Via Fenilpropanoide

MOLECULAR PLANT PATHOLOGY

(2002)

3

(5 ) 371ndash390

copy 2002 BLACKWELL SC IENCE LTD

371

Blackwell Science Ltd

Review

The phenylpropanoid pathway and plant defencemdasha genomics perspective

R ICHARD A D IXON LAHOUCINE ACHNINE PARVATH I KOTA CHANG- JUN L IU M S SR IN IVASA REDDY AND L IANGJ IANG WANG

Plant Biology Division Samuel Roberts Noble Foundation 2510 Sam Noble Parkway Ardmore OK 73401 USA

SUMMARY

The functions of phenylpropanoid compounds in plant defencerange from preformed or inducible physical and chemical barriersagainst infection to signal molecules involved in local andsystemic signalling for defence gene induction Defensive functionsare not restricted to a particular class of phenylpropanoidcompound but are found in the simple hydroxycinnamic acids andmonolignols through to the more complex flavonoids isoflavo-noids and stilbenes The enzymatic steps involved in the bio-synthesis of the major classes of phenylpropanoid compoundsare now well established and many of the corresponding geneshave been cloned Less is understood about the regulatory genesthat orchestrate rapid coordinated induction of phenylpropanoiddefences in response to microbial attack Many of the bio-synthetic pathway enzymes are encoded by gene families butthe specific functions of individual family members remain to bedetermined The availability of the complete genome sequence of

Arabidopsis thaliana

and the extensive expressed sequence tag(EST) resources in other species such as rice soybean barrelmedic and tomato allow for the first time a full appreciation ofthe comparative genetic complexity of the phenylpropanoidpathway across species In addition gene expression arrayanalysis and metabolic profiling approaches make possible com-parative parallel analyses of global changes at the genome andmetabolome levels facilitating an understanding of the relation-ships between changes in specific transcripts and subsequent

alterations in metabolism in response to infection

INTRODUCTION

Phenylpropanoids are natural products derived from the aminoacid

L

-phenylalanine via deamination by

L

-phenylalanine ammonia-

lyase (PAL) The simplest examples containing only the C

6

C

3

phenylpropane skeleton are the hydroxycinnamic acids suchas sinapic acid and the monolignols such as coniferyl alcoholMore complex phenylpropanoids are formed by condensation of aphenylpropane unit with a unit derived from acetate via malonylcoenzyme A these include the flavonoids isoflavonoids andstilbenes The C

6

C

1

benzoic acids of which salicylic acid (SA) is animportant example in relation to plant disease have been includedin discussions of phenylpropanoid natural products because oftheir presumed biosynthetic origin via side-chain shortening ofhydroxycinnamic acids However this may not be the only routefor their synthesis in plants The major biosynthetic routes to thevarious classes of phenylpropanoid compounds are summarizedin Fig 1 which also shows the primary metabolic pathways thatprovide precursors for phenylpropanoid biosynthesis Note theorganization of the pathways into a lsquocorersquo phenylpropanoidpathway from phenylalanine to an activated (hydroxy)cinnamicacid derivative via the actions of PAL cinnamate 4-hydroxylase(C4H) and 4-coumaratecoenzyme A ligase (4CL) and the specificbranch pathways for the formation of monolignolslignincoumarins benzoic acids stilbenes and flavonoidsisoflavonoids

All classes of phenylpropanoid compounds are not present inall plant species Although the hydroxycinnamic acid and flavo-noid classes are ubiquitous in higher plants members of theseclasses with specific substitution patterns may be peculiar tocertain genera or species Other phenylpropanoid classes suchas the isoflavonoids and stilbenes are limited to particular plantfamilies The isoflavonoids are mostly limited to the subfamilyPapilionoideae of the Leguminosae Their structural variation islarge involving the number and complexity of substituents onthe 3-phenylchroman framework different oxidation levels of theheterocycle and the presence of additional heterocyclic ringsNatural sources from which isoflavonoids have been isolatedhave been reviewed in detail (Dewick 1994) The stilbenes occursporadically in widely divergent species including peanut(Leguminosae) grapevine (Vitaceae) and pine (Pinaceae) Recentknowledge of the three-dimensional structures of stilbene

Correspondence

E-mail radixonnobleorg

MPP_131fm Page 371 Thursday August 22 2002 154 PM

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R A DIXON

et al


(2002)

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(5 ) 371ndash390 copy 2002 BLACKWELL SC IENCE LTD


Phenylpropanoids and plant defence

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(5 ) 371ndash390

synthases has indicated how their genes can evolve independ-ently from closely related chalcone synthase (

CHS

) genes that arefound ubiquitously in plants (Schroumlder 1997)

Natural products active in plant defence can be categorizedinto three broad groups phytoalexins phytoanticipins and signalmolecules Many phenylpropanoids exhibit broad-spectrumantimicrobial activity and are therefore believed to help the plantfight microbial disease Such compounds can be classified aspreformed lsquophytoanticipinsrsquo or inducible lsquophytoalexinsrsquo (VanEtten

et al

1994) The best-characterized phenylpropanoid-derivedphytoalexins are the pterocarpans isoflavans and isoflavanonesof legumes including bean alfalfa pea and soybean The prenylatedisoflavones of lupin which are synthesized during seedling devel-opment are a good example of phytoanticipins (Gagnon

et al

1995) Several reviews have summarized the criteria for theclassification of compounds as phytoalexins or phytoanticipinsas well as providing extensive details on the distribution andbiological activities of phenylpropanoid compounds involved in plantdefence (Dixon 2001 Dixon and Paiva 1995 Grayer and Harborne1994 Hammerschmidt 1999 Kuc

prime

1995 Mansfield 2000)It is becoming increasingly clear that phenylpropanoid natural

products may play important roles as signal molecules both in plantdevelopment and plant defence It is also possible that these rolesmay overlap such that genetic modification for improved diseaseresistance might affect developmental processes The best-knownexamples of regulatory roles for phenylpropanoids include theactivities of dehydrodiconiferyl glucosides (dimeric monolignolderivatives) and flavonoid glycosides as potential modulators ofcell division (Teutonico

et al

1991 Woo

et al

1999) flavonoidsas regulators of auxin transport (Jacobs and Rubery 1988) andSA as a regulator of both local and systemic pathogen-induceddefence gene activation the oxidative burst and pathogen-inducedcell death (Dempsey

et al

1999)

FUNCTIONS OF PHENYLPROPANOID COMPOUNDS IN PLANT DEFENCE

The early studies that led to the formulation of the so-calledlsquophytoalexin hypothesisrsquo demonstrated that a particular chemical

was induced in response to microbial attack and that it was ableto inhibit the growth of the particular pathogen when assayed

in vitro

Subsequent studies leading to the definition of manyhundreds of phytoalexins dispensed with the use of a pathogen asinducing agent when it was realized that more convenient pro-cedures such as exposure to copper ion or elicitors from microbialcell walls could induce the synthesis of natural products withantimicrobial activity It is only recently that more rigorousgenetic criteria have been used to determine whether specificnatural products do indeed play a role in disease resistance

in vivo

Such studies fall into three classes genetic modification ofthe pathogen to disrupt the mechanisms involved in phytoalexintolerance genetic modification of the host to increase ordecrease levels of a specific natural product or genetic introduc-tion of a novel antimicrobial compound into the plant Becauseof the relatively advanced knowledge of the molecular geneticsof the phenylpropanoid pathway many of the above studies haveinvolved phenylpropanoid compounds

Plant pathogenic fungi have evolved various mechanismsby which to either avoid or destroy induced chemical barriersto infection The most common mechanism of detoxification ofhost phenylpropanoid derivatives involves oxidative metabolismusually utilizing cytochrome P450 enzymes that in several plantpathogenic fungi are encoded by genes on supernumerary orlsquodispensablersquo chromosomes (Covert

et al

1996 Wasmann andVanEtten 1996) If the target substrate is important for resist-ance disruption of such genes will result in reduced virulenceThus disruption of the

MAK1

gene in the fungal pathogen

Nectria haematococca

leading to an inability to detoxify theisoflavonoid phytoalexin maackiain led to reduced virulence ofthe fungus on chickpea (Enkerli

et al

1998)Introduction of the lsquoforeignrsquo stilbene phytoalexin resveratrol

into tobacco or alfalfa by constitutive expression of a grapevinestilbene synthase gene resulted in greatly reduced symptomsfollowing infection of tobacco by the grey mould

Botrytis cinerea

(Hain

et al

1993) or of alfalfa by the leaf spot pathogen

Phomamedicaginis

(Hipskind and Paiva 2000) Constitutive over-expression of isoflavone

O

-methyltransferase (IOMT) in trans-genic alfalfa resulted in more rapid and increased production of

Fig 1

Biosynthetic pathways leading to phenylpropanoid natural products in plants The core reactions are shown in larger type Abbreviations BA benzoic acid BA2H benzoic acid 2-hydroxylase t-CA

trans

-cinnamic acid 4-CA 4-coumaric acid CA2H cinnamate 2-hydroxylase Calc coniferyl alcohol Cald coniferaldehyde CafCoA caffeoyl CoA 4-CCoA 4-coumaroyl CoA CGA chlorogenic acid C3H coumarate (coumaroyl quinateshikimate) 3-hydroxylase C4H cinnamate 4-hydroxylase ChA chorismic acid i-ChA isochorismic acid 4-CL 4-coumarateCoA ligase CHR chalcone reductase CHS chalcone synthase COMT caffeic acid

O

-methyltransferase Csh 4-coumaroyl shikimate Daid daidzein FerA ferulic acid FerCoA feruloyl CoA Gen genistein 5-HCald 5-hydroxyconiferaldehyde HQT hydroxycinnamoyl-CoAquinate hydroxycinnamoyl transferase ICS isochorismate synthase IFR isoflavone reductase IFS isoflavone synthase Il isoliquiritigenin IOMT isoflavone

O

-methyltransferase Liq liquiritigenin MCoA malonyl CoA Med medicarpin Nar naringenin Nc naringenin chalcone PAL

L

-phenylalanine ammonia-lyase

L

-phe

L

-phenylalanine PL pyruvate lyase SA salicylic acid Salc sinapyl alcohol Sald sinapaldehyde ShA shikimic acid Van vanillin VR vestitone reductase Note that the pathways are in several places over-simplified For example the pathway to lignin probably involves methylation and hydroxylation at the level of hydroxycinnamyl aldehydes and alcohols derived from the corresponding coenzyme A esters An additional pathway might operate at last partially at the level of the free acids Two key reactions of the shikimic acid pathway for the provision of aromatic amino acids (in this case phenylalanine) are shown in the box at the top left Il formed by the coaction of CHS and CHR is primarily involved in 5-deoxy-isoflavonoid biosynthesis in the Leguminosae Reactions not designated with an enzyme name may be catalysed by more than one enzyme


374

R A DIXON

et al


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3


the isoflavonoid phytoalexin medicarpin following infection by

Phoma medicaginis

with a resultant amelioration of symptoms(He and Dixon 2000)

Taken together the results of forward and reverse geneticapproaches indicate that phenylpropanoid compounds canindeed be effective in contributing to resistance

in vivo

but oneindividual compound or class of compound may not necessarilybe the sole factor imparting disease resistance consistent withthe multicomponent nature of plant defence responses Clearlythe diversity of plant natural products and hostndashpathogencombinations means that it is impossible to make any generalconclusions that might hold for the vast majority of systems not yetanalysed and it is this factor above all that has restricted interestin natural product pathways as targets for engineered resistance

A large body of physiological and genetic evidence supports arole for SA as a critical regulator of a number of plant defenceresponses although it now seems likely that the phenylpropa-noid pathway is not the only or even the most important routeto the biosynthesis of SA (see below) Several primary papers andrecent reviews have listed the evidence implicating SA as a signalfor the transcriptional regulation of pathogenesis-related proteingenes as a lsquogain-controlrsquo agonist for the oxidative burst and asa signal molecule for pathogen-induced host cell death (Dempsey

et al

1999 Kauss and Jeblick 1995 Klessig and Malamy 1994Malamy

et al

1996 Mur

et al

1997 Murphy

et al

1999Pierpoint 1997 Rao

et al

1997 Rate

et al

1999 Shirasu

et al

1997) SA is implicated in the above responses both locallyand systemically although it appears unlikely that SA is itselfthe mobile signal in systemic acquired resistance (Vernooij

et al

1994) Plants with drastically reduced SA levels resulting fromexpression of a bacterial salicylate hydroxylase gene haveseverely compromised disease resistance (Delaney

et al

1994)whereas the over-production of SA either via expression ofbacterial isochorismate synthase and isochorismate pyruvate lyasetransgenes (Verberne

et al

2000) or through general up-regulationof the phenylpropanoid pathway by over-expression of PAL (Felton

et al

1999) is associated with increased microbial resistance

REGULATORY ARCHITECTURE OF PHENYLPROPANOID BIOSYNTHESIS

Because of the extensive information available on its structuraland regulatory genes the phenylpropanoid pathway serves asan excellent system for developing an understanding of how togenetically manipulate complex natural product pathways inplants However we still lack important information concerningthe points of flux control at and within the various branchpathways depicted in Fig 1 and the potential cross-talk betweenpathways Also important is the extent to which sets of reactionsare organized in metabolic channels or lsquometabolonsrsquo resulting inthe sequestration of intermediates from diffusible cytosolic pools

