Journal of Plant Physiology 162 (2005) 1197—1209

www.elsevier.de/jplph

Impact and interaction of lipophilic antioxidants in

mutants and transgenic plants
Sonja Woitsch, Susanne Römer

University of Konstanz, Faculty of Biology, Universitätsstraße 10, 78457 Konstanz, Germany

Received 17 March 2005; accepted 13 April 2005

KEYWORDS Summary
Antioxidants;
Carotenoids and tocopherols are lipophilic antioxidants with important functions in
Carotenoids;
plants and humans. Due to their nutritional value and putative health benefits, they
Light stress;
have become a focus of intensive research. The identification of all genes of the
Mutants;
carotenoid and tocopherol biosynthesis has enabled the manipulation of their
Photoprotection;
biosynthetic pathways, aiming for quantitative and qualitative improvement. In
Tocopherol;
plants, carotenoids and tocopherols are of crucial importance because of their
Transgenic plants
protective abilities, which help to keep them alive even under light stress conditions.
A wealth of information has accumulated concerning the responses of plants to various
environmental stress factors. Here, we summarize some of the recent data
concentrating on the impact and possible interaction of lipophilic antioxidants in
mutants and transgenic plants with altered status of lipophilic antioxidants.
& 2005 Elsevier GmbH. All rights reserved.

Introduction Fraser and Bramley, 2004). Carotenoids are thought

to exercise protective functions against certain
In recent years, the importance of carotenoids cancers (Giovannucci, 2002) and prevent or ame-
and tocopherols has been increasingly recognized liorate some chronic disease states (Mayne, 1996;
due to the emerging knowledge of their health Mares-Perlman et al., 2002). Similarly, a role of
benefits. Because humans can synthesize neither tocopherol in health protection and improvement
carotenoids nor tocopherols, they rely on their of age-related ailments has been suggested
uptake through diet for the production of vitamin A (Nakagawa et al., 2004; Pfluger et al., 2004).
and the supply with vitamin E (Bramley et al., 2000; However, the protective function of carotenoids

Abbreviations: LHC, light harvesting complex; NPQ, non-photochemical quenching; PS II, photosystem II; ROS, reactive oxygen
species
Corresponding author. Tel.: +00 49 0 7531 882116; fax: +00 49 0 7531 883042.
E-mail address: [email protected] (S. Römer).

0176-1617/$ - see front matter & 2005 Elsevier GmbH. All rights reserved.
doi:10.1016/j.jplph.2005.04.007
1198 S. Woitsch, S. Römer

and tocopherols is not restricted to aspects of Biosynthesis of carotenoids

human health and nutrition, since both antioxi-
dants are also involved in plant stress protection Carotenoids are tetraterpenoids derived from a
(Demmig-Adams and Adams, 2002). C5 isoprene unit (isopentenyl diphosphate, IPP) and
In the following, we will focus on the impact and are therefore biosynthetically linked with the
role of carotenoids and tocopherols as lipophilic synthesis of other isoprenoid compounds such as
antioxidants in relevant mutants and transgenic tocopherols, chlorophylls and plastoquinones. De-
plants starting with a short overview of the tailed investigations of the isoprenoid biosynthesis
carotenoid and tocopherol biosynthesis. and functional genomics have shown that the IPP

Figure 1. Biosynthesis of carotenes and xanthophylls. The xanthophyll cycle is highlighted in light gray. Enzymes are
presented in gray, corresponding mutants are shown in brackets ( ). PSY: phytoene synthase, PDS: phytoene desaturase,
ZDS: x-carotene desaturase, LCY-b: b-lycopene cyclase, LCY-e: e-lycopene cyclase, HY-b: b-carotene hydroxylase, HY-e:
e-carotene hydroxylase, ZEP: zeaxanthin epoxidase, VDE: violaxanthin de-epoxidase, NXS: neoxanthin synthase.
Impact and interaction of lipophilic antioxidants 1199

used in the formation of carotenoids originates has finally disclosed the identity of the a-carotene
from the plastid localized mevalonic acid (MVA) hydroxylase gene as a P450 type monooxygenase
independent pathway (Rodrı́guez-Concepción and filling the final gap in the pathway (Tian et al.,
Boronat, 2002). The carotenoid biosynthetic path- 2004).
way has been recently reviewed (Fraser and
Bramley, 2004), and a schematic outline is given
in Fig. 1. The first step of carotenogenesis is the Tocopherol biosynthesis
formation of phytoene from two molecules of
geranylgeranyl diphosphate (GGDP) via the enzyme Vitamin E or tocochromanols ( ¼ tocols) comprise
phytoene synthase (PSY). In plants, four desatura- tocopherols and tocotrienols (Munné-Bosch and
tion steps yield the red colored lycopene that Falk, 2004). They are low molecular weight
serves as a substrate of cyclization reactions molecules of amphipathic nature. Their chemical
leading to a- and b-carotene, respectively. Intro- properties are favorable for their membrane-bound
duction of oxygen groups due to hydroxylation and location and enable an orientated position within
epoxidation gives rise to the b-carotene derived the membrane: embedding of the hydrophobic tail
xanthophylls (e.g. zeaxanthin, antheraxanthin and in the membrane and exposure of the redox-active
violaxanthin) and the a-carotene derivative lutein, head group to the membrane surface. A summary
the main xanthophyll in leaves. The genes encoding of tocopherol and tocotrienol biosynthesis is de-
carotenoid biosynthetic enzymes in higher plants picted in Fig. 2. Tocopherol biosynthesis requires
have been isolated by different approaches (Cun- hydroxyphenylpyruvate provided by the shikimate
ningham and Gantt, 1998), and map-based cloning pathway as a substrate for the production of the

