diversity and variability of plant secondary metabolism: a mechanistic view

Entomologia Experimentalis et Applicata 80: 177-188, 1996. @1996 Kluwer Academic Publishers. Printed in Belgium.

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Diversity and variability of plant secondary metabolism: a mechanistic view

T h o m a s H a r t m a n n Institut fiir Pharmazeutische Biologie der Technischen Universitiit Braunschweig, Mendelssohnstrasse 1, D-38106 Braunschweig, Germany

Accepted: November 3, 1995

Key words: structural diversity, biosynthesis, intraspecific variation, secondary plant metabolites, constitutive defence; pyrrolizidine alkaloids

Abstract

Based upon a brief historical view, the typical features of plant secondary metabolism and its role in chemical interactions between plants and their environment are discussed. Facts and arguments are presented favouring the hypothesis that secondary metabolism evolved under the selection pressure of a competitive environment. The high 'degree of chemical freedom' of secondary metabolism which, in contrast to primary metabolism, allows structural modifications with almost no restrictions, is stressed as mechanistic basis for the generation of chemical diversity. Biochemical and physiological properties of secondary metabolism are in accordance with such a view. It is suggested that the great chemical diversity and intraspecific variability of secondary metabolism is the result of processes of natural selection which act upon highly variable chemical structures. This view is exemplified by the pyrrolizidine alkaloids, a typical class of secondary compounds.

Introduction

Anybody dealing with plant secondary metabolism easily recognizes two of its most characteristic features: diverse chemical structures and high intraspecific variation. Traditionally these features were viewed as the strongest arguments favouring an incidental ori- gin of secondary metabolites 'as flotsam and jetsam on the metabolic beach' or as metabolic wastes and detoxification products (Haslam, 1986; Luckner, 1990). Today, of course, the idea that secondary metabolism is an essential part of the plant's biochemical equipment to cope with its often hostile environment is wide- ly accepted. In this article, structural diversity and intraspecific variability will be considered as inherent qualities of secondary metabolism. The first sec- tion outlines a general view, which then will be briefly exemplified by a selected but typical system of secondary metabolites (i.e., the pyrrolizidine alkaloids) studied in the author's laboratory.

What is secondary metabolism?

The phenomenon of secondary metabolism was recog- nized in the early phases of modern experimental botany. In his textbook published in 1873, Julius Sachs, one of the great pioneers of plant physiology, gave the following definition:

'Als Nebenprodukte des Stoffwechsels kann man solche Stoffe bezeichnen, welche w~lrend des Stoff- wechsels entstehen, aber keine weitere Verwendung ftir den Aufbau neuer Zellen finden .. . . . Irgend eine Bedeutung dieser Stoffe ffir die innere Okonomie der Pflanze ist bis jetzt nicht bekannt.' (Sachs, 1873: p. 641) Translation: 'We can designate as by-products of metabolism such compounds which are formed by metabolism but which are no longer used for the for- marion of new cells . . . . . Any importance of these compounds for the inner economy of the plant is yet unknown'.

This clear statement is still valid. Sachs did not refer to any functions of the by-products or, as we now say, the secondary products. This, however, was done by his contemporaries such as Ernst Stahl (18 8 8) with his

178

often quoted classical paper about chemical protection of plants against snails, or the Austrian physician and botanist Anton Kerner von Marilaun (1890) who devot- ed long chapters to the 'Schutzmittel' (protectives) of green leaves, roots or flowers against herbivores. Lro Errera (1886) in Belgium related the often observed localization of secondary compounds in peripheral tissues to their assumed defensive functions.

At the beginning of this century, these functional aspects of secondary metabolism were rejected or simply ignored by most plant physiologists and phytochemists. Secondary compounds were considered waste-products resulting from metabolic degradation or formed as by-products of plant metabolism (Moth- es, 1955; Luckner, 1990). Even Errera (1904) in his later years referred to secondary compounds as waste- products that may 'secondarily' be utilized for defence against animals. It was not until entomologists, such as Gottfried Fraenkel (1959), 'rediscovered' and empha- sized the ecological role of secondary metabolism in the interactions of plants with their environment (e.g., herbivorous insects). Among plant physiologists and phytochemists this view has been accepted slowly and hesitantly, sometimes it is still subject to controversial discussion (Haslam, 1986; Luckner, 1990). The idea was fully accepted by few and its outspoken advocates such as Swain (1977) were rare.

