MOLECULAR PLANT PATHOLOGY (2002) 3(5), 371390

Blackwell Science, Ltd

Review

The phenylpropanoid pathway and plant defence a genomics perspective

R I C H A R D A . D I X O N * , L A H O U C I N E A C H N I N E , PA R VA T H I KO T A , C H A N G - J U N L I U , M . S. S R I N I VA S A R E D D Y A N D L I A N G J I A N G WA N G
Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401, USA

SUMMARY
The functions of phenylpropanoid compounds in plant defence range from preformed or inducible physical and chemical barriers against infection to signal molecules involved in local and systemic signalling for defence gene induction. Defensive functions are not restricted to a particular class of phenylpropanoid compound, but are found in the simple hydroxycinnamic acids and monolignols through to the more complex avonoids, isoavonoids, and stilbenes. The enzymatic steps involved in the biosynthesis of the major classes of phenylpropanoid compounds are now well established, and many of the corresponding genes have been cloned. Less is understood about the regulatory genes that orchestrate rapid, coordinated induction of phenylpropanoid defences in response to microbial attack. Many of the biosynthetic pathway enzymes are encoded by gene families, but the specic functions of individual family members remain to be determined. 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, barrel medic, and tomato, allow, for the rst time, a full appreciation of the comparative genetic complexity of the phenylpropanoid pathway across species. In addition, gene expression array analysis and metabolic proling approaches make possible comparative parallel analyses of global changes at the genome and metabolome levels, facilitating an understanding of the relationships between changes in specic transcripts and subsequent alterations in metabolism in response to infection.

I N T RO D U C T I O N
Phenylpropanoids are natural products derived from the amino acid L-phenylalanine via deamination by L-phenylalanine ammonia*Correspondence: E-mail: radixon@noble.org

lyase (PAL). The simplest examples, containing only the C 6C3 phenylpropane skeleton, are the hydroxycinnamic acids, such as sinapic acid, and the monolignols, such as coniferyl alcohol. More complex phenylpropanoids are formed by condensation of a phenylpropane unit with a unit derived from acetate via malonyl coenzyme A; these include the avonoids, isoavonoids, and stilbenes. The C6C1 benzoic acids, of which salicylic acid (SA) is an important example in relation to plant disease, have been included in discussions of phenylpropanoid natural products because of their presumed biosynthetic origin via side-chain shortening of hydroxycinnamic acids. However, this may not be the only route for their synthesis in plants. The major biosynthetic routes to the various classes of phenylpropanoid compounds are summarized in Fig. 1, which also shows the primary metabolic pathways that provide precursors for phenylpropanoid biosynthesis. Note the organization of the pathways into a core phenylpropanoid pathway, from phenylalanine to an activated (hydroxy)cinnamic acid derivative via the actions of PAL, cinnamate 4-hydroxylase (C4H), and 4-coumarate:coenzyme A ligase (4CL), and the specic branch pathways for the formation of monolignols/lignin, coumarins, benzoic acids, stilbenes, and avonoids/isoavonoids. All classes of phenylpropanoid compounds are not present in all plant species. Although the hydroxycinnamic acid and avonoid classes are ubiquitous in higher plants, members of these classes with specic substitution patterns may be peculiar to certain genera or species. Other phenylpropanoid classes, such as the isoavonoids and stilbenes, are limited to particular plant families. The isoavonoids are mostly limited to the subfamily Papilionoideae of the Leguminosae. Their structural variation is large, involving the number and complexity of substituents on the 3-phenylchroman framework, different oxidation levels of the heterocycle, and the presence of additional heterocyclic rings. Natural sources from which isoavonoids have been isolated have been reviewed in detail (Dewick, 1994). The stilbenes occur sporadically in widely divergent species, including peanut (Leguminosae), grapevine (Vitaceae), and pine (Pinaceae). Recent knowledge of the three-dimensional structures of stilbene

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synthases has indicated how their genes can evolve independently from closely related chalcone synthase ( CHS ) genes that are found ubiquitously in plants (Schrder, 1997). Natural products active in plant defence can be categorized into three broad groups: phytoalexins, phytoanticipins, and signal molecules. Many phenylpropanoids exhibit broad-spectrum antimicrobial activity and are therefore believed to help the plant ght microbial disease. Such compounds can be classied as preformed phytoanticipins or inducible phytoalexins (VanEtten et al., 1994). The best-characterized phenylpropanoid-derived phytoalexins are the pterocarpans, isoavans, and isoavanones of legumes, including bean, alfalfa, pea, and soybean. The prenylated isoavones of lupin, which are synthesized during seedling development, are a good example of phytoanticipins (Gagnon et al., 1995). Several reviews have summarized the criteria for the classication of compounds as phytoalexins or phytoanticipins, as well as providing extensive details on the distribution and biological activities of phenylpropanoid compounds involved in plant defence (Dixon, 2001; Dixon and Paiva, 1995; Grayer and Harborne, 1994; Hammerschmidt, 1999; Kuc, 1995; Manseld, 2000). It is becoming increasingly clear that phenylpropanoid natural products may play important roles as signal molecules, both in plant development and plant defence. It is also possible that these roles may overlap, such that genetic modication for improved disease resistance might affect developmental processes. The best-known examples of regulatory roles for phenylpropanoids include the activities of dehydrodiconiferyl glucosides (dimeric monolignol derivatives) and avonoid glycosides as potential modulators of cell division (Teutonico et al., 1991; Woo et al., 1999), avonoids as regulators of auxin transport (Jacobs and Rubery, 1988), and SA as a regulator of both local and systemic pathogen-induced defence gene activation, the oxidative burst, and pathogen-induced cell death (Dempsey et al., 1999).