(Srere 1987) All of these factors may strongly impact the out-come of attempts to increase or decrease the level of a particularcompound by transgenic approaches Addressing these questionswill require interdisciplinary approaches involving molecularcellular and structural biology

Our understanding of flux control and cross-talk in phenyl-propanoid biosynthesis has come primarily from studies in whichspecific enzymes in the pathway have been over-expressed ordown-regulated in transgenic plants Such an approach hasshown that the entry point enzyme PAL is directly rate limiting forthe production of chlorogenic acid (CGA caffeoyl quinic acid) intobacco leaves but that factors in addition to PAL control fluxinto flavonoids and lignin (Howles

et al

1996) CGA has beenimplicated in resistance to both microbes and insects (Yao

et al

1995) although PAL over-expressing plants with elevated CGAappear to show impaired resistance to insect herbivory as a resultof cross-talk between the salicylate and jasmonate signalpathways (Felton

et al

1999)In potato tubers the creation of an artificial sink for tryp-

tophan through the transgenic expression of a tryptophan decar-boxylase gene resulted in lowered phenylalanine pools andreduced levels of wound-induced CGA and lignin with a result-ing increase in susceptibility to

Phytophthora infestans

(Yao

et al

1995) CGA levels are also reduced in tobacco by down-regulation of C4H the second enzyme in the phenylpropanoidpathway and this is accompanied by a feedback inhibition ofPAL activity possibly as a result of feedback inhibition of PALexpression by cinnamate or some derivative thereof (Blount

et al

2000) In contrast over-expression of C4H did not consistentlyresult in increased levels of CGA (Blount

et al

2000) confirmingthat PAL rather than C4H is the flux control point into the phenyl-propanoid pathway in tobacco leaves

Chalcone isomerase (CHI) catalyses a near-diffusion-limitedreaction that can also occur spontaneously at cellular pH andis not therefore generally viewed as a potential rate-limitingenzyme for flavonoid biosynthesis However over-expression ofCHI in tomato fruit peel leads to an 80-fold increase in the levelsof flavonols (Muir

et al

2001) and threefold increases in flavo-nol levels can be obtained by the expression of alfalfa CHI in

Arabidopsis

(CJ Liu and RA Dixon unpublished results) CHIwould therefore appear to be a component of flux control into theflavonoid branch of phenylpropanoid biosynthesis

The phenylpropanoid pathway presents some of the best-characterized examples of metabolic channelling in plant meta-bolism Metabolic channelling involves the physical organizationof successive enzymes in a metabolic pathway into complexesthrough which pathway intermediates are channelled withoutdiffusion into the bulk of the cytosol (Srere 1987) Such com-plexes are loose however and many of the enzymes involvedmay be operationally soluble The complexes allow for efficientcontrol of metabolic flux and protect unstable intermediates



375



(2002)

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from non-productive breakdown or access to enzymes frompotentially competing pathways Such complexes may involvedirect physical interactions between the various enzymes asrecently demonstrated for enzymes of flavonoid biosynthesisin

Arabidopsis

(Winkel-Shirley 1999) or may be associated withthe colocalization of enzymes on membranes or other surfaces(Liu and Dixon 2001) In both cases channelling can be demon-strated by double labelling or isotope dilution experiments inwhich exogenously applied intermediates are less efficientprecursors of downstream products than their upstream substratesSuch criteria have confirmed channelling between PAL and C4Hat the entry point into the phenylpropanoid pathway (Czichi andKindl 1975 Hrazdina and Jensen 1992 Hrazdina and Wagner1985 Rasmussen and Dixon 1999) and between isoflavonesynthase (IFS) and IOMT at the entry point into the isoflavonoidphytoalexin pathway (Liu and Dixon 2001) In both cases theinvolvement of a membrane-associated cytochrome P450enzyme (C4H or IFS) that might act to lsquoanchorrsquo the complex tothe endoplasmic reticulum should be noted

Metabolic channelling can impact plant defence responsesin two ways First it is possible that intermediates destinedto become a particular metabolic end product such as aphenylpropanoid-derived phytoalexin may be channelled in sucha way that they utilize different lsquopoolsrsquo of metabolic enzymes thanother products that may share some of the same biosyntheticsteps This could be achieved by utilizing different isoenzymic formsof the various pathway enzymes in different complexes Such amodel would predict that the multiple genes for many of thepathway enzymes described below might have both distinctand overlapping functions a hypothesis that remains to be testedIf this were true measurement of changes in gene transcriptsusing probes that do not distinguish between all possible formsof the encoded enzyme might lead to results that do notcorrelate with defence metabolism as observed for flavonoidisoflavonoid defences in bacterially infected alfalfa (Sallaud

et al

1997) Second although metabolic channelling might improvethe efficiency of induced defences it also presents a potentialbarrier to efficient metabolic engineering in that channelledintermediates may not be accessible to the enzyme productsof transgenes introduced in order to divert a pathway into theformation of a novel bioactive compound

COMPARATIVE GENOMICS OF PHENYLPROPANOID BIOSYNTHESIS

Our understanding of the complexity of gene families in plantshas increased rapidly in the past several years primarily becauseof the development of rapid expressed sequence tag (EST) andgenomic sequencing technologies For those species for whichextensive sequence information is available it is now possible toretrieve the sequences of the different members of gene families

by text and BLAST search in various Plant Gene Index databasessuch as those available at the TIGR website (httpwwwtigrorgtdbtgishtml) (Quackenbush

et al

2000) or the

Medicago

geneindex at the National Center for Genome Resources (httpsxgincgrorgmgi) (Bell

et al

2001) and to compute geneexpression patterns by counting the frequency of ESTs in variouscDNA libraries

We have begun a detailed bioinformatic analysis of phenylpro-panoid pathway gene complexity and expression (RA Dixon andL Wang unpublished results) Table 1 summarizes the apparentnumbers of gene family members for the various genes involvedin the core phenylpropanoid pathway and the lignin flavonoidand isoflavonoid branches in four dicot species [barrel medic(

Medicago truncatula

) and soybean from the Leguminosaetomato from the Solanaceae and


from theBrassicaceae] and two monocots (rice and maize) The sequenceidentifiers refer to tentative consensus sequences (TCs) thatrepresent EST contigs derived from clustering of the EST sequencesSingletons (EST sequences that only occur once and do not showoverlap to other sequences) are also included in the analysisEvery sequence annotated in the database as representing aspecific gene product was counted as such Gene annotation isbased on sequence similarity not function and this can lead toan overestimate of the number of genes with the specific functionas annotated (see below) The seven TCs for PAL from

Medicagotruncatula

most likely indicate the existence of seven different

PAL

or

PAL

-like gene transcripts from the libraries which havebeen sequenced to date with the caveat that this may be anoverestimate as some TCs may later be shown to cluster togetherHowever with over 140 000 ESTs now sequenced in

Medicagotruncatula

the data in Table 1 probably represent a fairly accur-ate picture of gene family complexity In the case of

Arabidopsis

the numbers are computed from the whole genome sequenceand can therefore be taken as validated

Several striking conclusions can be made from the data inTable 1 First in most of the species many of the genes exist asquite large gene families In the cases of

4CL

cinnamyl CoAreductase (

CCR

) cinnamyl alcohol dehydrogenase (

CAD

) laccaseand isoflavone reductase (

IFR

) these may have 10 or more mem-bers Second the levels of complexity differ between the differentspecies eg a single

4CL

gene in rice 10ndash16 in four of the otherspecies Third as would be predicted from metabolic analysis thekey genes of isoflavonoid biosynthesis are absent from the fournon-legume species Finally in spite of extensive EST sequencingsome genes that must exist have yet to be represented in the ESTdatabases (eg C4H in rice and maize)

The EST counting approach annotates genes based solely onsequence similarity This similarity is often less than would resultin physical detection on mid- to high-stringency DNA gel blotanalysis and should not be taken to imply proven function Thussome of the genes annotated as encoding a particular enzyme


376

R A DIXON

et al


(2002)

3


Table 1

Gene family members involved in the core phenylpropanoid pathway and the lignin flavonoid and isoflavonoid branches

Enzyme name

Tentative consensuses (TCs) or singletons in TIGR databases

M truncatula

Soybean Tomato

Arabidopsis

Rice Maize

Phenylalanine ammonia-lyase (PAL)TC28440 TC61607 TC84666 TC103728 TC48464 TC70927TC28441 TC73437 TC84677 TC115559 TC52373 TC70929TC35080 TC73439 TC93787 TC115700 TC52374 TC70930TC35727 TC95472 TC117801 TC52428 TC70931TC35728 AW035278 AA713237 TC52429 TC71742TC36057 BE462826 TC53734 TC80439TC37941 AW219744

BG735223Cinnamate 4-hydroxylase (C4H)

TC35724 TC73352 TC93282 TC115667TC35725 TC73353

4-Coumaratecoenzyme A ligase (4CL)TC29244 TC62684 TC85790 TC103592 TC55743 TC69073TC29487 TC63017 TC87087 TC104680 TC71566TC31279 TC63018 TC87740 TC105518 TC73077TC31821 TC64113 TC89636 TC109121 TC78593TC32992 TC66256 TC89693 TC109883 TC78929TC36008 TC69869 TC90983 TC110917TC37181 TC70573 TC91518 TC111771TC37802 TC71143 TC92146 TC116650TC38835 TC72975 TC93209 TC120152TC40006 TC73698 TC93567 TC124103TC40554 TC73700 TC93594 N96648TC42827 TC74240 TC94331TC42855 TC74241 AW031547

TC74245 AW616655TC75489 BE449653TC75671 AW039905

AW625022Caffeic acid

O

-methyltransferase (COMT)TC31891 TC62755 TC90236 TC109504 TC48357 TC77309TC31966 TC68824 TC94887 TC109505 TC48358 TC77890TC32648 TC112158 TC49029TC34905 TC117372TC39641 TC118345

TC121865TC121866NP236939

Caffeoyl coenzyme A

O

-methyl-transferase (CCOMT)TC30254 TC62082 TC85828 TC108307 TC48164 TC71157TC30408 TC62083 TC89798 TC117895 TC49289 TC71158TC32139 TC65887 TC93816 TC121427 NP001843TC32560 TC68488 TC93824 TC122589

TC73518 TC94433 AA394533TC73519TC75138

Ferulate 5-hydroxylase (F5H)TC28721 TC64463 TC86670 TC109653 TC54434TC38615 TC96360 TC120306

AI895344AW616986



377



(2002)

3

(5 ) 371ndash390

Table 1

continued

Enzyme name


M truncatula

Soybean Tomato

Arabidopsis

Rice Maize

Cinnamyl coenzyme A reductase (CCR)TC32087 TC68230 TC89868 TC103742 TC48219 TC71394TC32980 TC70793 TC91754 TC105238 TC48221 TC72304TC35837 TC70911 TC92006 TC107236 TC49671 TC78891TC36551 TC74702 TC96358 TC108680 TC50244 TC79954TC39655 TC77533 TC115959 TC51067 TC80830

TC115960 TC52858 NP003454TC117763TC118229TC121455TC125532

Cinnamyl alcohol dehydrogenase (CAD)TC29412 TC66049 TC85446 TC103635 TC52574 TC71268TC32920 TC66167 TC86190 TC103785 TC52613TC32921 TC66880 TC91305 TC105591 TC53411TC35882 TC68104 TC91547 TC108291TC39363 TC73412 TC94143 TC109690TC41505 TC73414 TC94740 TC109697AW696839 TC73524 TC95402 TC111929AW559294 TC74780 AW037980 TC115628

TC76785 TC116766TC116982TC119528TC120178TC122451TC123184TC126966TC126969

LaccaseTC31437 TC64439 TC96435 TC109933 TC49583TC34979 TC66286 TC97020 TC110163TC35170 TC69538 AI896093 TC111356TC36059 TC69683 AW032099 TC111531TC37979 TC71504 AW649943 TC111758TC40521 TC75229 AI782326 TC113955TC40531 TC75579 AW455342 TC115552TC40548 BE451044 TC120290TC40932 AW625159 TC120415TC42541 AW625489 TC120743AW691027 AW626092 TC122516AW691876 AW036325 TC123838

TC126250TC126968

Chalcone synthase (CHS)TC35574 TC61916 TC86565 TC106324 TC48400 TC71902TC29796 TC67543 TC87127 TC115490 TC54032TC31846 TC67544 TC90271 TC116475 NP252089TC31847 TC68628 TC118556 AU032872TC31848 TC73293 AU032888TC31850 TC75473 AU032899TC31852 AU032912TC31854TC31856


378

R A DIXON

et al


(2002)

3


Table 1

continued

Enzyme name


M truncatula

Soybean Tomato

Arabidopsis

Rice Maize

TC33667TC35573TC35575TC35576TC35577TC35803TC42671AW684295

Chalcone reductase (CHR)TC29099 TC62685 TC90973 TC54602TC29100 TC74221TC33979TC39402TC39403TC39404AW774745

