis
rosette development. Data are redrawn from Queval and Noctor (2007). (D) NAD
levels in oxidative stress conditions. The data show contents in an Arabidopsis
catalase-deficient oxidative stress mutant (cat2) and the corresponding wild-type
plants (Col-0).
173
their status, there has been little focus on whether these pools are modified by
environmental or other factors. Most of the attention has been paid to flexibility
or stability of redox states (reduced:oxidized ratios) in response to short-term
changes in metabolic conditions (e.g. Igamberdiev and Gardestrom, 2003;
Kasimova et al., 2006). The recognition of the potential importance of NAD
as a cleavable substrate begs the question of the flexibility of tissue contents.
How fixed are NAD pools in plants? A better understanding of this issue could
be relevant both to plant biology and to human nutrition.
Indications of the flexibility of pyridine nucleotide pools have come from
the analysis of mutants lacking dehydrogenases that consume NADH. In an
Arabidopsis mutant lacking cytosolic G3PDH, an appreciable increase in the
leaf NADH:NAD ratio was accompanied by changes in the antioxidative
system. In this case, the overall NAD pool size was the same as in wild-type
plants (Shen et al., 2006). The Nicotiana sylvestris CMSII mutant, which
lacks complex I activity (Gutierres et al., 1997; Pineau et al., 2005), has
proved to a very useful genetic system for analyzing in vivo responses to
perturbation of the mitochondrial electron transport chain. This mutant
respires through alternative NADH-oxidizing activities (Sabar et al., 2000)
and shows multiple effects on leaf metabolism, including perturbed photosynthesis, enhanced nitrogen assimilation and nitrogen-containing compounds, altered redox homeostasis, and modified responses to the
environment (Dutilleul et al., 2003a,b, 2005; Hager et al., 2010; Pellny
et al., 2008; Priault et al., 2006). Among the biochemical consequences of
the shift from complex I to alternative dehydrogenase activity are increases in
leaf contents of both NAD and NADH (Dutilleul et al., 2005; Hager et al.,
2010; Szal et al., 2008). Increases in NADH may be caused by the lower
substrate affinity of the alternative dehydrogenase compared to complex I
(Rasmusson et al., 2004). Strikingly, the NADH:NAD ratio was little or
not affected by the CMSII mutation, as both forms were increased to a
similar extent (Fig. 4B). Enhanced total NAD in response to a potential
over-reduction may reflect the importance of avoiding inhibition of flux
through glycolysis and the TCA cycle. However, there was no overall increase in NAD in the G3PDH mutants (Shen et al., 2006). Alongside NAD,
adenylates were also substantially increased in the CMSII line (Szal et al.,
2008). The flexibility of NAD contents in response to mutations has also been
revealed by studies of the old5 mutant, in which an increase in recycling
capacity acts to maintain and even increase the pool when de novo synthesis is
perturbed (Schippers et al., 2008).
Changes in NAD contents are not confined to mutants but are also
affected by development and the environment. In Arabidopsis leaves, NAD
contents can change throughout rosette development (Queval and Noctor,
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G. NOCTOR ET AL.
2007), with lower amounts in senescing leaves than in younger ones (Fig. 4C).
This may reflect senescence-linked changes in nitrogen nutrition and also,
perhaps, oxidative stress (Zimmermann et al., 2006). Tobacco plants grown
on abundant N have higher NAD and NADH contents than N-limited
plants (Fig. 4A). Since the NAD pyridine and purine rings contain a total of
seven N atoms (Fig. 1), this could be part of a two-way regulatory loop
between NAD synthesis and N assimilation (Hager et al., 2010). Oxidative
stress in a catalase-deficient Arabidopsis mutant also influences NAD contents to some extent (Fig. 4D). Increases in total NAD have also been linked
to oxidative stress resulting from loss of NADP-linked dehydrogenase function in yeast (Minard and McAlister-Henn, 2010).
V. PLANT PATHWAYS OF NAD SYNTHESIS

AND RECYCLING
Studies on the synthesis and metabolism of pyridine nucleotides and related
compounds, such as nicotine, ricinin, and trigonelline, have appeared over
the years (Frost et al., 1967; Mann and Byerrum, 1974a,b; Ryrie and Scott,
1969; Sinclair et al., 2000; Wagner et al., 1986; Waller et al., 1966). Annotations of plant genes based on homologies to genes in other organisms have
been performed only recently, as genomic information has become available
(Hunt et al., 2004; Katoh and Hashimoto, 2004). This advance has been
accompanied by reverse genetics studies to analyse the function of the
identified genes and detailed labelling studies to identify metabolic pathways
and intermediates. Together, this information has opened up new possibilities for understanding NAD synthesis and its regulation and has cleared the
way for rationally designed manipulation of this part of plant metabolism.
It has become clear that NAD synthesis and recycling pathways are
complex, with considerable variation between taxonomically different organisms. Similar to other nucleotides, the synthesis of NAD is divided into two
types of pathways: (1) de novo synthesis and (2) recycling routes that salvage
the pyridine ring released by NAD cleavage. To date, two de novo pathways
are well characterized while several recycling pathways have been described.
A. DE NOVO PRODUCTION OF NAMN IN BACTERIA AND PLANTS
The de novo pathway involves the production of NaMN from the pyridine
precursor, quinolinate. This compound is produced from aspartate or from
tryptophan. While many eukaryotes and even some prokaryotes have the
tryptophan pathway (Kurnasov et al., 2003), evidence that this pathway
175
operates in plants is limited. No predicted gene sequences are found in Arabidopsis, though they have been reported in rice (Katoh and Hashimoto, 2004;
Katoh et al., 2006). This observation, together with conflicting results obtained
from earlier labelling studies conducted in species such as tobacco and maize
(Arditti and Tarr, 1979; Henderson et al., 1959; Tarr and Arditti, 1982), may
possibly indicate differences in the de novo pathways of NAD synthesis between
different plant groups (Katoh et al., 2006).
Both rice and Arabidopsis genomes contain sequences that are homologous to the E. coli nadA, nadB, and nadC genes, encoding quinolinate
synthase (QS), L-aspartate oxidase (AO), and QPRT, respectively (Fig. 5).
The first enzyme of the pathway, AO, generates an unstable product, iminoaspartate, through an FAD-dependent reaction using either O2 or fumarate as the electron acceptor, producing either H2O2 or succinate as the
Fig. 5. Pathways of NAD synthesis and recycling in plants. ADPR-X, ADP-ribose

or derivative (see Fig. 3); NADK, NAD kinase; NADS, NAD synthetase; NaMN/
NMNAT, nicotinate mononucleotide/nicotinamide mononucleotide adenylyltransferase; NaPRT, nicotinate phosphoribosyltransferase; PRPP, 5-phosphoribosyl-1pyrophosphate; Ppi, pyrophosphate; QPRT, quinolinate phosphoribosyltransferase.
The names of the bacterial genes are also given for enzymes involved in NAD(P)
synthesis.
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G. NOCTOR ET AL.
reduced by-product (Marinoni et al., 2008). Iminoaspartate is then

condensed with DHAP by the second enzyme, QS, to produce quinolinate.
The presence of these genes confirms the earlier description of AO activity in
plants (Hosokawa et al., 1983).
Studies on E. coli NadA established that this protein was the site of O2
sensitivity. Based on a consensus binding sequence in the polypeptide, it was
proposed that this effect is due to oxidative inactivation of an ironsulphur
cluster (Draczynska-Lusiak and Brown, 1992; Gardner and Fridovich,
1991). Recent studies have confirmed the presence of an [4Fe4S] prosthetic
group (Cicchillo et al., 2005; Ollagnier de Choudens et al., 2005). Only three
of the iron atoms are coordinated to amino acids in the E. coli QS, leaving
one iron free to coordinate the reactants during catalysis (Rousset et al.,
2008; Saunders et al., 2008). Since this is reminiscent of other O2-sensitive
enzymes like aconitase, it may explain the sensitivity of QS to O2 (Rousset
et al., 2008; Saunders et al., 2008).
Single genes encoding homologues of NadA (At5g14760) and NadB
(At5g50210) have been identified in Arabidopsis. Expression of these genes
in the respective E. coli nadA- and nadB- mutants conferred the ability to
grow without exogenous nicotinic acid (Katoh et al., 2006). The protein
products of these genes were shown to be directed to plastids, and T-DNA
mutants to be embryo-lethal (Katoh et al., 2006). Arabidopsis QS consists of
two domains, a synthase domain and a SufE3 domain required for incorporation of the FeS cluster (Murthy et al., 2007). A non-lethal mutation in
the SufE domain is responsible for the early senescence phenotype of the old5
mutant (Schippers et al., 2008). Rather than decreased NAD, this effect was
associated with increased NAD, which was attributed to increased activities
of the recycling pathway in old5 (Schippers et al., 2008).
Quinolinate produced by AO and QS is converted to NaMN by QPRT,
which is homologous to the bacterial nadC gene. Like the first two enzymes,
only a single gene is predicted to encode this enzyme in Arabidopsis and TDNA knockout mutations described so far have been reported to be lethal
(Katoh et al., 2006). Also like the first two enzymes, the encoded protein has
been localized to the plastid (Katoh et al., 2006), suggesting that NaMN
synthesis through the de novo pathway may be restricted to this organelle, at
least in some plants.
B. CONVERSION OF NAMN TO NAD
The conversion of NaMN to NAD requires the final two enzymes of the Preiss
Handler pathway (Fig. 2). These enzymes, NaMN adenylyltransferase (NaMNAT) and NADS, are probably located in the cytosol, and so current concepts
177
suggest that the de novo pathway requires export of NaMN from the chloroplast. Like the enzymes that produce NaMN via quinolinate, NaMNAT is
encoded by a single gene in Arabidopsis (At5g55810) and homozygous mutant
plants cannot be recovered (Hashida et al., 2007). While heterozygote mutants
showed no obvious phenotype during the sporophyte (diploid) generation, the
development of haploid pollen (but not haploid embryo sacs) produced from
the mutant parent plants was perturbed (Hashida et al., 2007). This effect
involved disruption of pollen tube extension elongation, though pollen grain
germination was not affected. This perhaps suggests that NAD synthesis is
required to replenish the initial NAD complement of the pollen grain as this is
consumed during tube elongation (Hashida et al., 2007). NaMNAT has also
been implicated in guard cell responses in Arabidopsis (Hashida et al., 2010a).
Like the human enzymes, the Arabidopsis enzyme can use either NMN or
NaMN (Hashida et al., 2007).
The final enzyme of NAD synthesis from NaMN, NADS, is predicted also
to be encoded by a single gene in Arabidopsis (At1g55090). No detailed
studies of mutants have yet appeared to establish whether this gene is
indispensable or not. In the pathogenic bacterium Francisella tularensis, a
novel enzyme, named NMN synthetase, has recently been discovered that
can amidate NaMN to NMN (Sorci et al., 2009). In this case, in contrast to
the PreissHandler pathway (Fig. 2), the amidation step comes before the
adenylation step. Unlike NADS, NMN synthetase shows preference for the
mononucleotide NaMN over the dinucleotide NaAD while the F. tularensis
adenylyltransferase prefers NMN to NaMN (Sorci et al., 2009). Given the
uncertainties in plants over the terminal reactions of NAD synthesis, further
work might reveal whether any species- or tissue-specificity exists in the use
of NMN or NaMN to make NAD.
C. RECYCLING PATHWAYS
The well-characterized bacterial recycling pathway depends on nicotinamidase to produce nicotinic acid from nicotinamide formed by the activities
shown in Fig. 3 (right). This pathway clearly exists in plants, and nicotinamidase activity has been measured in several plant species (Katahira and
Ashihara, 2009; Schippers et al., 2008). Three Arabidopsis genes have been
shown to encode proteins with nicotinamidase activity and have been designated NIC1, NIC2, and NIC3 (Hunt et al., 2007; Wang and Pichersky, 2007).
The major nicotinamidase appears to be NIC1, and insertion mutants show
decreased pools of pyridine nucleotides in many tissues, pointing to a role for
the recycling pathway in maintaining NAD pools (Wang and Pichersky,
2007). The NIC2 gene is most strongly expressed in seeds: knockout mutants
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G. NOCTOR ET AL.
show normal vegetative growth but germination in the absence of seed

stratification was decreased (Hunt et al., 2007). Decreased germination was
not linked to changes in ABA but the observed increase in NAD contents
could reflect inhibition of PARP by accumulated nicotinamide. However,
not only nicotinamide but also NAD or NADP (but not NADH or
NADPH) was shown to inhibit seed germination (Hunt et al., 2007).
Conversion of nicotinate to NaMN occurs via NaPRT (Fig. 5). Two genes
encode this enzyme in Arabidopsis (Hunt et al., 2004). As yet, there is little
information on mutants for these genes, so it is not clear whether they are
essential or have any redundancy. NaPRT activity can be detected in potato
tubers (Katahira and Ashihara, 2009). Mammals lack nicotinamidase (although this enzyme is present in gut bacteria) and directly convert nicotinamide to NMN through a nicotinamide phosphoribosyltransferase (NPRT;
Fig. 2). However, there is no evidence for NPRT activity in plants (Katahira
and Ashihara, 2009; Wang and Pichersky, 2007; Zheng et al., 2005). Thus, the
dual activity of plant NaMN/NMNAT may be required to adenylate NaMN
produced by QPRT (de novo pathway), NaMN produced by NaPRT (recycling pathway from nicotinamide) and NMN generated by other sources.
NMN could be metabolized to nicotinamide prior to recycling to NAD
(Ashihara et al., 2005, 2008) or to NaMN via a nicotinamide-independent
pathway (Katahira and Ashihara, 2009). In potato tubers, no enzymes were
detected that were able to deamidate NMN to NaMN or convert NMN to
NPRT directly (Katahira and Ashihara, 2009). While the same study reported
clear labelling of NAD(P) from [3H]-quinolinate, the most heavily labelled
pool was nicotinate glycoside (NaG). Based on detection of the required
enzymes, it was suggested that NaG originates from a two-step degradation
of NaMN, and that it may be important as a temporary storage product. In
potato leaf discs the major products from [3H]-quinolinate were trigonelline
and, at a somewhat lower level, NAD(P) (Katahira and Ashihara, 2009).
Other reactions have been described, and significant inter-species differences in NAD recycling pathways appear to exist. For example, nicotinate
riboside kinase activity has been found in both mung bean seedlings and
potato tubers, providing an alternative route to NaMN (Katahira and
Ashihara, 2009; Matsui and Ashihara, 2008). A recent study reported that
isolated Jerusalem artichoke mitochondria were able to convert NMN to
NAD, though the enzyme responsible for the activity remains to be characterized (Di Martino and Pallotta, 2011).
D. REGULATION OF NAD SYNTHESIS
Measurements of extractable enzyme activities in rice seedlings, Arabidopsis,

and potato suggest that QPRT, NaMNAT, and NADS capacities are quite
similar (Hayashi et al., 2005; Katahira and Ashihara, 2009; Schippers et al.,
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2008). By comparison, a recent study of potato tubers reported that NaPRT

activity was 60-fold lower than that of NaMNAT (Katahira and Ashihara,
2009). Except in tissues producing ricinin or nicotine, NaPRT and QPRT
activities were within the same order of magnitude, though both were lower
than nicotinamidase (Mann and Byerrum, 1974b). The notion that phosphoribosyltransferases are important regulatory steps in the recycling pathway is consistent with information on the mammalian and bacterial
pathways, where NPRT or NaPRT activities are considered the rate-limiting
steps in NAD synthesis from nicotinamide and nicotinic acid, respectively
(Imai, 2009). Phosphoribosyltransferases use phosphoribosylpyrophosphate
(PRPP) as the ribose-phosphate donor. Interestingly, overexpression of
PRPP synthetase promoted biomass accumulation in both Arabidopsis and
tobacco (Koslowsky et al., 2008). Whether this effect is linked to synthesis of
NAD or other PRPP-requiring compounds is not yet clear.
Labelling studies are also consistent with nicotinamidase not being a
limiting enzyme in NAD recycling. In canola embryos and potato tuber
slices, exogenous nicotinamide is rapidly converted to nicotinic acid
(Ashihara et al., 2008; Katahira and Ashihara, 2009), consistent with high
nicotinamidase activity, which was more than 10-fold higher than NaPRT in
the potato tubers (Katahira and Ashihara, 2009). Studies of mutants show
that nicotinamidase is necessary for full maintenance of NAD pools (Hunt
et al., 2007; Wang and Pichersky, 2007) although whether this enzyme plays
an important regulatory role in wild-type plants remains unclear. Increased
NIC1 expression and nicotinamidase activity was part of the response of old5
mutants to perturbed activity of the de novo pathway (Schippers et al., 2008).
If allowed to accumulate, nicotinamide itself may play influential roles. It is a
competitive inhibitor of NAD-cleaving enzymes in yeast and mammals
(Bogan and Brenner, 2008) and has been shown to affect stress responses in
plants (Berglund et al., 1993, 1996). Studies in other organisms clearly point
to an important role for nicotinamidase (PNC1; Fig. 5). Increased copy
number of PNC1 affects ageing in yeast (Anderson et al., 2003) while in the
worm, Caenorhabditis elegans, mutations in PNC1 affect reproductive development (Vrablik et al., 2009).
NADS activity was increased in rice overexpressing a detoxifying enzyme
with NADPH-dihydroflavonol reductase activity (Hayashi et al., 2005) and
in the old5 Arabidopsis mutant (Schippers et al., 2008). In contrast, QPRT
was not affected by the old5 mutation (Schippers et al., 2008). This latter
observation might reflect specific stimulation of the recycling pathway in
conditions where the supply of quinolinate to QPRT by QS is impaired.
NAD synthesis genes have been reported to be strongly expressed in guard
cells, and NaMNAT proposed to be a regulator of ABA-induced changes in
NAD (Hashida et al., 2010a). A preliminary report suggests that overexpression of the NADS is not sufficient to increase NAD levels (Hashida et al.,
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2010b), consistent with the notion that enzymes earlier in the pathway are
more strongly limiting.
Little information is available on the capacities of the first two enzymes of
the de novo pathway, although both have been detected in plant extracts
(Hosokawa et al., 1983; Schippers et al., 2008). It seems reasonable to assume
that their capacities are significantly below those of the subsequent steps.
Indeed, the notion that quinolinate production is limiting for de novo NAD
synthesis is consistent with our recent data (see Section VI). The bacterial
NadA and NadB may function together as a complex. While there are several
arguments in favour of this notion, such as the instability of the product,
iminoaspartate, a physical association has not yet been conclusively demonstrated (Marinoni et al., 2008). Quinolinate production may be light-regulated
to some extent. Two vicinal cysteines in NadA (QS) that do not participate in
coordinating the FeS cluster were found to interact with thioredoxin (TRX),
suggesting they can form a disulfide bond (Rousset et al., 2008). An independent study reported that the enzyme activity is stimulated by disulfide bond
formation, that the activation/inactivation could be achieved by oxidized and
reduced TRX, respectively, and that mutated TRX with a single active site
cysteine formed a stable complex with NadA (Saunders and Booker, 2008).
While the importance of this potential regulatory mechanism remains to be
established in plants, it could have parallels with the oxidative activation of
chloroplast enzymes involved in cysteine and glutathione synthesis under
conditions of oxidative stress (Bick et al., 2001; Hothorn et al., 2006). Oxidative activation of NAD synthesis may be favoured under conditions in which
NAD is being consumed by stress-related reactions.
While nicotinamide is known to inhibit several NAD-cleaving enzymes,
NaMN and NAD have been reported to feedback-inhibit bacterial NaPRT
(Dulyaninova et al., 1998). In humans, however, NPRT but not NaPRT was
inhibited by NAD (Hara et al., 2007). Quinolinate may regulate NADK
activity in some bacteria (Garavaglia et al., 2003). In plants, analysis of
QPRT purified from castor bean endosperm revealed little or no inhibition
by nicotinate or NAD (Mann and Byerrum, 1974a). Now that plant genes
have been identified for the de novo and recycling pathways, analysis of the
purified recombinant enzymes should throw more light on their regulation.
VI. MANIPULATING NAD CONTENTS IN PLANTS

From a physiological point of view, it might be predicted that the de novo
pathway will be important in young tissues undergoing rapid growth with the
recycling pathway more influential in mature tissues, being solicited, in
181
particular, during increased use of NAD, for example, in stress conditions.

However, our knowledge of the interplay between the two pathways is still
limited. As Fig. 4 shows, leaf NAD contents are affected by environmental,
developmental, or genetic factors, though the variation is not much greater
than twofold. Our previous analyses established that increases of this order in
mitochondrial complex I mutants are associated with marked effects on
metabolism (Dutilleul et al., 2005). These plants also show enhanced stress
resistance, including resistance to pathogens (Dutilleul et al., 2003b). More
recently, a good correlation was observed between leaf NAD and leaf amino
acid contents, indicating tight coordination of pyridine nucleotide and plant
nitrogen status (Hager et al., 2010). To investigate the contribution of modified NAD status to these effects, we developed a project to enhance this status
in a targeted fashion. This section discusses initial results from this study.
A. NAD ENRICHMENT IN BACTERIA AND HUMAN CELLS
E. coli strains carrying additional copies of the nadA or nadB genes showed a
slight increase in quinolinate production, which was much greater when both
genes were present in higher copy number (Flachmann et al., 1988). Overexpression of the E. coli NaPRT produced a 25-fold increase in extractable
enzyme activity, but substantial increases in NAD were dependent on external supply of nicotinate, which enhanced NAD fivefold over control levels
(Wubbolts et al., 1990). Overexpression of a deregulated NAPRT in two
independent studies also increased NAD levels 1.5- to 2-fold (Berros-Rivera
et al., 2002; Heuser et al., 2007). Increases in NAD of up to sevenfold were
achieved by manipulation of multiple genes in E. coli, including inactivation
of two NUDIX hydrolase genes and overexpression of NaPRT (pncB) and
NADS (nadE; Heuser et al., 2007).
Work on mammalian cells offers a complex picture, possibly due to cellspecific differences in NAD metabolism (Bogan and Brenner, 2008). Most
studies have shown that nicotinate is more effective than nicotinamide in
enhancing cellular NAD (Hara et al., 2007; Micheli and Sestini, 1997).
However, a study on human liver tumour cells reported that NAD was
most strongly increased by nicotinamide with quinolinate being ineffective
in increasing NAD (Evans et al., 2002).
B. ENRICHING TISSUE NAD IN PLANTS
As a first step to testing the feasibility of manipulating NAD contents in

plants, we transformed Arabidopsis plants with bacterial nadB, nadA, or
nadC genes encoding the first three enzymes of the de novo synthesis pathway,
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G. NOCTOR ET AL.
or pncB, which produces NaMN in the recycling pathway (Fig. 5; Flachmann

et al., 1988; Wubbolts et al., 1990). Heterologous expression of bacterial
genes encoding enzymes already present in plants has been used for several
years to enhance compounds such as specific amino acids or glutathione
(Noctor et al., 1996, 1998; Shaul and Galili, 1992). An advantage of this
strategy is that the introduced gene is often not subject to strict control over
expression, allowing enzyme capacity to be increased manyfold. Similarly,
the introduced protein may be less sensitive to biochemical controls over its
activity than the endogenous enzyme. On the other hand, this approach may
be limited if the endogenous enzyme has requirements for its activity that the
introduced protein is unable to fulfil. One obvious example is a difference in
structure that affects interactions with other proteins. Because our aim was
to increase NAD in the compartment where it is probably formed in plants,
all expression cassettes were constructed for cytosolic expression.
After verification of transgene insertion and selection of homozygotes
through classical techniques, independent lines with good expression at the
level of transcript abundance were chosen for analysis of NAD contents.
Despite apparently strong expression of the transgenes, none of the lines
showed marked increases in NAD. However, when leaf discs from the nadC
overexpressors, with genetically enhanced capacity for QPRT, were given the
substrate, quinolinate (Fig. 6A), increases in leaf NAD of up to 10-fold could
be observed within 48 h. This system therefore allows inducible, marked
increases in NAD contents to be readily achieved. The results of a typical
experiment, shown in Fig. 6B, reveal that this dramatic enhancement of
NAD was associated with a smaller relative increase in NADP, presumably
due to stimulated NADK activity. The reduction states of both pools were
much less affected than their overall contents. While the NAD pool remained
highly oxidized, with NADH representing about 5% of the leaf pool,
NADPH and NADP were close to 50% reduced in all genotypes
(Fig. 6B). Supplying quinolinate to untransformed control plants had only
a slight effect on pyridine nucleotide pools.
These results suggest that the de novo path of NAD synthesis in plants is colimited by QPRT activity and quinolinate because simultaneous increases in
both are required to produce substantial increases in NAD. However, subcellular compartmentation could be a key factor. In our nadC transformants,
QPRT capacity is increased in the cytosol, whereas the de novo plant pathway
produces quinolinate in the plastid. Assuming that the plant QPRT is restricted to the same compartment as the first two enzymes (Katoh et al., 2006), there
would be no physiological requirement for quinolinate to cross the chloroplast envelope to allow NAD synthesis. However, our results clearly imply
that quinolinate can be taken up from the external milieu into the cytosol.
Fig. 6. Changes in gene expression triggered by targeted manipulation of endogenous NAD. Leaf discs from Arabidopsis Col-0 plants
transformed with a construct directing the product of the bacterial nadC gene to the cytosol (nadC-1, nadC-2) were incubated on buffer
containing quinolinate. Samples were harvested after 24-h incubation in the dark. Pyridine nucleotides were quantified as in Queval and
Noctor (2007) and parallel samples were used for microarray analysis with the CATMA arrays and approach reported in Mhamdi et al.
(2010b). Briefly, RNA from two biological replicates (leaf discs of two independent transformed lines supplied with quinolinate) were
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G. NOCTOR ET AL.
The failure of expression of NadB or NadC to increase NAD may be

explained either by co-limitation or by a need for functional association
between the proteins so that the unstable iminoaspartate can be efficiently
channelled from the first enzyme to the second. Substrate limitation in the
cytosol is unlikely to be an explanation. The KM values of the bacterial AO
(NadB) for aspartate and QS (NadA) for DHAP have been measured to be
11.7 and 0.36 mM, respectively (Marinoni et al., 2008), while the Arabidopsis QS has a KM for DHAP of 1.1 mM (Murthy et al., 2007). Aspartate
is quite an abundant amino acid, while high triose phosphate isomerase
activity keeps DHAP at substantial levels in cells. Both metabolites are
central to primary plant carbon and nitrogen metabolism. In barley and
spinach leaves, their cytosolic concentrations have been estimated to
be at least as high or higher than stromal concentrations (Winter et al.,
1993, 1994).
Homologous overexpression of the plant AO in the chloroplast could be
more successful in producing plants with constitutive increases in NAD, but
based on studies in bacteria, it seems unlikely that this alone would suffice to
produce the enrichment we report in Fig. 6B. More likely is that such marked
constitutive increases would require simultaneous overexpression of the first
three enzymes of the de novo pathway. We are currently exploring this
possibility. It will be interesting to see whether sustainable increases are
possible or whether degrading pathways would be up-regulated to
dye-swapped twice with control RNA samples from Arabidopsis transformed with an
empty vector. Only genes that showed significant same-direction changes in all four
analyses are shown. (A) Simple scheme showing the experimental strategy. (B) Leaf
NAD(H) and NADP(H) contents in the empty vector controls (Con) and lines
expressing nadC supplied with quinolinate (nadC-1 and nadC-2). White blocks,
NAD or NADP. Black blocks, NADH or NADPH. Similar results were obtained
in other independent experiments. (C) Overview of genes that were significantly
different between nadC lines supplied with quinolinate and the controls. Only
genes that were significant in all four analyses are shown. The colour bar at top
indicates expression fold change relative to controls. A full set of the data can be
consulted at http://urgv.evry.inra.fr/cgi-bin/projects/CATdb/consult_project.pl?
project_id161. (D) Comparison of the responses of the significant genes in (C)
with their expression in Arabidopsis mutants with targeted manipulation of glutathione or H2O2. Top pink frame, induced genes in nadC lines plus quinolinate. Bottom
green frame, repressed genes in nadC lines plus quinolinate. Expression of the 163
genes was analysed in T-DNA mutants deficient in glutathione reductase (gr1),
catalase (cat2), or both (cat2 gr1) (Mhamdi et al., 2010b). Red bars show induced
genes, green bars show repressed genes. For each bar, numbers indicate how many
of the induced or repressed genes in (C) were found to be significant in each line.
185
compensate for the enhanced synthetic capacity. Another interesting question is whether pyridine nucleotide redox states and associated metabolism
would be strongly affected in such lines.
C. INDUCIBLE INCREASES IN NAD(H) ARE ASSOCIATED WITH ACTIVATION

OF PATHOGENESIS-RELATED PATHWAYS
To provide an initial assessment of the impact of engineering increases in