F U N C T I O N S O F P H E N Y L P RO P A N O I D COMPOUNDS IN PLANT DEFENCE

The early studies that led to the formulation of the so-called phytoalexin hypothesis demonstrated that a particular chemical

was induced in response to microbial attack and that it was able to inhibit the growth of the particular pathogen when assayed in vitro. Subsequent studies leading to the denition of many hundreds of phytoalexins dispensed with the use of a pathogen as inducing agent when it was realized that more convenient procedures, such as exposure to copper ion or elicitors from microbial cell walls, could induce the synthesis of natural products with antimicrobial activity. It is only recently that more rigorous genetic criteria have been used to determine whether specic natural products do indeed play a role in disease resistance in vivo. Such studies fall into three classes: genetic modication of the pathogen to disrupt the mechanisms involved in phytoalexin tolerance, genetic modication of the host to increase or decrease levels of a specic natural product, or genetic introduction of a novel antimicrobial compound into the plant. Because of the relatively advanced knowledge of the molecular genetics of the phenylpropanoid pathway, many of the above studies have involved phenylpropanoid compounds. Plant pathogenic fungi have evolved various mechanisms by which to either avoid or destroy induced chemical barriers to infection. The most common mechanism of detoxication of host phenylpropanoid derivatives involves oxidative metabolism, usually utilizing cytochrome P450 enzymes that, in several plant pathogenic fungi, are encoded by genes on supernumerary or dispensable chromosomes (Covert et al., 1996; Wasmann and VanEtten, 1996). If the target substrate is important for resistance, disruption of such genes will result in reduced virulence. Thus, disruption of the MAK1 gene in the fungal pathogen Nectria haematococca , leading to an inability to detoxify the isoavonoid phytoalexin maackiain, led to reduced virulence of the fungus on chickpea (Enkerli et al., 1998). Introduction of the foreign stilbene phytoalexin resveratrol into tobacco or alfalfa by constitutive expression of a grapevine stilbene synthase gene resulted in greatly reduced symptoms following infection of tobacco by the grey mould Botrytis cinerea (Hain et al., 1993) or of alfalfa by the leaf spot pathogen Phoma medicaginis (Hipskind and Paiva, 2000). Constitutive overexpression of isoavone O-methyltransferase (IOMT) in transgenic 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 quinate/shikimate) 3-hydroxylase; C4H, cinnamate 4hydroxylase; ChA, chorismic acid; i-ChA, isochorismic acid; 4-CL, 4-coumarate:CoA ligase; CHR, chalcone reductase; CHS, chalcone synthase; COMT, caffeic acid Omethyltransferase; Csh, 4-coumaroyl shikimate; Daid, daidzein; FerA, ferulic acid; FerCoA, feruloyl CoA; Gen, genistein; 5-HCald, 5-hydroxyconiferaldehyde; HQT, hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase; ICS, isochorismate synthase; IFR, isoavone reductase; IFS, isoavone synthase; Il, isoliquiritigenin; IOMT, isoavone 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-simplied. 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-isoavonoid biosynthesis in the Leguminosae. Reactions not designated with an enzyme name may be catalysed by more than one enzyme.

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the isoavonoid 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 genetic approaches indicate that phenylpropanoid compounds can indeed be effective in contributing to resistance in vivo, but one individual compound or class of compound may not necessarily be the sole factor imparting disease resistance, consistent with the multicomponent nature of plant defence responses. Clearly, the diversity of plant natural products and hostpathogen combinations means that it is impossible to make any general conclusions that might hold for the vast majority of systems not yet analysed, and it is this factor above all that has restricted interest in natural product pathways as targets for engineered resistance. A large body of physiological and genetic evidence supports a role for SA as a critical regulator of a number of plant defence responses, although it now seems likely that the phenylpropanoid pathway is not the only, or even the most important, route to the biosynthesis of SA (see below). Several primary papers and recent reviews have listed the evidence implicating SA as a signal for the transcriptional regulation of pathogenesis-related protein genes, as a gain-control agonist for the oxidative burst, and as a signal molecule for pathogen-induced host cell death (Dempsey et al., 1999; Kauss and Jeblick, 1995; Klessig and Malamy, 1994; Malamy et al., 1996; Mur et al., 1997; Murphy et al., 1999; Pierpoint, 1997; Rao et al., 1997; Rate et al., 1999; Shirasu et al., 1997). SA is implicated in the above responses both locally and systemically, although it appears unlikely that SA is itself the mobile signal in systemic acquired resistance (Vernooij et al., 1994). Plants with drastically reduced SA levels resulting from expression of a bacterial salicylate hydroxylase gene have severely compromised disease resistance (Delaney et al., 1994), whereas the over-production of SA, either via expression of bacterial isochorismate synthase and isochorismate pyruvate lyase transgenes (Verberne et al., 2000) or through general up-regulation of the phenylpropanoid pathway by over-expression of PAL (Felton et al., 1999), is associated with increased microbial resistance.

R E G U L A T O R Y A RC H I T E C T U RE O F P H E N Y L P RO P A N O I D B I O S Y N T H E S I S
Because of the extensive information available on its structural and regulatory genes, the phenylpropanoid pathway serves as an excellent system for developing an understanding of how to genetically manipulate complex natural product pathways in plants. However, we still lack important information concerning the points of ux control at and within the various branch pathways depicted in Fig. 1 and the potential cross-talk between pathways. Also important is the extent to which sets of reactions are organized in metabolic channels or metabolons, resulting in the sequestration of intermediates from diffusible cytosolic pools

(Srere, 1987). All of these factors may strongly impact the outcome of attempts to increase or decrease the level of a particular compound by transgenic approaches. Addressing these questions will require interdisciplinary approaches involving molecular, cellular, and structural biology. Our understanding of ux control and cross-talk in phenylpropanoid biosynthesis has come primarily from studies in which specic enzymes in the pathway have been over-expressed or down-regulated in transgenic plants. Such an approach has shown that the entry point enzyme PAL is directly rate limiting for the production of chlorogenic acid (CGA, caffeoyl quinic acid) in tobacco leaves, but that factors in addition to PAL control ux into avonoids and lignin (Howles et al., 1996). CGA has been implicated in resistance to both microbes and insects (Yao et al., 1995), although PAL over-expressing plants with elevated CGA appear to show impaired resistance to insect herbivory as a result of cross-talk between the salicylate and jasmonate signal pathways (Felton et al., 1999). In potato tubers, the creation of an articial sink for tryptophan through the transgenic expression of a tryptophan decarboxylase gene resulted in lowered phenylalanine pools and reduced levels of wound-induced CGA and lignin, with a resulting increase in susceptibility to Phytophthora infestans (Yao et al., 1995). CGA levels are also reduced in tobacco by downregulation of C4H, the second enzyme in the phenylpropanoid pathway, and this is accompanied by a feedback inhibition of PAL activity, possibly as a result of feedback inhibition of PAL expression by cinnamate or some derivative thereof (Blount et al., 2000). In contrast, over-expression of C4H did not consistently result in increased levels of CGA (Blount et al., 2000), conrming that PAL rather than C4H is the ux control point into the phenylpropanoid pathway in tobacco leaves. Chalcone isomerase (CHI) catalyses a near-diffusion-limited reaction that can also occur spontaneously at cellular pH, and is not therefore generally viewed as a potential rate-limiting enzyme for avonoid biosynthesis. However, over-expression of CHI in tomato fruit peel leads to an 80-fold increase in the levels of avonols (Muir et al., 2001), and threefold increases in avonol levels can be obtained by the expression of alfalfa CHI in Arabidopsis (C.J. Liu and R.A. Dixon, unpublished results). CHI would therefore appear to be a component of ux control into the avonoid branch of phenylpropanoid biosynthesis. The phenylpropanoid pathway presents some of the bestcharacterized examples of metabolic channelling in plant metabolism. Metabolic channelling involves the physical organization of successive enzymes in a metabolic pathway into complexes through which pathway intermediates are channelled without diffusion into the bulk of the cytosol (Srere, 1987). Such complexes are loose, however, and many of the enzymes involved may be operationally soluble. The complexes allow for efcient control of metabolic ux, and protect unstable intermediates