The contemporary view of plant metabolism clearly supports the observation which Sachs made more than 100 years ago. We can distinguish between metabolites and metabolic pathways that are essential to growth and development (i.e., primary metabolism) and those that are non-essential in this context (i.e., secondary metabolism) (Table 1). In the past, the dispensabili- ty of secondary metabolism for growth and development provoked a number of hypothetical explanations about its function in plants (e.g., waste and detoxification products, expression of shunt and overflow metabolism, degradation products, storage products) (see Swain, 1977; Haslam, 1986; Luckner, 1990). But experimental support of any kind favouring one of these explanations has never been marshalled.

On the contrary, numerous examples reveal the function of secondary plant metabolites as chemical signals in the ecosystem (for review see Harborne, 1993; Rosenthal & Berenbaum, 1992; Fritz & Simms, 1992). Figure 1 illustrates the various ecological functions of secondary metabolism. We can regard secondary metabolism as the functional level of plant metabolism that is dispensable for growth and development but indispensable for the survival of the species

Table 1. Primary and secondary metabolism: two functional levels with entirely different characteristics

PRIMARY METABOLISM

Growth and development of the individual

�9 indispensable

�9 uniform

�9 universal

�9 conservative

SECONDARY METABOLISM

Interaction of the individual with its environment

�9 dispensable for growth and development

�9 indispensable for survival of a population

�9 unique

�9 diverse

�9 adaptive

(Table 1). To give just one example: sweet lupines, devoid of the bitter tasting quinolizidine alkaloids, grow well in gardens or protected fields, but they have no chance to survive in mixed populations with bitter lupines in the natural environment. Rabbits, for example, unerringly devour only the sweet specimens from such mixed populations and leave the bitter ones untouched. The difference between primary and secondary metabolism is perhaps better expressed func- tionally than structurally, since the same compound may have primary and secondary qualities. The arginine analogue canavanine, for example, is a power- ful seed protective in a number of legumes (Rosen- thai, 1992), but it is also an important nitrogenous seed storage-compound, which is completely mobi- lized during early stages of germination. It makes no sense to ask whether canavanine is a secondary compound (seed protection) or a primary compound (seed nitrogen storage). Instead one might ask whether during evolution the selection of canavanine as protective agent decreased the availability of seed storage nitrogen. The disadvantage of nitrogen shortage was com- pensated for by protection and concomitantly favoured selection of mechanisms allowing the remobilisation of canavanine-bound nitrogen.

The most characteristic features of secondary metabolism are its great structural diversity, restricted occurrence and high intraspecific variability. Some 100000 chemical structures have been isolated and identified from the plant kingdom and each species has its unique bouquet of secondary compounds, which generally exhibits high intraspecific variation. The her- itable nature of this chemical variation is well known

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D E F E N S E

Herbivores

Fungi

Bacteria

Viruses

Plants

ATTRACTION

And

STIMULATION

Pollination

Seed dispersal

Oviposition

Food-plant

Sequestration

Pharmacophagy

Symbiosis - N-Fixation - Mycorrhiza

PROTECTION AGAINST PHYSICAL EFFECTS

UV-Light

Evaporation

Cold

Figure 1. Secondary metabolites originate from common precursors of primary metabolism. Their functions concern all aspects of plants' chemical interactions with the environment.

(Berenbaum & Zangerl, 1992). The importance of reciprocal selective responses between ecologically closely linked organisms as basis of genetic variation is still best expressed by Ehrlich & Raven (1964) in their classical hypothesis on the coevolution of butterflies and plants: 'Indeed, the plant-herbivore 'interface' may be the major zone of interaction responsible for generating terrestrial organic diversity'. In a more general sense we just have to replace the term 'herbivore' to account for all ecologically linked factors, illustrated in Figure 1.

