Plant Science 176 (2009) 161–169

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Plant Science
journal homepage: www.elsevier.com/locate/plantsci

Review

Phytochemical diversity: The sounds of silent metabolism

Efraim Lewinsohn a,*, Mark Gijzen b
a
Department of Vegetable Crops, Newe Ya’ar Research Center, Agricultural Research Organization, P.O. Box 1021, Ramat Yishay 30095, Israel
b
Agriculture and Agri-Food Canada, 1391 Sandford Street, London, Ontario N5 V 4T3, Canada

A R T I C L E I N F O A B S T R A C T

Article history: Plants produce tens of thousands of different natural products also referred to as secondary metabolites.
Received 10 June 2008 These metabolites were once thought to be the result of aberrant metabolism, or a form of transient
Received in revised form 1 September 2008 storage of byproducts and intermediates thereof. Although the true role of such metabolites in plants
Accepted 27 September 2008
remains mostly unknown, it is evident that plants invest a great deal of resources in synthesizing,
Available online 21 October 2008
accumulating and sorting such metabolites, often produced through complex and highly regulated
biosynthetic pathways operating in multiple cellular and sub-cellular compartments. There is also
Keywords:
growing evidence indicating that many biosynthetic pathways leading to the accumulation of plant
Silent metabolism
Occult metabolic capacity
natural products are not fully active. Thus, occult enzymes exist, sometimes without any apparent
Plant secondary metabolism endogenous substrate or function, suggesting that plants have a reservoir of metabolic capabilities that
Natural product biosynthesis normally remains hidden or unused. It is often difficult to accurately guess what are the actual biological
Metabolic engineering roles of such enzymes solely based on bioinformatics, due to promiscuity towards substrates and the
Metabolic networks relatively ease to change substrate or product specificity by introducing minor changes in sequence of the
enzymes. It could be that such orphan enzyme activities are relics of a recent past that have not been fully
eliminated through selection and evolution. Additionally, it could be that such occult activities possess
unknown biochemical roles and coincidentally are able to accept novel substrates. We have coined the
term ‘‘silent metabolism’’ to describe occult metabolic capacities present or induced in plants. A few
examples illustrating silent metabolism in the terpenoid and phenylpropanoid pathways, as well as their
repercussion in the metabolic engineering of plant secondary metabolism are discussed in this review.
ß 2008 Elsevier Ireland Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
2. Occult terpenoid pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
2.1. Silent metabolism in carotenoid biosynthesis and degradation reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
3. Silent metabolism in the biosynthesis of volatile ethers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
4. Silent metabolism in the phenylpropanoid pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
4.1. Flavonoid biosynthetic pathways provide numerous examples of silent metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
4.2. Silent flavonoid metabolism in soybean seed coats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
4.3. Soybean seed coat anthocyanin pigments are substrates for the seed coat peroxidase enzyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
5. Mechanism of action of silent metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
5.1. Silent metabolism and relaxed selection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
6. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168

* Corresponding author.
E-mail addresses: twefraim@agri.gov.il (E. Lewinsohn), gijzenm@AGR.GC.CA (M. Gijzen).