Figure 2. Biosynthesis of tocopherols and tocotrienols. Tocopherol synthesis starting from geranylgeranyl diphosphate
(GGDP) is shown on the right-hand side of the figure, the conversion of GGDP to tocotrienols is presented on the left.
Enzymes are illustrated in gray, corresponding names are shown in brackets [ ]. Mutants are shown in brackets ( ). HPT:
homogentisate phytyltransferase, TC: tocopherol cyclase, g-TMT: g-tocopherol methyltransferase, MPBQ: methylphy-
tylbenzoquinone, MPBQMT: methylphytylbenzoquinone methyltransferase, DMPBQ: dimethylphytylbenzoquinone,
HGGT: homogentisic acid geranylgeranyl transferase, MGGBQ: methylgeranylgeranyl benzoquinone, MT: methyltrans-
ferase, DMGGBQ: dimethylgeranylgeranyl benzoquinol.
1200 S. Woitsch, S. Römer

aromatic precursor homogentisate catalyzed by soluble and lipophilic antioxidants. Ascorbate and
hydroxyphenylpyruvate dioxygenase (HPPD; Norris glutathione are required for the regeneration of
et al., 1998). The first step of tocopherol synthesis tocopherol, whereas ascorbate is a cofactor for
is the condensation of homogentisic acid (HGA) the enzyme violaxanthin de-epoxidase (Eskling
with phytyl diphosphate (PDP) to yield 2-methyl-6- et al., 1997; Smirnoff, 2000b). Additionally, ROS
phytyl-1,4-benzoquinol (MPBQ) by the membrane- are detoxified by a network of scavenging
bound enzyme homogentisate phytyltransferase enzymes. These include superoxide dismutase
(HPT1/VTE2). PDP is either derived via the non- (SOD), ascorbate peroxidase (APX), glutathione
mevalonic acid pathway from geranylgeranyl peroxidase (GPX) and catalase (CAT) (Mittler,
diphosphate or generated from phytol. MPBQ can 2002).
then be further methylated to 2,3-dimethyl-6- The protective function of carotenoids and
phytyl-1,4-benzoquinone (DMPBQ). Both MPBQ and tocopherols is rooted in their high antioxidative
DMPBQ are subsequently subjected to cyclization potential. Carotenoids (e.g. carotenes and xantho-
by tocopherol cyclase (TC/VTE1) to yield either phylls) are indispensable for plant photoprotection
d- or g-tocopherol (Porfirova et al., 2002). Methylation and their role has been comprehensively reviewed
of d- and g-tocopherol carried out by g-tocopherol (Niyogi, 1999; Baroli and Niyogi, 2000; Demmig-
methyltransferase (g-TMT/VTE4) leads to the forma- Adams and Adams, 1996, 2002). They are very
tion of the end products b- and a- tocopherol, efficient quenchers and scavengers of reactive
respectively. oxygen species (ROS), and, in particular, of 1O2. In
In contrast to tocopherol biosynthesis, the for- higher plants, a dynamic protection mechanism,
mation of tocotrienols makes direct use of GGDP the xanthophyll cycle, enhances plant photopro-
without prior modification. The condensation with tection (Demmig et al., 1987) by providing a way to
homogentisic acid leads to the formation of 2- safely dissipate excess light energy by non-photo-
methyl-6-geranylgeranyl benzoquinone. This step chemical quenching (NPQ). The xanthophyll cycle
in tocotrienol biosynthesis is catalyzed by the comprises the three xanthophylls zeaxanthin, an-
enzyme homogentisic acid geranygeranyl transfer- theraxanthin and violaxanthin and their reversible
ase (HGGT), whose gene sequence shows consider- interconversion. Non-photochemical quenching is
able sequence homology to hpt genes (Cahoon et correlated with the high light induced accumula-
al., 2003). The consecutive reactions are similar to tion of zeaxanthin as a consequence of violaxanthin
the tocopherol synthesis including methylations de-epoxidation at low pH and requires the presence
and cyclization. Interestingly, both pathways in- of the PsbS protein of photosystem II (Li et al.,
dicate a role of GGDP as one of the key substrate 2000; Demmig-Adams and Adams, 2002). Besides its
necessary for the production of tocopherols and functional role in thermal energy dissipation (Holt
tocotrienols. et al., 2005), an involvement of zeaxanthin in
stabilization of the thylakoid membrane and
protection against lipid peroxidation has been
proposed (Havaux, 1998). In the thylakoid mem-
Protective function of carotenoids and brane, carotenoids are essential structural compo-
tocopherols in plant stress protection nents of the light harvesting complexes where they
serve as accessory pigments (Horn and Paulsen,
In excess light, high levels of reactive oxygen 2002).
species (ROS) such as hydrogen peroxide (H2O2), Similar to carotenoids, tocopherols are also
superoxide (O
d
2 ), hydroxyl radicals ( OH) and involved in the quenching and scavenging of 1O2
1
singlet oxygen ( O2) accumulate in plants (Niyogi, (Neely et al., 1988) and act as highly efficient
1999). This accumulation can lead to severe recyclable chain reaction terminators for the
damage or even cell death, and represents a removal of polyunsaturated fatty acid (PUFA)
major threat to plant viability and productivity. radical species generated during lipid oxidation
Photoprotection is therefore an absolute neces- (Munné-Bosch and Alegre, 2002; Munné-Bosch and
sity. Besides carotenoids and tocopherols plant Falk, 2004). Furthermore, tocopherols contribute
protection involves the water-soluble low- to membrane stability by influencing its fluidity and
molecular weight molecules ascorbate (vitamin C; permeability (Fryer, 1992) and might participate in
Smirnoff, 2000a) and glutathione (Noctor and protection of the D1 protein against high light
Foyer, 1998), the main antioxidants in chloroplasts. (Trebst et al., 2002). The major tocochromanol
Ascorbate and glutathione are connected via in leaves is a-tocopherol, whereas seeds accumu-
the ascorbate–glutathione cycle. There is also late higher levels of tocotrienols (Grusak and
a functional relationship between these water- DellaPenna, 1999).
Table 1. Characteristics of carotenoid biosynthesis mutants and transgenics of higher plants