NAD on plant cell function, we conducted microarray analyses of RNA
isolated from the leaf discs accumulating high levels of NAD. At least at the
level of gene expression, there was no generalized perturbation of genes
involved in primary metabolism. Indeed, the transcriptomic signature
involved only a relatively small fraction of the genome (Fig. 6C). A gene
encoding NR was down-regulated, consistent with links that have been noted
between nitrate assimilation and NAD status (Dutilleul et al., 2005; Hager
et al., 2010; Kaiser et al., 2000, 2002; Rachmilevitch et al., 2004). However,
the most striking pattern emerging from the analysis was the effect on genes
known to be involved in phytohormone-mediated pathogenesis responses
(Table III). Among the most strongly induced genes were several associated
with SA synthesis and signalling (ICS1, FMO1, PAD4, BAP2; Bartsch et al.,
2006; Wildermuth et al., 2001). Repressed genes included wounding and
jasmonic acid (JA)-responsive genes (Table III). As SAJA antagonism is
well known to be part of plant responses to biotic stress (e.g. Koornneef
et al., 2008), our results suggest that increases in intracellular pyridine
nucleotides are sufficient to entrain some of these responses.
Changes in redox state are an integral part of plantpathogen interactions
(Chamnongpol et al., 1998; Vanacker et al., 2000). Thiol-sensitive components such as NPR1 are particularly important (Mou et al., 2003; Tada et al.,
2008). Recently, it was shown that infiltration of Arabidopsis leaves with
physiological concentrations of pyridine nucleotides can induce the pathogenesis-related (PR) genes PR1, PR2, and PR5 (Zhang and Mou, 2009). One
obvious potential link between pyridine nucleotide status and pathogenesis
responses is stimulation of NADPH oxidase activities (Torres et al., 2005).
However, induction of PR genes by exogenous pyridine nucleotides was little
or not affected in mutants for two of the main leaf NADPH oxidases and was
suggested to occur through an extracellular mechanism, possibly dependent
on calcium signalling (Zhang and Mou, 2009). Although no PR1 probe-set
was present on the array used in our experiment, we observed no induction of
the SA-associated genes PR2 or PR5. Hence, only a subset of pathogenesisassociated genes appears to be affected by enhanced NAD synthesis, and
TABLE III
Examples of Transcripts Showing Significantly Altered Expression in Plants with Enhanced NAD
Expression levels
NAD
H2O2
REPRESSED (111 genes in total)

At1g08830
CSD1, Cu/Zn superoxide dismutase, cytosol
At2g28190
CSD2, Cu/Zn superoxide dismutase, chloroplast
At1g12520
CCS1, SOD copper chaperone
At1g77760
NIA1, nitrate reductase1
At2g22500
DIC1/PUMP5, dicarboxylate carrier 1, plant uncoupling mitochondrial protein 5
At1g17420
LOX3, lipoxygenase3
At1g72520
Putative lipoxygenase
At2g34600
JAZ7, jasmonate/ZIM domain 7
At5g53750
Early wounding-induced jasmonic acid-dependent gene
At5g47230
AtEFR5, ethylene-response element binding factor 5
At2g44840
AtERF13, ethylene-response element binding factor 13
At3g02910
AVIG-like, avirulence-induced gene-like
At1g65390
Phloem protein 2A-5 (PP2A-5), innate immune response
1.16
1.50
1.00
1.71
1.57
1.99
1.89
1.14
1.64
1.06
1.39
1.03
1.11
Induced
Induced
Repressed
Repressed
Repressed
Repressed
Repressed
INDUCED (62 genes in total)

At1g74710
ICS1, isochorismate synthase1, salicylic acid synthesis
At1g33960
AIG1 (AVRRPT2-INDUCED GENE 1)
At1g19250
FMO1, flavin-dependent monoxygenase 1
At2g45760
BAP2 (BON ASSOCIATION PROTEIN 2), negative regulator of PCD
At3g52430
PAD4 (PHYTOALEXIN DEFICIENT 4)
At1g31580
ECS1, cell wall protein, defence response
At4g25100
FSD1 (FE SUPEROXIDE DISMUTASE 1), chloroplast
At3g20340
Paraquat-repressed gene
At3g50470
Hypersensitive response protein 3 (HR3)
2.68
2.55
1.84
1.20
1.48
2.24
1.50
1.19
1.13
Repressed
AGI code
Annotation
AGI, Arabidopsis gene identifier code. Expression levels are given as log2 treatment/control ratio (0 no expression change). Significant induction or repression
by short-term exposure to intracellular H2O2 in a catalase-deficient mutant (Mhamdi et al., 2010b) is indicated for comparison. For experimental details, see
legend of Fig. 6.
187
these are not necessarily those whose expression is modified by exogenous

pyridine nucleotides (Zhang and Mou, 2009). Further work is required to
elucidate this point.
D. LINKS BETWEEN NAD, ROS, AND THIOL STATUS?
Enhanced NAD or NADH could affect redox homeostasis through a

number of mechanisms. As well as affecting NADPH oxidases, increased
NADPH could modify thiol status by altering glutathione reductase (GR) or
TRX reductase activities. To assess whether the transcriptome changes we
report were linked to changes in intracellular oxidative stress or thiol-disulfide status, we first compared the microarray data with results recently
obtained using the same array in plants deficient in catalase (cat2) and/or
GR (gr1) (Mhamdi et al., 2010b). The comparison revealed only a partial
overlap, with the greatest overlap observed for double cat2 gr1 mutants
lacking both antioxidative enzymes. Overlap was most evident for JA-associated genes, whose expression has been linked to glutathione status
(Koornneef et al., 2008; Mhamdi et al., 2010b). Partial overlap between
the two studies could reflect the activation of ROS-dependent and ROSindependent signalling pathways by increases in NAD synthesis. Activation
of multiple pathways might be caused by simultaneous increases in NAD,
NADH, NADP, and NADPH (Fig. 6B) as these forms have been shownto impact signalling pathways differentially when supplied exogenously
to Arabidopsis leaves (Zhang and Mou, 2009). Direct analyses of
thiol redox status should throw further light on this question and are
underway.
Current concepts suggest that apoplastic exchange of pyridine nucleotides
between plant cells does not occur at very high rates under most conditions.
However, it has been proposed that these compounds could appear in the
extracellular milieu at significant concentrations when cellular integrity is
breached during pathogen attack or wounding, and that this might function
as an important signal (Zhang and Mou, 2009). Although we cannot exclude
the possibility that enhanced intracellular NAD contents cause increases in
extracellular NAD(P), it is noteworthy that expression of genes associated
with wounding and JA signalling were repressed, not induced, in our experiments (Table III). These genes are known to be affected by the intracellular
redox state (Koornneef et al., 2008; Mhamdi et al., 2010b) and were repressed
in response to both NAD enrichment and increased intracellular H2O2
(Table III). Despite this, it is evident that enhanced NAD does not merely
recapitulate an oxidative stress response (Fig. 6). In some cases, increased
NAD produced an antagonistic effect to those observed in cat2. Genes that
188
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showed this behaviour included two superoxide dismutases and a gene of

unknown function known to be repressed by the superoxide generator,
paraquat (Table III). An important point here could be that different ROS
such as superoxide, singlet O2 and H2O2 produce distinct transcriptomics
signatures in plants (Gadjev et al., 2006). Enhanced NAD and increased
H2O2 also affected the expression of NIA1, encoding an isoform of NR, in
opposite ways. The effects of enhanced NAD on NR expression could be
related to nitric oxide production (Rockel et al., 2002). It is also interesting
that although neither NaPRT was among the significant genes affected by
increased NAD, one of these genes (At2g23420) was among those induced by
oxidative stress in cat2 gr1 (Mhamdi et al., 2010b).
In conclusion, enhanced NAD synthesis triggers induction of specific
genes, a large number of which are associated with pathogenesis responses.
Although some interaction with oxidative stress responses is apparent, a
second major factor may be modification of processes related to the nonredox roles of NAD (Fig. 3). The concept of oxidative stress is a vague one
that is still poorly defined in plants (Foyer and Noctor, 2011). In particular,
the way in which enhanced ROS availability impacts on different redox
couples such as NAD, NADP, and glutathione remains to be elucidated
(Noctor, 2006). Model systems such as the one we describe here, which
allow inducible enhancement of cellular pyridine nucleotide pools, could be
useful in such studies.
VII. CONCLUSIONS AND PERSPECTIVES

Interest in pathways of NAD synthesis, traditionally driven by the vitamin
function of niacin, has been greatly stimulated by the recognition that NAD
plays important roles in cell signalling. Some of these roles involve cleavage
of the NAD molecule and could therefore be influenced by the activity of
NAD synthesis pathways. Several NAD-cleaving reactions, while not themselves involving redox exchange, appear to be tightly intertwined with redox
homeostasis and signalling processes.
Surprising gaps in our knowledge of NAD synthesis and metabolism remain. This is as true in plants as it is in other organisms. While ongoing studies
are likely to plug some of these gaps in knowledge quite quickly, it has become
apparent that NAD metabolism is complex and shows diversity both between
organisms and between different tissues of the same organism. Recent data,
including those shown here, clearly point to potential interactions between
NAD and plant defence signalling. These interactions could involve a number
of processes, including stress-related PARP activity and calcium signalling.
189
Recent analyses of NUDIX hydrolases in plants suggest that these components play critical roles in oxidative stress and pathogenesis responses. The
influence of NAD and NADP availability on NADPH oxidases and other
ROS-producing enzymes also remains to be established, as does the relationship between these nucleotides and intracellular thiols. The availability of
protocols that are able to provide information on the redox state and concentration of individual components in specific compartments will be useful here.
Our data suggest that it is feasible to manipulate the pathway of pyridine
nucleotide synthesis to achieve considerable increases in tissue NAD. A key
development in the systems biology era is the increasing erosion of notions of
compartmentalized housekeeping versus signalling functions, according to
which basic energetic processes were viewed as essential but of little importance in cell signalling functions. This has been replaced by an increasingly
sophisticated appreciation of the integrated, networking nature of cells and
tissues. Will manipulating a factor as central to cell function as NAD(H)
produce desirable effects on plant niacin contents, plant performance or stress
resistance? Can any such positive effects be separated from unwanted side
effects? It should now be possible to begin to answer this and related questions.
ACKNOWLEDGEMENTS
We thank Jean-Pierre Renou (URGV Evry, France) for microarray analyses,
Bertrand Gakie`re (IBP Orsay, France) for discussions and advice, and Fanta
Ouedraogo (IBP Orsay, France) for technical assistance. Funding from the
ANR-Genoplante project Redoxome is gratefully acknowledged by J. H.
and G. N. We are also grateful to the Universite de Paris sud 11 and the
French Embassy in China for funding towards the studies of S. L.
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Pantothenate Biosynthesis in Higher Plants
MICHAEL E. WEBB*,1 AND ALISON G. SMITH{
*School of Chemistry and Astbury Centre for Structural Molecular

Biology, University of Leeds, Leeds, United Kingdom
{
Department of Plant Sciences, University of Cambridge, Cambridge,
United Kingdom
I. Biological Function and Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A. Introduction ....................................................................
B. Biological Function of Pantothenate .......................................
C. Effects of Pantothenate Deficiency .........................................
D. Dietary Absorption and Metabolism in Mammals.......................
II. Distribution in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
III. Biosynthetic Pathway and Location of the Pathway . . . . . . . . . . . . . . . . . . . . . .
A. Ketopantoate Hydroxymethyltransferase .................................
B. Pantothenate Synthetase .....................................................
C. Reduction of Ketopantoate ..................................................
D. Source of -Alanine ...........................................................
IV. Regulation, Turnover, and Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Pantothenate Kinase ..........................................................
B. Phosphopantethenoylcysteine Synthetase..................................
C. Phosphopantethenoylcysteine Decarboxylase.............................
D. 40 -Phosphopantetheine Adenylyltransferase...............................
E. Dephospho-CoA Kinase .....................................................
F. Cellular and Tissue Patterns in Plants......................................
V. Major Differences to Eubacteria and Other Prototrophic Organisms. . . . .
A. Ketopantoate Reductase .....................................................
B. -Alanine Synthesis ...........................................................
C. Differences in the Archaeal Pathway .......................................
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0065-2296/11 $35.00
DOI: 10.1016/B978-0-12-386479-6.00001-9
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VI. Engineering the Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A. Engineering the Pathway in Bacteria .......................................
B. Engineering the Pathway in Plants .........................................
VII. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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ABSTRACT
Pantothenate (vitamin B5) is an essential metabolite for all organisms because it is the
precursor to the 40 -phosphopantetheine moiety of coenzyme A (CoA) and acyl carrier
protein. Pantothenate must be obtained from the diet by animals, but plants, bacteria,
and fungi can synthesise it de novo by the condensation of -alanine with pantoate,
which is synthesised in two steps from -ketoisovalerate, an intermediate in branched
chain amino acid biosynthesis. In plants, the first and the last enzymes in this
pathway, ketopantoate hydroxymethyltransferase and pantothenate synthetase,
have been identified and characterised, but the enzyme responsible for the intermediate step of reduction of ketopantoate to pantoate has not been identified. Similarly,
the source of -alanine for pantothenate biosynthesis in plants has not been established; we suggest that catabolism of the polyamine, spermidine, is the most likely
source of this metabolite. In contrast, all five plant enzymes required to convert
pantothenate into CoA have been identified and characterised. Strains of bacteria
producing increased quantities of pantothenate have been generated by metabolic
engineering, and knowledge of the bacterial pathway has been used in the first
attempts to engineer plants with similarly increased pantothenate production.
I. BIOLOGICAL FUNCTION AND REQUIREMENTS

A. INTRODUCTION
Pantothenate (Fig. 1A), or vitamin B5, is one of the structurally simplest of

the vitamins and is ubiquitous in nature. In all organisms, pantothenate is the
precursor for the 40 -phosphopantetheine moiety of both coenzyme A (CoA)
and the covalently linked phosphopantetheine prosthetic group of acyl carrier proteins (ACPs; Alberts and Vagelos, 1966). These cofactors play a vital
role in central carbohydrate and lipid metabolism, as well as being involved
in many secondary metabolite biosynthetic pathways. Pantothenate was first
identified by Williams et al. (1933) as an essential nutrient for yeast, Saccharomyces cerevisiae. This small molecule was later found to be identical to the
factors subsequently shown by Elvehjem and Koehn (1935) to prevent dermatitis in chicks and by Lythgoe et al. (1940) to be important for rat
nutrition. Hydrolysis of purified pantothenate was shown to produce alanine (Weinstock et al., 1939), and structural analysis of the resulting
pantoyl lactone (Stiller et al., 1940a) led to the determination of the structure
of pantothenate (Williams and Major, 1940) and two subsequent total
PANTOTHENATE BIOSYNTHESIS IN HIGHER PLANTS
Fig. 1.
205
Structure of pantothenate and coenzyme A.
syntheses (Stiller et al., 1940b; Williams et al., 1940). The pathway to pantothenate, via the condensation of pantoate with -alanine, is best characterised in bacteria (Fig. 2; Webb et al., 2004). In this review, we consider in
detail what is known of the synthesis of pantothenate in plants, its uptake
and subsequent conversion to the active cofactors in humans and other
organisms, and the prospects for engineering the pathway in plants.
B. BIOLOGICAL FUNCTION OF PANTOTHENATE
Coenzyme A (CoA, Fig. 1B) is so called because it was identified by Lipmann

et al. (1947) as the heat-stable cofactor for acetylation reactions, the A standing
for acetylation. The active part of the molecule is the terminal thiol group, which
is covalently linked via a thioester bond to acyl groups such as acetate, or longer
chain fatty acids. The CoA derivative is more soluble in the aqueous environment of the cell and is said to be activated because the DG of hydrolysis of the
thioester linkage is large and negative (e.g. 31.5 kJ mol 1 for acetyl CoA).
This then facilitates the formation of covalent bonds, such as citrate from acetyl
CoA and oxaloacetate in the Krebs cycle. CoA is involved in innumerable
reactions of central metabolism (e.g. fatty acid oxidation, and biosynthesis of
glycerolipids and sterols) as well as secondary metabolic pathways, including
those for polyketides, non-ribosomal protein synthesis, flavonoids, and lignin.
In Escherichia coli, it has been estimated that approximately 100 enzymes (over
4% of the total) use either CoA or a CoA ester as substrate (Begley et al., 2001).
206
Fig. 2. Biosynthetic pathway for pantothenate. Pantothenate is formed by the

condensation of -alanine and pantoate catalysed by the ATP-dependent pantothenate synthetase (PS). Pantoate is synthesis**ed in two steps from -ketoisovalerate
(-KIVA), the oxo-acid of valine, by ketopantoate hydroxymethyltransferase
(KPHMT), which has a 5,10-methylene tetrahydrofolate cofactor, followed by
NADPH-reduction by ketopantoate reductase (KPR).
ACPs have a much more restricted, although equally important, role in fatty
acid synthesis, and in E. coli, ACP is the most abundant soluble protein
constituting about 0.25% of the total soluble protein (Magnuson et al., 1993).
Again the acyl groups are attached via a thioester link to the terminal thiol.
Transthioesterification is readily achieved and this reactivity is central to the
chemistry of these thioesters. The pKa of the alpha proton is also reduced by
thioesterification, enabling Claisen ester condensation chemistry to occur readily in pathways of fatty acid biosynthesis.
C. EFFECTS OF PANTOTHENATE DEFICIENCY
Pantothenate is widespread in all foodstuffsindeed the name is derived

from the Greek panto then, which means from everywhereand so deficiency is not observed in the general population. A burning foot syndrome
207
exhibited by malnourished prisoners of war in the Second World War was

attributed to pantothenate deficiency although this was not confirmed
(Bender, 1999). However, in poultry chicks, dermatitis around the eyes and
beak was found to be alleviated by administering pantothenate. In adult
mice, pantothenate deficiency is associated with lower levels of total CoA and
an associated inability to maintain normal levels of glycogen (Smith et al.,
1987). In rats, mild pantothenate deficiency is associated with short-term
elevation in plasma triglyceride and free fatty acid levels before significant
weight loss occurs (Wittwer et al., 1990). These final symptoms are similar to
those observed after feeding with the L-enantiomer of the naturally occurring
vitamin D-pantothenate. The L-enantiomer has no biological activity but can
antagonise the incorporation of D-pantothenate when in excess (Sarett
and Barboriak, 1963). Moreover, maternal pantothenate deficiency led to
reduced food intake in neonatal pups and thereby to change in lipid composition (Rajalakshmi and Nakhasi, 1975). Finally, rats fed on a pantothenatedeficient diet exhibited loss of fur colour (Morgan and Simms, 1940): this
lends a weak pseudo-scientific basis for the widespread inclusion of pantothenol into hair care products, although the surfactant properties are probably
more important in this case (Hoezel et al., 1998).
A small number of clinical studies of pantothenate deficiency in man have
been performed with the antimetabolites o-methylpantothenate (Fry et al.,
1976) and homopantothenate (pantoyl--aminobutyric acid; Noda et al.,
1988). In both cases, a number of generalised symptoms were observed
including depression, frequent infection, fatigue, and minor neurological
disorders including numbness and the burning foot syndrome described in
earlier case studies.
Organism level effects of deficiency in a wide variety of animals of economic importance have been defined (Smith and Song, 1996 and references
therein). These organisms include pigs, birds, and fish. In all cases, pantothenate deficiency leads to loss of appetite and an associated decreased growth
rate. Additional symptoms include demyelination of motor neurons in chicks
associated with ataxia, extensive colitis and ulcers in swine, adrenal lesions in
rats and fused gill lamellae in fish (see Table I, reproduced from Smith and
Song, 1996). As a consequence, pantothenate is routinely added to animal
feeds and is the major market for the manufactured vitamin. The effect on
weight gain and maintenance in all organisms means that it is possible to
define minimum dietary requirements for a wide range of animals (see
Table II, reproduced from Smith and Song, 1996); however, no routine test
for pantothenate nutritional status has been identified. And in practice, the
ubiquity of pantothenate (either free or bound in CoA) in natural foodstuffs
means that no minimum dietary allowance is recommended.
208
TABLE I
Symptoms of Pantothenate Deficiency
Species
Visible signs
Histopathological abnormalities
Humans
Depression; personality changes;

frequent infection; fatigue; sleep
disturbances
Chicks
Dermatitis at corners of beak and

eyelids; rough feather; retarded
growth; ataxia
Swine
Loss of hair; reddening of skin;

excess nasal secretion; changes in
tongue; diarrhea; GI bleeding;
locomotor abnormalities in gait
Rats/
mice
Skin lesions; greying of hair and

bald patches; red porphyrin
whiskers; paralysis of hind legs
Clubbed gills; intralamellar
proliferative lesions; listless
Cardiac instability; abdominal

pains; neurological disorders;
Reye-like syndrome; muscle
weakness
Thymus involution; distended gall
bladder; demyelination of motor
neurons; moderate duodenal
ulcerations
Extensive colitis and small ulcers;
inflammatory changes in large
intestine; chromatolysis of dorsal
root ganglion cells;
demyelination of peripheral
nerves
Adrenal lesions; lipid depleted,
shrunk, and dying cells
Fish
Fused gill lamallae covered with

exudate; fatty liver; kidneys
deposited with glycogen and
hyaline bodies; clumped
mitochondria
Reproduced in part from Smith and Song (1996). In fact, given the widespread distribution of
pantothenate in most foodstuffs, this is confined to animals on artificial diets, or to experimentally induced treatments, such as with the antimetabolites o-methylpantothenate (Fry et al.,
1976), and homopantothenate (pantoyl--aminobutyric acid; Noda et al., 1988).
D. DIETARY ABSORPTION AND METABOLISM IN MAMMALS
Pantothenate is absorbed at low concentrations through the intestinal wall

via Na-dependent active transport (Fenstermacher and Rose, 1986; Prasad
et al., 1999), while at higher concentrations, it passively diffuses through the
cell membrane (Shibata et al., 1983). All forms of the vitamin act as dietary
nutrients; CoA can be cleaved by a non-specific nucleotide pyrophosphatase
(Skrede, 1973) and lysosomal acid phosphatase (Bremer et al., 1972) to
generate pantetheine. Pantetheine can then be degraded to generate pantothenate and cysteamine by pantetheinase (Dupre et al., 1973). Pantetheine is
also absorbed through the intestine; in fact, this process occurs more rapidly
than pantothenate transport (Ono et al., 1974; Turner and Hughes, 1962).
The protein responsible for this pantetheinase activity in mammals has
been identified. Using the N-terminal sequence of the protein isolated from
pig kidney, Maras et al. (1999) determined that this protein was found to be
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TABLE II
Recommended Dietary Requirements of Pantothenate
Species
Minimum
requirement
(mg kg 1 diet)
Determinant
Humans
Adults
Infants
47a
23a
Usual intake; urinary excretion

Human milk composition
Chickens
Chicks
Laying hens
7.810
1.98.8
Weight gain; feed efficiency; gain in fat

Egg production; maintenance of weight
Turkey
Poults (723 days)
Hens (13 years)
10.5
16.0
Weight gain; lack of dermatitis

Fertile eggs; hatchability
Swine
Breeding sows
11.912.5
Rats
8.010.0
Mice
Catfish
6.030.0
10.015.0
Litter birth weight; litter weanling

weights
Weight gain; acetylation of
sulfanilamide
Weight gain; absence of symptoms
Weight gain; lack of clubbed gills
mg day 1.
Reproduced in part from Smith and Song (1996).
similar to the GPI-anchored protein vanin-1. This protein and its human
homologues VNN1 and VNN2 are involved in lymphocyte migration.
Subsequent characterisation by Pitari et al. (2000) has shown that depletion
of the enzyme leads to lack of free cysteamine in tissue and has a role in
combating oxidative damage.
Once taken up, pantothenate is converted rapidly to phosphopantetheine
and CoA (Fig. 3). Pantothenate kinase (PanK) first phosphorylates pantothenate to generate 40 -phosphopantothenate. This undergoes CTP- or ATPdependent ligation with cysteine to generate phosphopantothenoylcysteine,
which is then decarboxylated by phosphopantethenoylcysteine decarboxylase (PPCDC) to produce 40 -phosphopantetheine. This intermediate can be
incorporated directly into ACPs by their cognate phosphopantetheinyl transferases (Walsh et al., 1997). Alternatively, transfer of an adenylyl group from
ATP, catalysed by phosphopantetheine adenylyltransferase (PPAT), leads to
dephosphocoenzyme A, and the final active form of the cofactor is produced
by dephosphocoenzyme A kinase (DPCK). The pathway from pantothenate
to CoA is conserved in all organisms, with the exception of some intracellular
parasites, such as Mycoplasma, Rickettsia, and Chlamydia, which appear to
encode orthologues only of the terminal gene, for DPCK. This suggests that
210
Fig. 3. Biosynthetic pathway from pantothenate to CoA. The conversion of

pantothenate to CoA takes place in five steps. Firstly, pantothenate is phosphorylated
by pantothenate kinase (PANK), and then it is ligated to cysteine by 40 -phosphopantethenoylcysteine synthetase (PPCS). Decarboxylation by 40 -phosphopantethenoylcysteine decarboxylase (PPCDC) produces the 40 -phosphopantetheine moiety, which
is either ligated to a serine residue in acyl carrier protein (ACP), or to adenosine
monophosphate by 40 -phosphopantetheine adenylyltransferase (PPAT) to produce
dephosphocoenzyme A. The last step is phosphorylation of the 30 -hydroxyl group by
dephosphocoenzyme A kinase (DPCK).
211
they have evolved an uptake system for CoA or dephosphoCoA. Defects in

the CoA biosynthesis genes are generally lethal due to the essential nature of
the products (as discussed in Section IV for plants). Moreover, for the
majority of organisms, neither CoA nor the intermediates can be taken up
by cells, and so auxotrophs are only known that are facultative, for example,
carrying temperature-sensitive mutations.
Metabolism of pantothenate is regulated chiefly at the level of the enzyme
PanK. In mammals, there are five isoforms of this enzyme with distinct
metabolic functions, and defects in each have different consequences. Two
of these isoforms, PanK1 and PanK1, are products of a single gene as a
result of alternative splicing (Rock et al., 2002a) and are chiefly expressed in
hepatic tissue. They are associated with the metabolic transition from glucose
utilisation/fatty acid synthesis to gluconeogenesis and oxidation of fatty
acids, as demonstrated by accumulation of lipid droplets in the livers of
PanK1/ mice (Leonardi et al., 2010). In contrast, PanK2 is chiefly
expressed in the mitochondria of neuronal tissues and point mutations in
this gene are associated with the condition PanK-associated neurodegenerative disorder (formerly known as HallervordenSpatz syndrome), which is a
rapidly progressing autosomal recessive disorder leading to dystonia, parkinsonism, and iron accumulation in the brain, and ultimately death (Hayflick
et al., 2003). In a mouse model of this condition, mutations were associated
with retinal degeneration and arrest of spermiogenesis, but the iron accumulation and neurodegenerative symptoms were not observed (Kuo et al.,
2005). Biochemical characterisation of the third isoform, PanK3 which is
also highly expressed in the liver (Zhou et al., 2001) shows that in contrast to
PanK1, it is strongly feedback regulated by both acetyl CoA and long chain
acyl-CoAs (Zhang et al., 2005) and this is consistent with a role in glycolysis
and fatty acid synthesis. Little is known about the final isoform, PanK4,
other than that it is expressed ubiquitously but is chiefly found in muscle
tissue (Zhou et al., 2001).
II. DISTRIBUTION IN PLANTS

