genetics and biochemistry secondary metabolism in plants

7/21/2019 Genetics and Biochemistry Secondary Metabolism in Plants

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Plants produce an amazing diversity of

low molecular weight compounds.

Although the structures of close to

50 000 have already been elucidated1, there

are probably hundreds of thousands of such

compounds. Only a few of these are part of

primary metabolic pathways (those common

to all organisms). The rest are termed sec-

ondary metabolites; this term is historical and

was initially associated with inessentiality but,

here, a secondary metabolite is defined as a

compound whose biosynthesis is restricted to

selected plant groups.

The ability to synthesize secondary

compounds has been selected throughout the

course of evolution in different plant lineages

when such compounds addressed specific

needs (Fig. 1). For example, floral scent

volatiles and pigments have evolved to attract

insect pollinators and thus enhance fertiliz-

ation rates2,3. The ability to synthesize toxic

chemicals has evolved to ward off pathogens

and herbivores (from bacteria and fungi to

insects and mammals) or to suppress thegrowth of neighboring plants47. Chemicals

found in fruits prevent spoilage and act as signals

(in the form of color, aroma and flavor) of

the presence of potential rewards (sugars,

vitamins and amino acids) for animals who eat

the fruit and thereby help to disperse the seeds.

Other chemicals serve cellular functions that

are unique to the particular plant in which they

occur (e.g. resistance to salt or drought8,9).

The chemical solutions to a common prob-

lem are often different in different plant lin-

eages. For example, the compounds that make

up floral scents vary widely from species to

species, even when the same class of pollina-

tors (e.g. moths) are attracted to the differing

bouquets10. The variety of herbivore-deterring

chemicals produced by plants also seems to be

vast, and individual plant lineages synthesize

only a small subset of such compounds11.

Each species contains only a subset of

genes for secondary metabolism

Although the pathways that produce most sec-

ondary compounds have not yet been eluci-

dated, it is clear that there are possibly

hundreds of thousands of different enzymes

involved in secondary metabolism in plants.

There are many known instances in secondary

metabolism in which the synthesis of multiple

products can be catalyzed by a single enzyme,

either from different substrates12,13 or, more

rarely, even from the same substrate14.

However, in most cases that have been investi-

gated, the enzymes in plant secondary metab-

olism are specific for a given substrate and

produce a single product.

Plant genomes are variously estimated to

contain 20 00060 000 genes, and perhaps

1525% of these genes encode enzymes

for secondary metabolism15,16. Clearly, thegenome of a given plant species encodes only

a small fraction of all the enzymes that would

be required to synthesize the entire set of

secondary metabolites found throughout the

plant kingdom. This article focuses on the

molecular evolutionary mechanisms that are

responsible for generating the great diversity

of plant secondary metabolites.

Gene duplication is not the only

mechanism of evolution of new genes

in secondary metabolism

It is believed that, at least in primary metab-

olism, new genes almost always arise by gene

duplication followed by divergence17,18. This

leaves the organism with one gene that

maintains the original function and a second

copy that is not restricted by natural selection.

This second copy can then accumulate

mutations until, rarely, it has acquired a new

function and might then become fixed in the

population. Domain swapping, with or without

prior gene duplications, can also create new,composite genes19.

How often do genes for secondary metab-

olism arise by gene duplication and diver-

gence, and how often do they arise by simple

allelic divergence? To resolve this issue, com-

parative analyses of orthologous loci from

related species that also include the identifi-

cation of gene function must be carried out

but such data are not yet available. Obviously,

if the original gene had an essential function,

as genes of primary metabolism would be

expected to have, gene duplication is a

necessary prerequisite. However, it is theo-

retically possible, for example, for a newallele in one of the plants genetic loci to be

selected for if it encodes the ability to make a

new defense compound, whereas the older

alleles specify the synthesis of another defense

compound that is no longer effective at deter-

ring the plants enemies. Thus, in secondary

metabolism, there is a potential for new genes

to evolve without a prior gene duplication

event. In such cases, orthologous genes in

related species might encode proteins with dif-

ferent functions.

