Plant Molecular Biology 24: 1-20, 1994. 1994 KIuwer Academic Publishers. Printed in Belgium.

Mini-review

Physiological roles for secondary metabolites in plants: some progress, many outstanding problems
M.J.C. Rhodes AFRC Institute of Food Research, Norwich Research Park, Colney, Norwich, Norfolk NR4 7UA, UK
Received and accepted 21 October 1993

Introduction

Kossel [45] was the first to introduce the term secondary metabolite over one hundred years ago, stating that 'whereas primary metabolites are present in every plant cell that is capable of division, secondary metabolites are present only "accidentally" and are not essential for plant life'. Kossel thus instigated the distinction which is still drawn between primary and secondary metabolism. Primary metabolites number, at most, a few hundred low-molecular weight compounds and are common to all plants. However, many thousands of secondary metabolites have been isolated from plants and their chemical structures elucidated. In contrast to primary metabolites, individual secondary compounds vary widely in their distribution between plant species and some may occur only in a single, or a few related, plant(s). Our present view of the complexity of the chemistry of plants has come largely from the activities of natural product chemists adept at extracting and purifying compounds to a sufficient level for their structure to be determined. As the methodology for the elucidation of chemical structure has evolved with improvements in GC and HPLC coupled to mass spectroscopy, and in NMR, it has been possible to characterise even the minor components of plants. Such studies have revealed the immense diversity and complexity of plant chemistry. Within each of the major groups of secondary metabolites such as alkaloids, phenylpropanoids and terpenoids, sev-

eral thousand individual compounds accumulating in plants have been characterised [52]. A distinction should be drawn between synthesis and accumulation of secondary metabolites. Our knowledge of the chemistry of the plants studied is based on compounds which accumulate to a detectable level. As virtually all secondary products undergo turnover, their accumulation is the resultant of synthesis and degradation. There are several examples where compounds were first detected in plant species where, uniquely, they accumulate to a high level and were subsequently shown to be universally present in plants even if at low levels. An example of this is ACC (1-aminocyclopentane- 1-carboxylic acid) which was isolated and characterised first from pear fruit where it accumulates to high levels [9], many years before its role in ethylene biosynthesis and its ubiquitous presence as a metabolic intermediate in plants was established [100]. It has been estimated that less than 10~o of known plant species have been studied chemically and even in many of these species the investigation has been limited to a few classes of compounds. Undoubtedly our present knowledge severely underestimates the number and range of secondary products made by plants. It is a feature of plant secondary metabolism within a particular species that a multiplicity of related compounds, often with common chiral (stereochemical) specificity are generated. Plants are extremely adept at complex chiral syntheses often routinely completing feats of synthesis at or

beyond the limits of the capabilities of current organic chemistry. This complexity and the limited distribution of some secondary metabolites has until recently led studies of secondary metabolism to be relegated from the mainstream of plant science. A factor leading to this has been the concentration of studies on economically important crops which have been bred for low levels of secondary products often with undesirable organoleptic, toxicological or anti-nutritional properties (tannins, glycoalkaloids, etc.). However in crop species such as Daucus carota (carrot) at least twelve different secondary pathways are known to operate [42]. It remains a major challenge to plant scientists to assimilate this diversity of chemistry within plant species into a broader appreciation of the roles secondary metabolites play in plant biology. Molecular biology has a key role to play in helping to elucidate the specificity of secondary metabolism and its regulation, and to probe the roles played by secondary metabolites in plant ontogeny. Much of the impetus to studies on the function of secondary metabolites at the molecular level stems from the pioneering investigations of the biochemistry of the phenylpropanoid and flavonoid pathways in the 1960s and 1970s [34]. In these studies the route of biosynthesis and enzymology of the flavonoid pathway were elucidated. The isolation and characterisation of most of the genes of the pathway followed leading to rapid advances in our understanding of its regulation at both the biochemical and genetic levels. Expression of these pathways is affected by a wide range of internal and external factors and regulation is predominantly effected by control at the transcriptional level [46]. However, control at both the transcriptional [ 50] and enzyme protein levels [5] mediated by metabolic intermediates also operates for at least some of the individual steps. The availability of these genes has enabled the role that phenylpropanoid-derived secondary products play in the plant to be investigated at the molecular level. However, progress in the understanding of other secondary pathways both at the biochemical and genetic levels generally lags far behind that of the phenylpropanoids. In this re-

view, some of the main features of secondary metabolism will be outlined and the contribution molecular techniques are making, and can make in the future, to clarifying the role and importance of individual secondary metabolites will be discussed. The chemical structures of many of the secondary compounds discussed in this review are shown in Fig. 1.

Some characteristics of secondary metabolic pathways

Several major pathways of secondary metabolism such as those of terpenoid and phenylpropanoid metabolism operate in essentially all higher plants. All secondary metabolic pathways originate from primary precursors and are often viewed as extensions of these primary pathways. However, such relationships are not always clearcut. The terpenoid pathway can be characterised as a primary pathway leading to essential metabolites such as membrane sterols and plant growth substances such as gibberellins and abscisic acid. This pathway may be represented as highly branched where intermediates are diverted at various levels to form terpenoid secondary products such as the monoterpene components of essential oils or sesquiterpenoid phytoalexins such as rishitin or capsidiol. However, although overall many pathways are branched, major arms of the pathway may show cell or organelle compartmentation and what may be represented as a single-branched or an extended pathway may, in reality, be several parallel pathways which are separately located and independently regulated. For example, the monoterpene components of essential oils are formed in specialised cells in oil glands on the leaf surface of such species as Mentha and Salvia and are secreted from the producing cells and stored exogenously in the space between the secretory cells and the overlying cuticle. Recent evidence suggests that plants have a range of prenyltransferases devoted to the formation of particular types ofterpenoids. Thus in Salvia, the epidermal layer rich in glandular tissue houses a prenyltransferase dedicated to the production of

FLAVANONFS H O H m , OH 0 FLAVONOLS
OH. ~

I~__R 2

Naringenin

R2=OH

.O,.

~//3'X~ R1

Kaempferol Quercetin Myricetin

OH O BENZOIC ACIDS

Isorhamnetin

R1 R2 R3 H OH H OH OH H OH OH OH OMeOH H

(OH
COOH
Salicylic a c i d

COOH 9

COOH ~OMe OH
Vanillic acid

COOH OMe ~ ~OMe OH OH

COOH

OH
p-Hydroxybenzoic acid

OH

Syringic acid

Protocatechuic acid

CH 3 O
Jasmonic acid

O
Pinosylvin

~ R=H R=OH

Resveratrol

TROPANE ALKALOIDS
N CH 3 N .CH3

O
Hyoscyamine

CH20H

- -OL
CH2OH

Scopolamine

Fig. 1. The chemical structures of some of the secondary metabolites mentioned in the text.