Because plants synthesise pantothenate de novo, they are a good dietary
source of the vitamin, although the highest levels are found in meat, dairy
products, and mushrooms (USDA, 2005). In order to measure bioavailable
pantothenate, it is first necessary to liberate it from the cofactors CoA and
ACP, and numerous studies have investigated the optimal way of doing this.
Since pantothenate is sensitive to both low and high pH, it is not possible to do
this by alkaline or acid hydrolysis, and instead enzymes are used, with
212
pantetheinase being the most suitable (Gonthier et al., 1998). Pantothenate

can then be quantified using a bioassay, in which growth of a bacterial
auxotroph such as Lactobacillus plantarum (Wyse et al., 1985) or E. coli
(Chakauya et al., 2008) is found to be related to pantothenate concentration.
However, it is essential to construct careful standard curves to determine the
linear range of sensitivity. Using these approaches, the estimated levels of
pantothenate level in most common vegetables and fruits have been
determined and range between 2 and 25 mM, equivalent to 525 nmol g 1
fresh weight (FW; Friedrich, 1988; USDA, 2005). The highest levels are found
in potatoes, grains and cereals, broccoli, and tomatoes. Sunflower kernels
have the highest pantothenate level measured to date in plants (301 nmol g 1
FW), while in cauliflower and broccoli florets, levels are 31 and 26 nmol g 1
FW, respectively; since these are consumed in larger amounts, they are
probably the best sources of dietary vitamin B5. However, processing (such
as freezing, canning, or refining) leads to a reduction in both free and bound
pantothenate. For example, Rychlik (2000) found that rice grains contain
95 nmol g 1 FW total pantothenate compared to 20 nmol g 1 FW in polished
rice. Interestingly, the free pantothenate was less affected with 25 nmol g 1
FW in the whole grain compared to 18 nmol g 1 FW in polished rice. Most
studies have concentrated on measuring pantothenate in the edible parts of
plants, but Rathinasabapathi and Raman (2005) quantified pantothenate in
young leaves from several higher plants and found a 10-fold variation in
amount from 0.73 nmol g 1 FW in Rosa sinensis to 7.3 nmol g 1 FW in
Citrus but in all cases much lower than that detected in the edible fruits,
flowers, and seeds.
A similar picture was seen in Brassica napus, in which levels in different plant
tissues were compared (Chakauya et al., 2008). Free pantothenate in leaves
was measured at 11 1 nmol g 1 FW compared with 15 and 41 nmol g 1
FW found in flowers and mature seeds, respectively. Similarly, that in green
seeds was half that of the pods (fruit and seed). This might indicate that the
vitamin comes from the fruit, possibly from biosynthesis or remobilisation
from other tissues. Assimilates for seed storage can be the result of one of two
physiological processes: utilisation of assimilates from current photosynthesis,
and remobilisation of substances accumulated pre-anthesis (Prasad et al.,
2002). There are reported cases of mobilisation of carbon from flag leaves
and stems to the grain in chickpeas and wheat (Davies et al., 2000). If this is the
case then pantothenate might be synthesised during an early stage of plant
growth and recycled throughout the plant to areas of high demand.
When combined, the results seem to suggest that pantothenate might be
mobilised from photosynthetic parts of the plant including older leaves
and the hull to nourish the developing seed. This is shown by the increase in
213
free pantothenate as the seed develops to the same level as in a fully imbibed
seed (Chakauya et al., 2008).
However, consideration of pantothenate levels in plants is complicated by
the incorporation of pantothenate into CoA and ACP. A metabolic profile
analysis of Arabidopsis thaliana looked at acetyl CoA levels and found that
there was several fold difference between seeds and leaves (Gibon et al., 2002).
They estimated that early developing seeds had about 2530 nmol g 1
dry weight falling to about 5 nmol g 1 dry weight at maturity. Using
HPLC analysis, Tumaney et al. (2004) estimated that early-mid-maturing
B. napus seeds have 25 nmol g 1 FW falling to 12.5 nmol g 1 at late maturing,
compared to just 5 nmol g 1 FW in leaves acetyl CoA levels in Arabidopsis
and spinach leaves were within the same range, 5 and 6.8 nmol g 1 FW,
respectively. It is likely that changes in the acetyl CoA pattern reflects
altered activity in lipid metabolism in the different tissues, with higher levels
required in seeds for the demands of triacylglyceride production or
mobilisation in development or germination, respectively. However, what is
not certain is whether the ratio of free to bound pantothenate remains the same
in all tissues.
At the subcellular level, acetyl CoA is found in all subcellular compartments in plants, including plastids, mitochondria, peroxisomes, and cytosol
where it is involved in several metabolic functions (Fatland et al., 2004;
Tumaney et al., 2004). The concentration has been measured at 3035 mM
in spinach stroma by Post-Beittenmiller et al. (1992), who estimated that 75%
of cellular acetyl CoA was in the chloroplast. However, it is widely accepted
that membranes are impermeable to CoA and its thioesters, implying that
either CoA is synthesised in all cellular compartments or made in one
compartment and then transported to others during biosynthesis. An example of such a CoA transporter is the Arabidopsis ATP-binding cassette (ABC)
transporter encoded by the PXA1 gene (Zolman et al., 2001). This transporter is involved in the import of long chain acyl-CoAs into the peroxisomes for
-oxidation. Further consideration of the subcellular distribution of pantothenate and CoA is given in Section IV.F describing the distribution of the
biosynthetic enzymes.
III. BIOSYNTHETIC PATHWAY AND

LOCATION OF THE PATHWAY
In all prototrophic organisms studied to date, pantothenate is synthesised
from 3-methyl-2-oxobutyric acid (-ketoisovalerate-KIVA) and -alanine (Fig. 2; Webb et al., 2004). The former compound is an intermediate in
214
valine biosynthesis, whereas -alanine can be derived from a number of

sources including decarboxylation of L-aspartate; degradation of uracil;
and degradation of the polyamines, spermine, and spermidine (discussed in
detail in Section V.B). The first enzyme of the pathway is ketopantoate
hydroxymethyltransferase (KPHMT), which converts -KIVA into ketopantoate. This is reduced to D-pantoate, which is then condensed with alanine to generate D-pantothenate, catalysed by pantothenate synthetase
(PS). It is only these two enzymes KPHMT and PS that have been characterised in plants. Putative enzymes responsible for the remaining steps are
discussed in Sections III.C and V.B.
A. KETOPANTOATE HYDROXYMETHYLTRANSFERASE
KPHMT is responsible for the first step in the pathway, the transfer of a
formaldehyde equivalent onto an enolate ion-derived from -KIVA to
generate ketopantoate (Fig. 4). The cellular formaldehyde equivalent is
provided by methylene tetrahydrofolate. This activity was first observed in
bacteria by McIntosh et al. (1957), who demonstrated the presence of the
Fig. 4. Outline mechanism of ketopantoate hydroxymethyltransferase

(KPHMT). (A) KPHMT catalyses the transfer of a formaldehyde equivalent from
methylene tetrahydrofolate (B) to -ketoisovalerate. (a) -Ketoisovalerate is bound
to an active-site magnesium ion which promotes formation of an enolate via deprotonation using an active-site base. (b) The enolate nucleophile attacks formaldehyde
with concerted proton transfer to generate ketopantoate. (B) Structure of the ringopen iminium form of methylene tetrahydrofolate.
215
activity in E. coli. Teller et al. (1976) purified this enzyme and characterised
its catalytic properties (Powers and Snell, 1976). The gene was subsequently
isolated by functional complementation (Jones et al., 1993), and this eventually led to the structural solution of the enzyme from both E. coli (von Delft
et al., 2003) and Mycobacterium tuberculosis (Chaudhuri et al., 2003). The
enzyme is a homodecameric structure with each protomer forming a ()8
barrel (Fig. 5A). The active site lies in a deep pocket formed by the C-termini
of the -strands (Fig. 5B). The catalytic properties of the bacterial enzyme
have been extensively characterised by Sugantino et al. (2003). Intriguingly,
Fig. 5. Crystal structure of bacterial ketopantoate hydroxymethyltransferase. (A)

E. coli KPHMT exists as a decamer of 10 ()8-barrel protomers (pdb 1m3u; von
Delft et al., 2003). (B) Pantoate is bound to a magnesium ion within the barrel of
each promoter.
216
the enzyme from this source does not have an absolute requirement for a
tetrahydrofolate cofactor for activity but will accept free formaldehyde as an
electrophilic partner for hydroxymethylation.
The existence of this enzymatic step in plants was first suggested in an
auxotrophic strain of Datura innoxia (thorn-apple), which required supplementation with pantothenate, pantoate, or ketopantoate for survival (Savage
et al., 1979). This suggested the presence of KPHMT in the plant, though no
activity could be measured in cell-free extracts (Sahl et al., 1988). KPHMT
activity in plants was then unambiguously demonstrated by Jones et al.
(1994) who fed 14C-labelled valine to pea-leaf discs, followed by HPLC
analysis of an extract. Radiolabel was found in peaks corresponding to KIVA, ketopantoyl-lactone, and pantoyl-lactone. The acid treatment during
extraction would have degraded any pantothenate in the sample.
Ottenhof et al. (2004) used the amino acid sequence of the E. coli enzyme to
identify two homologues of the enzyme in the complete genome sequence of
Arabidopsis, termed KPHMT1 (At2g46110) and KPHMT2 (At3g61530).
The coding sequence for both genes could be amplified from an Arabidopsis
cDNA library indicating that they are both transcribed, and both genes were
shown to encode functional enzymes from the fact that they complemented
the E. coli Hfr3000 YA139 panB mutant. Two gene products were also
identified by BLAST searching of the rice genome (2674.t00011 and 4685.
t00006), and in indeed, in all plant genomes publically available, there are
two PanB genes detectable. The predicted protein sequence of the plant
enzymes have 30% identity with the E. coli KPHMT protein with all the
residues conserved in bacteria being conserved in plants (Fig. 6). Both isoforms of the enzyme are synthesised with N-terminal extensions relative to
the E. coli protein. When these sequences from Arabidopsis KPHMT1 and
KPHMT2 were fused to GFP, they were able to target GFP to the mitochondria indicating that the KPHMT isoforms would similarly be located in
this organelle. This observation allowed the detection of enzyme activity in
plant extracts for the first time; mitochondria from both peas and Arabidopsis were isolated, and KPHMT activity could then be directly assayed in
extracts of these organelles. This was achieved using a coupled assay with the
next enzyme in the bacterial pathway, ketopantoate reductase (KPR), and
monitoring the oxidation of NADPH (Ottenhof et al., 2004).
By overexpressing the recombinant Arabidopsis enzymes in E. coli, both
isoforms KPHMT2 (Coxon, 2006) and KPHMT1 (Webb, unpublished
results) have been shown to be catalytically active. In the case of
KPHMT2, the methylene tetrahydrofolate cofactor was shown to be required for catalytic activity. This suggests that the precise enzymatic mechanism of the plant enzyme may be different to that of the bacterial enzyme for
which formaldehyde can be used as a substrate.
217
Fig. 6. Sequence alignment of ketopantoate hydroxymethyltransferases. The two

Arabidopsis KPHMTs are aligned with those from yeast (S. cerevisiae) and four
representative bacterial sequences. Conserved residues are highlighted.
B. PANTOTHENATE SYNTHETASE
PS catalyses the ATP-dependent ligation of pantoate and -alanine to form

pantothenate via a pantoyl adenylate intermediate (Fig. 7). PS activity was
first reported by Wieland and Moller (1941) in crude lysates of yeast. The
activity was subsequently identified in extracts of E. coli by Maas in 1952,
218
Fig. 7. Mechanism of pantothenate synthetase. Pantothenate synthetase catalyses

formation of a peptide bond between pantoate and -alanine by forming a pantoyl
adenylate intermediate from ATP and pantoate, generating pyrophosphate, followed
by displacement of AMP by -alanine.
and the protein responsible for this activity purified 50-fold by Pfleiderer
et al. (1960) and 300-fold by Kreiling et al. (1962) before purification to
homogeneity (2100-fold purification) by Miyatake et al. (1976). The protein
from both E. coli (von Delft et al., 2001) and M. tuberculosis (Zheng and
Blanchard, 2001) was subsequently overexpressed and purified and the crystal structures were determined (von Delft et al., 2001; Wang and Eisenberg,
2003).
The protein is dimeric (Fig. 8A) and each protomer comprises two
domains. The N-terminal domain has a Rossmann fold with the active site
at the interface formed between the N- and C-terminal domains. The protein
is structurally very similar to class I aminoacyl-tRNA synthetases and is thus
a member of the cytidylyltransferase superfamily. By analogy with these
enzymes, a hinge-bending mechanism has been proposed in which the Cterminal domain opens and closes over the active site during the enzymatic
reaction, because the catalytically essential residues are spatially separated
into two domains in the apo structure of the E. coli enzyme. In contrast,
structures for both the apo and pantoyl adenylate complex of the M. tuberculosis enzyme show little or no conformational change between them, being
essentially closed structures (Wang and Eisenberg, 2003). Recently, the
structures of both the apoenzyme and a pantoyl adenylate complex of
Staphylococcus aureus PS have been solved (Satoh et al., 2010). Structural
analysis indicated that the apoenzyme adopts an open and relatively mobile
structure, while the complex structure is closed and entirely rigid (Fig. 8B).
Structural comparison of the apoenzyme and the complex revealed how
S. aureus PS undergoes open/close conformational change and also
determined the key interactions with the adenine ring of PA (Fig. 8C) for a
hinge-bending domain closure.
219
Fig. 8. Structure of pantothenate synthetase. (A) Structure of the apoform of

pantothenate synthetase from S. aureus (pdb 3ag5; Satoh et al., 2010). The N-terminal
domain (left hand side) has a Rossmann fold, whilst the active site is at the interface
between this domain and the C-terminal part of the enzyme. (B) Structure of the same
enzyme in which the pantoyl adenylate intermediate is bound (pdb 3ag6), showing
that the conformation change in which the C-terminus has closed over the active site.
(C) Close-up of the active site with pantoyl adenylate structure.
The first report of PS in higher plants was that of Genschel et al. (1999)
who used functional complementation of an E. coli panC mutant AT1371 to
identify the genes for PS from Lotus japonicus, Oryza sativa, and also from
yeast, S. cerevisiae. By comparison of the plant enzyme with the bacterial
homologues, the protein appears to not to have an N-terminal extension
characteristic of a transit peptide for targeting to organelles, suggesting that
it would be located in the plant cytosol. The protein from L. japonicus was
overexpressed in E. coli and purified to 95% homogeneity. This L. japonicus
PS exhibited MichaelisMenten kinetics with respect to ATP, -alanine, and
D-pantoate although substrate inhibition was observed at higher concentrations of D-pantoate.
Ottenhof et al. (2004) subsequently used this gene sequence in BLAST
(sequence similarity) searches to identify the gene for PS in Arabidopsis
(At5g48840). The catalytic activity of this protein was then once again
confirmed by complementation of the E. coli panC mutant AT1371. Using
220
GFP-fusions and confocal fluorescence microscopy, the enzyme was shown

to localise to the cytosol, confirming the predictions from Genschel et al.
(1999). This protein was subsequently characterised further by Jonczyk and
Genschel (2006). In this study, they sought to understand the molecular basis
for the substrate inhibition by pantoate observed for the enzyme from
L. japonicus (Genschel et al., 1999). Analysis of the kinetics of the Arabidopsis
enzyme revealed that the variation in rate for different concentrations of
pantoate at fixed concentrations of ATP could not be accounted for by a
simple model for uncompetitive substrate inhibition (i.e. formation of a
dead-end S.E.S complex). The data did, however, fit well to an empirical
equation of the form v (1[S] 2[S]2)/(1 1[S] 2[S]2), and this equation was therefore used to interpret their data. This equation has the same
form as the analytical equation for partial uncompetitive inhibition (i.e. the
S.E.S complex is still able to form product but at a reduced rate), and the
data are consistent with the inhibited rate being ca. 10% of the uninhibited
rate. -Alanine utilisation was also negatively cooperative at high concentrations of pantoate.
Substrate inhibition of this type is not observed for the bacterial PSs, and
the molecular basis for the difference has been studied (Jonczyk and
Genschel, 2006). The simplest origin of substrate inhibition would be allosteric communication between the two subunits of PS. Sequence alignment of
plant and bacterial PSs revealed an amino acid insertion in the plant protein
N-terminal to the dimerisation region of the protein (Fig. 9). In order to
investigate the potential role for this part of the protein in allosteric substrate
inhibition, two mutated forms of the protein (named AtPTS) were generated.
The first had a single E132A alteration at end of the insertion, which led to a
reduction in substrate inhibition. A complete loss of substrate inhibition by
pantoate was seen in a second mutant in which residues P97-T133 were
replaced with P93-T107 of the E. coli protein, so that the entire insertion
had been removed. Intriguingly, the loss of substrate inhibition by pantoate
revealed increasing substrate inhibition by ATP which had previously been
masked.
The observation of substrate inhibition in this system may not be directly
relevant in vivo, since the total flux through the plant biosynthetic pathway
(discussed in Section VI) is low. The cellular concentration of pantoate is
therefore always below the concentration at which substrate inhibition
occurs. Jonczyk and Genschel (2006) proposed that instead allosteric communication between the two sites actually serves to enhance substrate binding at low substrate concentrations, and since higher substrate
concentrations are not encountered in planta there is no evolutionary pressure to reduce the negative effect of substrate inhibition.
221
Fig. 9. Sequence alignment of representative bacterial and plant sequences for

pantothenate synthetase. Highlighted residues (black) are conserved throughout
bacterial and plant sequences. The inserted sequence in plants and the corresponding
E. coli sequence identified by Jonczyk and Genschel (2006) as contributing to allosteric regulation of catalytic activity are highlighted in grey.
222
Jonczyk et al. (2008) further investigated the in planta function of PS. Two
T-DNA insertional mutants of At5g48840 were identified from the
Salk collection (Alonso et al., 2003), Salk_101909 (pts-1) and Salk_594477
(pts-2). Heterozygous mutant plants exhibited no phenotype, but it was not
possible to isolate homozygous plants of either knockout line. Examination
of the developing seeds in the siliques of heterozygous plants revealed that a
proportion were arrested at a pre-globular stage of development, and the
numbers of heterozygous and wild-type (WT) plants from the remaining
seeds were consistent with this hypothesis. The addition of exogenous pantothenate to growing heterozygous plants was sufficient to abolish the arrest
of seed development, and all seeds produced by these plants were able to
germinate on agar supplemented with pantothenate. Homozygous mutants
were able to grow into fertile plants on nutrient agar, although they were not
able to survive on soil. This was hypothesised to be due to a lack of active
transport into the roots of plants.
Jonczyk et al. (2008) used a combination of published transcript data
(Meyers et al., 2004) and promoter:GUS fusion assays to investigate the
expression patterns of Arabidopsis PanC gene during vegetative growth and
flower development. Both sets of data showed constitutive expression of the
gene across vegetative tissue types with very little variation. During flower
development, both the promoter:GUS fusion assays and microarray data
(Schmid et al., 2005) showed expression in the sepals and carpels with less in
the petals and stamen.
C. REDUCTION OF KETOPANTOATE
The identification and characterisation of KPR and the corresponding panE

gene in bacteria lagged behind that of the other three enzymes, in part
because of the presence of another enzyme able to catalyse the same reaction.
Acetohydroxyacid isomerase (AHIR, EC 1.1.1.86, encoded by ilvC), responsible for the reductive rearrangement of 2-acetolactate to yield 3-hydroxy-3methyl-2-oxobutyrate in branched chain amino acid biosynthesis (Fig. 10),
has been shown also to catalyse reduction of ketopantoate (Primerano,
1983), and indeed, an ilvC panE double mutation is required in order to
generate a pantothenate auxotroph (Frodyma and Downs, 1998a). In the
industrially important Corynebacterium glutamicum, panE is absent and only
AHIR catalyses this transformation (Merkamm et al., 2003).
The panE gene encoding KPR was eventually identified in Salmonella
typhimurium, where a gene previously called apbA was found to encode a
protein with KPR activity (Frodyma and Downs, 1998b). The enzyme preferentially uses NADPH rather than NADH as hydrogen donor (Frodyma
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Fig. 10. Alternative methods for ketopantoate reduction. Three distinct routes
for ketopantoate reduction have been identified: (A) Ketopantoate reductase
(KPR) catalyses the NADPH-dependent reduction of ketopantoate to generate
pantoate in E. coli. (B) Acetohydroxyacid isomeroreductase (AHIR, encoded by
ilvC) can also catalyse the NADPH-dependent reduction of ketopantoate to pantoate
in E. coli and is the sole pathway in C. glutamicum. (C) Reduction of ketopantolactone to pantolactone has also been proposed a possible route for this reduction in
plants.
and Downs, 1998a). Thereafter, the E. coli panE gene was isolated, the
enzyme protein overexpressed and purified, and the X-ray crystal structure
of the apoprotein solved (Fig. 11; Matak-Vinkovic et al., 2001). Several other
structures have been determined, including that of the ternary complex with
products NADP and pantoate bound (Ciulli et al., 2007).
To date, no dedicated enzyme for ketopantoate reduction has been characterised in plants. The sequence conservation between KPRs, even in bacteria, is low with only 16 residues completely conserved, all these lining the
active site (Fig. 11), so it was not surprising that sequence-based searches of
plant genomes have not identified a clear candidate. Instead there are 138
short-chain oxidoreductases, characterised by a Rossmann fold at the Nterminus. On the other hand, a KPR homologue was readily identifiable in
yeast (Ottenhof et al., 2004). Ottenhof et al. (2004) used a structure-led search
of the complete Arabidopsis proteome with the E. coli KPR structure a query.
One putative candidate (At5g34780) was identified, but the catalytic activity
of this protein has not been confirmed.
224
Fig. 11. Structure of ketopantoate reductase (KPR) from E. coli. At a sequence

level, KPR has very few conserved residues and all of these are found close to the
enzyme active site.
The branched chain amino acid biosynthetic pathway is well characterised

in plants (Binder, 2010). In Arabidopsis, acetohydroxyacid isomerase (or
ketolacid reductoisomeraseKARI) is encoded by At3g59610. The plant
enzymes were originally characterised from spinach (Dumas et al., 1992) and
barley (Durner et al., 1993), and the structure of enzymes from both spinach
(Biou et al., 1997) and rice (Leung and Guddat, 2009) has subsequently been
elucidatedboth proteins crystallised as dimers and showed an unusual knot
structure. Though the bacterial enzyme has been shown to catalyse the
transformation, the corresponding experiment has not yet been performed
with the plant enzyme.
A third possibility, reduction of ketopantolactone to form pantolactone
(Fig. 10), was postulated by Julliard (1994) who purified an 34-kDa monomeric protein from spinach leaf chloroplasts. This protein was able to catalyse the NADPH-dependent reduction of ketopantolactone in a manner
similar to two bacterial enzymes isolated by King et al. (1974). However, it
also accepted a range of other substrates, including the structurally unrelated
isatin and bornanedione (which also have higher affinity for the enzyme)
suggesting that this enzyme is a broad spectrum reductase rather than being
associated with pantothenate biosynthesis (Hata et al., 1989). Moreover,
plant PSs, in common with their bacterial counterparts, are unable to utilise
pantolactone, and a further enzyme would be required to convert it to the
225
open chain form. Although ketopantolactone readily ring-opens at room

temperature and neutral pH (t1/2 4 min), pantolactone is much more stable.
There is also an issue over compartmentation, discussed further in Section IV.F.
D. SOURCE OF -ALANINE
In bacteria, -alanine is synthesised by the catalytic decarboxylation of Laspartate by the pyruvoyl-dependent aspartate -decarboxylase (Williamson
and Brown, 1979). This enzyme, which is discussed in more detail in Section V.B, is homotetrameric with a double-c -barrel fold (Fig. 12; Albert
et al., 1998). Ottenhof et al. (2004), using both sequence- and structure-based
searches were unable to find a homologue of ADC present in the Arabidopsis
genome and proteome, nor in other plant genomes or yeast, and it is generally considered that this enzyme is confined to prokaryotes. Numerous other
routes to -alanine have been described, including the catabolism via oxidation of spermine and spermidine (White et al., 2001) and uracil (West et al.,
1985; described below in Section V.B). These are the principle pathways
operating in S. cerevisiae and Drosophila melanogaster, respectively. Both
pathways known to operate in plants but which is responsible for -alanine
formation for pantothenate biosynthesis have not been established. As discussed in Section V.B, polyamine degradation is currently the most likely
candidate source for this metabolite in plants.
Fig. 12. Structure of aspartate -decarboxylase (ADC). Aspartate -decarboxylase forms a tightly bound tetramer of double-c -barrels (Albert et al., 1998), which
auto-catalytically cleave to generate the pyruvoyl group required for catalysis.
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IV. REGULATION, TURNOVER, AND METABOLISM

As in all organisms, in plants pantothenate is rapidly converted to 40 -phosphopantetheine in CoA and ACP by an identical series of reactions (Fig. 3).
All five enzymes have now been identified in plants and the pathway reconstituted in vitro (Kupke et al., 2003). Several of the enzymes have also been
studied in greater detail.
A. PANTOTHENATE KINASE
PanK catalyses the ATP-dependent phosphorylation of pantothenate to

form phosphopantothenate and has been shown to be the major point of
regulation in CoA biosynthesis. Three distinct classes of PanKs are observed
in nature: Type I PanKs exemplified by E. coli CoaA are found exclusively in
prokaryotes, as are Type III PanKs (Brand and Strauss, 2005). The Type II
PanKs are generally only found in eukaryotes and as discussed in Section I.D
some are characterised by strong feedback inhibition by CoA and its esters
(Rock et al., 2002b). PanK activity in plants was first described by Falk and
Guerra (1993) who measured it in the stroma of spinach chloroplasts. This
activity could be chromatographically separated into two peaks suggesting
that distinct isoforms of the enzyme were present in the plant. Kupke et al.
(2003) identified two putative genes for this enzyme in Arabidopsis on the
basis of sequence similarity to other eukaryotic enzymes and demonstrated
that at least one of these proteins, AtPANK1 (At1g60440), was catalytically
active. Tilton et al. (2006) investigated the two genes at a genetic and
physiological level and demonstrated that both AtPANK1 and AtPANK2
(At4g32180) are functionally active in plants.
The N-terminal domain of AtPANK2 is homologous to full-length
AtPANK1 (50% sequence similarity); however, the gene for AtPANK2
also encodes an additional C-terminal domain of similar length, which is
also found in mammalian PANK4 (Zhou et al., 2001). The domain is also
found as a single protein in a variety of prokaryotes, and although its
function is unknown, the presence of a highly conserved cluster of charged
residues led to the proposal that it might constitute a metal-binding site.
Expression of the plant genes was investigated using real-time PCR analysis
(Tilton et al., 2006). AtPANK1 was found to be expressed in all tissues
examined, but expression was highest in the early stages of seed development
(5 days after flowering), whereas AtPANK2 transcript levels were highest
during the later stages of seed development (1011 days). This is consistent
with a role for CoA in fatty acid synthesis for storage lipids in the seeds.
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The functional role of the two proteins in plants was then investigated
using T-DNA insertion lines from the Salk institute collection (Alonso et al.,
2003). The full-length transcript levels of homozygous plants from both
pank1:pank1 and pank2:pank2 lines were shown to be reduced to < 0.5% of
WT levels by real-time PCR, although low levels (9%) of a truncated transcript for AtPANK2 could be detected. No phenotype could be observed for
either homozygous knockout, but it was not possible to isolate homozygous
double knockout pank1:pank1/pank2:pank2 plants following crossing. A
more detailed examination of developing seeds in the siliques of pank1:
pank1/PANK2:pank2 mutant plants revealed that approximately 25% of
seeds did not develop (as shown by shrivelled seed remnants). This suggests
that both homologues function in vivo to supply CoA. This observation was
further supported by analysis of fatty acid and CoA levels; only minor
decreases (1018%) in either were observed in single mutants suggesting
complementary and non-redundant roles for the two PanK isoforms.
B. PHOSPHOPANTETHENOYLCYSTEINE SYNTHETASE
The second step of CoA biosynthesis, ligation of phosphopantothenate and

cysteine to form phosphopantethenoylcysteine (PPC), is catalysed by PPC
synthetase. The Arabidopsis gene for this enzyme AtCoaB (At1g12350) was
identified by Kupke et al. (2003) on the basis of sequence similarity to the
human monofunctional protein (Daugherty et al., 2002). In bacteria, both
this step and the subsequent decarboxylation are catalysed by a single
bifunctional Dfp protein (Kupke et al., 2000), but both the plant and mammalian enzymes are monofunctional. Following identification of the candidate proteins, Kupke et al. (2003) overexpressed AtCoaB as an N-terminal
MBP fusion and demonstrated that this protein catalysed the ligation of
phosphopantothenate to cysteine in vitro. However, in contrast to the bacterial enzyme, which uses CTP to form a 40 -phosphopantothenoyl-CMP intermediate (Kupke, 2002), the plant enzyme was active with ATP. The human
enzyme, the structure of which has been determined (Manoj et al., 2003,
1p9o), has similarly been shown to use ATP, although it is considered that
both eukaryotic enzymes go via the same intermediate.
C. PHOSPHOPANTETHENOYLCYSTEINE DECARBOXYLASE
PPCDC, which catalyses the decarboxylation of PPC to form phosphopantetheine is the most extensively characterised enzyme in the plant CoA
biosynthetic pathway. Genes for two forms of this protein, AtHAL3A and
AtHAL3B, were initially identified by Espinosa-Ruiz et al. (1999) due to
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their sequence similarity to yeast HAL3, which is involved in tolerance to salt

stress through inhibition of the cell cycle-linked PPZ type-1 protein phosphatase. Similarity to a large family of prokaryotic flavoproteins was noted
at this time, although the function of these proteins had not yet been
established. The three-dimensional structure of this protein was then determined by Albert et al. (2000) who demonstrated that the trimeric protein was
likely to catalyse the ,-dehydrogenation of a peptidylcysteine (similar to
that observed in PPC).
Following the identification of the role of these proteins in bacterial CoA
synthesis (Kupke et al., 2000), one of the two homologues, AtHAL3A, was
shown to catalyse the decarboxylation of 40 -phosphopantothenoylcysteine to
generate 40 -phosphopantetheine (Kupke et al., 2001). AtHAL3A was overexpressed and purified and catalytic activity established via an HPLC-based
assay. Mutation of a single histidine residue (H90N) conserved between
AtHAL3A and the bacterial homologue, Dfp, was sufficient to abolish
decarboxylation. Similarly, mutation of a conserved cysteine residue
(C158A) in Dfp was sufficient to abolish its decarboxylation activity.
These site-directed mutagenesis studies were considerably expanded in the
subsequent study of Hernandez-Acosta et al. (2002), who characterised 11
site-directed mutants of AtHAL3A. Three of these mutations (N142D,
M145L, and C175S) led to loss of activity, whereas mutation of Lys34
(K34N, K34Q, and K34R) led to formation of an additional product that
was identified as the dephosphorylated product, pantetheine, demonstrating
that Lys34 is likely to be involved in recognition of the phosphate group of 40 PPC. This product was also observed in smaller amounts for all mutants
suggesting that pantothenoylcysteine is also a substrate. This was therefore
incubated with mutants and this led to observation of another product that
was identified as pantothenoyl aminoethenethiol. On the basis of this observation, they proposed that AtHAL3A catalyses the decarboxylation of PPC
via a two-step mechanism of oxidation (by FMN to form FMNH2) of
phosphopantothenoylcysteine to generate a thioaldehyde, which then spontaneously decarboxylates to generate a phosphopantothenoylaminoethenthiol intermediate that is reduced (by FMNH2 to form FMN). This
latter oxidation is dependent upon Cys175. Subsequent structural characterisation of AtHAL3A-C175S confirmed the existence of this intermediate:
oxidised pantothenoylcysteamine (pantothenoylaminoethenethiol) was resolved crystallographically (Steinbacher et al., 2003) supporting the proposed mechanism.
The in vivo function of both isoforms of PPCDC, AtHAL3A, and
AtHAL3B, has been investigated by Rubio et al. (2006) via characterisation
of three T-DNA knockout mutants. Two T-DNA insertion mutants of
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AtHal3a, hal3a-1 and hal3a-2, were identified in the Max-Planck Institute