Carotenoids Mutants/ Growth/ Car VAZ NPQ Fv/Fm Toco Hydrophilic Stress Ref.
transgenics stress antioxidants tolerance

Early steps dxs OE normal Carm n.d. n.d. n.d. ma
Toco n.d. n.d. 1
psy OE normal Carm Pm n.d. n.d. n.d. n.d. n.d. dwarfism . 2
psy AS normal Car. n.d. n.d. n.d. n.d. n.d. lethal 2
pds OE/ normal Car7 m n.d. n.d. n.d. n.d. norfl. resist. 2,3,4
(crtI OE)
pds AS normal Car(.) Pm Cb. n.d. n.d. n.d. n.d. n.d. lethal 2
Cyclization/ crtISO/ etiolated Lyccism n.d. n.d. n.d. n.d. (.) 5
isomerization (ccr1/2)
Hydroxylation hy-b OE HL/heat/UV-B Car(m) m 7 7 n.d. n.d. m 6,7
hy-b AS normal Car. Cbm V N. . . n.d. n.d. n.d. (.) 8,9
b1 normal Car(7) Lm V N. (.) (.) 7 n.d. n.d. 7 10
b2 normal Car(7) N. 7 7 7 n.d. n.d. 7 10
b1 b2 normal Car(.) Lm V N. . . (.) n.d. n.d. (.) 10
lut1 normal Car(7) ZeinoVm L. m . 7 n.d. n.d. (7) 11,12
lut2 norm./HL Car. Cb Am L. m . 7 n.d. n.d. 8,16
Impact and interaction of lipophilic antioxidants

lut1 b1 normal *Cb ZeinoVm L N. m . (.) n.d. n.d. (.) 10

lut1 b2 normal *Cb ZeinoVm L N. m . (.) n.d. n.d. (.) 10
lut1 b1 b2 normal *Cb Zeinom L V N. . . (.) n.d. n.d. (.) 10,13
Epoxidation npq1 HL/+cold Car7 Cb Z. (m) . ./7 m a
Toco YL n.d. PI. . 14,15,16
npq1 lut2 HL Car. Am Z L. m . 7 n.d. n.d. bleaching . 16
npq4 npq1 HL Car(7) Cb Z. 7 . . n.d. n.d. . 18
vtc2 npq1 HL Car. . . . (.)a
Toco .ASA mGSH . 19,20
npq2/(aba1) HL/+cold Car(7) Zm V (N). m 7 (.) n.d. n.d. PI. 14,17
lut1 aba1 seedlings Car7 CbZZeinomL. . n.d. n.d. vir.gr. (.) 12
lut2 aba1 seedlings HL/ Car. Cb Zm L. n.d. . 7 n.d. n.d. vir.gr. . 12,21
+cold
vde OE HL Car(7) Zm 7 m n.d. n.d. n.d. n.d. 22
vde AS HL/HL+W Car(7) Z. 7 . 7/. n.d. n.d. PIm 23,24,25

