in the induction of tyrosine aminotransferase and tocopherol biosynthesis in Arabidopsis thaliana

ORIGINAL ARTICLE

Iris Sandorf?Heike Holla nder-Czytko

Jasmonate is involved in the induction of tyrosine aminotransferase and tocopherol biosynthesis in Arabidopsis thaliana

Received:2May2002/Accepted:16July2002

óSpringer-Verlag2002

Abstract Coronatine-inducible tyrosine aminotransfer-ase(TAT),which catalyses the transamination from ty-rosine to p-hydroxyphenylpyruvate,is the?rst enzyme of a pathway leading via homogentisic acid to plastoqui-none and tocopherols,the latter of which are known to be radical scavengers in plants.TAT can be also induced by the octadecanoids methyl jasmonate(MeJA)and methyl-12-oxophytodienoic acid(MeOPDA),as well as by wounding,high light,UV light and the herbicide oxy-?uorfen.In order to elucidate the role of octadecanoids in the process of TAT induction in Arabidopsis thaliana(L.) Heynh.,the jasmonate-de?cient mutant delayed dehi-scence(dde1)was used,in which the gene for12-oxo-phytodienoic acid reductase3is disrupted.The amount of immunodetectable TAT was low.The enzyme was still fully induced by coronatine as well as by MeJA although induction by the latter was to a lesser extent and later than in the wild type.Treatment with MeOPDA,wounding and UV light,however,had hardly any e?ects.Tocoph-erol levels that showed considerable increases in the wild type after some treatments were much less a?ected in the mutant.However,starting levels of tocopherol were higher in non-induced dde1than in the wild type.We conclude that jasmonate plays an important role in the signal transduction pathway regulating TAT activity and the biosynthesis of its product tocopherol.

Keywords Arabidopsis?Jasmonate signalling?Reactive oxygen?Tocopherol?Tyrosine aminotransferase?Ultraviolet light Abbreviations dde:delayed dehiscence?FW:fresh weight?JA:jasmonate?MeJA:methyl jasmo-nate?MeOPDA:methyl-12-oxophytodienoic acid?OPDA:12-oxophytodienoic acid?OPR3:12-oxophy-todienoate-10,11-reductase-3?PUFA:polyunsaturated fatty acid?ROS:reactive oxygen species?TAT:tyro-sine aminotransferase?WT:wild type

Introduction

Plants respond to environmental changes,such as me-chanical wounding,pest attack or high light by inducing genes that can be activated by di?erent signal trans-duction pathways.Upon mechanical wounding,e.g.in Arabidopsis,the octadecanoid pathway is induced (Bowles1991;Laudert et al.1996).This pathway has been intensively studied because the product jasmonate (JA)and also the intermediate12-oxophytodienoic acid (OPDA)are themselves signalling molecules that trigger biological responses(for review,see Schaller2001).For example,jasmonate has been shown to play an impor-tant role in leaf senescence and pathogen defense (McConn et al.1997;Vijayan et al.1998;He et al.2002).

Treatment of Arabidopsis thaliana plants with the octadecanoid mimic coronatine led to the induction of a tyrosine aminotransferase(TAT)on the RNA and protein level.TAT was also induced by methyl jasmo-nate(MeJA)and methyl-12-oxophytodienoic acid (MeOPDA)and wounding(Lopukhina et al.2001).TAT catalyses the reaction from tyrosine to p-hydroxyphenyl-pyruvate.It is the?rst enzyme in the biosynthetic pathway leading via homogentisic acid to plastoquinone and tocopherols,which are known to function as radical scavengers in plants and thus protect the plant in a variety of di?erent stress situations.Increases in radicals and reactive oxygen species have been described after wounding of plants or exposure to high light and ultra-violet(UV)light(Fryer1992;Munne-Bosch and Alegre 2002a).As participation of octadecanoid signalling in response to UV light has been reported(Conconi et al.

Planta(2002)216:173–179

DOI10.1007/s00425-002-0888-0

Dedicated to Nikolaus Amrhein,Zu rich,on the occasion of his 60th birthday.

