Genetics and Biochemistry of Secondary Metabolites in Plants

trends in plant science Perspectives

maintains the original function and a second Genetics and biochemistry of copy that is not restricted by natural selection. This second copy can then accumulate mutations until, rarely, it has acquired a new secondary metabolites in function and might then become fixed in the population. Domain swapping, with or without prior gene duplications, can also create new, plants: an evolutionary composite genes19. How often do genes for secondary metabolism arise by gene duplication and diver- perspective gence, and how often do they arise by simple allelic divergence? To resolve this issue, com- Eran Pichersky and David R. Gang parative analyses of orthologous loci from related species that also include the identifi- The evolution of new genes to make novel secondary compounds in plants is an cation of gene function must be carried out – ongoing process and might account for most of the differences in gene function but such data are not yet available. Obviously, among plant genomes. Although there are many substrates and products in if the original gene had an essential function, plant secondary metabolism, there are only a few types of reactions. Repeated as genes of primary metabolism would be evolution is a special form of convergent evolution in which new enzymes with expected to have, gene duplication is a the same function evolve independently in separate plant lineages from a shared necessary prerequisite. However, it is theo- pool of related enzymes with similar but not identical functions. This appears to retically possible, for example, for a new be common in secondary metabolism and might confound the assignment of allele in one of the plant’s genetic loci to be gene function based on sequence information alone. selected for if it encodes the ability to make a new defense compound, whereas the older lants produce an amazing diversity of chemicals produced by plants also seems to be alleles specify the synthesis of another defense low molecular weight compounds. vast, and individual plant lineages synthesize compound that is no longer effective at deter- Although the structures of close to only a small subset of such compounds11. ring the plant’s enemies. Thus, in secondary P 1 50 000 have already been elucidated , there metabolism, there is a potential for new genes are probably hundreds of thousands of such Each species contains only a subset of to evolve without a prior gene duplication compounds. Only a few of these are part of genes for secondary metabolism event. In such cases, orthologous genes in ‘primary’ metabolic pathways (those common Although the pathways that produce most sec- related species might encode proteins with dif- to all organisms). The rest are termed ‘secondary compounds have not yet been eluci- ferent functions. ondary’ metabolites; this term is historical and dated, it is clear that there are possibly was initially associated with inessentiality but, hundreds of thousands of different enzymes Origin of new genes for secondary here, a ‘secondary’ metabolite is defined as a involved in secondary metabolism in plants. metabolism compound whose biosynthesis is restricted to There are many known instances in secondary A gene can be defined as new and distinct selected plant groups. metabolism in which the synthesis of multiple from its ancestral gene when: (1) it encodes an The ability to synthesize secondary products can be catalyzed by a single enzyme, enzyme that catalyzes a chemically similar compounds has been selected throughout the either from different substrates12,13 or, more reaction but on a different substrate than the course of evolution in different plant lineages rarely, even from the same substrate14. enzyme encoded by its progenitor gene; or (2) when such compounds addressed specific However, in most cases that have been investi- the encoded enzyme carries out a different needs (Fig. 1). For example, floral scent gated, the enzymes in plant secondary metab- chemical reaction on the same substrate. A volatiles and pigments have evolved to attract olism are specific for a given substrate and single-step change in both the substrate and insect pollinators and thus enhance fertiliz- produce a single product. the type of reaction is much less likely. How ation rates2,3. The ability to synthesize toxic Plant genomes are variously estimated to often do new genes of secondary metabolism chemicals has evolved to ward off pathogens contain 20 000–60 000 genes, and perhaps arise from other genes of secondary metab- and herbivores (from bacteria and fungi to 15–25% of these genes encode enzymes olism, and how often do they arise from genes insects and mammals) or to suppress the for secondary metabolism15,16. Clearly, the of primary metabolism? growth of neighboring plants4–7. Chemicals