0168-9452/$ – see front matter ß 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.plantsci.2008.09.018
162 E. Lewinsohn, M. Gijzen / Plant Science 176 (2009) 161–169
Hello darkness, my old friend
I’ve come to talk with you again
Because a vision softly creeping
Left its seeds while I was sleeping
And the vision that was planted in my brain
Still remains
Within the sound of silence
(P. Simon, 1964)
1. Introduction
Plants produce and accumulate a vast number of different
natural products, also called secondary metabolites. Although tens
of thousands of secondary metabolites have been chemically
identified [1–3], the biological roles of most of these compounds
remain obscure. Many natural compounds are of commercial and
industrial importance, imparting colors and scents to flowers,
fruits and vegetables, and are also key ingredients in medicinals
and nutraceuticals. Many studies have indicated that natural
products accumulated in plants have clear ecological roles such as
protection against predation, protection against fungal and
bacterial diseases or against adverse climatic conditions [1,2,4–
6]. Additionally, many natural products serve as signal molecules
to attract pollinators and seed-dispersers, or mediate pathogenic,
parasitic, or symbiotic interactions [1]. Still, the biological roles of
most specialized compounds in the plants producing them are
unknown [3]. According to the so called ‘‘Screening Hypothesis’’
theory [7], it is a rarity for a natural product to possess biological
activity. Therefore, evolution favors means to increase biodiversity
to provide novel compounds to be challenged by evolution. In this
context and according to the ‘‘screening hypothesis’’ many of the
compounds manufactured and stored by plants (and retained
through evolution) are part of the ‘‘screening’’ arsenal that might
become important in due evolutionary time. It is therefore
advantageous for occult and partial metabolic pathways to be
retained through evolution as means to readily provide the much
needed biochemical diversity.
Plant secondary metabolites were once thought to be the result
of aberrant or accidental metabolism or an odd form of storage of
nutrients. Still, numerous studies have demonstrated that plants
have developed intricate mechanisms to modulate the production
and accumulation of such metabolites. In most cases studied, it has
been found that natural products are usually formed by elaborate
arrays of enzymes, concertedly controlled by the expression of
their respective genes. Although the chemical structures of more
than 40,000 different terpenes, 20,000 phenolics and 5000
alkaloids are known [1], plants utilize a relatively small number
of biosynthetic pathways for the production of such an impressive
number of different compounds [3]. Thus, the vast phytochemical
diversity is paradoxically attained by relatively minor changes in
existing and ubiquitous metabolic pathways [8]. The reasons and
origins of such diversity in plant phytochemistry are still largely
unknown. Are the huge numbers of structures present merely
there for serendipitous reasons? Or is their presence directed and
favored by evolution. Relatively small chemical changes in a
molecule, can have a major impact in its biological function [9–11]
and minor changes in protein structure might bring about to
profound changes in enzymatic activity or substrate specificity
([12,13]).
Thus, very minor changes in the DNA of an organism are enough
to confer a change in a gene that will be reflected in the chemistry
of the organism that might provide adaptive advantages to the
variant organism, and thus favored by selection.
The much greater multitude of metabolites in living organisms as
compared to their unexpected low calculated number by the small
number of relevant genes identified during genome sequencing
projects has puzzled biologists [14]. It has been postulated that
promiscuity of enzymes, including multiple product and substrate
enzyme specificity as well of changes in compartmentation patterns
may also contribute to plant metabolome diversity [15]. Moreover,
the more knowledge we mine on specific biosynthetic pathways and
the accumulation of information on the complexity of biosynthetic
networks have revealed that the transcription patterns as a whole
are rarely directly reflected in the proteome of an organism [16] and
in turn, the proteome in many cases only vaguely reflects the
metabolome [17]. The more knowledge we extract on the regulation
of plant natural product biosynthesis, the more puzzling it gets.
Perhaps the old-fashioned unpopular and forgotten theory of being
natural products part of an ‘‘aberrant metabolism’’ was not so off-
line. It thus seems that the combination of enzymes and substrates
generated by serendipity may be a platform to generate the much
needed variation to attain effectiveness in plant defenses, and this
process might be favored by evolution [7]. In this scenario, it is
evolutionary advantageous to keep parts of the enzymatic
machinery active through generations, and this in turn is the basis
of what we have defined as ‘‘silent metabolism’’: Occult biosynthetic
capacities that is not easily detected but readily active when
challenged. ‘‘Silent metabolism’’ can be either constitutive or
induced by development, the environment and genetic manipula-
tion, and might seem to the naı̈ve spectator as if active enzymes and
biosynthetic pathways do not have any apparent role in the tissue
studied, but are uncovered when a change in metabolism is effected.
In this review we will discuss a few of many examples of ‘‘silent
metabolism’’ based on our own results and on other published
work. They all indicate that plants have practically a network of
occult biosynthetic capacities that confers them an unlimited
potential to produce a large array of different compounds that can
be activated when novel substrates become available.
2. Occult terpenoid pathways
Many monoterpenes are key constituents of essential oils and
impart unique aromas to flowers and fruits. The metabolic
engineering of the monoterpene pathway has been the focus of
many studies [18]. The Clarkia breweri floral gene linalool synthase
(LIS) encodes an enzyme that catalyzes the formation of (S)-linalool
from the monoterpene precursor geranyl diphosphate. Over-