C10 (geranyl diphosphate) rather than longerchain intermediates [27]. Enzymes of monoterpene biosynthesis are similarly localised in this tissue fraction suggesting that the monoterpene pathway is not a branch of a pathway leading to higher terpenoid products but rather is a separate pathway with distinct specialised enzyme machin-

ery [27]. Thus competition for geranyl diphosphate between the first enzyme devoted to monoterpene metabolism, limonene cyclase, and the prenyltransferase extending the C 10 intermediate to C15 and beyond to form higher terpenoids would not be a major factor of control. Such an organisation would allow for separate and unique

4 regulatory mechanisms to act independently of effects on production of other terpenoids. Another feature of secondary metabolism is that many secondary substances are synthesised in condensation reactions between intermediates which arise from different secondary pathways. For instance, the tropane alkaloid hyoscyamine is formed in a condensation reaction between a tropine moiety derived from arginine and an aromatic acid formed in a separate pathway from phenylalanine. A complicating feature of secondary metabolism is that a multiplicity of compounds are formed from a given chemical nucleus both within a species and, to a greater extent, across plant families. Typically a parent compound with highly defined stereochemistry is formed in a core pathway and a wide range of derivatives of this core compound are formed, accumulating to widely varying degrees, some at very low levels. These modifications are either substitutions around the parent compound by hydroxylation, methylation, oxidation etc. or the formation of conjugates. In one plant, Catharanthus roseus, over 100 indole alkaloids have been isolated [83 ]. The broader diversity of secondary metabolites can be illustrated by the phenylpropanoids and flavonoids which are formed in most, if not all, plant families. Cinnamic acids vary in the pattern of substitution around the aromatic nucleus but all have the same trans-configuration in the propane side chain. Free cinnamic acids are metabolic intermediates in plants but generally are present at only very low concentrations. However, cinnamic acid conjugates accumulate in most tissues to very significant levels. Cinnamic acids combine with a range of substances including sugars (glucose, rhamnose, fructose, rutinose etc.), organic acids (quinic, shikimic, tartaric acid, malic and malonic acids), amine compounds (putrescine, tryptamine), alkaloids (tropanes) and terpenoids (borneol). They also form conjugates with other phenolic compounds or are exported and covalently bound into the cell wall. Flavonoids are derived from the flavanone, 2-(S)-naringenin which arises from p-coumaroyl CoA and malonyl CoA by the concerted action of chalcone synthase (CHS) and chalcone-flavanone isomerase (CHI). The main classes of flavonoids, flavonols, flavones and anthocyanins arise from naringenin by modification of the B-ring by oxidation and hydroxylation. Flavonoid aglycones are important metabolic intermediates. These aglycones show significant differences in their patterns of substitution around the A and C rings forming a range of hydroxyl compounds and methyl ethers. However, the major ftavonoid compounds which accumulate in plants are the glycosides of these compounds and these are found in an almost bewildering diversity of forms. For instance, the flavonol quercitin has been shown to form as many as 80 different glycosides in plants [36]. Glycosylation in the 3 position occurs with glucose, the most common sugar, but rhamnose, galactose and arabinose glycosides are also abundant. Similarly, glycosylation of hydroxyl groups on both the A and C rings also occurs. In some cases, these sugars undergo acylation with organic acids such as malonic acid and with phenolic acids principally p-coumaric and ferulic acids. In addition to glycosides, some plant families accumulate sulphate esters of flavones and flavonols. These are formed by the action of a flavonol sulphotransferase which uses 3'-phosphoadenosine 5'-phosphosulphate as the sulphate donor, cDNAs for these sulphotransferases have recently been cloned from Flaveria chloraefolia [87]. In addition to the water-soluble ftavonoids, liphophilic flavonoids occur on leaf surfaces. Among these are the range of furanoflavonoids found on the leaf surface of Vellozia stipirata [93]. A fundamental characteristic of secondary metabolism is that secondary products are not found uniformly throughout the plant but are frequently limited to particular organs and to particular cells and tissues within that organ. The expression of secondary pathways is often a feature of cell specialisation and is integrated into the pattern of differentiation of those cell or tissue types. Such patterns of expression are most obvious with pigments but similar patterns of limited expression exist for all classes of products. Molecular studies of the phenylpropanoid path-

way have shown that this distribution of these secondary metabolites is controlled at the genomic level by organ- and tissue-specific sequences in the 5' regions of the genes coding for the enzymes of phenylpropanoid and flavonoid biosynthesis [4, 56] and by regulatory genes which co-ordinate the expression of individual structural genes [29]. The organ in which secondary products accumulate is often different from that in which their biosynthesis occurs. For instance, the capacity for nicotine biosynthesis is limited to the roots of tobacco plants even though nicotine accumulates in leaves as well as roots. The tropane alkaloid hyoscyamine is made exclusively in the roots of Dubiosia plants and exported to the aerial parts of the plant. However, its epoxidation to form scopolamine occurs in green tissues which lack the capacity for de novo tropane biosynthesis [39]. It is clear in this case that transport of an intermediate in the biosynthesis of tropanes occurs between the root and the shoot.

Biochemical specificity of secondary metabolic pathways

The tissue specificity of biosynthesis of secondary products and the production of compounds of defined stereochemistry suggests a tightly controlled and regulated biochemistry. There is growing knowledge of the routes and enzymology of many plant secondary pathways [69]. There is no evidence overall that enzymes of secondary metabolism are any less specific for their substrates than those of primary metabolism; both areas involve enzymes ranging in their degree of specificity for their substrates. It has been suggested [52] that enzymes of secondary metabolism can be divided into two classes, highly specific enzymes involved in the formation of the basic skeleton of a series of compounds often with defined stereochemistry, and those involved in the modification of this basic structure, such as dehydrogenases, mono-oxygenases, methyltransferases and glycosyltransferases which, although stringent for the stereochemistry of the parent compound, are