I.Sandorf?H.Holla nder-Czytko(&)

Ruhr-Universita t Bochum,Lehrstuhl fu r P?anzenphysiologie, Universita tsstr.150,44780Bochum,Germany

E-mail:heike.hollaender-czytko@ruhr-uni-bochum.de

Fax:+49-234-3214187

1996)and TAT is inducible by octadecanoids the question arose whether,in addition,OPDA and/or JA might also mediate induction of TAT upon stress.Mutants of Arabidopsis impaired in octadecanoid biosynthesis, perception or signalling are helpful tools to answer such questions(for review,see Berger2002).The male-sterile Arabidopsis mutants dde1(delayed dehiscence)and opr3 have a disruption in12-oxophytodienoate-10,11-reduc-tase-3(OPR3),the isoform that reduces9S,13S-OPDA to generate biologically active JA.Thus dde1showed no OPR3activity and no biologically active JA could be detected in opr3.(Sanders et al.2000;Stintzi and Browse 2000;Stintzi et al.2001).Induction of TAT under di?erent stress conditions was tested in the mutant dde1.In this paper we report that JA plays an important role in the induction of TAT and the biosynthesis of tocopherols upon environmental stress.

Materials and methods

Plant material and growth conditions

Seeds of Arabidopsis thaliana(L.)Heynh.,ecotype WS (Wassilewskija)and the mutant dde1(seeds obtained from R. Goldberg,UCLA,Los Angeles,Calif.,USA),were grown on sterile standard soil in a growth chamber at22°C and a8-h-light/16-h-dark cycle with a light intensity of100l mol photons m–2s–1(photosyn-thetically active radiation).Five-to six-week old plants were used for experiments.For wounding experiments,plant leaves were crushed with a hemostat twice across the midvein.For induction experiments, plants were sprayed with octadecanoids or herbicides at the given concentrations in40%(v/v)acetone containing0.1%(w/v)Tween-20.Control plants were sprayed using acetone/Tween-20only.UV irradiation was done for6h using a lamp at254and312nm(2·8W; K.Benda,Wiesloch,Germany).After the di?erent treatments, plants were further incubated under continuous white light of 100l mol photons m–2s–1for di?erent periods of time.Plant material was harvested,frozen in liquid nitrogen and either stored at–75°C or directly lyophilized.All experiments were done at least three times independently.

Protein extraction,PAGE and immunodetection

Protein was extracted as described in Lopukhina et al.(2001),ex-cept that only1g of plant material was used.Determination of protein concentrations was done according to Bradford(1976)with bovine serum albumin as standard.Proteins were separated on a 10%(w/v)SDS–PAGE gel(Laemmli1970).Transfer of proteins onto nitrocellulose was done according to Towbin et al.(1979). Immunodetection was performed using the alkaline-phosphatase detection system(Promega,Mannheim,Germany).Both the anti-TAT antiserum and the second antibody,goat-anti-rabbit IgG, were used at a dilution of1:5,000(v/v).Detection was with the substrates nitroblue tetrazolium(NBT)and5-bromo-4-chloro-3-indolyl phosphate(BCIP).

Tocopherol analysis

For tocopherol analysis,200mg of plant material(fresh weight) was frozen in liquid nitrogen and lyophilized for48h.After freeze-drying the material was either stored at–20°C or directly pulver-ized in liquid nitrogen.The powder was extracted four times with 350l l each of iso-hexane/iso-propanol(99:1,v/v)at4°C.The extracts were centrifuged at4°C and12,000g,and the superna-tants combined and evaporated under nitrogen in the dark(4°C). The residue was dissolved in1ml of iso-hexane/iso-propanol(99:1, v/v),centrifuged and the clear supernatant was used for HPLC analysis(model510;Waters,Milford,Mass.,USA)using a method modi?ed after Tailin et al.(1997).For each analysis,200l l of the supernatant was analyzed on a lichrosorb-100,NH2-column(5l m, 250mm·4mm i.d.;Knauer,Berlin,Germany)at a?ow rate of 2ml min–1with iso-hexane/iso-propanol(99:1,v/v)as the solvent. Quanti?cation of tocopherols was done with a?uorescence detec-tor(Waters,model470)at292nm ex and324nm em.Tocopherol standards were purchased from Sigma and Calbiochem.For each time point,four samples of plant material were taken.