(Cologne, Germany) collection via PCR-based screening. The insertions
were in the intron and first exon, respectively, after the ATG start codon
and in neither case could a functional transcript be identified in homozygous
seedlings. For AtHal3b, a T-DNA insertion mutant from the Salk collection
(SALK_045607) was identified. The expression level of the HAL3A and
HAL3B transcripts was then assessed by quantitative RT-PCR which confirmed that a fourfold higher expression level for HAL3A over HAL3B. The
expression of both transcripts was not detectable in the corresponding hal3a
and hal3b mutant plants, and the abundance of the other transcript was not
affected.
Neither the hal3a-1 nor the hal3b mutant plants exhibited a phenotype
with respect to growth rate, salt tolerance or response to osmotic stress,
despite the observations of Espinosa-Ruiz et al. (1999) who reported that
transformation with a full-length antisense HAL3A cDNA construct led to
plants with delayed growth rate and reduced tolerance of osmotic stress.
Rubio et al. (2006) suggested that this phenotype may have been due
to knockdown of both HAL3A and HAL3B transcripts in these plants,
since the genes are very similar at the nucleotide level and this was not
assayed in the original study. Rubio et al. were unable to generate homozygous hal3a-1 hal3b mutants, although they were able to identify hal3a-1/
hal3a-1:HAL3B/hal3b (aaBb) mutants. Examination of the seeds of these
plants indicated that approximately a quarter were arrested during embryogenesis. Analysis of the seedlings resulting from the remaining seeds revealed
aaBB and aaBb genotype in a 1:2 ratio as expected. This demonstrates that
the aabb genotype was embryo-lethal as expected but also that a low-abundance transcript for AtHAL3B alone is sufficient to provide sufficient CoA
for growth.
In the absence of a double homozygous mutant, Rubio et al. (2006)
investigated the aaBb mutant further. Whereas the single hal3a-1 and hal3b
mutant plants did not exhibit a phenotype, the aaBb mutants exhibited
delayed growth and reduced tolerance to osmotic stress. Sucrose-depleted
media was able to support the germination of the seeds of these plants but
seedling establishment was impaired, as shown by stunted plants with short
roots. This phenotype was attributed to a shortage in the supply of CoA to
allow the -oxidation of storage fatty acids to support growthdirect measurement of the lipid levels in aaBb seedlings grown in the absence of sucrose
was consistent with this hypothesis. The growth defect could be chemically
rescued by the addition of exogenous pantetheine but not pantothenate,
providing further support that the defect in this step was responsible for
the observed phenotype.
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D. 40 -PHOSPHOPANTETHEINE ADENYLYLTRANSFERASE
The gene encoding 40 -PPAT, which catalyses the penultimate step for CoA
biosynthesis, the adenylation of 40 -phosphopantetheine to generate dephosphocoenzyme A, was identified in the Arabidopsis genome by Kupke et al.
(2003) using the sequence of the human protein (Daugherty et al., 2002).
Unlike the mammalian enzyme, the plant PPAT is a monofunctional protein.
The identity of the Arabidopsis enzyme protein was verified by overexpression as an N-terminal MBP fusion protein, and characterisation of the
reaction products by HPLC. Together with the previous three enzymes in
the pathway, the purified protein catalysed the formation of dephosphocoenzyme A from pantothenate. With the addition of the fifth protein (AtCoaE
discussed below), CoA was formed. This demonstrated that the protein was a
functional 40 -PPAT.
Further biochemical analysis of the overexpressed enzyme was reported by
Rubio et al. (2008). This analysis was dependent upon measurement of the
kinetics of the reverse reaction from dephosphoCoA to generate phosphopantetheine and ATP, which was assayed by coupling to the activity of
hexokinase and glucose 6-phosphate dehydrogenase. The activity was
shown to be inhibited by CoA but not acetyl CoA, suggesting a regulatory
role for PPAT in plants.
Rubio et al. (2008) also characterised the role of PPAT in plants by
analysis of a T-DNA insertion mutant from the Salk collection
(SALK_093728) designated ppat-1, which has a T-DNA insert in the second
intron. Analysis of transcript abundance in this line by quantitative RT-PCR
showed a decrease of 90%. Homozygous ppat-1 seedlings were viable, although only 30% of germinated seeds survived transplanting. The seedlings
initially grew on medium both with and without sucrose supplementation,
the phenotype being less severe than that observed for the aaBb hal3 seedlings previously described (Rubio et al., 2006). Transformation of these
plants with a copy of the PPAT cDNA under the control of the 35S promoter
led to rescue of this phenotype, demonstrating that a lack of PPAT expression was responsible for the observed phenotype. More detailed analysis of
the overexpressing lines revealed increased levels of CoA and improved salt
and osmotic stress tolerance.
E. DEPHOSPHO-COA KINASE
The last required step for CoA biosynthesis is the phosphorylation of the 20 hydroxyl of the ribose moiety in dephosphocoenzyme A by DPCK. The
protein responsible for this step in Arabidopsis (AtCoaE) was identified by
231
Kupke et al. (2003) on the basis of similarity to putative monofunctional

CoaE enzymes from other eukaryotes and the monofunctional DPCK from
E. coli (Mishra et al., 2001). The function of the protein in the transformation
of dephosphocoenzyme A to CoA was confirmed by analysis of the reconstituted biosynthetic pathway by HPLC as described above for PPAT, but it
has not been studied further.
F. CELLULAR AND TISSUE PATTERNS IN PLANTS
Although the complete pantothenate biosynthetic pathway has not been

elucidated, knowledge of the subcellular distribution of the first and last
enzymes of the pathway enables considerable insight into the organisation
of pantothenate biosynthesis within plant cells (Fig. 13). KPHMT is confined
Fig. 13. Schematic of a plant cell indicating the known location of the enzymes of
pantothenate and CoA biosynthesis, and the possible transporters. KPHMT is mitochondrial, whereas PS is found in the cytosol. Thus either pantoate or ketopantoate must
be exported from the mitochondrion. The source of -alanine is unknown but polyamine
degradation occurs in the peroxisome and transport to the cytosol is therefore
required. PANK is most likely cytosolic, and although the location of the other CoA
biosynthesis enzymes have not been studied directly, there is no evidence that they are
synthesised with transit peptides, so they too are likely to be in this compartment. This
means that there must be CoA transporters on the membranes of the peroxisomes/
glyoxysomes, mitochondria, and chloroplasts. There may also be another transporter
for 40 phosphopantetheine on the chloroplast for the production of ACP.
232
to plant mitochondria, which are also the site of the last steps of synthesis of
tetrahydrofolate (Neuburger et al., 1996; Ravanel et al., 2001), the essential
cofactor for this enzyme. On the other hand, PS is located in the cytosol, so
either ketopantoate or pantoate needs to be exported across the mitochondrial membranes. Pantoate and -alanine can both be taken up by plant
tissue (Rathinasabapathi and Raman, 2005), as can pantothenate itself
(Jonczyk et al., 2008), indicating that transporters are present, but currently
there is no further biochemical or molecular data available.
Some insights have come from the extensive study of transport of pantothenate across membranes in bacteria, where pantothenate permease,
encoded by panF, was first identified in E. coli (Jackowskii and Alix, 1990),
and shown to catalyse the Na-dependent import of pantothenate across the
plasma membrane. The protein has 12 membrane-spanning domains, a
similar topological profile to the superfamily of cation-dependent carriers.
The mammalian pantothenate transporter, which was first cloned from
rabbit intestine by Prasad et al. (1999), is a member of the same group of
proteins, although the intestinal protein from rabbits has been shown to
transport biotin in addition to pantothenate. In contrast, in budding yeast
(S. cerevisiae), the pantothenate transporter in the plasma membrane was
identified as FEN2, a protein with no sequence similarity to either the
bacterial or the mammalian carriers (Stolz and Sauer, 1999). Moreover, it
was shown to be a H-dependent symporter, rather than using Na. A
homologue is found in fission yeast, Schizosaccharomyces pombe, and intriguingly, the malarial parasite, Plasmodium falciparum, also has a Hpantothenate transporter (Saliba and Kirk, 2001), thus offering a potential
target for anti-malarial therapeutics. However, there have been no studies on
pantothenate transporters in higher plants. Sequence similarity searches with
the rabbit Na-pantothenate transporter as query identify At5g45380, annotated as a high-affinity urea-sodium-symporter (Liu et al., 2003), as the best
hit but with only 19% identical (39% similar) residues. In contrast, there are
no convincing homologues of the yeast FEN2 protein.
The first committed step towards CoA synthesis is catalysed by PanK,
activity of which was found to be predominantly localised in chloroplasts
from spinach (Falk and Guerra, 1993). This would necessitate transport of
pantothenate from its site of synthesis into the chloroplast. However, molecular studies of the two Arabidopsis PANK isoforms suggest that they are in
fact localised to the cytosol, since neither is synthesised with N-terminal
extensions suggestive of transit peptides (Kupke et al., 2003). This has been
confirmed for AtPANK1 by GFP-fusion experiments (H. M. Whitney and
A. G. Smith, unpublished observations). CoA itself is found in all cellular
compartments, and ACP for de novo fatty acid synthesis is in plastids. This
233
multicompartmentation of enzymes involved in pantothenate and CoA synthesis in plant cells implies that there must be transporters present, responsible for shuttling intermediates of these pathways between the different
compartments. A transporter for CoA into mitochondria has been reported
for potato mitochondria (Neuburger et al., 1984). A complete understanding
of pantothenate metabolism in plants will therefore require the study of these
transporters.
V. MAJOR DIFFERENCES TO EUBACTERIA AND

OTHER PROTOTROPHIC ORGANISMS
The chief differences between plant pantothenate biosynthesis and that in
other organisms rest in the enzymes responsible for ketopantoate reduction
and for the synthesis of -alanine.
A. KETOPANTOATE REDUCTASE
Ketopantoate reductase is responsible for the reversible reduction of

ketopantoate to generate D-pantoate in the eubacterial pantothenate pathway.
This small monomeric protein is a prototypical short-chain ketoreductase,
using NADPH as a cofactor (Frodyma and Downs, 1998a; Shimizu et al.,
1988). The mechanisms of the enzyme from E. coli (Zheng and Blanchard,
2000a, 2000b, 2003) have been well characterised and the three-dimensional
structure elucidated (Matak-Vinkovic et al., 2001) as described in Section III.C.
No close homologue of this enzyme exists in plants, and as yet no enzyme has
been shown to unambiguously carry out the required catalytic activity (see
Section III.C). Nonetheless, [14C]-valine feeding experiments to pea-leaf disks,
which resulted in the incorporation of radiolabel into ketopantolactone and
pantolactone (Jones et al., 1993) clearly demonstrates that plants are able to
catalyse this transformation. Nonetheless, given the fact that other enzymes
have been characterised that can catalyse this reaction (described in detail in
Section III.C), it remains possible that a direct homologue of bacterial KPR is
not present in plants.
B. -ALANINE SYNTHESIS
Five distinct pathways for -alanine synthesis have been described in the
literature. In bacteria, this molecule is synthesised via the action of the
enzyme L-aspartate -decarboxylase, encoded by panD (Fig. 14 route (i);
Williamson and Brown, 1979). This enzyme is unusual in that it is a member
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Fig. 14. -Alanine synthesis routes. Three major routes to -alanine have been
characterised. Decarboxylation of aspartate (i) to generate -alanine is required to
support pantothenate biosynthesis in bacteria in the absence of supplementation of
growth media with uracil. Uracil can be catabolised to generate -alanine via a threestep pathway starting with uracil dehydrogenase (ii); however, this pathway is unable
to support prototrophic growth. The third pathway, the FAD-dependent oxidation of
spermidine (iii) to generate 3-aminopropanal, was identified by White et al. (2003) in
yeast, and is the most likely candidate for formation of -alanine in plants.
of the small class of pyruvoyl-dependent decarboxylases (Van Poelje and

Snell, 1990). This class of enzymes, which includes S-adenosylmethionine
decarboxylase and phosphatidylserine decarboxylase, contains a covalently
bound pyruvoyl group that is involved in catalysis, rather than the more
widespread pyridoxal- or FMN-dependent decarboxylases. This is formed
via the intramolecular rearrangement of a serine residue to form an ester
intermediate (Ramjee et al., 1997; Schmitzberger et al., 2003) before chain
cleavage yields a dehydroalanyl residue that is then hydrolysed to form the
pyruvoyl group. The pyruvoyl group binds to the amino acid substrate to
form an iminium intermediate, which can then stabilise the negative charge
formed as a result of decarboxylation. The enzyme is observed in all eubacteria but is not present in any eukaryotic organisms.
235
In the yeast, S. kluyveri and other organisms including D. melanogaster

(which uses -alanine in cuticle formation) -alanine is made from the
degradation of pyrimidines. This three-step pathway leads from uracil via
dihydrouracil to -ureidopropionate and finally -alanine and urea (Fig. 14
route (ii)). As described above this pathway is also found in E. coli but
without supplementation with excess pyrimidine bases, the flux through the
pathway is insufficient to support prototrophic growth by panD mutant cells.
The enzymes for both the synthesis and degradation of pyrimidines to alanine are present in plants (Zrenner et al., 2006), and this pathway has
recently been characterised (Zrenner et al., 2009). Walsh et al. (2001) have
reported the overexpression and functional characterisation of the last enzyme required for this pathway in Arabidopsis, -ureidopropionase. Walsh
and coworkers showed that the protein was functionally active, suggesting
that plants are able to synthesise -alanine via this route. Zrenner et al.
(2006) report a detailed consideration of the three enzymes noting that the
enzymes are all predicted to be localised to different organelles and therefore
(as for pantothenate biosynthesis) there is a requirement for membrane
transport of intermediates. They also considered the expression pattern of
these genes and showed that they were highest in senescent leaves. However,
homozygous knockout mutants of all three genes are able to grow and show
no phenotypic differences to WT plants (Zrenner et al., 2009). Given the
severe growth defects observed as a result of mutation in the pathway from
pantothenate to CoA, this suggests that pyrimidine degradation is not essential for -alanine, and thus pantothenate biosynthesis in plants.
In S. cerevisiae, an alternative pathway via oxidation of spermine and
spermidine to generate 3-aminopropanal is observed (Fig. 14 route (iii)).
White et al. (2001) demonstrated that a putative amine oxidase, FMS1 was
rate-limiting for both -alanine and pantothenate accumulation in this organism. Deletion of this protein leads to auxotrophs requiring either alanine or pantothenate to support growth, demonstrating that this enzyme
catalyses the only pathway for -alanine in this organism. In contrast, overexpression of the protein led to excretion of pantothenate into the growth
media. In fact, deletion of any of the four genes for the polyamine biosynthetic pathway also leads to auxotrophs requiring pantothenate or -alanine
supplementation for growth. Landry and Sternglanz (2003) subsequently
characterised the recombinant protein and showed that it was a flavoprotein
binding FMN in a 1:1 stoichiometry and that the enzyme oxidised spermine
to generate spermidine and 3-aminopropanal. The structural basis for formation of these products rather than 1,3-diaminopropane and 4-(4-aminobutyl)-aminobutanal was resolved by Huang et al. (2005), who determined
the structure of FMS1 in complex with spermine and FMN. The second
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product of this oxidation, spermidine is also required for hypusine biosynthesis (an essential modification of eukaryotic initiation factor 5A
(Chattopadhyay et al., 2003). Indeed, Chattopadhyay et al. (2008) have
subsequently shown that over 50% of spermidine is used for this purpose
alone in polyamine-limiting conditions.
The polyamine biosynthetic pathway in plants is distinct from that in other
eukaryotes in that direct decarboxylation of arginine is the principle pathway
for putrescine formation. Arginine decarboxylase forms agmatine, which is
hydrolysed to form N-carbomoylputrescine and the precursor putrescine
(Fuell et al., 2010). In other eukaryotes, decarboxylation of ornithine yields
putrescine directly, but the required enzyme ornithine decarboxylase is not
found in Arabidopsis. Formation of spermidine is essential for plant survival
(Imai et al., 2004b), and a homozygous double knockout of the two spermidine synthase genes is embryo-lethal. It is possible that a lack of the hypusine
modification on eIF5A in plants has a similar effect to that observed in other
eukaryotes. In contrast, spermine synthase is not essential (Imai et al., 2004a)
and this therefore puts the role of polyamine degradation in formation of alanine for pantothenate into question. In plants there are five homologous
isoforms of the polyamine oxidase FMS1 (PAO15) that have distinctive
tissue (Takahashi et al., 2010), and substrate (Fincato et al., 2011) specificities. Three of these isoforms catalyse the oxidation of spermidine in addition to spermine (see Table III, reproduced from Fincato et al., 2011), indeed
one isoform PAO3 catalyses this oxidation preferentially over that of spermine. Fincato et al. also demonstrated that both PAO2 and PAO4 formed
putrescine from spermidine (rather than diaminopropane) suggesting that
the second product was 3-aminopropanal. This therefore supports a role for
these enzymes in -alanine biosynthesis. Of particular note is that this
particular isoform is most strong expressed in flowers, in a similar pattern
to that observed for other enzymes of pantothenate biosynthesis (Takahashi
et al., 2010). The crystal structure of the homologous polyamine oxidase
from maize has recently been elucidated in complex with spermidine suggesting that this protein will function in this pathway (3kpf, 3lir, 3ku9).
The second step for -alanine production in yeast is the oxidation of 3aminopropanal to form -alanine directly. White et al. (2001) initially
assigned this function to one of the wide range of aldehyde dehydrogenases,
but subsequent work demonstrated that, in fact, only two of the available
dehydrogenases, Ald2p and Ald3p, are required (White et al., 2003).
Tylichova et al. (2010) reported the structural and functional characterisation of two aminoaldehyde dehydrogenases from Pisum sativum. These
NAD-dependent dehydrogenases have broad substrate specificity and catalyse the oxidation of a wide range of C3 and C4 aminoaldehydes to the
TABLE III
Kinetic Constants of Substrate Oxidation by Recombinant Polyamine Oxidases (PAO) from Arabidopsis
AtPAO1
kcat (s
Spermine
Spermidine
1
2.5 0.4
0
AtPAO2
1
Km (mM)
kcat (s
120 20
0
4.2 1.2
4.6 1.5
AtPAO3
1
Km (mM)
kcat (s
270 30
409 40
1.7 0.5
3.4 1.4
AtPAO4
1
Km (mM)
kcat (s
580 40
274 50
4.6 1.0
0.1 0.03
Km (mM)
47 5
139 18
The data clearly show that while spermine is the preferred substrate for isoforms 1, 2, and 4, spermidine is the preferred substrate for AtPAO3 though this can
still oxidise spermine. The values given are the mean standard error of at least three independent experiments.Reproduced from Fincato et al. (2011).
238
TABLE IV
Kinetic Parameters of AMADH2 from Pisum sativum
PsAMADH2
Ligand
4-Trimethylaminobutanal
4-Guanidinobutanal
3-Aminopropanal
4-Aminobutanal
Km (mM)
Vmax (nmol s 1 mg 1)
Vmax/Km (relative)
21
7
10
29
140
78
190
57
0.35
0.59
1
0.01
Measurement of enzyme activity of P. sativum AMADH2 with different substrates clearly shows
that it preferentially oxidises 3-aminopropanal to form -alanine, with a threefold selectivity
over 4-trimethylaminobutanal, the preferred substrate for the other isoform AMADH1. Thus
AMADH2 is a good candidate for involvement in -alanine production in plants via oxidation
of polyamines (Fig. 14, route (iii)). Data reproduced from Tylichova et al. (2010).
corresponding o-amino acids. Both isoforms oxidised 3-aminopropanal

to generate -alanine; however, one isoform (PMADH2) preferentially oxidised this substrate as shown in Table IV reproduced from Tylichova et al.
(2010) suggesting that this pathway of -alanine synthesis is fully extant in
plants.
Two further pathways to -alanine, catabolism of propionate (Kupiecki,
1957) or cyanide (Maruyama et al., 2000), have also been proposed, but there
is little molecular evidence to support their involvement in the plant pantothenate pathway. The conversion of propionate (via propionyl CoA) to alanine was first observed by Kupiecki in humans in 1957, the final step,
transamination of malonic semialdehyde with -aminobutyric acid has subsequently been observed by Arst (1978) in Aspergillus nidulans. The existence
of such a pathway in plants has been tested by the use of radiotracers in
Limonium latifolium (Plumbaginaceae). This plant accumulates -alanine
betaine as an osmoprotectant. Feeding with [2-14C]-propionate led to accumulation of radiolabelled -alanine (Rathinasabapathi, 2002), but it was not
established whether this was then incorporated into pantothenate.
Subsequent feeding studies by Duhaze et al. (2003) have shown with a series
of feeding experiments that the pathways of polyamine and uracil degradation can also contribute to the cellular pool of -alanine in this organism. The
final pathway, that of cyanide detoxification, is dependent upon the initial
reaction of cyanide with cysteine to form cyanoalanine, which can subsequently be decarboxylated and hydrolysed to form -alanine (Bruskova
et al., 1988; Maruyama et al., 2000). There is no evidence to suggest that
this pathway is a significant source of -alanine in the absence of unusually
high concentrations of cyanide.
239
In conclusion, there are a several pathways to -alanine in different

organisms, but it seems most likely that, as in yeast, polyamine degradation
provides the major source of -alanine for pantothenate biosynthesis in
plants.
C. DIFFERENCES IN THE ARCHAEAL PATHWAY
The pantothenate biosynthesis pathway in the archaea is distinct from that in

all other organisms. The earliest attempt to reconstruct this pathway was that
of Genschel (2004) who used a sequence-database mining approach to identify candidate proteins to catalyse the formation of pantoate (KPHMT and
KPR) but this was not able to identify direct homologues of either PS or
PanK. Chromosomal proximity was therefore used as a marker to identify
candidate proteins for these two activities. Linkage to the gene for KPHMT
pointed to two conserved archaeal proteins (COG1701, a protein of unknown function and COG1829, a predicted kinase). These were also linked
to the chromosomal loci for candidate bifunctional PPCS/PPCDC.
The function of the putative PPCS and PPCDC was confirmed by Kupke
and Schwarz (2006) who purified the isolated candidate CoaB and CoaC
domains of the archaeal Dfp protein. As expected both domains were functionally active although the archaeal protein catalyses the CTP-dependent
formation of 40 -phosphopantothenoylcysteine as observed for the eubacterial proteins (rather than ATP as observed for eukaryotic homologues). The
authors also demonstrated that the second domain catalysed the oxidative
decarboxylation of PPC to form 40 -phosphopantetheine, although key residues implicated in the mechanism of the plant enzyme are missing in
this protein, including the cysteine residue involved in the oxidation/reduction cycle.
Ronconi et al. (2008) performed the first characterisation of a candidate
archaeal PS from Methanosarcina mazei. The overexpressed protein was
inactive and did not exhibit PS activity on its own. Addition of E. coli
PanK, however, led to formation of 40 -phosphopantothenate. Ronconi
et al. suggest that this is due to strong product inhibition of their candidate
PS by pantothenate, which is relieved when the production concentration is
depleted by the action of PanK. On the basis of predominant formation of
ADP in their assays and isotope exchange experiments, they concluded that
the archaeal PS was an ADP-forming synthetase.
Yokooji et al. (2009) developed an alternative hypothesis following their
overexpression and purification of a candidate PanK from Thermococcus
kodakaraensis, TK2141. PanK activity could not be detected in cell-free
lysates of this organism, and although TK2141 had PanK activity, it was
240
Fig. 15. Pathway to 40 -phosphopantothenate in archaea. In archaea, pantothenate synthetase and pantothenate kinase are absent. Instead, pantoate is phosphorylated by pantoate kinase to generate 4-phosphopantoate and this is then a substrate
for phosphopantothenate synthetase, which generates 40 -phosphopantothenate
directly.
more effective as a pantoate kinase (Vmax sevenfold higher) suggesting that

this was the true activity. Yokooji et al. then investigated the linked TK1686
gene and demonstrated that this protein catalysed the ATP-dependent ligation of 4-phosphopantoate and -alanine to form 40 -phosphopantothenate
directly (with accompanying formation of AMP) in contrast to the pathway
in other organisms (Fig. 15). These enzymes are the homologues of those
identified by Genschel (2004), and the difference in observed behaviour is
therefore surprising. They hypothesised that the observation of phosphopantothenate synthesis by Ronconi et al. (2008) might have been due to low
levels of pantoate phosphorylation by E. coli panK.
VI. ENGINEERING THE PATHWAY