genome of a given plant species encodes only The recent advances in whole-genome found in fruits prevent spoilage and act as signals a small fraction of all the enzymes that would sequencing EST databases have provided (in the form of color, aroma and flavor) of be required to synthesize the entire set of important information for this question, but no the presence of potential rewards (sugars, secondary metabolites found throughout the definitive answers. In general, the order of ori- vitamins and amino acids) for animals who eat plant kingdom. This article focuses on the gin of different genes in primary metabolism the fruit and thereby help to disperse the seeds. molecular evolutionary mechanisms that are can be inferred from their level of relatedness Other chemicals serve cellular functions that responsible for generating the great diversity to each other (i.e. their level of sequence iden- are unique to the particular plant in which they of plant secondary metabolites. tity). Current sequencing projects are uncover- occur (e.g. resistance to salt or drought8,9). ing many gene ‘families’ whose existence and The chemical solutions to a common prob- Gene duplication is not the only extent was only suspected before (Table 1). lem are often different in different plant lin- mechanism of evolution of new genes These families are defined by their shared eages. For example, the compounds that make in secondary metabolism ‘motifs’ in the encoded proteins (which up floral scents vary widely from species to It is believed that, at least in primary metab- might constitute the active site and/or bind- species, even when the same class of pollina- olism, new genes almost always arise by gene ing domains of substrates and co-factors). tors (e.g. moths) are attracted to the differing duplication followed by divergence17,18. This However, because the true functions of most bouquets10. The variety of herbivore-deterring leaves the organism with one gene that members of plant gene families are not yet

possible. For example, some of the acylated anthocyanin derivatives that are synthesized (a) OH by enzymes belonging to the aforementioned acyltransferase family might be synthesized HO O (b) OH by all plants, at least under some specific but presently unknown conditions, but this has O not yet been ascertained. Even if they are not uniformly found in all plants, the ability OH O OH H O O O to make such compounds might be an O ancestral trait that has been lost in various HO O O OH plant lineages; this means that, at one point in time, these compounds were primary OH H O metabolites (produced by all plant groups). HO OH OMe Thus, although comparisons of available OH OMe sequence information indicate that many genes of secondary metabolism have evolved Rutin Rotenone directly from other genes known or presumed to be involved in secondary metabolism, it (c) (d) O is reasonable to assume that, in most cases, OH the ultimate (and sometimes the proximate) N ancestor was a gene involved in primary O metabolism. Indeed, genes of primary metab- OMe olism can serve as a pool from which similar genes of secondary metabolism could evolve OMe over and over again.

Linalool Berberine Gain and loss of genes for specific secondary compounds are continuing processes (e)MeO O OH (f) N There are many examples of a specific sec- S ondary compound that is restricted to one N O plant lineage and is not found in related lin- N OH H eages, especially the ancestral one (such an observation should always be considered pro- DIMBOA Brassilexin visional because it is of course possible that other lineages will later be found to make such a compound). This represents prima facie evi- Fig. 1. Examples of plant secondary metabolites and their proposed function in the plant from dence that the ability to synthesize this com- which they were isolated. (a) Rutin, obtained from Forsythia intermedia, thought to act as a visual pound arose within this lineage. pollinator attractant. (b) Rotenone, obtained from Derris elliptica, thought to act as an Molecular evidence for the origin of a insect feeding deterrent. (c) Linalool, obtained from Clarkia breweri, thought to act as an olfactory pollinator attractant. (d) Berberine, obtained from Berberis wilsoniae, thought to act as a new gene encoding the enzyme that catalyzes defense toxin. (e) DIMBOA, obtained from Zea mays, thought to act as a defense toxin. the formation of this compound requires (f) Brassilexin, obtained from Brassica spp., thought to act as an antifungal toxin. analysis of the presence of the gene in this and related plant lineages, as well as a com- parison of its sequence similarity to other related genes. For example, the gene from known, it remains difficult to answer the metabolism glycosyl transferases that also Clarkia breweri (family Onagraceae) that questions posed above completely. contains