less specific for the pattern of substitution of that parent compound. It is suggested that the action of this group of relatively non-specific enzymes could, in part, explain the proliferation of related secondary metabolites in plant tissues. For instance, Datura and related solanaceous species produce tropane alkaloids: the parent compound is tropine which has the 3c~-hydroxyl configuration and combines to form a range of esters; the main ones are derivatives of tropic acid, hyoscyamine (atropine) and scopolamine but these are found together with a range of acylation products with other acids such as phenyllactic, tiglic, acetic, methylbutyric, isovaleric and substituted cinnamic acids. In transformed root cultures of a Datura hybrid as many as 28 different acyl derivatives of tropine were described [ 70 ]. The type and quantities of these acylated derivatives vary considerably between tropane-producing species. In addition, many of these species also form an isomer of tropine with the 3/? configuration (pseudotropine) and a series of acylated derivatives retaining the stereochemistry of this parent compound. The o~-tropine and /?-tropine arise from a common precursor, tropinone, by two separate dehydrogenases (TR1 and TR11) with opposite stereospecificity, TR1 producing e-tropine and T R l l producing /?-tropine [40, 63]. Two acyltransferases specific for either :~- and /?-tropine involved in the formation of aliphatic esters of tropine have been partially purified from extracts of transformed roots of D. stramonium [71]. Although these transferases are specific for the tropine moiety they are less so for the acylating group and their activity can explain the accumulation of the wide range of aliphatic esters of both c~- and/?-tropine found in Datura [72]. The biochemical specificity and complexity of secondary metabolism is well illustrated by studies on the formation of the alkaloid berberine in cell cultures of two genera, Berberis and Coptis [98, 103]. This pathway is relatively limited in its distribution among plant species, yet involves very specific enzyme proteins and has sophisticated organisational and regulatory features. The biosynthetic pathway (see Fig. 2) originates from the condensation of two tyrosine-derived intermedi-

Ho.fx-U Ho.<.r
)<- ~ NH2 At-.. J/. .5I--H CHO~

c.3o
k II -- n e

c.3o.c.Uh
"--H ~ ~ ~N--CH3 ~ OH ~ ( S~ - N i M ~ O )

Z
OH" ~ ~j,,,~ OH" ~ .,'>{"

(S)-Scoulerine ~

Ha

(s)-eeticuline

"<7
~

"OCH3

V/'OH

CH30~
CH2\ < . ~ . ~ O C H " ] ~
q ~ OCH3
"OCH 3

(S)-Tetrahydrocolumbamine CH30 -

COrnS ~""OCH~ (S)-Tetrahydroberberine :

I

BERBERIS

/o CH~ ~

Columbamine
Fig. 2.

The pathway of berberine biosynthesis in Berberis and Coptis cell cultures. The divergence at the final two steps is indicated by ~ Berberis route and . . . . . ~- for the Coptis route.

ates, dopamine and 4-hydroxyphenylacetaldehyde to form (S)-norcoclaurine. The (S)configuration established during this condensation is retained throughout the pathway. The conversion of this precursor to berberine involves the combined action of three O-methyltransferases and one N-methyltransferase. Although these enzymes are catalytically very similar using S-adenosylmethionine as the methyl donor, their specificities for their alkaloid substrates are very high such that it is clear that the enzymology determines the sequence of the methylation reactions; 6-0 methylation proceeds N-methylation and then 4'-O methylation and finally methyla-

tion of 9-0. These four methylations involve four separate enzyme proteins. The specificity is illustrated by the 9-0 methyltransferase from Coptis which has only very low activity with substrates for the other three transferases [73]. In addition to these four methyltransferases, the pathway from (S)-norcoclaurine to berberine involves four other enzymes. One of these is the berberine bridge enzyme which catalyses a unique reaction in which the N-methyl group of (S)-reticuline is involved in an oxidative cyclisation reaction to form (S)-scoulerine. The berberine bridge enzyme has recently been cloned from cell suspension cultures of Eschscholtzia californica elicited with a

yeast cell preparation [16]. The c-DNA has an ORF of 538 amino acids of which 22 are absent from the N-terminus of the mature protein and probably constitute a signal peptide directing the pro-enzyme to cytoplasmic vesicles. In Berberis, although the enzymes of (S)-reticuline synthesis are soluble, the four enzymes responsible for the conversion of (S)-reticuline to berberine are located in cell particles which band on a sucrose gradient at a density of 1.14 [103]. In Coptis the enzymes converting (S)-reticuline to berberine are also present in a particle of similar density [ 103] but the route of biosynthesis is different in that the final two steps in the pathway are reversed, the methyleneoxy bridge being formed before the oxidation step in Coptis [98]. The intermediate between (S)-tetrahydrocolumbamine and berberine is thus (S)-tetrahydroberberine in Coptis but columbamine in Berberis [103]. In four species of Thalictrum tested, the Coptis route rather than that operating in the Berberis was used [26]. The localisation of the final steps in berberine biosynthesis in vesicular structures provides a mechanism for transporting cytotoxic quaternary alkaloids, such as berberine, into the vacuole for storage [ 103 ]. These findings indicate that the berberine pathway is under sophisticated control based on specific enzymes targeted to particular cell organelles controlling a pathway leading to products of high stereochemical selectivity. The stereoselectivity is important since in other plants (S)-reticuline isomerises to form (R)-reticuline which is the parent compound of morphane alkaloids of Papaver such as morphine and codeine. The work on berberine biosynthesis illustrates the specificity of secondary metabolite formation with highly specific enzyme proteins catalysing reactions in a defined order. Extrapolating such studies to secondary metabolism in general would suggest that there are many specific genes devoted to secondary pathways which, for the whole plant kingdom, must greatly outnumber the total of genes devoted to primary metabolism. There have, so far, been few comparative studies of genes of secondary metabolism. The gene for strictosidine synthase, an

enzyme controlling an important step in indole alkaloid biosynthesis, has been cloned from two different species in the genus Rauvolfla, R. serpentina and R. mannii, which originate in different continents. These two genes show 100 ~o homology at the nucleotide level within the coding of sequence [6]. PCR studies of ten Rauvolfia species with origins across the tropics indicated a high degree of conservation of the coding sequence of strictosidine synthase [7]. Although these studies indicated selective pressure for retention of the coding sequence and optimisation of strictosidine synthase activity, some differences in the upstream sequences were observed which may indicate that the pattern of expression of the gene may be regulated differentially to meet local conditions [6]. Understanding how at the molecular level the co-ordinated expression of such genes is controlled during plant development is a major challenge for the future.