Results

Regulation of TAT

Tyrosine aminotransferase was isolated from coronatine-treated Arabidopsis by the RNA di?erential display technique(Lopukhina et al.2001).In non-treated leaves the basal levels of TAT as detected by immunodetection were low.By3–4h after application of the phytotoxin coronatine there was a detectable increase in TAT pro-tein;this was followed by a massive increase up to24h to a level that was stable for at least48h(Fig.1a).The octadecanoids MeJA and MeOPDA were able to induce the protein as well,although not as strongly as coronatine (Lopukhina et al.2001).Induction by MeJA could be measured4–5h after application;however,induction by MeOPDA was delayed by a few hours and was weaker (Fig.1a).In both cases,protein levels were stable for at least48h.Wounding a plant leads to an up-regulation of the octadecanoid biosynthetic pathway and to an in-crease in endogenous JA and OPDA levels.Wounding triggers the activation of enzymes not only locally but also systemically,as could be shown for the enzyme allene oxide synthase and proteinase inhibitors(Farmer and Ryan1990;Laudert et al.1996).A rise in TAT protein could be detected as early as3.5h after wounding,fol-lowed by a transient increase for24h and a subsequent slow decline(latter data not shown).The induction of TAT in response to wounding was mainly local,if at all; very little systemic reaction could be detected(Fig.1b).

Tyrosine aminotransferase is the?rst enzyme in the biosynthesis of tocopherols,which are known to be rad-ical scavengers.Even in untreated plants,prolonged growth in continuous white light leads to an increase in TAT activity(Lopukhina et al.2001)and TAT protein (Fig.1a,48h).Expression of TAT was tested in plants exposed to UV light.UV light is known to induce radical formation and reactive oxygen species in plants.Induc-tion of TAT occurred as early as1.5h after exposure to UV light and increased drastically over the following hours(Fig.1b).When oxy?uorfen,a diphenyl ether herbicide that leads to photobleaching in plants via inhi-bition of the protoporphyrinogen oxidase(Camadro et al. 1995),was applied a transient increase in TAT protein could be detected with a maximum at24h(Fig.1b). Tocopherol content in stress situations

The most abundant tocopherol in Arabidopsis was a-to-copherol with levels in non-induced plants that varied

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between 10and 15l g per gram fresh weight (FW).The corresponding levels of c -tocopherol,the biosynthetic precursor,were 0.15–0.3l g g –1,reaching only between 1.5and 3%of those of a -tocopherol.When untreated plants were kept under continuous illumination with white light,a slow rise in a -tocopherol levels could be seen,so that after 48h levels had increased by 30–40%(Fig.2a).Plants sprayed with oxy?uorfen showed no di?erence.Treatment with MeJA,MeOPDA,coronatine and wounding led to a more pronounced increase of 65–75%for a -tocopherol within 48h after application (Fig.2a).During the ?rst 10–12h (data not shown)there was no di?erence from untreated plants,but after 24h a signi?cant enhancement caused by the chemicals was clearly measurable.Levels of c -tocopherol were much lower in the wild-type (WT)plant;however,in stress sit-uations caused by wounding or chemicals,levels changed very quickly.Continuous light alone doubled the con-centration and the octadecanoids MeJA,MeOPDA and the mimic coronatine led to a further increase (Fig.2b).Wounding of the plant had a drastic e?ect and resulted in an almost 10-fold increase of c -tocopherol up to more than 1l g (g FW)–1.Signi?cant di?erences from the un-treated plant could already be measured 6h after wounding,considerably earlier than after octadecanoid treatment (Fig.2b).Oxy?uorfen showed similar kinetics to wounding,except that concentrations of c -tocopherol after 48h were in the range of 4l g (g FW)–1,a 25-fold increase from starting levels (Fig.2b).

Exposing plants to UV light for 6h with subsequent continuous illumination had only minor e?ects on a -tocopherol levels,but led to considerable enhancement of c -tocopherol concentrations up to 1.5l g (g FW)–1(Fig.3).The increase was only moderate as long as plants were exposed to UV light,but accelerated after transfer to normal light.