Origin of new genes for secondary

metabolism

A gene can be defined as new and distinct

from its ancestral gene when: (1) it encodes an

enzyme that catalyzes a chemically similar

reaction but on a different substrate than the

enzyme encoded by its progenitor gene; or (2)

the encoded enzyme carries out a different

chemical reaction on the same substrate. A

single-step change in both the substrate and

the type of reaction is much less likely. How

often do new genes of secondary metabolism

arise from other genes of secondary metab-

olism, and how often do they arise from genes

of primary metabolism?The recent advances in whole-genome

sequencing EST databases have provided

important information for this question, but no

definitive answers. In general, the order of ori-

gin of different genes in primary metabolism

can be inferred from their level of relatedness

to each other (i.e. their level of sequence iden-

tity). Current sequencing projects are uncover-

ing many gene families whose existence and

extent was only suspected before (Table 1).

These families are defined by their shared

motifs in the encoded proteins (which

might constitute the active site and/or bind-

ing domains of substrates and co-factors).

However, because the true functions of most

members of plant gene families are not yet

439

trends in plant science

Perspectives

October 2000, Vol. 5, No. 101360 - 1385/00/$ see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(00)01741-6

Genetics and biochemistry of

secondary metabolites in

plants: an evolutionaryperspective

Eran Pichersky and David R. Gang

The evolution of new genes to make novel secondary compounds in plants is anongoing process and might account for most of the differences in gene function

among plant genomes. Although there are many substrates and products inplant secondary metabolism, there are only a few types of reactions. Repeatedevolution is a special form of convergent evolution in which new enzymes with

the same function evolve independently in separate plant lineages from a shared

pool of related enzymes with similar but not identical functions. This appears tobe common in secondary metabolism and might confound the assignment of

gene function based on sequence information alone.


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known, it remains difficult to answer the

questions posed above completely.Thus, the large plant gene family of

cytochrome-p450s-dependent oxygenases con-

tains only a few members currently recognized

to be involved in primary metabolism, such

as in steroid and phenylpropanoid biosynthe-

sis20. This large gene family also contains

members already identified as being

involved in secondary metabolism (e.g. the

formation of menthol and carvone21). A

similar situation also exists in the family

of genes encoding O-methyltransferase

enzymes, which are involved in primary

metabolism (e.g. lignin formation) as well

as in secondary metabolism (e.g . phenyl-

propene and alkaloid biosynthesis1,22).

Another example is a family of secondary

metabolism glycosyl transferases that also

contains the gene for carboxypeptidase ofprimary metabolism23. By contrast, several

large families of genes have recently been

identified that contain only a few members

with a defined function, all involved in sec-

ondary metabolism. For example, a large

gene family, with an estimated70 members in

Arabidopsis alone, encodes enzymes for

acyl transferases involved in the synthesis

of various scent, pigment and defense

compounds24. Some members of this gene

family might be involved in primary meta-

bolism but none has yet been identified.

The distinction between primary and

secondary metabolism is difficult to make

with our present knowledge and, in some

cases, such a distinction is simply not

possib le. For example, some of the acylated

anthocyanin derivatives that are synthesized

by enzymes belonging to the aforementioned

acyltransferase family might be synthesized

by all plants, at least under some specific but

presently unknown conditions, but this has

not yet been ascertained. Even if they are

not uniformly found in all plants, the ability

to make such compounds might be an

ancestral trait that has been lost in various

plant lineages; this means that, at one point

in time, these compounds were primary

metabolites (produced by all plant groups).

Thus, although comparisons of available

sequence information indicate that many

genes of secondary metabolism have evolved

directly from other genes known or presumed

to be involved in secondary metabolism, it

is reasonable to assume that, in most cases,

the ultimate (and sometimes the proximate)

ancestor was a gene involved in primarymetabolism. Indeed, genes of primary metab-

olism can serve as a pool from which similar

genes of secondary metabolism could evolve

over and over again.