the gene for carboxypeptidase of encodes the enzyme IEMT [which catalyzes Thus, the large plant gene family of primary metabolism23. By contrast, several the methylation of (iso)eugenol to give cytochrome-p450s-dependent oxygenases con- large families of genes have recently been (iso)methyleugenol and is involved in floral tains only a few members currently recognized identified that contain only a few members scent biosynthesis] has been shown to have to be involved in primary metabolism, such with a defined function, all involved in sec- arisen from the gene encoding the enzyme as in steroid and phenylpropanoid biosynthe- ondary metabolism. For example, a large COMT (which methylates caffeic acid to sis20. This large gene family also contains gene family, with an estimated 70 members in give ferulic acid and is involved in lignin members already identified as being Arabidopsis alone, encodes enzymes for biosynthesis) some time after the divergence involved in secondary metabolism (e.g. the acyl transferases involved in the synthesis of the order Myrtales22 (Fig. 2). However, formation of menthol and carvone21). A of various scent, pigment and defense such data are rare. similar situation also exists in the family compounds24. Some members of this gene The number of changes in the primary of genes encoding O-methyltransferase family might be involved in primary meta- sequence of an enzyme that are required to enzymes, which are involved in primary bolism but none has yet been identified. alter its substrate specificity or its mode of metabolism (e.g. lignin formation) as well The distinction between primary and action can vary. Sequence comparisons of as in secondary metabolism (e.g. phenyl- secondary metabolism is difficult to make related extant enzymes do not address this propene and alkaloid biosynthesis1,22). with our present knowledge and, in some issue directly because enzymes accumulate Another example is a family of secondary cases, such a distinction is simply not neutral changes over time, making the

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amino acid substitutions critical for change of function difficult to identify. This is Table 1. Selected plant gene families with at least some members that are especially true because, in most cases, the involved in plant secondary metabolism active site and binding site of the enzyme, as well as other functionally important Enzyme gene family Example from secondary No. of copies domains, are not well defined22. Nonetheless, metabolisma in Arabidopsis examples are known in which pairs of enzymes with different substrates differ 2-Oxoglutarate-dependent Flavone synthase Ͼ10 at one or a few positions25. In addition, in dioxygenases vitro mutagenesis experiments have shown Acyl transferases Acetyl-CoA:benzylalcohol acetyl Ͼ70 that the substrate preference of O-methyltransferase transferases and the type of reaction catalyzed Carboxymethyl methyltransferases S-adenosylmethionine:salicylic acid Ͼ20 by a fatty acid desaturase can be changed methyl transferase by as few as 5–7 amino acid substitutions (turning the fatty acid desaturase into a Cytochromes p450 DIBOA hydroxylase Ͼ100 hydroxylase)22,26. Finally, in the terpene Glutathione-S-transferases Petunia An9 gene Ͼ20 synthase (TPS) family (in which exon shuf- Methylene bridge-forming enzymes Berberine bridge enzyme Ͼ10 fling could have been involved in the evolution of some members27), domain- NADPH-dependent dehydrogenases Isoflavone reductase Ͼ50 swapping experiments with sesquiterpene O-Methyl transferases (Iso)eugenol O-methyltransferase Ͼ20 epi-aristolochene synthases have shown that the exchange of a single small segment Polyketide synthases Stilbene synthase Ͼ10 could result in new substrate preference or Terpene synthases Linalool synthase Ͼ20 in different products being made from the original substrate28. aNot from Arabidopsis There are currently not enough data to cal- culate how frequently such changes have resulted in new enzymes of secondary metabolism. However, several factors seem to facil- the plant, the recently evolved enzyme that above, the new enzyme is likely to be a itate this process. The new substrate (new for catalyzes its formation need not initially be variation of an existing enzyme that uses a the newly evolved enzyme, but not necessarily efficient. However, if the production of the similar substrate and catalyzes the formation new to the plant) often closely resembles the new chemical confers a selective advantage of a similar product. It is probably more old substrate, so that one or a few amino acid to the plant, genetic changes will be likely to arise from a gene that is already substitutions can allow the altered enzyme to selected for over time that favor increased spatially and temporally expressed in the recognize the new substrate (while maintaining synthesis. Such changes