Chalcone isomerase (CHI)TC35835 TC62667 TC89245 TC110376 TC48677 TC72293TC39443 TC63639 TC94706 TC112674 TC78271TC39717 TC69262 TC95516 TC113988TC40174 TC74465 AW928395 TC115647

TC74468 NP281215H36669

Flavanone 3-

β

-hydroxylase (F3

β

H)TC36151 TC67927 TC95171 TC115605 TC50019 TC78946TC37458 TC74581 TC86916 TC121953 TC55099TC38104 TC87110 T44308

TC91452TC94340TC97192

Flavonoid 3

prime

-hydroxylase (F3

prime

H)TC31717 AW933742 TC121490

Flavonoid 3

prime

5

prime

-hydroxylase (F3

prime

5

prime

H)TC33338 TC76586 TC87512 TC112562TC36887 TC88431 TC115032TC42130 AW034237 TC121970

TC122245Dihydroflavonol reductase (DFR)

TC28514 TC66100 TC88191 TC105710 TC50901 TC69820TC37214 TC67453 TC94998 TC112835 TC50971 TC75299AW981263 TC67457 NP000412 TC115766 TC53190 TC77854

TC68957 TC119438 TC78297TC69984 NP240316TC75004TC76010

Anthocyanidin synthase (ANS)TC69143 TC104059 TC56535

Isoflavone synthase (IFS)TC32250 TC61958TC36522 TC61959TC36523

Isoflavone

O

-methyl-transferase (IOMT)TC29273 TC69577



379



(2002)

3

(5 ) 371ndash390

may in fact encode related enzymes with different functions Forexample the many

4CL

genes in the four dicot species listed inTable 1 most likely encode either true isoforms of 4CL or otherenzymes that utilize a similar reaction mechanism involving theactivation of an acidic function by the formation of an acyladenylate (Cukovic

et al

2001 Ehlting

et al

2001) In severalspecies distinct isoforms of 4CL have been characterized at theenzymatic level (Knobloch and Hahlbrock 1975 Lee and Douglas1996 Vincent and Nicholson 1987) although their biochem-ical properties do not necessarily suggest differential functionsin lignification or flavonoid biosynthesis The activation of

4CL

genes is however often associated with induced defence(Uhlmann and Ebel 1993) In wheat wounding or elicitationspecifically leads to the induction of a CAD isoform with substratepreference for sinapyl alcohol consistent with the syringyl-richlignin that accumulates under these conditions (Mitchell

et al

1999) The situation with

CHS

genes is particularly interestingCHS is the prototypical enzyme representative of a class ofhomodimeric polyketide synthases that catalyse condensationof a lsquostarterrsquo coenzyme A ester (4-coumaroyl CoA in the case ofCHS and stilbene synthase) with one to three molecules of malonylCoA It is now known that some genes were at first incorrectlyannotated as encoding CHS for example the pyrone synthase of

Gerbera hybrida

that uses acetyl CoA as the starter molecule formalonyl condensation (Eckerman

et al

1998) Classical molecu-lar hybridization analysis has demonstrated the presence ofmore than eight

CHS

genes in tetraploid alfalfa (

Medicago sativa

)

(Junghans

et al

1993) but only a single true

CHS

gene in

Arabi-dopsis

(Feinbaum and Ausubel 1992) in contrast to the 16 TCsannotated as CHS in diploid

Medicago truncatula

and the fourTCs annotated as CHS in

Arabidopsis

In the case of these dimericpolyketide synthases sequence similarities are in some casessufficiently close that genes encoding enzymes with differentfunctions may cross-hybridize on gel blot analysis This is animportant point because RNA gel blot analysis of CHS transcriptshas been used in many studies as a measure of induced defence(Dhawale

et al

1989 Lawton

et al

1983 Sallaud

et al

1997)

IFR

genes were first cloned from legumes (Paiva

et al

19911994 Tiemann

et al

1991) and were selected for study in view ofthe involvement of IFR specifically in the branch of isoflavonoidmetabolism leading to isoflavan and pterocarpan phytoalexinsHowever many species that do not accumulate isoflavonoidscontain genes with high sequence identity to legume IFRs It nowappears that IFR is just one member of a large family of NADPH-dependent oxidoreductases that includes the phenylcoumaranbenzylic ether and pinoresinol-lariciresinol reductases of lignanbiosynthesis (Gang

et al

1999 Karamloo

et al

2001) andseveral other genes that are developmentally regulated or inducedduring redox shifts and oxidative stress (Babiychuk

et al

1995Lers

et al

1998 Petrucco

et al

1996 van Eldik

et al

1997)Thus the non-legume species in Table 1 all express genes fallinginto TCs annotated as encoding IFR-like proteins but appear toexpress no other genes of isoflavonoid biosynthesis and have notbeen shown to accumulate isoflavonoid natural products

Table 1 continued

Enzyme name


M truncatula Soybean Tomato Arabidopsis Rice Maize

TC37053TC40736TC40780AW686089

Isoflavone 2prime-hydroxylase (I2primeH)TC33268 TC94137TC39922

Isoflavone reductase (IFR)TC31930 TC62478 TC87096 TC115941 TC48979 TC77262TC28549 TC63010 TC95230 TC117817 TC51843 TC80585TC31929 TC69565 TC96920 TC118151 TC53547 NP003471TC32401 TC69853 BE462550 TC54779TC33160 TC69984 NP273546TC36748 TC73558 NP274174TC39922TC36918 TC73885TC39622 TC73886AW686812 TC74059AW687254 TC74060AW688509 TC75734


380

R A DIXON

et al


(2002)

3


What is the biological significance of the multigene familiesencoding many of the genes of phenylpropanoid biosynthesisAn obvious hypothesis is that there is a need to independentlyregulate the production of different phenylpropanoid productsin the same or different cells and that different gene family membersare somehow involved in the production of different classes ofcompounds Legumes in particular use phenylpropanoid compoundsas both phytoalexins and signal molecules for the attraction ofsymbiotic microbes and the independent regulation of suchpathways would clearly be necessary An alternative hypothesis isone of gene dosage In the legumes which use isoflavonoids asphytoalexins there may be a need for rapid and massive accumu-lation of these compounds immediately following infection andamplification of genes encoding enzymes at key flux control

points (eg PAL and CHS) may have allowed plants to achievethis Whatever the reason definitive information as to why manyof the gene families in Table 1 are so complex will require specificdown-regulation of the individual gene forms This has been prob-lematical in the past owing to the often very high DNA sequenceconservation between family members such that the use of anti-sense or gene silencing with large sequence fragments wouldresult in the down-regulation of several or maybe all of the genesRecent advances in plant gene silencing technology based on anunderstanding of RNA-interference (RNAi) (Wesley

et al

2001)should now facilitate the molecular dissection of the functions ofindividual members of phenylpropanoid pathway gene families

Figure 2A shows a dendrogram of the seven PAL TCs from

Medicago truncatula

in relation to the most closely related full-length

Fig 2 Sequence comparisons and expression patterns of Medicago truncatula L-phenylalanine ammonia-lyase (PAL) genes (A) Dendrogram of M truncatula PAL tentative consensus sequences (TCs) in the TIGR MtGI database aligned with plant PAL sequences The dendrogram was created using the Clustal Sequence Alignment program of the Lasergene software package (DNASTAR Madison WI USA) The amino acid sequences were aligned using the following Multiple Alignment Parameters Gap Penalty = 50 and Gap Length Penalty = 50 The Pair-wise Alignment Parameters were ktuple = 3 Gap Penalty = 5 Window = 5 and Diagonal Saved = 5 (B) In silico expression analysis of M truncatula PAL TCs The tissue sources refer to one or more cDNA libraries in which expressed sequence tags (ESTs) belonging to a particular TC were found EST counts are normalized to a per 10 000 ESTs basis The insect herbivory library is from leaf tissue isolated from plants that had been grazed by Spodoptera exigua (beet armyworm) for 24 h The infected leaf library is from leaves infected with Colletotrichum trifolii AM root is a library from roots colonized by the arbuscular mycorrhizal fungus Glomus versiforme Elicited cells are root-derived suspension culture exposed to crude yeast elicitor


Phenylpropanoids and plant defence 381

copy 2002 BLACKWELL SC IENCE LTD MOLECULAR PLANT PATHOLOGY (2002) 3 (5 ) 371ndash390

plant PAL sequences in the NCBI GENBANK A group of fivesequences clusters with other functionally characterized legumePALs TC36057 is more closely related to Arabidopsis PALs 1 and2 and two bean PAL genes and TC35080 is more distant andrelated to Arabidopsis PAL 3 The in silico expression pattern ofthe seven putative PAL or PAL-like genes in different tissues isshown in Fig 2B It can be seen that three TCs correspond togenes that are expressed in stems and are therefore candidatesfor involvement in stem lignification whereas the four others areapparently not expressed in stems Three TCs correspond to genesthat are very strongly expressed in elicitor-treated cell suspensioncultures conditions that result in the accumulation of isoflavo-noid phytoalexins There is no relation between the dendrogramshown in Fig 2A and the expression pattern in Fig 2B forexample TC28440 and TC35727 are the most strongly expressedin stems but do not cluster together based on sequenceFigure 2B also shows the effects of infection insect herbivory

symbiotic association and abiotic factors on EST numberscomputed from cDNA libraries of control and challenged tissuesFor three of the TCs the highest expression level was in elicitedcell cultures The PAL encoded by TC35727 is expressed in healthyleaves but its expression is reduced following infection whereasTC28440 appears to be down-regulated by insect herbivoryTC37941 appears to be expressed only in roots following nitro-gen starvation or nodulation This complex pattern of PAL genesand their expression in legumes contrasts with the relativelysimple organization of PAL in tobacco (two families each withtwo very closely related genes Nagai et al 1994 Pellegrini et al1994) raspberry (two genes with 88 identity but in differentclusters within the plant PAL gene phylogeny Kumar and Ellis2001) and some of the other species shown in Table 1

Figure 3A shows a dendrogram of the Medicago truncatulacaffeoyl coenzyme A (CCOMT) gene sequences CCOMT wasoriginally proposed to be specifically involved in the formation of

Fig 3 Sequence analysis and expression patterns of Medicago truncatula caffeoyl coenzyme A (CCOMT ) genes (A) Dendrogram showing the five CCOMT tentative consensus sequences (TCs) in the TIGR MtGI database in relation to functionally characterized CCOMT gene sequences from GENBANK (B) In silico expression analysis of M truncatula CCOMT TCs Details as in the legend to Fig 2


382 R A DIXON et al

MOLECULAR PLANT PATHOLOGY (2002) 3 (5 ) 371ndash390 copy 2002 BLACKWELL SC IENCE LTD

cell wall esterified ferulic acid as a pathogen defence response(Pakusch et al 1989) although the enzyme is now believed toplay a key role in the biosynthesis of lignin during vascular devel-opment (Ye et al 1994) There are five CCOMT TCs in Medicagotruncatula four of which are more closely related to the Arabi-dopsis CCOMT than to alfalfa CCOMT Three of the five TCs areexpressed in stems and therefore potentially involved in lignifica-tion in that organ (Fig 3B) One TC (TC32560) is strongly inducedin elicited cell cultures (but not roots or stems) Of the threeCCOMT TCs that are modulated by infection herbivory or elicita-tion the patterns are quite distinct Thus it is clear that plant defencemakes use of the selective expression of particular members

of the gene families encoding phenylpropanoid biosyntheticenzymes a finding inconsistent with the simple lsquogene dosagersquomodel proposed above

Unlike PAL and CCOMT IFS is a branch point enzyme specificfor the formation of a single class of natural product the isofla-vonoids It might therefore be expected that the genomiccomplexity and expression patterns of IFS genes would be simplerthan those of PAL CCOMT or CHS genes IFS is a cytochromeP450 of the CYP93C class (Jung et al 2000 Steele et al 1999)Figure 4A shows a dendrogram of the three CYP93 genesrevealed as TCs in the Medicago truncatula gene index They areclosely related to CYP93s with IFS activity characterized from the

Fig 4 Sequence analysis and expression patterns of Medicago truncatula isoflavone synthase (IFS ) genes (A) Dendrogram showing the three IFS tentative consensus sequences (TCs) in the TIGR MtGI database in relation to all known IFS gene sequences from GENBANK (B) In silico expression analysis of M truncatula IFS TCs Details as in the legend to Fig 2




other legumes Lotus japonicus licorice (Glycyrrhiza) and cowpea(Vigna) The tissue-specific expression pattern of the threeputative IFS genes from Medicago truncatula (Fig 4B) shows veryclearly that these genes are only expressed in the below-groundorgans of the plant The lack of expression in infected leafmaterial (Fig 4B) is perhaps surprising but may reflect the pathogenused (Colletotrichum trifolii) and the time of harvest of thematerial for library construction IFS genes are for example inducedin alfalfa leaves infected with the fungal pathogen Phomamedicaginis (He and Dixon 2000) All three TCs are expressedin elicited cell cultures that have been validated as producingisoflavonoid phytoalexins Interestingly TC36522 the closestorthologue of the functionally characterized IFS genes from soybeanis not the most strongly expressed in any of the tissues analysedRather TC32250 has the highest expression level and this geneis specifically and highly expressed in roots in response to phos-phate starvation It is not known whether this has any physiolog-ical significance for processes associated with phosphate nutritionsuch as the establishment of mycorrhizal interactions Neverthe-less this observation points to the dramatic impact of nutritionalphysiological status on the expression of genes that canmistakenly be thought of as responding primarily to infection