Relating plant chemical defence to secondary metabolism we can distinguish three basic strategies (Hartmann, 1985):

�9 Induction (i.e., formation of defences in response to microbial or herbivore attack) (see Baldwin, this volume).

�9 Production of metabolically safe pro-toxins that are enzymatically ignited upon attack (e.g., cyanogenic glycosides producing toxic cyanide

and carbonyls or glucosinolates producing mustard oils).

�9 Constitutive accumulation of defence compounds (i.e., compounds that are always present in the target-tissue in concentrations needed to fulfil their functions).

There are, of course, no strict border-lines between these strategies. Thus, more recently the number of examples has been increasing, indicating that constitutive defence can be reinforced by induction (Bald- win, this volume). Nevertheless, constitutively produced secondary metabolites provide the most typical examples of secondary metabolism in plants. Most phenolics, saponines, essential oils, resins, and alkaloids are constitutively produced. In fact, plants appear to have evolved passive, anticipatory, and chemically creative strategies in the form of constitutively accu- mulated metabolites to escape herbivore or pathogen attack. In addition to such preventive measures, more active and aggressive defence mechanisms have been

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selected in the form of inducible responses (e.g., phy- toalexins). In the following, I will restrict discussion to constitutively synthesized secondary compounds.

Chemical diversity of secondary metabolism

Chemical diversity in plants is not incidental but is brought about by specifically organized and controlled biogenetic pathways which are well integrated into metabolism. The great majority of secondary constituents is produced via a few basic biogenetic routes, leading to one or a few key-metabolites, from which numerous derivatives are formed by usually simple enzymatic transformation. Examples are the polyke- tides, phenylpropanoids, isoquinoline alkaloids, or indole alkaloids, each with a major pathway and sev- eral thousands of individual products in the periph- ery. Figure 2 illustrates this principle for the phenylpropanoids. The key-intermediate is cinnamic acid derived from the proteinaceous amino acid phenylala- nine. Cinnamic acid and its hydroxylated derivatives are transformed to a second level of key-intermediates by four mechanisms: side-chain elongation (addition of acetate units) and substitution (e.g., piperine); chain elongation and cyclization (e.g. flavonoids, stilbenes, xanthones); chain shortening (e.g. benzoic acids, acetophenones, styrenes) and side chain reduction (e.g., cinnamyl alcohols as lignin precursors, coumarines, and other simple derivatives). These second level key- metabolites are further diversified by specific hydroxylation, O-methylation, dehydrogenation, phenolox- idation, esterification, glycosylation etc. In case of the flavonoids alone these simple strategies have been combined to produce over 5000 structures.

The predisposition to chemical diversification seems to be an inherent quality ofkey-metabolites. The monoterpenes are one of the most impressive exam- pies. More than four decades ago Leopold Ruzicka proposed the 'biogenetic isoprene rule' (Ruzicka et al., 1953) which set the foundation for nearly all further biosynthetic investigations (Croteau, 1987). More recently it has been shown that the reactions which gen- erate the various cyclic key-intermediates are catalyzed by enzymes collectively termed monoterpene cyclases. Some twenty cyclases have been reported which synthesize various monoterpene olefins (Figure 3). The enzymatic mechanism proceeds via ionic intermediates almost exactly as Ruzicka postulated (Croteau, 1987). Common substrate of the cyclases is geranyl diphosphate, the unique precursor of all monoterpenes.

Table 2. Intraspecific features of secondary metabolism

Biosynthesis is specifically i n t eg ra t ed into

the developmental programme of the plant. It is usually defined: �9 in space - restricted to particular cells, t issues or organs

�9 in t ime - restricted to particular stages of development

and often accompanied by specific: �9 ma in t enance -turnover, degradation, conversion, conjugation,

polymerization

�9 long .d is tance t rans loca t ion - via phloem or xylem

�9 t issue a l locat ion - cell to cell translocation

�9 accumula t ion - in vacuoles, ducts, laticifers, glands etc.