expression of Clarkia’s LIS in tomato fruit caused the accumulation
of (S)-linalool [19] as expected, but also the unexpected formation
of 8-hydroxylinalool, a compound absent in control fruits (Fig. 1).
The formation of 8-hydroxylinalool is thought to be mediated by
an endogenous ‘‘occult’’ hydroxylase activity able to act on (S)-
linalool. The actual biological role of this hydroxylase in tomato
fruit metabolism is presently unknown, but the novel availability
of substrate due to expression of a foreign gene enabled us to
uncover this activity. The Clarkia LIS gene was also over-expressed
in petunia flowers. In this case, (S)-linalool was formed but was
rapidly glycosylated (Fig. 1) [20]. In turn, when the Clarkia LIS gene
was over-expressed in carnation flowers, linalool was also
detected in the transgenic flowers but it was apparently further
metabolized to linalool oxides (Fig. 1) [21]. Thus, the over-
expression of an identical gene in different target tissues and
organisms gave rise to distinct phenotypes, according to the silent
metabolism present or induced in the target plant. Therefore, in
metabolic engineering experiments, one has to take into account
not only the potential functions of the genes manipulated, but also
the patterns of the silent metabolism in the target organism that
might interact with the novel products generated.
 E. Lewinsohn, M. Gijzen / Plant Science 176 (2009) 161–169
Fig. 1. Silent linalool metabolism revealed by metabolic engineering. Overexpression of the Clarkia breweri (S)-linalool synthase (LIS) gene in different target tissues and
organisms gives rise to different phenotypes. Linalool and 8-hydroxylinalool were detected in transgenic tomato fruit (top arrows [19]. Linalool glycoside was detected in
petunia flowers [20] and linalool oxides were accumulated in carnation flowers (bottom arrows [21]). The phenotypes reflect gene overexpression as modulated by the
existing or induced silent metabolism of the target organism.
Subsequent metabolic engineering of the terpenoid pathway in
tomato fruit further demonstrated the existence of a very active
grid of seemingly ‘‘occult’’ enzymes. The lemon basil (Ocimum
basilicum L. cv. Sweet Dani) geraniol synthase (GES) gene was over-
expressed in tomato fruits in attempts to modify their aroma and
flavor [22]. The GES gene encodes a protein able to synthesize
geraniol, a rose-scented monoterpene alcohol from geranyl
diphosphate. Diversion of the plastidial terpenoid pathway of
tomato to the formation of geraniol was achieved as evidenced by
the accumulation of relatively high levels of geraniol in transgenic
tomato fruit [22]. Nevertheless, transgenic fruit accumulated at
least eleven novel additional metabolites that share a common
chemical backbone and most likely are derived from geraniol.
These volatiles included the monoterpene alcohols nerol and
citronellol, the monoterpene aldehydes geranial, neral and
citronellal, the monoterpenol esters geranyl, neryl and citronellyl
acetate, geranic and neric acid and rose oxide (Fig. 2). The
conversion of geraniol to geranial and neral is catalyzed by alcohol
dehydrogenase, an enzyme long known to be active in tomato
fruits and characterized by a broad substrate specificity able to
accept ethanol (and also geraniol) as substrates [23]. Geraniol
reductase activity, able to generate citronellol from geraniol and
NADP+, was readily detected in nontransgenic tomato fruit [22].
Additionally, alcohol acetyltransferase activity able to act on
geraniol, nerol or citronellol and acetyl CoA to generate geranyl
acetate, neryl acetate, and citronellyl acetate, respectively, was
also readily detected in ripe control tomatoes [22]. The actual role
of these enzymes can only be speculated, but the above evidence
suggests that tomato fruits possess active but occult enzymatic
activities that can readily accept novel substrates if the substrates
become available. To conclude, metabolic engineering of the
plastidial terpenoid pathway revealed that a ‘‘silent’’ array of
enzymes is present or can be readily induced in tomato fruit. The
metabolic flow through this occult, ‘‘silent’’ (but not silenced) grid
can be rapidly triggered and detected when a novel substrate is
present.
163
2.1. Silent metabolism in carotenoid biosynthesis and degradation
reactions
Carotenoids are nutritionally important tetraterpenoid pig-
ments that that are also part of the photosynthetic apparatus
and are needed in human nutrition as pro-vitamin A. Attempts
to improve the nutritional value of rice have focused on
increasing the levels of b-carotene in the endosperm yielding
what we term ‘‘Golden Rice’’. This was attained by the ectopic
expression of PSY (phytoene synthase and CTRL1 a bacterial gene
coding for carotenoid desaturase [24,25]. In the initial experi-
ments, a construct directing the expression of lycopene b-
cyclase was also employed, to further direct carotenoid
biosynthesis to b-carotene but shown to be unnecessary, as
transgenes overexpressing PSY and CTRL1 only displayed a
similar carotenoid pattern as transgenes overexpressing the
three genes [24]. The product expected to be formed by the
action of the two carotenoid biosynthesis transgenes PSY and
CTRL1 is lycopene, a red pigment. Still, Golden Rice accumulates
b-carotene and xanthophyll derivatives. The absence of lyco-
pene in Golden Rice shows that the pathway proceeds beyond
the transgenic end point and thus that an endogenous pathway
was also acting. Moreover, by using realtime PCR, it was
elegantly shown that in wild-type rice endosperm the mRNA
expression of the relevant carotenoid biosynthetic enzymes
encoding phytoene desaturase, z-carotene desaturase, carotene
cis-trans-isomerase, b-lycopene cyclase, and b-carotene hydro-
xylase were all active. Only the mRNA corresponding to PSY was
virtually absent in wild type rice indicating that this was the
limiting step for carotenoid accumulation [26]. Thus, an occult,
silent, but potentially active biosynthetic pathway was revealed
in wild-type rice. This suggests that the wild ancestor of rice
contained a pigmented endosperm. This trait was probably
selected against early during rice domestication and this was
attained by incorporating mutants deficient in the expression of
PSY in cultivated rice.
164 E. Lewinsohn, M. Gijzen / Plant Science 176 (2009) 161–169