Mutants and transgenic plants were grouped according to the step of the carotenoid biosynthetic pathway in which they are affected. AS: anti-sense, OE: over-expression. The following
parameters were considered: carotenoid content and composition (Car), pool of violaxanthin+antheraxanthin+zeaxanthin (VAZ), non-photochemical quenching (NPQ), PSII efficiency (Fv/Fm),
tocopherol content and composition (Toco) as well as hydrophilic antioxidants and the observed stress tolerance. m: increase, (m): slight increase, .: reduction, (.): slight reduction, 7:
similar to wild type control, n.d.: not determined, normal: growth conditions (ranging from 70 to 150 mmol m
2 s
1), HL: high light conditions (ranging from 700 to 2000 mmol m
2 s
1), heat:
heat stress (40 1C), UV-B: UV-B treatment, cold: cold stress (10 1C), W: water stress, *:carotenoid content was based on various parameters (compare references). P: phytoene, Lyccis: cis-
lycopene, Cb: b-carotene, Zeino: zeinoxanthin, L: lutein, Z: zeaxanthin, A: antheraxanthin, V: violaxanthin, N: neoxanthin, ASA: total ascorbate, GSH: glutathione, YL: young leaves, norfl.
resist.: herbicide resistance to norflurazone, PI: photoinhibition, vir. gr.: virescent greening. References (Ref.): (1) Estévez et al., 2001; (2) Busch et al., 2002; (3) Wagner et al., 2002; (4)
Misawa et al., 1994; (5) Park et al., 2002; (6) Davison et al., 2002; (7) Götz et al., 2002; (8) Pogson and Rissler, 2000; (9) Rissler and Pogson, 2001; (10) Tian et al., 2003; (11) Pogson et al.,
1996; (12) Pogson et al., 1998; (13) Tian et al., 2004; (14) Niyogi et al., 1998; (15) Havaux et al., 2000; (16) Niyogi et al., 2001; (17) Leon-Klosterziel et al. (1996); (18) Havaux and Niyogi,
1999; (19) Müller-Moulé et al., 2003; (20) Müller-Moulé et al., 2004; (21) Havaux et al., 2004; (22) Hieber et al., 2002; (23) Chang et al., 2000; (24) Verhoeven et al., 2001; (25) Sun et al.,
2001.
1201
 1202

Table 2. Characteristics of mutants and transgenic plants modified in tocopherol biosynthesis

Tocopherols Mutants/ Growth/ Car VAZ NPQ Fv/Fm Toco Hydrophilic Stress Ref.
transgenics stress antioxidants tolerance

Condensation hppd OE Normal n.d. n.d. n.d. n.d. mseeds n.d. n.d. 1
hpt1 /(vte2) OE HL Car7 n.d. n.d. n.d. mseeds n.d. n.d. 2,3
hpt1y-tmt OE normal n.d. n.d. n.d. n.d. mseeds+leaves n.d. n.d. 2
Methylation mpbqmt /(vte3) OE normal n.d. n.d. n.d. n.d. ma
Toco n.d. n.d. 4
y-tmt /(vte4) OE normal n.d n.d. n.d. n.d shift g-a n.d. n.d. 4
Cyclization vte1 HL Car7 7 m n.d. 7/. mASA mGSH n.d. 5,6
tc /(vte1) OE HL n.d. n.d. n.d. n.d. m .ASA .GSH n.d. 6
vte1 vtc1 HL Car7 7 7 n.d. n.d. .ASA mGSH 7 6
vte1 cad2 HL Car. . 7 n.d. n.d. mASA .GSH . 6
sxd1 HL n.d. n.d. n.d. n.d. .a+g
Toco n.d. . 7
.Tocotrienol

Mutants and transgenic plants were grouped according to the step of the tocopherol biosynthetic pathway in which they are affected. As: anti-sense, OE: over-expression. The following
parameters were considered: carotenoid content and composition (Car), pool of violaxanthin+antheraxanthin+zeaxanthin (VAZ), non-photochemical quenching (NPQ), PSII efficiency (Fv/Fm),
tocopherol content and composition (Toco) as well as hydrophilic antioxidants and the observed stress tolerance. m: increase, (m): slight increase, .: reduction, (.): slight reduction; 7:
similar to wild type control, n.d.: not determined, normal: growth conditions (ranging from 70 to 150 mmol m
2 s
1), HL: high light conditions (ranging from 700 to 2000 mmol m
2 s-1), ASA:
total ascorbate, GSH: glutathione. References (Ref.): (1) Falk et al., 2003; (2) Collakova and DellaPenna, 2003a; (3) Collakova and DellaPenna, 2003b; (4) van Eenennaam et al., 2003; (5)
Porfirova et al., 2002; (6) Kanwischer et al., 2005; (7) Hofius et al., (2004).
S. Woitsch, S. Römer
Impact and interaction of lipophilic antioxidants 1203