The importance of pantothenate both as an essential nutrient and as a
commodity chemical in its own right means that considerable efforts have
been made to engineer the pathway to enhance pantothenate concentrations
in bacteria and in plants. About 4000 tonnes of pantothenate is produced
annually for cosmetics, vitamin preparations, and feed additives
(Vandamme, 1992). Current production is by bulk chemical synthesis and
this requires optical resolution to separate the L-isomer from the biologically
active D-isomer. Since free pantothenate is unstable, most commercial preparations are in the form of calcium pantothenate (for feeds) or the more
241
stable alcohol form (panthenol). Both panthenol and other various forms of
pantothenate are absorbed by animals and then subsequently converted to
the acid form. BASF (http://www.products.basf.com) reports that vitamin
demand is increasing by 4% annually. To meet this demand other production
routes are sought either to substitute for the chemical synthesis or to complement it.
A. ENGINEERING THE PATHWAY IN BACTERIA
Microorganisms such as E. coli produce 15 times more pantothenate than

their cellular requirements. Because of this, there have been many attempts to
tap this product through fermentation processes (Marx et al., 2002; Sahm
and Eggeling, 1999). Studies of the whole pathway in both E. coli and the
industrially important C. glutamicum have been undertaken with a view to
improving the yields of pantothenate from industrial scale fermentation. PS
is highly expressed in E. coli and this suggests that the supply of one of its
substrates is limiting. Powers and Snell (1976) proposed that pantoate was
limiting, whereas evidence for limitation by -alanine has also been reported
(Cronan, 1980). Subsequently, Jackowskii and Rock (1981) demonstrated
that while -alanine was limiting for pantothenate synthesis, neither compound was limiting for CoA synthesis, due to regulation of subsequent steps.
A -alanine auxotroph was used to determine the limiting concentration
(8 mM) of exogenous -alanine required to trigger excretion of excess pantothenate and it was calculated that 15-fold more pantoate is formed than is
required for either pantothenate or CoA production. Elischewski et al. (1999)
subsequently demonstrated that overexpression of KPR leads to enhanced
excretion of pantothenate suggesting that this enzyme rather than KPHMT
is the limiting enzyme in this branch of the pathway.
In C. glutamicum, -alanine supply is also the limiting factor for pantothenate production and supplementation with -alanine leads to an increase in
pantothenate production (Dusch et al., 1999). This effect could also be
obtained by transformation with C. glutamicum panD (though intriguingly
not with E. coli panD, possibly due either to poor protein expression or to
slow post-translational activation of this enzyme). Subsequent studies have
shown that the chief limitation on the flux through the pantoate branch is
competition between the pantothenate pathway and that for the branched
chain amino acids (Fig. 16). Many of the enzymes in this pathway are
multifunctional and function in branches for both isoleucine and valine
synthesis. Overexpression of both panB and panC, both with and without
overexpression of the ilvB, ilvC, ilvN, and ilvD gene products, leads to an
increase in pantothenate production (Sahm and Eggeling, 1999). More
242
Fig. 16. Relationship between pantothenate biosynthesis and the branched chain
amino acid biosynthesis pathway. The branched chain amino acid pathway uses a
common set of enzymes (shown in bold) to transform pyruvate and 2-ketobutyrate
(generated by threonine deaminaseTDH, encoded by ilvA) into valine and isoleucine respectively. In C. glutamicum, the second common enzyme in this pathway,
acetohydroxyacid isomeroreductase (AHIR, encoded by ilvC) is required both to
form -ketoisovaleric acid (-KIVA) and to reduce ketopantoate to pantoate. Competitive substrate inhibition by the other substrates reduces the flux through the
pantothenate biosynthesis pathway. Deletion of TDH is therefore an effective strategy to increase the flux through the pathway. DHAD, dihydroxyacid dehydratase;
AHAS, acetohydroxyacid synthase.
243
interestingly, deletion of ilvA (only in the isoleucine pathway) leads to an

increase in pantothenate production possibly due to an increased cellular
concentration of -KIVA. It has subsequently been shown that AHIR
(encoded by ilvC) is in fact the only functional enzyme for ketopantoate
reduction in this organism and so this deletion would also serve to allow an
increased flux through the pantothenate pathway (Merkamm et al., 2003).
The cellular requirement for branched chain amino acids for protein
synthesis is generally much higher than that for CoA and this is reflected
by the large intrinsic difference in the fluxes of the two pathways
(Chassagnole et al., 2002, 2003). From -KIVA the flux to valine is 10-fold
that to pantothenate. Overproduction of pantothenate in prokaryotes is
probably fundamentally limited at this branchpoint; there are limitations in
the supply of the 5,10-methylene tetrahydrofolate cofactor for KPHMT, and
overexpression of panB leads to considerable glycine accumulation (from the
action of serinehydroxymethyltransferase to form 5,10-methyleneTHF). Engineering in glycine catabolising enzymes and feeding with serine may be able
to overcome this limitation. Competition for AHIR between the two pathways will also limit the flux through the pathway in C. glutamicum. This
could be overcome by expression of a dedicated KPR.
B. ENGINEERING THE PATHWAY IN PLANTS
There is increasing interest in using plants for biotransformation processes,

and industrial support of vitamin phytofarming has been increasing in the past
decade (Herbers, 2003). For example, BASF has already invested about 600
million in developing capacities to increase vitamin production in plants.
This follows successful expression of enzymes for -carotene (provitamin A)
biosynthesis in the rice endosperm achieving a yield of about 2 mg g 1
-carotene to produce the golden rice (Ye et al., 2000). This has been followed
by extensive refining and optimisation, for example, by tissue specific promoters, such that Diretto et al. (2007) report total carotenoid levels of 114 mg g 1
dry weight in potato tubers. These studies have included both feeding and
genetic modification-based approaches.
A range of approaches, similar to those explored in bacterial systems, have
been taken to enhance the production of pantothenate in plant systems.
Rathinasabapathi and Raman (2005) first investigated which branch of the
pantothenate biosynthetic pathway was limiting for formation of pantothenate in L. latifolium, Lycopersicon esculentum, Phaseolus vulgaris, and Citrus
x paradisi by direct measurement of pantothenate in leaves after treatment
with either pantolactone or -alanine. Only treatment with pantolactone was
observed to increase concentration of total pantothenate. In order to confirm
244
that this observation was not due to poor uptake of -alanine, they demonstrated that 14C-labelled -alanine was accumulated by the leaves but that
this accumulation was not accompanied by additional production of
pantothenate.
In contrast, -alanine appears to be limiting for pantothenate production
in tobacco. Fouad and Rathinasabapathi (2006) generated transgenic tobacco in which E. coli L-aspartate decarboxylase was expressed constitutively.
This led to a 1.2- to 4-fold increase in cellular -alanine, an accompanying
3.2- to 4.1-fold increase in pantothenate and an accompanying increase in
total amino acid concentration. This expression was also found to enhance
the plants ability to cope with heat-induced stress. This work has now been
extended by Fouad and Altpeter (2009) who investigated the effect of transplastomic expression on the heat-stress tolerance of these plants. In this case,
the gene for E. coli panD was inserted into the chloroplast genome by sitespecific recombination and the resultant plants were shown to have increased
tolerance to high temperature stress. This phenotype was, however, attributed to -alanine rather than to increased production of pantothenate.
In Arabidopsis, Jonczyk et al. (2008) demonstrated that while expression of
the gene for PS is necessary for pantothenate biosynthesis, it is not limiting.
Transformation of WT plants with a construct for constitutive cytosolic
expression of E. coli panC did not lead to an increase in plant pantothenate
biosynthesis although it did lead to a 300-fold increase in the measured PS
activity. Chakauya et al. (2008) investigated the effect of transformation of
oil seed rape (B. napus) with the genes encoding either PS (panC) or KPHMT
(panB) under the control of the strong constitutive CaMV35S promoter. No
significant differences in the concentration of pantothenate could be observed for the transgenic lines expressing either Arabidopsis or E. coli PS.
Expression of E. coli KPHMT, however, led to a 1.5- to 2.5-fold increase in
pantothenate levels in leaves, flowers, siliques, and seed. This suggests that in
oil seed rape, as in the organisms studied by Rathinasabapathi and Raman
(2005), the supply of pantoate is limiting for pantothenate biosynthesis.
However, the limited increase in pantothenate production suggests that the
supply of both substrates for pantothenate synthesis is tightly controlled in
this organism.
VII. CONCLUSION
Despite being one of the simplest vitamins, with only four enzymatic steps in
its biosynthesis, the pathway leading to pantothenate in plants still remains
unclear. Only two of the four enzymes of the bacterial pathway, KPHMT
245
and PS, have been shown to be unambiguously present in plants. The

involvement of polyamine degradation now appears likely to be the pathway
for of -alanine synthesis: the enzyme is functionally active in Zea mays, and
the best candidate for a competing pathway, pyrimidine degradation, is nonessential. The final remaining step required for pantothenate synthesis, the
reduction of ketopantoate to pantoate can be catalysed by the eubacterial
acetohydroxyacid reductoisomerase (AHIR), and plant AHIRs have now
been overexpressed and characterised. If these enzymes do catalyse this
transformation to any degree, then this reaction would be sufficient to
support pantothenate biosynthesis.
Little is known about the regulation of the pathway to pantothenate in
plants, the pathway is undeniably a low flux pathway, and enzyme activities
in crude cell lysates can only be detected after enrichment of the lysates by
first isolating the organelles in which the enzymes are found. Nonetheless,
although this makes it challenging to study the pathway, prospects for
developing biotransformation systems in plants with enhanced levels of the
vitamin are more straightforward, particularly by reference to studies in
bacteria (Chakauya et al., 2006).
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AUTHOR INDEX
A
Abadia, A., 129
Abadia, J., 129, 130
Abdel-Ghany, S.E., 176, 184
Abdou, M., 2324
Abell, C., 204205, 211216, 217218, 219
220, 222223, 225, 233234, 244,
245
Abelmann, K., 123, 124
Abeydeera, N.D., 52
Abian, J., 129
Abiko, Y., 208
Abrams, G.D., 216
Abrassart, D., 165166
Abratti, G., 7374
Abrigo, E., 2122
Acharya, C.B., 2324
Adamek-Swierczynska, S., 65
Adams, J.L., 2122
Adams-Phillips, L., 166
Adato, A., 5556, 5758, 6869
Agrawal, V., 222
Agrimi, G., 171172
Agyei-Owusu, K., 5657
Aharoni, A., 45, 5558, 6869
Ahlfors, R., 166
Ahn, I.P., 6667, 7172
Ahn, T.K., 56
Ahrazem, O., 17
Aida, R., 2021
Ajjawi, I., 5254, 6667
Ajlani, G., 7
Akita, M., 161, 174175, 176, 182
Akiyama, K., 15
Akiyama, T., 236
Akkermans, A.D., 5051, 52, 55, 66
Alabadi, D., 1213
Alamgir, M., 2122
Alan, L.K., 49
Alawady, A., 19, 20
Al-Babili, S., 8, 1819, 2022, 23, 24
Albert, A., 225, 227228
Albertini, A.M., 175176, 180, 184
Alberts, A.W., 204205
Alejandro, S., 228229, 230
Alfred, S., 1415
Ali, M.S., 2122
Alix, J.H., 232
Allendorf, D., 100
Allred, C.C., 213
Almeida, R.S., 130
Alonso, J.M., 222, 227

Altmann, B., 164165
Altpeter, F., 244
Aluru, M., 22
Alvarez-Fernandez, A., 130
Alva, V., 127
Alworth, W.L., 96, 98, 121
Amat, J.A., 6
Ammelburg, M., 127
Amorim, E.P., 24
Amorim, V.B.O., 24
Amrhein, N., 49, 55, 66, 6869
Anantha, S., 161, 174175
Andaluz, S., 129, 130
Andersen, P.E., 161162, 172173
Anderson, I., 56
Anderson, M.D., 6263, 213
Anderson, R.M., 179
Angelini, R., 236, 237
Angenot, L., 3940, 5455
An, R., 163164
Anyango-Oyunga, M., 24
Aogaichi, T., 118, 119121, 126
Apel, K., 187188
Apel, W., 20
Apetrei, R., 173, 176, 177180
Araki, N., 8
Araki, T., 207, 208
Arango, J., 8, 1819, 23
Araujo, W.L., 62
Arditti, J., 174175
Argyrou, A., 106
Arigoni, D., 49, 55, 66, 6869
Arilla-Luna, S., 127, 128
Arisi, A.C.M., 181182
Arite, T., 1516
Arjunan, P., 59
Armstrong, C.M., 165166
Armstrong, G.A., 8
Arnoux, P., 12
Arora, A., 70
Arrese-Igor, C., 64
Arst, H.N., 238
Asada, K., 154155
Asai, M., 117
Asai, S., 133
Ashihara, H., 177179
Ashurst, J.L., 216, 219220, 222223, 225,
233, 245
Askin, D., 6
Atienza, S.G., 2223
258
AUTHOR INDEX
Atomi, H., 239240

Augustin, M., 121, 126
Auldridge, M.E., 15, 20
Ausubel, F.M., 185
Avenson, T.J., 56
AverYanov, A.A., 132
B
Bacher, A., 9899, 100, 101102, 103, 104,
105, 106, 107, 109, 110113, 114
119, 120, 121122, 123, 124125,
126, 127, 131, 132, 134, 135, 136
Bachmann, H., 13
Bachmann, L., 111
Bachmann, M., 56
Backhausen, J.E., 154155, 170171
Backhaus, R.A., 11
Back, S.L., 62
Bacot, K.O., 108110, 117118, 128
Badur, R., 63, 70
Bai, L., 7, 1011, 12, 19, 20, 22
Bailey, S., 5
Bainbridge, K., 15
Baisakh, N., 2122
Baker, J.C., 6162
Baker, L.J., 48
Balakumar, P., 4445
Balamurug, K., 4143
Ballesteros, J., 2223
Ball, L.J., 101102
Ballottari, M., 56
Balmer, Y., 55
Banas, A., 70
Bandarian, V., 98
Bandyopadhyay, A., 2122
Ban, N., 5557
Barajas, P., 222, 227
Barboriak, J.J., 206207
Barbosa, A.C., 5051, 72
Bar, C., 8, 1819, 20, 23
Barends, T.R., 133134, 136
Barile, M., 43, 54, 128
Barone, M., 64
Bar-Or, C., 21
Barra, D., 208209
Barras, F., 176
Barrero, J.M., 1213
Barrette, T.R., 810
Barrick, J.E., 5556, 5758
Barroso, J.B., 70
Barsby, T., 211213, 244
Bartel, B., 213
Bartels, D., 63, 70
Bartels, K., 111
Bartley, G., 67
Bartosz, C.E., 235
Bartsch, M., 166167, 185
Bartunik, H.D., 111
Bassi, R., 56, 12, 13

Bass, J., 165166
Battaini, F., 166
Bauer, S., 101102, 127
Baumlein, H., 129, 130
Baur, R., 110
Bautor, J., 166167, 185
Bauwe, H., 63
Baxter, C.J., 68, 70, 71
Beach, R.L., 118121, 126
Beale, M.H., 15
Becard, G., 15
Bechoff, A., 24
Becker, U., 241
Beck, S., 206207
Bednarczyk, K., 65
Bednarek, P., 166167, 185
Beer, S.V., 132
Beevers, H., 63
Begley, T.P., 4041, 45, 4647, 48, 5051,
5253, 55, 57, 6465, 161, 174175,
205206, 227
Beguerie, S., 16
Behal, R.H., 62
Beisel, K.G., 20
Belanger, F., 5051, 52, 66
Belenky, P., 159160, 167168
Belles-Boix, E., 166
Belles, J.M., 227228, 229
Bell, H., 41
Bellido, M.L., 2021
Bellussi, L., 208
Belmonte, M., 178, 179
Beltran, J., 23
Bender, D.A., 206207
Bennett, E.M., 5253
Bennett, G.N., 181
Ben-Shaul, Y., 111, 112113, 115116
Bent, A.F., 166
Berberich, T., 236
Bergaentzle, M., 157, 158159, 161
Berglund, T.L., 179
Bergmuller, E., 178179
Bermel, W., 101102
Bernacchia, G., 63, 70
Bernasconi, P., 64
Berny-Seigneurin, D., 7
Berros-Rivera, S.J., 181
Berry, S., 7374
Bertram, J.S., 7071
Bessman, M.J., 166167
Bettendorff, L., 3940, 48, 5455
Beveridge, C.A., 1516
Bewell, M.A., 165166
Beyer, P., 810, 1819, 2022, 23, 24
Bick, J.A., 180
Bieganowski, P., 159160
Biesecker, L.G., 66
Bieseler, B., 228
AUTHOR INDEX
Binder, M., 241
Binder, S., 224
Biou, V., 64, 224
Birch, C.D., 174
Birch, L.M., 214216
Bisaccia, F., 66
Bisseling, T., 5051, 52, 55, 66
Bitsch, R., 7
Bitterman, K.J., 179
Bjorkman, O., 56
Blaesse, M., 228
Blake, C.J., 157, 161
Blanchard, J.S., 106, 214216, 217218, 233
Blanco, E.A., 171172
Blanco-Portales, R., 2021
Blasesse, M., 227, 228
Blass, J.P., 4344
Blazquez, M.A., 1213
Blevins, D.G., 130
Bligny, R., 164
Block, M.A., 7
Blondeau, K., 127
Bloom, A.J., 170171, 185
Blundell, T.L., 214216, 217218, 219220,
222223, 225, 233234
Bock, R., 20
Bocobza, S., 45, 5558, 6869
Bogan, K.L., 156157, 158160, 168170,
179, 181
Boger, P., 20, 64, 224
Bohmer, T., 41
Bohm, V., 7
Boiteux, S., 5051, 72
Boldt, R., 235
Bonetta, D., 1415
Bonomi, H.R., 111112
Booker, J., 1516
Booker, S.J., 4647, 5051, 176, 180
Bor, M., 164, 173
Boronat, A., 6465
Bos, M., 4041
Botran, L., 13
Bottger, L.H., 175176, 180, 184
Bouchereau, A., 238
Bours, R., 16
Boutin, J.P., 1213
Boutry, M., 173
Bouvier, F., 6, 11, 1617
Bouwmeester, H.J., 15, 16, 235238
Bowerman, A.F., 7, 1819, 2223
Bown, D.H., 106, 110111, 118119
Bowsher, C.G., 6162, 70
Boyang, C., 49
Brace, C.S., 165166
Bracher, A., 103
Bradbury, L., 2021, 22
Braden, B.C., 111113
Braga, O., 162
Brand, L.A., 226
Breaker, R.R., 5558, 6869, 128129

Breen, M., 115
Breitenbach, J., 22
Breitenbach, M., 70
Breithaupt, C., 111112
Bremer, J., 208
Brenner, C., 156157, 158160, 161,
167170, 179, 181
Brew, B.J., 162
Brewer, P.B., 15
Briggs, A.G., 166
Bringer-Meyer, S., 6465, 181
Briozzo, P., 236238
Britton, G., 6, 11, 20
Brizio, C., 128
Brook, J.M., 214216, 233
Brosche, M., 166
Brown, G.M., 98, 105, 225, 233234
Brown, O.R., 176
Browse, J., 226
Brugger, R., 13
Bruniard, J.M., 7374
Bruning, J.C., 133
Bruskova, R.K., 238
Brutnell, T.P., 7, 1011, 12, 19, 20, 22
Buchanan, B.B., 55
Buck, D., 214216, 233
Buckler, E.S., 7, 1011, 12, 19, 20, 22
Bugg, T.D.H., 16
Bugos, R.C., 56
Buhr, E.D., 165166
Bunik, V.I., 62
Burgdorf, L.T., 133134
Burkhardt, P.K., 8
Burkle, L., 49, 55, 66, 6869
Burow, M., 68, 70
Burrows, R.B., 105
Bushman, B.S., 7374
Butterworth, R.F., 4344, 72
Byerrum, R.U., 174175, 178179, 180
Bykhovsky, V.Y., 180
Bykova, N.V., 165
Byrne, K.L., 5051, 72
C
Caballero, J.L., 2021
Cage, D.A., 13, 1415
Cahill, S.M., 106
Calabrese, J.C., 101102, 121, 123
Calcagnile, V.M., 66
Camara, B., 6, 11, 1617
Campobasso, N., 4647, 48, 6465
Campos, N., 6465
Camp, P.J., 62
Cantero, A., 8, 6465
Capell, T., 22
Capobianco, L., 66
Cappello, A.R., 66
259
260
AUTHOR INDEX
Carell, T., 133134, 136

Carlson, T.J., 108110, 128
Carlton, J.M., 6
Carreras, A., 70
Carrisi, C., 66
Carver, T.L.W., 185187
Casati, P., 164165
Castegna, A., 66, 171
Cauerhff, A.A., 111113
Cavallani, D., 208
Cavanagh, C.R., 7, 1819, 2223
Cazzaniga, S., 13
Cazzonelli, C.I., 7, 1819, 20, 2223
Ceballos, H., 23
Chabregas, S.M., 52
Chabret, C., 208209
Chaggar, S., 2122
Chai, C.L., 10, 228229
Chai, M.F., 163164
Chakauya, E., 211213, 244, 245
Chaki, M., 70
Chamnongpol, S., 185187
Chander, S., 22
Chang, A.K., 64
Changeux, J.P., 3940
Chang, S.I., 64
Chang, S.Z., 161
Chang, Y.C., 106, 108
Chang, Y.J., 6465
Chantraine, F., 3940, 48
Chaouch, S., 164, 183, 187188
Charnikhova, T., 15, 16
Chassagnole, C., 222, 241243
Chatterjee, A., 45, 4647, 5051, 55, 57
Chattopadhyay, M.K., 235236
Chatwell, L., 106, 109
Chaudhary, N., 1819
Chaudhuri, B.N., 214216
Chavarriaga, P., 23
Chaykin, S., 174
Cheah, M.T., 57
Chen, A., 66
Chen, C.T., 235
Cheng, G., 5253
Cheng, X., 49, 55, 66, 130
Chen, H.C., 106, 110111
Chen, H.L., 4344
Chen, H.M., 222, 227
Chen, J., 115, 163164
Chen, L., 21, 2223
Chen, Q.J., 163164
Chen, S.C., 106, 108
Chen, Y.M., 810, 163164, 180
Che, P., 6263
Cherest, H., 231232
Chernikevich, I.P., 65
Chernov, B.K., 100
Chetrit, P., 173, 180181
Cheuk, R., 222, 227
Chimento, A., 66
Ching, K.H.L., 211
Chipman, D.M., 59
Chiragdze, D.Y., 222223, 233234
Chistensen, K.C., 159160, 167168
Chiu, H.J., 4647, 48, 5253, 6465
Cho, H.S., 17
Choi, D., 17
Choi, J.D., 64, 7374
Chong, J.L., 165166
Chow, T., 1415
Christmann, A., 1415
Christou, P., 22
Chua, N.H., 165166
Chu, B., 5051, 52, 66
Chubb, A.C., 19, 20
Chung, I.K., 5253, 55, 66
Chung, M.C.M., 233
Cicchillo, R.M., 176
Cichon, M., 133134
Ciulli, A., 222223
Cline, J.K., 38
Cline, K., 13
Cohen-Addad, C., 224
Cohen-Chalamish, S., 128129
Cohen, K.O., 24
Cohen, N., 4143
Colabroy, K., 161, 174175
Colburn, S., 1819
Colella, M., 128
Coles, M., 123, 124, 127
Collingro, A., 171
Cong, L., 2223
Cong, R., 236
Conley, T.R., 70
Conlin, B.J., 67, 21
Connolly, E.L., 130
Contreras-Moreira, B., 127, 128
Cooper, J., 65
Copeland, L., 63, 64
Copeland, W.C., 171172
Cordoba, E., 6465, 7374
Cornard, J.P., 6
Cornelissen, M., 166
Cornic, G., 173
Corpas, F.J., 70
Corso, T., 43
Cosman, K.M., 67, 21
Cossetto, S.B., 19, 20
Couee, I., 70
Coumans, B., 3940, 5455
Cousins, A.B., 185
Coxon, K.M., 211213, 216, 244, 245
Craig, P.O., 111113
Crisp, P., 19, 20
Cronan, J.E., 205206, 241
Crossley, R.A., 130
Croteau, R., 8, 7071
Cruz, J.A., 56
AUTHOR INDEX
Cryle, M.J., 133134, 136
Cui, L.W., 213
Culianez-Macia`, F.A., 226, 227228, 229,
230231, 232233
Cunningham, F.X. Jr., 67, 1011
Curcio, R., 66
Cushman, J., 5253, 6667
Cushman, M., 104, 111113, 114115, 116,
120, 121, 122, 123, 124125,
126, 132
Cuttriss, A.J., 7, 10, 12, 19, 20, 22
Czerniecki, J., 3940, 48, 5455
Czihal, A., 129, 130
D
Dabrowska, Z., 168170, 173
Dahlgran, A.L., 98
Dahmer, M.L., 64, 7374
Dai, L., 132133
DAlexandri, F.L., 6
Daley, M., 1819
DallOsto, L., 13
Dancer, J.E., 216
Dangl, J.L., 185187
Daniels, L., 135136
Danoun, S., 15
Datta, K., 2122
Datta, S.K., 2122
Daugherty, M., 227, 230
Dauk, M., 170171, 173
Davey, M.W., 24
Davidson, F.R., 3
Davidson, P.A., 56
Davidson, R., 206207
Davies, S.L., 212213
Davis, C.R., 3
Davis, E.J., 6263
Day, D.A., 71, 161162, 170171, 232233
De Almeida, A., 13
de Almeida, J.D., 52, 55, 6869
Debey, S., 166167, 185
De Block, M., 166
Debnam, P.M., 63, 70
De Brouwer, D., 166
De Cottet, B.V., 166
Decottignies, P., 55
De Crecy-Lagard, V., 227, 230
De Filippis, G., 66
Defossez, P.A., 165166
Degand, H., 173
de Gara, L., 128, 166
De Graaf, A.A., 6465
de Groot, H.J.M., 6
De Kouchkovsky, Y., 173
De Las Rivas, J., 129
De Leonardis, F., 66
Deli, J., 11
261
DellaPenna, D., 46, 7, 811, 19, 20

Del Ro, L.A., 70
Delumeau, O., 163164
del Val, G., 55
Delvaux, D., 3940
Deng, S., 132
Deng, W.T., 13
Dennis, E.S., 64, 71
Den Otter, F.C., 185, 187188
Denu, J.M., 166167
de Oliveira, R.C., 5051, 72
de Oliveira, R.L., 5051, 72
De Paepe, R., 161, 164, 172, 173174,
180181, 185
De Pauw, E., 3940, 5455
de Pauw, M., 64, 71
de Pinto, M.C., 128
de Rosso, V.V., 6
Detry, O., 3940, 48
Devoe, L.D., 208, 232
Dewdney, J., 185
Dhanaraj, V., 214216, 217218, 225
Dhanoa, P.K., 154155, 163164, 171172
Dhuique-Mayer, C., 24
Diano, A., 243
Diaz, C., 6869
Diaz, G.A., 4143
Dickinson, G., 165166
Diercks, T., 123, 124
Dieuaide-Noubhani, M., 63
Dijkema, C., 63
Dijkwel, P.P., 173, 176, 177180
Dilley, R.A., 12
Di Martino, C., 171172, 178
Dimuzio, E.M., 67, 21
Ding, X., 5051, 7172
Diolez, P., 173
Diretto, G., 24, 243
Disch, A., 7071
Dive, G., 3940
Djuranovic, S., 127
Dogbo, O., 6, 16
Dolce, V., 66
Dolferus, R., 64, 71
Dolnikowski, G.G., 2122
Domagalska, M.A., 16
Dominick, O.C., 171172
Domratcheva, T., 133134
Dong, H., 128, 132
Dong, J., 6162
Dong, X., 185187
Do, P.T., 171172
Dorne, A.J., 7
Dorocke, J.A., 48
Dorrestein, P.C., 47
dos Reis, R.V., 24
dos Santos-Serejo, J.A., 24
dos Santos, V.J., 24
262
AUTHOR INDEX
Douce, R., 7, 49, 52, 62, 63, 64, 154155,

161162, 164, 170171, 224,
231233
Douchkov, D., 129, 130
Douglas, B.J., 111113, 114, 115116
Downs, D.M., 4647, 48, 5253, 55, 6465,
66, 222223, 233
Draczynska-Lusiak, B., 176
Drake, R., 2122
Dressen, U., 63
Drew, M.C., 70
Driscoll, S., 173
Druce, S., 164165
Drueckhammer, D.G., 230231
Dufourc, E.J., 63
Dufour, D., 24
Duggleby, R.G., 5859, 63, 64, 7374
Duhaze, C., 238
Dulinski, R., 65
Dulyaninova, N.G., 180
Dumas, R., 64, 224
Duncan, J.L., 211
Dun, E.A., 15
Dupre, S., 208209
Dupuis, I.I., 64, 70, 71
Duque, P., 165166
Durner, J., 64, 224
Dusch, N., 241243
Dutilleul, C., 164, 172, 173, 180181, 185
Dyar, R.E., 224225
E
Ealick, S.E., 45, 4647, 48, 5051, 5253, 55,
57, 6465, 227
Eberhardt, S., 98, 100, 103, 105, 117118,
123, 124, 126, 131
Echave, J., 113
Echt, S., 101102
Eggeling, L., 241243
Eggers, U., 110
Eide, D.J., 130
Eirich, L.D., 133134
Eisenberg, D., 217218
Eisenreich, W., 98, 101, 103, 104, 105, 106,
107, 109, 120, 121122, 123, 124
125, 126, 131, 132, 134, 135, 136
Eker, A.P.M., 133134
El Amrani, A., 70
Elias, B., 3940
Elischewski, F., 241
Ella, E.S., 64
Elliott, K.A., 236
El Moualij, B., 3940, 48
Elthon, T.E., 154155, 170171, 173
Elvehjem, C.A., 156157, 204205
Emeric, G., 224
Emes, M.J., 63, 70
Emmerlich, V., 154155, 170171
Ermler, U., 5859