could involve same manner in which production of the the same catalytic domain). Sometimes, the additional amino acid substitutions that new chemical is advantageous, even if enzyme recognizes only a small part of the increase the catalytic efficiency of the the new and old substrates and new and old substrate to begin with, although as long as enzyme. However, an alternative way to products are not as structurally similar as that part of the molecule is similar between increase fitness would be to increase the they otherwise could have been. This is because the old and the new substrate, a small change expression of the gene encoding the new descent from an enzyme that recognizes a more in the substrate-binding site of the enzyme is enzyme. Indeed, the turnover numbers of similar substrate but is not expressed in the sufficient. many enzymes of secondary metabolism right tissue or at the right time will require It should be remembered that many are many orders of magnitude lower than mutations in both coding regions and pro- enzymes of secondary metabolism can those of enzymes of primary metabolism, moter elements (i.e. in two separate parts of already recognize more than one substrate, even for enzymes from the same gene fami- the gene). If, on the other hand, modifica- although they often have different catalytic lies. As a consequence, plants often achieve tion of only the coding region need occur, rates toward them29. In addition, the existence high synthesis rates of some secondary genes encoding new enzymes can evolve of large families of enzymes, which are metabolites by expressing their genes at more rapidly. themselves the product of repeated cycles high levels in a given tissue and under BEAT (acetyl-CoA–benzylalcohol acetyl- of gene duplications and divergence, given conditions (e.g. following pathogen transferase, which is involved in floral scent increases the probability that a small change attack), to the point that the enzymes can biosynthesis) and GAT4 (anthocyanin 5- in one or another member of the family will constitute 0.1–1.0% (or more) of the total aromatic acyltransferase, which is involved result in an enzyme that can carry out the protein in the cell30. in floral pigment biosynthesis) might be an same type of reaction on a new substrate, example of this24. They are acyltransferases or carry out a different reaction on an old New genes are likely to be expressed that share significant sequence similarity and substrate. This ‘snowball effect’ (the more in specific tissues or cells, or at a show coincident expression in flower petal genes there are in the family, the faster new specific time epidermal cells, although the substrates for members arise) can probably explain in The biosynthesis of secondary metabolites the two enzymes differ greatly (small benze- part the large size of the O-methyltrans- is often restricted to a particular tissue and noid versus larger glycosylated flavonoid, ferase, terpene synthase, cytochrome p450 occurs at a specific stage of development. respectively). It is therefore not surprising and dehydrogenase–reductase gene families For a new gene of secondary metabolism to that, in secondary metabolism, there is little (Table 1), to name just a few. provide an adaptive advantage, it therefore discernable correlation between the relatedness Furthermore, because the new product needs to be expressed in a specific tissue or of enzymes and the relatedness of the corre- would not be essential for the survival of type of cell at a specific time. As described sponding substrates and products.

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evolved. Instead, the expression pattern of existing biosynthetic genes must have changed (e.g. through an altered promoter or transcription factor). subsp. trichocarpa × Evolution of new pathways In discussing the origin of new enzymes that catalyze the formation of new products, we should not lose sight of the fact that biochemical ‘pathways’ do not run in parallel, independently of each other, but instead are more accurately represented as intercon- nected networks of reactions. Although a new reaction in secondary metabolism, possibly more so than in primary metabolism, Myrtales usually gives rise to an end product that is not further metabolized by the plant, the substrate on which the new enzyme acts could in IEMT principle be an intermediate in an existing pathway and not necessarily an end product by itself. Indeed, an ‘instant’ new pathway could be created when a new enzyme con- verts an intermediate in one pathway into an intermediate of another pathway, thus linking the two (Fig. 3). An example of this concept was recently Fig. 2. Phylogenetic tree consisting of COMT (which methylates caffeic acid to give ferulic demonstrated for plant primary metabolism acid and is involved in lignin biosynthesis) sequences from several