Role and significance of the accumulation of secondary metabolites

The development of theories to describe the physiological importance of plant secondary metabolism has been outlined by Luckner [52]. An early theory saw secondary metabolites as 'waste products' of metabolism ('overflow' or 'shunt' metabolites): a feature of multicellular plants lacking an excretion system. Many secondary products show significant phytotoxicity and since metabolic processes are unbalanced, all organisms have to deal with the potential accumulation of certain metabolites to toxic levels. Microbial systems can deal with such problems by releasing metabolites into the medium while higher animals have well developed excretory systems. Plants, in contrast, have to store their accumulating metabolic products. They sequester toxic hydrophilic compounds in the vacuole and detoxify lipophilic compounds by rendering them more hydrophilic by conjugation and then transporting them to the vacuole. Toxic lipophilic products may be synthesised in gland Cells and stored extracellularly, or exported as components of suberins or the cuticular waxes.

However, to view secondary products in plants merely as waste materials fits uneasily with our knowledge of the biochemical specificity of secondary metabolism, the strict regulation of its expression at the genetic level and the precise temporal and spatial regulation of expression of secondary metabolic pathways. Stahl [80] was the first to suggest that secondary metabolites, rather than being metabolic by-products, have a role in the plant's interaction with its environment and with other organisms to provide a defence against infection, predation and environmental stress. This theory has gained wide acceptance although until relatively recently the experimental support for it has been largely based on correlative rather than any more direct evidence. It is easy to ignore the waste product theory but the detoxification of certain metabolites has to be achieved and the accumulation of some products may reflect this fact. Many of the defence mechanisms available to animals are not available to sessile organisms such as plants [95]. Morphological and physical barriers are important components of the plants' defences but the importance of secondary metabolites in defence is being increasingly appreciated. If it is accepted that defence based on low-molecular-weight chemicals operates, given the range of challenges to which a plant in the field is exposed - from microbial pathogens, herbivorous animals and insects, and competing plants to environmental stresses, extremes of temperature, UV light, mechanical damage, drought etc. -, it is not surprising that a defence strategy to cover a wide range of eventualities should lead to the complex chemical constitutions we observe in plant species today. In considering the role of individual compounds the possiblilty of a single compound having more than one role or that two or more compounds may act synergistically must be considered in trying to ascribe functions to secondary metabolites. In such considerations, it may not be necessary to ascribe a role or a benefit to each and every secondary product. Some, for instance, may be inactive compounds which are merely intermediates in the pathways leading to end-products having impor-

tant defence roles, others may be derivatives or conjugated forms of such compounds while others still may result from lack of absolute specificity among enzymes involved in the pathways [3]. It must be assumed that if secondary metabolites play an effective defensive role, their production must have evolved under selection pressures to optimise that defensive role. Jones and Firn [41] have examined the hypothesis that a welldefended plant might be expected to contain a moderate diversity of highly active compounds, with few if any inactive ones. They contrast this with their perception that most plant secondary products are of low defensive value. They argue that the present diversity of low-activity compounds is retained only because it raises the probability of further mutations leading to the accumulation of rare, yet active compounds. Assessing the value to a plant of a secondary product is very difficult given the possibility of multiple roles and synergistic interactions. Among these metabolites there is a range of potency. Alkaloids, for instance, have high toxicity in animals and are highly targeted in their mode of action so it is clearly wrong to consider such compounds as being of low activity. Against this, some phenolic defence compounds such as chlorogenic acid have relatively low fungistatic activity, yet show some activity against a wide range of organisms and rely on the establishment of high local concentrations for their effectiveness. It seems likely that there is pressure to maintain pathways that lead to compounds which confer benefit, with the observed diversity of related compounds resulting from modification of the selected lead compound by substitution or conjugation using existing or mutated activities. The phytoalexins are one group of compounds, for which there is good evidence for a role in defence against fungi. However, fungal pathogens have evolved the ability to metabolise phytoalexins and thus the complex mixture of related phytoalexins produced by the plant during fungal attack may reflect the production of new phytoalexin derivatives to counteract the capacity of the fungus for detoxification [86]. It is beyond the scope of this review to address in detail the role of secondary metabolites in the

9 complex interactions with other organisms. There are a number of reviews available which discuss current understanding of the roles played by plant secondary metabolites in the defence of the plant against herbivory by insects [2, 11, 62], mammals [91, 94], in the competition between plant species [64] In addition, there is an excellent monograph on the ecological importance of secondary products [37]. In contrast, the present review will concentrate on those areas where a combination of biochemical and molecular techniques are being used to explore the role played by individual secondary metabolites in plant physiological processes. The two areas chosen are the role of phytoalexins in defence and the role of secondary products as signalling molecules. with suppressed phenylpropanoid metabolism show increased susceptibility to the pathogen Cerospora nicotianae [47]. The pathogen-induced activation of the phenylpropanoid pathway leads to the accumulation of phenolic compounds such as chlorogenic acid and coumarins, the formation of cinnamic acids esters in the cell wall and the biosynthesis and deposition of lignin and suberin. In addition to changes in phenylpropanoids, there is the induction of the de novo biosynthesis of species-specific defence compounds, the phytoalexins [61]. Thirdly, there is a system to transmit a signal to activate defence in organs distant from the original point of attack [18]. Among potential low-molecular-weight plant defence compounds, the phytoalexins have been investigated in some detail. The phytoalexins are essentially absent from uninfected plant tissues and accumulate on infection. In a number of cases, this biosynthesis has been shown to result from increased transcription of the relevant m R N A s and de novo synthesis and processing of the biosynthetic enzymes [46]. Phytoalexins represent many different chemical classes (sesquiterpenes, flavonoids, furanocoumarins, polyacetylenes, glucosinolates). Some phytoalexins are characteristically formed in a particular plant family, for example sesquiterpenes in Solanaceae and isoflavonoids in Leguminosae. These compounds have anti-microbial properties and are implicated as part of an inducible defence within the plant [ 61 ]. A range of unrelated plants, such as grape (Vitis vinifera), peanut (Arachis hypogaea) and Scots pine (Pinus sylvestris) produce stilbenes, phenylpropanoid-derived phytoalexins, in response to attack by pathogens. Stilbene synthase (STS) is a polyketide synthase closely related to CHS, which catalyses the condensation of p-coumaroyl CoA and malonyl CoA to form the stilbene resveratrol. The gene for STS has been cloned from Arachis hypogaea where resveratrol is the major phytoalexin [75]. It has been possible to express this gene in Nicotiana under the control of its endogenous promoter and establish a pathway in which intermediates in flavonoid biosynthesis are diverted into the formation of a novel phytoalexin