FUNCTIONAL GENOMICS APPROACHES TO THE INVOLVEMENT OF PHENYLPROPANOID BIOSYNTHESIS IN PLANT DEFENCE

The evidence for the induction of specific phenylpropanoidpathway gene family members during induced defence arguesfor more gene-selective approaches to expression profiling thanthe often non-discriminatory RNA gel blot analyses previouslyapplied The increasingly popular cDNA micro- or macro-arraytechniques while undoubtedly powerful lack selectivity forclosely related gene sequences Oligonucleotide-based DNA chiptechnology makes it possible to profile in parallel large numbersof transcripts with a selectivity that allows for independent meas-urement of different gene family members Oligonucleotide chipscontaining the various Medicago truncatula phenylpropanoidgene family members summarized in Table 1 have been producedas part of the Noble Foundationrsquos Medicago truncatula functionalgenomics program (httpwwwnobleorgmedicagoindexhtm)

A limited number of studies on gene expression profilingin plantndashmicrobe interactions have been reported to date(Reymond 2001) It is almost certain that application of in depthexpression profiling techniques to plantndashmicrobe interactionswill reveal more widespread alterations in host gene expressionthan originally foreseen In relation to systems in which phenyl-propanoid biosynthesis is induced there is already strong evidencefor the gene activation of enzymes of primary metabolism suchas the pentose phosphate and shikimate pathways (Fahrendorfet al 1995 Somssich and Hahlbrock 1998) which feed into

the secondary metabolic pathways Indeed elicitor treatment ofparsley cell cultures leading to the accumulation of phenylpropanoid-derived furanocoumarin phytoalexins is accompanied by avery extensive re-programming of gene expression (Somssichand Hahlbrock 1998) It will be interesting by coupling geneexpression array analysis with proteomic and metabolomicapproaches to determine the extent to which the changes intranscription are mirrored by changes in protein translation andconsequently linked metabolic alterations

Until recently studies on induced phenylpropanoid biosynthe-sis during plant defence monitored changes in either singlecompounds with known antifungal activity or particular classesof compounds such as isoflavonoids or stilbenes generallyutilizing high performance liquid chromatography (HPLC) with UVdetection In some cases such approaches might indeed identifythe major compound or compounds correlated with diseaseresistance as seen for example in the case of soluble 4-coumaroyl-hydroxyagmatine that accumulates during resistance of barleydetermined by the Mlo resistance gene (von Roumlpenack et al 1998)However minor components that act synergistically with moremajor components might be missed and targeted profiling willoften provide no information on changes in precursor pools that maygive important hints as to sites of flux control Recently describedtechnologies for broader metabolic profiling using mass spectro-metric detection (Fiehn et al 2000 Roessner et al 2000Trethewey et al 1999) provide a means to monitor many hundredsof metabolites in a single experiment and applications of thesetechniques will allow a better understanding of the metabolicconsequences of activation of particular gene family members indifferent tissues and in response to different biotic stresses Inparticular as transgenic plants with altered phenylpropanoidmetabolism for improved disease resistance paper pulping orproduction of speciality chemicals enter commercialization indepth metabolic profiling for the demonstration of lsquosubstantialequivalencersquo will become an important requirement of the feder-ally mandated regulatory process

NOVEL GENES OF PHENYLPROPANOID BIOSYNTHESIS

The basic core pathways shown in Fig 1 have been known formany years The enzymes and their genes were discovered bya combination of time-consuming biochemical and geneticapproaches using tractable model systems A major challenge forthe future will be to discover the many genes involved specificallyin the biosynthesis of useful bioactive phenylpropanoids limitedonly to certain species such as the pterocarpan 6a-hydroxylaseand flavonoid 6-hydroxylase cytochrome P450 enzymes recentlycharacterized from soybean (Latunde Dada et al 2001 Schopferet al 1998) This discovery process will doubtless be acceleratedby the application of bioinformatics tools to the ever-increasing


384 R A DIXON et al


amount of gene sequence information becoming available formany plant species Critical to the ability to make better predictionsof gene function from sequence information will be the paralleldevelopment of protein structure databases (Norin andSundstrom 2002) Such information on the relation betweenprimary sequence and enzyme function will allow by comparisonof protein structures rather than primary sequence per seimproved functional annotation of gene sequences This is ofparticular importance in the case of natural product pathways suchas the phenylpropanoid pathway by which different speciesproduce very different compounds but using conserved classesof enzymes An example of the value of this approach is thestructure-based prediction modelling of the Gerbera hybridapyrone synthase which although performed after the true functionof the enzyme had been determined (Eckerman et al 1998)demonstrated by structural criteria that this enzyme could notpossibly encode a CHS as previously annotated (Jez et al2000b) Detailed structural information is now appearing forenzymes of phenylpropanoid biosynthesis (Ferrer et al 1999 Jezet al 2000a Zubieta et al 2001 2002) and will facilitate theprediction of potential activities for enzymes that fall within well-studied classes such as polyketide synthase O-methyltransferase(Schroeder et al 2002) or glucosyl transferase

A good example of both the unreliability of sequence-only-based functional annotation and the evolutionary flexibility ofplant phenylpropanoid biosynthesis is the discovery that anacyltransferase involved in the biosynthesis of the major leafhydroxycinnamate ester sinapoyl malate is encoded in Arabidopsisby a gene with high sequence identity to serine carboxypeptidases(Lehfeldt et al 2000) of which there are numerous annotatedyet not functionally characterized family members in the Arabi-dopsis genome

The data in Figs 2ndash4 clearly illustrate the value of EST-basedapproaches to studies on defence gene expression Such studiescan reveal potential new functions for gene products in well-characterized pathways based on unexpected expressionpatterns of individual gene family members that can then be testedby reverse genetics approaches coupled to metabolic profilingand defence response phenotyping This type of approach willalso be helpful for resolving the functions of genes whose rolesin phenylpropanoid-based defences are currently less clear Oneexample of such a gene is the pea defence response geneDRR206 This gene is strongly induced in pea in response to bothfungal and bacterial infection (Riggleman et al 1985) and whenexpressed in transgenic Brassica napus confers resistance toboth blackleg stem canker Leptosphaeria maculans and Rhizoc-tonia solani and delayed disease development with Sclerotiniasclerotiorum (Wang and Fristensky 2001 Wang et al 1999)DRR206 exhibits about 60 sequence identity to the lsquodirigentproteinsrsquo that are involved in directing stereoselective phenolicradical coupling in the biosynthesis of lignans from two molecules

of coniferyl alcohol (Davin et al 1997) It is interesting tonote that although lignans have antifungal antibacterial andanti-insect activities (Davin and Lewis 1992) they have attractedless attention than other classes of phenylpropanoids in relationto possible roles in defence The techniques now exist to deter-mine the metabolic phenotypes of transgenic plants protected bythe expression of DRR206 and it will be interesting to discoverwhether DRR206 is indeed a true dirigent protein involved in theformation of an antimicrobial lignan

THE BIOSYNTHESIS OF SALICYLIC ACID

The biosynthesis of SA continues to remain something of a para-dox It now appears that there are several routes to benzoic acidderivatives in plants (El-Mawla and Beerhues 2002 El-Mawlaet al 2001 Verberne et al 1999) and that different routes maybe used in different species or even in the same species depend-ing on the response in question Until recently SA formation inplants was believed to occur via a branch of phenylpropanoidmetabolism involving side-chain shortening of cinnamic acidby either an oxidative route analogous to the β-oxidation of fattyacids (Loumlscher and Heide 1994) or a non-oxidative route via thecorresponding chain-shortened aldehyde a reaction previouslyshown to occur during the formation of benzoic acid derivativesin several species (Schnitzler et al 1992 Yazaki et al 1991)Recent labelling studies have provided good evidence for theoperation of the former pathway for the biosynthesis of SA incucumber and Nicotiana attenuata although the plants used inthese feeding experiments had not been induced for local orsystemic disease resistance responses (Jarvis et al 2000) A recentstudy in tobacco led to the conclusion that the free benzoic acidfound in leaves and cell cultures was unlikely to be involved inSA biosynthesis but that benzoyl glucose was likely to be anintermediate (Chong et al 2001) Genes encoding enzymes forneither of the chain-shortening pathways have yet beenunequivocally identified in plants Irrespective of the chain-shortening pathway the final step in SA biosynthesis from phenyl-propanoid precursors appears to involve the 2-hydroxylationof benzoic acid A benzoate 2-hydroxylase was purified fromtobacco and suggested to be a high molecular weight solublecytochrome P450 similar to bacterial P450s (Leoacuten et al 1995)However the gene encoding this enzyme has yet to be clonedand there are therefore no gene probes currently available forstudying SA biosynthesis from L-phenylalanine in plants

It has recently been confirmed that plants can also synthesizeSA from the shikimate pathway intermediate chorismate via theenzyme isochorismate synthase (ICS) (Wildermuth et al 2001)(Fig 1) and the same pathway operates for the biosynthesis of23-dihydroxybenzoic acid in Catharanthus roseus (Muljonoet al 2002) Arabidopsis contains two ICS genes one of whichencodes a plastid-targeted enzyme that is induced during fungal




and bacterial infection (Wildermuth et al 2001) The enhanceddisease susceptibility sid2ndash2 mutant of Arabidopsis harbours asignificant deletionrearrangement in the ICS1 gene does notaccumulate ICS1 transcripts and produces significantly reducedlevels of SA in response to infection However ICS mutants stillproduce the low constitutive levels of SA found in wild-type plantsand it has been suggested that this SA and perhaps the SAassociated with pathogen-induced cell death might still be formedvia PAL (Wildermuth et al 2001) It will be interesting to studyICS gene expression in species such as tobacco in which the localand systemic production of SA associated with resistance responseshas been previously ascribed to the phenylpropanoid pathway(Lee et al 1995 Pallas et al 1996 Verberne et al 1999Yalpani et al 1993) Because of the close association of the shiki-mate and phenylpropanoid pathways it is possible that geneticmanipulation of PAL might result in feedback effects on ICS

TRANSCRIPTIONAL REGULATION OF PHENYLPROPANOID BIOSYNTHESIS DURING PLANT DEFENCE

It has generally been assumed that the appearance of phenyl-propanoid metabolites during a plantrsquos response to infection is aresult of the transcriptional activation of the various biosyntheticpathway genes This assumption must be qualified by noting thatin most cases this has been inferred from the measurement ofsteady state transcript levels an approach that does not distin-guish between increased transcription or increased mRNAstability Nevertheless there are several examples directlydocumenting increases in transcription rates of phenylpropanoidpathway genes following the elicitation of infection as measuredby nuclear transcript run-on assays (Ni et al 1996 Rushton andSomssich 1999) and there is considerable interest in defining

the different transcription factors involved in the co-ordinatedup-regulation of defence response pathways It is likely that someof these factors are also involved in the transcriptional control ofthe same pathways during plant development

Several reviews have described the types of transcriptionfactors that regulate the expression of genes including those ofthe phenylpropanoid pathway in plants (Liu et al 1999 Meshi andIwabuchi 1995 Weisshaar and Jenkins 1998) Recent informa-tion pertaining to phenylpropanoids that may be involved indefence responses is summarized in Table 2 Several distinctclasses of transcription factor appear to operate in the overallcontrol of phenylpropanoid biosynthesis of which the mybfactors have perhaps received the most attention There are at least100 (e-value cut-off = 100E-10) myb family members in Medi-cago truncatula and 175 annotated as myb genes in ArabidopsisIn Medicago truncatula at least 11 myb genes are up-regulatedduring leaf infection and at least 28 are up-regulated during rootnodulation and arbuscular mycorrhizal symbiosis

PROSPECTS FOR METABOLIC ENGINEERING OF PHENYLPROPANOID BIOSYNTHESIS FOR IMPROVED DISEASE RESISTANCE

Some of the disease problems in highly bred cultivated crops mayhave resulted from the successive loss of natural products duringyears of selection for food quality traits and at least some ofthese pathways can now be restored by transgenic approachesHowever it has been argued that the levels of natural productsrequired may be impractically high (Stuiver and Custers 2001) Asecond argument commonly used against developing naturalproduct engineering as a strategy for improving disease resist-ance is the ability of pathogens to overcome the effects ofantimicrobial compounds by the evolution of detoxification