Interactions of antioxidants and putative transgene due to shortage of precursor supply for
compensation mechanisms interconnected isoprenoid pathways such as gib-
berellic acid and chlorophyll biosynthesis (Fray
The assumption that carotenoids and tocopherols et al., 1995). Interestingly, the over-expression of
are indispensable for protection against high light 1-deoxy-D-xylulose-5-phosphate synthase (dxs) in
and antioxidative stress, and may functionally Arabidopsis resulted in an enhanced flux through
replace each other is far too simplistic. A good the pathway and led to an increase in carotenoids
comparison of the impact and interdependence of and a-tocopherol (Estévez et al., 2001).
these lipophilic antioxidants is often hampered by
the availability of relevant data, differences in Mutants and transgenic plants with
stress conditions and variations in experimental alterations in cyclization and isomerization
design. The complexity and variety of plant stress
protective systems adds to complicate the picture. Cyclization of lycopene seems also to be crucial
Tables 1 and 2 summarize some current results for the survival of photosynthetic organisms. This
focusing on the effects of altered carotenoid or step is a key regulatory point of carotenoid
tocopherol contents on plant stress tolerance and biosynthesis, channeling metabolites either towards
putative compensatory mechanisms. the a- or the b-branch of the pathway (Cunningham
For the purpose of increasing the amount of et al., 1996). To date, no photoautotrophic mutants
specific antioxidants, over-expression by genetic could be isolated which are completely devoid of
manipulation of the pathway is most desirable. e- and b-lycopene cyclase (LCY) activity demonstrat-
Readers are referred to several excellent reviews ing the enormous importance of carotenes with
considering recent achievements and develop- ionone ring structures (e.g. b-carotene) for photo-
ments (Ajjawi and Shintani, 2004; Fraser and protection and plant viability. Co-suppression of b-
Bramley, 2004; Sattler et al., 2004). However, this lcy in tobacco decreased the amount of b-carotene
does not diminish the importance of mutants and its derivatives and led to an augmentation of
defective in certain steps of the biosynthetic a-tocopherol, putatively as a compensation for the
pathways and the usefulness of transgenic anti- loss of carotenoids (Römer, personal communica-
sense/RNAi plants with altered sets of lipophilic tion). Plants over-expressing lycopene cyclases in
antioxidants to obtain a more thorough insight in a fruit-specific manner were outside the scope
function and interaction of these components in of this article and not considered. A defect in
plants (cf. Baroli and Niyogi, 2000; Demmig-Adams the isomerization blocked the formation of all-
and Adams, 2002). trans-lycopene, whereupon cis-lycopene accumu-
lated in etiolated Arabidopsis plants (Park et al.,
Mutants and transgenic plants with 2002, Table 1).
alterations in early steps of carotenoid
biosynthesis Mutants and transgenic plants with altered
carotene hydroxylation
Mutants and transgenic plants impaired in early
steps of carotenoid biosynthesis (e.g. phytoene Hydroxylation reactions are responsible for the
synthase and phytoene desaturase, and carotenoid formation of the xanthophylls lutein and zeax-
isomerase) are devoid of colored carotenoids or anthin. Deficiencies in the carotene hydroxylation
accumulate only limited amounts of precursors steps can be partially overcome (Table 1). This was
(Sandmann, 1991; Römer, 1999; Park et al., 2002). elegantly shown using specific mutants of Arabi-
They are unable to survive under normal growth dopsis termed lut1 and lut2 (Pogson et al., 1996,
and high light conditions and suffer severe photo- 1998). The lut1 mutation disrupts e-ring hydroxyla-
oxidation. Down-regulation of phytoene synthase tion without affecting b-hydroxylation, and pro-
(psy) or phytoene desaturase (pds) in antisense vides first genetic evidence for the existence of a
tobacco plants had a lethal effect (Busch et al., distinct e-ring specific hydroxylase (Pogson et al.,
2002). These findings illustrate the absolute neces- 1996,1998) whose gene was recently cloned (Tian
sity of carotenoids (Table 1). On the contrary, et al., 2004). As a consequence, the mutants were
transgenic plants over-expressing psy (Busch et al., unable to synthesize the main leaf xanthophyll
2002) or pds (Misawa et al., 1994; Wagner et al., lutein (cf. Table 1). However, plant viability and
2002) were viable. As shown previously, constitu- photosynthetic capacity were not drastically af-
tive over-expression of psy caused a dwarf pheno- fected. The absence of lutein can be clearly
type in plants with high expression levels of the compensated for by the accumulation of nearly
1204 S. Woitsch, S. Römer