Espinosa-Ruiz, A., 227228, 229
Estevez, J.M., 8, 6465
Estler, M., 241
Evans, J., 181
Evans, R.R., 64, 7374
Eyobo, Y., 177
F
Faber, M., 24
Fachmann, W., 94
Fairhurst, S.A., 47
Faith, C.B., 49
Falk, K.L., 226, 232233
Fanceschetti, M., 236
Fang, J., 10
Fanning, K.J., 22
Fan, W., 185187
Fanwick, P.E., 114, 115
Farias, L.P., 52, 55, 66, 6869
Farrell, M., 227, 230
Fatland, B.L., 213
Fayol, V., 211212
Federico, R., 236, 237
Feicht, R., 117118, 124
Fei, Y.J., 208, 232
Fenstermacher, D.K., 208
Fermas, S., 15
Fernandez, M.G.S., 12, 19, 22, 2324
Fernandez-Ocana, A., 70
Fernie, A.R., 15, 49, 55, 62, 66, 6869, 70,
71, 171172, 173, 176, 177180
Ferreira, S., 6869, 70
Ferro, M., 21
Feth, F., 174
Fey, S., 6869, 70
Fidler, A., 106, 109, 121, 126
Fiehn, O., 130
Fiermonte, G., 66, 171172
Fincato, P., 236, 237
Finkelstein, J., 204205
Finnegan, E.J., 19, 20
Fischer, M., 98, 101102, 103, 104, 105, 106,
107, 109, 110113, 114115, 114,
116118, 120, 121, 122, 123,
124125, 126, 127, 131, 132
Fites, R.C., 5354
Fitzpatrick, T.B., 49, 55, 66, 6869
Flachmann, R., 63, 70, 181182
Flechner, A., 63
Fleming, G.R., 56
Fleming, J.C., 4143
Floss, H.G., 106, 110111, 118119, 135
Flugel, R.S., 209211
Flugge, U.I., 163, 167171
Flyvbjerg, H., 161162, 172173
Folkers, K., 204205
Fontecave, M., 176, 180, 184
AUTHOR INDEX
Foor, F., 98
Forlani, G., 64
Fornasari, M.S., 113
Fouad, W.M., 244
Fox, H.M., 207, 208
Foyer, C.H., 154155, 161, 164, 166,
170171, 172, 173174, 180182,
185187, 188
Frachisse, J.M., 164
Frago, S., 126127
Frankel, N., 113
Frank, R.A., 3940, 58
Franssen, M.C.R., 15
Franzblau, S.G., 115
Frederich, M., 3940, 5455
Frederick, R.L., 17
Freeling, M., 52
Frey, A., 13
Frick, D.N., 166167
Fricker, M.D., 68, 70
Fridovich, I., 173, 176, 180181
Friedlein, A., 13
Friedrich, W., 211212
Frodyma, M.E., 222223, 233
Frommer, W.B., 232
Frost, G.M., 174
Fry, P.C., 207, 208
Fuchs, R.T., 5556
Fuell, C., 236
Fufezan, C., 12
Fujii, H., 1415
Fujii, I., 110111
Fujiki, Y., 6263
Fujimori, T., 162, 163164
Fujiwara, M., 4445
Fukuda, A., 64
Fukusaki, E., 166167
Fuller, L., 161
Fung, P., 1415
Funk, C., 38
Furey, W., 59
Fu, Z.M., 1516
Fu, Z.Y., 12, 19, 22
G
Gadal, P., 164165
Gadjev, I., 187188
Gadrinab, C., 222, 227
Gagneul, D., 238
Gai, J., 6465
Gakiere, B., 155
Galhardo, R.S., 5152, 66
Galili, G., 181182
Galione, A., 165166
Galizzi, A., 180
Gallagher, C.E., 12, 19, 22
Gallais, S., 164
Galland, F., 208209
263
GallardoGuerrero, L., 6
Gallego, F., 6465
Galluccio, M., 128
Galpaz, N., 21
Gamborg, O.L., 216
Ganapathy, V., 208, 232
Gangolf, M., 3940, 48, 5455
Gantt, E., 67, 1011
Gao, F., 4143
Garavaglia, S., 180
Garcia-Limones, C., 2021
Gardestrom, P., 165, 167170, 172173
Gardner, P.R., 176
Garin, J., 21
Garmier, M., 173, 180181
Garnier, P., 166
Garrido, A., 6
Garrido-Fernandez, J., 6
Garvin, D.F., 67, 21
Gas, E., 8, 18
Gaskin, D.J., 130
Gassen, H.G., 181182
Gass, N., 64
Gautam, R., 101102
Gazarini, M.L., 6
Gazzaniga, F., 159160, 161, 167168
Gechev, T.S., 187188
Geddes, W.F., 6667
Gehring, A.M., 209211
Geigenberger, P., 213
Gelb, B.D., 4143
Gelfand, M.S., 5556, 128129
Gelpi, E., 129
Gemel, J., 6162
Genoud, D., 157, 161
Genschel, U., 219220, 221, 222, 225, 231
232, 233234, 239240, 244
Genty, B., 154155
Georg, G.I., 114, 115
Gerard-Hirne, C., 173
Gerdes, S., 161, 174175
Gerhardthaase, S., 111112
Gerhardt, S., 111113, 115, 121, 123, 124,
126
Gerisch, B., 70
Gerstenschlager, I., 105
Geserick, C., 235
Ge, X., 166167
Ghashghaie, J., 173
Ghazal, H., 222
Gholson, R.K., 174, 175176, 180
Giacometti, G.M., 12
Giancaspero, T.A., 128
Giavalisco, P., 15
Gibbs, M., 164165
Gibon, Y., 213
Gibson, G.E., 4344
Gichuki, S.T., 24
Gigliobianco, T., 3940, 5455
264
Gilmore, A.M., 12
Giorgetti, A., 12
Gitschier, J., 211, 226
Giuliano, G., 19, 24
Glaab, J., 154155, 185
Glagotskaia, T., 64
Glas, A.F., 133134, 136
Gobbato, E., 166167, 185
Goddard, P., 1516
Godoi, P.H., 5152
Gogorcena, Y., 129
Gohlke, J., 164165
Golbik, R., 59
Golda, A., 45, 5354, 6566
Goldbaum, F.A., 111113
Golding, A., 70
Golding, G.B., 6869
Goldman, I.D., 4143
Golova Iu, B., 100
Gomez-Casati, D.F., 164165
Gomez-Gomez, L., 17
Gomez, M.D., 17
Gomez-Rodrguez, M.V., 70
Gomez-Roldan, V., 15
Gong, X., 6162
Gonthier, A., 211212
Gonzalez, E.M., 64
Gonzalez-Guzman, M., 228229, 230
Good, A.G., 64, 71
Goodwin, T.W., 56
Goodyear, G.H., 204205
Goral, V., 161, 174175
Goryachenkova, E.V., 238
Goss, R., 12
Goto, F., 176177, 179180
Gouesbet, G., 70
Gouia, H., 164, 183, 187188
Goulet, C., 15
Goussot, M., 15
Gout, E., 164
Goyer, A., 45
Graham, D.E., 98, 134, 135136
Graham, I.A., 228229, 230
Granell, A., 17
Grant, R.S., 162
Gray, G.R., 6869
Gray, J.E., 164, 177178, 179
Graziani, G., 166
Graziewicz, M.A., 171172
Green, P.J., 5556, 5758, 6869
Greiner, A., 224
Griffith, M., 6869
Griffiths, A.E., 176
Griffith, T., 174175
Grigiene, J., 161162, 172173
Grill, E., 1415
Grimm, B., 19, 20
Grimshaw, C., 70
Grisar, T., 3940, 48, 5455
AUTHOR INDEX
Grishin, N.V., 127
Grochowski, L.L., 98
Grolle, S., 6465
Gromes, R., 180
Gross, C.J., 208
Grosse, H., 163, 167171
Grossman, A.R., 56
Grosso, E.D., 208
Groten, K., 164, 173
Grove, B.C., 5556, 5758, 6869
Grove, T.L., 4647, 5051
Grundy, F.J., 5556, 57
Grunwald, D., 231232
Grusak, M.A., 2122
Gryczka, C., 129, 130
Guarante, L., 165166
Guddat, L.W., 64, 7374, 224
Guerinot, M.L., 130
Guern, J., 164
Guerra, D.J., 226, 232233
Guerra-Lopez, D., 135136
Guevara, D., 6869
Guillemin, G.J., 162
Guillon, B., 55
Guimaraes, B.G., 111112
Guirard, B.M., 205206
Gulyai, I.E., 3940, 5455
Gunning, B., 7
Gunyuzlu, P.L., 225, 235238
Guo, A.L., 71, 128, 132
Guo, R., 22
Guo, X.L., 10
Guo, Y.Q., 22
Gurmu, D., 106
Gusarov, I., 57
Guse, A.H., 165166
Gutierres, S., 173
Gutlich, M., 181182
Guyonvarch, A., 222, 241243
Gweon, H.S., 216, 219220, 223, 225
H
Haase, I., 106, 110113, 114115, 116,
117118, 121
Hadwiger, L.A., 174
Haferkamp, I., 171
Haft, D.H., 135
Hagedorn, P.H., 161162, 172173
Hagenimana, V., 24
Hager, A.G., 12, 7374
Hager, J., 161, 164, 172, 173174, 180181,
183, 185, 187188
Hakim, A.M., 43
Hamblin, M.T., 2324
Hamill, J.D., 174
Hanada, A., 1516
Hand, D.B., 8
Handler, P., 159
AUTHOR INDEX
Hanes, J.W., 45, 48, 55
Hanfrey, C.C., 236
Hanhart, C., 4849
Hanna-Rose, W., 179
Hannich, M., 206207
Hansen, R.G., 206207, 211212
Han, Y., 164, 183, 187188
Hanzawa, Y., 236
Hao, Q., 235236
Harada, K., 166167
Hara, N., 162, 180, 181
Harbinson, J., 154155
Harders, H.D., 110
Harding, M., 66
Harding, S.A., 163164
Haris, A., 212213
Harjes, C.E., 7, 1011, 12, 19, 20, 22
Harper, A.E., 157
Harris, R.A., 48, 6263
Harris, S.A., 204205
Hartig, E., 175176, 180, 184
Hartmann, D.J., 211212
Hartmann, M.D., 127
Harvey, R.A., 118, 121
Hashida, S.N., 155, 162, 163164, 176177,
179180
Hashimoto, T., 155, 161, 174175, 176, 180,
181, 182
Hasselmann, C., 157, 158159, 161
Hata, H., 224225
Hattori, S., 224225
Haudenschild, C.D., 222
Haurogne, K., 15
Havaux, M., 56
Hawkins, A.R., 103, 104, 105
Hayaishi, O., 155
Hayakawa, H., 100
Hayashibe, E., 178
Hayashi, H., 236
Hayashi, M., 164, 178180
Hayflick, S.J., 211, 226
Hazell, A.S., 4344, 72
Hazra, A., 45, 55
Heathcote, P., 175176, 180, 184
Heazlewood, J.L., 66, 71
Heber, U., 163, 168170
Heeren, G., 70
He, G.Y., 2223
Heineke, D., 163, 167171
Heinlein, C., 173174
Heintz, D., 11
Heldt, H.W., 163, 167171, 184
Heller, C., 222, 227
Hemmerich, P., 118119
Henderson, L.M., 155, 157, 158159,
174175, 208
Henderson, S.W., 43
Henkes, S., 63, 70
Henkin, T.M., 5556, 57
265
Hennig, J., 71
Hentschel, V., 7
Henze, K., 63
Herald, V., 71
Herbers, K., 243
Herguedas, B., 127
Hermoso, J.A., 127
Hernandez-Acosta, P., 226, 227, 228,
230231, 232233
Heroux, M., 43
Herz, S., 98, 100, 103, 131
Hessels, J.K.C., 133134
Heupel, R., 170171
Heuser, F., 181
He, X.Y., 2223
Heyes, M.P., 181
He, Z.H., 2223
Higa, A., 129
Hilker, D.M., 4041
Hille, J., 173, 176, 177180
Hiller-Sturmhofel, S., 43
Hill, R.E., 6465
Hill, S.A., 62
Himmeldirk, K., 6465
Hinchliffe, E., 2122
Hirabayashi, T., 162, 163164, 179180
Hiromasa, Y., 6162
Hirosue, E., 166167
Hirschberg, J., 10, 19, 20, 21
Hix, L.M., 7071
Hjerno, K., 6869, 70
Hodges, M., 164165
Hodges, T.K., 64
Hoezel, H., 206207
Hoferichter, P., 163, 167171
Hoffart, L.M., 176
Hofmann, D., 20
Hofte, M., 132
Hoglund, A.-S., 1819
Hohmann, S., 54
Holaday, D., 204205
Holdsworth, M.J., 177178, 179
Hollander, I., 105
Hollmann, J., 7
Holmes, K., 130
Holocher, K., 12
Holtgrefe, S., 154155, 164165, 170171
Holtzapffel, R., 71
Hong, H.K., 165166
Hong, Z.Q., 63
Horgan, R., 56, 7
Hori, T., 20
Hornero-Mendez, D., 2223
Horn, M., 171
Horton, P., 56
Horwitt, M.K., 157
Hosokawa, Y., 175176, 180
Hossain, M.A., 64, 154155
Hothorn, M., 180
266
AUTHOR INDEX
Howe, J.A., 3, 23
Howells, R.M., 2122
Howitt, C.A., 7, 1819, 2223
Hsu, P.-C., 130
Huang, H.M., 4344
Huang, J., 164, 178180
Huang, L., 165166, 179
Huang, Q., 235236
Huang, W., 208, 232
Huang, Z.J., 10
Hube, B., 130
Huber, R., 101102, 106, 109, 111113,
114115, 116, 121, 123, 124, 126,
127, 228
Hubner, G., 59, 64
Hug, E., 64
Hughes, D.E., 208
Hugueney, P., 18
Hunt, L., 155, 164, 174, 177178, 179
Hunziker, W., 13
Huq, E., 18
Hutzenlaub, W., 118119
Hyashi, K., 5455
I
Iacopetta, D., 66
Iba, K., 8
Igamberdiev, A.U., 165, 167170, 172173
Iglesias, A.A., 164165
Ilg, A., 2021
Illarionov, B., 98, 104, 105, 107, 111113,
114, 115, 120, 121122, 123,
124125, 126, 131, 132
Imai, A., 236
Imai, K., 96
Imai, S., 165166, 171172, 178179
Imanaka, T., 239240
Inaba, K., 65
Indiveri, C., 128
Inoue, T., 214216, 218, 219, 222223, 233
Inze, D., 166, 185188
Isaacson, T., 10, 19, 20
Ishikawa, K., 166167
Ishikawa, S., 1516
Ishil, N., 207, 208
Ishizawa, K., 238
Ismail, A.M., 64
Ismond, K.P., 64, 71
Issakidis-Bourguet, E., 164, 183, 187188
Itami, T., 162, 164, 176177, 179180
Itokawa, N., 65
Ito, M., 6263
Iwai, K., 6667
Iwami, K., 5354
Iwashima, A., 47, 48, 6566
Izawa, N., 64
Izmailov, S.F., 238
J
Jabrin, S., 231232
JackowskiI, S., 232, 241
Jackowski, S., 205206, 211, 226
Jackson, B.J., 165
Jacobsen, N.E., 98
Jacob-Wilk., 52
Jadid, N., 11
Jaenchen, R., 135
Jagoe, M., 7, 226
Jahnke, S., 20
Jaillard, D., 168170
Jakobs, C., 70
Jakobs, W.P., 6667
Jakoby, M., 129, 130
Jambunathan, N., 166167
Jansen, F.J., 6
Jarvik, T., 6
Jaspers, P., 166
Jemiola-Rzeminska, M., 12
Jenkins, A.H., 4041
Jensen, D., 1415
Jiang, B., 1516
Jimenez, L.F., 8
Jin, G., 111113, 115
Job, D., 224
Johnson, M.A., 211, 226
Johnson, M.P., 56
Johnston, J.L., 213
Johnston, M.L., 6162
Jomantas, J., 128
Jonczyk, R., 219220, 221, 222, 231232,
239240, 244
Jones, C.E., 214216, 233
Jones, J.D.G., 185187
Jordan, B.R., 179
Jordan, D.B., 101102, 108110, 117118,
121, 123, 128
Jordan, F., 5455, 59
Joseph, L.M., 13
Joshi, M., 120, 122, 124125
Jouan, C., 3940, 48
Jouanin, L., 181182
Jourdain, A., 231232
Journet, E.-P., 63, 170171
Joyard, J., 7, 21
Julliard, J.H., 49, 52, 224225
Jung, B., 171
Jung, G., 227, 228
Jung, S.M., 7374
Jurgenson, C.T., 45, 47, 5051, 57
K
Kabira, J., 24
Kaeberlein, M., 165166
Kairies, N., 123, 124
Kaiser, J.T., 103, 104, 111113, 121, 132
AUTHOR INDEX
Kaiser, W.M., 154155, 185, 187188
Kakefuda, G., 64
Kaku, K., 64
Kalbin, G., 179
Kalinowski, J., 241243
Kameda, K., 208
Kamiya, Y., 8, 15
Kandianis, C.B., 7, 1011, 12, 19, 20, 22
Kandlbinder, A., 154155, 185
Kaneko, M., 100
Kaneko, Y., 48
Kanellis, A.K., 7071
Kangasjarvi, J., 166
Kang, M.K., 64
Kang, Y.W., 17
Kaplan, N.O., 205206
Kapran, I., 2324
Karim, M.A., 211
Karlsen, J., 41
Karthikeyan, S., 101102, 112113, 127
Kasajima, I., 162, 164
Kashiwabara, P.M., 52, 55, 6869
Kashkar, H., 133
Kasimova, M.R., 161162, 172173
Kasten, S.A., 6162
Katagiri, H., 96
Katahira, R., 177179
Kataoka, M., 233
Katoh, A., 155, 161, 174175, 176, 182
Kato-Noguchi, H., 64, 71
Kato, T., 236
Katzenmeier, G., 100
Katzin, A.M., 6
Kawaide, H., 8
Kawai, F., 117118, 121
Kawai, K., 64
Kawai-Yamada, M., 162, 163164, 176177,
178180
Kawasaki, Y., 47, 48, 5253
Kaya, E., 133134, 136
Keasling, J.D., 56
Keating, V., 22
Ke, D.Y., 1819
Ke, J., 62, 213
Keller, J., 127
Kellermann, J., 123
Keller, P.J., 106, 110111, 118119, 121, 135
Kelly, D.J., 130
Kelly, G.J., 164165
Kelly, M.J., 101102
Kemter, K., 123, 124, 127
Kendrick, Z.V., 206207
Kennedy, I.A., 6465
Kennedy, M.J., 2122
Kenyon, G.L., 59
Keresztesy, J.C., 204205
Kerr, S.J., 162
Keryer, E., 55
Kessel, M., 108110, 128
267
Kessler, H., 123, 124

Keulemans, J., 24
Khailova, L., 59
Khan, G.L., 225
Khurana, J.P., 1819
Khurana, P., 1819
Kiener, A., 135
Kilkenny, M.L., 233234
Kim, B.R., 6465
Kim, C.J., 222, 227
Kim, C.Y., 214216
Kim, E.H., 1011, 12, 19, 22
Kim, J., 11, 6869
Kim, R.N., 17, 127
Kim, R.R., 120, 122, 124125
Kim, S.H., 6667, 7172, 127
Kim, S.U., 6465, 106, 110111
Kimura, E.A., 6
Kim, W.T., 17
Kim, Y.S., 5253, 55, 66
Kim, Y.T., 7374
King, H.L., 224225
King, J., 216
Kingma, J., 181182
Kinsland, C., 4647, 48, 6465, 205206
Kirchberger, S., 171172
Kirk, J.T.O., 7
Kirk, K., 232
Kirz, A., 5051, 52, 66
Kishimoto, S., 2021
Kishi, T., 117
Kis, K., 98, 99, 110111, 114115, 116
Kitamura, Y., 129
Kitaoka, S., 217218
Klee, H.J., 15, 20
Kleinig, H., 810, 18
Kleinridders, A., 133
Klessig, D.F., 71
Klinke, S., 111113
Klipp, E., 70
Klocke, S., 164165
Klose, K.E., 177
Kloti, A., 8, 2122, 227228, 243
Knaff, D.B., 180
Knapp, S.J., 7374
Knight, J.S., 70
Knorzer, O., 224
Kobayashi, M., 100
Kobayashi, Y., 165166
Kochian, L.V., 67, 21
Kochibe, N., 5455
Koch, M., 111112
Koehn, C.J., 204205
Koenig, A., 9899
Kohlen, W., 15, 16
Kohler, P., 117118
Kohnle, A., 9899, 100, 106, 110111
Kolb, H.A., 3940
Kolkman, J.M., 7374
268
AUTHOR INDEX
Koller, A., 55
Kollist, H., 166
Kolotilin, I., 21
Koltai, H., 21
Komeda, Y., 236
Kondo, K., 6667
Kong, D., 49, 55, 66
Konig, S., 64
Konishi, S., 218, 219
Koornneef, A., 185, 187188
Koornneef, M., 4849
Kopecny, D., 236238
Koppchen, S., 20
Kopriva, S., 63
Koretke, K.K., 127
Korn, S., 126, 134, 135, 136
Korotchkina, L., 59
Korte, A., 1415
Koshiba, T., 1415
Koslowsky, S., 178179
Kotaka, M., 103, 104, 105
Koussevitzky, S., 6869
Kowald, A., 70
Kowalska, E., 45, 55, 6869, 70
Kozik, A., 4041, 45, 49, 5253, 54, 55,
6566, 6869, 70
Kozlov, Y.I., 128
Krab, K., 161162, 172173
Kraev, A.S., 100, 128
Kramer, D.M., 56
Kranl, K., 7
Kraut, H., 94, 94
Krebs, C., 4647, 5051, 176
Kreiling, A., 217218
Kreneva, R.A., 57
Kresovich, S., 2324
Kreunen, S.S., 7, 10, 20
Krieger, C., 9899, 100, 101102, 105
Kriek, M., 47
Krishan, P., 4445
Krobitsch, S., 70
Krojer, T., 106, 109, 126
Krook, J., 63
Kruger, N.J., 63
Kruszewski, S.P., 6667
Krut, O., 133
Kugelbrey, K., 110111, 115
Kuhlemeier, C., 64, 70, 71
Kuhne, R., 101102
Kujda, M., 45, 5354, 66, 70
Kumar, P., 101102, 112113
Kundu, L.M., 133134
Kunert, K.-J., 181182
Kuntz, M., 11
Kunz, N., 181182
Kuo, Y.M., 211
Kupiecki, F.P., 238
Kupke, T., 226, 227, 228, 230231, 232233,
239
Kupper, H., 67, 21

Kurnasov, O., 161, 174175
Kursteiner, O., 64, 70, 71
Kusano, T., 236
Kusumi, K., 8
Kuwada, S., 117
Kuzuyama, T., 8
Kwagh, J.G., 64
Kwak, J.M., 5253, 55, 66
Kylen, A., 6566
Kyozuka, J., 1516
L
Labbe, A., 6869
Labischinski, H., 111
Ladenstein, R., 98, 111113, 114115, 116,
123, 124, 126
Laforenza, U., 4143
Lahely, S., 157, 158159, 161
Lahm, H.W., 9899, 100
Lakaye, B., 3940, 48, 5455
Laloi, C., 187188
Lamb, H.K., 103, 104, 105
Lancien, M., 164165
Lan, C.-Y., 130
Landry, J., 235236
Langebartels, C., 185187
Lange, B.M., 8, 7071
Lange, P.R., 235
Lange, S.E., 179
Langlais, P.J., 43
Lang, S., 166
Lapikova, V.P., 132
Laplagne, D.A., 113
Larher, F.R., 238
Larsen, M., 6869, 70
Larsen, P., 6869, 70
Larson, T.R., 228229, 230
Latowski, D., 12
Lattanzio, G., 130
Laufer, A., 181182
Laval-Martin, D.L., 164
Laxa, M., 68, 70
Leaver, C.J., 62, 71
Le, D.T., 7374
Lee, C.Y., 120, 122, 124125, 165166
Lee, D.K., 17
Lee, H.C., 165166
Lee, J., 124
Lee, K.H., 176
Lee, M., 4647, 5051
Leeper, F.J., 3940, 5657, 58
Lee, S., 135, 166167
Lee, Y.H., 6667, 7172
Lee, Y.T., 64
Lefebvre, V., 11
Lehmann, M., 68, 70
Lehrach, H., 70
AUTHOR INDEX
Leibach, F.H., 208, 232
Leibundgut, M., 5557
Leisse, A., 70
Leisse, T.J., 222, 227
Lelandais, C., 173
Lelarge, C., 164, 172, 173, 180181, 185
Lelarge-Trouverie, C., 161, 164, 172,
173174, 180181, 185
Lemaire, S.D., 55
Le Marechal, P., 55
Lenobel, R., 236238
Leon, A., 7374
Leonardi, R., 47, 211
Leon, P., 8, 6465, 7374
Leon-Reyes, A., 185, 187188
Leport, L., 238
Lerner, F., 155, 174, 178
Lesuisse, E., 129, 130
Letisse, F., 15, 243
Leulliot, N., 127
Leung, E.W.W., 224
Leustek, T., 5051, 52, 66, 180
le Van, Q., 106, 110111, 135
Levine, M.N., 6667
Levin, I., 21
Levinson, B., 211, 226
Lewendon, A., 217218
Leyser, C., 15
Leyser, H.M.O., 1516
Leyser, O., 1516
Liang, H., 49, 55, 66
Liao, D.I., 101102, 121, 123
Liaw, S.H., 106, 108
Licciardello, N., 12, 19, 22
Lichtenthaler, H.K., 6465, 7071, 7374
Li, C.L., 10, 165166
Li, F.Q., 810, 1819
Li, G.J., 166167
Li, J.S., 22
Li, J.Y., 1516
Li, K., 6465
Li, L., 67, 21, 2324, 130
Lin, C.H., 106, 108
Lindermayr, C., 164165
Lindgren, L., 1819
Lindhauer, M.G., 7
Lindhurst, M.J., 66
Lindley, N.D., 222, 241243
Lindqvist, Y., 5859
Lingens, F., 105
Ling, H.Q., 49, 55, 66, 130
Lin, H., 1516
Linka, N., 171
Lin, S.J., 165166
Lintig, J., 810
Lin, Y.H., 106, 108
Lipmann, F., 205206
Li, Q.B., 12, 13, 19, 22
Li, S.S., 22
Liu, F., 132133

Liu, H.J., 2223
Liu, J., 68, 70, 71
Liu, L.H., 13, 232
Liu, M., 10
Liu, Q., 16, 235236
Liu, W.P., 22
Li, X.M., 5051, 7172, 128, 132
Li, Y., 4647, 5051, 6869
Li, Z.R., 56
Lobley, C.M.C., 233234
Locato, V., 128, 166
Lockwood, S.F., 7071
Lockyer, M., 103, 104, 105
Loewen, M.C., 15
Loiseau, L., 176
Lois, L.M., 6465
Lombard, C., 24
Longley, M.J., 171172
Lonsdale, D., 40
Lopez, A., 67, 21
Lopez, L.E., 57
Lopez-Millan, A.F., 129, 130
Lopez-Raez, J.A., 16
Lopez, R.C., 17
Lottspeich, F., 63, 70, 100, 123, 126
Loukanina, N., 178
Lowe, D.J., 47
Low, J., 24
Lucca, P., 2122, 243
Luche, D.D., 5152
Ludewig, U., 232
Ludwig, H.C., 111, 112113, 115116
Ludwig, W., 217218
Luethy, M.H., 6162
Lugtenburg, J., 6
Luisi, B.F., 3940, 58
Luit, B., 178, 179
Lukienko, P.I., 72
Lumba, S., 1415
Lupas, A.N., 127
Lu, Q.T., 10
Lu, S., 67, 21
Luttgen, H., 105, 123, 124
Lutz, S., 181
Lu, X.Q., 22
Lykidis, A., 227, 230
Lyman, C.M., 204205
Lythgoe, B., 204205
Lytovchenko, A., 15
M
Maass, D., 1819
Maas, W.K., 217218
MacDonald, M.V., 211213, 244
Machado, C.R., 5051, 72
Mackill, D.J., 64
Maclaren, J.A., 96
269
270
Macrae, T.F., 204205
Ma, C.X., 2223
Madden, R.J., 156157
Maekawa,M., 1516
Maes, N., 7374
Magalhaes, M.L., 106
Magallanes-Lundback, M., 11
Magni, G., 177
Magnuson, K., 205206
Magome, H., 15
Mahalingam, R., 166167
Maiga, I.H., 2324
Mailander, B., 110
Major, R.T., 204205
Makarchikov, A.F., 3940, 5455
Malamy, J., 71
Malergue, F., 208209
Maley, G.F., 96, 117
Malmberg, G., 168170, 173
Mandal, M., 129
Mandel, T., 5556, 5758, 64, 6869
Man, H.M., 154155
Manieri, W., 55
Mann, D.F., 174, 178179, 180
Manoj, N., 227
Mansoorabadi, S.O., 4647
Manstein, D.J., 126127
Mantelli, M., 64
Maras, B., 208209
Marchant, J.S., 4143
Marcheva, B., 165166
Marechal, E., 7
Marinoni, I., 175176, 180, 184
Marion-Poll, A., 1213
Markey, S.P., 181
Markham, R., 24
Markovic, J., 166
Marobbio, C.M., 66
Marquard, C.R., 235
Martin, A., 2223
Martin, D., 3940, 48
Martinez-Gomez, N.C., 4647
Martinez-Julvez, M., 127
Martinez-Ripoll, M., 227228
Martin, F., 208209
Martin, I., 22
Martin, J., 127
Martin, P.R., 43
Martins, F., 47
Martin, W., 8, 63
Martynowski, D., 177
Maruyama, A., 238
Marx, A., 241
Masamoto, K., 8, 20
Mase, K., 133
Mashhadi, Z., 126
Masselon, C., 21
Massucci, M.T., 208209
Masuda, T., 96, 117
AUTHOR INDEX
Matak-Vinkovic, D., 222223, 233234
Mathews, C.K., 66
Mathieu, C.D., 173, 180181
Matsubara, S., 20
Matsuda, M., 65
Matsui, A., 178
Matsumara, H., 218, 219
Matsumura, M.Y., 6
Matsuyama, T., 236
Matusova, R., 15
Matvienko, M., 222
Maul, M.J., 133134, 136
Mauve, C., 161, 164, 172, 173174,
180181, 185
Mavandadi, F., 115
Ma, Y., 1415
Mayer, M., 810
Ma, Z., 115
Maziya-Dixon, B., 22, 23
Mazzucchelli, G., 3940, 5455
McAlister-Henn, L., 173174
McCarty, D.R., 13, 1415, 20
McCaskill, D., 7071
McCourt, J.A., 64, 7374
McCready, R.M., 6566
McCulloch, K.M., 48
McDonald, K., 11, 20
McIntosh, E.N., 214216
Mc Lafferty, F.W., 4647, 6465
McLafferty, F.W., 47
McLeish, M.J., 59
McLennan, A.G., 166167
McLilley, R., 168
McPeek, M.A., 161
McSteen, P., 52
Meacock, P.A., 5051, 52, 54, 55, 66, 72
Medina, J., 19
Medina, M., 126127, 128
Medvedik, O., 179
Meesters, H.A.R., 181182
Meining, W., 111113, 114115, 116, 123,
124
Meir, A., 21
Mellema, S., 64
Melnichenko, N.G., 72
Melzer, M., 63
Menck, C.F., 5052, 55, 66, 6869, 72
Menda, N., 21
Mengel, R., 118119
Meng, X.B., 1516
Menkir, A., 22
Mentzen, W.I., 213
Menya, G., 24
Mercadante, A.Z., 6
Mercer, E.I., 56
Merino, E.F., 6
Merkamm, M., 222, 241243
Merlin, J.C., 6
Merzlyak, M.N., 67, 21
AUTHOR INDEX
Metzlaff, M., 166
Meyer, B., 111
Meyers, B.C., 222
Mhamdi, A., 164, 183, 187188
Mialoundama, A.S., 11
Michael, A.J., 236
Micheli, V., 181
Micol, J.L., 1213
Miege, C., 7
Miernyk, J.A., 6062
Miginiac-Maslow, M., 55
Migliore, M., 24
Miguel, R.N., 222223, 233
Mihalic, J.T., 115
Millar, A.H., 62, 66, 71
Miller, G., 6869
Miller, J.F., 7374
Mimica, J.L., 7, 1819, 2223
Minard, K.I., 173174
Minkov, I.N., 187188
Miranda-Ros, J., 45, 5657
Mironom, A.S., 57
Mironov, A.A., 5556, 128129
Mironov, V.N., 100, 128
Misawa, N., 20
Mishra, P., 230231
Misra, A.N., 6869
Mitchell, E., 175176, 180
Mitchell, H.K., 204205
Mitchell-Olds, T., 222
Mitchell, S., 12, 19, 22
Mitsuda, H., 5354, 6667, 117118, 121
Mitsunaga, T., 6566
Mittler, R., 6869, 187188
Miyamoto, E., 129
Miyatake, K., 217218
Miyazawa, T., 64
Mizote, T., 47
Moche, M., 106
Mock, H.P., 129, 130
Moeder, W., 185187
Moes, D., 1415
Moffatt, B.A., 6869
Molin, W.T., 5354
Moller, B.L., 68, 70
Moller, E.F., 217218
Moller, I.M., 161162, 164165, 167168,
172173
Money, B.P., 6061
Monte, D.C., 24
Monteferrante, C., 175176, 180, 184
Montrichard, F., 163164
Mooney, B.P., 6162
Moore, B., 100101
Morales, F., 129
Moran, N.A., 6
Morera, S., 236238
Morgan, A.F., 206207
Morgunova, E., 111113, 115
271
Mori, S., 130