Gain and loss of genes for specific

secondary compounds are continuing

processes

There are many examples of a specific sec-

ondary compound that is restricted to one

plant lineage and is not found in related lin-

eages, especially the ancestral one (such an

observation should always be considered pro-

visional because it is of course possible that

other lineages will later be found to make such

a compound). This represents prima facie evi-

dence that the ability to synthesize this com-

pound arose within this lineage.

Molecular evidence for the origin of a

new gene encoding the enzyme that catalyzes

the formation of this compound requires

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

Clarkia breweri (family Onagraceae) that

encodes the enzyme IEMT [which catalyzesthe methylation of (iso)eugenol to give

(iso)methyleugenol and is involved in floral

scent biosynthesis] has been shown to have

arisen from the gene encoding the enzyme

COMT (which methylates caffeic acid to

give ferulic acid and is involved in lignin

biosynthesis) some time after the divergence

of the order Myrtales22 (Fig. 2). However,

such data are rare.

The number of changes in the primary

sequence of an enzyme that are required to

alter its substrate specificity or its mode of

action can vary. Sequence comparisons of

related extant enzymes do not address this

issue directly because enzymes accumulate

neutral changes over time, making the

440


Perspectives

October 2000, Vol. 5, No. 10

Fig. 1. Examples of plant secondary metabolites and their proposed function in the plant fromwhich they were isolated. (a) Rutin, obtained fromForsythia intermedia, thought to act as a visualpollinator attractant. (b) Rotenone, obtained from Derris elliptica, thought to act as aninsect feeding deterrent. (c) Linalool, obtained from Clarkia breweri, thought to act as an olfactorypollinator attractant. (d) Berberine, obtained from Berberis wilsoniae, thought to act as adefense toxin. (e) DIMBOA, obtained from Zea mays, thought to act as a defense toxin.(f) Brassilexin, obtained fromBrassica spp., thought to act as an antifungal toxin.

N

O

O

OH

OH

MeO

DIMBOA

O

O

O

O

OH

OH

OH

OH

O

OH

OH

OH

O

OH

OH

OH

OH

Rutin

Brassilexin

NH

S

N

OH

Linalool

O OO

OMe

OMe

O

H

H

Rotenone

N

OMe

O

O

OMe

Berberine

(a)

(c)

(b)

(d)

(e) (f)


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amino acid substitutions critical for change

of function difficult to identify. This is

especially true because, in most cases, the

active site and binding site of the enzyme,

as well as other functionally important

domains, are not well defined22. Nonetheless,

examples are known in which pairs of

enzymes with different substrates differ

at one or a few positions25. In addition, in

vitro mutagenesis experiments have shown

that the substrate preference of O-methyl-

transferases and the type of reaction catalyzed

by a fatty acid desaturase can be changed

by as few as 57 amino acid substitutions

(turning the fatty acid desaturase into a

hydroxylase)22,26. Finally, in the terpene

synthase (TPS) family (in which exon shuf-

fling could have been involved in the

evolution of some members27), domain-

swapping experiments with sesquiterpene

epi-aristolochene synthases have shown thatthe exchange of a single small segment

could result in new substrate preference or

in different products being made from the

original substrate28.

There are currently not enough data to cal-

culate how frequently such changes have

resulted in new enzymes of secondary metab-

olism. However, several factors seem to facil-

itate this process. The new substrate (new for

the newly evolved enzyme, but not necessarily

new to the plant) often closely resembles the

old substrate, so that one or a few amino acid

substitutions can allow the altered enzyme to

recognize the new substrate (while maintaining

the same catalytic domain). Sometimes, the

enzyme recognizes only a small part of the

substrate to begin with, although as long as

that part of the molecule is similar between

the old and the new substrate, a small change

in the substrate-binding site of the enzyme is

sufficient.