Jasmonate plays a role in TAT induction

The experiments described above clearly show that TAT is octadecanoid-inducible.As the dde1mutant contains no detectable amount of 3R,7S-JA,but OPDA can still be induced,e.g.by wounding (Stintzi et al.2001),it

is

Fig.1a,b.Regulation of TAT in WT Arabidopsis thaliana plants.Immunodetection of TAT in total protein extracts (10l g per lane),after separation by SDS–PAGE.a U ,untreated;C ,50l M coronatine;J ,100l M MeJA;O ,100l M MeOPDA.b W ,wounded plants:s ,systemic and l ,local leaves;OX ,100l M oxy?uorfen;UV ,exposed to UV light.The time after treatment is given in hours.Equal loading was checked by staining the blots with Ponceau S (not shown).Data shown are a representative example of three di?erent,independent

experiments

Fig.2a,b.Regulation of tocopherol content in WT Arabidopsis plants.a a -Tocopherol,b c -tocopherol.Results are given for control plants (diamonds ),wounded plants (open squares ),and plants treated with 50l M coronatine (solid triangles ),100l M MeJA (open circles ),100l M MeOPDA (open triangles )and 100l M oxy?uorfen (solid circles ).Time after treatment is given in hours.A typical experiment is shown.Data are means of four di?erent measurements.For greater clarity only the SD of the control is indicated with bars.SD values were in a similar range for all measurements

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the ideal candidate to di?erentiate between MeJA and MeOPDA as inducers and,in addition,to investigate whether the octadecanoids not only regulate TAT di-rectly but also mediate,for example,wound-or UV-induction of TAT.In non-treated mutant plants the immunodetectable amount of TAT was slightly lower than in the WT.No di?erences in coronatine-induction of TAT were detected between the WT and the mutant:up-regulation of the protein showed comparable time courses and intensities (Fig.4a).Induction by exoge-nous MeJA was weaker and time-delayed with a maxi-mum at 24h and a following decrease.For MeOPDA,no enhancement was observed during the ?rst 24h and only after 48h could a slight rise in the amount of protein be seen (Fig.4a).Wounding of mutant plants as well as exposing them to UV light resulted in no in-duction of TAT (Fig.4b).With oxy?uorfen a slight up-regulation of TAT was observed,weaker but with a similar time to that of WT plants (Fig.4b).Tocopherol contents in JA-de?cient plants

In the mutant dde1,tocopherol levels were higher than in WT plants.Whereas a -tocopherol contents were be-tween 15and 20l g (g FW)–1,50%higher than for the WT,c -tocopherol levels in untreated plant material were between 2.5and 4l g (g FW)–1,10–20%of a -tocopherol

levels.Exposing plants to continuous white light or spraying them with MeJA,MeOPDA or coronatine led to a 30–40%decrease in a -tocopherol during the ?rst 6h.Afterwards,a -tocopherol levels recovered and were slightly above starting concentrations by 24h (Fig.5a).For oxy?uorfen and wounding no sharp decrease was observed.After oxy?uorfen treatment,levels showed a continuous small decrease,whereas after wounding a continuous minor increase was observed (Fig.5a).The decline in levels during the ?rst 6h could also be seen for levels of c -tocopherol after treatment with coronatine and MeJA,whereas for control plants,MeOPDA-treated and wounded plants,levels did not change ap-preciably.Oxy?uorfen treatment led to a decrease (Fig.5b).Exposing JA-de?cient plants to UV light caused a decrease in the contents of a -and c -tocopherol (Fig.6).

Discussion

Tocopherols belong to a group of lipid-soluble com-pounds called ‘‘vitamin E’’.As essential micronutrients for animals and possessing antioxidant properties,their biosynthesis and regulation in plants is of considerable interest.E?orts have been made to enhance the amount of tocopherols in plants through metabolic engineering with the aim of improving the nutritional values

of

Fig.3a,b.Tocopherol concen-trations in WT Arabidopsis plants after exposure to UV light for 6h (t =6h)and fur-ther incubation in the light for another 18h (t =24h).Levels of untreated plants are shown at t =0h.a a -Tocopherol,b c -tocopherol.Data represent the means of four di?erent measurements of three inde-pendent experiments.Bars in-dicate SD

values

Fig.4a,b.Regulation of TAT in the Arabidopsis mutant dde1.Legend as in Fig.1,except that TAT was not measured in sys-temic leaves after wounding and 15l g protein per lane was used for gel electrophoresis.Data shown are a representative

example of four di?erent,inde-pendent experiments

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plants (Shintani and DellaPenna 1998;for review,see Grusak and DellaPenna 1999).Tyrosine aminotrans-ferase is the ?rst enzyme in the pathway leading from tyrosine to prenylquinones,and has been cloned only recently,while looking for coronatine-induced tran-scripts of Arabidopsis (Lopukhina et al.2001).

