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Author(s): Cristina Prandi (editor), Francesca Cardinale (editor)
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Language: english
Methods in
Molecular Biology 2309
Cristina Prandi
Francesca Cardinale Editors
Strigolactones
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK
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For over 35 years, biological scientists have come to rely on the research protocols and
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Strigolactones
Methods and Protocols
Edited by
Cristina Prandi
Department of Chemistry, University of Turin, Turin, Italy
Francesca Cardinale
DISAFA-PlantStressLab, University of Turin, Grugliasco, Italy
Editors
Cristina Prandi Francesca Cardinale
Department of Chemistry DISAFA-PlantStressLab
University of Turin University of Turin
Turin, Italy Grugliasco, Italy
ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-1428-0 ISBN 978-1-0716-1429-7 (eBook)
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Preface
Plants produce and release various chemicals into the environment, as well as primary and
secondary metabolites. Abiotic and biotic stresses affect the composition and the amount of
these compounds by promoting or suppressing their biosynthesis and/or efflux. Many of
the key chemical players involved in plant-plant, plant-microbe, and plant-insect chemical
communication are as yet unidentified.
Strigolactones (SLs) are typical examples of such signaling molecules. Plants release only
very small amounts of SLs into the soil, and these molecules decompose rapidly in the
rhizosphere. SLs can only be analyzed and quantified using recently developed, highly
sensitive mass spectrometry methods and were originally isolated as germination stimulants
for seeds of parasitic weeds in the Orobanchaceae family. Being detrimental to the producing
plant, SLs were initially regarded as harmful secondary metabolites. However, it was subse-
quently shown that they act as crucial chemical signals for root colonization by symbiotic
arbuscular mycorrhizal (AM) fungi and became then recognized as beneficial plant meta-
bolites. Yet more recently, SLs were identified as hormones that regulate different aspects of
plant development and responses to biotic and abiotic stress.
Needless to say, this led the scientific community to dramatically increase their interest in
these new phytohormones and to actively research their perception, signal transduction,
molecular mechanisms of action, biosynthesis, evolution and genetic regulation, as well as
formulations for agriculture applications. This blooming research covers as different aspects
as the molecular evolution underlying parasitic life habits in plants, the redefinition of
hormonal crosstalk networks and their effects on plant development and responses to stress,
the definition of novel core signaling pathways including, for example, the
neo-functionalization of small-molecule receptors in plants, and the hacking of signaling
pathways for endogenous small molecules to the purpose of perceiving exogenous ones.
Another important aspect of the burgeoning SL research is the identification of families of
SLs which share similar molecular structures but at the same time are structurally and
stereochemically finely tuned to the various SLs functions. This led to design synthetic
analogues for multiple scopes, ranging from the elucidation of mechanisms of action to the
preparation in bulk, thus paving the way to a variety of potential applications in agriculture
and medicine.
Due to the multiple biological roles of SLs and their impact in rather different disciplin-
ary areas such as chemistry, biochemistry, plant physiology, plant and root development,
mycology, agronomy, and even medicine, undertaking research in this field implies coping
with numerous, diverse and innovative experimental procedures. Indeed, the thriving
scientific activity on SLs entails the application and implementation of new experimental
procedures and protocols that need to be standardized and promoted within the broader
scientific community in order to have reliable and comparable scientific results. Our and
other scientists’ experience suggested that many newcomers in the field are not completely
aware of the fine details and working tricks in SL-related experimental procedures. This may
jeopardize the advancement in our understanding of SL biology and application potential.
Furthermore, the fact that SL-like compounds are produced in the plant and share both
overlapping and distinct functions with SLs makes it necessary to provide reliable methods
to assess their levels and functions as well and to distinguish them from “canonical” SLs.
v
vi Preface
Finally, both natural and synthetic analogues of SLs are rather unstable and easily hydrolyze
in aqueous solution. This may then lead to underestimate or misinterpret SL effects and
activity.
For all of the above-cited reasons, we believe that as SLs attract more and more
numerous researchers from different fields, the “historical” SL community should provide
a blueprint of consolidated experimental protocols that are trusted to generate reliable
results. This led to the idea of confecting a book clearly presenting the most useful labora-
tory protocols in SL research. The challenge in undertaking this project has been that the
content develops around expertise from very different disciplinary fields; this is reflected in
the different sections of this book.
First of all, as natural SLs are produced in very tiny amounts, specific analytical methods
are presented. For the same reason, when fairly large amounts of pure SLs are needed in
research, chemically synthesized SL analogues or mimics are the only real option; thus,
wet-lab paths to their synthesis are described. Also, issues around stability are addressed. In a
different section, the main protocols to evaluate SL activity and effects toward soil inhabi-
tants such as parasitic plants, mycorrhizal and non-mycorrhizal fungi, and nodulating
bacteria are covered. Finally, protocols to assess effects on plant development are discussed,
along with different biochemical and in planta assays to quantify SLs, and the main pitfalls
and tricks to obtain crystals of purified proteins in SL signaling.
To conclude, with this book we and the authors meant to help boosting SL research by
delivering a clear-cut and standardized set of experimental protocols to a broad scientific
community. In our vision, this should contribute to speed up the understanding and
potential of applications of these multifaceted, challenging and fascinating molecules.
Turin, Italy Cristina Prandi

Grugliasco, Italy Francesca Cardinale
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
PART I STRIGOLACTONE CHEMISTRY
1 Evaluation and Quantification of Natural Strigolactones

from Root Exudates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Xiaonan Xie, Kaori Yoneyama, Takahito Nomura,
and Koichi Yoneyama
2 Isolation and Identification of Naturally Occurring Strigolactones . . . . . . . . . . . . 13
Kotomi Ueno, Takatoshi Wakabayashi, and Yukihiro Sugimoto
3 Chemical Synthesis of Triazole-Derived Suppressors of Strigolactone
Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Shisanku Ito, Ko Kikuzato, Hidemitsu Nakamura,
and Tadao Asami
4 Synthesis of Simple Strigolactone Mimics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Tomáš Pospı́šil
5 Synthesis of Analogs of Strigolactones and Evaluation of Their Stability
in Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Daniel Blanco-Ania and Binne Zwanenburg
PART II STRIGOLACTONES AND SOIL BIOTA
6 Strigolactone-Like Bioactivity via Parasitic Plant Germination Bioassay . . . . . . . . 59

Jean-Bernard Pouvreau, Lucie Poulin, Sarah Huet,
and Philippe Delavault
7 Evaluation of the Effect of Strigolactones and Synthetic Analogs on Fungi . . . . . 75
Valentina Fiorilli, Mara Novero, and Luisa Lanfranco
8 Analyzing the Effect of Strigolactones on the Motility Behavior
of Rhizobia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Lydia M. Bernabéu-Roda, Juan Antonio Lopez-Ráez,
and Marı́a J. Soto
9 Chemotropic Assay for Testing Fungal Response to Strigolactones
and Strigolactone-Like Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Rocı́o Pineda-Martos, Antonio Di Pietro, and David Turrà
PART III STRIGOLACTONES AS PLANT HORMONES
10 Methods for Phenotyping Shoot Branching and Testing Strigolactone

Bioactivity for Shoot Branching in Arabidopsis and Pea. . . . . . . . . . . . . . . . . . . . . . 115
Aitor Muñoz, Jean-Paul Pillot, Pilar Cubas, and Catherine Rameau
vii
viii Contents
11 Bioassays for the Effects of Strigolactones and Other Small Molecules

on Root and Root Hair Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
José Antonio Villaécija-Aguilar, Sylwia Struk, Sofie Goormachtig,
and Caroline Gutjahr
12 Methods for Medium-Scale Study of Biological Effects
of Strigolactone-Like Molecules on the Moss
Physcomitrium (Physcomitrella) patens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Ambre Guillory and Sandrine Bonhomme
13 Controlled Assays for Phenotyping the Effects of Strigolactone-Like
Molecules on Arbuscular Mycorrhiza Development . . . . . . . . . . . . . . . . . . . . . . . . . 157
Salar Torabi, Kartikye Varshney, José A. Villaécija-Aguilar,
Andreas Keymer, and Caroline Gutjahr
14 Application of Strigolactones to Plant Roots to Influence Formation
of Symbioses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
Eloise Foo
PART IV BIOACTIVITY ASSAYS
15 Evaluation of Bioactivity of Strigolactone-Related Molecules

by a Quantitative Luminometer Bioassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
Elena Sánchez, Pilar Cubas, Francesca Cardinale, and Ivan Visentin
16 A Protoplast-Based Bioassay to Quantify Strigolactone Activity
in Arabidopsis Using StrigoQuant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
Justine Braguy, Sophia L. Samodelov, Jennifer Andres, Rocio Ochoa-
Fernandez, Salim Al-Babili, and Matias D. Zurbriggen
17 Synthesis of Profluorescent Strigolactone Probes for Biochemical
Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
Alexandre de Saint Germain, Guillaume Clavé,
and François-Didier Boyer
18 The Use of Differential Scanning Fluorimetry to Assess Strigolactone
Receptor Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
Cyril Hamiaux, Bart J. Janssen, and Kimberley C. Snowden
19 Structural Analysis of Strigolactone-Related Gene Products . . . . . . . . . . . . . . . . . . 245
Inger Andersson, Gunilla H. Carlsson, and Dirk Hasse
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
Contributors
SALIM AL-BABILI • Division of Biological and Environmental Science and Engineering,