equimolar levels of b-carotene derived xantho- lut1 b2, lut1 b1 b2) was partially counteracted
phylls (Pogson et al., 1998), thus structurally and by a shift to higher levels of b-carotene derived
functionally replacing this pigment. The capability xanthophylls.
to shift between the different branches of xantho- Over-expression of hy-b of plant or bacterial
phyll formation seems to be a general phenomenon origin resulted in an improvement of high light
in higher plants emphasizing the flexibility and stress resistance and UV-B tolerance in Arabidopsis
adaptive ability of the photosynthetic apparatus. and tobacco, respectively (Davison et al., 2002;
This assumption is corroborated by results of Götz et al., 2002). These results provide further
reconstitution experiments revealing that some of evidence for the importance of zeaxanthin in plant
the binding sites in reconstituted light harvesting protection. Unfortunately, tocopherol levels of
complexes (LHC) could be occupied by various these transgenic plants were not determined.
xanthophylls (Bassi and Caffari, 2000). Whether the
tocopherol content of these mutants is changed
remains to be examined. Mutants and transgenic plants with
The impact of alterations in the synthesis of b- alterations in carotenoid epoxidation and de-
carotene derived xanthophylls is more complex. epoxidation
Hydroxylation of b-carotene leads to the produc-
tion of zeaxanthin by b-carotene hydroxylase. Epoxidation reactions are responsible for the
Several structural b-carotene hydroxylase genes production of violaxanthin from zeaxanthin under
have been isolated (Tian and DellaPenna, 2004). low light conditions whereas de-epoxidation reac-
Complementing previous investigations using var- tions are converting violaxanthin back to zeax-
ious carotenoid mutants, the generation of T-DNA anthin upon high light exposure. The reversible
knockout mutants of Arabidopsis for b-carotene interconversion of these xanthophyll pigments via
hydroxylases (b1, b2) and the availability of the the intermediate antheraxanthin is caused by
lut1 mutant together with the production of b- xanthophyll cycle activity (Demmig-Adams and
carotene hydroxylase antisense plants (Rissler and Adams, 1996).
Pogson, 2001) have enabled a detailed composi- Several mutants defective in the epoxidation of
tional carotenoid analysis (Tian et al., 2003; Tian zeaxanthin (npq2, aba1/2) and de-epoxidation of
and DellaPenna, 2004). Surprisingly, the b1, b2 violaxanthin (npq1) have been isolated in Arabi-
single mutants, as well as the corresponding dopsis (see Table 1). The inability to synthesize
double mutants, were viable and little affected in violaxanthin and neoxanthin due to defects in the
plant growth under normal conditions, a fact that structural gene encoding zeaxanthin epoxidase
illustrates a certain degree of functional redun- (Marin et al., 1996; Niyogi et al., 1998) resulted
dancy within the b-carotene hydroxylases. Addi- in the accumulation of zeaxanthin as the only
tionally, the hy-b antisense plants exhibited only a b-carotene derived xanthophyll in npq2 and aba1
small reduction of NPQ, although they contained mutants of Arabidopsis. The high level of zeax-
less zeaxanthin after high light illumination (Rissler anthin in npq2 affected the kinetics of induction
and Pogson, 2001). The relative stress tolerance and relaxation but not the extent of NPQ. Despite
could also be ascribed to a compensatory increase their high zeaxanthin contents, the stress tolerance
of other antioxidants with related functions. of these mutants was not significantly ameliorated
Supporting this hypothesis, analysis of antioxidant (Hurry et al., 1997; Niyogi et al., 1998).
levels in tobacco hy-b AS plants revealed an A defect in violaxanthin de-epoxidation as
elevation of tocopherol content (Woitsch and described in the npq1 mutant of Arabidopsis
Römer, personal communication). The b1 b2 double inhibited the high light induced zeaxanthin forma-
mutant showed a decrease in b-carotene derived tion and caused a remarkable decrease in NPQ.
xanthophylls containing two hydroxylated b-rings Photodamage of the npq1 mutant was dependent
which was compensated by an increase in the a- on the developmental leaf stage. Younger leaves of
carotene-derived xanthophyll lutein with only one the npq1 mutant were relatively unaffected by
hydroxylated b-ring. Therefore, just one hydroxy- high light stress and showed significantly higher
lated b-ring seems to be necessary for the assembly a-tocopherol contents and VAZ (total of violax-
of a functional light harvesting complex (LHC II). In anthin, antheraxanthin and zeaxanthin) levels
accordance, Phillip et al. (2002) showed that the than did the wild type (Havaux et al., 2000)
binding of xanthophylls to LHC II requires 3- putatively compensating for the lack of zeaxanthin.
hydroxy-b-ring groups, but not necessarily specific No increase of the water-soluble antioxidants
b-carotene derivatives. The lack of lutein in the glutathione and ascorbate was detected in the
lut1 mutants and double/triple mutants (lut1 b1, mutant compared to the wild type. Acclimation of
Impact and interaction of lipophilic antioxidants 1205