The nature of the product formed is determined by the specificity of the cyclase. Similar cyclases are responsible for the formation of the more complex olefinic key- intermediates of the sesquiterpenes (15 carbon atoms) and diterpenes (20 carbons). The diversification of the basic olefinic structures again occurs by simple enzymatic reactions, e.g. hydroxylation, dehydrogenation, double-bond reduction, isomerization etc.

Biochemical basis of intraspecific variation in secondary metabolism

In any plant species, a given secondary pathway is specifically integrated into the developmental programme (Table 2). Biosynthesis usually occurs in an organ and tissue specific manner and is often temporally restricted during development. In Mentha (mint) species (Lamiaceae), for example, monoterpenes are primarily produced in the glandular trichomes of the leaf (Gershenzon et al., 1989). The biosynthesis was found to occur only during the first two weeks of leaf development. The activities of the seven enzymes involved in the biosynthesis from active isoprenes to menthone are high during the first two weeks of leaf development but decline to very low levels thereafter. Similar patterns are encountered for other secondary compounds such as flavone glycosides in Secale cereale (Poaceae), indole alkaloids in Catharanthus roseus (Apocynaceae) or cyanogenic glycosides in Sorghum bicolor (Poaceae) (Gershenzon, 1994).

Biosynthesis of secondary metabolites is often accompanied by specific translocation and storage (Table 2). Long-distance movements via the phloem are known from the quinolizidine alkaloids synthesized in the leaves of lupines or the pyrrolizidine alkaloids

- - _ ~ C O O H . ~. . . . .~ /COOH ~ C O O H ~ C O O H

H o " ~ J Ho- y H o - ' ~ OH OCH 3

Cinnamic acid ~ 4-Coumaric acid ~ Caffeic acid ~.- Ferulic acid

II R ~ / ~ C-O-R

Cinnamoyl esters

o \ tl

Cinnamoyl amides

R- L ~ Coumarins

i l

O I1 R _ ~ ~ C-x

Di.ydroci.namicacids / /

R_Cr Styrenes

R . _ ~ -~jCH2OH

Cinnamyl alcohols

C H 3 0 ~ C O O H

HO~ y OCH 3

--~ Sinapic acid

R ~ , . ' ~ - v , ~ COOH

Chain elongation OH

:"C' f R ~ Styrylpyrones

\ \ O H

R . _ ~ C-CH3

Acetophenones

Flavonoids

OH

Xanthones

181

~ C O O H

Benzoic acids

X = OH, CoA (thioester) or glucose (1-O-acyl glucoside)

Figure 2. Schematic representation of the phenylpropanoid pathways. Cinnamic acid and its simple derivatives are the common precursors of key-intermediates of the various phenylpropanoid classes illustrated. The class-specific key-intermediates in turn are structurally diversified to yield many thousand individual compounds. (Courtesy of Dr. Dieter Strack)

which are synthesized in the roots of Senecio species (see below), processes that require specific loading and unloading mechanisms. Root-to-shoot translocation following the transpiration stream via the xylem vessels are known from tropane alkaloids and nicotine. Within an organ, short distance transport is needed to translocate the compounds specifically to their pre-

ferred sites of storage (e.g., epidermal tissues) (Hart- mann, 1991).

Specific sequestration in elaborately fashioned compartments is one of the most remarkable features of constitutive secondary metabolism. Water soluble secondary compounds are typically stored in cell vacuoles. Specific membrane-carriers and other biochem-

182

a-Terpinene

a-Pinene

"Active isoprenes"

--~AOp P

Geranyl diphosphate

"OPP f Myrcene

i• ~ Umonene

~Plnene ~ ~OPP ~_. S / ~ Borny, d/phosphate Camphene O~ ~ ~0

1,8-Cineole Sabinene Camphor

I

Figure 3. The various types of cyclic monoterpenes are synthesized from the common precursor geranyl diphosphate by action of specific monoterpene cyclases.

ical mechanisms are needed to maintain the often extremely high vacuolar concentrations (e.g., >250 mM of berberine alkaloids in Chelidonium majus, Papaveraceae) (Wink, 1987). Lipophilic compounds accumulate in glandular trichomes (e.g., the monoterpenes of mints), in resin ducts (e.g., pine trees) or secretory cavities (e.g., in orange peels). All these compartments contain a large intercellular space, lined by specialized epithelial cells which synthesize and

specifically secrete the lipophilic compounds into the extracellular space.