Fig. 2. Silent metabolism of geraniol in tomato fruit. The geraniol produced by the overexpression of the Ocimum basilicum geraniol synthase (GES) gene was isomerized,
oxidized, reduced and/or esterified to the monoterpene alcohols nerol and citronellol, the monoterpene aldehydes geranial, neral and citronellal, the monoterpenol esters
geranyl, neryl and citronellyl acetate, the monoterpene acids geranic and neric acid and to rose oxide. Some of the enzymes able to catalyze these reactions can readily be
measured in nontransformed fruit, including geraniol dehydrogenase, geraniol reductase, and an alcohol acetyltransferase activity readlily acting on geraniol, nerol or
citronellol and acetyl CoA to generate geranyl acetate, neryl acetate and citronellyl acetate, respectively [22].

Carotenoid content not only influences fruit color, but indirectly
affects the volatile composition of fruits. Pleiotropic effects on
aroma and flavor caused by genes affecting carotenogenesis have
been detected by utilizing defined mutants and near-isogenic lines
of tomatoes that solely differ in carotenoid patterns [27,28].
Similar effects of carotenoid content and composition on the aroma
apocarotenoid volatiles of watermelon, melon, bell peppers and
carrots [27–29] and [Azulay, Y., Tadmor, Y., and Lewinsohn E.,
unpublished results] have been noted. Carotenoids give rise to
apocarotenoid (norisoprene) aroma volatiles upon oxidative
cleavage [30], and thus carotenoid composition dictates the
composition of apocarotenoid volatiles. This relationship is
probably mediated by the oxidative cleavage of carotenoids into
apocarotenoid volatiles [19,27]. A group of carotenoid cleavage
dioxygenase enzymes (CCD’s) catalyze such cleavages and accept a
variety of carotenoids as substrates [19,27,29,31–33]. b-Carotene
is the predominant pigment in orange-fleshed melon (Cucumis
melo) varieties, while pale-green and white cultivars have much
lower b-carotene levels. In parallel, the potent odorant b-ionone,
the 9, 10 (90 , 100 ) cleavage product of b-carotene, is present in
orange-fleshed melons and in much lower levels in pale green and
white fleshed varieties. A search for a gene putatively responsible
for the cleavage of b-carotene into b-ionone was carried out in
annotated melon fruit EST databases yielding a sequence
(CmCCD1) similar to other plant carotenoid cleavage dioxygenase
genes [29]. Interestingly, the sequence originated from ‘Tam Dew’
melons, a pale green fleshed variety that lacks either b-carotene
and its oxidative cleavage product b-ionone. Still, the CmCCD1
gene is up-regulated upon maturation, similarly to the corre-
sponding CCD genes in orange-fleshed varieties that contain high
levels of both b-carotene and its degradation product b-ionone
[29]. To assess the biochemical functionality of CmCCD1, the clone
was overexpressed in E. coli strains previously engineered to
produce b-carotene. The CmCCD1 gene product efficiently cleaved
b-carotene at the 9, 10 and 90 , 100 positions and b-ionone was
released from the bacterial cultures. Further examination revealed
that the CmCCD1 gene product cleaves many other carotenoids,
but only at positions 9, 10 and 90 , 100 , generating geranylacetone
from phytoene; pseudoionone from lycopene; as well as a-ionone
and pseudoionone from d-carotene. These acyclic and monocyclic
carotenoids are normally absent in melons, still the CCD enzyme
efficiently accepted them as substrates generating novel apocar-
otenoid volatiles. Thus, it seems that the broad substrate specificity
of the CmCCD1 allows the formation of novel volatile metabolites
when the proper carotenoid substrate is available. A similar CCD
enzyme with a broad substrate-specificity was also discovered in
tomatoes [33]. These findings easily explain the pleiotropic effects
on aroma previously observed in tomato carotenoid mutants and
the general correlation between carotenoid composition and
apocarotenoid aroma chemicals observed [27,28]. However, the
question of why is an active CCD enzyme is needed in the fruits of a
melon genotype that lacks b-carotene, the apparent substrate of
this enzyme.
Carotenoids are biosynthesized and accumulated within
plastids, yet the carotenoid cleavage dioxygenases are apparently
cytosolic. Thus, it is likely that subcellular compartmentation
limits the access of the CCD enzymes to carotenoid substrates and
limits carotenoid cleavage. This might explain the vast difference
(about a thousand fold) between the carotenoid levels (measured
in mg/g FW) and apocarotenoid aroma levels (measured in mg/g
 E. Lewinsohn, M. Gijzen / Plant Science 176 (2009) 161–169 165

