trends in plant science

Perspectives

Genetics and biochemistry of

secondary metabolites in
plants: an evolutionary
perspective
Eran Pichersky and David R. Gang
The evolution of new genes to make novel secondary compounds in plants is an
ongoing process and might account for most of the differences in gene function
among plant genomes. Although there are many substrates and products in
plant secondary metabolism, there are only a few types of reactions. Repeated
evolution 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 to
be common in secondary metabolism and might confound the assignment of
gene function based on sequence information alone.
lants 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 secondary 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 fertilization 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 the
growth 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 problem are often different in different plant lineages. For example, the compounds that make
up floral scents vary widely from species to
species, even when the same class of pollinators (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 secondary compounds have not yet been elucidated, 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 investigated, the enzymes in plant secondary metabolism 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, the
genome 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 metabolism, new genes almost always arise by gene
duplication followed by divergence17,18. This
leaves the organism with one gene that

1360 - 1385/00/$ see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(00)01741-6

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 metabolism arise by gene duplication and divergence, and how often do they arise by simple
allelic divergence? To resolve this issue, comparative analyses of orthologous loci from
related species that also include the identification 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 theoretically possible, for example, for a new
allele 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 deterring 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 different 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 metabolism, 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 origin of different genes in primary metabolism
can be inferred from their level of relatedness
to each other (i.e. their level of sequence identity). Current sequencing projects are uncovering 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 binding domains of substrates and co-factors).
However, because the true functions of most
members of plant gene families are not yet
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(a)

OH
HO

(b)

OH
O

OH

HO

OH

OH
OH
O

OH

HO

H
OMe
OMe

OH
Rutin

Rotenone

(c)

(d) O

OH

N
OMe
OMe

Linalool

(e)

MeO

Berberine

OH

(f)

Gain and loss of genes for specific

secondary compounds are continuing
processes
N
S

OH
DIMBOA

N
H
Brassilexin

Fig. 1. Examples of plant secondary metabolites and their proposed function in the plant from
which they were isolated. (a) Rutin, obtained from Forsythia intermedia, thought to act as a visual
pollinator attractant. (b) Rotenone, obtained from Derris elliptica, thought to act as an
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
defense toxin. (e) DIMBOA, obtained from Zea mays, thought to act as a defense toxin.
(f) Brassilexin, obtained from Brassica spp., thought to act as an antifungal toxin.

known, it remains difficult to answer the

questions posed above completely.
Thus, the large plant gene family of
cytochrome-p450s-dependent oxygenases contains only a few members currently recognized
to be involved in primary metabolism, such
as in steroid and phenylpropanoid biosynthesis20. This large gene family also contains
members already identified as being
involved in secondary metabolism (e.g. the
formation of menthol and carvone 21). 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. phenylpropene and alkaloid biosynthesis1,22).
Another example is a family of secondary
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October 2000, Vol. 5, No. 10

possible. 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 primary
metabolism. Indeed, genes of primary metabolism can serve as a pool from which similar
genes of secondary metabolism could evolve
over and over again.

metabolism glycosyl transferases that also

contains the gene for carboxypeptidase of
primary 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 secondary metabolism. For example, a large
gene family, with an estimated 70 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 metabolism 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

There are many examples of a specific secondary compound that is restricted to one
plant lineage and is not found in related lineages, especially the ancestral one (such an
observation should always be considered provisional because it is of course possible that
other lineages will later be found to make such
a compound). This represents prima facie evidence that the ability to synthesize this compound 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 comparison of its sequence similarity to other
related genes. For example, the gene from
Clarkia breweri (family Onagraceae) that
encodes the enzyme IEMT [which catalyzes
the 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

trends in plant science

Perspectives
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-methyltransferases 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 shuffling could have been involved in the
evolution of some members27), domainswapping experiments with sesquiterpene
epi-aristolochene synthases have shown that
the 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 calculate how frequently such changes have
resulted in new enzymes of secondary metabolism. However, several factors seem to facilitate 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 are
themselves 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-methyltransferase, 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

Table 1. Selected plant gene families with at least some members that are
involved in plant secondary metabolism
Enzyme gene family

Example from secondary

metabolisma

No. of copies
in Arabidopsis

2-Oxoglutarate-dependent
dioxygenases

Flavone synthase

.10

Acyl transferases

Acetyl-CoA:benzylalcohol acetyl
transferase

.70

Carboxymethyl methyltransferases

S-adenosylmethionine:salicylic acid
methyl transferase

.20

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

Not from Arabidopsis

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 families. As a consequence, plants often achieve
high synthesis rates of some secondary
metabolites by expressing their genes at
high 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 cell30.
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 promoter elements (i.e. in two separate parts of
the gene). If, on the other hand, modification of only the coding region need occur,
genes encoding new enzymes can evolve
more rapidly.
BEAT (acetyl-CoAbenzylalcohol acetyltransferase, which is involved in floral scent
biosynthesis) and GAT4 (anthocyanin 5aromatic 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 benzenoid 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 corresponding substrates and products.
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subsp. trichocarpa

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

Myrtales

IEMT

Fig. 2. Phylogenetic tree consisting of COMT (which methylates caffeic acid to give ferulic
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
after the origination of the order Myrtales. Modified from Ref. 22.