Center for Desert Agriculture, the BioActives Lab, King Abdullah University of Science
and Technology, Thuwal, Saudi Arabia
INGER ANDERSSON • Laboratory of Molecular Biophysics, Department of Cell and Molecular
Biology, Uppsala University, Uppsala, Sweden; Arctic University of Norway, Tromsø,
Norway; Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, Vestec, Czech
Republic
JENNIFER ANDRES • Institute of Synthetic Biology and CEPLAS, University of Düsseldorf,
Düsseldorf, Germany
TADAO ASAMI • Graduate School of Agricultural and Life Sciences, The University of Tokyo,
Tokyo, Japan
LYDIA M. BERNABÉU-RODA • Department of Soil Microbiology and Symbiotic Systems,
Estacion Experimental del Zaidı́n (CSIC), Granada, Spain
DANIEL BLANCO-ANIA • Department of Organic Chemistry, Radboud University Nijmegen,
Institute for Molecules and Materials, Nijmegen, The Netherlands
SANDRINE BONHOMME • Université Paris-Saclay, INRAE, AgroParisTech, Institut Jean-
Pierre Bourgin, Versailles, France
FRANÇOIS-DIDIER BOYER • Université Paris-Saclay, CNRS, Institut de Chimie des Substances
Naturelles, Gif-sur-Yvette, France
JUSTINE BRAGUY • Division of Biological and Environmental Science and Engineering,
Center for Desert Agriculture, the BioActives Lab, King Abdullah University of Science
and Technology, Thuwal, Saudi Arabia; Institute of Synthetic Biology and CEPLAS,
University of Düsseldorf, Düsseldorf, Germany
FRANCESCA CARDINALE • DISAFA-PlantStressLab, University of Turin, Grugliasco (TO),
Italy
GUNILLA H. CARLSSON • Laboratory of Molecular Biophysics, Department of Cell and
Molecular Biology, Uppsala University, Uppsala, Sweden
GUILLAUME CLAVÉ • Université Paris-Saclay, CNRS, Institut de Chimie des Substances
Naturelles, Gif-sur-Yvette, France
PILAR CUBAS • Centro Nacional de Biotecnologı́a-CSIC, Madrid, Spain
ALEXANDRE DE SAINT GERMAIN • Institut Jean-Pierre Bourgin, INRAE, AgroParisTech,
Université Paris-Saclay, Versailles, France
PHILIPPE DELAVAULT • Laboratory of Plant Biology and Pathology (LBPV), University of
Nantes, Nantes Cedex 3, France
ANTONIO DI PIETRO • Department of Genetics, University of Cordoba, Cordoba, Spain
VALENTINA FIORILLI • Department of Life Sciences and Systems Biology (UNITO-DBIOS),
University of Turin, Turin, Italy
ELOISE FOO • Discipline of Biological Sciences, School of Natural Sciences, University of
Tasmania, Hobart, TAS, Australia
SOFIE GOORMACHTIG • Department of Plant Biotechnology and Bioinformatics, Ghent
University, Ghent, Belgium; Center for Plant Systems Biology, VIB, Ghent, Belgium
AMBRE GUILLORY • Université Paris-Saclay, INRAE, AgroParisTech, Institut Jean-Pierre
Bourgin, Versailles, France
ix
x Contributors
CAROLINE GUTJAHR • Plant Genetics, TUM School of Life Sciences Weihenstephan, Technical
University of Munich (TUM), Freising, Germany
CYRIL HAMIAUX • The New Zealand Institute for Plant & Food Research Limited, Auckland,
New Zealand
DIRK HASSE • Laboratory of Molecular Biophysics, Department of Cell and Molecular Biology,
Uppsala University, Uppsala, Sweden
SARAH HUET • Laboratory of Plant Biology and Pathology (LBPV), University of Nantes,
Nantes Cedex 3, France
SHISANKU ITO • Graduate School of Agricultural and Life Sciences, The University of Tokyo,
Tokyo, Japan
BART J. JANSSEN • The New Zealand Institute for Plant & Food Research Limited, Auckland,
New Zealand
ANDREAS KEYMER • Plant Genetics, TUM School of Life Sciences Weihenstephan, Technical
KO KIKUZATO • Graduate School of Agricultural and Life Sciences, The University of Tokyo,
Tokyo, Japan
LUISA LANFRANCO • Department of Life Sciences and Systems Biology (UNITO-DBIOS),
JUAN ANTONIO LÓPEZ-RÁEZ • Department of Soil Microbiology and Symbiotic Systems,
Estacion Experimental del Zaidı́n (CSIC), Granada, Spain
AITOR MUÑOZ • Centro Nacional de Biotecnologı́a-CSIC, Madrid, Spain
HIDEMITSU NAKAMURA • Graduate School of Agricultural and Life Sciences, The University
of Tokyo, Tokyo, Japan
TAKAHITO NOMURA • Utsunomiya University, Utsunomiya, Japan
MARA NOVERO • Department of Life Sciences and Systems Biology (UNITO-DBIOS),
ROCIO OCHOA-FERNANDEZ • Institute of Synthetic Biology and CEPLAS, University of
Düsseldorf, Düsseldorf, Germany; iGRAD Plant Graduate School, University of
Düsseldorf, Düsseldorf, Germany
JEAN-PAUL PILLOT • Université Paris-Saclay, INRAE, AgroParisTech, Institut Jean-Pierre
ROCÍO PINEDA-MARTOS • School of Agricultural Engineering, Area of Agroforestry
Engineering, University of Seville, Seville, Spain
TOMÁŠ POSPÍŠIL • Faculty of Science, Department of Chemical Biology, Palacký University
Olomouc, Olomouc, Czech Republic
LUCIE POULIN • Laboratory of Plant Biology and Pathology (LBPV), University of Nantes,
Nantes Cedex 3, France
JEAN-BERNARD POUVREAU • Laboratory of Plant Biology and Pathology (LBPV), University
of Nantes, Nantes Cedex 3, France
CATHERINE RAMEAU • Université Paris-Saclay, INRAE, AgroParisTech, Institut Jean-Pierre
SOPHIA L. SAMODELOV • Institute of Synthetic Biology and CEPLAS, University of
Düsseldorf, Düsseldorf, Germany; Clinical Pharmacology and Toxicology,
Universit€ atsspital Zürich, Zürich, Switzerland
ELENA SÁNCHEZ • Centro Nacional de Biotecnologı́a-CSIC, Madrid, Spain
KIMBERLEY C. SNOWDEN • The New Zealand Institute for Plant & Food Research Limited,
Auckland, New Zealand
Contributors xi
MARÍA J. SOTO • Department of Soil Microbiology and Symbiotic Systems, Estacion

Experimental del Zaidı́n (CSIC), Granada, Spain
SYLWIA STRUK • Department of Plant Biotechnology and Bioinformatics, Ghent University,
Ghent, Belgium; Center for Plant Systems Biology, VIB, Ghent, Belgium
YUKIHIRO SUGIMOTO • Graduate School of Agricultural Science, Kobe University, Nada,
Kobe, Japan
SALAR TORABI • Plant Genetics, TUM School of Life Sciences Weihenstephan, Technical
DAVID TURRÀ • Department of Agricultural Sciences, University of Naples Federico II,
Naples, Italy
KOTOMI UENO • Faculty of Agriculture, Tottori University, Koyama, Tottori, Japan
KARTIKYE VARSHNEY • Plant Genetics, TUM School of Life Sciences Weihenstephan, Technical
JOSÉ ANTONIO VILLAÉCIJA-AGUILAR • Plant Genetics, TUM School of Life Sciences
Weihenstephan, Technical University of Munich (TUM), Freising, Germany
IVAN VISENTIN • DISAFA-PlantStressLab, University of Turin, Grugliasco (TO), Italy
TAKATOSHI WAKABAYASHI • Graduate School of Agricultural Science, Kobe University, Nada,
Kobe, Japan
XIAONAN XIE • Utsunomiya University, Utsunomiya, Japan
KAORI YONEYAMA • Ehime University, Matsuyama, Japan; PRESTO, Japan Science and
Technology, Kawaguchi, Japan
KOICHI YONEYAMA • Utsunomiya University, Utsunomiya, Japan; Ehime University,
Matsuyama, Japan
MATIAS D. ZURBRIGGEN • Institute of Synthetic Biology and CEPLAS, University of
Düsseldorf, Düsseldorf, Germany; iGRAD Plant Graduate School, University of
Düsseldorf, Düsseldorf, Germany
BINNE ZWANENBURG • Department of Organic Chemistry, Radboud University Nijmegen,
Institute for Molecules and Materials, Nijmegen, The Netherlands
Part I
Strigolactone Chemistry
Chapter 1
Evaluation and Quantification of Natural Strigolactones

from Root Exudates
Xiaonan Xie, Kaori Yoneyama, Takahito Nomura, and Koichi Yoneyama
Abstract
Strigolactones (SLs) in the root exudates can be detected by germination assays with root parasitic weed
seeds, but precise and accurate evaluation and quantification are possible only by chemical analysis with the
liquid chromatography–tandem mass spectrometry (LC-MS/MS). Here we describe methods for root
exudate collection, sample preparation, and LC-MS/MS analysis of SLs.
Key words LC-MS/MS, Hydroponic culture, Solid-phase extraction (SPE) cartridge, Solvent parti-
tioning, Parent ion, Diagnostic product ion, Confirming product ion
1 Introduction
Strigolactones (SLs) were originally identified as germination sti-

mulants for root parasitic weeds [1] and germination tests with root
parasitic weed seeds are highly sensitive and specific to SLs
[2]. However, not only SLs but other host-derived chemicals have
been shown to induce germination of parasite seeds [2]. In addi-
tion, plants produce not a single but mixtures of SLs, it is quite
difficult to evaluate quantitative and/or qualitative differences in
SL profile in root exudates by germination tests [3–5]. Further-
more, germination inhibitors in the root exudates often mask the
activity of SLs.
Large amounts of various plant metabolites in the root exu-
dates hamper direct analysis of SLs, very minor components, by
HPLC with UV-visible or diode-array detectors. SLs could be
analyzed by gas chromatography–mass spectrometry (GC-MS)
but only after extensive purifications [6]. In 2003, liquid
chromatography-tandem mass spectrometry (LC-MS/MS) was
first introduced as a highly specific analytical tool for SLs [7] and
now has become a standard and indispensable instrument for
chemical analyses of SLs in root exudates and plant tissues. An
Cristina Prandi and Francesca Cardinale (eds.), Strigolactones: Methods and Protocols, Methods in Molecular Biology, vol. 2309,
https://doi.org/10.1007/978-1-0716-1429-7_1, © Springer Science+Business Media, LLC, part of Springer Nature 2021
3
4 Xiaonan Xie et al.
ultra-high-pressure liquid chromatography (UHPLC) system,

equipped with a 25–150 mm long column packed with micrometer
particles of octadecylsilylated (ODS)-silica, connected to an elec-
trospray ionization (EI)-MS can analyze more than 20 natural SLs
at sub-picomolar levels within 15–20 min. In addition, this analyti-
cal method enables the detection of SL-related compounds that
have not yet been discovered [8]. Here we describe standard pro-
tocols for root exudate collection, sample preparation, and
LC-MS/MS analysis of SLs. Purification by solid phase extraction
(SPE) cartridges may be skipped if samples are clean enough for
LC-MS/MS analysis.
2 Materials
Glassware should be cleaned thoroughly to avoid contamination.

All reagents are of analytical grade and solvents including water for
LC-MS are of LC-MS grade. For accurate determination and
quantification of SLs, 2H- or 13C-labled SL standards shall be
used. Pure water (Milli-Q) should be used for culture media and
other solutions.
2.1 Hydroponic 1. Petri dishes (i.d. 9 cm).

Culture 2. Filter paper (i.d. 8.5 cm).
3. 1% NaClO solution containing 0.1% Tween 20 and/or 70%
ethanol. Dilute commercial 5% NaClO with pure water to 1%
NaClO and add 0.1% (vol/vol) Tween 20. Dilute 95% ethanol
with pure water to 70% ethanol.
4. Paper cups (500 mL) (see Note 1).
5. Plastic cups (500 mL) (see Note 1).
6. Styrofoam (1.8 cm thick): Prepare disks which are slightly
smaller than the mouth of the plastic cup. Make 2–4 holes in
the disk to support seedlings (see Fig. 1).
7. Sponge sheet (ca. 1 cm thick): Cut into 2 cm wide and 3 cm
long belts.
8. Nutrient solution: Hoagland or other standard nutrient solu-
tions (see Note 2).
9. Phosphate free nutrient solution: Hoagland and other standard
nutrient solutions without phosphate (see Note 3).
Evaluation and Quantification of Natural Strigolactones from Root Exudates 5
Sponge belt
Styrofoam
floating lid
with holes
500 mL plastic cup containing

350 mL culture medium
500 mL paper cup
Fig. 1 Hydroponic culture for root exudate collection
2.2 Sample 1. Ethyl acetate.

Preparation 2. Separately funnel.
2.2.1 Solvent Partitioning 3. Anhydrous sodium sulfate (Na2SO4) or magnesium sulfate
(MgSO4).
4. Filter paper (No. 2).
5. Funnel.
6. Flasks.
7. Rotary evaporator (SpeedVac) (see Note 4).
8. Sample vials.
2.2.2 Concentration with 1. ODS or HLB cartridge (see Note 5).

Solid-Phase Extraction 2. Pure water (Milli-Q water).
(SPE) Cartridges
3. Acetonitrile.
4. Sample vials.
5. Nitrogen gas.
2.2.3 Sample Purification 1. SPE cartridges (ODS, HLB, DEAE) (see Note 5).
2. Solvents (see Note 6).
3. Sample vials.
4. Nitrogen gas.
3 Methods
3.1 Hydroponic 1. Sterile seeds with 1% NaClO containing 0.1% Tween

Culture 20 and/or 70% ethanol.
2. Germinate seeds on a filter paper wetted with sterile pure water
in 9 cm i.d. Petri dishes and grow them for a week (see Note 7).
3. Wrap a healthy seedling with a sponge band and place it into a
hole in the Styrofoam lid (Fig. 1).
4. Place the lid carrying seedlings on 350 mL of culture medium
in a 500 mL plastic cup. Then, place these plastic cups in
500 mL paper cups (Fig. 1).
5. Place the seedlings in a growth chamber maintained at appro-
priate temperature and light regime. Refresh culture medium
every 2–3 days (see Note 8).
3.2 Extraction of 1. Collect culture medium containing root exudates and extract
Root Exudates with equal volume of ethyl acetate three times by using a
separatory funnel. Collect upper phase (ethyl acetate) (see
3.2.1 Solvent Partitioning
Note 9).
2. Combine ethyl acetate solutions and dry over anhydrous
Na2SO4 or MgSO4.
3. After filtration, evaporate the solvent to nearly dryness under
reduced pressure (see Note 10).
4. Immediately dissolve the residue in a small volume of acetoni-
trile and transfer to a vial (see Note 11).
5. Store the vial at or below 4 C until use (see Note 12).
3.2.2 Concentration with 1. Activate SPE cartridges according to the manufacturer’s

Solid-Phase Extraction instructions.
(SPE) Cartridges 2. Collect culture medium containing root exudate and inject to
the cartridge (see Note 13). Wash the cartridge with pure
water.
3. Elute trapped root exudates with 80–100% acetonitrile (see
Note 14) and evaporate the solvent under nitrogen gas flow
(see Note 15).
trile and transfer to a vial (see Note 11).
5. Store the vial at or below 4 C until use (see Note 12).
3.2.3 Sample Purification 1. Activate SPE cartridges according to the manufacturer’s

instructions.
2. Dissolve sample with an appropriate organic solvent (see Note
16).
3. Inject sample solution to the cartridge and wash with the same
solvent.
4. Elute SLs with a solvent (mixture) of stronger elution power
(see Note 17) and evaporate the solvent under nitrogen gas
flow (see Note 15).
trile and transfer to an LC-MS sample vial.
3.3 LC-MS/MS A typical LC-MS/MS analysis of proton adduct ions of SLs is