Mori, Y., 129
Morosinotto, T., 12
Mortl, S., 111113, 114115
Moschou, P.N., 236, 237
Mou, Z., 185188
Mozafar, A., 6667
Mseeh, F., 127
Mucke, U., 64
Muhitch, M.J., 64
Mulder, P., 16
Mulholland, F., 130
Mullen, R.T., 154155, 163164, 171172
Muller, C., 7374
Muller, F., 118, 121
Muller, M., 133134
Muller, N., 49, 55, 66, 6869
Munne-Bosch, S., 7071
Munoz-Blanco, J., 2021
Murakami, T., 207, 208
Murfet, I.C., 1516
Murillo, C., 10
Murphy, K.J., 174
Murthy, U.M.N., 176, 184
Musetti, V., 11
Mutterer, J., 1617
N
Nagano, M., 176177, 179180
Nagayama, K., 64
Nahon, S., 21
Nahvi, A., 5557
Naik, R., 173
Nakajima, K., 5354
Nakamura, S., 231232
Nakano, Y., 217218
Nakayama, H., 47
Nakayama, I., 64
Nakayama, Y., 166167
Nakazawa, T., 47
Nakhasi, H.L., 206207
Nambara, E., 70
Namgoong, S.K., 64
Nam, H.G., 5253, 55, 66
Naquet, P., 208209
Naqvi, S., 22
Narrow, C.M., 206207
Nassar, N., 23
Navarro, D., 43
Navazio, L., 165166
Negri, A., 175176, 180, 184
Neill, S.J., 56
Neims, A., 98
Nelson, O.E., 6667
Nemeria, N.S., 59
Nestel, P., 24
Neuberger, G., 110111
Neuburger, J., 170171
272
AUTHOR INDEX
Neuburger, M., 62, 154155, 161162,

170171, 231233
Neufeldt, E.J., 4143
Neuhaus, H.E., 171172
Nezu, Y., 64
Nghiem, H.O., 3940
Nicewonger, R., 48
Nielsen, E., 64
Nielsen, J., 68, 70, 71
Nielsen, P., 110111
Niere, M., 163
Nievelstein, V., 810
Niitsu, M., 236
Nijhawan, A., 1819
Nikolaev, E.V., 177
Nikolaev, O.N., 132
Nikolau, B.J., 6263, 213
Ning, J., 222
Nishimura, H., 47, 48, 65
Nishimura, M., 63
Nishimura, N., 1415
Nishino, K., 65
Nishino, Y., 65
Nisolle, M., 3940, 48
Nitschke, W., 810
Niwa, Y., 8
Nixon, P.F., 63, 64, 7374
Niyogi, K.K., 56, 11
Njdroge, S.M., 24
Nkeng, P., 11
Noctor, G., 155, 161, 164, 168171, 172,
173174, 180182, 183, 185, 187188
Noda, S., 207, 208
Nonnis, S., 175176, 180, 184
Nordlund, P., 106
Norris, S.R., 810
North, H.M., 13
Nosaka, K., 45, 47, 48, 5253, 55, 66
Novelli, G.D., 205206
Nudler, E., 5556, 5758, 6869
Nuin, P., 6869
Nune, G., 211
Nunes-Nesi, A., 49, 55, 62, 66, 6869,
171172, 173, 176, 177180
O
Obata, T., 68, 70
ODonovan, G.A., 225
Oertli, J.J., 6667
Ogawa, T., 166167
Ohad, I., 10, 19
OHalloran, D.M., 67, 21
OHandley, S.F., 166167
Ohara-Nemoto, Y., 100
OHare, T., 22
Ohlrogge, J.B., 213
Ohlsson, A.B., 179
Ohmiya, A., 2021
Oh, S.H., 163164

Ohtani, T., 20
Okuzaki, A., 64
Olczak, M., 49, 5253, 55, 66, 6869
Oldrogge, J., 226
Oliva, G., 5152
Oliva, N., 2122
Oliveira e Silva, S., 24
Oliver, D.J., 62
Ollagnier-de-Choudens, S., 176, 180, 184
Olney, J.W., 43
Olsen, C.E., 68, 70
Olungu, C., 7374
Onodera, H., 162, 164
Ono, S., 208
Onozuka, M., 47
Ontiveros-Palacios, N., 57
Orendi, G., 173174
Ortholand, J.Y., 224
Orzaez, D., 17
Osago, H., 162, 180, 181
Oschkinat, H., 101102, 124
Osterman, A.L., 127, 161, 174175, 177, 227,
230
Ostrovsky, Y.M., 48
Ostrowska, K., 49, 5253, 54, 55, 6566,
6869, 70
Ottenhof, H.H., 214216, 219220, 223, 225
Ott, K.H., 64
Otto, M.K., 110, 114
Ovadis, M., 67
Overmyer, K., 166
Owori, C., 24
P
Padmasree, K., 170171
Paes, N.S., 24
Pai, E.F., 126127
Pai, H.S., 17
Paine, J.A., 2122
Pajerowska-Mukhtar, K., 185187
Pallardo, F.V., 166
Palloix, A., 11
Pallotta, M.L., 171172, 178
Palmieri, F., 66, 171172
Palta, J.A., 212213
Palva, E.T., 166
Paolillo, D.J., 67, 21
Papacchioli, V., 24, 243
Papini-Terzi, F.S., 52, 66
Paradies, E., 171172
Park, D., 5253, 55, 66
Parker, J.E., 166167, 185
Park, H., 7, 10, 20
Parkhi, V., 2122
Park, H.W., 226, 228229, 243244
Park, M.H., 235236
Park, M.S., 214216
AUTHOR INDEX
Park, P.K., 230231
Park, S., 1415
Parry, A.D., 56, 7
Parthasarathy, M.V., 67, 21
Passarella, S., 43, 54
Patel, D.J., 5657
Patel, H.H., 115
Patel, M.S., 59
Paterami, I., 7071
Paterson, T., 118, 120, 121
Patil, K.R., 68, 70, 71
Patrick, D.A., 115
Paul, D., 52
Pauloski, R.M., 6465
Pawlowski, K., 5051, 52, 55, 66
Paxton, R., 6263
Peapus, D.H., 48
Pebay-Peyroula, E., 224
Pedrajas, J.R., 70
Peleato, M.L., 129
Pellny, T.K., 161, 164, 166, 172, 173174,
180181, 185
Penaganti, A., 166167
Peng, H.P., 70
Peng, T., 6162
Peng, W., 132133
Penuelas, J., 7071
Peres, V.J., 6
Perez-Galvez, A., 6
Persson, K., 111113, 114, 115116
Perumov, D.A., 57, 128
Pestana, K.N., 24
Petersen, J.L., 133134, 136
Peterson, M., 206207
Peter, U., 163, 167171
Pfleiderer, G., 217218
Pfleiderer, W., 118119
Pfundel, E.E., 12
Pham, C., 67
Phan, A.T., 5657
Philippe, J.M., 208209
Phillips, K., 52
Phillips, M.A., 6465
Pichersky, E., 177178, 179
Pieterse, C.M.J., 185, 187188
Pignol, D., 12
Piippo, M., 166
Piletskaya, T.P., 72
Pillot, J.P., 15
Pilon, M., 176, 184
Pilon-Smits, E.A.H., 176, 184
Pineau, B., 173
Pires, O.R., 23
Piros, J.R., 65
Pitari, G., 208209
Pitcher, L.H., 180
Plaut, G.W.E., 96, 98, 117, 118121, 126
Pleiss, J., 5859
Pletnev, A.A., 159160, 167168
273
Plummer, J.A., 212213

Podlepa, E.M., 180
Pogson, B.J., 46, 7, 10, 11, 1819, 20, 2223
Polanuyer, B., 227, 230
Polikarpov, I., 111113
Pollak, N., 163
Pollard, M., 213
Polonskaia, A., 5657
Ponce, M.R., 1213
Pongratz, C., 133
Popov, A., 111112
Portais, J.C., 15
Post-Beittenmiller, D., 213
Potot, S., 4041
Potrykus, I., 8, 2122
Pou de Crescenzo, M.A., 164
Powell, C.A., 219220
Powers, S.G., 214216, 241
Poyner, R.R., 4647
Pradet, A., 64
Praekelt, U., 5051, 52, 55, 66
Praekelt, U.M., 5051, 72
Prasad, L.K., 212213
Prasad, P.D., 208, 232
Pratt, E.F., 204205
Preiss, J., 159
Priault, P., 173
Primerano, D.A., 222
Prioul, J.L., 161, 164, 172, 173174,
180181, 185
Procter, C.M., 130
Puech-Pages, V., 15
Pugin, A., 164
Puhler, A., 241243
Pulido, R., 225
Pun, S., 22
Purko, M., 214216
Puttmer, S., 103
Pye, W., 19, 20
Q
Qian, Q., 10, 1516
Qin, F., 165166
Qin, J., 2122
Qin, X.Q., 13, 15
Qiu, D., 5051, 7172
Quadri, L.E., 209211
Quagliariello, E., 43, 54
Quesada, V., 1213
Queval, G., 155, 161, 164, 168171, 172,
173174, 183, 187188
Quevillon-Cheruel, S., 127
Quinlan, R.F., 12, 19, 22
R
Rachmilevitch, S., 185
Radermecker, M., 3940, 48
274
AUTHOR INDEX
Radloff, R., 5859

Rafikov, R., 57
Raghavendra, A.S., 170171
Rahier, A., 11
Rai, M., 2122
Raines, C.A., 63
Rajalakshmi, R., 206207
Rajan, K., 212213
Rakel, D., 40
Ralph, S.G., 6465
Ralser, M., 70
Raman, S.B., 211212, 231232, 243244
Rambla, J.L., 17
Ramjee, M.K., 225, 233234
Ramos-Onsins, S., 222
Rampling, L., 7, 1819, 2223
Ramsey, K.M., 165166
Ramsperger, A., 121, 126
Randall, D.D., 6062
Randolph, L.F., 8
Rani, K., 15
Rao, D.R., 174175
Rapala-Kozik, M., 45, 49, 5254, 55, 6566,
6869, 70
Raschke, M., 49, 55, 66, 6869
Rasmusson, A.G., 154155, 164165,
167168, 170171, 173
Rathinasabapathi, B., 211212, 231232,
238, 243244
Rausch, T., 180
Ravanel, S., 231232
Rawat, M., 135136
Raymond, P., 63
Rebeille, F., 231232
Reddick, J.J., 4647, 48, 5253, 6465
Redestig, H., 68, 70, 71
Reed, G.H., 4647
Rehana, S., 2122
Rehg, J.E., 211
Reichler, S., 6465
Reindl, A., 6465
Rellan-Alvarez, R., 130
Renard, M., 163164
Rendon, M.A., 6
Rendon- Martos, M., 6
Renganathan, M., 12
Ren, H., 128, 132
Ren, J., 103, 104, 105
Rennenberg, H., 181182
Renou, J.P., 164, 183, 187188
Repsilber, D., 68, 70, 71
Reuke, B., 134, 135, 136
Reumann, S., 170171
Reuveni, M., 21
Revuelta, J.L., 96
Reynolds, J.J., 98
Reyss, A., 164, 173
Ribeiro, A.F., 5051, 52, 55, 66, 6869
Ribeiro, D.T., 52, 55, 6869
Ricard, B., 64
Ricci, V., 41
Richardson, D.R., 129, 130
Richter, G., 9899, 100, 101102, 103, 105,
107, 121, 123, 124, 126, 131
Rieder, B., 171172
Riegler, H., 178179, 235
Riens, B., 163, 167171
Rindi, G., 4143
Ringys-Beckstein, H., 222
Rissler, H.M., 20
Ritsema, T., 185, 187188
Ritsert, K., 114115
Ritz, H., 100, 103
Rius, S.P., 164165
Rivoal, J., 64
Rizzi, M., 180
Roach, P.L., 47
Roberti, M., 128
Roberts, D.A., 163164
Robertson, D., 56
Robinson, G., 168170, 184
Robinson, N.J., 130
Rocheford, T.R., 3, 7, 8, 1011, 12, 1819,
20, 22
Roche, T.E., 6162
Rock, C.O., 205206, 211, 226, 241
Rockel, A., 187188
Rockel, P., 187188
Rodermel, S., 22
Rodionov, D.A., 5556, 128129
Rodlonov, D.A., 177
Rodrigues, A., 1415
Rodriguez-Celma, J., 130
Rodriguez-Concepcion, M., 78, 18
Rodriguez Milla, M.A., 5253, 6667
Rodriguez, P.L., 1213, 228229, 230
Rodriguez-Villalon, A., 8, 18
Roepstorff, P., 6869, 70
Roessner-Tunali, U., 64
Rohdich, F., 104, 105, 107, 131, 132
Rohilla, A., 4445
Rohmer, M., 7071
Roje, S., 127, 128, 155
Rolin, D., 63
Rolland, N., 7, 21
Romero, C., 8
Romisch-Margl, W., 106
Romisch, W., 101, 105, 106, 107, 109, 121,
126, 131
Ronan, P.J., 133134, 136
Ronconi, S., 222, 231232, 239240, 244
Ronen, G., 10, 20
Rontein, D., 63
Rooney, W.L., 2324
Rosei, M.A., 208
Rosen, B.A., 64
Rose, R.C., 208
Rose-Sallin, C., 157, 161
AUTHOR INDEX
Ross, J.J., 1516
Rothlisberger, U., 9899, 100
Roubelakis-Angelakis, K.A., 236, 237
Roughan, G., 213
Rousset, C., 176, 180, 184
Rowan, T., 121
Royuela, M., 64
Ruban, A.V., 56
Rubio, A., 17
Rubio-Moraga, A., 17
Rubio, S., 228229, 230
Rujan, T., 8
Russell, R.M., 2122
Ruyter- Spira, C., 16
Rychlik, M., 211212, 222, 231232, 244
Rychter, A., 168170, 173
Rydstrom, J., 179
Ryrie, I.J., 174
S
Saalbach, I., 129, 130
Sabar, M., 173
Saga, G., 12
Sagor, G.H.M., 236
Saha, B., 212213
Sahl, S.V., 216
Sahm, H., 6465, 181, 241243
Sahoo, G., 2122
Said, H.M., 4143
Saindrenan, P., 164, 183, 187188
Sairam, R.K., 70
Saji, H., 236
Salamini, F., 63, 70
Salazar, B., 23
Saldanha, S.A., 214216, 219220, 222223,
225, 233
Saliba, K.J., 232
Saller, S., 104, 105, 107, 111112, 131, 132
Salmi, M., 6465, 7374
Salome Pais, M., 6869, 70
Salvi, D., 21
Sambaiah, T., 111112, 115
Sanakis, Y., 176, 184
Sancar, A., 134135
Sanchez-Baeza, F., 129
SanchezCasas, P., 71
Sanchez, J.P., 165166
Sandalova, T., 111113, 114, 115116
Sande, A., 41
Sandermann, H., 185187
Sanders, D., 165166
Sandmann, G., 20, 22
Sandoval, F.J., 127, 128
San, K.Y., 181
Santaella, M., 17
Santiago, J., 1415
Santoro, A., 66
Sarett, H.P., 206207
275
Satoh, A., 218, 219

Sato, S., 236
Sato, T., 6263
Sauer, G., 127
Sauer, N., 232
Saunders, A.H., 176, 180
Savage, A.D., 216
Sawaya, M.R., 214216
Saxena, P.K., 216
Sayer, B.G., 47, 6465
Scanlon, M.J., 52
Schaefer, G., 206207
Schaller, S., 12
Schaub, P., 2021
Schauer, N., 68, 70, 71
Scheffzek, K., 180
Scheibe, R., 154155, 164165, 170171
Schenk, G., 63
Scheuring, J., 98, 115, 124
Schieberle, P., 117118
Schiffmann, S., 101102, 103
Schippers, J.H.M., 173, 176, 177180
Schledz, M., 8
Schlichting, I., 133134, 136
Schlicker, S.A., 95, 95
Schmid, D.G., 227, 228
Schmid, K.J., 222
Schmidt-Base, K., 114115
Schmieder, P., 101102
Schmitzberger, F., 214216, 219220, 223,
225, 233234
Schmitz-Esser, S., 171
Schnabele, K., 2021
Schnarrenberger, C., 63
Schneider, G., 5859, 111113, 114, 115116
Schneider, M., 111
Schneider, S., 133134, 136
Schnepple, H., 110, 111, 112113, 115116
Schoffeniels, E., 3940
Scholle, M., 227, 230
Schonheit, P., 135
Schorderet, D.F., 4143
Schorken, U., 6465
Schott, A.K., 121, 123, 124, 126
Schott, K., 111, 115, 123
Schramek, N., 103, 110111, 114115, 116,
117118, 121
Schramm, M., 133
Schroeder, F.C., 47, 5051, 57
Schroeder, J.I., 70
Schroer, K., 181
Schultze, J.L., 166167, 185
Schumann, U., 154155, 163164, 171172
Schurmann, P., 55
Schurr, U., 20
Schuster, M., 103
Schutze, S., 133
Schwab, W., 2021
Schwall, G., 63, 70
276
AUTHOR INDEX
Schwartzberg, P.L., 66
Schwartz, C.A., 23
Schwartz, S.H., 13, 1415
Schwarzkopf, B., 134, 135, 136
Schwarzlander, M., 68, 70
Schwarz, W., 239
Schwender, J., 7071, 7374
Schyns, G., 4041
Scislowski, P.W., 6263
Scolnik, P., 67
Scossa, F., 24
Scott, K.J., 174
Sebela, M., 236238
Sedlmaier, H., 118119, 121
Seifert, J., 181182
Seigneurin-Berny, D., 21
Sekiguchi, M., 100
Selbig, J., 68, 70, 71
Selengut, J.D., 135
Selvaraj, G., 170171, 173
Sempuku, K., 65
Seo, M., 1415
Serganov, A., 5557
Sergeant, M.J., 16
Serrano, R., 227229, 230
Sestini, S., 181
Seto, H., 8
Setterdahl, A.T., 180
Seulberger, H., 96
Sfinchez-Baeza, F., 129
Shaanan, B., 59
Shaner, D.L., 64, 7374
Shanley, M.S., 225
Shapira, M., 5556, 5758, 6869
Shatalin, K., 57
Shaul, O., 181182
Sheehy, J.A., 1819
Shen, W., 170171, 173
Shewmaker, C.K., 1819
Shibata, D., 236
Shibata, K., 208
Shibata, T., 162, 180, 181
Shigeoka, S., 166167
Shih, M.C., 70
Shimamoto, K., 6667
Shimizu, M., 6566
Shimizu, S., 224225, 233
Shimizu, T., 64
Shimizu-Takahama, M., 163, 168170
Shinn, P., 222, 227
Shintani, D.K., 5254, 5556, 5758, 6667,
6869
Shipton, C.A., 2122
Shirano, Y., 236
Shirasu, K., 15
Shlomo, H., 21
Shoji, K., 176177, 179180
Shulaev, V., 187188
Sibanda, B.L., 225
Siddiqua, A., 165166

Siddique, K.H.M., 212213
Sidorov, V., 64
Sieberer, T., 1516
Sieburth, L.E., 17
Siehl, D.L., 64
Silva-Filho, M.C., 52, 55, 6869
Silva, I.D., 213
Silverstone, A.L., 2122
Simkin, A.J., 15
Simms, H.D., 206207
Sinclair, D.A., 179
Sinclair, S.J., 174
Singh, B.K., 64, 7374
Singh, M., 101102, 112113
Singh, S.R., 212213
Singleton, C.K., 43
Sirikantaramas, S., 68, 70
Skatrud, P.L., 234, 236238
Skinner, D.J., 12, 19, 22
Skrede, S., 208
Skriabin, K.G., 128
Slabaugh, M.B., 7374
Slocum, R.D., 235
Smith, A.G., 204205, 211216, 215, 217
218, 219220, 219, 222223, 225,
233234, 244, 245
Smith, A.M., 5556, 57
Smith, C.M., 96, 98, 121, 206207, 208, 209
Smith, D.D., 47
Smith, T.A., 1415
Smythe, G.A., 162
Snedden, W.A., 154155, 163164,
171172
Snegaroff, J., 236238
Snell, E.E., 204205, 214216, 233234, 241
Soberon, M., 57
Sodek, A., 6869
Solairaj, P., 4445
Solovchenko, A.E., 67, 21
Someroski, J.F., 174175
Sommer, A., 3
Somogyi, J.C., 4041
Song, J., 6263, 185187
Song, L., 6869
Song, Q., 43
Song, S., 66
Song, T.M., 22
Song, W.O., 207, 208, 209, 211212
Song, Y., 114, 115
Sonnewald, U., 63, 70, 235
Soole, K.L., 154155, 170171, 173
Sorci, L., 177
Sorefan, K., 15
Sotta, B., 13
Souci, S.W., 94, 94
Souley, S., 2324
Southan, M.D., 64
Sowinski, S.G., 7, 1011, 12, 19, 20, 22
AUTHOR INDEX
Spedaletti, V., 236, 237
Spenser, I.D., 47, 6465
Spoel, S.H., 185187
Spoonamore, J.E., 98
Sprenger, G.A., 6465
Srinivasan, M., 1516
Srivastava, G.C., 70
Stahlberg, K.G., 1819
Stahmann, K.P., 96
Stammers, D.K., 103, 104, 105
Stanley, R.H., 204205
Stapleton, A.E., 7, 1011, 12, 19, 20, 22
Starmann, J., 164165
Starosta, A., 49, 5253, 55, 66, 6869
Stasolla, C., 178, 179
Stebbins, R., 161
Steffen, C., 206207
Steinbacher, S., 101102, 103, 111113, 121,
123, 124, 127, 227, 228
Steinkami, M.P., 4143
Steinmuller, D., 67, 21
Stenmark, P., 106
Stepanova, A.N., 222, 227
Stepanov, A.I., 128, 132
Stepuro, A.I., 72
Stepuro, I.I., 72
Sternglanz, R., 235236
Stevenson, D.K., 222, 227
Stickland, H.G., 218, 219
Stidham, M.A., 64
Stiller, E.T., 204205
Stirnberg, P., 1516
Stitt, M., 63, 70, 168, 213, 235
Stockton, G.W., 64
Stoimenova, M., 154155, 185
Stolz, J., 232
Strack, D., 15
Strand, M.K., 171172
Strand, S., 241
Strauss, E., 205206, 226, 227
Streb, P., 173
Strid, A., 179
Strohm, S., 181182
Stromberg, J.A., 176
Strong, F.M., 156157
Strube, F., 187188
Struys, E.A., 66, 70
Strzalka, K., 12
Stuart, G.R., 171172
Stuurman, J., 64
Stuwe, T., 180
Subramanian, M.V., 64
Subramanian, V.S., 4143
Sudarsan, N., 5556, 5758
Sugantino, M., 214216
Suh, S.C., 6667
Suire, C., 1617
Suitor, C.W., 95
277
Sulmon, C., 70
Sumitomo, K., 2021
Sun, C.H., 10
Sundstrom, M., 5859
Sun, F., 132
Sun, G., 4041
Sun, L., 10
Sun, M., 132
Sun, Q., 165166
Sun, Z., 11
Surdin-Kerjan, Y., 231232
Susin, S., 129
Suss, K.H., 63
Sutak, R., 129, 130
Su, Z., 1516
Suzuki, Y., 117118, 121
Swanson, R.A., 166
Sweetlove, L.J., 68, 70, 71
Swennen, R., 24
Szal, B., 168170, 173
Szostkiewicz, I., 1415
Szyniarowski, P., 53, 54, 6566
T
Tabata, S., 236
Tabor, C.W., 235236
Tabor, H., 235236
Tachezy, J., 129, 130
Taconnat, Y., 164, 183, 187188
Tada, Y., 185187
Tadege, M., 71
Tadera, K., 117118
Tadmor, Y., 21
Tadros, M., 810
Tagliaferri, E.G., 157, 161
Taheri, P., 132
Takagi, T., 238
Takahama, U., 163, 168170
Takahara, K., 162, 163164, 176177,
179180
Takahashi, H., 155, 162, 163164, 176177,
178180
Takahashi, J.S., 165166
Takahashi, T., 236
Takahashi, Y., 236
Takeda-Kamiya, N., 15
Takii, Y., 5354
Tallaksen, C.M., 41
Talukdar, A., 115
Tamaoki, M., 236
Tamoi, M., 166167
Tamura, H., 218, 219
Tamura, K., 164, 178180
Tanaka, Y., 64
Tan, B.C., 13, 1415
Tang, G.W., 2122
Tang, J.Y., 10
278
Tang, Y., 166167
Tan, J., 2122
Tan, S., 64, 7374
Tanumihardjo, S.A., 3, 23, 24
Tan, Y., 170171, 173
Tao, H.G., 207, 208
Tarighi, S., 132
Tarr, J.B., 174175
Tartaglini, E., 4143
Tauriainen, A., 166
Tavazza, R., 24, 236, 237, 243
Tavernier, E., 164
Tavladoraki, P., 236, 237
Taylor, D.C., 170171, 173
Taylor, H.F., 1415
Taylor, N.L., 71
Taylor, S.V., 4647, 6465
Tcherkez, T., 173
Tchikov, V., 133
Tedeschi, G., 175176, 180, 184
Teige, M., 63
Tej, S.S., 222
Teller, J.H., 214216
Terpstra, P., 181182
Terwilliger, T.C., 214216
Tevini, M., 67, 21
Teyssier, E., 7
Tezuka, T., 164, 178180
Thangathirupathi, A., 4445
Thauer, R.K., 135
Thelen, J.J., 6162
Thiry, M., 3940, 48
Thomas, L., 49
Thompson, A.J., 16
Thompson, P., 6162
Thore, S., 5557, 56
Thornalley, P.J., 4445
Thorpe, T.A., 178
Tian, L., 11
Tice, A.B., 67
Tiedemann, J., 129, 130
Tiessen, A., 213
Tiliney-Bassett, R.A.E., 7
Tilton, G.B., 226
Timm, D.E., 48
Tittmann, K., 59
Tjaden, J., 171172
To, A., 13
Tobin, A.K., 6162
Toda, T., 65
Todd, A.R., 204205
Todd, K.G., 43
Todisco, S., 171
Tohge, T., 68, 70
Tohme, J., 23
Toki, S., 162, 164
Toledo-Ortiz, G., 18
Tolosa, E.A., 238
AUTHOR INDEX
Tomita, H., 239240
Tomlins, K., 24
Tong, L., 166167
Tonhosolo, R., 6
Torchetti, E.M., 128
Toriyama, K., 64
Torres, M.A., 185187
Toulokhonova, L.V., 180
Toyn, J.H., 225, 234, 235238
Tranel, P.J., 7374
Trauth, A.U., 171172
Traut, T.W., 225
Trautwein, A.X., 175176, 180, 184
Treharne, K.J., 56
Trenerry, V.C., 157, 161
Trenkamp, S., 62
Trentmann, O., 171
Trucco, F., 7374
Truda, M., 47
Truesdail, J.H., 204205
Truffault, V., 123, 124, 127
Truong, M., 11, 20
Tsegaye, Y., 5254, 6667
Tsuchiya, M., 162, 180, 181
Tsujihara, T., 47, 48
Tuinstra, M.P., 2324
Tu, L., 176
Tumaney, A.W., 213
Tunc-Ozdemir, M., 5556, 5758, 6869
Tuominen, H., 166
Turkan, A., 6162
Turk, D., 114115
Turnbull, C., 1516, 19, 20
Turner, J.B., 208
Turner, N.C., 212213
Turner, W.L., 163164
Tuttle, L.C., 205206
Tylichova, M., 236238
U
Uchimaya, H., 155, 162, 163164, 176177,
178180
Uebele, M., 227, 228
Ueda, K., 155
Ueda, Y., 166167
Uehara, Y., 65
Uenohara, K., 161, 174175, 176, 182
Uhlemann, K., 59
UlIanov, A.V., 100
Ulryck, N., 127
Umehara, M., 1516
Umezaki, H., 207, 208
Undas, A.K., 16
Underwood, B.A., 3
Urbany, C., 171
UR Rahman, L., 129
Utermohlen, O., 133
AUTHOR INDEX
V
Vagelos, P.R., 204205
Vainstein, A., 67
Valderrama, R., 70
Vallabhaneni, R., 7, 8, 1011, 12, 1819,
2021, 22, 7071
Vanacker, H., 185187
Van Aken, O., 164, 173
Van Beilen, J.B., 181182
Van Breusegem, F., 164, 166, 173, 187188
Van Camp, W., 185187
Vandamme, E.J., 240241
Vandekerchove, J., 810
Van den Bergh, I., 24
van den Brulle, J., 104, 132
Vanderauwera, S., 166, 187188
Vanderbeld, B., 163164
van der Plas, L.H.W., 63
Vander Velde, D., 114, 115
van de Sande, K., 1516
Van de Steene, N., 166
van de Velde, J., 133134
Van Eck, J., 67, 21
van Jaarsveld, P., 24
van Kammen, A., 5051, 52, 55, 66
Van Loon, A.P., 4647, 6465
Van Loon, L.C., 185, 187188
Van Montagu, M., 185187
Van Norman, J.M., 17
Van Poelje, P.D., 233234
van Sluys, M.A., 5152, 55, 66, 6869
van Tilbeurgh, H., 127
van Vliet, A.H., 130
Vedel, F., 173
Vega, D.R., 111112
Velazquez-Campoy, A., 126127
Velikovsky, C.A., 111113
Ventrella, A., 171172
Verduyn, C., 166
Vergara, G.V., 64
Verhage, A., 185, 187188
Verhoeven, A.S., 56
Verhoeven, N., 66
Vernon, G., 2122
Verstappen, F.W.A., 15, 16, 235236
Vigeolas, H., 213
Viitanen, P.V., 101102, 108110, 111113,
114, 115116, 117118, 121,
123, 128
Vilarinhos, A.D., 24
Vinkovic, M., 222223, 233234
Viollet, J., 211212
Vishnevetsky, M., 67
Vitreschak, A.G., 5556, 128129
Vivancos, P.D., 166
Vizzotto, C.S., 23
Voegel, T., 19
Vogel, J.T., 15
279
Vogels, G.D., 133134