species and including the Clarkia breweri IEMT sequence, showing that Clarkia IEMT evolved from Clarkia COMT when sweetgum (Liquidambar styraciflua) after the origination of the order Myrtales. Modified from Ref. 22. coniferyl aldehyde 5-hydroxylase (CAld5H) and 5-hydroxyconiferyl aldehyde O-methyltransferase (COMT isoform) were shown to convert coniferyl aldehyde to sinapyl alde- Changes in location of new enzymes regulatory mechanism has evolved post facto hyde via 5-hydroxyconiferyl aldehyde, sug- If, however, a new enzyme does arise in a cell to take advantage of the need to shuttle inter- gesting that a CAld5H–COMT-mediated or organelle separated from where the new mediates in the pathway, such a mechanism pathway to sinapic acid might be functional reaction can impart benefits to the plant or cannot be assumed ipso facto. in some plants31. This is in contrast with the from where the new substrate is present, there generally accepted route to sinapic acid from are several possible scenarios that could Evolution of gene expression ferulic acid through 5-hydroxyferulic acid. result in selective advantage. In one scenario, As discussed above, changes in gene express- A new pathway might be formed even with- additional mutations in the control region of the ion are often crucial, although not sufficient out the creation of any new enzyme simply by gene (including the coding part that specifies by themselves, for the evolution of subcellular location) or in other genes new genes (and new pathways). encoding regulatory proteins could alter the However, such changes can often be (a) enzyme’s distribution. In a second scenario, confused with the origin of a new ABC DE additional changes elsewhere in the genome gene. For example, C. breweri syn- could result either in the de novo synthesis thesizes linalool in its petals whereas New enzyme of the substrate in the appropriate location or its close relative Clarkia concinna in the transport of the substrate into the cellu- does not, even though C. concinna VWXYZ lar or subcellular location of the new enzyme. possesses the same enzyme respon- The latter changes might explain the obser- sible for linalool synthesis, linalool (b) vation that biosynthetic pathways for sec- synthase (LIS), as C. breweri does. AB DE ondary metabolism are sometimes split over However, in C. concinna, LIS is more than one subcellular compartment or found only in the stigma and at a C across two or more types of cells or even much lower level of expression than tissues (the biosynthesis of alkaloids provides in C. breweri2,27. Thus, if a plant VW YZ examples of all of these1). Such shuttling of species is found to synthesize a sec- substrates between different cellular compart- ondary compound in a particular ments or different cells is often cited as exam- organ and its relatives do not synthe- Fig. 3. Two methods by which new biochemical ples of a mechanism for the ‘regulation’ of the size this compound in that same pathways can originate: (a) through the formation pathway. However, the possibility that such an organ, it is important to verify of a new enzyme that links two pre-existing path- arrangement is the result of the contingent whether these relatives produce ways; (b) through co-expression in the same com- nature of the evolution of new enzymes (and such a compound elsewhere in the partment of selected enzymes from two pathways that share the same intermediate. therefore of the new pathways) should not be plant. If they do, this probably means ignored. Thus, although it is possible that a that no new biosynthetic genes have

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expressing in the same compartment two sets related to oxidoreductases, some being related and TPS families that catalyze reactions in (or partial sets) of enzymes belonging to two to carboxypeptidase and others being of primary metabolism or in related branches different pathways that share at least one inter- unknown provenance34. of secondary metabolism might provide a mediate (Fig. 3) but that have not previously Even more intriguing is the observation that pool from which new enzymes can evolve, operated in the same compartment. As this many examples of convergent evolution in sometimes more than once. discussion shows, as long as we continue to plant secondary metabolism are of a special An important consequence of repeated evo- think of pathways as linear arrays of reactions, case, termed repeated evolution, in which a lution is that the catalytic function of a newly it is difficult for us to determine the sequence new genetic function arises independently but described gene or protein cannot be assigned of events that gave rise to them. Only a from orthologous or paralogous genes27,29. solely on its degree of sequence identity to detailed examination of the presence or For example, the repeated evolution