FW) in many fruits [19,27]. However, this leaves the question open
for speculation if carotenoids are the bona fida substrate for CCD’s
or whether these genes actually have a different role in the cell
metabolism and serendipitously accept carotenoids as substrates.
One could speculate that this activity was important in the melon
wild progenitors, and the gene was retained without any apparent
function during domestication.
3. Silent metabolism in the biosynthesis of volatile ethers
The scent of roses is a very complex trait that is a result of the
presence of dozens of aroma chemicals that have distinct
biosynthetic origins; among those are the mono- and sesquiter-
penes, phenylalanine derived compounds, fatty acid derivatives
and phenolic methyl ether compounds. The modern hybrid tea
rose cultivars emit phenolic methyl ethers, a group compounds
that are only present in the Chinese ancestral species and not in the
European ancestral species [34,35]. The last steps in the formation
of these compounds are the sequential methylation of orcinol (3,5-
dihydroxytoluene) and 3-hydroxy, 5-methoxytoluene to give rise
to orcinol dimethyl ether (3,5-dimethoxytoluene). The reactions
are catalyzed by O-methyltransferases (OMT’s) that utilize S-
adenosyl methionine as a methyl donor and an alcoholic substrate
as an acceptor. Hybrid roses possess two types of orcinol
methyltransferase genes with similar but not identical substrate
preferences OOMT1 and OOMT2 [34,36]. Both gene products
perform both of the methylations involved, but OOMT1 is much
more active towards orcinol than towards 3-hydroxy, 5-methox-
ytoluene. Interestingly, OOMT2-like genes seem to be present in all
rose types, while OOMT1 types are only present in Chinese types
and their progenies [35]. Still, only those roses that possess OOMT1
emit orcinol dimethyl ether. European roses, that do not emit
orcinol dimethyl ether, still express OOMT2 and possess enzymatic
capability in vitro to produce orcinol methyl ether from orcinol,
indicating that another biosynthetic step must be preventing
orcinol dimethyl ether accumulation. Thus, if OOMT2 is not
operational in European roses, why is it active in cell-free extracts?
It could therefore be that the biological function of OOMT2 is not
related to orcinol dimethyl ether formation, providing another
example of occult metabolic capabilities driven by ‘‘silent’’
metabolism.
Chemical diversity with respect to volatile monoterpene and
phenylpropene contents has been documented and rationalized in
sweet basil (Ocimum basilicum), using genomic and metabolomic
tools [12,16,37]. A sweet basil type that contains predominantly
the phenylpropene derivative estragole, possess OMT activities
able to methylate chavicol to estragole, while a type of basil that
accumulates only methyl eugenol (a 3 methoxylated estragole
derivative) possesses an activity able to methylate eugenol to
methyl eugenol (Fig. 3). However, if chavicol is offered as a
substrate it will be methylated to estragole in cell-free extracts,
although the plant itself does not contain estragole. Moreover,
some of the lines able to O-methylate chavicol in vitro, do not
accumulate estragole in vivo, and some of the lines are able to O-
methylate isoeugenol in vitro to generate methylisoeugenol, a
compound that has not been detected in sweet basil. Interestingly,
the ‘‘lemon-scented’’ basil line 197 contains citral (a mixture of the
monoterpene aldehydes geranial and neral) but lacks volatile
phenylpropenes in its essential oil (Fig. 3). Still, cell-free extracts
derived from this lemon-scented line can readily O-methylate
chavicol to estragole, eugenol to methyl eugenol or isoeugenol to
methyl-isoeugenol (Fig. 3) although none of these compounds are
present in the plant. This further indicates the presence of ‘‘silent’’
O-methyltransferase activities in basil lines that may readily

Fig. 3. Silent metabolism in phenylpropene metabolism in basil (Ocimum basilicum). Essential oil compositions (left panels) were compared to phenylpropene O-
methyltransferase towards various substrates (right panels). ‘‘Exotic’’ basil contains almost exclusively methyl chavicol, but displays enzymatic activities able to act on
chavicol, eugenol and isoeugenol Line R3 contains almost exclusively estragole and linalool, and is able to methylate chavicol at a high rate as expected; but unable to
methylate eugenol or isoeugenol. Line 145 possesses almost exclusively estragole, and displays a specific chavicol O-methyltransferase activity generating estragole, but
unable to act efficiently on either eugenol or isoeugenol. Line 149 possesses methyl eugenol and some methyl chavicol in its essential oil and displays O-methyltransferase
activity towards chavicol, eugenol, and isoeugenol. Line ‘‘4’’ Contains eugenol and linalool (lacks p-methylated volatile phenylpropenes, and possess no detectable O-
methyltransferase activity as expected. Line 197 is a lemon-scented basil and contains almost exclusively the monoterpene aldehydes citral, still it displays high chavicol,
eugenol and isoeugenol O-methyltransferase activity. Adapted from [55].
166 E. Lewinsohn, M. Gijzen / Plant Science 176 (2009) 161–169