Analysis performed with a triple quadrupole/linear ion trap instrument
(LIT) (QTRAP5500; AB Sciex) with an electrospray source. MS/
MS spectra are recorded in product ion scan mode using LIT. Ion
source is maintained at 400 C with curtain gas at 20 psi, collision-
ally activated dissociation (CAD) gas at 7 psi (12 psi for LIT), ion
source gas at 80 psi, and ion source gas2 at 70 psi. Ionspray voltage
is set at 5500 V in positive ion mode and 4500 V in negative ion
mode. Declustering, entrance, and collision cell exit potentials are
maintained at 60, 10, and 15 V, respectively. Collision energy is
optimized for each SL. HPLC separation of natural SLs listed in
Table 1 was performed on a UHPLC (Nexera X2; Shimadzu)
equipped with an ODS column (Kinetex C18, ϕ 2.1 150 mm,
1.7 μm; Phenomenex). The column oven temperature was main-
tained at 30 C. The mobile phase consisted of acetonitrile and
water, both of which contained 0.1% (vol/vol) acetic acid. HPLC
separation was conducted with a linear gradient of starting from
35% acetonitrile for 1 min and rising to 95% acetonitrile at 19 min,
followed by a 0.1 min gradient to 100% acetonitrile, which was
maintained for 3 min, before going back to 35% acetonitrile using a
0.5 min gradient, prior to the next run. Finally, the column was
equilibrated for 6 min, using this solvent composition. Retention
times along with monitoring transitions of natural SLs (Fig. 2) are
listed in Table 1.
4 Notes
1. Two to four seedlings can be grown hydroponically using

500 mL cups. In case to collect larger amounts of root exu-
dates, strainers and containers can be used as in [9, 10]. In the
case of woody plant species including poplar, cutting shoots are
grown in pots filled with mold and red granular soil for
1 month and then transfer to hydroponics [11]. Root exudates
can directly be collected from plants in pot culture by washing
with water or culture media as in [12]. For plant species
Table 1
Retention time (min) and monitoring transitions (m/z) of natural strigolactones in LC-MS/MS analysis
Monitoring transitions (m/z)
Retention time Precursor Diagnostic Confirming

Strigolactone (min) ion product ion product ion
7-Hydroxyorobanchyl acetate (1) 4.47 [M + H–60]+ 345 97 231
+
Avenaol (2) 4.80 [M + H] 377 97 235
Fabacol (3) 5.02 [M + H]+ 363 97 203
+
Solanacol (4) 5.48 [M + H] 343 97 201
+
Medicaol (5) 5.85 [M + H] 345 97 231
+
7-Oxo-orobanchyl acetate (6) 6.12 [M + H] 403 97 247
Strigol (7) 6.62 [M + H]+ 347 97 215
+
Sorgomol (8) 6.62 [M + H] 347 97 233
+
Orobanchol (9) 6.63 [M + H] 347 97 205
Strigone (10) 6.85 [M + H]+ 345 97 203
+
Solanacyl acetate (11) 9.12 [M + H–60] 325 97 228
+
Strigyl acetate (12) 9.68 [M–60] 328 97 215
+
Fabacyl acetate (13) 9.78 [M + H] 405 97 231
Zealactone (14) 9.78 [M + H]+ 377 97 231
+
Orobanchyl acetate (15) 10.57 [M–42] 347 97 205
+
5-Deoxystrigol (16) 12.07 [M + H] 331 97 217
+
4-Deoxyorobanchol (17) 12.27 [M + H] 331 97 217
Carlactonoic acid (18)a 12.27 [M–H] 331 69 113
[M + H]+ 333 97 269
Methyl carlactonoate (19) 16.48 [M + H]+ 347 97 315
+
Carlactone (20) 18.24 [M + H] 303 97 207
HPLC separation was performed on a UHPLC (Nexera X2; Shimadzu) equipped with an ODS column (Kinetex C18, ϕ
2.1 150 mm, 1.7 μm; Phenomenex). The column oven temperature was maintained at 30 C. The mobile phase
consisted of acetonitrile and water, both of which contained 0.1% (vol/vol) acetic acid. HPLC separation was conducted
with a linear gradient of starting from 35% acetonitrile for 1 min and rising to 95% acetonitrile at 19 min, followed by a
0.1 min gradient to 100% acetonitrile, which was maintained for 3 min, before going back to 35% acetonitrile using a
0.5 min gradient, prior to the next run. Finally, the column was equilibrated for 6 min, using this solvent composition
a
Carlactonoic acid can be detected by both positive and negative modes with similar sensitivities. For tissue samples,
LC-MS/MS analysis operated in negative mode affords clearer results.
Fig. 2 Structures of natural strigolactones. See Table 1 for full compound names
producing relatively large amounts of SLs, root exudates can be

collected by placing a few seedlings in a test tube containing
pure water [13].
2. Include 1 mM 2-(N-morpholino)ethanesulfonate (MES) if pH
of culture solution decreases below 6.0 during cultivation [14].
3. Sterile tap water can be used if plants grow well.
4. Evaporation should be done below 40 C and do not concen-
trate to dryness.
5. Recovery rates of SLs appear to be slightly better with HLB as
compared to ODS. DEAE (Diethylethanolamine) can be used
for purification of acidic compounds including
carlactonoic acid.
6. Acetonitrile and pure water are used for ODS and HLB car-
tridges. 2-Propanol and 2-propanol containing 1% acetic acid
are used for DEAE cartridge.
7. This incubation period may be longer for smaller plant species.
8. Change culture medium with low-phosphate or phosphate-free
media 1 week prior to the sampling of root exudates. Promo-
tion of SL production under phosphate starvation starts within
a few days but SL production may be unstable during this
acclimation period.
9. Add internal standards (2H- or 13C-labeled SLs) to the col-
lected medium. Synthetic SL GR24 may be used as an internal
standard. However, recovery rates of more unstable natural SLs
would be lower than that of GR24 and the effects of matrices in
LC-MS/MS analysis are different as their retention times are
different. For large volume of culture media, absorption of root
exudates by activated charcoal is convenient as in [9, 15]. The
absorbed root exudates can be eluted by acetone from the
charcoal.
10. Do not dry completely to avoid degradation of SLs, in particu-
lar, noncanonical SLs.
11. If necessary, the solvent can be evaporated under nitrogen gas
flow. However, samples should be stored as solutions.
12. Store the sample vials in tight boxes containing drying agent
(silica gel).
13. Collected culture medium shall be filtered through filter paper
if necessary.
14. 80% acetonitrile is enough to elute most SLs trapped in the
cartridge.
15. Evaporation of the solvent under nitrogen gas flow appears to

cause no significant degradation of SLs.
16. 10–20% acetonitrile is suitable to dissolve samples.
17. 80% acetonitrile can elute most SLs. Stepwise elutions with
40%, 60%, and 80% acetonitrile afford partial separation of
natural SLs.
Acknowledgments
This study was supported by the Japan Science and Technology

Research Promotion Program for Agriculture, Forestry, Fisheries
and Food Industry, the Japan Society for the Promotion of Sciences
(KAKENHI 15K07093, 16K07618, 16K18560, 17K07650), and
the Japan Science and Technology Agency PRESTO
(JPMJPR17QA).
References
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(3753):1189–1190 e00219
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(9):2219–2230 7-Oxoorobanchyl acetate and
5. Yoneyama K, Xie X, Yoneyama K, Kisugi T, 7-oxoorobanchol as germination stimulants
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CSP (2018) Which are the major players, tatissimum). Biosci Biotechnol Biochem 73
canonical or non-canonical strigolactones? J (6):1367–1370
Exp Bot 69(9):2231–2239 11. Yoneyama K, Mori N, Sato T, Yoda A, Xie X,
6. Yokota T, Sakai H, Okuno K, Yoneyama K, Okamoto M, Iwanaga M, Ohnishi T,
Takeuchi Y (1998) Alectrol and orobanchol, Nishiwaki H, Asami T, Yokota T, Akiyama K,
germination stimulants for Orobanche minor, Yoneyama K, Nomura T (2018) Conversion of
from its host red clover. Phytochemistry carlactone to carlactonoic acid is a conserved
49:1967–1973 function of MAX1 homologs in strigolactone
biosynthesis. New Phytol 218(4):1522–1533
7. Sato D, Awad AA, Chae SH, Yokota T,
Sugimoto Y, Takeuchi Y, Yoneyama K (2003) 12. López-Ráez JA, Kohlen W, Charnikhova T,
Analysis of strigolactones, germination stimu- Mulder P, Undas AK, Sergeant MJ,
lants for Striga and Orobanche, by high- Verstappen F, Bugg TD, Thompson AJ,
performance liquid chromatography/tandem Ruyter-Spira C, Bouwmeester H (2010) Does
mass spectrometry. J Agric Food Chem abscisic acid affect strigolactone biosynthesis?
51:1162–1168 New Phytol 187(2):343–354
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Kawada M, Haruta S, Nishiwaki H, sen MCR, Beale MH, Bouwmeester HJ (2005)
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the plant-parasitic Striga and Orobanche spp. germination stimulant for root parasites. Planta
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signal for mycorrhizal symbionts and
Chapter 2
Isolation and Identification of Naturally Occurring

Strigolactones
Kotomi Ueno, Takatoshi Wakabayashi, and Yukihiro Sugimoto
Abstract
The accurate structure determination of strigolactones (SLs) that are produced by plants leads to the precise
understanding of the biosynthesis and functions of their molecules. SLs need to be isolated and purified
from the plant roots or root exudates in a hydroponic solution using appropriate methods in order to
determine the structures. In this chapter, we describe a small-scale extraction method for chromatographic
analysis of known SLs and a large-scale purification method for isolation of unknown SLs, together with
methods for the hydroponic culture of plants and collection of root exudates. Finally, we present spectro-
scopic data that are helpful in identifying SLs.
Key words Hydroponic culture, Solid-phase extraction, Silica gel column chromatography, Prepara-
tive HPLC, Spectroscopic analysis
1 Introduction
Strigolactones (SLs) are a new class of phytohormones that func-

tion as signaling molecules, and are secreted by plant roots into the
rhizosphere [1]. Over 25 different SLs have been discovered that
commonly contain a methylbutenoride (D-ring) but display varia-
tions in structure due to their differences in stereochemistry and
functional groups [2, 3]. Strigol and its related compounds are
called canonical SLs, that is, they consist of a tricyclic lactone
(ABC-ring) connected to the D-ring via an enol ether bridge. On
another note, their congeners that have an unclosed BC-ring struc-
ture are called noncanonical SLs. SLs are produced not only by
several angiosperms but also by gymnosperms, ferns, and mosses
[4]. Different plant species produce different SLs and exude mix-
tures of several SLs. The composition of a mixture may even differ
between cultivars within the same species [5, 6]. Furthermore, the
amounts and ratios of SLs in a plant can fluctuate under different
growth conditions and developmental stages [7]. Therefore, opti-
mizing the culture conditions of plants is important not only to
13
14 Kotomi Ueno et al.
isolate and identify SLs but also to elucidate their biosynthesis and
structural diversity.
Identifying SLs in root exudates is generally performed by
liquid chromatography–tandem mass spectrometry (LC-MS/MS)
analysis. Prior to the analysis, SLs must be extracted, concentrated,
and purified from the hydroponic solution, since SLs are produced
by plants in extremely low quantities [8, 9]. Known SLs can be
identified through comparing chromatographic behavior and prod-
uct ions with those of standard compounds (see Chapter 1). How-
ever, for novel SLs, structure determination is required. They are
continuously secreted from roots under phosphorus-deficient con-
ditions, although production of SLs is only in small quantities
[9]. Accordingly, a large amount of SLs can be collected through
continuous adsorption from the hydroponic solution. Special tech-
niques for manipulating SLs are not required since they are neutral
molecules with moderate polarity. However, SLs easily decompose
under alkaline conditions, and they are unstable in nucleophilic
solvents, such as water or alcohol [10]. As such, quick and appro-
priate operation is needed. In this chapter, we describe simple SL
purification methods from the hydroponic solution and plant roots
for LC-MS/MS analysis, and elaborate the large-scale purification
methods for structure elucidation.
2 Materials
1. Purchase the plant seeds and organic solvents (guaranteed