Phytoalexins in plant defences against pathogens

Advances in genetic manipulation techniques and the increasing availability of relevant genes open the prospect of making specific single gene alterations in secondary pathways and have allowed the role of secondary metabolites in vivo to be investigated directly. As indicated earlier, the defence of a plant against pathogens is multilayered [18]. It involves morphological features and physical barriers and the production of specific defence proteins (e.g. proteinase inhibitors, extracellular glucanases and chitinases). In addition, it is proposed that low-molecular-weight secondary metabolites provide a chemical defence against infection. Plant chemistry can contribute to defence at a number of levels. Firstly, there are constitutive compounds, often in peripheral organs, such as simple phenols, tannins etc., which may provide a primary chemical barrier to infection. Secondly, there are localised inducible chemical changes, which are activated by the presence of the pathogen and provide a means of limiting its further ingress into the plant tissue. This usually involves a general activation of phenylpropanoid metabolism. Evidence for the importance ofphenylpropanoids in such defences is strong. For instance, transgenic tobacco plants

10 [32]. The peanut STS gene in tobacco was induced by UV light and by elicitation with a watersoluble preparation of Phytophthora megasperma and the accumulation of resveratrol detected by HPLC. However, the concentration of resveratrol accumulating (30-50 ng/g fresh weight) was less than 10~o of that found in elicited Arachis cells [ 32] and this small accumulation of resveratrol in tobacco did not lead to any significant change in resistance to infection [33]. However, in further experiments the STS gene Vst 1 from Vitis vinifera was transformed into tobacco which led to the rapid appearance of high levels of STS m R N A and to the accumulation of very high levels of resveratrol at up to 400 #g/g fresh weight. An inverse correlation was found between the levels of resveratrol accumulating in the leaves of transgenic tobacco containing the grape STS gene and incidence of infection with the fungal pathogen Botrytis cinerea. This work represents the first successful use of recombinant D N A techniques to increase the disease resistance of a plant by establishment of a novel production of a phytoalexin [33] and provides strong evidence for the role of at least one class of phytoalexin, the stilbenes, in plant defence. In Pinus sylvestris at least two STSs occur as well as CHS. These STSs differ in their substrate specificity from the STS of Arachis in that cinnamoyl CoA or dihydrocinnamoyl CoA are the preferred substrates (pinosylvin and dihydropinosylvin synthases respectively; see Fig. 1). Unstressed plantlets of Pinus had appreciable CHS activity, induction with yeast extract led to a transitory increase in CHS activity after 16 h and to an accumulation of flavonoid compounds. STS shows a different pattern, it is absent in unstressed plantlets and increases in activity after a lag of at least 6 h to a peak after about 30 h [24]. The main product accumulating during this period was pinosylvin which has been shown to have fungistatic properties [ 38 ]. The dihydropinosylvin synthase, but not the pinosylvin synthase, has been cloned from Pinus [24]. The dihydropinsylvin synthase shows a high degree of homology to Pinus CHS (73.2~o) and to the STS of Arachis (65.5~o) at the protein level. The availability of these genes and their heterologous expression would allow the role of the pinosylvin and its derivatives in stress metabolism to be investigated. There are other opportunities where expression of a single gene would lead to the appearance of a novel phytoalexin and allow the roles of other phytoalexins to be probed. Casbene synthase catalyses the synthesis of the C20 diterpene phytoalexin, casbene, from geranylgeranyl diphosphate and the gene for this enzyme has recently been cloned from Ricinus [ 51 ]. Expression of this gene in a heterologous system will allow the role of casbene in defence to be studied. The gene for sesquiterpene cyclase [20] which catalyses the first step in sesquiterpene phytoalexin biosynthesis from the C15 precursor, farnesyl diphosphate, in solanaceous species has been cloned and is a useful toot to investigate the role of sesquiterpenes in plant defence. However, more than one gene would in this case have to be expressed if novel pathways to form phytoalexins such as rishitin, phytuberin and capsidiol were to be established in heterologous systems. An alternative approach to study the role of phytoalexins in defence would be to express relevant genes in the antisense orientation to deplete the capacity of the plant to produce phytoalexins to see if this correlated with increased susceptibility to infection. Isoflavone 2'-hydroxylase (formononetin 2'hydroxylase) has been identified as a major factor in the accumulation of pterocarpan phytoalexins in Cicer cell suspension cultures challenged by elicitors from the fungus Ascochyta rabiei [30]. Manipulation of the activity of this enzyme in vivo would enable the role of pterocarpans in the susceptibility of the plant to the fungus to be to be investigated. The opportunities for improving plant defences by genetic manipulation of pathways leading to defence compounds have been reviewed [47]. Most pathways leading to the formation of such compounds are multigenic and manipulation must involve either increasing the activity of a rate-limiting gene or modifying the expression of regulatory genes which co-ordinate the expres-

11 sion of the structural genes coding for the individual pathway enzymes. leaves on the same plant [54]. The concentration in infected leaves may reach 1.0 #g/g fresh weight while leaves on untreated control plants contain less than 0.01 #g/g fresh weight [ 54]. Exogenously supplied SA is readily transported within plants via the phloem system. Concentrations of SA in the phloem exudates of up to 300 #M have been observed 18 h upon inoculation of tobacco leaves with Pseudomonas syringae [ 66]. In TMV-infected tobacco leaves, Enyedi et al. [ 17] showed that in leaves bearing lesions, the main product accumulating was a conjugate of SA, tentatively identified as O-fl-D-glucosyl-SA (GSA). GSA was not present in either the phloem or in uninoculated leaves. A UDP-G, salicylic acid glucosyltransferase (GTase), has been purified 176-fold from Arena roots and shows high specificity towards SA [96]. It is inactive towards p-hydroxybenzoic acid and methylsalicylate but shows a low activity towards m-hydroyxbenzoic acid [97]. GTase activity is induced in tobacco leaves infected with TMV [19, 96]. The induction of GTase shows specificity for SA; only 2,6-dihydroxybenzoic acid among a wide range of benzoic acid derivatives tested was as active as SA [ 19]. Healthy tobacco leaves have a low basal level of GTase activity but in infected leaves this level is elevated by about 7-fold after 72 h. No significant levels of GSA or GTase activity were found in a healthy leaf immediately above the infected leaves [19]. GTase is induced in oat leaves treated exogenously with SA and leads to GSA accumulation. Such reactions probably represent detoxification. In roots of Vicia faba exogenously supplied SA is detoxifled to GSA but in Fagopyrum SA is first oxidised to 2,5-dihydroxybenzoic acid and then glycosylated in position 5. In both cases SA appears to induce the detoxifying enzymes [76]. It is likely that SA is transported as the free acid and that this is the active form of the signalling molecule. Glycosylation by the inducible GTase could be a mechanism for controlling SA levels by detoxification. There is some evidence that GSA is less biologically active than SA; for instance it is less inhibitory to chloride ion uptake than is SA [ 18]. Further experiments in which a bacterial gene encoding an enzyme which degrades SA, was ex-