accept novel substrates if the ability to produce those substrates is

acquired by breeding or mutation, easily yielding to novel
chemotypes.
Two types of genes coding for phenylpropene specific OMT’s
are found in sweet basil. Both gene product types O-methylate
phenylpropene alcohols at the 4 position. CVOMT1 readily
accepts chavicol to generate methylchavicol (estragole), while
EOMT1 accepts eugenol to generate methyleugenol, but still
retains the ability to methylate chavicol to estragole at a
significant rate. A single T-to-C mutation, causing a phenyla-
lanine residue at position 260 changed to a serine controls this
substrate specificity [12]. Nevertheless, plants possessing EOMT
activity still have the ‘‘silent’’ ability to O-methylate chavicol to
estragole if chavicol is offered as a substrate. Breeders can
predict that if the trait to generate the substrate chavicol was
introduced by crosses or metabolic engineering the resulting
progenies will contain estragole by the action of silent
metabolism.

4. Silent metabolism in the phenylpropanoid pathways

The flavonoid phenylpropanoid biosynthetic pathways of

plants have been studied from many angles due to the importance,
ubiquity, and the visibility of the products.

4.1. Flavonoid biosynthetic pathways provide numerous examples of

silent metabolism

Phenotypes that are controlled by flavonoid pigments are

powerful tools for genetic investigations because of they can easily
be measured. The coloration of flowers, seeds, and other plant
organs captures our attention, even if one is not a biochemist,
geneticist, or plant biologist. It also seems that flavonoid pathways
determine traits that are subject to heavy selection pressures; at
least this is clearly so for domesticated crop and ornamental plants.
The flavonoid pathway provides a rich source of examples to
illustrate silent metabolism because it is such a large and well-
Fig. 4. Wild and cultivated soybean seed coat. (A) Schematic illustration of the
soybean seed coat, in cross section. Anthocyanin pigments accumulate in the
palisade epidermis while peroxidase is concentrated in the hourglass cells.
Hourglass cells are approximately 75 mm in length. (B) A photograph of commonly
grown yellow-seeded soybeans, lacking seed coat pigmentation due to the
dominant I locus (Glycine max cv. Lindarin). (C) A photograph of wild-type
soybeans with a black-seeded phenotype due to accumulation of anthocyanin
pigments (Glycine max PI437654).