reagent) from local suppliers.
2. Hydroponic culture media: prepare the individual stock solu-
tions as summarized in Tables 1 and 2 (see Note 1); weigh the
chemical components as described in Tables 1 and 2 and dis-
solve them in distilled water; mix and dilute the stock solutions
with distilled water before use.
3. Extraction of root exudates. Solid-phase extraction cartridges
(for small-scale extraction): Short cartridges containing a uni-
versal polymeric reversed-phase sorbent (see Note 2). For
example, Oasis HLB 6 cc (60 μm, 150 mg); Activated charcoal
bag (for large-sale extraction): Charcoal activated from coco-
nut shell (30–60 mesh) and disposable tea filter bags (approxi-
mately 9.5 cm 7.0 cm). Pack the activated charcoal (12 g) in
the tea filter bag (see Fig. 1).
4. Silica gel for column chromatography.
For canonical SLs: Particle size, 75–150 μm (100–200
mesh); pH 5.5–7.0 (e.g., Wakogel® C-200); For noncanonical
SLs: Spherical neutral; particle size, 63–210 μm (65–220
mesh); pH 6.5–7.5 (e.g., Silica gel 60 N) (see Note 3).
Isolation and Identification of Natural SLs 15
Table 1
Chemical composition of 1/2 Hoagland medium
Stock solution Preparation of medium (1 L)
Components M.W. (g/L) (mM) Add (mL) stock soln. Final conc. (μM)
1 KNO3 101.10 126.4 1250 2 2500
Ca(NO3)2 4H2O 236.15 295.2 1250 2500
NH4NO3 80.04 20.01 250 500
2 MgSO4 7H2O 246.47 123.2 500 2 1000
3 Fe(III)EDTA 3H2O 421.09 2.105 5 2 500
a
4 KH2PO4 136.09 34.02 250 2 10
(mg/100 mL)
5 H3BO3 61.83 284.4 46 0.5 23
MnCl2 4H2O 197.91 197.9 10 5
ZnSO4 7H2O 287.58 23.0 0.8 0.4
CuSO4 5H2O 249.69 8.0 0.32 0.16
Na2MoO4 2H2O 241.97 4.8 0.2 0.1
a
Reduce the added volume to 0.2 mL when a low-phosphorus medium is prepared
5. HPLC columns.
Reverse phase: general ODS, 5 μm (or 3 μm) 250 20 mm
i.d. (e.g., CAPCELLPAK C18 UG120 S5).
Direct chiral phase: chiral, 5 μm 250 4.6 mm i.d. (CHIR
ALPAK IC).
3 Methods
3.1 Small-Scale 1. Sow the seeds on the moist paper towels that are placed in Petri
Extraction dishes. Wrap the dishes in aluminum foil and incubate the seeds
and Purification of SLs in the dark at the appropriate temperature (20–30 C) (see
from Roots and Root Note 4).
Exudates 2. Put the germinated seeds in the cut sponge cubes (hydroponic
sponges). Place the sponge cubes on top of a 50-mL tube filled
with hydroponic solution (see Note 5).
3. Grow the plants hydroponically for 2–4 weeks. Replace the
hydroponic solution with a new one every 3–4 days.
4. In terms of promoting SL biosynthesis, replace the hydroponic
solution with tap water or a hydroponic solution without a
phosphate and/or nitrogen source.
5. For extraction and purification of SLs from plant root exudates,
collect the hydroponic solution (ca. 40 mL) and add an internal
standard.
6. Filter the solution through the filter paper.
Table 2
Chemical composition of Long Ashton medium
Stock solution Preparation of medium (1 L)
Components M.W. (g/L) (mM) Add (mL) stock soln. Final conc. (μM)
1 K2SO4 174.26 21.78 125 6.4 800
2 CaCl2 2H2O 147.02 73.5 500 3.2 1600
3 MgSO4 7H2O 246.47 46 187 3.2 600
4 Na2HPO4 12H2O 358.14 29.75 83 6.4 530
5 NH4NO3 80.04 50.25 628 1.6 1000
6 Fe(III)EDTA 3H2O 421.09 8.42 20 2 40
7 NaCl 58.44 5.84 100 0.4 40
H3BO3 61.83 3.10 50 20
MnSO4 4H2O 223.06 2.26 10 4
(mg/L)
ZnSO4 7H2O 287.58 287 1 0.4
Na2MoO4 2H2O 241.97 120 0.5 0.2
CuSO4 5H2O 246.69 24.9 0.1 0.04
Seedling
Sponge cube
Activated charcoal
Tea filter bag
Foam board
Internal filter
Hydroponic culture system
Fig. 1 Hydroponic culture for the large-scale extraction of SL
7. Load the filtrate on the Oasis HLB 6 cc cartridge conditioned

and equilibrated with acetone and water, respectively.
8. Wash the cartridge with 4 mL of 20% acetone (v/v), then elute
the SLs with 4 mL of 80% acetone (v/v) (see Note 6).
9. Evaporate the acetone in the eluate under nitrogen gas, leaving
a small amount of water (see Note 7).
10. Add acetonitrile to the aqueous residue up to a concentration

of 50% (v/v), and subject the sample to LC-MS/MS analysis.
11. For the extraction and purification of the SLs from roots,
harvest the root tissues (ca. 0.1–1.0 g FW) and immediately
immerse them in acetone with a minimum volume that is ten
times the weight of the roots (v/w) (see Note 8).
12. Add an internal standard in the acetone solution.
13. Cut the root tissues into small pieces with scissors while
submerged in the acetone solution.
14. Keep the suspension at a temperature of 4 C for 1 h (see Note
9).
15. Filter the aqueous acetone solution through the filter paper.
16. Concentrate the solution with a rotary evaporator to 1–5 mL.
Then, evaporate the remaining acetone under nitrogen gas,
leaving 100–200 μL of aqueous residue (see Note 7).
17. Add 10 mL of water to the residue, mix well, and collect the
supernatant after centrifugation.
18. Load the supernatant on the Oasis HLB cartridge conditioned
and equilibrated with acetone and water, respectively.
19. Prepare a sample for LC-MS/MS analysis following the steps
8–10 as described above.
3.2 Hydroponic 1. Germinate the seeds as described at step 1 (see Note 10).
Culture and Collection 2. Put the germinated seeds in the cut sponge cubes (planted
of Root Exudates sponges). Set the sponge cubes with seeds to the hydroponic
for Large-Scale culture system (deep water culture system) (Fig. 1).
Extraction 3. Prepare the activated charcoal bag. Put the bag in an internal
filter for aquariums. Set the filter in the hydroponic culture
container (Fig. 1, see Note 11).
4. Grow the plants hydroponically in the container filled with
modified Hoagland solution or Long Ashton nutrient solution
for 2–8 weeks. Replenish the hydroponic solution container to
keep the water at the appropriate level.
5. Collect the charcoal bag and replace it with a new one in the
internal filter every week.
6. Soak the collected charcoal with acetone (80 mL) and store at a
temperature of 4 C for more than 2 days (see Note 12).
7. Filter the acetone solution through the filter paper. Remove
the acetone in the filtrate using a rotary evaporator at a temper-
ature of 30 C (see Note 13).
8. Perform a liquid–liquid partition between the residual aqueous
solution (ca. 20 mL) and EtOAc (7 mL) three times using a
separatory funnel.
9. Combine the EtOAc solution and wash the organic solution

with 0.2 M K2HPO4 (pH 8.3, 1 mL 2) and then H2O
(1 mL 2) (see Note 14).
10. Dehydrate the organic solution using Na2SO4 (or MgSO4).
Concentrate the solution using a rotary evaporator after
removing Na2SO4 using absorbent cotton or filter paper.
11. Apply the residue to the silica gel column.
3.3 Open Silica Gel 1. Suspend the silica gel (20 g) in n-hexane (ca. 50 mL). Pour the
Column silica gel slurry into the glass column (Φ 1.5 cm) (see Note 15).
Chromatography 2. Dissolve the residue (prepared in Subheading 3.2) in a small
amount of CHCl3. Add a small amount of hexane to the
solution. Gently add the solution to the top of the column
using a pipette (see Note 16).
3. Gently pour hexane (50 mL) to the column and start collecting
the column effluent into an Erlenmeyer flask.
4. Allow the effluent to drain before pouring the additional hex-
ane (50 mL) (see Note 17).
5. After draining the hexane into another flask, gently pour the
hexane–EtOAc (9:1, 50 mL 2) mixture. Collect the column
effluent separately (50 mL per fraction).
6. Allow the solvent (50 mL 2) to flow while increasing the
concentration of EtOAc in the solvent stepwise every 10% until
the concentration reaches 100%.
7. After draining the 100% EtOAc (50 mL 2) solution, add
MeOH (50 mL) and allow to drain. Collect a total of
23 fractions.
8. Identify the fractions containing the germination stimulants
through bioassays (see Note 18).
3.4 Preparative HPLC 1. Depending on the purity of the active fraction and the instru-
ments available in the laboratory, perform the preparative
HPLC procedure in the reversed-phase mode or the normal-
phase mode. Conditions for reverse phase: 40–70% MeOH in
H2O (e.g., 60% MeOH for heliolactone [11]), Flow rate
3–5 mL/min, column temp. room temp. (ca. 25 C), detection
235 nm. Condition for normal phase: 25–100% EtOH in
hexane (e.g., 50% EtOH for Orobanchyl acetate [12]), Flow
rate 0.5–1 mL/min.
2. Collect the column effluent fractions every minute or as per the
peak detected by UV absorption.
3. Dilute each fraction with water (approximately ten times) or
remove MeOH in vacuo at a temperature of 30 C. Treat the
aqueous solution with EtOAc, wash the organic layer with
H2O, dehydrate the EtOAc layer with Na2SO4, and concen-

trate the solution after filtration (see Subheading 3.2).
4. Identify the fractions that contain the germination stimulants
through bioassays.
5. Perform the preparative HPLC procedure in different condi-
tions if the active fractions contain contaminants or at least two
active substances.
3.5 Nuclear 1. Prepare the maximum amount of pure material, ideally in

Magnetic Resonance milligrams (see Note 19).
(NMR) Analysis 2. Dissolve the material in 0.6 mL CDCl3 (see Note 20) and
for Determining transfer the solution to an NMR tube (5 mm o.d.) by filtration
the Relative Structure using a Pasteur pipette packed with a small piece of glass wool.
of SLs 3. Record the 1H NMR spectra using an available spectrometer. If
the compound is an SL, signals assignable to the D-ring and the
enol ether bridge will be observed at approximately δ 7.5, 6.9,
6.2, and 2.0. Figure 2 shows the typical 1H NMR chemical
shifts of SLs in CDCl3 and C6D6 solution.
4. Measure 13C NMR, 1H-1H COSY, NOESY, HMQC, and
HMBC spectra in addition to the 1H NMR spectra to deter-
mine the relative structure if the active substance is novel.
3.6 Mass 1. Prepare 100 μL of the sample solution in acetonitrile with a

Spectrometry concentration of approximately 10 μg/mL.
(MS) Analysis 2. Inject ca. 5 μL of the solution into the mass spectrometer with
for Determining a soft ionization system, such as electrospray ionization (ESI)
the Molecular Weight coupled with LC (see Note 21). SLs are ionized in a positive ion
mode. The ion at m/z 97 corresponding to the D-ring will be
detected in addition to the pseudomolecular ions [M + H]+ and
[M + Na]+.
3.7 Ultraviolet– 1. Prepare 3 mL of the sample solution in acetonitrile with a

Visible (UV-Vis) concentration of approximately 50 μM in a quartz cuvette
and Circular Dichroism (1 cm) (see Note 22).
(CD) Spectroscopy 2. Measure an absorbance spectrum of 200–400 nm using
for Determining ultraviolet-visible spectrometer. Table 3 shows the general
the Absolute measurement conditions.
Configuration 3. Calculate the exact molar concentration of the solution from
the molar attenuation coefficient (ε) and the absorbance at the
wavelength of peak absorption (λmax) (see Note 23).
4. Place the cuvette in a circular dichroism spectropolarimeter and
measure an absorbance spectrum under the conditions
described in Table 3. Measure an absorbance of the solvent
that is used to dissolve the sample (blank).
1.1 5.5 (5.1)