The protein was induced also by the octadecanoids MeJA and MeOPDA as well as wounding;induction was strong with coronatine and MeJA,but weaker and slightly delayed after MeOPDA treatment (Fig.1).Im-munodetection with the antibody usually leads to the detection of two bands,one at 47kDa and one that is 2–3kDa smaller.Young plants predominantly show the 47-kDa band;upon maturation or induction by stimuli,as discussed below,both bands intensify (data not shown).Whether the second band is a truncated form of the ?rst or another isoform still has to be clari?ed.As six genes have been identi?ed by the Arabidopsis genomic sequencing project (see Lopukhina et al.2001),another isoform would be reasonable,allowing the plant a more subtle regulation upon di?erent stimuli.In the jasmo-nate-de?cient mutant dde1,MeOPDA was not able to induce TAT;even after 48h the amount of protein was not higher than in untreated control plants,indicating that jasmonate is required for up-regulation of TAT.Exogenous MeJA was still active although its e?ect was seen later and was weaker than in the WT (Fig.4),which might be due to the fact that in WT plants the biosynthesis of octadecanoids is triggered by the prod-ucts themselves,as could be shown by enhancement of,for example,allene oxide synthase mRNA after appli-cation of MeOPDA or MeJA (Laudert and Weiler 1998).In the mutant the biosynthesis of JA is impaired,thus the autocatalytic stimulation and production of JA does not happen.In dde1,TAT was fully induced by coronatine (Fig.4),showing that in its activation of TAT coronatine mimics jasmonate and not OPDA.This is in agreement with statements of Lopukhina et al.(2001),who deduced this from the kinetics of activation and maxima of TAT activities in plants after treatment with the octadecanoids.Coronatine regulates the syn-thesis of OPDA and JA in plants (Laudert and Weiler 1998),but in the mutant activation of TAT by corona-tine obviously must be caused by additional mecha-nisms.As coronatine induces chlorosis and ethylene (Mitchell and Young 1978;Ferguson and Mitchell 1985),it has to be checked if one of these processes is involved.Wounding of plants led to induction of

TAT

Fig.5a,b.Regulation of tocopherol contents in the Arabidopsis mutant dde1.a a -Tocopherol,b c -tocopherol.Legend as in Fig.2.A typical experiment is shown.Data are means of four di?erent measurements.For greater clarity,only the SD of the control is indicated with bars.SD values were in a similar range for all

measurements

Fig.6a,b.Tocopherol concen-trations in UV-treated plants of the Arabidopsis mutant dde1.a a -Tocopherol,b c -tocopherol.Legend as in Fig.3.Data rep-resent the means of four di?er-ent measurements of three independent experiments.Bars indicate SD values

177

in local but not in systemic leaves.Obviously no sys-temic signal is involved in the up-regulation of TAT (Fig.1b).UV light led to a very strong induction of TAT(Fig.1b).It has been shown that wounding,as well as exposing plants to UV light,results in induction of the octadecanoid pathway and octadecanoid pathway-regulated proteins(Conconi et al.1996;Laudert and Weiler1998;Mu ssig et al.2000).