Class Genepathway regulated Reference

WRKY PhenylpropanoidsPR proteins Eulgem et al (1999 2000)MYBNtmyb2 PALdefence response genes Sugimoto et al (2000)PAP1-D Phenylpropanoid pathway Borevitz et al (2001)TT2 Condensed tannins Nesi et al (2001)AmMYB308330 Phenylpropanoidslignin Tamagnone et al (1998)BHLHTT8 DFR BAN Nesi et al (2000)LIM protein familyNtlim1 PAL 4CL and CAD in tobacco Kawaoka et al (2000)bZIP familyGHBF-1 CHS in soybean Droumlge-Laser et al (1997)Ku-likeKAP2 CHS Lindsay et al (2002)

BAN Banyuls CAD cinnamyl alcohol dehydrogenase CHS chalcone synthase 4CL 4-coumaratecoenzyme A ligase DFR dihydroflavonol reductase PAL L-phenylalanine ammonia-lyase PR pathogenesis-related

Table 2 Classes of transcription factors that regulate andor interact with phenylpropanoid pathway biosynthetic genes potentially involved in defence See Weisshaar and Jenkins (1998) for references to earlier literature


386 R A DIXON et al


pathways These pathways often require only single cytochromeP450 enzymes that can evolve quite rapidly (Covert et al 1996)It is possible to get around this problem by the introduction oftwo or more unrelated novel antimicrobial compounds andindeed such a strategy might also lead to synergistic effects thatcan obviate the potency question This is facilitated by the factthat there are several single enzyme reactions that can generateantimicrobial phenylpropanoid compounds from commonmetabolic intermediates Examples include O-methylation of theubiquitous flavanone naringenin to yield sakuranetin (Rakwalet al 2000) isoprenylation of isoflavones (LaFlamme et al 1993)or the production of stilbenes and other polyketides from malonylCoA and various starter molecules (Schroumlder 1997)

A further objection to metabolic pathway engineering con-cerns the large numbers of genes that may have to be transferredand coordinately regulated in order to introduce many of the mosteffective antimicrobial compounds The increasing production ofan endogenous antimicrobial compound through the over-expressionof a rate-limiting enzyme is a simpler strategy However in most casesthe flux control points in the pathway are not understoodImproved fungal disease resistance of alfalfa over-expressingisoflavone O-methyltransferase is associated with coordinated over-expression of all the other genes in the biosynthesis of the phyto-alexin medicarpin from L-phenylalanine but only in response toinfection (He and Dixon 2000) Although the reason for this pheno-menon remains unclear it provides an example of how it is possibleto engineer an improved inducible phytoalexin response withoutpotentially deleterious constitutive production of phytoalexins

As outlined above significant progress has been made inelucidating the three-dimensional structures of several key enzymesinvolved in the biosynthesis of monolignols flavonoids andisoflavonoid phytoalexins Such structural studies will facilitatestructure-based rational re-design of enzymes such as polyketidesynthases and O-methyltransferases for the transgenic introduc-tion of novel phenylpropanoid natural products for plant defenceThus structure-based mutational re-design of pyrone synthasehas yielded a novel enzyme with chalcone synthase activity (Jezet al 2000a) and it has been possible by the same approachto alter the starter molecule specificity of alfalfa CHS (Jezet al 2002) Mutations around the active site of caffeic acid O-methyltransferase (COMT) lead to forms of the enzyme withaltered kinetic preferences for acid aldehyde and alcoholsubstrates potentially involved in lignin or lignan biosynthesis(Zubieta et al 2002) It should therefore be possible in the futureto design lsquonewrsquo enzymes for more efficient pathway flux or the intro-duction of novel natural products for improved disease resistance

ACKNOWLEDGEMENTS

We thank Drs Fang Chen Dianjing Guo Xian-Zhi He Joseph NoelShashi Sharma and Chloe Zubieta for helpful discussions on

various aspects of phenylpropanoid biosynthesis and Cuc Ly forartwork Work in the corresponding authorrsquos laboratory wasfunded by the Samuel Roberts Noble Foundation Forage GeneticsInternational and David Michael and Company

REFERENCES

Babiychuk E Kushnir S Bellesboix E Van Montagu M and Inzeprimeprimeprimeprime D(1995) Arabidopsis thaliana NADPH oxidoreductase homologs confertolerance of yeast toward the thiol-oxidizing drug diamide J Biol Chem270 26 224ndash26 231

Bell C Dixon RA Farmer AD Flores R Inman J Gonzales RAHarrison MJ Paiva NL Scott AD Weller JW and May GD(2001) The Medicago genome initiative a model legume database NuclAcids Res 29 114ndash117

Blount JW Korth KL Masoud SA Rasmussen S Lamb C andDixon RA (2000) Altering expression of cinnamic acid 4-hydroxylasein transgenic plants provides evidence for a feedback loop at the entrypoint into the phenylpropanoid pathway Plant Physiol 122 107ndash116

Borevitz J Xia Y Blount JW Dixon RA and Lamb C (2001) Activa-tion tagging identifies a conserved MYB regulator of phenylpropanoidbiosynthesis Plant Cell 12 2383ndash2393

Chong J Pierrel MA Atanassova R WerckReichhart D Fritig Band Saindrenan P (2001) Free and conjugated benzoic acid intobacco plants and cell cultures Induced accumulation upon elicitationof defense responses and role as salicylic acid precursors Plant Physiol 125318ndash328

Covert SF Enkerli J Miao VPW and VanEtten HD (1996) A genefor maackiain detoxification from a dispensable chromosome of Nectriahaematococca Mol Gen Genet 251 397ndash406

Cukovic D Ehlting J VanZiffle J and Douglas CJ (2001) Structureand evolution of 4-coumaratecoenzyme A ligase (4CL) gene familiesBiol Chem 382 645ndash654

Czichi U and Kindl H (1975) Formation of p-coumaric acid and o-coumaric acid from L-phenylalanine by microsomal membrane fractionsfrom potato evidence of membrane-bound enzyme complexes Planta125 115ndash125

Davin LB and Lewis NG (1992) Phenylpropanoid metabolism Bio-synthesis of monolignols lignans and neolignans lignins and suberinsRec Adv Phytochem 26 325ndash375

Davin LB Wang H-B Crowell AL Bedgar DL Martin DMSarkanen S and Lewis NG (1997) Stereoselective bimolecular cou-pling by an auxiliary (dirigent) protein without an active center Science275 362ndash366

Delaney TP Uknes S Vernooij B Friedrich L Weymann KNegrotto D Gaffney T Gut-Rella M Kessmann H Ward Eand Ryals J (1994) A central role of salicylic acid in plant disease resist-ance Science 266 1247ndash1250

Dempsey DA Shah J and Klessig DF (1999) Salicylic acid anddisease resistance in plants Crit Rev Plant Sci 18 547ndash575

Dewick PM (1994) The isoflavonoids In The Flavonoids Advancesin Research Since 1986 (Harborne JB ed) London Chapman amp Hallpp 117ndash238

Dhawale S Souciet G and Kuhn DN (1989) Increase of chalconesynthase mRNA in pathogen-induced soybeans with race-specific resistanceis different in leaves and roots Plant Physiol 91 911ndash916




Dixon RA (2001) Natural products and disease resistance Nature 411843ndash847

Dixon RA and Paiva NL (1995) Stress-induced phenylpropanoidmetabolism Plant Cell 7 1085ndash1097

Droumlge-Laser W Kaiser A Lindsay WP Halkier B Loake GADoerner PW Dixon RA and Lamb CJ (1997) Rapid stimulationof a soybean protein-serine kinase that phosphorylates a novel bZIPtranscription factor GHBF-1 in the induction of early transcription-dependent defenses EMBO J 16 726ndash738

Eckerman S Schroumlder G Schmidt J Strack D Edrada RAHelariutta Y Elomaa P Kotilainen M Kilpelaumlinen I Proksch PTeeri TH and Schroumlder J (1998) New pathway to polyketides inplants Nature 396 387ndash390

Ehlting J Shin JJK and Douglas CJ (2001) Identification of 4-coumaratecoenzyme A ligase (4CL) substrate recognition domains PlantJ 27 455ndash465

van Eldik GJ Ruiter RK Colla PHWN van Herpen MMASchrauwen JAM and Wullems GJ (1997) Expression of an isofla-vone reductase-like gene enhanced by pollen tube growth in pistils ofSolanum tuberosum Plant Mol Biol 33 923ndash929

El-Mawla AMAA and Beerhues L (2002) Benzoic acid biosynthesisin cell cultures of Hypericum androsaemum Planta 214 727ndash733

El-Mawla AMAA Schmidt W and Beerhues L (2001) Cinnamicacid is a precursor of benzoic acids in cell cultures of Hypericum andro-saemum L but not in cell cultures of Centaurium erythraea RAFNPlanta 212 288ndash293

Enkerli J Bhatt G and Covert SF (1998) Maackiain detoxificationcontributes to the virulence of Nectria haematococca MP VI on chickpeaMol PlantndashMicrobe Interact 11 317ndash326

Eulgem T Rushton PJ Robatez S and Somssich IE (2000) TheWRKY superfamily of plant transcription factors Trends Plant Sci 5199ndash206

Eulgem T Rushton PJ Schmelzer E Hahlbrock K and Somssich IE(1999) Early nuclear events in plant defence signalling rapid geneactivation by WRKY transcription factors EMBO J 18 4689ndash4699

Fahrendorf T Ni W Shorrosh BS and Dixon RA (1995) Stressresponses in alfalfa (Medicago sativa L) XIX Transcriptional activation ofoxidative pentose phosphate pathway genes at the onset of the isofla-vonoid phytoalexin response Plant Mol Biol 28 885ndash900

Feinbaum RL and Ausubel FM (1992) Transcriptional regulation ofthe Arabidopsis thaliana chalcone synthase gene Mol Cell Biol 81985ndash1992

Felton GW Korth KL Bi JL Wesley SV Huhman DVMathews MC Murphy JB Lamb C and Dixon RA (1999)Inverse relationship between systemic resistance of plants to micro-organisms and to insect herbivory Curr Biol 9 317ndash320

Ferrer J-L Jez JM Bowman ME Dixon RA and Noel JP (1999)Structure of chalcone synthase and the molecular basis of plantpolyketide biosynthesis Nature Struct Biol 6 775ndash784

Fiehn O Kopka J Trethewey RN and Willmitzer L (2000) Identifi-cation of uncommon plant metabolites based on calculation of elementalcompositions using gas chromatography and quadrupole massspectrometry Anal Chem 72 3573ndash3580

Gagnon H Tahara S and Ibrahim RK (1995) Biosynthesis accumu-lation and secretion of isoflavonoids during germination and develop-ment of white lupin (Lupinus albus L) J Exp Bot 46 609ndash616

Gang DR Kasahara H Xia ZQ Mijnsbrugge KV Bauw GBoerjan W Van Montagu M Davin LB and Lewis NG (1999)

Evolution of plant defense mechanisms relationships of phenylcoumaranbenzylic ether reductases to pinoresinol-lariciresinol and isoflavonereductases J Biol Chem 274 7516ndash7527

Grayer RJ and Harborne JB (1994) A survey of antifungal compoundsfrom higher plants Phytochemistry 37 19ndash42

Hain R Reif H-J Krause E Langebartels R Kindl H Vornam BWeiese W Schmelzer E Schrier PH Stocker RH and Stenzel K(1993) Disease resistance results from foreign phytoalexin expression ina novel plant Nature 361 153ndash156

Hammerschmidt R (1999) Phytoalexins what have we learned after 60years Annu Rev Phytopathol 37 285ndash306

He X-Z and Dixon RA (2000) Genetic manipulation of isoflavone 7-O-methyltransferase enhances the biosynthesis of 4prime-O-methylated isoflavonoidphytoalexins and disease resistance in alfalfa Plant Cell 12 1689ndash1702

Hipskind JD and Paiva NL (2000) Constitutive accumulation of aresveratrol-glucoside in transgenic alfalfa increases resistance to Phomamedicaginis Mol PlantndashMicrobe Interact 13 551ndash562

Howles PA Paiva NL Sewalt VJH Elkind NL Bate Y Lamb CJand Dixon RA (1996) Overexpression of L-phenylalanine ammonia-lyase in transgenic tobacco plants reveals control points for flux intophenylpropanoid biosynthesis Plant Physiol 112 1617ndash1624

Hrazdina G and Jensen RA (1992) Spatial organization of enzymes inplant metabolic pathways Annu Rev Plant Physiol Plant Mol Biol 43241ndash267

Hrazdina G and Wagner GJ (1985) Metabolic pathways as enzymecomplexes evidence for the synthesis of phenylpropanoids and flavo-noids on membrane associated enzyme complexes Arch BiochemBiophys 237 88ndash100

Jacobs M and Rubery PH (1988) Naturally occurring auxin transportregulators Science 241 346ndash349

Jarvis AP Schaaf O and Oldham NJ (2000) 3-Hydroxy-3-phenylpropanoic acid is an intermediate in the biosynthesis ofbenzoic acid and salicylic acid but benzaldehyde is not Planta 212119ndash126

Jez JM Austin MB Ferrer J-L Bowman ME Schroumlder J andNoel JP (2000a) Structural control of polyketide formation in plant-specific polyketide synthesis Chem Biol 7 919ndash930