(1.1)
1.0 3.7 (3.3)
1.4 6.8
(1.2) 7.5 (7.4) 7.5 (7.4)
1.4 6.1 (4.9)
1.7 3.5 6.1 (5.1) 6.1
(1.4) (3.1)
1.9 2.5 1.6
(1.6) (2.3) 2.0 1.7
6.9 6.9
(5.7) 2.0 (1.3) (5.6) 2.0 (1.3)
Canonical SLs Non-canonical SLs
Fig. 2 1H NMR chemical shifts of the basic skeleton of canonical and noncanonical SLs. The approximate ppm
values observed in CDCl3 are given with those observed in C6D6 in parentheses. These values are referenced
to refs. 5, 12–15
Table 3
UV and CD measurement conditions
UV
Measurement range 190–340 nm Scan speed 200 nm/min
Bandwidth 2.0 nm Data interval 0.2 nm
Response Medium
CD
Measurement range 190–340 nm Scan speed 100 nm/min
Spectral band width 1 nm Response time 1s
Data accumulation interval 1 nm Accumulations Three times
a b
10 10
5 5 (R)-MeCLA
Mol.CD
Mol.CD
0 0
-5 -5
(S)-MeCLA
-10 -10
200 250 300 350 200 250 300 340
Wavelength [nm] Wavelength [nm]
Fig. 3 CD spectra of heliolactone (a) and MeCLA (b) Refer to ref. 16 for CD spectra of canonical SLs
5. Subtract the absorbance spectrum of the blank from that of the

sample to obtain the CD spectrum of the compound. Convert
ellipticity ([mdeg], the vertical axis of the spectrum) to molar
ellipticity using the molar concentration determined by UV
spectroscopy. As an example, CD spectra of noncanonical
SLs, such as heliolactone and MeCLA, are shown in Fig. 3.
4 Notes
1. Select the media suitable for each plant. Poaceae plants (e.g.,
sorghum and rice) prefer Long Ashton solution, whereas other
plants prefer 1/2 or 1/4 Hoagland solution.
2. A silica-based C18 cartridge is not suitable for collecting
organic compounds in large amounts of aqueous solution.
3. Noncanonical SLs may be less stable compared with canonical
SLs under acidic conditions.
4. The small seeds are directly sown on a wet gauze or absorbent
cotton spread on a mesh sink strainer (7.5–12 cm diameter).
Put the strainer with the germinated seeds on a disposable
plastic cup filled with culture solution. Cover the cup with
aluminum foil to shield the germinated seeds from light.
5. Grow the plants in tap water for approximately a week if root
elongation is slow.
6. Most SLs cannot be eluted and can be recovered using washing
and elution solvents, respectively. Specific SLs can be purified
by changing the acetone concentration in the solvent in a
stepwise manner.
7. Noncanonical SLs, such as carlactone, decompose when con-
centrated to dryness.
8. Do not freeze the root tissues to minimize the possible degra-
dation of SLs.
9. The extraction time varies from several hours to several days
depending on the target SLs and plant species; however, an
extraction period beyond 1 week can lead to degradation
of SLs.
10. The small seeds are directly sown on a wire mesh basket cov-
ered with a wet gauze or absorbent cotton. The basket with the
germinated seeds should be placed in a deep tray filled with tap
water or culture solution.
11. If an ebb and flow (flood and drain) subirrigation hydroponic
culture system is used and the charcoal bag can be set into the
drain tube, the internal filter for aquariums will be unnecessary.
When the activated charcoal bags are not used, the huge
amount of hydroponic solution (approximately 100 L) needs

to be treated with a similar volume of EtOAc [11].
12. Some non-SL germination stimulants (e.g., costunolide) may
not be eluted in acetone. Such germination stimulants need to
be extracted from the hydroponic solution with EtOAc using
liquid–liquid partition method.
13. After the removal of acetone, a considerable amount of water
held in the collected charcoal is left.
14. Remove the acidic contaminants using 0.2 M K2HPO4. Wash-
ing is needed especially when SLs are directly extracted from
the hydroponic solution with EtOAc. Remove OH by wash-
ing the organic layer with a small amount of distilled water, as
SLs are unstable even under a weak alkaline condition.
15. Note that 20 g of silica gel is approximately 45 cm2 in volume,
and the length of the column is approximately 25 cm in a glass
tube with 1.5 cm diameter. The column length should be a
minimum of 20 cm for a satisfactory purification of the frac-
tions containing SLs.
16. Alternatively, the residue is dissolved in a small amount of
EtOAc, and silica gel powder is added to the solution. EtOAc
is removed using a rotary evaporator until the gel becomes dry.
The dried gel is suspended in hexane, and the slurry is applied
on top of the silica gel column.
17. The volume of hexane (i.e., the volume of individual fractions)
should be proportional to that of the silica gel column.
18. Conduct bioassays using parasitic weed seeds as described in
later chapters. Pretreat (condition) the surface-sterilized seeds
on 8-mm glass fiber paper discs moistened with distilled water
for an appropriate period (6 days or more depending on the
species and seed conditions) at 20–30 C in the dark. Place a
disc with the conditioned seeds upon another disc with a test
sample. Count the number of germinated seeds under a micro-
scope after incubation for 1–4 days in the dark.
19. Generally, the quantity of the required material is approxi-
mately 5 mg for 1H NMR measurement. However, it is hard
to obtain naturally occurring SLs in such a large quantity.
20. CD2Cl2 and C6D6 are also used. To avoid contamination of
other solvent molecules, dissolve the sample in the 1H solvent,
CHCl3 (CH2Cl2 or C6H6) and then, remove the solvent using
a rotary evaporator before dissolving in the deuterated solvent,
CDCl3 (CD2Cl2 or C6D6). Reduce the volume of the deuter-
ated solvent if using Shigemi Symmetrical MICRO NMR
Tubes or 5-mm Micro Bottom Tubes.
21. The molecular ion [M]+ is hardly detected using the electron
ionization method because its hydroxyl and acetyl groups are
desorbed by ionization.
22. Prepare a solution with the correct concentration (approxi-
mately 50 μM) if the new compound can be accurately
weighed.
23. 5-DS (MeCN): λmax 234 nm, ε 17,000 [10]; 4-DO (MeCN):
λmax 234 nm, ε 15,600 [10]; CL (CHCl3): λmax 272 nm, ε
12,700 (personal communication from Mark Bruno); Z–CL
(MeCN): λmax 270 nm, ε 12,900 (unpublished data); and E–
CL (MeCN): λmax 259 nm, ε 13,400 (unpublished data).
References
1. Al-Babili S, Bouwmeester HJ (2015) Strigolac- promotes exudation of orobanchol, the signal
tones, a novel carotenoid-derived plant hor- for mycorrhizal symbionts and germination
mone. Annu Rev Plant Biol 66:161–186 stimulant for root parasites. Planta
2. Wang Y, Bouwmeester HJ (2018) Structural 225:1031–1038
diversity in the strigolactones. J Exp Bot 10. Akiyama K, Ogasawara S, Ito S et al (2010)
69:2219–2230 Structural requirements of strigolactones for
3. Yoneyama K, Xie X, Yoneyama K et al (2018) hyphal branching in AM fungi. Plant Cell
Which are the major players, canonical or Physiol 51:1104–1117
non-canonical strigolactones? J Exp Bot 11. Sugimoto Y, Ueyama T (2008) Production of
69:2231–2239 (+)-5-deoxystrigol by Lotus japonicus root cul-
4. Xie X (2016) Structural diversity of strigolac- ture. Phytochemistry 69:212–217
tones and their distribution in the plant king- 12. Ueno K, Furumoto T, Umeda S et al (2014)
dom. J Pestic Sci 41:175–180 Heliolactone, a non-sesquiterpene lactone ger-
5. Ćavar S, Zwanenburg B, Tarkowski P (2015) mination stimulant for root parasitic weeds
Strigolactones: occurrence, structure, and from sunflower. Phytochemistry 108:122–128
biological activity in the rhizosphere. Phyto- 13. Ueno K, Nomura S, Muranaka S et al (2011)
chem Rev 14:691–711 Ent-20 -epi-orobanchol and its acetate, as ger-
6. Ueno K, Nakashima H, Mizutani M et al mination stimulants for Striga gesinerioides
(2018) Bioconversion of 5-deoxystrigol stereo- seeds isolated from cowpea and red clover. J
isomers to monohydroxylated strigolactones Agric Food Chem 59:10485–10490
by plants. J Pestic Sci 43:198–206 14. Abe S, Sado A, Tanaka K et al (2014) Carlac-
7. Motonami N, Ueno K, Nakashima H et al tone is converted to carlactonic acid by MAX1
(2013) The bioconversion of 5-deoxystrigol in Arabidopsis and its methyl ester can directly
to sorgomol by the sorghum, Sorghum bicolor interact with AtD14 in vitro. Proc Natl Acad
(L.) Moench. Phytochemistry 93:41–48 Sci U S A 11:18084–18089
8. Sato D, Awad AA, Takeuchi Y et al (2005) 15. Xie X, Kisugi T, Yoneyama K et al (2017)
Confirmation and quantification of strigolac- Methyl zealactonoate, a novel germination
tones, germination stimulants for root parasitic stimulant for root parasitic weeds produced by
plants Striga and Orobanche, produced by cot- maize. J Pestic Sci 42:58–61
ton. Biosci Biotechnol Biochem 69:98–102 16. Ueno K, Sugimoto Y, Zwanenburg B (2015)
9. Yoneyama K, Yoneyama K, Takeuchi Y et al The genuine structure of alectrol: end of a long
(2007) Phosphorus deficiency in red clover controversy. Phytochem Rev 14:835–847
Chapter 3
Chemical Synthesis of Triazole-Derived Suppressors

of Strigolactone Functions
Shisanku Ito, Ko Kikuzato, Hidemitsu Nakamura, and Tadao Asami
Abstract
Triazole is a five-membered heteroring consists of two carbon atoms and three nitrogen atoms and exhibits
a wide range of biological activities. The basic heterocyclic rings are 1,2,3-triazole and 1,2,4-triazole. Here
we describe the chemical synthetic methods for triazole derivatives that can suppress the function of SL by
inhibiting SL biosynthesis pathway or SL perception sites such as D14.
Key words Triazole, Strigolactone, Cytochrome P450, Carotenoid cleavage dioxygenase, Strigolac-
tone receptor, α/β-Hydrolase
1 Introduction
Triazole is an organic heterocyclic compounds containing a five-

membered bis-unsaturated ring structure composed of three nitro-
gen atoms and two carbon atoms. The triazole scaffold has been
used for a number of cytochrome P450 monooxygenase inhibitors.
The lone pair in sp2 hybridized nitrogen atom in triazole can bind to
heme iron in cytochrome P450s [1]. There have been a number of
[H]-1,2,4-triazole-type plant growth regulators that target cyto-
chrome P450s in plant hormonebiosynthesis pathway [2]. For
example, cocrystal structures between triazole-type plant growth
regulators and cytochrome P450 demonstrate that unicolazole and
brassinazole bind to CYP90B1, a cytochrome P450 in brassinos-
teroid biosynthesis pathway through the lone pair–heme interac-
tions [3]. As cytochrome P450 is also involved in SL biosynthesis
pathway, we prepared triazole derivatives in expectation of new SL
biosynthesis inhibitors and eventually found TIS108 as a potent SL
biosynthesis inhibitor [4]. Ten times more active inhibitor KK5
than TIS108 was found through further structure–activity relation-
ship studies [5]. The target sites of these two triazole-type SL
biosynthesis inhibitors have not been elucidated, but they are
25
26 Shisanku Ito et al.
expected to bind to CYP770A, a cytochrome P450 in SL biosyn-

thesis pathway.
In addition to the control of cytochrome P450 function, tria-
zole has been used for the control of α/β-hydrolase function. It has
been reported that [H]-1,2,3-triazole moiety is a good leaving
group of triazole ureas upon the interaction with α/β-hydrolase
to form covalent binding [6]. After intensive structure–activity
relationship studies, we found that KK094, a triazole-urea, cova-
lently binds to D14, an α/β-hydrolase-type rice strigolactone
receptor. Its covalent biding was confirmed by LC/MSMS analysis
and cocrystallization [7]. In these researches intensive chemical
synthesis of versatile triazole derivatives played an important role
in finding both SL biosynthesis inhibitors and SL receptor inhibi-
tors. Here we described how they can be synthesized in a short
pathway so that several investigators can obtain similar results.
2 Materials
2.1 SL Biosynthesis Phenacyl bromide, 1,2,4-triazole, (4-chlorobutoxy)benzene, bis

Inhibitors (2-bromoethyl)ether, and 4-phenoxybutyl chloride were purchased
from Tokyo Chemical Inc. (TCI). K2CO3, acetone, and DMF
super dehydrated were purchased from Fujifilm Wako Pure
Chemical Co.
2.2 SL Receptor Indoline, triphosgene, N,N-diisopropylethylamine, dimethylami-

Inhibitor KK094 nopyridine and 1,2,4-triazole were purchased from Tokyo
Chemical Inc.
3 Methods
3.1 Biosynthesis Biosynthesis Inhibitor TIS108 (6-Phenoxy-1-phenyl-2-(1H-1,2,4-