No enhancement of TAT by either treatment was detected in dde1plants(Fig.4b),supporting the fact that the octadecanoid JA and not OPDA mediates reg-ulation of TAT.Treatment with the herbicide oxy?u-orfen led to a pronounced transient increase of TAT levels in WT plants that was not completely abolished in the mutant,but was weaker,indicating the participation of other mechanisms in addition to jasmonate.The?nal e?ect of the herbicide is photobleaching of plants (Camadro et al.1995),which might be related to cor-onatine-induced chlorosis.In both cases TAT is still induced.It has been reported that wounding of plants, exposure to UV light and oxy?uorfen treatment lead to an increase of radicals in plants,which can be either radicals of polyunsaturated fatty acids(PUFA)or re-active oxygen species(ROS)or both.Tocopherols are known to be scavengers of lipid peroxy radicals,reactive oxygen species and singlet oxygen(overview in Fryer 1992;Munne-Bosch and Alegre2002a).In Chlamydo-monas reinhardtii it has been shown that tocopherols are essential for photosynthesis as they are scavengers of singlet oxygen and thus prevent degradation of the D1 protein of photosystem II(Trebst et al.2002).Also, Gra?es et al.(2001)showed that in transgenic Arabid-opsis with reduced geranylgeranyl reductase activity the enhanced susceptibility of photosystem II to photoin-hibition and photooxidative stress was correlated with a reduced tocopherol content in thylakoids.Thus,induc-tion of TAT under stress situations where PUFA radi-cals and ROS are generated could be the basis for new production of tocopherols and thereby protect the plant. Tocopherol levels,i.e.a-and c-tocopherol,were mea-sured in Arabidopsis to see whether induction of TAT was accompanied by a corresponding increase in the products of the biosynthetic pathway.Light dependency of tocopherol biosynthesis was obvious as continuous white light already led to a35–40%increase in a-to-copherol.High light doubled the amount(data not shown).All other treatments resulted in,at maximum,a 75%increase(Fig.2a).E?ects were more pronounced and di?erentiated when c-tocopherol was considered. Again,continuous illumination led to a slight increase in control plants,but whereas treatment with octadeca-noids and their mimic coronatine only doubled the amount of c-tocopherol after48h,wounding,oxy?u-orfen and UV-light led to a10-to25-fold increase (Figs.2b,3b).Obviously,conditions under which the formation of PUFA radicals and especially ROS are favoured lead to much higher c-tocopherol concentra-tions than treatment with octadecanoids where hydro-peroxy molecules are formed.Enhanced tocopherol formation has been shown for di?erent forms of stress, such as drought,high light and UV exposure(DeLong and Ste?en1997;Munne-Bosch et al.1999;Trebst et al. 2002).Also,aging of plants leads to considerable in-duction of TAT and massive increases in a-and c-to-copherol levels(data not shown).This is in accordance with data for barley leaves,which showed accumulation of both a-and c-tocopherol accompanied by increased expression of4-hydroxyphenylpyruvate dioxygenase, the enzyme that follows TAT in the biosynthetic path-way to tocopherols and catalyses the reaction from 4-hydroxyphenylpyruvate to homogentisate(Chrost et al.1999).Plant aging increases oxidative stress in chloroplasts as chlorophyll is degraded,lipid peroxida-tion enhanced and the membrane destabilized,and thus tocopherols have a role as radical scavengers as well as in stabilizing the membrane(Munne-Bosch and Alegre 2002b).When tocopherol contents in the JA-de?cient mutant dde1were examined,the situation turned out to be more complicated.In untreated plants,contents were higher;the content of c-tocopherol was in the range of 2.5l g(g FW)–1,a value only reached in induced WT plants48h after treatment with the herbicide oxy?uor-fen.Thus,in this case the lack of JA results in a massive increase in tocopherol compared to the WT.As there is a basic amount of TAT present,this could either be due to enhanced substrate availability and?ow through the pathway or to a decrease in tocopherol degradation. Also,the c-tocopherol methyltransferase forming a-to-copherol might be the bottleneck.Upon treatment with octadecanoids,?rst a decrease in tocopherols could be observed followed by a recovery.Although coronatine still induced TAT there was no corresponding increase in tocopherol levels.For oxy?uorfen and UV treat-ments,a continuous decrease in the concentration of both tocopherols was observed(Figs.5,6).Possibly, sacri?cial scavenging and degradation of tocopherols under these conditions was more rapid than biosynthe-sis.Thus,although MeJA has an important role in the induction of TAT,the situation on the level of toc-opherols is more complicated as the regulation of other enzymes of the biosynthetic pathway has to be taken into consideration,as well as the antioxidant properties of other plant products, e.g.isoprene,ascorbic acid,?avonoids and the xanthophylls(Packer et al.1979; Demmig-Adams and Adams1992;Ma?ei et al.1998; A?ek and Yakir2002).Transgenic plants and mutants with altered expression of di?erent enzymes of tocoph-erol biosynthesis have been created and tested under stress conditions,and represent new tools with which to investigate regulation of and by tocopherols(e.g. Shinatani and DellaPenna1998;Tanaka et al.1999; Collakova and DellaPenna2001;Gra?es et al.2001). Transgenic plants with altered amounts of TAT are underway and are currently being tested in order to elucidate the role of TAT in tocopherol biosynthesis and the role of tocopherols under stress conditions.

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Acknowledgements We thank Prof.Dr.E.W.Weiler,Ruhr-Uni-versita t Bochum,for helpful discussions,critical reading of the manuscript and?nancial assistance.Supported by the Deutsche Forschungsgemeinschaft,Bonn.

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