Jez JM Bowman ME Dixon RA and Noel JP (2000b) Structureand mechanism of the evolutionarily unique plant enzyme chalconeisomerase Nature Struct Biol 7 786ndash791

Jez JM Bowman ME and Noel JP (2002) Expanding the biosyntheticrepertoire of plant type III polyketide synthases by altering startermolecule specificity Proc Natl Acad Sci USA 99 5319ndash5324

Jung W Yu O Lau S-MC OrsquoKeefe DP Odell J Fader G andMcGonigle B (2000) Identification and expression of isoflavonesynthase the key enzyme for biosynthesis of isoflavones in legumesNature Biotechnol 18 208ndash212

Junghans H Dalkin K and Dixon RA (1993) Stress responses inalfalfa (Medicago sativa L) XV Characterization and expression patternsof members of a subset of the chalcone synthase multigene family PlantMol Biol 22 239ndash253

Karamloo F Wangorsch A Kasahara H Davin LB Haustein DLewis NG and Vieths S (2001) Phenylcoumaran benzylic ether andisoflavonoid reductases are a new class of cross-reactive allergens inbirch pollen fruits and vegetables Eur J Biochem 268 5310ndash5320

Kauss H and Jeblick W (1995) Pretreatment of parsley suspensioncultures with salicylic acid enhances spontaneous and elicited productionof H2O2 Plant Physiol 108 1171ndash1178


388 R A DIXON et al


Kawaoka A Kaothien P Yoshida K Endo S Yamada K andEbinuma H (2000) Functional analysis of tobacco LIM protein Ntlim1involved in lignin biosynthesis Plant J 22 289ndash301

Klessig DF and Malamy J (1994) The salicylic acid signal in plantsPlant Mol Biol 26 1439ndash1458

Knobloch KH and Hahlbrock K (1975) Isoenzymes of p-coumarateCoAligase from cell suspension cultures of Glycine max Eur J Biochem52 311ndash320

Kucprimeprimeprimeprime J (1995) Phytoalexins stress metabolism and disease resistance inplants Annu Rev Phytopathol 33 275ndash297

Kumar A and Ellis BE (2001) The phenylalanine ammonia-lyase genefamily in raspberry Structure expression and evolution Plant Physiol127 230ndash239

LaFlamme P Khouri H Gulick P and Ibrahim R (1993) Enzymaticprenylation of isoflavones in white lupin Phytochemistry 34 147ndash151

Latunde Dada AO Cabello Hurtado F Czittrich N Didierjean LSchopfer C Hertkorn N WerckReichhart D and Ebel J (2001)Flavonoid 6-hydroxylase from soybean (Glycine max L) a novel plantP-450 monooxygenase J Biol Chem 276 1688ndash1695

Lawton MA Dixon RA Hahlbrock K and Lamb CJ (1983) Elicitorinduction of mRNA activity rapid effects of elicitor on phenylalanineammonia-lyase and chalcone synthase mRNA activities in bean cells EurJ Biochem 130 131ndash139

Lee D and Douglas CJ (1996) Two divergent members of a tobacco 4-coumaratecoenzyme A ligase (4CL) gene family Plant Physiol 112193ndash2205

Lee HI Leon J and Raskin I (1995) Biosynthesis and metabolism ofsalicylic acid Proc Natl Acad Sci USA 92 4076ndash4079

Lehfeldt C Shirley AM Meyer K Ruegger MO Cusumano JCViitanen PV Strack D and Chapple C (2000) Cloning of the SNG1gene of Arabidopsis reveals a role for a serine carboxypeptidase-likeprotein as an acyltransferase in secondary metabolism Plant Cell 121295ndash1306

Leoacuten J Shulaev V Yalpani N Lawton MA and Raskin I (1995)Benzoic acid 2-hydroxylase a soluble oxygenase from tobacco catalyzessalicylic acid biosynthesis Proc Natl Acad Sci USA 92 10 413ndash10 417

Lers A Burd S Lomaniec E Droby S and Chalutz E (1998) Theexpression of a grapefruit gene encoding an isoflavone reductase-likeprotein is induced in response to UV irradiation Plant Mol Biol 36847ndash856

Lindsay WP McAlister FM Zhu Q He X-Z Droge-Laser WHedrick S Doerner P Lamb C and Dixon RA (2002) KAP-2 aprotein that binds to the H-box in a bean chalcone synthase promoter isa novel plant transcription factor with sequence identity to the largesubunit of human Ku autoantigen Plant Mol Biol 49 503ndash514

Liu C-J and Dixon RA (2001) Elicitor-induced association of isoflavoneO-methyltransferase with endomembranes prevents formation and 7-O-methylation of daidzein during isoflavonoid phytoalexin biosynthesisPlant Cell 13 2643ndash2658

Liu LS White MJ and MacRae TH (1999) Transcription factorsand their genes in higher plantsmdashfunctional domains evolution andregulation Eur J Biochem 262 247ndash257

Loumlscher R and Heide L (1994) Biosynthesis of p-hydroxybenzoatefrom p-coumarate and p-coumaroyl-coenzyme A in cell-free extracts ofLithospermum erythrorhizon cell cultures Plant Physiol 106 271ndash279

Malamy J Sanchez-Casas P Hennig J Guo A and Klessig DF(1996) Dissection of the salicylic acid signaling pathway in tobacco MolPlantndashMicrobe Interact 9 474ndash482

Mansfield JW (2000) Antimicrobial compounds and resistance The roleof phytoalexins and phytoanticipins In Mechanisms of Resistance toPlant Diseases (Slusarenko A Fraser RSS and van Loon LC eds)Dordrecht Kluwer Academic Publishers pp 325ndash370

Meshi T and Iwabuchi M (1995) Plant transcription factors Plant CellPhysiol 36 1405ndash1420

Mitchell HJ Hall SA Stratford R Hall JL and Barber MS(1999) Differential induction of cinnamyl alcohol dehydrogenase duringdefensive lignification in wheat (Triticum aestivum L) Characterisationof the major inducible form Planta 208 31ndash37

Muir SR Collins GJ Robinson S Hughes S Bovy A De Vos CHRvan Tunen AJ and Verhoeyen ME (2001) Overexpression ofpetunia chalcone isomerase in tomato results in fruit containingincreased levels of flavonols Nature Biotechnol 19 470ndash474

Muljono RAB Scheffer JJC and Verpoorte R (2002) Isochoris-mate is an intermediate in 23-dihydroxybenzoic acid biosynthesis inCatharanthus roseus cell cultures Plant Physiol Biochem 40 231ndash234

Mur LAJ Bi YM Darby RM Firek S and Draper J (1997)Compromising early salicylic acid accumulation delays the hypersensitiveresponse and increases viral dispersal during lesion establishment inTMV-infected tobacco Plant J 12 1113ndash1126

Murphy AM Chivasa S Singh DP and Carr JP (1999) Salicylicacid-induced resistance to viruses and other pathogens a parting of theways Trends Plant Sci 4 155ndash160

Nagai N Kitauchi F Shimosaka M and Okazaki M (1994) Cloningand sequencing of a full-length cDNA coding for phenylalanine ammonia-lyase from tobacco cell culture Plant Physiol 104 1091ndash1092

Nesi N Debeaujon I Jond C Pelletier G Caboche M andLepiniec L (2000) The TT8 gene encodes a basic helix-loop-helix domainprotein required for expression of DFR and BAN genes in Arabidopsissiliques Plant Cell 12 1863ndash1878

Nesi N Jond C Debeaujon I Caboche M and Lepiniec L (2001)The Arabidopsis TT2 gene encodes an R2R3 MYB domain proteinthat acts as a key determinant for proanthocyanidin accumulation indeveloping seed Plant Cell 13 2099ndash2114

Ni W Fahrendorf T Ballance GM Lamb CJ and Dixon RA(1996) Stress responses in alfalfa (Medicago sativa L) XX Transcrip-tional activation of phenylpropanoid pathway genes in elicitor-treatedcell suspension cultures Plant Mol Biol 30 427ndash438

Norin M and Sundstrom M (2002) Structural proteomics developmentsin structure-to-function predictions Trends Biotechnol 20 79ndash84

Paiva NL Edwards R Sun Y Hrazdina G and Dixon RA (1991)Stress responses in alfalfa (Medicago sativa L) XI Molecular cloningand expression of alfalfa isoflavone reductase a key enzyme of isoflavonoidphytoalexin biosynthesis Plant Mol Biol 17 653ndash667

Paiva NL Sun Y Dixon RA VanEtten HD and Hrazdina G(1994) Molecular cloning of isoflavone reductase from pea (Pisumsativum L) Evidence for a 3R-isoflavanone intermediate in (+)-pisatinbiosynthesis Arch Biochem Biophys 312 501ndash510

Pakusch AE Kneusel RE and Matern U (1989) S-adenosyl-L-methioninetrans-caffeoyl-coenzyme A 3-O-methyltransferase fromelicitor-treated parsley cell suspension cultures Arch Biochem Biophys271 488ndash494

Pallas JA Paiva NL Lamb CJ and Dixon RA (1996) Tobaccoplants epigenetically suppressed in phenylalanine ammonia-lyaseexpression do not develop systemic acquired resistance in response toinfection by tobacco mosaic virus Plant J 10 281ndash293




Pellegrini L Rohfritsch O Fritig B and Legrand M (1994) Pheny-lalanine ammonia-lyase in tobacco Molecular cloning and gene expres-sion during the hypersensitive reaction to tobacco mosaic virus and theresponse to a fungal elicitor Plant Physiol 106 877ndash886

Petrucco S Bolchi A Foroni C Percudani R Rossi GL andOttonello S (1996) A maize gene encoding a NADPH binding enzymehighly homologous to isoflavone reductases is activated in response tosulfur starvation Plant Cell 8 69ndash80

Pierpoint WS (1997) The natural history of salicylic acid InterdisciplinarySci Rev 22 45ndash52

Quackenbush J Liang F Holt I Pertea G and Upton J (2000) TheTIGR gene indices reconstruction and representation of expressed genesequences Nucl Acids Res 28 141ndash145

Rakwal R Agrawal GK Yonekura M and Kodama O (2000)Naringenin 7-O-methyltransferase involved in the biosynthesis ofthe flavanone phytoalexin sakuranetin from rice (Oryza sativa L) PlantSci 155 213ndash221

Rao MV Paliyath C Ormrod DP Murr DP and Watkins CB(1997) Influence of salicylic acid on H2O2 production oxidative stressand H2O2-metabolizing enzymesmdashSalicylic acid-mediated oxidativedamage requires H2O2 Plant Physiol 115 137ndash149

Rasmussen S and Dixon RA (1999) Transgene-mediated and elicitor-induced perturbation of metabolic channeling at the entry point into thephenylpropanoid pathway Plant Cell 11 1537ndash1551

Rate DN Cuenca JV Bowman GR Guttman DS and Greenberg JT(1999) The gain-of-function Arabidopsis acd6 mutant reveals novelregulation and function of the salicylic acid signaling pathway incontrolling cell death defenses and cell growth Plant Cell 11 1695ndash1708

Reymond P (2001) DNA microarrays and plant defence Plant PhysiolBiochem 39 313ndash321

Riggleman RC Fristensky B and Hadwiger LA (1985) The diseaseresistance response in pea is associated with increased levels of specificmRNAs Plant Mol Biol 4 81ndash86

Roessner U Wagner C Kopka J Trethewey RN and Willmitzer L(2000) Simultaneous analysis of metabolites in potato tuber by gaschromatography-mass spectrometry Plant J 23 131ndash142

von Roumlpenack E Parr A and Schulze-Lefert P (1998) Structuralanalyses and dynamics of soluble and cell wall-bound phenolics in abroad spectrum resistance to the powdery mildew fungus in barleyJ Biol Chem 273 9013ndash9022

Rushton PJ and Somssich IE (1999) Transcriptional regulation of plantgenes responsive to pathogens and elicitors In PlantndashMicrobe Interactions4 (Stacey G and Keen NT eds) St Paul MN American PhytopathologicalSociety pp 251ndash274

Sallaud C Zuanazzi J El-Turk J Leymarie J Breda C Buffard Dde Kozak I Ratet P Husson P Kondorosi A and Esnault R(1997) Gene expression is not systemically linked to phytoalexin produc-tion during alfalfa leaf interaction with pathogenic bacteria Mol PlantndashMicrobe Interact 10 257ndash267

Schnitzler JP Madlung J Rose A and Seitz HU (1992) Biosynthe-sis of p-hydroxybenzoic acid in elicitor-treated carrot cell cultures Planta188 594ndash600

Schopfer CR Kochs G Lottspeich F and Ebel J (1998) Molecularcharacterization and functional expression of dihydroxypterocarpan 6a-hydroxylase an enzyme specific for pterocarpanoid phytoalexinbiosynthesis in soybean (Glycine max L) FEBS Lett 432 182ndash186

Schroumlder J (1997) A family of plant-specific polyketide synthases factsand predictions Trends Plant Sci 2 373ndash378