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from non-productive breakdown or access to enzymes from potentially competing pathways. Such complexes may involve direct physical interactions between the various enzymes, as recently demonstrated for enzymes of avonoid biosynthesis in Arabidopsis (Winkel-Shirley, 1999), or may be associated with the colocalization of enzymes on membranes or other surfaces (Liu and Dixon, 2001). In both cases, channelling can be demonstrated by double labelling or isotope dilution experiments in which exogenously applied intermediates are less efcient precursors of downstream products than their upstream substrates. Such criteria have conrmed channelling between PAL and C4H at the entry point into the phenylpropanoid pathway (Czichi and Kindl, 1975; Hrazdina and Jensen, 1992; Hrazdina and Wagner, 1985; Rasmussen and Dixon, 1999), and between isoavone synthase (IFS) and IOMT at the entry point into the isoavonoid phytoalexin pathway (Liu and Dixon, 2001). In both cases, the involvement of a membrane-associated cytochrome P450 enzyme (C4H or IFS), that might act to anchor the complex to the endoplasmic reticulum, should be noted. Metabolic channelling can impact plant defence responses in two ways. First, it is possible that intermediates destined to become a particular metabolic end product, such as a phenylpropanoid-derived phytoalexin, may be channelled in such a way that they utilize different pools of metabolic enzymes than other products that may share some of the same biosynthetic steps. This could be achieved by utilizing different isoenzymic forms of the various pathway enzymes in different complexes. Such a model would predict that the multiple genes for many of the pathway enzymes described below might have both distinct and overlapping functions, a hypothesis that remains to be tested. If this were true, measurement of changes in gene transcripts, using probes that do not distinguish between all possible forms of the encoded enzyme, might lead to results that do not correlate with defence metabolism, as observed for avonoid/ isoavonoid defences in bacterially infected alfalfa (Sallaud et al., 1997). Second, although metabolic channelling might improve the efciency of induced defences, it also presents a potential barrier to efcient metabolic engineering, in that channelled intermediates may not be accessible to the enzyme products of transgenes introduced in order to divert a pathway into the formation of a novel bioactive compound.

C O M P A RA T I V E G E N O M I C S O F P H E N Y L P RO P A N O I D B I O S Y N T H E S I S
Our understanding of the complexity of gene families in plants has increased rapidly in the past several years, primarily because of the development of rapid expressed sequence tag (EST) and genomic sequencing technologies. For those species for which extensive sequence information is available, it is now possible to retrieve the sequences of the different members of gene families

by text and BLAST search in various Plant Gene Index databases, such as those available at the TIGR website (http://www.tigr.org/ tdb/tgi.shtml) (Quackenbush et al., 2000) or the Medicago gene index at the National Center for Genome Resources (https:// xgi.ncgr.org/mgi/) (Bell et al., 2001), and to compute gene expression patterns by counting the frequency of ESTs in various cDNA libraries. We have begun a detailed bioinformatic analysis of phenylpropanoid pathway gene complexity and expression (R.A. Dixon and L. Wang, unpublished results). Table 1 summarizes the apparent numbers of gene family members for the various genes involved in the core phenylpropanoid pathway and the lignin, avonoid, and isoavonoid branches in four dicot species [barrel medic (Medicago truncatula) and soybean from the Leguminosae, tomato from the Solanaceae, and Arabidopsis thaliana from the Brassicaceae] and two monocots (rice and maize). The sequence identiers refer to tentative consensus sequences (TCs) that represent EST contigs derived from clustering of the EST sequences. Singletons (EST sequences that only occur once and do not show overlap to other sequences) are also included in the analysis. Every sequence annotated in the database as representing a specic gene product was counted as such. Gene annotation is based on sequence similarity, not function, and this can lead to an overestimate of the number of genes with the specic function as annotated (see below). The seven TCs for PAL from Medicago truncatula most likely indicate the existence of seven different PAL or PAL-like gene transcripts from the libraries which have been sequenced to date, with the caveat that this may be an overestimate as some TCs may later be shown to cluster together. However, with over 140 000 ESTs now sequenced in Medicago truncatula, the data in Table 1 probably represent a fairly accurate picture of gene family complexity. In the case of Arabidopsis, the numbers are computed from the whole genome sequence and can therefore be taken as validated. Several striking conclusions can be made from the data in Table 1. First, in most of the species, many of the genes exist as quite large gene families. In the cases of 4CL, cinnamyl CoA reductase (CCR), cinnamyl alcohol dehydrogenase (CAD ), laccase and isoavone reductase (IFR), these may have 10 or more members. Second, the levels of complexity differ between the different species e.g. a single 4CL gene in rice, 1016 in four of the other species. Third, as would be predicted from metabolic analysis, the key genes of isoavonoid biosynthesis are absent from the four non-legume species. Finally, in spite of extensive EST sequencing, some genes that must exist have yet to be represented in the EST databases (e.g. C4H in rice and maize). The EST counting approach annotates genes based solely on sequence similarity. This similarity is often less than would result in physical detection on mid- to high-stringency DNA gel blot analysis, and should not be taken to imply proven function. Thus, some of the genes annotated as encoding a particular enzyme

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Table 1 Gene family members involved in the core phenylpropanoid pathway and the lignin, avonoid, and isoavonoid branches Tentative consensuses (TCs) or singletons in TIGR databases Enzyme name

M. truncatula

Soybean TC61607 TC73437 TC73439

Tomato TC84666 TC84677 TC93787 TC95472 AW035278 BE462826 AW219744 BG735223 TC93282