Secondary metabolites as cell signalling compounds

A role proposed for some secondary metabolites is that they act as signal molecules and participate in transduction pathways leading to physiological and developmental responses. To carry out such a role, a secondary metabolite must be synthesised rapidly, be transported to the target site, carry sufficient structural information to be selectively recognised by a receptor and then rapidly degraded so that it does not persist at the target site once it has effected its responses [53 ]. Many of the potential signal molecules now under investigation are products of the phenylpropanoid pathway. This may be a feature of the wide distribution of these compounds in plants but may also be a reflection of our narrow knowledge of secondary metabolism. It has been proposed that salicylic acid (o-hydroxybenzoic acid, SA) acts as a messenger in the process of systemic acquired resistance (SAR) of tobacco and cucumber in which signals from infected leaves are transmitted to other undamaged leaves where they induce changes that enhance resistance [54, 58]. Salicyclic acid is one of a group of hydroxybenzoic acids, which are commonly found in plants, (p-hydroxybenzoic, protocatechuic, vanillic and syringic acids) but generally at lower concentrations than the corresponding cinnamic acid derivatives to which they are biosynthetically related. The free acids are generally minor components, but conjugates are widely found, in particular the glucosides although glucose esters can also occur [43]. Exogenously supplied SA stimulates resistance to pathogens and induces accumulation of the same defence proteins (chitinase, glucanase, peroxidase, superoxide dismutase) which are induced by the presence of pathogens [18, 66]. Infection of tobacco with TMV leads to a 20-fold stimulation of the concentration of SA in infected leaves and a 5-fold stimulation in uninfected upper

12 pressed in plants suggests that SA is essential for the development of systemic acquired resistance [25]. The enzyme involved was the salicyclate hydroxylase of Pseudomonas putida which catalyse the formation of catechol from SA. Expression of the salicyclate hydroxylase gene in transgenic tobacco blocked SA accumulation in TMVtreated plants and the acquisition of resistance in leaves distant to the point of attack [25]. Exogenously supplied SA modifies gene expression in a way which parallels that of the presence of a pathogen. Treatment of tobacco with SA will lead to the accumulation of pathogenesis-related proteins (PR) such as PR1 and glycine-rich proteins such as GPRP8 by a mechanism which involves stimulation of transcription of their genes and the accumulation of the related mRNAs [54, 58, 90]. The accumulation of SA and the induction of PR genes in tobacco infected with TMV was inhibited at high temperatures (32 C) [55]. However, supplying SA at the higher temperature restored the ability to induce expression of the PR genes. The biosynthesis of benzoic acids is closely related to that of the general phenylpropanoid pathway. Benzoic acids are formed from their corresponding cinnamic acids by a side-chain shortening reaction which leads to the loss of an acetate unit [74, 101]. Two separate routes for this elimination have been shown to operate in plants [101]; one via the cinnamoyl CoA thioesters and the elimination of acetyl CoA by a sequence of reactions analogous to fi-oxidation of fatty acids and the other via a non-oxidative pathway with the release of acetic acid. The biosynthetic route of SA formation is unknown. Two possibilities exist, one by ortho-hydroxylation of t-cinnamic acid to form o-coumaric acid [28] which would be the substrate for the chainshortening reactions or the other in which cinnamic acid is the substrate for chain-shortening and o-hydroxylation of benzoic acid occurs subsequently. Evidence that the second of these two routes might operate, at least in tobacco, has come from the detection of a benzoic acid 2-hydroxylase activity which is induced in tobacco infected with TMV [49]. SA has also been implicated as an endogenous inducer of thermogenesis [65] in flowers of aroid species which leads to the volatilisation of amines which attract insect pollinators. Heat production in thermogenesis is mediated by the alternate (cyanide-insensitive) oxidase of the mitochondria. A cDNA and the corresponding genomic clone aox 1 which encodes a 42 kDa alternative oxidase precursor protein has been isolated and characterised from Saurornatum guttaturn [67]. SA applied to immature spadix tissue in the light stimulates accumulation of the 1.6 kb transcripts for the alternate oxidase within 4 h leading to the accumulation of proteins products and enhanced activity of the alternate pathway of respiration [68]. There are several regions in the promoter of aox 1 which show homology to regions of the promoters of other SA activated genes such as P R l a and GRP8 and it may be that one or more of these sequences plays a role in the potential SA-controlled regulation of expression of these genes. This evidence suggests that SA fits the requirements as a signal compound, as it is readily synthesised de novo as a response to the stimulus, in this case the presence of the pathogens, from a branch of an active pathway, it is transported in the phloem, it influences the pattern of gene expression both locally and at distant regions of the plant and it is readily detoxified as its glycoside. Whether SA is the primary signal in SAR is, however, unclear [66]. Other phenylpropanoid-derived secondary products have been implicated as signal compounds in other situations but the evidence is often less complete. Petunia was transformed with an antisense chs gene under the control of a modified CaMV 35S promoter containing up to 8 copies of an anther box sequence conferring enhanced tapetal specificity of expression [57]. Five transformants showed reduced pigmentation in the anthers, associated with reduced levels of endogenous chs expression, and were male-sterile. CHS-deficient Petunia plants produce abnormal anthers, devoid of flavonoid pigments, producing pollen which although viable shows severely reduced pollen germination and tube growth [84].