Schroeder G Wehinger E and Schroeder J (2002) Predicting thesubstrates of cloned plant O-methyltransferases Phytochemistry 591ndash8

Shirasu K Nakajima H Rajasekhar VK Dixon RA and Lamb CJ(1997) Salicylic acid potentiates an agonist-dependent gain control thatamplifies pathogen signals in the activation of defense mechanismsPlant Cell 9 261ndash270

Somssich IE and Hahlbrock K (1998) Pathogen defence in plantsmdasha paradigm of biological complexity Trends Plant Sci 3 86ndash90

Srere PA (1987) Complexes of sequential metabolic enzymes Annu RevBiochem 56 89ndash124

Steele CL Gijzen M Qutob D and Dixon RA (1999) Molecularcharacterization of the enzyme catalyzing the aryl migration reaction ofisoflavonoid biosynthesis in soybean Arch Biochem Biophys 367147ndash150

Stuiver MH and Custers JHHV (2001) Engineering disease resistancein plants Nature 411 865ndash868

Sugimoto K Takeda S and Horochika H (2000) MYB-relatedtranscription factor NtMYB2 induced by wounding and elicitors is aregulator of the tobacco retrotransposon Tto1 and defense-related genesPlant Cell 12 2511ndash2528

Tamagnone L Merida A Parr A Mackay S Culianez-Macia FARoberts K and Martin C (1998) The AmMYB308 and AmMYB330transcription factors from antirrhinum regulate phenylpropanoid andlignin biosynthesis in transgenic tobacco Plant Cell 10 135ndash154

Teutonico RT Dudley MW Orr JD Lynn DG and Binns AN(1991) Activity and accumulation of cell division-promoting phenolics intobacco tissue cultures Plant Physiol 97 288ndash297

Tiemann K Inzeacute D Van Montagu M and Barz W (1991) Ptero-carpan phytoalexin biosynthesis in elicitor-challenged chickpea (Cicerarietinum L) cell cultures Purification characterization and cDNAcloning of NADPHisoflavone oxidoreductase Eur J Biochem 200751ndash757

Trethewey RN Krotzky AJ and Willmitzer L (1999) Metabolicprofiling a Rosetta Stone for genomics Curr Opin Plant Biol 2 83ndash85

Uhlmann A and Ebel J (1993) Molecular cloning and expression of4-coumaratecoenzyme A ligase an enzyme involved in the resistanceresponse of soybean (Glycine max L) against pathogen attack PlantPhysiol 102 1147ndash1156

VanEtten HD Mansfield JW Bailey JA and Farmer EE (1994)Two classes of plant antibiotics phytoalexins versus lsquophytoanticipinsrsquoPlant Cell 6 1191ndash1192

Verberne MC Muljono RAB and Verpoorte R (1999) Salicylic acidbiosynthesis In Biochemistry and Molecular Biology of Plant Hormones(Hooykaas PJJ Hall MA and Libbenga KR eds) AmsterdamElsevier Science Publishers pp 295ndash312

Verberne MC Verpoorte R Bol JF MercadoBlanco J andLinthorst HJM (2000) Overproduction of salicylic acid in plants bybacterial transgenes enhances pathogen resistance Nature Biotechnol18 779ndash783

Vernooij B Friedrich L Morse A Reist R Kolditz-Jawhar RWard E Uknes S Kessmann H and Ryals J (1994) Salicylic acidis not the translocated signal responsible for inducing systemic acquiredresistance but is required in signal transduction Plant Cell 6 959ndash965

Vincent JR and Nicholson RL (1987) Evidence for isoenzymes of 4-hydroxycinnamic acidCoA ligase in maize mesocotyls and their responseto infection by Helminthosporium maydis race O Physiol Mol PlantPathol 30 121ndash129


390 R A DIXON et al


Wang Y and Fristensky B (2001) Transgenic canola lines expressing peadefense gene DRR206 have resistance to aggressive blackleg isolatesand to Rhizoctonia solani Mol Breed 8 263ndash271

Wang Y Nowak G Culley D Hadwiger LA and Fristensky B(1999) Constitutive expression of pea defense gene DRR206 confersresistance to blackleg (Leptosphaeria maculans) disease in transgeniccanola (Brassica napus) Mol PlantndashMicrobe Interact 12 410ndash418

Wasmann CC and VanEtten HD (1996) Transformation-mediatedchromosome loss and disruption of a gene for pisatin demethylasedecrease the virulence of Nectria haematococca on pea Mol PlantndashMicrobe Interact 9 793ndash803

Weisshaar B and Jenkins GI (1998) Phenylpropanoid biosynthesis andits regulation Curr Opin Plant Biol 1 251ndash257

Wesley VS Helliwell CA Smith NA Wang MB Rouse DT Liu QGooding PS Singh SP Abbott D Stoutjesdijk PA Robinson SPGleave AP Green AG and Waterhouse PM (2001) Constructdesign for efficient effective and high-throughput gene silencing inplants Plant J 27 581ndash590

Wildermuth MC Dewdney J Wu G and Ausubel FM (2001)Isochorismate synthase is required to synthesize salicylic acid for plantdefence Nature 414 562ndash565

Winkel-Shirley B (1999) Evidence for enzyme complexes in the phenyl-propanoid and flavonoid pathways Physiol Plant 107 142ndash149

Woo HH Orbach MJ Hirsch AM and Hawes MC (1999) Meristem-localized inducible expression of a UDP-glycosyltransferase gene isessential for growth and development in pea and alfalfa Plant Cell 112303ndash2315

Yalpani N Leacuteon J Lawton MA and Raskin I (1993) Pathway ofsalicylic acid biosynthesis in healthy and virus-inoculated tobacco PlantPhysiol 103 315ndash321

Yao KN Deluca V and Brisson N (1995) Creation of a metabolic sinkfor tryptophan alters the phenylpropanoid pathway and the susceptibilityof potato to Phytophthora infestans Plant Cell 7 1787ndash1799

Yazaki K Heide L and Tabata M (1991) Formation of p-hydroxybenzoicacid from p-coumaric acid by cell free extract of Lithospermum erythrorhizoncell cultures Phytochemistry 30 2233ndash2236

Ye ZH Kneusel RE Matern U and Varner JE (1994) An alternativemethylation pathway in lignin biosynthesis in Zinnia Plant Cell 6 1427ndash1439

Zubieta C Dixon RA and Noel JP (2001) Crystal structures ofchalcone O-methyltransferase and isoflavone O-methyltransferase revealthe structural basis for substrate specificity in plant O-methyltransferasesNature Struct Biol 8 271ndash279

Zubieta C Kota P Ferrer J-L Dixon RA and Noel J (2002) Structuralbasis for the modulation of lignin monomer methylation by caffeic acid5-hydroxyferulic acid 35-O-methyltransferase Plant Cell 14 1265ndash1277


372

R A DIXON

et al


(2002)

3




373



(2002)

3

(5 ) 371ndash390


CHS



et al


et al


prime



et al

1991 Woo

et al


et al

1999)




in vitro


in vivo



et al


MAK1




et al



Botrytis cinerea

(Hain

et al


Phomamedicaginis


O


Fig 1


trans


O


O


L


L

-phe

L



374

R A DIXON

et al


(2002)

3



Phoma medicaginis



in vivo



et al


et al

1996 Mur

et al

1997 Murphy

et al


et al

1997 Rate

et al

1999 Shirasu

et al


et al


et al


et al


et al






et al


et al


et al




(Yao

et al


et al


et al



et al


Arabidopsis





375



(2002)

3

(5 ) 371ndash390


Arabidopsis



et al





et al

2000) or the

Medicago


et al



Medicago truncatula




Medicagotruncatula


PAL

or

PAL


Medicagotruncatula


Arabidopsis



4CL


CCR


CAD


IFR


4CL




376

R A DIXON

et al


(2002)

3


Table 1


Enzyme name


M truncatula

Soybean Tomato

Arabidopsis

Rice Maize



TC35724 TC73352 TC93282 TC115667TC35725 TC73353


TC74245 AW616655TC75489 BE449653TC75671 AW039905


O


TC121865TC121866NP236939

Caffeoyl coenzyme A

O


TC73518 TC94433 AA394533TC73519TC75138


AI895344AW616986



377



(2002)

3

(5 ) 371ndash390

Table 1

continued

Enzyme name


M truncatula

Soybean Tomato

Arabidopsis

Rice Maize


TC115960 TC52858 NP003454TC117763TC118229TC121455TC125532




TC126250TC126968



378

R A DIXON

et al


(2002)

3


Table 1

continued

Enzyme name


M truncatula

Soybean Tomato

Arabidopsis

Rice Maize




TC74468 NP281215H36669

Flavanone 3-

β

-hydroxylase (F3

β


TC91452TC94340TC97192

Flavonoid 3

prime

-hydroxylase (F3

prime

H)TC31717 AW933742 TC121490

Flavonoid 3

prime

5

prime

-hydroxylase (F3

prime

5

prime




TC68957 TC119438 TC78297TC69984 NP240316TC75004TC76010



Isoflavone

O




379



(2002)

3

(5 ) 371ndash390


4CL


et al

2001 Ehlting

et al


4CL


et al


CHS


Gerbera hybrida


et al


CHS


Medicago sativa

)

(Junghans

et al


CHS

gene in

Arabi-dopsis


Medicago truncatula


Arabidopsis


et al

1989 Lawton

et al

1983 Sallaud

et al

1997)

IFR


et al

19911994 Tiemann

et al


et al

1999 Karamloo

et al


et al

1995Lers

et al

1998 Petrucco

et al

1996 van Eldik

et al


Table 1 continued

Enzyme name



TC37053TC40736TC40780AW686089




380

R A DIXON

et al


(2002)

3




et al



Medicago truncatula











382 R A DIXON et al


















384 R A DIXON et al
























386 R A DIXON et al





ACKNOWLEDGEMENTS



REFERENCES























































388 R A DIXON et al










































































390 R A DIXON et al


















373



(2002)

3

(5 ) 371ndash390


CHS



et al


et al


prime



et al

1991 Woo

et al


et al

1999)




in vitro


in vivo



et al


MAK1




et al



Botrytis cinerea

(Hain

et al


Phomamedicaginis


O


Fig 1


trans


O


O


L


L

-phe

L



374

R A DIXON

et al


(2002)

3



Phoma medicaginis



in vivo



et al


et al

1996 Mur

et al

1997 Murphy

et al


et al

1997 Rate

et al

1999 Shirasu

et al


et al


et al


et al


et al






et al


et al


et al




(Yao

et al


et al


et al



et al


Arabidopsis





375



(2002)

3

(5 ) 371ndash390


Arabidopsis



et al





et al

2000) or the

Medicago


et al



Medicago truncatula




Medicagotruncatula


PAL

or

PAL


Medicagotruncatula


Arabidopsis



4CL


CCR


CAD


IFR


4CL




376

R A DIXON

et al


(2002)

3


Table 1


Enzyme name


M truncatula

Soybean Tomato

Arabidopsis

Rice Maize



TC35724 TC73352 TC93282 TC115667TC35725 TC73353


TC74245 AW616655TC75489 BE449653TC75671 AW039905


O


TC121865TC121866NP236939

Caffeoyl coenzyme A

O


TC73518 TC94433 AA394533TC73519TC75138


AI895344AW616986



377



(2002)

3

(5 ) 371ndash390

Table 1

continued

Enzyme name


M truncatula

Soybean Tomato

Arabidopsis

Rice Maize


TC115960 TC52858 NP003454TC117763TC118229TC121455TC125532




TC126250TC126968



378

R A DIXON

et al


(2002)

3


Table 1

continued

Enzyme name


M truncatula

Soybean Tomato

Arabidopsis

Rice Maize




TC74468 NP281215H36669

Flavanone 3-

β

-hydroxylase (F3

β


TC91452TC94340TC97192

Flavonoid 3

prime

-hydroxylase (F3

prime

H)TC31717 AW933742 TC121490

Flavonoid 3

prime

5

prime

-hydroxylase (F3

prime

5

prime




TC68957 TC119438 TC78297TC69984 NP240316TC75004TC76010



Isoflavone

O




379



(2002)

3

(5 ) 371ndash390


4CL


et al

2001 Ehlting

et al


4CL


et al


CHS


Gerbera hybrida


et al


CHS


Medicago sativa

)

(Junghans

et al


CHS

gene in

Arabi-dopsis


Medicago truncatula


Arabidopsis


et al

1989 Lawton

et al

1983 Sallaud

et al

1997)

IFR


et al

19911994 Tiemann

et al


et al

1999 Karamloo

et al


et al

1995Lers

et al

1998 Petrucco

et al

1996 van Eldik

et al


Table 1 continued

Enzyme name



TC37053TC40736TC40780AW686089




380

R A DIXON

et al


(2002)

3




et al



Medicago truncatula











382 R A DIXON et al


















384 R A DIXON et al
























386 R A DIXON et al





ACKNOWLEDGEMENTS



REFERENCES























































388 R A DIXON et al










































































390 R A DIXON et al

















374

R A DIXON

et al


(2002)