Arabidopsis
TC103728 TC115559 TC115700 TC117801 AA713237

Rice TC48464 TC52373 TC52374 TC52428 TC52429 TC53734

Maize TC70927 TC70929 TC70930 TC70931 TC71742 TC80439

Phenylalanine ammonia-lyase (PAL) TC28440 TC28441 TC35080 TC35727 TC35728 TC36057 TC37941 Cinnamate 4-hydroxylase (C4H) TC35724 TC35725 4-Coumarate:coenzyme A ligase (4CL) TC29244 TC29487 TC31279 TC31821 TC32992 TC36008 TC37181 TC37802 TC38835 TC40006 TC40554 TC42827 TC42855

TC73352 TC73353 TC62684 TC63017 TC63018 TC64113 TC66256 TC69869 TC70573 TC71143 TC72975 TC73698 TC73700 TC74240 TC74241 TC74245 TC75489 TC75671

TC115667

TC85790 TC87087 TC87740 TC89636 TC89693 TC90983 TC91518 TC92146 TC93209 TC93567 TC93594 TC94331 AW031547 AW616655 BE449653 AW039905 AW625022 TC90236 TC94887

TC103592 TC104680 TC105518 TC109121 TC109883 TC110917 TC111771 TC116650 TC120152 TC124103 N96648

TC55743

TC69073 TC71566 TC73077 TC78593 TC78929

Caffeic acid O-methyl- transferase (COMT) TC31891 TC31966 TC32648 TC34905 TC39641

TC62755 TC68824

TC109504 TC109505 TC112158 TC117372 TC118345 TC121865 TC121866 NP236939 TC108307 TC117895 TC121427 TC122589 AA394533

TC48357 TC48358 TC49029

TC77309 TC77890

Caffeoyl coenzyme A O-methyl-transferase (CCOMT) TC30254 TC30408 TC32139 TC32560

TC62082 TC62083 TC65887 TC68488 TC73518 TC73519 TC75138 TC64463

TC85828 TC89798 TC93816 TC93824 TC94433

TC48164 TC49289 NP001843

TC71157 TC71158

Ferulate 5-hydroxylase (F5H) TC28721 TC38615

TC86670 TC96360 AI895344 AW616986

TC109653 TC120306

TC54434

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Table 1 continued Tentative consensuses (TCs) or singletons in TIGR databases Enzyme name

M. truncatula

Soybean TC68230 TC70793 TC70911 TC74702 TC77533

Tomato TC89868 TC91754 TC92006 TC96358

Arabidopsis
TC103742 TC105238 TC107236 TC108680 TC115959 TC115960 TC117763 TC118229 TC121455 TC125532 TC103635 TC103785 TC105591 TC108291 TC109690 TC109697 TC111929 TC115628 TC116766 TC116982 TC119528 TC120178 TC122451 TC123184 TC126966 TC126969 TC109933 TC110163 TC111356 TC111531 TC111758 TC113955 TC115552 TC120290 TC120415 TC120743 TC122516 TC123838 TC126250 TC126968 TC106324 TC115490 TC116475 TC118556

Rice TC48219 TC48221 TC49671 TC50244 TC51067 TC52858

Maize TC71394 TC72304 TC78891 TC79954 TC80830 NP003454

Cinnamyl coenzyme A reductase (CCR) TC32087 TC32980 TC35837 TC36551 TC39655

Cinnamyl alcohol dehydrogenase (CAD) TC29412 TC32920 TC32921 TC35882 TC39363 TC41505 AW696839 AW559294

TC66049 TC66167 TC66880 TC68104 TC73412 TC73414 TC73524 TC74780 TC76785

TC85446 TC86190 TC91305 TC91547 TC94143 TC94740 TC95402 AW037980

TC52574 TC52613 TC53411

TC71268

Laccase TC31437 TC34979 TC35170 TC36059 TC37979 TC40521 TC40531 TC40548 TC40932 TC42541 AW691027 AW691876 TC64439 TC66286 TC69538 TC69683 TC71504 TC75229 TC75579 TC96435 TC97020 AI896093 AW032099 AW649943 AI782326 AW455342 BE451044 AW625159 AW625489 AW626092 AW036325 TC49583

Chalcone synthase (CHS) TC35574 TC29796 TC31846 TC31847 TC31848 TC31850 TC31852 TC31854 TC31856 TC61916 TC67543 TC67544 TC68628 TC73293 TC75473 TC86565 TC87127 TC90271 TC48400 TC54032 NP252089 AU032872 AU032888 AU032899 AU032912 TC71902

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Table 1 continued Tentative consensuses (TCs) or singletons in TIGR databases Enzyme name

M. truncatula
TC33667 TC35573 TC35575 TC35576 TC35577 TC35803 TC42671 AW684295

Soybean

Tomato

Arabidopsis

Rice

Maize

Chalcone reductase (CHR) TC29099 TC29100 TC33979 TC39402 TC39403 TC39404 AW774745 Chalcone isomerase (CHI) TC35835 TC39443 TC39717 TC40174 TC62667 TC63639 TC69262 TC74465 TC74468 TC89245 TC94706 TC95516 AW928395 TC110376 TC112674 TC113988 TC115647 NP281215 H36669 TC115605 TC121953 T44308 TC48677 TC72293 TC78271 TC62685 TC74221 TC90973 TC54602

Flavanone 3--hydroxylase (F3H) TC36151 TC37458 TC38104

TC67927 TC74581

TC95171 TC86916 TC87110 TC91452 TC94340 TC97192 AW933742

TC50019 TC55099

TC78946

Flavonoid 3-hydroxylase (F3H) TC31717 Flavonoid 3,5-hydroxylase (F35H) TC33338 TC36887 TC42130 Dihydroavonol reductase (DFR) TC28514 TC37214 AW981263

TC121490 TC112562 TC115032 TC121970 TC122245 TC105710 TC112835 TC115766 TC119438 NP240316 TC50901 TC50971 TC53190 TC69820 TC75299 TC77854 TC78297

TC76586

TC87512 TC88431 AW034237

TC66100 TC67453 TC67457 TC68957 TC69984 TC75004 TC76010 TC69143

TC88191 TC94998 NP000412

Anthocyanidin synthase (ANS) TC104059 TC56535 Isoavone synthase (IFS) TC32250 TC36522 TC36523 Isoavone O-methyl-transferase (IOMT) TC29273 TC61958 TC61959

TC69577

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Table 1 continued Tentative consensuses (TCs) or singletons in TIGR databases Enzyme name