Inhibitor TIS108 triazol-1-yl)hexan-1-one)
1.
Phenacyl bromide (10 mmol, 1.99 g), 1H-1,2,4-triazole

(10 mmol, 0.69 g), and K2CO3 (excess, 5 g) are mixed in
acetone (10 ml) and stirred for 2 h at room temperature. The
reaction mixture is then poured into 30 ml of water and
extracted three times with 10 ml of ethyl acetate. The organic
Chemical Synthesis of Triazole-Derived Suppressors of Strigolactone Functions 27
layer was dried with Na2SO4 and concentrated under reduced

pressure to give white residue, which was then washed with n-
hexane to give enough pure 1-phenyl-2-(1H-1,2,4-triazol-1-
yl)ethenone for the next reaction (85% yield).
2.
1-Phenyl-2-(1H-1,2,4-triazol-1-yl)ethanone (6.8 mmol,

1.3 g) and sodium hydride (10 mmol, 0.4 g) were dissolved
in dimethylformamide (4 ml) and stirred for 30 min, and then
(4-chlorobutoxy)benzene (10 mmol, 1.85 g) was added under
nitrogen atmosphere and stirred for 12 h at room temperature.
The reaction mixture was quenched with distilled water. The
aqueous layer was extracted three times with ethyl acetate. The
organic layer was dried with Na2SO4 and concentrated under
reduced pressure. The resulting oil was purified by column
chromatography (n-hexane: ethyl acetate ¼ 3∶2, v/v). The
product was isolated as a white solid in 12.7% yield (see
Note 1). 1H NMR (CDCl3, 500 MHz) δ 1.43–1.61 (2H,
m), 1.75–1.90 (2H, m), 2.15–2.36 (2H, m), 3.92 (2H, t J
¼ 6.3 Hz), 6.09 (1H, dd J ¼ 4.8, 10.3 Hz), 6.83 (2H, d J ¼
9.0 Hz), 6.93 (1H, t J ¼ 6.8 Hz), 7.26 (2H, t J ¼ 8.0 Hz),
7.51 (2H, t J ¼ 6.5 Hz), 7.63 (1H, t J ¼ 6.5 Hz), 7.95 (1H,
s), 7.99(2H, d J ¼ 8.3 Hz), 8.38 (1H, s); 13C NMR (CDCl3,
300 MHz) δ 194.2, 158.8, 151.4, 143.0, 134.4, 134.3, 129.5,
129.2, 128.8, 120.8, 114.4, 67.1, 63.9, 32.7, 28.6, 22.8;
HRMS (ES+) calcd. for C20H23N3O2+ (M + H) 336.1712,
found 336.1707; Melting Point 107–108 C.
3.2 Biosynthesis Biosynthesis Inhibitor KK5 (4-(2-Phenoxyethoxy)-1-phenyl-

Inhibitor KK5 2-(1H-1,2,4-triazol-1-yl)butan-1-one)
1.
Phenol (10 mmol, 0.94), bis(2-bromoethyl)ether

(10 mmol, 2.32 g) and K2CO3 (excess amount, 5 g) were
mixed in acetone (15 ml) and refluxed for 2 h with vigorous
stirring. The solvent was removed under reduced pressure to
give residue, which was then dissolved into 20 ml of ethyl

acetate and 30 ml of water. After separation of organic layer
water layer was extracted three times with 10 ml of ethyl
acetate. The combined organic layer was dried with NaSO4
and concentrated under reduced pressure to give residue,
which was then recrystallized with n-hexane: ethyl acetate to
give white crystal of (2-(2-bromoethoxy)ethoxy)benzene (79%
yield).
2.
To a suspension of sodium hydride (36 mmol, 0.85 g) in

dimethylformamide (5 ml) was added 1-phenyl-2-(1H-1,2,4-
triazol-1-yl)ethanone (5.8 mmol, 1.08 g) in dimethylforma-
mide (5 ml) at 0 C under nitrogen. After the solution is stirred
at 0 C for 10 min, (2-(2-bromoethoxy)ethoxy)benzene
(8.5 mmol, 2.06 g) in dimethylformamide (5 ml) is added at
0 C. The mixture was warmed to 70 C and stirred for 5 h.
The reaction was quenched by adding distilled water on ice.
The aqueous phase was extracted with ethyl acetate three times.
The combined organic phases were dried over anhydrous
Na2SO4 and concentrated in vacuo. Purification by silica gel
column chromatography (hexane–ethyl acetate as the eluent)
gave KK5 as a white solid (5.5% yield) (see Note 1).
1
H NMR (CDCl3): δ 8.31 (s, 1H), 7.94 (d, J ¼ 7.5 Hz,
2H), 7.91 (s, 1H), 7.56 (t, J ¼ 7.3 Hz, 1H), 7.41 (t,
J ¼ 7.7 Hz, 2H), 7.29 (t, J ¼ 7.9 Hz, 2H), 7.00–6.89 (m,
3H), 6.31 (dd, J ¼ 9.9 and 5.2 Hz, 1H), 4.11 (t, J ¼ 4.6 Hz,
2H), 3.81–3.68 (m, 2H), 3.68–3.59 (m, 1H), 3.33–3.24 (m,
1H), 2.62–2.50 (m, 1H), 2.41–2.30 (m, 1H). 13C NMR
(400 MHz, CDCl3): δ 194.1, 159.2, 151.8, 143.7, 134.2,
134.3, 134.2, 129.6, 129.1, 128.8, 121.2, 114.7, 69.9, 67.2,
66.4, 60.4, 32.7. HRMS (m/z): [M + H]+ calculated for
C20H22N3O3+, 352.1656; found, 352.1662.
3.3 Receptor KK094: mixture of indolin-1-yl(1H-1,2,3-triazol-1-yl)methanone

Inhibitor KK094 N1 and indolin-1-yl(2H-1,2,3-triazol-2-yl)methanone N2.
Chemical Synthesis of Triazole-Derived Suppressors of Strigolactone Functions 29
1. Indoline (3.4 mmol, 0.4 g) and N,N-diisopropylethylamine

(10.1 mmol, 1.301 g,) were added to the triphosgene
(1.7 mmol, 498 mg,) solution in THF (5 ml) (see Note 2)
keeping the temperature below <10 C and cooled to 0 C.
The reaction was stirred for 15 min on ice. After adding iced
water, the reaction solution was extracted with ethyl acetate
twice. The ethyl acetate layers were combined, dehydrated with
Na2SO4, the solvent was removed under vacuum and the
carbamoyl chloride intermediate was obtained. This residue
was used without further purification for the next step.
2. The resulting residue was dissolved in THF (10 ml) and N,N-
diisopropylethylamine (10.1 mmol, 1.301 g,), 1H-1,2,3-tria-
zole (4.0mmol, 278 mg,) and dimethylaminopyridine (cat.)
were added on ice and stirred overnight at room temperature
(rt). Then the solvent was removed under vacuum and the
resulting residue was dissolved and extracted with ethyl acetate
and water twice. The ethyl acetate layers were combined, the
solvent was removed under vacuum and the resulting residue
was purified on a silica gel column (Hexane:CH2Cl2:EtOAc
gradient from 1:4:0.4 to 0:4:0.4) (see Note 3) giving KK094-
N1 (190 mg, 26%) as a white solid and KK094-N2 (97 mg,
14%) as a white solid (see Note 4).
KK094-N1: 1H NMR (500 MHz, CDCl3): δ 8.36 (d,
J ¼ 1.1 Hz, 1H), 8.11 (br s, 1H), 7.78 (d, J ¼ 1.1 Hz, 1H),
7.33–7.27 (m, 2H), 7.16 (t, J ¼ 7.4 Hz, 1H), 4.67 (t, J ¼ 8.3 Hz,
2H), 3.26 (t, J ¼ 8.3 Hz, 2H). 13C NMR (125 MHz, CDCl3): δ
145.92, 141.88, 132.91, 132.19, 127.68, 125.45, 125.00,
124.96, 117.49, 51.77, 28.55. HRMS (m/z): calcd. for
C11H10N4O, [M + H]+: 215.0927; found, 215.0927.
KK094-N2: 1H NMR (500 MHz, CDCl3): δ 8.10 (s, 1H),
7.88 (s, 2H), 7.35–7.20 (m, 2H), 7.13 (t, J ¼ 7.4 Hz, 1H), 4.47 (t,
J ¼ 8.3 Hz, 2H), 3.22 (t, J ¼ 8.3 Hz, 2H). 13C NMR (125 MHz,
CDCl3): δ 146.35, 141.95, 136.27, 132.03, 127.67, 125.04,
124.84, 117.48, 51.39, 28.45. HRMS (m/z): calcd. for
C11H10N4O [M + Na]+:237.0747; found, 237.0757.
4 Notes
1. In the final alkylation process in SL biosynthesis inhibitors

TIS108 and KK5, DMF should be anhydrous, otherwise the
yield of the reaction is drastically reduced.
2. Triphosgene is very toxic. This reaction should be performed in
a well ventilated fume hood. Any object that comes into con-
tact with triphosgene should be rinsed with 10% NaOH
solution.
3. Separation of N1 and N2 isomers of KK094 with aminosilica
TLC worked better than with silica gel TLC, but it did not
work well for column chromatography, maybe due to the
degradation of the products.
4. Recrystallization of KK094 with MeOH gave worse yield.
Acknowledgments
This work was supported in part by a grant from the Core Research
for Evolutional Science and Technology (CREST) Program of the
Japan Science and Technology Agency (JST) (to T.A.); a JSPS
Grant-in-Aid for Scientific Research (grant number 26520303 to
H.N., 18H03939 to T.A.); a JSPS Grant-in-Aid for Scientific
Research on Innovative Areas (grant number 18H04608) “Frontier
Research on Chemical Communications” (no. 2906) (to H.N.).
References
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Chapter 4
Synthesis of Simple Strigolactone Mimics

Tomáš Pospı́šil
Abstract
Strigolactones (SLs) are natural compounds occurring in plants which have a numerous functions in plant
development; therefore, they are plant hormones. Unfortunately, their natural abundance is very low and
because of their structure complexity it is difficult to prepare them in big quantities; alternatives with
simpler structures and similar biological activity was developed. SLs mimics are compounds with simple
synthesis. Methods for preparation of basic SLs mimics are described here.
Key words Strigolactones, SL mimics, Plant hormones, Debranones
1 Introduction
Strigolactones (SLs) are plant hormones with a broad range of

biological activity. For example, it was discovered that SLs regulate
plant architecture, wherein they inhibit bud outgrowth and shoot
branching [1, 2]. They are also involved in plant response to abiotic
stress [3]. Furthermore, SLs are known as a branching factor for
arbuscular mycorrhizal fungi [4] and as a germination stimulant for
parasitic plants [5]. The family of SLs is nowadays divided into two
groups: canonical and noncanonical SLs. Canonical SLs have three
annulated rings (the ABC scaffold) connected via an enol-ether to
the fourth ring (D-ring). Noncanonical SLs have an enol ether
D-ring moiety, but they miss the A, B, or C part (Fig. 1).
Isolation of SLs from natural sources is difficult because they
are produced in small amounts in plants (15 pg/day/plant)
[6]. Total synthesis of SLs is made in a linear fashion through
several steps and therefore it is costly to prepare them in a multi-
gram scale. The alternative is to prepare compounds with simplified
structures but similar activity. There are two groups of such com-
pounds, SL analogs and SL mimics. SL analogs have a modified
ABC part connected with the D-ring through an enol ether bridge.
Depending on the modification of the ABC part they are similar to
canonical or noncanonical SLs (Fig. 2). On the other hand, SL
31
32 Tomáš Pospı́šil
Fig. 1 General structures of canonical and noncanonical SL
Fig. 2 Structures of some SL analogs
mimics have only the D-ring part with a substituent on C-5 posi-
tion (Fig. 3). Such compounds is easy to prepare in only 1 or
2 steps. Synthesis of two basic types of SL mimics derived from
phenol (debranones) [7] and carboxylic acid [8–10], together with
preparation of bromobutenolide (Br-D-ring) [11] as a starting
material, will be described (Scheme 1).
2 Materials
All commercial chemicals are stored according to the manufac-

turer’s description and are used without further purification. All
the compounds are prepared in fume hood and stored in a freezer
(20 C). For disposing of waste material, follow all waste disposal
regulations. All operations with dry solvents are performed in argon
atmosphere.
2.1 Synthesis 1. The dried apparatus for synthesis consists of a two-necked

of Br-Butenolide round-bottomed flask (250 mL) and a condenser which are
(Br-D Ring) anhydrified by flame annealing of the apparatus under argon
atmosphere. Eventually, the apparatus is heated in the oven at
120 C for 12 h and cooled down in a desiccator prior to
the use.
2. Dry CCl4 is prepared by adding CaH2 (5 g) to commercial
CCl4 (0.5 L). After standing for 12 h, CCl4 is distilled under
argon atmosphere.
3. TLC plate: silica gel coated with fluorescent indicator F254.
Synthesis of Simple SL Mimics 33
Fig. 3 Structures of some SL mimics
Scheme 1 (a) Preparation of bromobutenolide; (b) synthesis of SL mimics from phenols, (c) synthesis of SL
mimics from carboxylic acids
2.2 Synthesis of SL 1. Silica gel for column chromatography (40–63 μm mesh).