3



Phoma medicaginis



in vivo



et al


et al

1996 Mur

et al

1997 Murphy

et al


et al

1997 Rate

et al

1999 Shirasu

et al


et al


et al


et al


et al






et al


et al


et al




(Yao

et al


et al


et al



et al


Arabidopsis





375



(2002)

3

(5 ) 371ndash390


Arabidopsis



et al





et al

2000) or the

Medicago


et al



Medicago truncatula




Medicagotruncatula


PAL

or

PAL


Medicagotruncatula


Arabidopsis



4CL


CCR


CAD


IFR


4CL




376

R A DIXON

et al


(2002)

3


Table 1


Enzyme name


M truncatula

Soybean Tomato

Arabidopsis

Rice Maize



TC35724 TC73352 TC93282 TC115667TC35725 TC73353


TC74245 AW616655TC75489 BE449653TC75671 AW039905


O


TC121865TC121866NP236939

Caffeoyl coenzyme A

O


TC73518 TC94433 AA394533TC73519TC75138


AI895344AW616986



377



(2002)

3

(5 ) 371ndash390

Table 1

continued

Enzyme name


M truncatula

Soybean Tomato

Arabidopsis

Rice Maize


TC115960 TC52858 NP003454TC117763TC118229TC121455TC125532




TC126250TC126968



378

R A DIXON

et al


(2002)

3


Table 1

continued

Enzyme name


M truncatula

Soybean Tomato

Arabidopsis

Rice Maize




TC74468 NP281215H36669

Flavanone 3-

β

-hydroxylase (F3

β


TC91452TC94340TC97192

Flavonoid 3

prime

-hydroxylase (F3

prime

H)TC31717 AW933742 TC121490

Flavonoid 3

prime

5

prime

-hydroxylase (F3

prime

5

prime




TC68957 TC119438 TC78297TC69984 NP240316TC75004TC76010



Isoflavone

O




379



(2002)

3

(5 ) 371ndash390


4CL


et al

2001 Ehlting

et al


4CL


et al


CHS


Gerbera hybrida


et al


CHS


Medicago sativa

)

(Junghans

et al


CHS

gene in

Arabi-dopsis


Medicago truncatula


Arabidopsis


et al

1989 Lawton

et al

1983 Sallaud

et al

1997)

IFR


et al

19911994 Tiemann

et al


et al

1999 Karamloo

et al


et al

1995Lers

et al

1998 Petrucco

et al

1996 van Eldik

et al


Table 1 continued

Enzyme name



TC37053TC40736TC40780AW686089




380

R A DIXON

et al


(2002)

3




et al



Medicago truncatula











382 R A DIXON et al


















384 R A DIXON et al
























386 R A DIXON et al





ACKNOWLEDGEMENTS



REFERENCES























































388 R A DIXON et al










































































390 R A DIXON et al


















375



(2002)

3

(5 ) 371ndash390


Arabidopsis



et al





et al

2000) or the

Medicago


et al



Medicago truncatula




Medicagotruncatula


PAL

or

PAL


Medicagotruncatula


Arabidopsis



4CL


CCR


CAD


IFR


4CL




376

R A DIXON

et al


(2002)

3


Table 1


Enzyme name


M truncatula

Soybean Tomato

Arabidopsis

Rice Maize



TC35724 TC73352 TC93282 TC115667TC35725 TC73353


TC74245 AW616655TC75489 BE449653TC75671 AW039905


O


TC121865TC121866NP236939

Caffeoyl coenzyme A

O


TC73518 TC94433 AA394533TC73519TC75138


AI895344AW616986



377



(2002)

3

(5 ) 371ndash390

Table 1

continued

Enzyme name


M truncatula

Soybean Tomato

Arabidopsis

Rice Maize


TC115960 TC52858 NP003454TC117763TC118229TC121455TC125532




TC126250TC126968



378

R A DIXON

et al


(2002)

3


Table 1

continued

Enzyme name


M truncatula

Soybean Tomato

Arabidopsis

Rice Maize




TC74468 NP281215H36669

Flavanone 3-

β

-hydroxylase (F3

β


TC91452TC94340TC97192

Flavonoid 3

prime

-hydroxylase (F3

prime

H)TC31717 AW933742 TC121490

Flavonoid 3

prime

5

prime

-hydroxylase (F3

prime

5

prime




TC68957 TC119438 TC78297TC69984 NP240316TC75004TC76010



Isoflavone

O




379



(2002)

3

(5 ) 371ndash390


4CL


et al

2001 Ehlting

et al


4CL


et al


CHS


Gerbera hybrida


et al


CHS


Medicago sativa

)

(Junghans

et al


CHS

gene in

Arabi-dopsis


Medicago truncatula


Arabidopsis


et al

1989 Lawton

et al

1983 Sallaud

et al

1997)

IFR


et al

19911994 Tiemann

et al


et al

1999 Karamloo

et al


et al

1995Lers

et al

1998 Petrucco

et al

1996 van Eldik

et al


Table 1 continued

Enzyme name



TC37053TC40736TC40780AW686089




380

R A DIXON

et al


(2002)

3




et al



Medicago truncatula











382 R A DIXON et al


















384 R A DIXON et al
























386 R A DIXON et al





ACKNOWLEDGEMENTS



REFERENCES























































388 R A DIXON et al










































































390 R A DIXON et al

















376

R A DIXON

et al


(2002)

3


Table 1


Enzyme name


M truncatula

Soybean Tomato

Arabidopsis

Rice Maize



TC35724 TC73352 TC93282 TC115667TC35725 TC73353


TC74245 AW616655TC75489 BE449653TC75671 AW039905


O


TC121865TC121866NP236939

Caffeoyl coenzyme A

O


TC73518 TC94433 AA394533TC73519TC75138


AI895344AW616986



377



(2002)

3

(5 ) 371ndash390

Table 1

continued

Enzyme name


M truncatula

Soybean Tomato

Arabidopsis

Rice Maize


TC115960 TC52858 NP003454TC117763TC118229TC121455TC125532




TC126250TC126968



378

R A DIXON

et al


(2002)

3


Table 1

continued

Enzyme name


M truncatula

Soybean Tomato

Arabidopsis

Rice Maize




TC74468 NP281215H36669

Flavanone 3-

β

-hydroxylase (F3

β


TC91452TC94340TC97192

Flavonoid 3

prime

-hydroxylase (F3

prime

H)TC31717 AW933742 TC121490

Flavonoid 3

prime

5

prime

-hydroxylase (F3

prime

5

prime




TC68957 TC119438 TC78297TC69984 NP240316TC75004TC76010



Isoflavone

O




379



(2002)

3

(5 ) 371ndash390


4CL


et al

2001 Ehlting

et al


4CL


et al


CHS


Gerbera hybrida


et al


CHS


Medicago sativa

)

(Junghans

et al


CHS

gene in

Arabi-dopsis


Medicago truncatula


Arabidopsis


et al

1989 Lawton

et al

1983 Sallaud

et al

1997)

IFR


et al

19911994 Tiemann

et al


et al

1999 Karamloo

et al


et al

1995Lers

et al

1998 Petrucco

et al

1996 van Eldik

et al


Table 1 continued

Enzyme name



TC37053TC40736TC40780AW686089




380

R A DIXON

et al


(2002)

3




et al



Medicago truncatula











382 R A DIXON et al


















384 R A DIXON et al
























386 R A DIXON et al





ACKNOWLEDGEMENTS



REFERENCES























































388 R A DIXON et al










































































390 R A DIXON et al


















377



(2002)

3

(5 ) 371ndash390

Table 1

continued

Enzyme name


M truncatula

Soybean Tomato

Arabidopsis

Rice Maize


TC115960 TC52858 NP003454TC117763TC118229TC121455TC125532




TC126250TC126968



378

R A DIXON

et al


(2002)

3


Table 1

continued

Enzyme name


M truncatula

Soybean Tomato

Arabidopsis

Rice Maize




TC74468 NP281215H36669

Flavanone 3-

β

-hydroxylase (F3

β


TC91452TC94340TC97192

Flavonoid 3

prime

-hydroxylase (F3

prime

H)TC31717 AW933742 TC121490

Flavonoid 3

prime

5

prime

-hydroxylase (F3

prime

5

prime




TC68957 TC119438 TC78297TC69984 NP240316TC75004TC76010



Isoflavone

O




379



(2002)

3

(5 ) 371ndash390


4CL


et al

2001 Ehlting

et al


4CL


et al


CHS


Gerbera hybrida


et al


CHS


Medicago sativa

)

(Junghans

et al


CHS

gene in

Arabi-dopsis


Medicago truncatula


Arabidopsis


et al

1989 Lawton

et al

1983 Sallaud

et al

1997)

IFR


et al

19911994 Tiemann

et al


et al

1999 Karamloo

et al


et al

1995Lers

et al

1998 Petrucco

et al

1996 van Eldik

et al


Table 1 continued

Enzyme name



TC37053TC40736TC40780AW686089




380

R A DIXON

et al


(2002)

3




et al



Medicago truncatula











382 R A DIXON et al


















384 R A DIXON et al
























386 R A DIXON et al





ACKNOWLEDGEMENTS



REFERENCES























































388 R A DIXON et al










































































390 R A DIXON et al

















378

R A DIXON

et al


(2002)

3


Table 1

continued

Enzyme name


M truncatula

Soybean Tomato

Arabidopsis

Rice Maize




TC74468 NP281215H36669

Flavanone 3-

β

-hydroxylase (F3

β


TC91452TC94340TC97192

Flavonoid 3

prime

-hydroxylase (F3

prime

H)TC31717 AW933742 TC121490

Flavonoid 3

prime

5

prime

-hydroxylase (F3

prime

5

prime




TC68957 TC119438 TC78297TC69984 NP240316TC75004TC76010



Isoflavone

O




379



(2002)

3

(5 ) 371ndash390


4CL


et al

2001 Ehlting

et al


4CL


et al


CHS


Gerbera hybrida


et al


CHS


Medicago sativa

)

(Junghans

et al


CHS

gene in

Arabi-dopsis


Medicago truncatula


Arabidopsis


et al

1989 Lawton

et al

1983 Sallaud

et al

1997)

IFR


et al

19911994 Tiemann

et al


et al

1999 Karamloo

et al


et al

1995Lers

et al

1998 Petrucco

et al

1996 van Eldik

et al


Table 1 continued

Enzyme name



TC37053TC40736TC40780AW686089




380

R A DIXON

et al


(2002)

3




et al



Medicago truncatula











382 R A DIXON et al


















384 R A DIXON et al
























386 R A DIXON et al





ACKNOWLEDGEMENTS



REFERENCES























































388 R A DIXON et al










































































390 R A DIXON et al


















379



(2002)

3

(5 ) 371ndash390


4CL


et al

2001 Ehlting

et al


4CL


et al


CHS


Gerbera hybrida


et al


CHS


Medicago sativa

)

(Junghans

et al


CHS

gene in

Arabi-dopsis


Medicago truncatula


Arabidopsis


et al

1989 Lawton

et al

1983 Sallaud

et al

1997)

IFR


et al

19911994 Tiemann

et al


et al

1999 Karamloo

et al


et al

1995Lers

et al

1998 Petrucco

et al

1996 van Eldik

et al


Table 1 continued

Enzyme name



TC37053TC40736TC40780AW686089




380

R A DIXON

et al


(2002)

3




et al



Medicago truncatula











382 R A DIXON et al


















384 R A DIXON et al
























386 R A DIXON et al





ACKNOWLEDGEMENTS



REFERENCES























































388 R A DIXON et al










































































390 R A DIXON et al

















380

R A DIXON

et al


(2002)

3




et al



Medicago truncatula











382 R A DIXON et al


















384 R A DIXON et al
























386 R A DIXON et al





ACKNOWLEDGEMENTS



REFERENCES























































388 R A DIXON et al










































































390 R A DIXON et al
























382 R A DIXON et al


















384 R A DIXON et al
























386 R A DIXON et al





ACKNOWLEDGEMENTS



REFERENCES























































388 R A DIXON et al










































































390 R A DIXON et al

















382 R A DIXON et al


















384 R A DIXON et al
























386 R A DIXON et al





ACKNOWLEDGEMENTS



REFERENCES























































388 R A DIXON et al










































































390 R A DIXON et al




























384 R A DIXON et al
























386 R A DIXON et al





ACKNOWLEDGEMENTS



REFERENCES























































388 R A DIXON et al










































































390 R A DIXON et al

















384 R A DIXON et al
























386 R A DIXON et al





ACKNOWLEDGEMENTS



REFERENCES























































388 R A DIXON et al










































































390 R A DIXON et al































386 R A DIXON et al





ACKNOWLEDGEMENTS



REFERENCES























































388 R A DIXON et al










































































390 R A DIXON et al

















386 R A DIXON et al





ACKNOWLEDGEMENTS



REFERENCES























































388 R A DIXON et al










































































390 R A DIXON et al
























































388 R A DIXON et al










































































390 R A DIXON et al

















388 R A DIXON et al










































































390 R A DIXON et al






















































390 R A DIXON et al

















390 R A DIXON et al

















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