It should be remembered that many

enzymes of secondary metabolism can

already recognize more than one substrate,

although they often have different catalytic

rates toward them29. In addition, the existence

of large families of enzymes, which arethemselves the product of repeated cycles

of gene duplications and divergence,

increases the probability that a small change

in one or another member of the family will

result in an enzyme that can carry out the

same type of reaction on a new substrate,

or carry out a different reaction on an old

substrate. This snowball effect (the more

genes there are in the family, the faster new

members arise) can probably explain in

part the large size of the O-methyltrans-

ferase, terpene synthase, cytochrome p450

and dehydrogenasereductase gene families

(Table 1), to name just a few.

Furthermore, because the new product

would not be essential for the survival of

the plant, the recently evolved enzyme that

catalyzes its formation need not initially be

efficient. However, if the production of the

new chemical confers a selective advantage

to the plant, genetic changes will be

selected for over time that favor increased

synthesis. Such changes could involve

additional amino acid substitutions that

increase the catalytic efficiency of the

enzyme. However, an alternative way to

increase fitness would be to increase the

expression of the gene encoding the new

enzyme. Indeed, the turnover numbers of

many enzymes of secondary metabolism

are many orders of magnitude lower than

those of enzymes of primary metabolism,

even for enzymes from the same gene fami-

lies. As a consequence, plants often achieve

high synthesis rates of some secondary

metabolites by expressing their genes athigh levels in a given tissue and under

given conditions (e.g. following pathogen

attack), to the point that the enzymes can

constitute 0.11.0% (or more) of the total

protein in the cell 30.

New genes are likely to be expressed

in specific tissues or cells, or at a

specific time

The biosynthesis of secondary metabolites

is often restricted to a particular tissue and

occurs at a specific stage of development.

For a new gene of secondary metabolism to

provide an adaptive advantage, it therefore

needs to be expressed in a specific tissue or

type of cell at a specific time. As described

above, the new enzyme is likely to be a

variation of an existing enzyme that uses a

similar substrate and catalyzes the formation

of a similar product. It is probably more

likely to arise from a gene that is already

spatially and temporally expressed in the

same manner in which production of the

new chemical is advantageous, even if

the new and old substrates and new and old

products are not as structurally similar as

they otherwise could have been. This is because

descent from an enzyme that recognizes a more

similar substrate but is not expressed in the

right tissue or at the right time will require

mutations in both coding regions and pro-

moter elements (i.e. in two separate parts of

the gene). If, on the other hand, modifica-

tion of only the coding region need occur,

genes encoding new enzymes can evolve

more rapidly.BEAT (acetyl-CoAbenzylalcohol acetyl-

transferase, which is involved in floral scent

biosynthesis) and GAT4 (anthocyanin 5-

aromatic acyltransferase, which is involved

in floral pigment biosynthesis) might be an

example of this24. They are acyltransferases

that share significant sequence similarity and

show coincident expression in flower petal

epidermal cells, although the substrates for

the two enzymes differ greatly (small benze-

noid versus larger glycosylated flavonoid,

respectively). It is therefore not surprising

that, in secondary metabolism, there is little

discernable correlation between the relatedness

of enzymes and the relatedness of the corre-

sponding substrates and products.

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Perspectives


Table 1. Selected plant gene families with at least some members that are

involved in plant secondary metabolism

Enzyme gene family Example from secondary No. of copiesmetabolisma in Arabidopsis

2-Oxoglutarate-dependent Flavone synthase 10dioxygenases

Acyl transferases Acetyl-CoA:benzylalcohol acetyl 70transferase

Carboxymethyl methyltransferases S-adenosylmethionine:salicylic acid 20methyl transferase

Cytochromes p450 DIBOA hydroxylase 100

Glutathione-S-transferases Petunia An9 gene 20

Methylene bridge-forming enzymes Berberine bridge enzyme 10

NADPH-dependent dehydrogenases Isoflavone reductase 50

O-Methyl transferases (Iso)eugenol O-methyltransferase 20

Polyketide synthases Stilbene synthase 10

Terpene synthases Linalool synthase 20

aNot fromArabidopsis


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Changes in location of new enzymes

If, however, a new enzyme does arise in a cell

or organelle separated from where the new

reaction can impart benefits to the plant or

from where the new substrate is present, there

are several possible scenarios that could

result in selective advantage. In one scenario,

additional mutations in the control region of the

gene (including the coding part that specifies

subcellular location) or in other genes

encoding regulatory proteins could alter the

enzymes distribution. In a second scenario,

additional changes elsewhere in the genome

could result either in the de novo synthesis

of the substrate in the appropriate location or

in the transport of the substrate into the cellu-lar or subcellular location of the new enzyme.