The most evident differences between primary and secondary metabolism are the different metabolic characteristics (Figure 4). Since secondary compounds often do not undergo rapid turnover (i.e., actual degradation in balance with synthesis) their formation is characterized by low steady state dynamics and thus low specific activities of the enzymes involved (Fig-

ure 4). This appears reasonable, since constitutive secondary compounds must just be synthesized to be stored at the right place and time in concentrations needed to fulfil their function (e.g., defending against herbivores or pathogens). There is no real need for turnover. Unfortunately, in many studies the simple disappearance of secondary compounds is taken as evidence for turnover. Disappearance of a metabolite, however, may include biosynthetic interconversions, conjugation reactions, or polymerization; it says noth- ing about the actual metabolic fate of the compound (Gershenzon, 1994). In addition, studies in which precursors or products are exogenously fed to plant tissue cultures or isolated organs may produce artefacts. The only way to actually document turnover is to follow the fate of the endogenously synthesized secondary metabolites. Two of the most prominent and often quoted examples for 'turnover' later turned out to be artefacts: (1) monoterpenes synthesized with labelled 14CO2 in detached mint shoots exhibit pro- nounced turnover, but in the same experiment per- formed with rooted plants the labelled monoterpenes synthesized remain stable for at least 40 days (Mihaliak et al., 1991); (2) experiments with endogenously produced nicotine usinglSNO3 as biosynthetic precursor provided no evidence for nicotine turnover in Nico- tiana sylvestris (Baldwin et al., 1994). This result con- trasts with studies reporting the half-life of nicotine as < ld (Robinson, 1974). But the high estimates of nicotine turnover are derived from studies in which labelled nicotine was introduced to the plant, rather than its biosynthetic precursors, and hence the high rates may reflect nicotine detoxification or other types of salvage metabolism, rather than normal nicotine turnover (Baldwin et al., 1994). These examples by no means categorically exclude the occurrences of turnover in secondary metabolism. But they demon- strate that turnover and degradation are not essential for secondary metabolism. Clearly some secondary metabolites, particularly those playing dual roles in defence and storage in seeds (e.g., canavanine), are readily metabolized during germination. Despite lack of turnover, many secondary metabolites are dynamic in respect to transformation (i.e., structural diversification) (Figure 4). As already discussed, secondary pathways often constitute a basic route, leading to one or few key-metabolites (P in Figure 4) that are diversified by enzymatic transformation (P1 to P5 in Figure 4). Basic routes and transformations are often spatially and temporally separated. Menthone produced during the early stages of mint leaf development, for exam-

183

pie, is further transformed into menthol, menthylesters and menthofurane in maturing mint leaves, which no longer de novo synthesize monoterpenes. Nicotine produced in Nicotiana roots is subsequently converted into nornicotine by demethylation in the stem. Gener- ally these transformations are simple chemical modifications which are catalyzed by substrate specific enzymes. An impressive example is the transformation of quercitin, the key-intermediate of the flavonols, into polymethylated flavonol glucosides in Chrysos- plenium americanum (Saxifragaceae) (Ibrahim et al., 1988). The methylation occurs in a stepwise fashion as given in Figure 5. One methyl group is added after the other by catalysis of a series of distinct, substrate- specific O-Methyltransferases. Although only a lim- ited number of secondary pathways has so far been characterized enzymatically, high substrate specificity seems to be a general feature of the enzymes involved. In the laboratory of M. H. Zenk the entire biosynthetic pathway leading from two molecules of tyrosine to the most highly oxidized benzophenantridine alkaloid, macarpine was elucidated (Kutchan & Zenk, 1993). This pathway, with its 20 specifically catalyzed enzymatic conversions, is the longest secondary metabolic sequence so far completely elucidated at the enzyme level. We must realize that each secondary pathway with all its diverging side branches and products is specifically brought about in a unique manner by a number of highly specific enzymes. Thus, biosynthesis of the many thousand secondary products should be accomplished by an even higher number of distinctive enzymes governed by a respective number of genes. This is easily seen in Figure 5: assuming that the products P1 - P5 are formed from P by five distinctive enzymes and the basic pathway (A to P) requires three enzymes, we need a total of eight specific enzymes to produce the five final products.