of the known enzymes27,37. It is currently a common absence of particular reactions in related plant enzyme homospermidine synthase, which practice (as can be seen by perusing the species whose true phylogeny is known can catalyzes the committed step in the synthesis sequence databases) to assign function to allow us to determine which reactions came of pyrrolizidine alkaloids, from the ubiquitous newly obtained sequences based solely on first (based on parsimony analysis). Such eukaryotic enzyme deoxyhypusine synthase homology to other sequences in the database, analyses are yet to be attempted. (which catalyzes the first step in the acti- and, less often, on the determination of ex- Nevertheless, new pathways can also be vation of a translation initiation factor) has pression level and cell-type location. These created by repeated cycles of gene duplication been invoked to explain the sporadic occurrence approaches carry a high risk of misidentifi- and divergence. Two examples illustrate this of the pyrrolizidine alkaloids throughout the cation of the true role of enzymes involved in point. First, in the pathway leading to the syn- angiosperms35. plant secondary metabolism. thesis of the defense compound DIMBOA, In another example, the cyanidin-3-gluco- Thus, it was recently shown biochemically four sequential hydroxylation reactions are side–gluthathione-S-transferases from maize that a methyltransferase gene from Arabidopsis carried out by similar cytochrome-p450- and petunia each arose independently from that had originally been thought to encode dependent mono-oxygenases32. For such a paralogous members of the gluthathione COMT, based on its sequence similarity to pathway to evolve over time, the intermedi- S-transferase (GST) family36. Similarly, other known COMTs, actually encodes an ates must have conferred some selective limonene synthases in both gymnosperms enzyme that methylates quercetin, a flavonol, advantage by themselves. Alternatively, the and angiosperms are each more similar to and is not active with caffeic acid38. Similarly, original mono-oxygenase of the DIMBOA other terpene synthases within their lineages an Arabidopsis TPS gene initially identified pathway might initially have been able to than to each other (Fig. 4), but the terpene as ‘limonene synthase’ based on sequence catalyze all (or some) of the four hydroxyl- synthases from gymnosperms and angiosperms comparisons alone has now been shown to ation reactions, with the high substrate speci- are also related to each other37. This indicates encode myrcene synthase39. Clearly, actual ficities observed32 in the current enzymes that specific limonene synthase enzymatic biochemical data demonstrating catalytic evolving later. activities evolved in plants more than function will be required to uncover the true A second example is found in the flavonoid once but, in all cases, they evolved from a function. Experiments yielding biochemical biosynthetic pathway, in which two homolo- member of the terpene synthase family. It data demonstrating catalytic function are not gous enzymes (flavone synthase and antho- appears that the universal presence of always straightforward and can be difficult to cyanidin synthase) are core enzymes in two several related enzymes in each of the GST carry out in practice, especially for large distinct pathways that diverge from the product of flavanone 3-hydroxylase. All three of these enzymes are 2-oxoglutarate-dependent δ-Selinene synthase ( ) dioxygenases and share high sequence simi- larity33. Anthocyanidin synthase is in the path- (Ð)-4 -limonene/(Ð)-α-pinene synthase ( ) way leading to the anthocyanins, whereas flavone synthase is a branch-point enzyme, (Ð)-4 -limonene synthase ( ) shunting flux to the formation of flavonol glucosides instead. Thus, by multiple du- Myrcene synthase ( ) plication events and subsequent divergence of Pinene synthase ( ) Gymnosperms the flavanone 3-hydroxylase or its precursor gene, multiple new enzymes and (+)-Bornyl diphosphate synthase ( ) two pathways evolved. (+)-Sabinene synthase ( ) Convergent and repeated evolution in secondary metabolism 1,8-Cineole synthase ( ) One of the most remarkable observations about the evolution of secondary meta- (Ð)-4 -limonene synthase ( ) bolism in plants is clearly the many cases (Ð)-4 -limonene synthase ( ) that appear to represent convergent evolution. For example, cyanogenesis (the release (Ð)-4 -limonene synthase ( ) Angiosperms of hydrogen cyanide as a defense compound) appears to have arisen several times during plant evolution. Recently, it has been shown that the enzymes that catalyze the Fig. 4. Phylogenetic tree of terpene synthases from gymnosperms and angiosperms showing release of HCN from cyanogenic glycosides that limonene synthases evolved separately in these two plant lineages. Modified from (hydroxynitrile lyases) have arisen inde- Ref. 37. pendently several times, with some being