the npq1 mutant at 1500 mmol m
2 s
1 for 3 days different types of antioxidants. Previously, a link
resulted in similar values to those of the wild type between the formation of zeaxanthin due to
(Havaux et al., 2000). Short-term exposure to xanthophyll cycle activity, non-photochemical
combinatorial light and cold stress caused a more quenching and the availability of ascorbate has
pronounced photoinhibition in the npq1 mutant. been postulated based on the finding that ascor-
Stress was counteracted by the accumulation of bate deficiency in the single ascorbate mutant vtc2
carotenoids, tocopherols and flavonoids during is limiting violaxanthin de-epoxidation (Müller-
photoacclimation (Havaux and Kloppstech, 2001). Moulé et al., 2002). Analysis of mutants with lower
Moreover, various double mutants were generated, ascorbate contents, in combination with either a
such as npq1 lut2 (Niyogi et al., 2001) or lut2 aba1 lack in non-photochemical quenching (vtc2 npq4)
(Pogson et al., 1998), that are impaired in the due to the absence of the PsbS protein or a lack in
synthesis of lutein as well as certain b-carotene zeaxanthin formation (vtc2 npq1), revealed signs of
derived xanthophylls (zeaxanthin or antherax- persistent photooxidative stress. Accumulation of
anthin and violaxanthin, respectively). After trans- significantly more glutathione than the wild type
fer to high light, the npq1 lut 2 mutant suffered implied a compensation of the reduced ascorbate
more photobleaching than the single mutant npq1 levels (Müller-Moulé et al., 2004).
(Havaux and Niyogi, 1999). Despite the deficiency
in lutein and zeaxanthin this double mutant
displayed an astonishing acclimation to high light Mutants and transgenic plants with altered
conditions (Niyogi et al., 2001). The lut2 npq2 tocochromanol synthesis
double mutant contains no other xanthophyll but
zeaxanthin and is unable to form trimeric light Unlike the lack of carotenoids, the complete
harvesting complexes thus compromising the struc- absence of tocopherols does not seem to be
tural integrity of the LHC complex. Although deleterious to plants. Mutants of the cyanobacteria
photoinhibition was similar in wild type and Synechocystis as well as the vte1 mutant of
mutant, the double mutant exhibited a greater Arabidopsis thaliana are able to survive under
resistance to photooxidation and was less prone to normal and high light conditions (Collakova and
lipid peroxidation (Havaux et al., 2004). DellaPenna, 2001; Dähnhardt et al., 2002). How-
In summary, the results obtained from experi- ever, tocopherol levels are strongly increased upon
ments using mutants impaired in carotenoid epox- unfavorable environmental conditions, e.g. high
idation or de-epoxidation reactions showed the light stress and drought or due to senescence
flexibility of the light harvesting system with (Chrost et al., 1999; Havaux et al., 2000; Munné-
respect to its xanthophyll composition. Only under Bosch and Alegre, 2000, 2003; Collakova and
specific experimental or environmental conditions DellaPenna, 2003b) indicating a physiologically
were the disadvantages of the alteration of the significant role. A growing body of evidence
normal full set of xanthophylls clearly visible. suggests that tocopherols can be functionally
Whereas a defect in violaxanthin de-epoxidation replaced by other antioxidants. The antioxidative
negatively affected NPQ and stress tolerance under defense system in younger leaves seems to be
high light conditions, the substitution of all especially adaptable and flexible in this respect.
xanthophylls by zeaxanthin significantly reduced For example, the reduced amount of tocopherols
the size of the light harvesting antenna of PS II and due to down-regulation of geranylgeranyl reduc-
diminished the photosynthetic capacity under low tase (Graßes et al., 2001) was counteracted by an
light. These findings demonstrate that the selective increase in xanthophyll cycle pigments in trans-
gain of a higher proportion of one xanthophyll with genic tobacco plants (Havaux et al., 2003). Vice
certain properties beneficial under a specific versa, a reduction in xanthophyll cycle pigments in
condition can be accompanied by a partial loss of the npq1 mutant of A. thaliana caused an elevation
plant performance under a different situation. of a-tocopherol during long-term high light illumi-
Optimal use and balance of light energy in nation (Havaux et al., 2000). Although the toco-
fluctuating environmental conditions seems there- pherol and tocotrienol content was successfully
fore to require the whole range of a- and b- enhanced by genetic engineering of different steps
carotene derived xanthophylls and the presence of of the biosynthetic pathway, the majority of
a functional xanthophyll cycle as a dynamic over-expressing lines were not tested for the
protection mechanism. level of other antioxidants or alterations of plant
To unravel the networking of antioxidative stress tolerance. So far, the most comprehensive
defense components, additional mutants were analysis was performed by Kanwischer et al. (2005).
studied including double mutants impaired in The consequences of antioxidant deficiencies in
1206 S. Woitsch, S. Römer