Vogt, K., 13
Volk, R., 9899, 100, 101, 110111, 116
Von Delft, F., 214216, 215, 217218,
222223, 225, 233
von Lintig, J., 3, 8, 13, 14, 18, 19
von Schaewen, A., 63
von Wiren, N., 232
Vorwieger, A., 129, 130
Voskoboyev, A.I., 48
Vozza, A., 66
Vrablik, T.L., 179
Vrebalov, J., 67, 21
Vreugdenhil, D., 63
Vu, T.H., 222
W
Wachter, A., 5556, 5758, 6869, 180
Wagner, K.G., 174
Wagner, M., 171
Wagner, R., 174
Waldo, G.S., 214216
Walker, D.W., 162
Walker, J.E., 66
Waller, G.R., 174
Waller, J.C., 154155, 163164, 171172
Walsh, C.T., 209211
Walsh, J.H., 211212
Walter, M.H., 15, 6465
Walton, N.J., 130
Wamelink, M.M., 70
Wan, B., 115
Wang, C., 2223, 185187
Wang, D., 43
Wang, G., 5051, 7172, 177178, 179
Wang, H., 208, 232
Wang, K., 22
Wang, Q., 21
Wang, R.X., 1516
Wang, S.B., 5051, 7172, 166167
Wang, S.S., 217218
Wang, T.C., 181
Wang, W., 127
Wang, X.C., 128, 132, 163164, 166167
Wang, Y.H., 1516, 4143, 128, 132
Wang, Z.G., 22, 132133
Warburton, J.D., 6
Ward, C.M., 157, 161
Watanabe, A., 6263
Waters, B.M., 130
Wawrzak, Z., 101102, 121, 123
Webb, E., 48
Webb, M.E., 204205, 213214, 233234
Weber, S., 111112
Wedemeyer, W.J., 226
Weesie, R.J., 6
Wege, C., 241
280
AUTHOR INDEX
Weiland, T., 217218

Weilersbacher, G.S., 43
Wei, M., 211213, 244
Weinkauf, S., 98
Weinreb, P.H., 209211
Weinstock, H.H., 204205
Wei, P.C., 163164
Weisshaar, B., 129, 130, 222
Wei, Y., 170171, 173
Welch, W., 13
Wells, J.M., 130
Welsch, R., 8, 1819, 20, 23
Weretilnyk, E.A., 6869
Westaway, S.K., 211, 226
Westby, A., 24
Westhoff, P., 63
West, T.P., 225
Whelan, J., 19, 20
Whitehead, L., 230
White, R.H., 98, 126, 134, 135136
White, W.H., 22, 225, 234, 235238
White, W.S., 22
Whitney, H.M., 216, 218, 219220, 223,
225, 245
Whitty, B., 6869
Widmann, M., 5859
Wiegert, T., 6465
Wiegmann, K., 133
Wieland, T., 217218
Wientjes, F.J., 181182
Wildermuth, M.C., 185
Wildt, J., 187188
Wildung, M.R., 7071
Wilhelm, C., 12
Wilkin, D.R., 224225
Willekens, H., 185187
Willett, B., 1516
Williams, M., 7, 1011, 12, 19, 20, 22
Williamson, J.M., 98, 225, 233234
Williamson, L., 1516
Williams, R.J., 204205
Williams, R.R., 38
Wills, S., 15
Wilson, D.E., 130, 206207
Wilson, K., 111
Windahl, K.L., 157, 161
Wingsle, G., 6869, 70
Winkler, W.C., 5557, 128129
Wins, P., 3940, 48, 5455
Winter, H., 168170, 169, 184
Wirtzfeld, B., 3940, 5455
Wirtz, G., 13
Wiskich, J.T., 170171
Witholt, B., 181182
Wittwer, C.T., 206207
Witty, M., 214216, 215, 225, 233234
Woggon, W., 13
Wohlgemuth, G., 130

Wojtczak, A., 208
Wojtera, J., 164165
Wolak, N., 70
Wolf, E., 6465
Wolfe, R.S., 133134
Wolff, T., 164, 173
Wong, C.E., 6869
Wong, L., 22
Wong, W.C., 115
Wood, H.C.S., 118, 120, 121
Wood, J.G., 179
Wood, W.A., 214216
Woodward, J.B., 52
Woodworth, A.R., 64
Woolley, D.W., 156157
Work, C.E., 204205
Wright, S.Y., 2122
Wright, W., 1516
Wubbolts, M.G., 181182
Wuest, F., 1819
Wu, G., 185
Wu, H., 49, 55, 66
Wunderlich, G., 6
Wunderlich, T., 133
Wunn, J., 8
Wu, P.H.L., 174175
Wurtele, E.S., 6263, 213
Wurtzel, E.T., 56, 7, 811, 12, 1819,
2021, 22, 7071
Wust, F., 8, 1819, 20, 23
Wu, T., 128, 132
Wu, Y.P., 2223
Wynn, R.M., 6263
Wyse, B.W., 211212
Wyss, A., 13
Wyss, M., 13
X
Xiao, S., 132133
Xiao, Y.G., 2223
Xia, X.C., 2223
Xia, Y., 166167
Xie, D., 132133
Xie, W., 161162
Xi, J., 4647, 6465
Xu, A., 161162
Xu, C., 5051, 7172
Xue, Z., 234, 236238
Xu, H., 98, 126, 134, 135136
Xu, Y., 22
Y
Yamada, H., 96, 224225, 233
Yamada, K., 162, 180, 181
Yamaguchi, S., 1516
AUTHOR INDEX
Yamamoto, H.Y., 56, 12
Yamamoto, I., 207, 208
Yamamoto, K.T., 100, 236
Yamaya, T., 162, 163164
Yanagisawa, S., 162, 163164
Yan, C.Y., 1516
Yang, C.-Y., 130
Yang, D., 115
Yang, G.X., 2223
Yang, H., 211
Yang, K.S., 174
Yang, S., 163164
Yang, X.H., 12, 19, 22
Yang, Y., 1415
Yan, J.B., 7, 1011, 12, 19, 20, 22
Yan, M.X., 1516
Yan, X., 6162
Yan, Y., 59
Yasuda, Y., 64, 71
Yasumoto, K., 5354
Yates, A.A., 95
Yazdanpanah, B., 133
Yeaman, S.J., 6263
Yeates, T.O., 214216
Ye, H., 176, 184
Yenush, L., 227228
Ye, Q.Z., 114, 115
Yeung, E.S., 161162
Ye, X., 2122, 243
Ying, W., 166
Yin, Y., 178
Yokooji, Y., 239240
Yokota, H., 127
Yoneyama, K., 15
Yoon, M.Y., 7374
Yoon, S.S., 7374
Yoshida, S., 15
Yoshida, T., 65
Yoshihara, T., 176177, 179180
Yoshimoto, S., 117118, 121
Yoshimura, K., 166167
Yoshino, J., 165166
Yoshioka, H., 133
Yoshioka, S., 2021
Young, D.W., 98
Yruela, I., 127, 128
Yuan, M., 5051, 7172
Yu, D., 6465
Yu, H.C., 106, 108
Yu, J., 8, 1819
Yujun Xu, E., 211
Yu, L.H., 164, 178180
Yu, M., 214216
Yu, Q.J., 2021
Yusa, T., 5455
Yu, Y., 101102
281
Z
Zabalza, A., 64
Zabrodskaya, S.V., 72
Zaharieva, M., 12, 19, 22
Zambelli, A., 7374
Zamir, D., 10, 20, 21
Zechmann, B., 168170
Zeevaart, J.A.D., 13, 1415
Zeidler, J., 47, 7374
Zentgraf, U., 173174
Zeth, K., 127
Zhai, H., 47
Zhang, C., 6465
Zhang, H., 4344, 126, 127, 177
Zhang, J., 2122, 166, 243
Zhang, M., 6465
Zhang, S.X., 43, 59, 132
Zhang, X., 111112, 113, 185188
Zhang, Y.L., 10, 2223, 4647, 5051, 114,
115, 127, 128
Zhang, Y.M., 211
Zhang, Z., 59
Zhao, D., 128, 132
Zhao, R., 4143
Zhao, Y., 1415, 115, 128, 132, 165166
Zheng, R.J., 214216, 217218, 233
Zheng, X., 178
Zheng, Y.J., 101102
Zheng, Z., 170171
Zhou, B., 211, 226
Zhou, D.X., 165166
Zhou, L., 59
Zhou, Q., 127
Zhou, X., 67, 21
Zhu, C.F., 22
Zhu, H., 166167
Zhu, J.K., 70
Zhu, T., 166167
Zhu, Y., 49, 55, 66
Ziegler, M., 155, 163, 174, 178
Zilinskas, B., 180
Zimmerman, J., 222, 227
Zimmermann, P., 173174
Zingler, N., 123, 124
Zocchi, G., 130
Zogaj, X., 177
Zolman, B.K., 213
Zorzi, W., 3940
Zou, J., 170171, 173
Zou, Y., 59
Zrenner, R., 178179, 235
Zuker, A., 67
Zuo, J., 185187
Zverinskii, I.V., 72
Zwingmann, C., 43
Zylberman, V., 111113
SUBJECT INDEX
A
b-Alanine
source, 225
synthesis
AMADH2, Pisum sativum, 236238
PAO, Arabidopsis, 236, 237
propionate/cyanide catabolism, 238
pyrimidine degradation, 235
routes, 233234
spermine and spermidine oxidation,
235236
B
Biosynthesis, carotenoids
carotene (see Carotene, biosynthesis)
catabolism
Arabidopsis, 20
CCD1 expression levels, 2021
14
CO2 uptake data, 20
cleavage products
beta-ionone, 17
bixin, 16
diverse roles, 14
novel-signalling molecules, 17
phytohormones, 1416
saffron, 1617
vitamin A, 1314
isoprenoids/terpenoids, 78
metabolite feedback
lutein levels, 20
PSY3 gene expression, 20
storage capacity, 21
transcriptional regulation
allelic variation, 19
ATCTA, 19
CRITSO and SDG8, 19
lutein levels, 20
phytoene biosynthesis, 18
PSY and DXS, 18
xanthophylls
hydroxylases, 1112
NXS, 13
ZEP and VDE, 1213
Burning foot syndrome, 206207
C
Carotene, biosynthesis
cyclases, 1011
desaturases (PDS and ZDS), 810
isomerases (Z-ISO and CRTISO), 10

phytoene synthase, 8, 9
Carotenoid-cleavage dioxygenases (CCD)
catalysed degradation, 20
CCD7 and CCD8 mutants, 1516
gene family, 13
Carotenoids
biosynthesis
carotene, 811
catabolism, 2021
cleavage products, 1317
isoprenoids/terpenoids, 78
metabolite feedback, 20
storage capacity, 21
transcriptional regulation, 1719
xanthophylls, 1113
dietary
b-carotene, 3
precursor structures, 4
vitamin A deficiency, 3
distribution
apicoplast, 6
astaxanthin, 6
colourless amyloplasts, 7
flamingos, 6
leucoplasts and elaioplasts, 7
transformation, chromoplast, 67
nutrition
banana and plantain, 24
cassava, 23
maize, 22
potato, 24
rice, 2122
sorghum, 2324
sweet potato, 24
wheat, 2223
photosynthetic organisms
non-photochemical
quenching (NPQ), 5
photoprotective, 5
photosystem assembly, 45
xanthophylls, 56
zeaxanthin, 56
Catabolism, carotenoid biosynthesis
Arabidopsis, 20
CCD1 expression levels, 2021
14
CO2 uptake data, 20
Cleavage products, carotenoids
beta-ionone, 17
bixin, 16
diverse roles, 14
284
SUBJECT INDEX
Cleavage products, carotenoids (cont.)
novel-signalling molecules, 17
phytohormones
ABA, 1415
CCD7 and CCD8, 15
d27, 1516
strigolactones, 15, 16
saffron, 1617
vitamin A
b-carotene, 13
b-ionone ring, 14
F
Flavocoenzyme biosynthesis
cellular topology, plants
chloroplasts, 128
putative organelle-targeting
sequences, 128
enzyme evolution, plants
bacterial origin, 131
eubacteria, 131
riboflavin pathway, 131
H
Heavy riboflavin synthase, 110
K
Ketopantoate hydroxymethyltransferase
(KPHMT)
acid treatment, extraction, 216
crystal structure, 214216
genome sequence, Arabidopsis, 216, 217
mechanism, 214216
Ketopantoate reduction
34-kDa monomeric protein, 224225
apbA, 222223
plant enzymes, spinach and barley, 224
routes, 222, 223
structure, E. coli, 223, 224
L
Lyases
acetohydroxyacid synthase, 64
1-deoxy-d-xylulose-5-phosphate
synthase, 6465
pyruvate decarboxylase, 64
TDP-dependent, 63
M
Metabolite feedback, carotenoid biosynthesis
lutein levels, 20
PSY3 gene expression, 20
Mitochondrial carrier family (MCF), 171
N
Nicotinamide adenine dinucleotide (NAD)
biosynthesis and manipulation,
plants
annotations, genes, 174
conversion, NaMN, 176177
enrichment, bacteria and human cells,
181
humans
eukaryotic pathway, 161
pathways, 159, 160
pyridine ring, dietry source, 159160
quinolinate, 160
intracellular distribution
fractionation and filtration,
protoplast, 168
NAD(H) pools, response, 170, 172
pyridine nucleotide concentrations,
168170
measurement
acid extraction, 161
enzymatic cycling assays, 161
fluorescent properties, pyridine,
161162
GC-MS, LC-MS and CE-MS method,
162
HPLC, 161
and NADP interconversion, NADK
ATP-dependent phosphorylation, 163
G6PDH, 164
NADK3, 163164
overexpression, 164
phosphatase activity, 164
and NADP pools, redox links
non-phosphorylating, 164165
transhydrogenase activity, 165
NaMN de novo production, bacteria
O2-sensitive enzymes, 176
QS, AO and QPRT, 175176, 175
quinolinate, 174175
synthase and SufE3 domain, 176
niacin (vitamin B3)
canine black-tongue disease, 156157
digestive enzymes, gut, 158159
dosage, 159
sources, 157, 158
tryptophan, 157
non-redox roles
enzymes, turnover, 165166
nucleotide concentration, 167168
NUDIX hydrolases, 166167
PARP, 166
pathogenesis-related pathways, NAD(H)
NADPH oxidase stimulation, 185187
transcripts, altered expression, 185,
186
plasticity, NAD(H) tissue contents,
172174
SUBJECT INDEX
recycling pathways
inter-species differences, 178
NaPRT activity, 178
nicotinamidase, 177178
regulation
NaPRT and QPRT activities, 178179
nicotinamidase, 179
overexpression, NADS, 179180
TRX, disulfide bond formation, 180
and ROS, thiol status, 187188
structure, NAD(P) and NAD(P)H, 156
subcellular transport
mitochondrial G3PDH, 170171
AtNDT1 and AtNDT2, 171172
NTT and MCF, 171
tissue enrichment
Arabidopsis, nadB, nadA and nadC
genes, 181182
gene expression, changes, 182, 183
overexpression, AO, 184185
QPRT activity and quinolinate, 182
substrate limitation, cytosol, 184
Non-photochemical quenching (NPQ), 56
Nucleotide trasporter-type (NTT), 171
O
Oxidorductases
branched-chain a-ketoacid
dehydrogenase, 6263
a-ketoacid dehydrogenase, 6061
a-ketoglutarate dehydrogenase, 62
mtPDH, 6162
plastidial pyruvate dehydrogenase
complex, 62
P
Pantothenate biosynthesis
bacteria, pathway engineering
C. glutamicum, 241243
E. coli, 241
glycine accumulation, 243
biological function, 205206
biosynthetic pathway and location
b-alanine, source, 225
ketopantoate reduction, 222225
KPHMT, 214216
3-methyl-2-oxobutyric acid and
b-alanine, 213214
PS, 217222
and coenzyme A, structure, 204205
deficiency, effects
burning foot syndrome, 206207
dietary requirements, 207, 209
o-methylpantothenate and
homopantothenate, 207
symptoms, 207, 208
dietary absorption and metabolism
285
conversion, CoA, 209211

N-terminal sequence, protein, 208209
PanK, 211
pantetheine, 208
distribution, plants
bioavailability measurement, 211212
CoA and ACP, 213
subcellular distribution, 213
tissues comparison, 212213
eubacteria vs. prototrophic organisms
b-alanine synthesis, 233239
archaeal pathway, 239240
ketopantoate reductase, 233
pathway, 204205, 206
plants, pathway engineering
Arabidopsis, PS, 244
14
C-labelled b-alanine accumulation,
243244
transgenic tobacco, 244
regulation, turnover, and metabolism
cellular and tissue patterns, plants,
231233
decarboxylase, PPC, 227229
dephospho-CoA kinase, 230231
kinase, 226227
4-phosphopantetheine
adenylyltransferase, 230
PPC synthetase, 227
Pantothenate synthetase (PS)
bacterial and plant sequences, 220, 221
E. coli panC mutant AT1371, 219220
expression patterns, Arabidopsis PanC, 222
mechanism, 217218
seed development, 222
structure, 218, 219
substrate inhibition, 220
Phosphopantethenoylcysteine (PPC)
decarboxylase
aaBb, 229
AtHAL3A, mutation, 228
hal3a-1 and hal3b, 229
PPCDC, 227228
T-DNA insertion mutants, 228229
synthetase, 227
Plant thiamine biosynthesis
genes and protein
auxotrophic mutants, 4849
coupling, pyrimidine and thiazole,
5253
pyrimidine component, 49
TDP, 5354
thiazole, 5052
regulation, THIC and THI1, 5558
Pyrimidine
biosynthesis
A. thaliana, 49
compartment localization, 50
THIC transcript levels, 49
biosynthetic precursors, 45
286
SUBJECT INDEX
Pyrimidine (cont.)
component synthesis
Saccharomyces cerevisiae, 47
S-adenosyl methionine (SAM), 4647
condensation, 48
coupling, 5253
R
Riboflavin
biosynthesis
B. subtilis, regulation, 129
condensation, pyrimidine derivative, 99
flavinogenic strains and microbial
flavinogenesis, 9698
GTP cyclohydrolase II, 98
microorganisms, 96, 97
rib operon, regulation, 128129
xylene ring, 9899
biosynthetic enzyme, potential herbicide
anti-infective drugs, 132
high-throughput screening (HTS), 132
commercial, 96
content, food, 94
deaminase/reductase
deamination and reduction
mechanisms, 108
E. coli, 106
GTP cyclohydrolase II
transformation, 105
in vivo study, 106
plastid-targeting sequence, 106, 107
putative genes, A. thaliana and Oryza
sativa, 106, 109
RibG-protein, B. subtilis, 108
5-deaza-7,8-didemethyl-8-hydroxyriboflavin
Chlamydomonas reinhardtii, 136
cofG and cofH gene, 135
covalent adduct, 136
DNA photolyases, 133134
FADH2 chromophore, 134135
FbiC protein, 136
F420-dependent enzymes, 135
mechanism, 134
Mycobacterium smegmatis, 135136
deficiency, 95
de novo, 99
dietary reference intake, 95
excretion and enhanced formation,
iron-deficient roots
A. thaliana, 130
ferric and ferrous iron, 129
Medicago trunculata roots, 130
membrane proteins, 130
strategies, iron deprivation, 129
sugar beets, 129
yeasts and bacteria, 130
flavocoenzyme
cellular topology, biosynthesis,
127128
evolution, plants, 131

kinase and FAD synthetase
A. thaliana, 127
Brevibacterium ammoniagenes, 126127
5-phosphorylated precursors, 126
x-ray structures, 127
lumazine synthase
cistrans isomerisation, 115
human-and plant-pathogenic
microorganisms, 112
icosahedral, capsid, 111
inhibitors, 114
inorganic phosphate, 114115
1-MDa protein, 111
mutagenesis study, 116
pentacyclic inhibitor, 115
protein subunit, 110
pseudomaturated, 114
quaternary structures, 112113
rate acceleration, 116
reaction mechanism, 110111
sequence comparison, 113
space filling model, B. subtilis, 112
structural superposition, monomers,
113
x-ray structures, 111112
plant resistance
lipoxygenase and phenylalanine
ammonia lyase upregulation, 132
rice lumazine synthase, 132133
ROS, 132, 133
virus-induced silencing, 133
redox reactions, 94
ribab
A. thaliana, 100101
bifunctional RibA proteins, 101
cognate genes, E. coli, 100
3,4-dihydroxy-2-butanone
4-phosphate synthase, 101, 102, 103
dimers, 102103
GTP cyclohydrolase II, 103, 104, 105
M. tuberculosis, 101102
synthase
amino acid sequence, 123
archaeal enzymes, 126
B. subtilis, 123
Cannizzaro reaction, 125
carbon atoms, xylene ring, 118
4-carbon segment, 116117
cloning and expression, genes,
117118
13
C NMR, 124125
covalent adduct, 121122
deprotonation, 118119
6,7-dimethyl-8-ribityllumazine,
118119
E. coli, 124
enzyme-catalysed and-uncatalysed
reactions, 121
exomethylene anion, 119121
green fluorescent substance, 117
SUBJECT INDEX
homotrimer structure, 124
isotope effects, 126
ligand molecules, 123124
pentacyclic intermediate, 122
quasi-quinoid dehydrolumazine
structure, 125
reaction mechanisms, 120
redox process, 122
S. pombe, 123, 124
stereochemistry, 6,7-dimethyl-8ribityllumazine conversion, 122
T
TDP-dependent enzymes
binding, 5657
catalytic mechanisms, plants
aldehyde intermediate, 60
decarboxylation and transferase-type
reaction, 60
thiazolium ring, 59
ylide-like carbanion generation, 59
classification and localization
lyases, 6365
oxidoreductases, 6063
transferases, 63
feedback inhibitor role, 49
formation, 48
mammalian cells, 42
mitochondrial transporters, 66
plant stress, 69
synthesis, 5354
TDP-binding riboswitch (THI-BOX), 56
transketolase (TK), 70
Thiamine
abiotic stress responses
Arabidopsis roots, 68
CalvinBenson cycle and pentose
phosphate pathway, 70
carotenoids, 7071
cytosolic enzymes, 70
ethanolic fermentation pathway, 71
metabolic analyses, 6768
TDP-dependent pathways, 69
THI1 gene, 6869
tricarboxylic acid cycle and acetylCoA mitochondrial production, 71
Z. mays, 6869
biosynthesis
bacteria and yeast, 4648
genes and protein, plant, 4855
regulation, plant, 5558
biotic stress
signalling processes, 7172
systemic acquired resistance (SAR), 71
deficiency symptoms, mammals
damage, uptake/transport, 4143
diabetes and diabetic complications,
4445
287
functional disorders, mitochondria,

4344
levels, cell, 4041
phosphorylated analogues, structure and
biological functions
alarmone, 3940
content, plant food, 41
heterocyclic moieties, 38
human, 40
plant tissues
absorption, soil, 6667
chloroplasts, 66
leaves, 6667
seed content, 6566
thiamine-binding proteins, 65
rescue, stressed plants, 72
TDP-dependent enzymes
catalytic mechanisms, 5960
classification and localization, plant
cell, 6065
Thiamine diphosphate (TDP)
damage, uptake/transport, 4143
description, 3940
enzymes (see TDP-dependent enzymes)
Thiazole
biosynthesis, component
Arabidopsis, 52
sequence analysis, THI1 protein, 51
subfunctionalization, 52
synthesizing protein structure, 51
THI4, 5051
biosynthetic precursors, 45
component synthesis, 47
condensation, 48
coupling, 5253
Transcriptional regulation, carotenoid
biosynthesis
allelic variation, 19
ATCTA, 19
CRITSO and SDG8, 19
lutein levels, 20
phytoene biosynthesis, 18
PSY and DXS, 18
Transferases, 63
Transketolase (TK), 63
V
Vitamin A. See carotenoids
Vitamin B1. See Thiamine
Vitamin B2. See Riboflavin
Vitamin B3. See NAD
Vitamin B5. See panthotenate
X
Xanthophylls, synthesis
hydroxylases, 1112
neoxanthin synthase (NXS), 13
ZEP and VDE, 1213

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