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numbers of genes with vast numbers of poten- References 18 Pichersky, E. (1990) Nomad DNA – a model for tial substrates. However, if analysis of the 1 De Luca, V. and St Pierre, B. (2000) The cell and movement and duplication of DNA sequences in sequence homology is coupled to a detailed developmental biology of alkaloid biosynthesis. plant genomes. Plant Mol. Biol. 15, 437–448 understanding of the metabolite composition Trends Plant Sci. 5, 168–173 19 Doolittle, R.F. (1995) The multiplicity of for a given species (‘biochemical genomics’), 2 Dudareva, N. and Pichersky, E. (2000) domains in proteins. Annu. Rev. Biochem. 64, these experiments do become possible. Biochemical and molecular genetic aspects of 287–314 floral scent. Plant Physiol. 122, 627–633 20 Szekeres, M. and Koncz, C. (1998) Biochemical Future prospects 3 Mol, J. et al. (1998) How genes paint flowers and and genetic analysis of brassinosteroid In future work, researchers will hopefully seeds. Trends Plant Sci. 3, 212–217 metabolism and function in Arabidopsis. Plant examine the provenance of new genes of sec- 4 Bennet, R.N. and Wallsgrove, R.M. (1994) Physiol. Biochem. 36, 145–155 ondary metabolites. This should be done by Secondary metabolism in plant defense – 21 Lupien, S. et al. (1999) Regiospecific cytochrome comparing them with the ancestral genes in mechanisms. New Phytol. 127, 617–633 p450 limonene hydroxylases from mint (Mentha) related species, using established statistical 5 Harborne, J.B. (1999) The comparative species: cDNA isolation, characterization, and methods that have been used extensively in biochemistry of phytoalexin induction in plants. functional expression of (Ϫ)-4S-limonene-3- taxonomic studies40. As detailed above, many Biochem. Syst. Ecol. 27, 335–367 hydroxylase and (Ϫ)-S-limonene-6-hydroxylase. of the questions raised here – how often new 6 Dixon, R.A. et al. (1996) Metabolic engineering: Arch. Biochem. Biophys. 368, 181 genes of secondary metabolism arise, which prospects for crop improvement through the 22 Wang, J. and Pichersky, E. (1999) Identification genes are more likely to serve as the source of genetic manipulation of phenylpropanoid of specific residues involved in substrate new genes and what the specific changes are biosynthesis and defense responses – a review. discrimination in two plant O-methyltransferases. that occur when new genes of secondary metab- Gene 179, 61–71 Arch. Biochem. Biophys. 368, 172–180 olism evolve – can be better addressed when 7 Mitchel-Olds, T. et al. (1998) Chemical ecology 23 Li, A.X. and Steffens, J.C. (2000) An data of such comparisons are available. in the molecular era. Trends Plant Sci. 3, acyltransferase catalyzing the formation of The increasing availability of plant genome 362–365 diacylglucose is a serine carboxypeptidase-like sequences and EST data sets makes this 8 Trossat, C. et al. (1998) Salinity promotes protein. Proc. Natl. Acad. Sci. U. S. A. 97, approach feasible, but there is a need to carry accumulation of 3-dimethylsulfoniopropionate 6902–6907 out additional EST data acquisition from plant and its precursor S-methylmethionine in 24 St Pierre, B. and De Luca, V. Evolution of species other than the standard crop plants of chloroplasts. Plant Physiol. 116, 165–171 acyltransferase genes. In Recent Advances in the Western world, because it is such non- 9 Nuccio, M.L. et al. (1999) Metabolic engineering Phytochemistry – Evolution of Metabolic standard plants that hold most of the diversity of plants for osmotic stress resistance. Curr. Pathways (Vol. 34), Plenum Press (in press) of secondary plant metabolites. Moreover, Opin. Plant Biol. 2, 128–134 25 Frickk, S. and Kutchan, T.M. (1999) Molecular EST projects that are concerned with sec- 10 Knudsen, J.T. and Tollsten, L. (1993) Trends in cloning and functional expression of ondary metabolism should strive to analyze floral scent chemistry in pollination syndromes: O-methyltransferases common to isoquinoline tissues that are especially active in the syn- floral scent composition in moth-pollinated taxa. alkaloid and phenylpropanoid biosynthesis. thesis of such metabolites, to increase the Bot. J. Linn. Soc. 113, 263–284 Plant J. 17, 329–333 probability of obtaining as many sequences as 11 Ananthakrishnan, T.N. (1999) Induced 26 Broun, P. et al. (1998) Catalytic plasticity of fatty possible from mRNAs that are normally found responses, signal diversity and plant defense: acid modification enzymes underlying chemical in low abundance in generalized tissues. The implications in insect phytophagy. Curr. Sci. diversity of plant lipids. Science 282, 1315–1317 recent report of an EST data set acquired from 76, 285–290 27 Cseke, L. et al. (1998) Structure and evolution of the peltate glands of mint (specialized tissue 12 Allina, S.M. et al. (1998) 4-Coumarate:coenzyme linalool synthase. Mol. Biol. Evol. 15, 1491–1498 for the synthesis of monoterpenes) is a good A ligase in hybrid poplar. Properties of native 28 Back, K. and Chappell, J. (1996) Identifying example41. enzymes, cDNA cloning and analysis of functional domains within terpene cyclases using Finally, there is a pressing need to develop recombinant enzymes. Plant Physiol. 