13 The main site of pigment deposition in anthers is in the tapetal layer. TLC analysis of extracts from white anthers showed a severe reduction in ftavonoid content accompanied by 2- to 3-fold higher amounts of phenylpropanoid precursors such as ferulic and p-coumaric acids compared to control purple anthers [57]. Pollen from the white anthers were unable to grow in in vitro culture in contrast to pollen from normal purple anthers [84]. These results may indicate that flavonoids play an essential role in male gametophyte development or function. An in vilro pollen-germination rescue assay was developed [59] in which pollen from chs mutant plants was restored in its ability to germinate and grow in culture by extracts of wild-type stigmas. Subsequent experiments showed that this activity was due to the presence of free kaempferol in the stigma extracts. Free kaempferol was a minor component of these extracts while the major ftavonoids present were the 3-O-glycosides of kaempferol and quercetin. The major flavonoids of petunia pollen are the 3-O-glycosides of the flavonols, kaempferol and quercetin, 4,2',6'tetrahydroxychalcone and a dihydroflavonol, taxifolin. A detailed analysis of the structure/ activity relationships for the restoration of pollen function showed that activity was observed with flavonols, but not with cinnamic or benzoic acids, or with other flavonoids such as flavones, flavanones, chalcones, anthocyanins, catechins or isoflavonoids. The lowest concentration for full restoration was given by kaemperol at 1 #M and by two related flavonols, galangin and isorhamnetin. The other major flavonol of Petunia anther tissue, quercetin, was somewhat less active (10 #M for maximal response). It is interesting that the 3-O-glycosides, the major forms in which kaempferol and quercetin occur in both the anther and stigma tissues are inactive. In parallel studies, it was shown that diffusates of mature pollen of tobacco strongly increased germination frequency and growth of immature pollen of tobacco in in vitro culture [ 102]. Again, the factors responsible for the stimulation were shown to be the flavonol aglycones quercitin, kaempferol and myricetin. In both sets of experiments very low levels (0.1-1 #M) of flavonols were required for the observed effect. Little is known of the mechanism of action of the flavonols but it is clear that they affect patterns of pollen development at very low concentrations, indicative of a signalling role. It is interesting that both for SA and the flavonols it is the free phenolic rather that the conjugate which is physiologically active and conjugation may represent a mechanism for detoxifiying and controlling levels of the active compound. Other examples where phenolic compounds may be acting as signalling compounds are the flavonoids which can act as either inducers or inhibitors of expression of the nod genes of Rhizobiurn [23]. Acetophenones such as acetosyringone (AS) are secondary products produced on wounding of plants and have been characterised as inducers of the vir genes of Agrobacterium tumefaciens and A. rhizogenes [79]. However, acetosyringone is only found in significant amounts in solanaceous plants [78] and in Agrobacteriumsusceptible plants of other families, other phenolic compounds such as benzoic acid and cinnamic acid derivatives may act as the main vir inducers [77]. An example of a secondary metabolite, other than a phenylpropanoid, which may play a role as a signalling compound is jasmonic acid (JA, [3-oxo-2-(2'-cis-pentenyl)-cyclopentane- 1-acetic acid]). This compound and its methyl ester were first discovered in plants as components of the essential oils of Jasminurn [15] and Rosmarinus [12]. JA is synthesised from lipids via the ~-linolenic acid cascade (see Fig. 3) upon membrane damage as a result of the activity of fatty acyl hydrolase to release long-chain fatty acids such as linolenic acid (C18:3). Linolenic acid then undergoes oxidation by lipoxygenase to form its 13-hydroperoxide [88]. The 13-hydroperoxide undergoes cyclisation under the influence of a hydroperoxide cyclase to yield 12-oxo-cis-cis-lO, 15phytodienoic acid (12-oxo-PDA) [14, 104]. 12oxo-PDA is then reduced in the C5 side chain to form the cis-unsaturated derivative and then undergoes a series of three /3-oxidation cycles to form epi-jasmonic acid which isomerises to JA. MeJA arises by subsequent esterification of JA.

14 described [8]. However, the relative biological activity of such conjugates is unknown. JA is ACYLHYDROLASE l transported in the phloem [18] and since it induces expression of proteinase inhibitors in unJ~---~ ,COOH treated leaves distal to the site of application, it can move through the plant and elicit changes in LIPOXYGENASE l Linolenicacid(C18:3) metabolism [ 18]. OOH Exogenous treatment of plant tissues with either JA or MeJA, which is degraded by plant ~ C O O H [ Linolenic13-hydroperoxide esterases to yield JA, induces senescence [85], proteinase inhibitor formation [22], tuber formaHYDROPEROXIDE CYCLASE tion in potato [44] and acts as a trigger in tendril coiling [21, 92]. Potato contains tuber-inducing ,'" " ~ / ~ C O O H compounds which are related to JA but chemically distinct from it. One of the natural inducers 12-oxo-PDA O is the glucose ester of 'tuberonic acid' (3-oxo-2[ 5- hydroxy-2- cis- pentenyl ]-cyclopentane- 1-aceREDUCTASE tic acid) [44]. Jasmonate has been implicated as a mediator in the wound responses of plants [ 13] ~ C O O H and as a signal transducer in elicitation of secondary product formation [31, 60]. MeJA, being volatile, may play a role in inter-plant communiO cation [22 ]. Wounding of soyabean hypocotyl tisBETA-OXIDATION l sue led to increases in the endogenous levels of jasmonates from a basal level of about 90 ng/g fresh weight to a peak of about 500 ng/g fresh 'qq~ weight after 8 h and remained elevated for up to O epi-jasmonicacid 24 h after wounding [13]. Elicitation of cell suspension cultures of Rauvolfia canescens or Eschscholtzia californica with a purified yeast cell wall elicitor fraction led to a rise in JA level from a basal level in untreated cells of about 25 ng/g dry weight to more than 1250 ng/g dry weight jasmonicacid within 45 rain of addition of the elicitor. The reO sponse in MeJA was smaller and did not occur Fig. 3. The proposed pathway ofjasmonic acid biosynthesis. until some 55 min later [31]. These stimulations lead to average internal concentrations of jasThe presence of enzymes catalysing the formation monate of the order of 1 micromolar, within the of 12-oxo-PDA from 18:3 fatty acid and its subrange of concentrations of JA and MeJA sufficient to induce physiological changes in plants sequent conversion to JA has been described in a number of plant species [88, 89, 104]. JA for[18]. In E. californica cells treated with a yeast mation, unlike that of SA, is induced by woundelicitor, it was shown that the rise in JA was asing as well as by pathogen attack [18]. It is unsociated with significant accumulations of interclear whether the JA biosynthetic pathway is mediates in JA biosynthesis, free c~-linolenic acid constitutive or induced by these stresses. Little is and 12-oxo-PDA [60]. known of the degradation of JA but conjugation Jasmonates act to alter the pattern of gene exof JA with amino acids such as tyrosine has been pression within plant tissues. In soybean cell susMEMBRANE L1PIDS

,""