M. truncatula

Soybean

Tomato

Arabidopsis

Rice

Maize

TC37053 TC40736 TC40780 AW686089 Isoavone 2-hydroxylase (I2H) TC33268 TC39922 Isoavone reductase (IFR) TC31930 TC28549 TC31929 TC32401 TC33160 TC36748 TC39922 TC36918 TC39622 AW686812 AW687254 AW688509

TC94137

TC62478 TC63010 TC69565 TC69853 TC69984 TC73558 TC73885 TC73886 TC74059 TC74060 TC75734

TC87096 TC95230 TC96920 BE462550

TC115941 TC117817 TC118151

TC48979 TC51843 TC53547 TC54779 NP273546 NP274174

TC77262 TC80585 NP003471

may in fact encode related enzymes with different functions. For example, the many 4CL genes in the four dicot species listed in Table 1 most likely encode either true isoforms of 4CL or other enzymes that utilize a similar reaction mechanism involving the activation of an acidic function by the formation of an acyl adenylate (Cukovic et al., 2001; Ehlting et al., 2001). In several species, distinct isoforms of 4CL have been characterized at the enzymatic level (Knobloch and Hahlbrock, 1975; Lee and Douglas, 1996; Vincent and Nicholson, 1987), although their biochemical properties do not necessarily suggest differential functions in lignication or avonoid biosynthesis. The activation of 4CL genes is, however, often associated with induced defence (Uhlmann and Ebel, 1993). In wheat, wounding or elicitation specically leads to the induction of a CAD isoform with substrate preference for sinapyl alcohol, consistent with the syringyl-rich lignin that accumulates under these conditions (Mitchell et al., 1999). The situation with CHS genes is particularly interesting. CHS is the prototypical enzyme representative of a class of homodimeric polyketide synthases that catalyse condensation of a starter coenzyme A ester (4-coumaroyl CoA in the case of CHS and stilbene synthase) with one to three molecules of malonyl CoA. It is now known that some genes were at rst incorrectly annotated as encoding CHS, for example the pyrone synthase of Gerbera hybrida that uses acetyl CoA as the starter molecule for malonyl condensation (Eckerman et al., 1998). Classical molecular hybridization analysis has demonstrated the presence of more than eight CHS genes in tetraploid alfalfa ( Medicago sativa)

(Junghans et al., 1993), but only a single true CHS gene in Arabidopsis (Feinbaum and Ausubel, 1992), in contrast to the 16 TCs annotated as CHS in diploid Medicago truncatula and the four TCs annotated as CHS in Arabidopsis. In the case of these dimeric polyketide synthases, sequence similarities are in some cases sufciently close that genes encoding enzymes with different functions may cross-hybridize on gel blot analysis. This is an important point because RNA gel blot analysis of CHS transcripts has 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 rst cloned from legumes (Paiva et al., 1991, 1994; Tiemann et al., 1991), and were selected for study in view of the involvement of IFR specically in the branch of isoavonoid metabolism leading to isoavan and pterocarpan phytoalexins. However, many species that do not accumulate isoavonoids contain genes with high sequence identity to legume IFRs. It now appears that IFR is just one member of a large family of NADPHdependent oxidoreductases that includes the phenylcoumaran benzylic ether and pinoresinol-lariciresinol reductases of lignan biosynthesis (Gang et al., 1999; Karamloo et al., 2001) and several other genes that are developmentally regulated or induced during redox shifts and oxidative stress (Babiychuk et al., 1995; Lers et al., 1998; Petrucco et al., 1996; van Eldik et al., 1997). Thus, the non-legume species in Table 1 all express genes falling into TCs annotated as encoding IFR-like proteins, but appear to express no other genes of isoavonoid biosynthesis and have not been shown to accumulate isoavonoid natural products.

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What is the biological signicance of the multigene families encoding many of the genes of phenylpropanoid biosynthesis? An obvious hypothesis is that there is a need to independently regulate the production of different phenylpropanoid products in the same or different cells, and that different gene family members are somehow involved in the production of different classes of compounds. Legumes, in particular, use phenylpropanoid compounds as both phytoalexins and signal molecules for the attraction of symbiotic microbes, and the independent regulation of such pathways would clearly be necessary. An alternative hypothesis is one of gene dosage. In the legumes, which use isoavonoids as phytoalexins, there may be a need for rapid and massive accumulation of these compounds immediately following infection, and amplication of genes encoding enzymes at key ux control

points (e.g. PAL and CHS) may have allowed plants to achieve this. Whatever the reason, denitive information as to why many of the gene families in Table 1 are so complex will require specic down-regulation of the individual gene forms. This has been problematical in the past owing to the often very high DNA sequence conservation between family members, such that the use of antisense or gene silencing with large sequence fragments would result in the down-regulation of several or maybe all of the genes. Recent advances in plant gene silencing technology based on an understanding of RNA-interference (RNAi) (Wesley et al., 2001) should now facilitate the molecular dissection of the functions of individual 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.

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plant PAL sequences in the NCBI GENBANK. A group of ve sequences clusters with other functionally characterized legume PALs, TC36057 is more closely related to Arabidopsis PALs 1 and 2 and two bean PAL genes, and TC35080 is more distant and related to Arabidopsis PAL 3. The in silico expression pattern of the seven putative PAL or PAL-like genes in different tissues is shown in Fig. 2B. It can be seen that three TCs correspond to genes that are expressed in stems and are therefore candidates for involvement in stem lignication, whereas the four others are apparently not expressed in stems. Three TCs correspond to genes that are very strongly expressed in elicitor-treated cell suspension cultures, conditions that result in the accumulation of isoavonoid phytoalexins. There is no relation between the dendrogram shown in Fig. 2A and the expression pattern in Fig. 2B; for example, TC28440 and TC35727 are the most strongly expressed in stems, but do not cluster together based on sequence. Figure 2B also shows the effects of infection, insect herbivory,

symbiotic association, and abiotic factors on EST numbers computed from cDNA libraries of control and challenged tissues. For three of the TCs, the highest expression level was in elicited cell cultures. The PAL encoded by TC35727 is expressed in healthy leaves, but its expression is reduced following infection, whereas TC28440 appears to be down-regulated by insect herbivory. TC37941 appears to be expressed only in roots following nitrogen starvation or nodulation. This complex pattern of PAL genes and their expression in legumes contrasts with the relatively simple organization of PAL in tobacco (two families, each with two very closely related genes; Nagai et al., 1994; Pellegrini et al., 1994), raspberry (two genes with 88% identity but in different clusters within the plant PAL gene phylogeny; Kumar and Ellis, 2001), and some of the other species shown in Table 1. Figure 3A shows a dendrogram of the Medicago truncatula caffeoyl coenzyme A (CCOMT) gene sequences. CCOMT was originally proposed to be specically involved in the formation of

Fig. 3 Sequence analysis and expression patterns of Medicago truncatula caffeoyl coenzyme A (CCOMT ) genes. (A) Dendrogram showing the ve 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.