One of the unique features of secondary metabolism is the high 'degree of freedom' of its components. A certain constituent can vary qualitatively (structurally) and quantitatively (in concentration) or may even disappear without disastrous consequences for growth and development of the producing organism (see Table 1). On the contrary, primary metabolism and all its components which are needed for growth and development, must be stringently maintained stable to ensure the structural und functional integrity of the cell or organism. In fact, the degree of freedom (in the chemical sense) of secondary metabolism is the mechanistic basis for chemical variation, and consequently the prerequisite for diversification under selective pres-

184

Primary Pathways

A = B = C

High steady-state dynamics High turnover High enzyme activity (nkat/mg range) Feedback regulation

G ~ e .~

Reversible storage

Macro- molecules

P1 Secondary Pathways Transformation/,.~ P ~

A--~ B ---~ C --~ P ~ ~ P4 - ' - " - " ~

~ P5 1 Accumulation Low steady state-dynamics Often absence of turnover Low enzyme activity (pkat/mg range) Rarely feedback regulation

Figure 4. The differences in dynamics between primary and secondary pathways. A to C = intermediates of basic pathways; P = product/key- intermediate; P1 to P5 transformation products.

sure (i.e., herbivores, pathogens, physical effects) of a competing and continuously changing environment.

The pyrrolizidine alkaloids: a case study

Pyrrolizidine alkaloids (PAs) are a typical class of secondary constituents well suited to exemplify some of the general aspects of secondary metabolism discussed above. In this chapter I briefly discuss some recent research in the PA field from our laboratory intending to combine biochemical, physiological and functional (chemoecological) aspects.

PAs encompass a diverse group of about 360 structures with restricted occurrence in certain higher plant taxa such as the genera Senecio (Asteraceae, tribe Senecioneae), Eupatorium (Asteraceae, tribe Eupatorieae), Heliotropium (Boraginaceae), Crotalar- ia (Fabaceae). PAs are important components in the chemical defence of the producing species, which are usually avoided by herbivores. PAs are feeding deter- rents and per se non-toxic, but they are pro-toxins that are readily transformed into toxic pyrrolic metabolites by the action of mixed function oxidases (e.g., in the

liver of vertebrates) (Mattocks, 1986). PAs are pow- erful defence compounds. This is evidenced by the fact that a number of insects from diverse taxa have evolved adaptations not only to overcome PA-plant defence, but also to sequester and utilize PAs for their own defence against insectivores (for review see Bop- prr, 1986; Hartmann, 1991; Hartmann & Witte, 1995).

Tissue distribution, translocation, storage. In Senecio plants, PAs are detectable in roots, shoots and inflorescences at all developmental stages. The PA concentrations between organs, however, are quite different (Hartrnann & Zimmer, 1986). In flowering specimens of Senecio vulgaris, for example, the inflorescences contain 60-80% of total PAs at a tissue concentration which is 10- to 30-fold higher than in the vegeta- tive parts. Thus, PAs are maintained in defined tissue- specific distribution patterns.