116, a domain-swapping strategy. Proc. Natl. Acad. an efficient system to test en masse the 743–754 Sci. U. S. A. 93, 6841–6845 catalytic activities of the enzymes whose 13 Maury, S. et al. (1999) Tobacco 29 Wang, J. and Pichersky, E. (1998) sequences are being revealed by the ongoing O-methyltransferases involved in Characterization of S-adenosyl-L- and future mass-sequencing EST projects, phenylpropanoid metabolism. The different methionine:(iso)eugenol O-methyltransferase because it is clear that, in secondary metab- caffeoyl-coenzyme A/5-hydroxyferuloyl- involved in scent production in Clarkia breweri. olism, only the demonstration of enzymatic coenzyme A 3/5-O-methyltransferase and Arch. Biochem. Biophys. 349, 153–160 activity can unambiguously identify the func- caffeic acid/5-hydroxyferulic acid 30 White, W.L.B. et al. (1998) Cyanogenesis in tion of the protein. It is also clear that, with all 3/5-O-methyltransferase classes have distinct Cassava: the role of hydroxynitrile lyases in root the new tools of molecular biology and bio- substrate specificities and expression patterns. cyanide production. Plant Physiol. 116, 1219–1225 chemistry being put to use to address these Plant Physiol. 121, 215–223 31 Osakabe, K. et al. (1999) Coniferyl aldehyde exciting questions, our understanding of the 14 Phillips, M.A. et al. (1999) Monoterpene 5-hydroxylation and methylation direct syringyl evolution of plant secondary metabolism is synthases of loblolly pine (Pinus taeda) produce lignin biosynthesis in angiosperms. Proc. Natl. poised for major advances42. pinene isomers and enantiomers. Arch. Biochem. Acad. Sci. U. S. A. 96, 8955–8960 Biophys. 372, 197–204 32 Glawischnig, E. et al. (1999) Cytochrome p450 Acknowledgements 15 Bevan, M. et al. (1998) Analysis of 1.9 Mb of monooxygenases of DIBOA biosynthesis: Research in our laboratory was funded by contiguous sequence from chromosome 4 of specificity and conservation among grasses. NSF grant MCB-9974436 and by a Margaret Arabidopsis thaliana. Nature 391, 485–488 Phytochemistry 50, 925–930 and Herman Sokol Post-doctoral Fellowship 16 Somerville, C. and Somerville, S. (1999) 33 Rosati, C. et al. (1999) Molecular in the Sciences to D.R.G. We thank Plant functional genomics. Science characterization of the anthocyanidin synthase Jonathan Gershenzon, Leslie D. Gottlieb and 285, 380–383 gene in Forsythia ϫ intermedia reveals organ- three anonymous reviewers for their useful 17 Ohno, S. (1970) Evolution by Gene Duplication, specific expression during flower development. comments on the manuscript. Springer-Verlag Plant Sci. 149, 73–79

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34 Hickel, A. et al. (1996) Hydroxylnitrile lyases: 38 Muzac, I. et al. (2000) Functional expression of evaluation of expressed sequence tags from mint functions and properties. Physiol. Plant 98, an Arabidopsis cDNA clone encoding a flavonol glandular trichomes. Proc. Natl. Acad. Sci. 891–898 3Ј-O-methyltransferase and characterization of U. S. A. 97, 2934–2939 35 Ober, D. and Hartmann, T. (1999) Homospermidine the gene product. Arch. Biochem. Biophys. 375, 42 Facchini, P.J. (1999) Plant secondary synthase, the first pathway-specific enzyme of 385–388 metabolism: out of the evolutionary abyss. pyrrolizidine alkaloid biosynthesis, evolved from 39 Bohlmann, J. et al. (2000) Terpenoid secondary Trends Plant Sci. 4, 382–384 deoxyhypusine synthase. Proc. Natl. Acad. Sci. metabolism in Arabidopsis thaliana: cDNA U. S. A. 96, 14777–14782 cloning, characterization, and functional 36 Alfenito, M.R. et al. (1998) Functional expression of a myrcene/(E)-␤-ocimene synthase. Eran Pichersky* and David R. Gang are at complementation of anthocyanin sequestration Arch. Biochem. Biophys. 375, 261–269 the Biology Dept, University of Michigan, in the vacuole by widely divergent glutathione 40 Rogers, J.S. and Swofford, D.L. (1998) A fast Ann Arbor, MI 48109-1048, USA. S-transferases. Plant Cell 10, 1135–1149 method for approximating maximum likelihoods 37 Bohlmann, J. et al. (1998) Plant terpenoid of phylogenetic trees from nucleotide sequences. *Author for correspondence (tel ϩ1 734 synthases: molecular and phylogenetic Syst. Biol. 47, 77–89 936 3522; fax ϩ1 734 647 0884; e-mail analysis. Proc. Natl. Acad. Sci. U. S. A. 95, 41 Lange, B.M. et al. (2000) Probing essential oil [email protected]). 4126–4133 biosynthesis and secretion by functional

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