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Fig. 4 Sequence analysis and expression patterns of Medicago truncatula isoavone 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.

cell wall esteried ferulic acid as a pathogen defence response (Pakusch et al., 1989), although the enzyme is now believed to play a key role in the biosynthesis of lignin during vascular development (Ye et al., 1994). There are ve CCOMT TCs in Medicago truncatula, four of which are more closely related to the Arabidopsis CCOMT than to alfalfa CCOMT. Three of the ve TCs are expressed in stems and therefore potentially involved in lignication in that organ (Fig. 3B). One TC (TC32560) is strongly induced in elicited cell cultures (but not roots or stems). Of the three CCOMT TCs that are modulated by infection, herbivory, or elicitation, the patterns are quite distinct. Thus, it is clear that plant defence makes use of the selective expression of particular members

of the gene families encoding phenylpropanoid biosynthetic enzymes, a nding inconsistent with the simple gene dosage model proposed above. Unlike PAL and CCOMT, IFS is a branch point enzyme specic for the formation of a single class of natural product, the isoavonoids. It might therefore be expected that the genomic complexity and expression patterns of IFS genes would be simpler than those of PAL, CCOMT, or CHS genes. IFS is a cytochrome P450 of the CYP93C class (Jung et al., 2000; Steele et al., 1999). Figure 4A shows a dendrogram of the three CYP93 genes revealed as TCs in the Medicago truncatula gene index. They are closely related to CYP93s with IFS activity characterized from the

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other legumes Lotus japonicus, licorice (Glycyrrhiza), and cowpea (Vigna). The tissue-specic expression pattern of the three putative IFS genes from Medicago truncatula (Fig. 4B) shows very clearly that these genes are only expressed in the below-ground organs of the plant. The lack of expression in infected leaf material (Fig. 4B) is perhaps surprising, but may reect the pathogen used (Colletotrichum trifolii ) and the time of harvest of the material for library construction; IFS genes are, for example, induced in alfalfa leaves infected with the fungal pathogen Phoma medicaginis (He and Dixon, 2000). All three TCs are expressed in elicited cell cultures that have been validated as producing isoavonoid phytoalexins. Interestingly, TC36522, the closest orthologue of the functionally characterized IFS genes from soybean, is not the most strongly expressed in any of the tissues analysed. Rather, TC32250 has the highest expression level, and this gene is specically and highly expressed in roots in response to phosphate starvation. It is not known whether this has any physiological signicance for processes associated with phosphate nutrition, such as the establishment of mycorrhizal interactions. Nevertheless, this observation points to the dramatic impact of nutritional/ physiological status on the expression of genes that can mistakenly be thought of as responding primarily to infection.

F U N C T I O N A L G E N O M I C S A P P RO A C H E S T O T H E I N V O L V E M E N T O F P H E N Y L P RO P A N O I D BIOSYNTHESIS IN PLANT DEFENCE

The evidence for the induction of specic phenylpropanoid pathway gene family members during induced defence argues for more gene-selective approaches to expression proling than the often non-discriminatory RNA gel blot analyses previously applied. The increasingly popular cDNA micro- or macro-array techniques, while undoubtedly powerful, lack selectivity for closely related gene sequences. Oligonucleotide-based DNA chip technology makes it possible to prole, in parallel, large numbers of transcripts with a selectivity that allows for independent measurement of different gene family members. Oligonucleotide chips containing the various Medicago truncatula phenylpropanoid gene family members summarized in Table 1 have been produced as part of the Noble Foundations Medicago truncatula functional genomics program (http://www.noble.org/medicago/index.htm). A limited number of studies on gene expression proling in plantmicrobe interactions have been reported to date (Reymond, 2001). It is almost certain that application of in depth expression proling techniques to plantmicrobe interactions will reveal more widespread alterations in host gene expression than originally foreseen. In relation to systems in which phenylpropanoid biosynthesis is induced, there is already strong evidence for the gene activation of enzymes of primary metabolism, such as the pentose phosphate and shikimate pathways (Fahrendorf et al., 1995; Somssich and Hahlbrock, 1998), which feed into

the secondary metabolic pathways. Indeed, elicitor treatment of parsley cell cultures leading to the accumulation of phenylpropanoidderived furanocoumarin phytoalexins is accompanied by a very extensive re-programming of gene expression (Somssich and Hahlbrock, 1998). It will be interesting, by coupling gene expression array analysis with proteomic and metabolomic approaches, to determine the extent to which the changes in transcription are mirrored by changes in protein translation and consequently linked metabolic alterations. Until recently, studies on induced phenylpropanoid biosynthesis during plant defence monitored changes in either single compounds with known antifungal activity or particular classes of compounds, such as isoavonoids or stilbenes, generally utilizing high performance liquid chromatography (HPLC) with UV detection. In some cases, such approaches might indeed identify the major compound or compounds correlated with disease resistance, as seen, for example, in the case of soluble 4-coumaroylhydroxyagmatine that accumulates during resistance of barley determined by the Mlo resistance gene (von Rpenack et al., 1998). However, minor components that act synergistically with more major components might be missed, and targeted proling will often provide no information on changes in precursor pools that may give important hints as to sites of ux control. Recently described technologies for broader metabolic proling using mass spectrometric detection (Fiehn et al., 2000; Roessner et al., 2000; Trethewey et al., 1999) provide a means to monitor many hundreds of metabolites in a single experiment, and applications of these techniques will allow a better understanding of the metabolic consequences of activation of particular gene family members in different tissues and in response to different biotic stresses. In particular, as transgenic plants with altered phenylpropanoid metabolism for improved disease resistance, paper pulping, or production of speciality chemicals enter commercialization, in depth metabolic proling for the demonstration of substantial equivalence will become an important requirement of the federally mandated regulatory process.