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 cellular or subcellular location of the new enzyme.
The latter changes might explain the observation that biosynthetic pathways for secondary 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 compartments or different cells is often cited as examples 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
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regulatory mechanism has evolved post facto

to take advantage of the need to shuttle intermediates in the pathway, such a mechanism
cannot be assumed ipso facto.
Evolution of gene expression

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 interconnected networks of reactions. Although a
new reaction in secondary metabolism, possibly more so than in primary metabolism,
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
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 converts 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-methyltransferase (COMT isoform) were shown to
convert coniferyl aldehyde to sinapyl aldehyde via 5-hydroxyconiferyl aldehyde, suggesting 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 without the creation of any new enzyme simply by

As discussed above, changes in gene expression are often crucial, although not sufficient
by themselves, for the evolution of
new genes (and new pathways).
(a)
However, such changes can often be
confused with the origin of a new
A
B
D
E
C
gene. For example, C. breweri synthesizes linalool in its petals whereas
New enzyme
its close relative Clarkia concinna
V
W
X
Y
Z
does not, even though C. concinna
possesses the same enzyme respon(b)
sible for linalool synthesis, linalool
synthase (LIS), as C. breweri does.
A
B
D
E
However, in C. concinna, LIS is
found only in the stigma and at a
C
much lower level of expression than
V
W
Y
Z
in C. breweri2,27. Thus, if a plant
species is found to synthesize a secondary compound in a particular
Fig. 3. Two methods by which new biochemical
organ and its relatives do not synthepathways can originate: (a) through the formation
size this compound in that same
of a new enzyme that links two pre-existing pathorgan, it is important to verify
ways; (b) through co-expression in the same comwhether these relatives produce
partment of selected enzymes from two pathways
such a compound elsewhere in the
that share the same intermediate.
plant. If they do, this probably means
that no new biosynthetic genes have

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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 intermediate (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 synthesis of the defense compound DIMBOA,
four sequential hydroxylation reactions are
carried out by similar cytochrome-p450dependent mono-oxygenases32. For such a
pathway to evolve over time, the intermediates 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 hydroxylation reactions, with the high substrate specificities observed32 in the current enzymes
evolving later.
A second example is found in the flavonoid
biosynthetic pathway, in which two homologous enzymes (flavone synthase and anthocyanidin synthase) are core enzymes in two
distinct pathways that diverge from the product of flavanone 3-hydroxylase. All three of
these enzymes are 2-oxoglutarate-dependent
dioxygenases and share high sequence similarity33. Anthocyanidin synthase is in the pathway leading to the anthocyanins, whereas
flavone synthase is a branch-point enzyme,
shunting flux to the formation of flavonol
glucosides instead. Thus, by multiple duplication events and subsequent divergence of
the flavanone 3-hydroxylase or its precursor gene, multiple new enzymes and
two pathways evolved.

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 activation 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-glucosidegluthathione-S-transferases from maize
and 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 evolution 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 expression level and cell-type location. These
approaches carry a high risk of misidentification of the true role of enzymes involved in
plant secondary metabolism.
Thus, it was recently shown biochemically
that a methyltransferase gene from Arabidopsis
that 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,
an Arabidopsis 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

-Selinene synthase (

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

()-4 -limonene synthase (

)
)

Myrcene synthase (
Pinene synthase (

Gymnosperms

(+)-Bornyl diphosphate synthase (

)

(+)-Sabinene synthase (
Convergent and repeated evolution in
secondary metabolism

One of the most remarkable observations

about the evolution of secondary metabolism in plants is clearly the many cases
that appear to represent convergent evolution. For example, cyanogenesis (the release
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
release of HCN from cyanogenic glycosides
(hydroxynitrile lyases) have arisen independently several times, with some being

1,8-Cineole synthase (

()-4 -limonene synthase (

()-4 -limonene synthase (

()-4 -limonene synthase (

Angiosperms

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

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numbers of genes with vast numbers of potential 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 secondary 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 metabolism evolve can be better addressed when
data 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 nonstandard plants that hold most of the diversity
of secondary plant metabolites. Moreover,
EST projects that are concerned with secondary metabolism should strive to analyze
tissues that are especially active in the synthesis 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 metabolism, only the demonstration of enzymatic
activity can unambiguously identify the function of the protein. It is also clear that, with all
the new tools of molecular biology and biochemistry 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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October 2000, Vol. 5, No. 10

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Trends Plant Sci. 5, 168173
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Biochemical and molecular genetic aspects of
floral scent. Plant Physiol. 122, 627633
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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 11 734
936 3522; fax 11 734 647 0884; e-mail
lelx@umich.edu).

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