The latter changes might explain the obser-

vation that biosynthetic pathways for sec-

ondary metabolism are sometimes split over

more than one subcellular compartment or

across two or more types of cells or even

tissues (the biosynthesis of alkaloids provides

examples of all of these1). Such shuttling of

substrates between different cellular compart-

ments or different cells is often cited as exam-

ples of a mechanism for the regulation of the

pathway. However, the possibility that such an

arrangement is the result of the contingent

nature of the evolution of new enzymes (and

therefore of the new pathways) should not be

ignored. Thus, although it is possible that a

regulatory mechanism has evolvedpost facto

to take advantage of the need to shuttle inter-

mediates in the pathway, such a mechanism

cannot be assumed ipso facto.

Evolution of gene expression

As discussed above, changes in gene express-

ion are often crucial, although not sufficient

by themselves, for the evolution of

new genes (and new pathways).

However, such changes can often be

confused with the origin of a new

gene. For example, C. breweri syn-

thesizes linalool in its petals whereas

its close relative Clarkia concinna

does not, even though C. concinnapossesses the same enzyme respon-

sible for linalool synthesis, linalool

synthase (LIS), as C. breweri does.

However, in C. concinna, LIS is

found only in the stigma and at a

much lower level of expression than

in C. breweri2,27. Thus, if a plant

species is found to synthesize a sec-

ondary compound in a particular

organ and its relatives do not synthe-

size this compound in that same

organ, it is important to verify

whether these relatives produce

such a compound elsewhere in the

plant. If they do, this probably means

that no new biosynthetic genes have

evolved. Instead, the expression pattern of

existing biosynthetic genes must have

changed (e.g. through an altered promoter or

transcription factor).

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 bio-

chemical 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, poss-

ibly more so than in primary metabolism,

usually gives rise to an end product that is not

further metabolized by the plant, the sub-

strate on which the new enzyme acts could in

principle be an intermediate in an existing

pathway and not necessarily an end product

by itself. Indeed, an instant new pathwaycould 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

demonstrated for plant primary metabolism

when sweetgum (Liquidambar styraciflua)

coniferyl aldehyde 5-hydroxylase (CAld5H)

and 5-hydroxyconiferyl aldehyde O-methyl-

transferase (COMT isoform) were shown to

convert coniferyl aldehyde to sinapyl alde-

hyde via 5-hydroxyconiferyl aldehyde, sug-

gesting that a CAld5HCOMT-mediated

pathway to sinapic acid might be functional

in some plants31. This is in contrast with the

generally accepted route to sinapic acid from

ferulic acid through 5-hydroxyferulic acid.

A new pathway might be formed even with-

out the creation of any new enzyme simply by

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Perspectives


Fig. 2. Phylogenetic tree consisting of COMT (which methylates caffeic acid to give ferulicacid and is involved in lignin biosynthesis) sequences from several species and including theClarkia breweri IEMT sequence, showing that Clarkia IEMT evolved from Clarkia COMTafter the origination of the order Myrtales.Modified from Ref. 22.

IEMT

subsp. trichocarpa

Myrtales

Fig. 3. Two methods by which new biochemicalpathways can originate: (a) through the formationof a new enzyme that links two pre-existing path-ways; (b) through co-expression in the same com-

partment of selected enzymes from two pathwaysthat share the same intermediate.