N O V E L G E N E S O F P H E N Y L P RO P A N O I D BIOSYNTHESIS
The basic core pathways shown in Fig. 1 have been known for many years. The enzymes and their genes were discovered by a combination of time-consuming biochemical and genetic approaches, using tractable model systems. A major challenge for the future will be to discover the many genes involved specically in the biosynthesis of useful bioactive phenylpropanoids limited only to certain species, such as the pterocarpan 6a-hydroxylase and avonoid 6-hydroxylase cytochrome P450 enzymes recently characterized from soybean (Latunde Dada et al., 2001; Schopfer et al., 1998). This discovery process will doubtless be accelerated by the application of bioinformatics tools to the ever-increasing

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amount of gene sequence information becoming available for many plant species. Critical to the ability to make better predictions of gene function from sequence information will be the parallel development of protein structure databases (Norin and Sundstrom, 2002). Such information on the relation between primary sequence and enzyme function will allow, by comparison of protein structures rather than primary sequence per se, improved functional annotation of gene sequences. This is of particular importance in the case of natural product pathways, such as the phenylpropanoid pathway, by which different species produce very different compounds but using conserved classes of enzymes. An example of the value of this approach is the structure-based prediction modelling of the Gerbera hybrida pyrone synthase, which, although performed after the true function of the enzyme had been determined (Eckerman et al., 1998), demonstrated by structural criteria that this enzyme could not possibly encode a CHS as previously annotated (Jez et al., 2000b). Detailed structural information is now appearing for enzymes of phenylpropanoid biosynthesis (Ferrer et al., 1999; Jez et al., 2000a; Zubieta et al., 2001, 2002), and will facilitate the prediction of potential activities for enzymes that fall within wellstudied classes, such as polyketide synthase, O-methyltransferase (Schroeder et al., 2002), or glucosyl transferase. A good example of both the unreliability of sequence-onlybased functional annotation and the evolutionary exibility of plant phenylpropanoid biosynthesis is the discovery that an acyltransferase involved in the biosynthesis of the major leaf hydroxycinnamate ester, sinapoyl malate, is encoded in Arabidopsis by a gene with high sequence identity to serine carboxypeptidases (Lehfeldt et al., 2000), of which there are numerous annotated yet not functionally characterized family members in the Arabidopsis genome. The data in Figs 2 4 clearly illustrate the value of EST-based approaches to studies on defence gene expression. Such studies can reveal potential new functions for gene products in wellcharacterized pathways based on unexpected expression patterns of individual gene family members that can then be tested by reverse genetics approaches coupled to metabolic proling and defence response phenotyping. This type of approach will also be helpful for resolving the functions of genes whose roles in phenylpropanoid-based defences are currently less clear. One example of such a gene is the pea defence response gene DRR206. This gene is strongly induced in pea in response to both fungal and bacterial infection (Riggleman et al., 1985) and, when expressed in transgenic Brassica napus, confers resistance to both blackleg stem canker Leptosphaeria maculans and Rhizoctonia solani and delayed disease development with Sclerotinia sclerotiorum (Wang and Fristensky, 2001; Wang et al., 1999). DRR206 exhibits about 60% sequence identity to the dirigent proteins that are involved in directing stereoselective phenolic radical coupling in the biosynthesis of lignans from two molecules

of coniferyl alcohol (Davin et al., 1997). It is interesting to note that, although lignans have antifungal, antibacterial, and anti-insect activities (Davin and Lewis, 1992), they have attracted less attention than other classes of phenylpropanoids in relation to possible roles in defence. The techniques now exist to determine the metabolic phenotypes of transgenic plants protected by the expression of DRR206, and it will be interesting to discover whether DRR206 is indeed a true dirigent protein involved in the formation of an antimicrobial lignan.

THE BIOSYNTHESIS OF SALICYLIC ACID

The biosynthesis of SA continues to remain something of a paradox. It now appears that there are several routes to benzoic acid derivatives in plants (El-Mawla and Beerhues, 2002; El-Mawla et al., 2001; Verberne et al., 1999), and that different routes may be used in different species, or even in the same species depending on the response in question. Until recently, SA formation in plants was believed to occur via a branch of phenylpropanoid metabolism, involving side-chain shortening of cinnamic acid by either an oxidative route analogous to the -oxidation of fatty acids (Lscher and Heide, 1994), or a non-oxidative route via the corresponding chain-shortened aldehyde, a reaction previously shown to occur during the formation of benzoic acid derivatives in several species (Schnitzler et al., 1992; Yazaki et al., 1991). Recent labelling studies have provided good evidence for the operation of the former pathway for the biosynthesis of SA in cucumber and Nicotiana attenuata, although the plants used in these feeding experiments had not been induced for local or systemic disease resistance responses (Jarvis et al., 2000). A recent study in tobacco led to the conclusion that the free benzoic acid found in leaves and cell cultures was unlikely to be involved in SA biosynthesis, but that benzoyl glucose was likely to be an intermediate (Chong et al., 2001). Genes encoding enzymes for neither of the chain-shortening pathways have yet been unequivocally identied in plants. Irrespective of the chainshortening pathway, the nal step in SA biosynthesis from phenylpropanoid precursors appears to involve the 2-hydroxylation of benzoic acid. A benzoate 2-hydroxylase was puried from tobacco and suggested to be a high molecular weight, soluble cytochrome P450 similar to bacterial P450s (Len et al., 1995). However, the gene encoding this enzyme has yet to be cloned, and there are therefore no gene probes currently available for studying SA biosynthesis from L-phenylalanine in plants. It has recently been conrmed that plants can also synthesize SA from the shikimate pathway intermediate chorismate via the enzyme isochorismate synthase (ICS) (Wildermuth et al., 2001) (Fig. 1), and the same pathway operates for the biosynthesis of 2,3-dihydroxybenzoic acid in Catharanthus roseus (Muljono et al., 2002). Arabidopsis contains two ICS genes, one of which encodes a plastid-targeted enzyme that is induced during fungal

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and bacterial infection (Wildermuth et al., 2001). The enhanced disease susceptibility sid22 mutant of Arabidopsis harbours a signicant deletion/rearrangement in the ICS1 gene, does not accumulate ICS1 transcripts, and produces signicantly reduced levels of SA in response to infection. However, ICS mutants still produce the low constitutive levels of SA found in wild-type plants, and it has been suggested that this SA, and perhaps the SA associated with pathogen-induced cell death, might still be formed via PAL (Wildermuth et al., 2001). It will be interesting to study ICS gene expression in species such as tobacco, in which the local and systemic production of SA associated with resistance responses has been previously ascribed to the phenylpropanoid pathway (Lee et al ., 1995; Pallas et al ., 1996; Verberne et al ., 1999; Yalpani et al., 1993). Because of the close association of the shikimate and phenylpropanoid pathways, it is possible that genetic manipulation of PAL might result in feedback effects on ICS.