,'"'<"COOH

15 pension cultures, MeJA addition stimulated increases in the steady state levels of m R N A for wound-induced genes such as chalcone synthase: 20 #M MeJA treatment led to the appearance of chsl m R N A within 1 h of treatment and a maximum level o f m R N A between 4 and 12 h [ 13]. JA treatment led to the accumulation of m R N A coding for a Kunitz-type proteinase inhibitor in tissue slices of stored potato tubers [99]. In cell cultures of Glycine max treatment with 250 #M MeJA led the induction of phenylalanine ammonia-lyase (PAL) m R N A accumulation and subsequent accumulation of isoflavonoid phytoalexins. In Eschscholtzia californica MeJA treatment led to 5-fold increase in m R N A for the berberine bridge enzyme, a key enzyme in benzo(c)phenanthridine biosynthesis [16], 6 h after the start of treatment. This preceded a peak in enzyme activity, 12-fold higher than the control after 17 hour and finally, the accumulation of the benzo(c)phenanthridine alkaloids [31 ]. Growth of the primary root of Arabidopsis thaIiana was inhibited by 5070 by 0.1 #M MeJA. An Arabidopsis mutant, jarl, showed decreased sensitivity to MeJA [81]. The mutation is recessive and controlled by a single Mendelian factor. It is pleiotropic and the results suggest that the lesion in jar1 affects a general j asmonate response pathway. JA meets the requirements of a signal compound in having stringent requirements for activity; saturation or hydroxylation of the side chain leads to a complete loss of activity [92]. The active form appears to be ( + ) epi-jasmonate which readily isomerises to jasmonate [31]. It is synthesised in response to wounding, it is transported and has high structural specificity. JA structurally and biosynthetically shows a strong similarity with the animal hormones, the prostaglandins which are derived from C2o:4 fatty acids by the enzyme cyclo-oxygenase. In mammalian systems, prostaglandins modulate inflammatory reactions. The mechanism by which small molecules such as salicylic acid and jasmonates influence patterns of gene expression in plants is unknown. The analogy between prostaglandins and jasmonates may be revealing in this respect. Prostaglandins act as agonists for membrane signal transduction pathways by binding to specific membrane-bound receptor proteins and effecting changes in intracellular Ca 2 + concentrations, patterns ofinositide phosphorylation and in protein phosphorylation leading to changes in the activity of DNA-binding factors [82]. Aspirin (methylsalicylic acid), which mimics SA effects in plants, inhibits the effects of prostaglandins in animal systems by inhibiting the fatty acid cyclo-oxygenase enzyme involved in their biosynthesis [82]. At present there is no evidence in plants that SA and JA interact in this way. The investigation of how small molecules interact at the plant genome level is in its infancy but they are thought to initiate signal transduction cascades upon binding of the small molecule to a receptor protein. Binding proteins for phenolic signal molecules involved in the activation of the vir genes ofA. turnefaciens have been investigated using ct-bromoacetosyringone (ASBr), an analogue of AS, which is a selective and irreversible inhibitor of vir gene activation. ASBr was labelled through replacement of one of the methoxy groups with 125I and used to investigate AS-binding proteins [48]. Two soluble nuclear-encoded proteins of 10 and 21 kDa bind the labelled inhibitor and are proposed to mediate an early step in the signal transduction pathway [48]. Salicylic acidbinding proteins have been isolated from tobacco leaves. SA binding is associated with a large, soluble protein (650 kDa) and shows a K d of 14 #M, which is within the physiological range of SA [10]. Whether this protein is involved in signal propagation induced by SA remains to be seen. Investigation of the interaction between JA and binding proteins will be greatly enhanced by the availability of mutants in the jasmonate signal pathway [81].

Molecular approaches to secondary metabolism

It is clear that the application of molecular biological approaches is contributing to our understanding of the role played by some plant secondary metabolites in plant physiology. It is now

16 possible to demonstrate more directly the role of phytoalexins as defence compounds, substantiating the conclusions derived from a great body of correlative evidence which has been amassed over the fifty years since the presence of phytoalexins in plants was first postulated by Muller and Borger [61]. Molecular biological studies have themselves highlighted the role of individual secondary metabolites as internal signalling compounds which influence patterns of gene expression by initiating signal transduction pathways. The sheer diversity of the secondary products that accumulate in plants has been a major impediment to an understanding of their role in plant physiology. Typically, for any one class of compound an almost bewildering array of variants around a parent molecule can co-exist in a plant. Such compounds are likely to play a wide range of roles in the plant which offer it a selective advantage in its environment and in its interactions with competitors. It is likely that a given role may be played by different secondary metabolites in different families. An example is the role of acetosyringone which may be the major vir gene inducer in solanaceous species, but this role is undertaken by related phenolic compounds in other families [77, 78]. Molecular studies have shown a role for compounds such as SA and JA as internal signal compounds. However there is more general role for individual secondary metabolites as signalling compounds at a macromolecular level operating between species. Examples range from flower pigments, flavonoids acting arranged in flowers as pollen guides, to volatile compounds acting as attractants for pollinating insects and as antifeedants. In a sense, the spectrum of secondary products in a plant is a signature for the plant in its interaction with other organisms. How the complexity of secondary metabolism in plants has evolved remains an enduring problem. It seems very likely that there are selective pressures in evolution for the retention of secondary pathways yielding products which conferred advantage to the plant rather than selective pressures to maintain formation of individual products. It is therefore not necessary for every product of a pathway to have a beneficial role as long as at least one confers sufficient benefit to the plant for the maintenance and operation of the pathway. There have been several attempts to estimate the cost/benefit to a plant of the accumulation of secondary metabolites but it is doubtful if this is a useful approach at our present state of knowledge, given the possibilities for multiple functions and synergism [ 1]. Indeed it is possible to argue that plants usually have ample supplies of energy from sunlight and of their carbon substrate, CO> and that a degree of profligacy in the elaboration of complex carbon compounds might occur without major detriment. However, in sulphur- or nitrogen-containing secondary products there may be limitations to such profligacy in some circumstances. There are relatively few secondary pathways, other than the main routes of phenylpropanoid and flavonoid formation, which have been fully described and fewer still in which more than one or two genes are available. The berberine pathway discussed above has been extensively studied at the biochemical level but here only one of the genes has been isolated and characterised so far [16]. For most other pathways we have only a partial knowledge of their route and enzymology. A number of individual genes representing diverse secondary pathways other than that of the phenylpropanoids have been isolated and characterised (for review see [35]). The availability of these and other related genes will allow the regulation of other secondary pathways to be investigated at the molecular level. The investigations of phenylpropanoids have revealed their importance as physiological regulators. As the investigation of secondary metabolism broadens we can expect further examples of secondary products which influence fundamental aspects of plant development and physiology.

Acknowledgements
The author wishes to thank his colleagues Drs Bachmann, Michael, Parr, Robins and Walton for their critical reading of this manuscript and for

17

their many valuable suggestions for its improvement.

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