Mimics from Phenol 2. TLC plate: silica gel coated with fluorescent indicator F254.
2.3 Synthesis of SL 1. The dried apparatus for synthesis consists of a two-necked

Mimics from round-bottomed flask (250 mL) and a condenser which are
Carboxylic Acid anhydrified by flame annealing of the apparatus under an argon
atmosphere. Eventually, the apparatus is heated in the oven at
120 C for 12 h and cooled down in a desiccator prior to
the use.
34 Tomáš Pospı́šil
2. Dry acetone is prepared by adding K2CO3 (15 g) to commer-

cial acetone (500 mL). After 12 h, acetone is distilled under
argon atmosphere. Additional drying over molecular sieve 3 Å
can be used [12].
3. Silica gel for column chromatography (40–63 μm mesh).
4. TLC plate: silica gel coated with fluorescent indicator F254.
3 Methods
3.1 5-Bromo-3- 1. Take a dry two-necked round-bottomed flask (250 m capacity)

Methylfuran-2(5H)- equipped with a condenser and load with dry CCl4 (150 mL)
One (Bromobutenolide, under argon atmosphere (see Notes 1 and 2).
Br-D Ring) 2. To the flask with CCl4 add 3-methylfuran-2(5H)-one (5 g,
51 mmol) followed by AIBN (10 mg).
3. Heat the reaction mixture to reflux for 2 h and irradiate at the
same time with a 250 W UV-lamp (see Note 3). The cloudy
mixture slowly becomes clear, turns to yellow and again some
precipitation will be formed.
4. Control progress of the reaction on TLC (ethyl acetate–petro-
leum ether ¼ 1/1) (see Note 4). When the reaction is com-
plete, cool the mixture to 0 C (see Note 5).
5. Filter off the precipitated succinimide and remove the solvent
by rotavap in vacuo. The resulting yellow oil is bromobuteno-
lide. The typical yield is about 8 g (87%). This product is used
without further purification.
3.2 SL Mimics from 1. In a round-bottomed flask (50 mL) dissolve the corresponding
Phenol (Debranones) phenol (2 mmol) in CH2Cl2 (5 mL).
2. In a beaker dissolve K2CO2 (2 mmol) and tetra-n-butylammo-
nium bromide (0.2 mmol) in water (10 mL).
3. Add water solution to organic solution and stir vigorously for
10 min at room temperature.
4. Dissolve bromobutenolide (2.5 mmol) in CH2Cl2 (5 mL) and
add the solution dropwise to the reaction mixture during a
period of 15 min.
5. Stir the two-phase mixture at room temperature and follow the
progress of the reaction by TLC (ethyl acetate–petroleum
ether ¼ 1/1).
6. When the reaction is complete (see Note 6), separate the
organic phase and the aqueous phase extract with CH2Cl2
(3 5 mL).
Synthesis of Simple SL Mimics 35
7. Combine the organic phase wash with brine (10 mL), dry over
Na2SO4, filter, and evaporate the solvent in vacuo.
8. Purify the crude product on column chromatography (silica
gel, ethyl acetate–petroleum ether) (see Note 7).
3.3 SL Mimics 1. In a dry two-necked round-bottomed flask (25 mL) under

from Acids argon atmosphere dissolve carboxylic acid (0.57 mmol) in dry
acetone (6 mL).
2. Add 5-bromo-3-methylfuran-2(5H)-one (0.62 mmol) to the
reaction mixture and stir for 5 min at room temperature.
3. Finally add triethylamine to the reaction mixture (0.62 mmol)
dropwise and let the reaction mixture be stirred for 12 h, at
room temperature. The reaction mixture turns to a brown
solution.
4. Dilute the mixture with water (5 mL) and extracted with
CH2Cl2 (3 10 mL).
5. Wash combined organic layers with water (10 mL), then brine
(10 mL), dry over Na2SO4, and filter.
6. Evaporate the organic solvent under vacuum and purify the
residue by column chromatography on silica gel (ethyl acetate–
petroleum ether) (see Note 8).
4 Notes
1. CCl4 is on the list of ozone-depleting compounds. Depending

on the country you are in, you may need some authorization to
use it.
2. 1,2-dichloroethane can be used instead of CCl4.
3. You may avoid to use UV lamp. The reaction then needs more
time to complete.
4. Use KMnO4 (5% solution in water) stain for visualization. The
starting material is not well visible under UV.
5. Heptane (150 mL) can be added before cooling to speed up
the precipitation.
6. Reaction time can vary from 2 to 12 h.
7. Depending on the starting phenol, the typical yield can vary
from 40 to 80%.
8. Depending on the starting carboxylic acid, the yield ranges
from 30 to 80%.
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És gonddal nézek minden elmenőre:
Ó életem, enyém.
Golyózáporban haladok előre,

Ki minden tűzzel, karddal ismerős
S úgy csókolódzom és dőlök a sírba,
Mint bátor, büszke hős.
Hatalmas ősz.
Hatalmas ősz,
Reám lehelltél s érzem, egyre érzem,
Hogy nagyra nősz.
Te jó, te nagy,
A mi akartam lenni, régen, egykor,
Most az te vagy.
Igaz, egész,
Mely önnön-magát bátran koronázza
S az éjbe néz.
Azt hittem én,

Gőgös és hervadó virág leszek csak,
Szép és szegény.
Bús szemű bölcs,

Itt állok, ember, s karomon a gyermek.
A lágy gyümölcs.
A kar, a kar,
Egy ember ága, lombos és gyümölcsös,
Mit is akar?
A sors vagyok,
A méz, a tűz, a köny, a szív, az élet,
Mondd, ki nagyobb?
Vágytam vakon
É
És ami vágyam itt-ott megmaradt még,
Eléd rakom.
Arany-idő,
Vígasztaló, mosolylyal szomorító,
Kibékítő.
Hatalmas ősz,
Reám lehelltél s érzem, egyre érzem,
Hogy nagyra nősz.
Limlomok.
1914 – – – – – – – –
––––––––––
––––––––––
Siessünk,
Hogy elmondhassunk mindent, ami kell még,
Vén limlomok vannak szivünkbe zárva,
Mint sifonérban fájó, ócska kelmék.
Terítsd ki őket, lelkem, még ma emlék,
Holnapra rongy lesz, megcsufolt és árva,
Le is gázolják, szótlanul, a sárba.
Ha járok mostan véres lobogók közt,

Eszembe jutnak fájó, ócska képek,
És kérdezem a tűzbe robogók közt
Emlékeimtől: jaj, csak ez az élet?
Eszembe jut, ami szép, ami fáj is,
Egy régi arc, egy régi-régi táj is.
Eszembe jut a kisgyermek panassza,
A palatábla és az első skála
Szegény Anyám bús, gyöngyvirágos arca,
Szegény Apám sötét Kossuth-szakálla.
Eszembe jut, hogy ültem néha éjjel
Virrasztva álmosan az őszi széllel.
Eszembe jut, a bor, a málna íze,
A hosszu-hosszu-hosszu téli esték,
A hó fehérje, cseresznye pirossa,
Egy képeskönyv és egy gyerek-betegség.
Eszembe jut, egy halvány beteg angyal,
Vásott kis ördög, kivel kergetőztem,
Csatakos aszfalt, pezsgő, keserű könny,
S kórházban halt meg csúnyán, csúnya őszben.
Eszembe jut, hogy irtam néha verset,
Diákkoromban, olajlámpa mellett,
Öcsémmel a tavaszt remegve lestük,
Dermedt kezünkön vastag, téli keztyük
Eszembe jut Nagyapa arca, holtan.
Egy viola, egy fény a könnyes égen,
Egy bárány, egy galamb, a Feleségem,
Hogy boldog voltam és szomorú voltam.
Eszembe jut, hogy nézett rám a nyáron
A Duna vize hajnaltájba, hívón,
Ballagtam haza a redakcióból,
Ugy sírtam ott a Ferencz-József-hídon.
Eszembe jut sok perc ürömje, méze,
Egy szőnyeg, egy kilincs, egy régi csésze.
Eszembe jut, egykor vidéken vőfény
Voltam s cilinderemen a verőfény
Táncolt – – – – – – – –
––––––––––
––––––––––
Öcsém.
Az én öcsém mostan katona a határon,
A szerb hegyek mentén Ferencz József bakája.
Szívdobbanásommal mérek fel minden éjet,
Lámpákat gyújtok és így gondolok reája.
Ezer szurony között, szekerek éjjelében

Az ő szeme ragyog, kiáltó-ismerősen.
Ki tudja merre néz, ki tudja, hol világít
Sok százezer szem közt? – nem tudja senki, ő sem.
Öcsém, én kisöcsém a háború zajába

Egy csókot küldök én, a lőporfüstbe: versem,
Hogy érezd a bátyád, gondolj játékainkra,
Ragyogj, kis hős, ragyogj és a lelked nevessen.
Emlékszel édesem a játékháborúnkra,

Mint villogott kardunk és porzott a határdomb?
Ugy-e ma is csattog a kard és döng az ágyú,
S a véres trombita ma is trarát-trará-t mond?
Mert téged hivlak, Kard, kisgyermek drága Kardja,

Játékos ismerős, ragyogjon régi pengéd
A nyári dombokon s hogy az öcsémre fordulsz,
Ismerd meg édesen és légy hozzája gyengéd.
Jó Puska ne feledd, hogy gyermekpuska voltál

Egykor az ő kezén s ha forgatnak a szerbek,
Tagadd meg önmagad, nevess barátaidra,
Légy játékpuska csak, légy te is újra gyermek.
Ami golyó kiszáll, száz angyal fogja össze,
Mint röpködő lepkét, kacagjon fel az élet,
S te Isten, akiben gyermekkoromba hittem,
Hajolj le csöndesen és védd meg az öcsémet.
Arckép.
A palaszürke égben áll.
Hull rá a hó, az esti hó.
Nehéz, meleg ruháitól
Oly vastag, mint egy eszkimó.
Kunyhóba faggyúgyertya ég.

A szalma benn a térdig ér.
Cigaretták. Csajkába rum.
A földön rongyok, vatta, vér.
Körötte hó, hó, egyre hó,

Erdők, hegyek, tábortüzek.
Rövidlátó szegény szemén
Csillámló, éles szemüveg.
Egy női aranyóralánc

Lóg a zsebéből. Hajdanán
Üvegszekrényünk dísze volt.
Nászára kapta az anyám.
Ezüst órája oly kopott.

Még a nagyapja vette meg.
Negyvennyolcban ő véle volt
S azt tiktakolja: »Isaszeg«.
Az óra nem feledte el,

Bár sok év szállt felette el.
Hallgatja ő is csendesen.
Ő sem, ő sem feledte el.
Hallgatja elhagyott szíve
S a hóban valami forog.
Ágyúk ugatnak mérgesen
Veszett, dühös komondorok.
Egy-egy halott fölé hajol.

Itt a ravatal pár fa-léc.
Egy messze hang hahóz neki
És szalutál egy szanitéc.
Sovány kis arca halovány,

De oly nyugodt, de oly setét,
Ki tudja, megfordulna-e,
Ha kimondanám a nevét?
Közönyös, furcsa, idegen.