TRA N S C R I P T I O N A L RE G U L A T I O N O F P H E N Y L P RO P A N O I D B I O S Y N T H E S I S D U R I N G PLANT DEFENCE

It has generally been assumed that the appearance of phenylpropanoid metabolites during a plants response to infection is a result of the transcriptional activation of the various biosynthetic pathway genes. This assumption must be qualied by noting that, in most cases, this has been inferred from the measurement of steady state transcript levels, an approach that does not distinguish between increased transcription or increased mRNA stability. Nevertheless, there are several examples directly documenting increases in transcription rates of phenylpropanoid pathway genes following the elicitation of infection, as measured by nuclear transcript run-on assays (Ni et al., 1996; Rushton and Somssich, 1999), and there is considerable interest in dening

the different transcription factors involved in the co-ordinated up-regulation of defence response pathways. It is likely that some of these factors are also involved in the transcriptional control of the same pathways during plant development. Several reviews have described the types of transcription factors that regulate the expression of genes, including those of the phenylpropanoid pathway, in plants (Liu et al., 1999; Meshi and Iwabuchi, 1995; Weisshaar and Jenkins, 1998). Recent information pertaining to phenylpropanoids that may be involved in defence responses is summarized in Table 2. Several distinct classes of transcription factor appear to operate in the overall control of phenylpropanoid biosynthesis, of which the myb factors have perhaps received the most attention. There are at least 100 (e -value cut-off = 1.00E-10) myb family members in Medicago truncatula and 175 annotated as myb genes in Arabidopsis. In Medicago truncatula, at least 11 myb genes are up-regulated during leaf infection and at least 28 are up-regulated during root nodulation and arbuscular mycorrhizal symbiosis.

P RO S P E C T S F O R M E T A B O L I C E N G I N E E R I N G O F P H E N Y L P RO P A N O I D B I O S Y N T H E S I S F O R I M P RO V E D D I S E A S E RE S I S T A N C E
Some of the disease problems in highly bred cultivated crops may have resulted from the successive loss of natural products during years of selection for food quality traits, and at least some of these pathways can now be restored by transgenic approaches. However, it has been argued that the levels of natural products required may be impractically high (Stuiver and Custers, 2001). A second argument commonly used against developing natural product engineering as a strategy for improving disease resistance is the ability of pathogens to overcome the effects of antimicrobial compounds by the evolution of detoxication

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

Class WRKY MYB Ntmyb2 PAP1-D TT2 AmMYB308/330 BHLH TT8 LIM protein family Ntlim1 bZIP family G/HBF-1 Ku-like KAP2

Gene/pathway regulated Phenylpropanoids/PR proteins

Reference Eulgem et al. (1999, 2000) Sugimoto et al. (2000) Borevitz et al. (2001) Nesi et al. (2001) Tamagnone et al. (1998) Nesi et al. (2000) Kawaoka et al. (2000) Drge-Laser et al. (1997) Lindsay et al. (2002)

PAL /defence response genes Phenylpropanoid pathway Condensed tannins Phenylpropanoids/lignin DFR, BAN
PAL, 4CL, and CAD in tobacco CHS in soybean CHS

BAN, Banyuls; CAD, cinnamyl alcohol dehydrogenase; CHS, chalcone synthase; 4CL, 4-coumarate:coenzyme A ligase; DFR, dihydroavonol reductase; PAL, L-phenylalanine ammonia-lyase; PR, pathogenesis-related.

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pathways. These pathways often require only single cytochrome P450 enzymes that can evolve quite rapidly (Covert et al., 1996). It is possible to get around this problem by the introduction of two or more unrelated novel antimicrobial compounds and, indeed, such a strategy might also lead to synergistic effects that can obviate the potency question. This is facilitated by the fact that there are several single enzyme reactions that can generate antimicrobial phenylpropanoid compounds from common metabolic intermediates. Examples include O-methylation of the ubiquitous avanone naringenin to yield sakuranetin (Rakwal et al., 2000), isoprenylation of isoavones (LaFlamme et al., 1993), or the production of stilbenes and other polyketides from malonyl CoA and various starter molecules (Schrder, 1997). A further objection to metabolic pathway engineering concerns the large numbers of genes that may have to be transferred, and coordinately regulated, in order to introduce many of the most effective antimicrobial compounds. The increasing production of an endogenous antimicrobial compound through the over-expression of a rate-limiting enzyme is a simpler strategy. However, in most cases, the ux control points in the pathway are not understood. Improved fungal disease resistance of alfalfa over-expressing isoavone O-methyltransferase is associated with coordinated overexpression of all the other genes in the biosynthesis of the phytoalexin medicarpin from L-phenylalanine, but only in response to infection (He and Dixon, 2000). Although the reason for this phenomenon remains unclear, it provides an example of how it is possible to engineer an improved inducible phytoalexin response without potentially deleterious constitutive production of phytoalexins. As outlined above, signicant progress has been made in elucidating the three-dimensional structures of several key enzymes involved in the biosynthesis of monolignols, avonoids, and isoavonoid phytoalexins. Such structural studies will facilitate structure-based rational re-design of enzymes, such as polyketide synthases and O-methyltransferases, for the transgenic introduction of novel phenylpropanoid natural products for plant defence. Thus, structure-based mutational re-design of pyrone synthase has yielded a novel enzyme with chalcone synthase activity (Jez et al., 2000a), and it has been possible, by the same approach, to alter the starter molecule specicity of alfalfa CHS (Jez et al., 2002). Mutations around the active site of caffeic acid Omethyltransferase (COMT) lead to forms of the enzyme with altered kinetic preferences for acid, aldehyde, and alcohol substrates potentially involved in lignin or lignan biosynthesis (Zubieta et al., 2002). It should therefore be possible in the future to design new enzymes for more efcient pathway ux or the introduction of novel natural products for improved disease resistance.

various aspects of phenylpropanoid biosynthesis, and Cuc Ly for artwork. Work in the corresponding authors laboratory was funded by the Samuel Roberts Noble Foundation, Forage Genetics International, and David Michael and Company.

R E F E RE N C E S
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ACKNOWLEDGEMENTS
We thank Drs Fang Chen, Dianjing Guo, Xian-Zhi He, Joseph Noel, Shashi Sharma and Chloe Zubieta for helpful discussions on

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