A B C D E

V W X Y Z

New enzyme

A B

C

D E

V W Y Z

(a)

(b)


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expressing in the same compartment two sets

(or partial sets) of enzymes belonging to two

different pathways that share at least one inter-

mediate (Fig. 3) but that have not previously

operated in the same compartment. As this

discussion shows, as long as we continue to

think of pathways as linear arrays of reactions,

it is difficult for us to determine the sequence

of events that gave rise to them. Only a

detailed examination of the presence or

absence of particular reactions in related plant

species whose true phylogeny is known can

allow us to determine which reactions came

first (based on parsimony analysis). Such

analyses are yet to be attempted.

Nevertheless, new pathways can also be

created by repeated cycles of gene duplication

and divergence. Two examples illustrate this

point. First, in the pathway leading to the syn-

thesis of the defense compound DIMBOA,

four sequential hydroxylation reactions arecarried out by similar cytochrome-p450-

dependent mono-oxygenases32. For such a

pathway to evolve over time, the intermedi-

ates must have conferred some selective

advantage by themselves. Alternatively, the

original mono-oxygenase of the DIMBOA

pathway might initially have been able to

catalyze all (or some) of the four hydroxyl-

ation reactions, with the high substrate speci-

ficities observed32 in the current enzymes

evolving later.

A second example is found in the flavonoid

biosynthetic pathway, in which two homolo-

gous enzymes (flavone synthase and antho-

cyanidin synthase) are core enzymes in two

distinct pathways that diverge from the prod-

uct of flavanone 3-hydroxylase. All three of

these enzymes are 2-oxoglutarate-dependent

dioxygenases and share high sequence simi-

larity33. Anthocyanidin synthase is in the path-

way leading to the anthocyanins, whereas

flavone synthase is a branch-point enzyme,

shunting flux to the formation of flavonol

glucosides instead. Thus, by multiple du-

plication events and subsequent divergence of

the flavanone 3-hydroxylase or its pre-

cursor gene, multiple new enzymes andtwo pathways evolved.

Convergent and repeated evolution in

secondary metabolism

One of the most remarkable observations

about the evolution of secondary meta-

bolism in plants is clearly the many cases

that appear to represent convergent evolu-

tion. For example, cyanogenesis (the release

of hydrogen cyanide as a defense com-

pound) appears to have arisen several times

during plant evolution. Recently, it has been

shown that the enzymes that catalyze the

release of HCN from cyanogenic glycosides

(hydroxynitrile lyases) have arisen inde-

pendent ly several times, with some being

related to oxidoreductases, some being related

to carboxypeptidase and others being of

unknown provenance34.

Even more intriguing is the observation that

many examples of convergent evolution in

plant secondary metabolism are of a special

case, termed repeated evolution, in which a

new genetic function arises independently but

from orthologous or paralogous genes27,29.

For example, the repeated evolution of the

enzyme homospermidine synthase, which

catalyzes the committed step in the synthesis

of pyrrolizidine alkaloids, from the ubiquitous

eukaryotic enzyme deoxyhypusine synthase

(which catalyzes the first step in the acti-

vation of a translation initiation factor) has

been invoked to explain the sporadic occurrence

of the pyrrolizidine alkaloids throughout the

angiosperms35.

In another example, the cyanidin-3-gluco-

sidegluthathione-S-transferases from maizeand petunia each arose independently from

paralogous members of the gluthathione

S-transferase (GST) family36. Similarly,

limonene synthases in both gymnosperms

and angiosperms are each more similar to

other terpene synthases within their lineages

than to each other (Fig. 4), but the terpene

synthases from gymnosperms and angiosperms

are also related to each other37. This indicates

that specific limonene synthase enzymatic

activities evolved in plants more than

once but, in all cases, they evolved from a

member of the terpene synthase family. It

appears that the universal presence of

several related enzymes in each of the GST

and TPS families that catalyze reactions in

primary metabolism or in related branches

of secondary metabolism might provide a

pool from which new enzymes can evolve,

sometimes more than once.