Előtte köd, utána köd.
Csak néz és ezt gondolja tán:
Ezerkilencszáztizenöt.
Alázatos, fiúi fohász
az aggokhoz.
Ti aggok, kik az ablakokban ültök
S a szemüvegetek vigyázva, lassan
Sikáljátok a szarvasbőrrel s enyves
Szemetek elkalandoz a magasban,
Ahol a nap dalol, gondoljatok ránk,

Kik mélybe hörgünk, vérpárázta füstök
Sötét gomolyán, lángoló özönben,
S ne rezzenjen meg a haragos üstök
Szigorú főtökön, mikor biráltok,

Kegyetlen aggok, mostoha apáink,
Irgalmat esdünk, ifjak, a pokolban,
Ne lássatok a jó Ábelbe Káint,
Itélő bírák, szakállas urak ti,

Olyanok, mint az öregistent festik,
Vén képeken, halljátok a szavunkat,
Még reggel van, hadd éljünk mi is estig,
Csak estig éljünk, csendesen megérve,

Mint fán az alma, mezőn a kalászok,
Aztán hulljunk le – ó szent, szent az élet –
Százszor szent – ezt kiáltjuk megalázott
Szívünkből, ezt ordítjuk, szent az élet,

Legyen áldása mindétig mi vélünk,
Ti sárga aggok, gyeplő-igazítók,
Bocsássatok meg most nekünk, hogy élünk,
S ítéljétek meg, mily szép a rigó-fütty

A lármás nyári erdőn, tiszta vízbe
A fürge pisztráng, halvány lány az ágyon,
A vaj, a füge, a piros ribiszke,
Mert szent a föld is, jertek zengedezzünk,

Költők, az élet papjaivá kentek,
Gyalázatos, ki káromolni merte,
Az élet szent és nincsen nála szentebb,
Felétek nyújtjuk ifjú karjainkat,

Ártatlan testünk, árva-árva vének,
Húsz-harminc évünk, a mi bús bűnünket,
Hogy szálljon szívetekbe ez az ének.
Ádám.
Most gyakran gondolok arcodra, Ádám,
Bús ősapám, mert fáj, hogy létezem
S a nevem: ember. A gond szolgaágyán
Feléd lóbázom csüggeteg kezem.
Papagály s tigris közt csúf emberállat,

Kire kövér kenyérfa bólogat,
Körömmel vakarod vörös szakállad,
S úgy képzelem, hogy véres a fogad.
Ó első ember… Ó fájdalmak útja…

Te vagy a szennyes óceánnak kútja,
Amelyből fájó sorsomat öröklöm.
Bár láthatnálak szinről-szinre téged,

Hogy most könny, átok és jaj éget,
Őrjöngve az arcodba vágjam öklöm.
Régi idill 1895-ben.
Jaj, hova lettek a zongorás estek,
A végtelenből hova-hova estek?
Most oly sötét a házunk, mint a rab-lak

És fekete és fekete az ablak.
Ki itt, ki ott. Az élet olyan olcsó,

A zongoránk is mint bezárt koporsó.
Csak nézem egyre és mit úgy szerettem

Egy képet látok ódon képkeretben.
Itt ültek ők, e vidéki szalonba,

És apám játszott nékik andalogva.
Hangok zümmögtek, babonás varázzsal

Szivünkre szálltak, fulánkos darázs-raj.
Úgy szúrt a szépség és úgy fájt a bánat,

Hogy futni kellett, ki az éjszakának
Sápadt arccal és túlvilági hangon

Szólitottunk meg lányokat, a gangon.
Benn az olajos függőlámpa égett,

Lábújjhegyen járkáltak a cselédek.
Nénik kötöttek és a tiszta csöndbe

Egy-egy kötőtű rebbent meg zörögve.
A bácsik előtt pettyes, puha sárga
Dohány aranylott a dohányszitánkba.
Dús ételek és cukrok kisded halma,

Sötét rum és bor, datolya meg alma.
Az öregek méláztak csöndbe szépen,

Kossúth Lajos is felfigyelt a képen,
Deák Ferenc meg intett véle szembe

S e nyájas körre néztek elmerengve.
Jaj, hova lettek a zongorás estek?

Kihullt a kép már. A keret üres lett.
Óda.
(Távoli beteg feleségemnek.)
Én feleségem, jó és drága-drága,
Eddig neked dalt alig-alig írtam,
Mert nem bíztam tintában és papírban,
S féltem, hogy félrebillen gyenge ága.
Nem is vittelek tornyos frizurával
Fényes parkettre, kart-a-karba-öltve,
Ki csöndesen jöttél hozzám, e Földre,
Sok fogcsikorgatáson, könnyön által.
Külvárosokban jártunk, bús ebek közt,

Mikor az ősz nyugalmas, tiszta, síma
Mosollyal vérzett – áldott heroína –
S sírtunk a ködben, mélypiros sebek közt.
Mit tudta a kultúr, fekélyes, úri
Népség, mi vagy te s az a rongy, ki fennen
Hordozza a fejét a bálteremben,
S karján egy gyémánt-fülü, buta húri.
Én nem hiszek a nőbe. Nem a kézbe,

Mely ad-vesz, a szájba – kis piros ajtó,
Most nevető, most bánatos-sohajtó –
A karba, amely integet igézve:
De hittem a te két jó-jó szemedben,
A két szemed mélyén horgonyt vetettem,
Én mindég a lélekbe-szembe hittem,
Most is hiszek, megálltam, várok itten.
Nekem a jóság, a jó szerelem vagy,
Az életemmel járkálsz, ha velem vagy,
De ha távol vagy, olyan szomorú vagy,
Akkor nekem a szenvedés, a bú vagy.
Most is megmozdul e polgári otthon,
Olyan bitang már a költő kabátja
Éjjel két nyomorék-karját kitárja
S feléd ölel, ő is – szegény – búsongón.
Mert tett engem az élet hősi-vaddá,

Adott nekem aranyat, myrrhát, lázat,
Volt kenyerem a gőg meg az alázat,
De csak te tettél krisztusi lovaggá,
Ki köntösét is megfelezi azzal,
Akit szeret. Érted téptem le álcám.
A fájdalmat hoztad szépmívű tálcán,
A szegénységet, kinccsel és vigasszal.
Világok lázát mérik most a népek

S én egy higany-pont rebbenését várom
37·2°… vagy… 37·3°…?
S a pesti utcán is remegve lépek.
Remegve nézek innen messze, hátra,
Oda, hol hóban ível föl a Tátra
S betegek fekszenek, lágyan merengve,
Szájukba hőmérővel, téli csendbe.
Őszi síp.
Ősz
Kullog a hegyben, a ravaszdi
Csősz.
Vén
Puskás öreg, jól ismerem már
Én.
Néz.
A szeme kancsal, botja vége
Réz.
Kürt
Lóg vállán. Mindenkit halálba
Küld.
Gaz,
Himlőhelyes, fegyenc paraszt csak.
Az.
Vár.
Jaj, hogy vigyáz. Az arca, inge
Sár.
Jő
És puskaport robbant szüretre
Ő.
Bor
Csorog a csapról, gyöngyös mustja
Forr.
Mondd,
Mért ül mégis puttonyodba
Gond,
Mér
Áztatja zöldes, lomha bajszod
Vér?
Szentbeszéd.
Én, ki tavaly még könnyű szívvel jártam,
Forró sugárban,
Most járok itt a sápadt bús fagyok
Fenyérein
És felordítok, hol vagyok?
Testvéreim.
Volt egy kutyám

S nem vertem ki, mikor haragos éjjel
A hóvihar acsarkodott a széllel,
Csunyán.
S mikor beteg lett,
Köré sereglett
A bánatom, a gondom.
Felkeltem, hogy reá tekintsek,
Úgy néztem mint egy árva kincset.
Egész éjszaka virrasztottam otthon.
Aztán vásott és buta lázban
Nem madarásztam,
Mert szántam a madárfiókot, a picinykét.
Vigan kaszáló,
Zöld, könnyű háló
Zacskójával nem fogtam soha pillét.
Sohase ültem lesve régi cser-tőn,
Vörös vadat nem űztem havas erdőn.
Kikerültem a gyikok napos árkát.
El nem tapostam
A réteken a szentjános-bogárkát.
Mostan
Elmondom nektek, emberek
Csak ezt, csak ezt – könnyezzetek.
Unalom.
Lelkem éjén rosz malom
Zakatol: az unalom.
Régi, rozzant, rosz malom
És úgy hivják: unalom.
Régi, rozzant kereke
Álmos is és fekete
A garatja végtelen
S a búzája életem,
Elhagyott és végtelen
Tompa- csöndes életem.
Lisztje: semmi, szárnya: éj,
Csönd: zúgása, árnya: mély.
S nézem egyre – árva tiszt –
Hogy szitál a lomha liszt.
Bús időm azzal ölöm,
Ezt a semmit őrölöm,
Árva molnár őrölöm
S lisztem nem bú, nem öröm,
Színtelen köd, csunya lom,
Fájó semmi: unalom.
Szerzőnek
a Tevan-kiadásban megjelent
egyéb munkái:
Mágia
(Versek 1912) Ára 3 K
A szegény kisgyermek panaszai

Ára 4 K
Mécs
(Tevan-könyvtár 34-35) Ára 60 f
Öcsém
(Tevan-könyvtár 68) Ára 40 f
TARTALOM:
Ének Virág Benedekről 3
Bús pesti nép 6
Szonett azokhoz, akik még húsz évesek 7
Ó régi kávéház 9
Szeszélyes akkordok a holdról 12
Pléhkoszorú 15
Lánc, lánc, eszterlánc 16
Séta a városon kivűl, vidéken 18
Ének régi dolgokról és egy régi fájdalomról 20
Nem szabad feledni 24
Sötét, nehéz tavaszi ég 25
Bácskai rajz 27
Mély éjeken 28
Egy új poéta panasza a régi holdhoz 30
Délutáni meditáció 32
Keserű ének 34
Mérgek litániája 35
A jó hold 36
Skála 38
Hideg 41
Kis, kósza vágyak 43
Csatakos virradat 44
Rózsa 46
A rosz élet 49
Egy kézre vágyom 52
Akarsz-e játszani? 53
Reggeli 55
A jó élet 57
Csöndes, ünnepi óda az élethez 59
Hatalmas ősz 61
Limlomok 63
Öcsém 65
Arckép 67
Alázatos, fiúi fohász az aggokhoz 69
Ádám 71
Régi idill 1895-ben 72
Óda 74
Őszi síp 77
Szentbeszéd 79
Unalom 81
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Methods and Protocols

ISSN 1064-3745 ISSN 1940-6029 (electronic)

© Springer Science+Business Media, LLC, part of Springer Nature 2021

Turin, Italy Cristina Prandi

PART I STRIGOLACTONE CHEMISTRY

1 Evaluation and Quantification of Natural Strigolactones

PART II STRIGOLACTONES AND SOIL BIOTA

6 Strigolactone-Like Bioactivity via Parasitic Plant Germination Bioassay . . . . . . . . 59

PART III STRIGOLACTONES AS PLANT HORMONES

10 Methods for Phenotyping Shoot Branching and Testing Strigolactone

11 Bioassays for the Effects of Strigolactones and Other Small Molecules

PART IV BIOACTIVITY ASSAYS

15 Evaluation of Bioactivity of Strigolactone-Related Molecules

SALIM AL-BABILI • Division of Biological and Environmental Science and Engineering,

MARÍA J. SOTO • Department of Soil Microbiology and Symbiotic Systems, Estacion

Evaluation and Quantification of Natural Strigolactones

Strigolactones (SLs) were originally identified as germination sti-

ultra-high-pressure liquid chromatography (UHPLC) system,

Glassware should be cleaned thoroughly to avoid contamination.

2.1 Hydroponic 1. Petri dishes (i.d. 9 cm).

500 mL plastic cup containing

500 mL paper cup

Fig. 1 Hydroponic culture for root exudate collection

2.2 Sample 1. Ethyl acetate.

2.2.2 Concentration with 1. ODS or HLB cartridge (see Note 5).

3.1 Hydroponic 1. Sterile seeds with 1% NaClO containing 0.1% Tween

3.2.2 Concentration with 1. Activate SPE cartridges according to the manufacturer’s

3.2.3 Sample Purification 1. Activate SPE cartridges according to the manufacturer’s

3.3 LC-MS/MS A typical LC-MS/MS analysis of proton adduct ions of SLs is

1. Two to four seedlings can be grown hydroponically using

Monitoring transitions (m/z)

Retention time Precursor Diagnostic Confirming

producing relatively large amounts of SLs, root exudates can be

15. Evaporation of the solvent under nitrogen gas flow appears to

This study was supported by the Japan Science and Technology

Isolation and Identification of Naturally Occurring

Strigolactones (SLs) are a new class of phytohormones that func-

1. Purchase the plant seeds and organic solvents (guaranteed

Stock solution Preparation of medium (1 L)

Stock solution Preparation of medium (1 L)

Tea filter bag

Hydroponic culture system

Fig. 1 Hydroponic culture for the large-scale extraction of SL

7. Load the filtrate on the Oasis HLB 6 cc cartridge conditioned

10. Add acetonitrile to the aqueous residue up to a concentration

9. Combine the EtOAc solution and wash the organic solution

H2O, dehydrate the EtOAc layer with Na2SO4, and concen-

3.5 Nuclear 1. Prepare the maximum amount of pure material, ideally in