studied pathway that has evolved a variety of functions that serve

plants. With regard to seed crops, for most species the selection has
been for lack of color. Wild wheat, rice, barley, soybean and many
other crops have colored seed while most domesticated strains
lack color.
4.2. Silent flavonoid metabolism in soybean seed coats
Seed coat pigmentation in soybean is determined by the
accumulation of anthocyanin and proanthocyanidin molecules in
the palisade cells of epidermis [38]. Nearly all commonly grown
soybean cultivars are yellow-seeded but may vary with regard to
pigmentation of the hilum (point of connection of the seed to the
ovary wall). Conversely, wild-type soybeans are deeply pigmented
and black-seeded (Fig. 4). Over the years, through genetic crossing
and inheritance studies, a bewildering array of seed coat
pigmentation phenotypes have been uncovered and described
[39]. There are many genes that can influence seed coat
pigmentation, but three major ones, I, R, and T, have the greatest
effects [40]. Among these, the I locus is the most important. This
gene controls the expression and distribution of chalcone synthase
in the seed coat tissues, and thus it effectively operates as a
bottleneck that can restrict entry of substrate into various
branches of the flavonoid pathway [41]. The I locus exerts
powerful epistatic effects that can mask phenotypes conditioned
by R and T, because these other genes operate in pathways that are
downstream from chalcone synthase. The I locus is also tissue
specific, only altering flavonoid metabolism in the seed coat. This is
likely because flavonoids play essential roles in other plant tissues,
such as in reproductive tissues where they are essential for
fertility.
Even though most commonly grown soybeans are yellow-
seeded due to a lack of chalcone synthase in the seed coat tissues,
genes corresponding to steps in the downstream pathways
continue to be expressed at high levels. For example, dihydro-
flavonol-4-reductase (DFR) and flavonoid 30 hydroxylase (F30 H)
transcripts are highly expressed in seed coats where they could
have participated in the biosynthesis of anthocyanin pigments
during the late stages of seed development. Moreover, these genes
are also expressed in yellow-seeded phenotypes that do not
accumulate any pigments [42,43]. The isoflavonoid branch also
appears to be constitutively active in seed coats, regardless of
chalcone synthase levels, as evidenced by the expression of
isoflavone synthase [44]. But in this case, isoflavonoid products can
be detected in seed coat tissues, whereas anthocyanins clearly are
not present. How does this occur? There are several possibilities,
but it seems most likely that the isoflavonoids present in
developing seed coats were synthesized in other plant organs
and simply transported to the seed coat. In any case, it is apparent
that the seed coat is programmed to make anthocyanins,
isoflavonoids, and other phenylpropanoids that are dependent
upon the key entry point enzyme, CHS, regardless of whether there
is a sufficient amount of this enzyme to ensure flux through the
pathway.
 E. Lewinsohn, M. Gijzen / Plant Science 176 (2009) 161–169
4.3. Soybean seed coat anthocyanin pigments are substrates for the
seed coat peroxidase enzyme
Another example of silent metabolism in soybean seed coats
that is apparently linked to anthocyanin pigmentation relates to
the accumulation of peroxidase. The soybean peroxidase is a class
III plant peroxidase and is the most abundant protein in mature
seed coats, where it accounts for approximately 5% of the total
soluble protein [45]. The occurrence of soybean peroxidase in the
seed coat is controlled by a single dominant gene, Ep. Homozygous
recessive epep genotypes do not accumulate soybean peroxidase
due to a deletion mutation in the structural gene encoding the
enzyme [46]. Despite this difference in the amount of soybean
peroxidase enzyme in Ep- versus ep ep genotypes, there is no
known phenotypic effect such as change in seed color, perme-
ability, lignification, longevity, or hardness that can be associated
with the Ep locus. Thus, the function of SBP in the seed coat remains
enigmatic. Perhaps this is because its role is dependent on the
presence of anthocyanin pigments that are absent from virtually all
modern soybean cultivars.
The participation of anthocyanin pigments and other flavonoid
molecules in oxidative reactions is gaining more attention, since
this may constitute a significant portion of their functionality.
With regards to soybean seed coats, wild-type (black) soybeans
accumulate large amounts of anthocyanin pigments and soybean
peroxidase in separate but adjacent cell layers of the seed coat. The
anthocyanins are concentrated in the palisade cells of the
epidermis of the seed coat, whereas soybean peroxidase accumu-
lates within the hourglass cells of the subepidermis [45,47]. The
vacuolar targeting signal of the soybean peroxidase is somewhat
unusual among class III plant peroxidases, as most other enzymes
of this type are secreted into the apoplast. Nonetheless, maceration
of the seed or imbibition of water simultaneously releases these
two soluble seed coat constituents. In fact, seed coat anthocyanin
pigments are substrates for soybean peroxidase-catalyzed reac-
tions, as shown in Fig. 5. The polymerization of seed coat
anthocyanin pigments by soybean peroxidase into insoluble
products may provide defense or protection for the seed. It is
reminiscent of binary systems where enzymes and substrates are
spatially separated in different cell types or layers, such as
glucosinolates and cyanogenic glycosides [48]. Thus, it is plausible
that function of SBP is related to its activity towards anthocyanin
Fig. 5. Polymerization of anthocyanin pigments by seed coat peroxidase.
Anthocyanins purified from soybean seed coats were dissolved in 250 mM
NaH2PO4 buffer (pH 7.0), and H2O2 was added to 0.01% (v/v), alone or in
combination with purified soybean seed coat peroxidase (SBP) enzyme. Photos
were taken 5 min and 24 h after additions. This figure illustrates that SBP catalyzes a
rapid polymerization of anthocyanin pigments into insoluble products. The
addition of H2O2 alone, without SBP, can de-colorize solutions of anthocyanin
pigments over long periods, but no precipitate forms under these circumstances.
167
pigments, and that this has become obscured as a consequence of
domestication and selection of yellow-seeded types.
5. Mechanism of action of silent metabolism
5.1. Silent metabolism and relaxed selection
It is instructive to consider the idea of silent metabolism
together with the well-known genetic concepts of epistasis and
relaxed selection, as well as the related topics of sub- and neo-
functionalization of gene products. In cases where genes interact to
produce particular phenotypes, epistatic genes may either hide or
reveal the phenotypic expression of other genes. This differs from
dominant and recessive interactions because the genes are not
allelic. For example, epistatic interactions are commonly observed