mutants with drastically reduced amounts of glu- Busch M, Seuter A, Hain R. Functional analysis of the
tathione (cad2), ascorbate (vtc1) and tocopherols early steps of carotenoid biosynthesis in tobacco.
(vte1), as well in transgenic plants with elevated Plant Physiol 2002;128:439–53.
tocopherol levels, were investigated. Tocopherol Cahoon EB, Hall SE, Ripp KG, Gantke TS, Hith WD,
deficiency in the vte1 mutant resulted in an increase Coughlan SJ. Metabolic redisign of vitamin E bio-
of total ascorbate and glutathione content, whereas synthesis in plants for tocotrienol production and
increased antioxidant content. Nat Biotechol 2003;21:
wild-type plants over-expressing vte1 exhibited lower
1082–7.
levels of these water-soluble antioxidants relative to
Chang S-H, Burgos RC, Sun W-H, Yamamoto HY. Antisense
non-transformed wild-type controls (Table 2). In suppression of violaxanthin de-epoxidase in tobacco
the vte1 vtc1 double mutant the elevation of does not affect plant performance in controlled
glutathione was even more pronounced than in the growth conditions. Photosynth Res 2000;64:95–103.
vte1 mutant. Only in the vte1 cad2 double mutant Chrost B, Falk J, Kernebeck B, Mölleken H, Krupinska K.
(devoid in tocopherol and glutathione) a marked Tocopherol biosynthesis in senescing chloroplasts—a
decrease in photosynthetic pigments was observed. mechanism to protect envelope membranes against
RNAi-mediated silencing of the potato SXDS1 gene oxidative stress and a prerequisite for lipid remobili-
caused a drastic reduction of tocopherol and toco- zation? In: Argyroudi-Akoyunoglou JH, Senger H,
trienols in source leaves due to a disruption of editors. The chloroplast: from molecular biology to
tocopherol cyclization and led to a large decrease in biotechnology. Dordrecht: Kluwer; 1999. p. 171–6.
photosynthetic capacity (Hofius et al., 2004). Collakova E, DellaPenna D. Isolation and functional
Taken together, the data obtained from various analysis of homogentisate phytyltransferase from
Synechocystis sp. PCC6803 and Arabidopsis. Plant
carotenoid and tocopherol mutants and transgenic
Physiol 2001;127:1113–24.
plants emphasize the complexity of the plant
Collakova E, DellaPenna D. Homogentisate phytyltrans-
defense system and the intricate relation and ferase activity is limiting for tocopherol biosynthesis
delicate balance between various components of in Arabidopsis. Plant Physiol 2003a;131:632–42.
the antioxidative defense system. The detailed Collakova E, DellaPenna D. The role of homogentisate
dissection of plant stress responses in wild type, phytyltransferase and other tocopherol pathway en-
mutants and transgenics bringing together exper- zymes in the regulation of tocopherol synthesis during
tise from different areas of research and the use of abiotic stress. Plant Physiol 2003b;133:930–40.
more standardized experimental conditions will Cunningham Jr FX, Gantt E. Genes and enzymes of
certainly further our understanding of the fascinat- carotenoid biosynthesis in plants. Annu Rev Plant
ing ways in which plants are able to protect Physiol Plant Mol Biol 1998;49:557–83.
themselves from biotic or abiotic stresses. Cunningham Jr FX, Pogson B, Sun ZR, McDonald KA,
DellaPenna D, Gantt E. Functional analysis of the b
and e lycopene cyclase enzymes of Arabidopsis reveals
a mechanism for control of cyclic carotenoid forma-
Acknowledgments tion. Plant Cell 1996;8:1613–26.
Dähnhardt D, Falk J, Appel J, van-der-Kooij TAW, Schulz-
We thankfully acknowledge financial support by Friedrich R, Krupinska K. The hydroxyphenylpyruvate
the Deutsche Forschungsgemeinschaft (Grant No dioxygenase from Synechocystis sp PCC 6803 is not
2047/2-2) and are grateful for help and support by required for plastoquinone biosynthesis. FEBS Lett
Prof. Dr. P. Böger at University of Konstanz. 2002;532:177–81.
Davison PA, Hunter CN, Horton P. Over-expression of
b-carotene hydroxylase enhances stress tolerance in
Arabidopsis. Nature 2002;418:203–6.
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