An important consequence of repeated evo-

lution is that the catalytic function of a newly

described gene or protein cannot be assigned

solely on its degree of sequence identity to

known enzymes27,37. It is currently a common

practice (as can be seen by perusing the

sequence databases) to assign function to

newly obtained sequences based solely on

homology to other sequences in the database,

and, less often, on the determination of ex-

pression level and cell-type location. These

approaches carry a high risk of misidentifi-

cation of the true role of enzymes involved in

plant secondary metabolism.

Thus, it was recently shown biochemically

that a methyltransferase gene fromArabidopsisthat had originally been thought to encode

COMT, based on its sequence similarity to

other known COMTs, actually encodes an

enzyme that methylates quercetin, a flavonol,

and is not active with caffeic acid38. Similarly,

anArabidopsis TPS gene initially identified

as limonene synthase based on sequence

comparisons alone has now been shown to

encode myrcene synthase39. Clearly, actual

biochemical data demonstrating catalytic

function will be required to uncover the true

function. Experiments yielding biochemical

data demonstrating catalytic function are not

always straightforward and can be difficult to

carry out in practice, especially for large

443


Perspectives


Fig. 4. Phylogenetic tree of terpene synthases from gymnosperms and angiosperms showingthat limonene synthases evolved separately in these two plant lineages. Modified fromRef. 37.

()-4 -limonene synthase ( )



1,8-Cineole synthase ( )

(+)-Sabinene synthase ( )

(+)-Bornyl diphosphate synthase (

)

Pinene synthase ( )

Myrcene synthase ( )


()-4 -limonene/()--pinene synthase ( )

Gymnosperms

Angiosperms

-Selinene synthase ( )


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numbers of genes with vast numbers of poten-

tial substrates. However, if analysis of the

sequence homology is coupled to a detailed

understanding of the metabolite composition

for a given species (biochemical genomics),

these experiments do become possible.

Future prospects

In future work, researchers will hopefully

examine the provenance of new genes of sec-

ondary metabolites. This should be done by

comparing them with the ancestral genes in

related species, using established statistical

methods that have been used extensively in

taxonomic studies40. As detailed above, many

of the questions raised here how often new

genes of secondary metabolism arise, which

genes are more likely to serve as the source of

new genes and what the specific changes are

that occur when new genes of secondary metab-

olism evolve can be better addressed whendata of such comparisons are available.

The increasing availability of plant genome

sequences and EST data sets makes this

approach feasible, but there is a need to carry

out additional EST data acquisition from plant

species other than the standard crop plants of

the Western world, because it is such non-

standard plants that hold most of the diversity

of secondary plant metabolites. Moreover,

EST projects that are concerned with sec-

ondary metabolism should strive to analyze

tissues that are especially active in the syn-

thesis of such metabolites, to increase the

probability of obtaining as many sequences as

possible from mRNAs that are normally found

in low abundance in generalized tissues. The

recent report of an EST data set acquired from

the peltate glands of mint (specialized tissue

for the synthesis of monoterpenes) is a good

example41.

Finally, there is a pressing need to develop

an efficient system to test en masse the

catalytic activities of the enzymes whose

sequences are being revealed by the ongoing

and future mass-sequencing EST projects,

because it is clear that, in secondary metab-

olism, only the demonstration of enzymaticactivity can unambiguously identify the func-

tion of the protein. It is also clear that, with all

the new tools of molecular biology and bio-

chemistry being put to use to address these

exciting questions, our understanding of the

evolution of plant secondary metabolism is

poised for major advances42.

Acknowledgements

Research in our laboratory was funded by

NSF grant MCB-9974436 and by a Margaret

and Herman Sokol Post-doctoral Fellowship

in the Sciences to D.R.G. We thank

Jonathan Gershenzon, Leslie D. Gottlieb and

three anonymous reviewers for their useful

comments on the manuscript.

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Eran Pichersky* and David R. Gang are at

the Biology Dept, University of Michigan,

Ann Arbor, MI 48109-1048, USA.

*Author for correspondence (tel 1 734

936 3522; fax 1 734 647 0884; e-mail

[email protected]).

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