among genes that encode enzymes within a linear biochemical
pathway. The catalytic efficiency, substrate specificity, or other
characteristics of a particular enzyme in the pathway can alter the
final products or the metabolic flux through the entire path. A
mutation in a gene that results in a non-functional enzyme would
effectively block the pathway at that step, possibly leading to
accumulation of substrate upstream and a depletion of substrate
downstream. For individuals that carry such an epistatic mutation,
the enzymes that operate downstream are biochemical orphans
and become part of the silent metabolism of the plant cell. Thus,
epistatic genetic effects may provide a context for silent
metabolism that is otherwise cryptic or puzzling.
Epistatic genetic effects that become fixed in a population may
result in the degeneration of a biochemical pathway [49] or supply
opportunities for enzymes that have become biochemical orphans
to evolve new capabilities. Genetically, this is considered a form of
relaxed selection, a reduction or removal of selective pressure that
operates on a gene. Gene duplication is another genetic mechanism
that results in relaxed selection and offers similar prospects. The
sub-functionalization of an enzyme, so that it acquires character-
istics that are somewhat different from its ancestral type, is an
adaptation that is facilitated by removing any conservative selective
pressure. Likewise, neo-functionalization is the acquisition of a
completely new property that is distinct from its ancestral type. This
occurs more rarely but potentially offers the organism novel traits
that may drastically alter its fitness and success. Therefore, the silent
metabolism of a plant cell represents a biochemical manifestation of
the genetic notion called relaxed selection.
The impact of gene duplication on primary and secondary
metabolic pathways has been estimated using the model plant
Arabidopsis [50]. The relatively high levels of intraspecific variation
observed in pathways of secondary metabolism contrast with the
general conservation of function in primary metabolism. The
flexibility of secondary metabolism is supposed to be enabled by a
greater genetic redundancy of these pathways compared to
primary metabolic routes [50]. It is a compelling hypothesis that
plant populations maintain a diverse reservoir of secondary
metabolic capability to meet the ever changing selective pressures
in their environment.
6. Concluding remarks
It has become evident that the initial Darwinian view of
biological systems that have been optimized by evolution is much
more complex that we initially thought. The large phytochemical
diversity that we find in nature is a result of the different selection
pressures that plants have successfully coped with during
evolution. The ubiquity of plant silent metabolism may be a great
advantage in a changing and evolving world. It is likely that plants
have evolved means of preserving the biochemical information to
168 E. Lewinsohn, M. Gijzen / Plant Science 176 (2009) 161–169
generate chemicals without actually generating them. Moreover,
the availability of plant complete genomes and the advancement in
our understanding of gene evolution, has led to the discovery that
plants that do not normally accumulate a certain natural product
still posses homologous genes to those present in plants that
actively synthesize them [51].
It has long been known by geneticists that through hybridization
it is possible to augment the generation of qualitative and
quantitative variation in secondary chemistry. Qualitatively, hybrids
may express all of the secondary chemicals of the parental taxa, may
fail to express certain parental chemicals, or may express novel
chemicals that are absent in each parent. Quantitatively, concentra-
tions of parental chemicals may vary markedly among hybrids. This
phenomenon has been termed ‘‘additive inheritance’’ [52], and it
might be a reflection or operate in a similar or related way as the
phenomenon long known as ‘‘heterosis’’ or ‘‘hybrid vigor’’. In essence,
each parent may contribute previously silent but complementary
enzymes to generate complementary networks, and thus the
hybrid’s phytochemistry might be very different to that of their
parents. Recently, a combined metabolomic–genomic set of tools
have been employed to rationalize flavonoid biosynthesis in poplar
and has provided interesting insights about this phenomenon [53].
Most of the biochemical, molecular and genetic studies on
secondary metabolism have been conducted using domesticated
plants. All cultivated crops originated from wild relatives and
many of the original traits present in the progenitors have been lost
through crop domestication [54]. Cultivated plants have been
subjected to directed hybridization and selected taking into
account commercially important or practical traits. Thus, breeding
and crop improvement has allowed for a limited set of important
traits to prevail and be overrepresented in our modern crops. The
number of generations in which selection pressure has been
attained is very small as compared to the number of generations
that wild plants have experienced selection pressures throughout
natural evolution. Silent metabolism could be a relic of important
and valuable traits or functions that the ancestral wild plants
acquired and preserved during their evolution, but their cultivated
offspring have lost. If the expression of a silent partial pathway or
biochemical orphan do not have harmful effects and do not
significantly affect the overall fitness of the plant, only very little
selection pressure is applied towards eliminating those traits, and
therefore they will be conserved during the domestication process.
Another possibility of the widespread occurrence of silent
metabolism is that many of the enzymes we detect as ‘‘silent’’ do
have an important yet unknown metabolic function. Due to the
broad substrate specificity displayed by many of these enzymes,
novel substrates can be readily utilized by serendipitously
recruited enzymes and novel phenotypes can be generated that
are subjected to natural or human selection. Thus, if the new traits
generated provide an evolutionary value, they will be retained.
Increasing and retaining phytochemical diversity is key to the
survival and adaptation of plants, and ‘‘silent metabolism’’ might
be one of the key factors by which plants cost-effectively retain
their flexibility to defend themselves and communicate with other
organisms. In this context, silent metabolism is not at all silenced,
but is a loud reservoir of metabolic pathways that can easily be
activated, conferring fitness benefits to the bearing organism.
Silent metabolism constitutes an evolutionary asset and thus
widespread the plant kingdom, contributing to the impressive
phytochemical diversity that we presently experience.
Acknowledgements
We thank Yaron Sitrit, Kobi Tadmor and Ilan Levin for useful
comments.
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