Studies with plant in vitro cultures and intact rooted plants revealed that in S. vulgaris and a number of related species PAs are exclusively synthesized in the roots (Hartmann & Toppel, 1987). PAs (e.g., senecionine) are synthesized in the form of their polar salt-like N-oxides (see Figure 6). These are exported from roots

OH[6]

[ 2 ] H O ~ O I ~ . ~ OH[3]

[5] [4 ]~, - . .~ 0 H [11 OH Q

Transformation proceeds in stepwise fashion and precise order

Oglucose I~OCH3

H3CO ~ " ~ O i ~

H3CO" Y -'~ -OCH3 OH O

Figure 5. The final steps in the biosynthesis of 3,6,7,4'- tetramethylquercetin-3-O-glucoside from quercetin. The reactions proceed precisely in the order given by the numbers in parentheses (lbrahim et al., 1988).

via the phloem path into shoots, and are efficiently allo- cated to the sites of preferential storage (Hartmann et al., 1989). Phloem loading and unloading of the polar PA N-oxides are assumed to be specific processes that predict the existence of selective membrane carriers. Plants that do not produce PAs (e.g., Galinsoga and Achillea, Asteraceae) are unable to translocate PAs via the phloem (Hartmann et al., 1989). A specific carrier- system responsible for the selective uptake and safe storage of PA N-oxides in cell vacuoles could be characterized from cell cultures of S. vulgaris (Ehmke et al., 1988).

Biosynthesis and maintenance. Since the roots are the sites of PA biosynthesis, root cultures of S. vulgaris and other S. species were found to be excellent in vitro systems to elucidate the biosynthetic pathway on the enzyme level (Hartmann & Toppel, 1987). The pyrrolizidine moiety (necine base) of PAs is derived from arginine via putrescine and the enzyme catalyzing the first reaction of the alkaloid specific pathway could be identified as a homospermidine synthase (Btttcher

185

et al., 1993, 1994). This unique enzyme links the secondary pathway to primary metabolism (putrescine). Alkaloid formation is strictly linked to growth velocity (Hartmann et al., 1988; Sander & Hartmann, 1989). Furthermore root cultures accumulate PAs as stable products that undergo neither turnover nor degradation. In root cultures of S. erucifolius, for example, a 'population' of 14C-labelled alkaloid molecules produced in pulse-chase experiments with labelled arginine or putrescine, remains entirely stable over a growth peri- od of 15-19 days. The absence of significant turnover and degradation of PAs requires rigorous control mechanisms that adjust the total amount of PAs found in a plant to its growth velocity. The nature of these mechanisms are unknown, but certainly the decision con- cerning how much alkaloid is to be produced must be made at the actual site of alkaloid synthesis and is most likely associated with homospermidine formation.

Transformation of senecionine N-oxide: generating diversity. Although various root cultures of Senecio spp. have quite different patterns of PAs, they all produce senecionine N-oxide as primary product of biosynthesis (Hartmann, 1994). Extensive tracer studies with different Senecio spp. revealed that shoots and leaves of these species are able to convert [14C] senecionine N-oxide into the species-specific PA derivatives. These conversions comprise simple reactions including position-specific hydroxylations, dehydro- genations and epoxidations (Figure 6). (Dierich, 1992; Hartmann, unpubl, results).

Although the enzymes (and genes) responsible for this diversification have to be characterized, it is reasonable to assume that variation in the presence and/or activities of these peripheral enzymes are causal to variation observed in PA patterns between populations of, for example, S. vulgaris and S. vernalis (von Bors- tel et al., 1989) or S. jacobaea (Witte et al., 1992). Spontaneously occurring changes in the activities of peripheral enzymes, i.e., mutations of the underlying genes provide the mechanistic basis for chemical diversity upon which processes of natural selection may act.

Conclusions

Structural diversity and intraspecific variability are the most striking characteristics of plant secondary metabolism. Following the hypothesis that secondary metabolism evolved under selection pressure, chemical diversification is one of the essential needs for

186

........ oo o O o" O O

Acetylerucifoline N-oxide Erucifoline N-oxide Seneciphylline N-oxide Jacozine N-oxide

HO H O ~ 0

O" Erucifiorine N-oxide

\ / HO CH3 I OH HO~z/

H 3 c ~ O O ~ , o ~ O 3 O ~ CI/0~O~~00

O" O" Senecionine N-oxide Jaconine N-oxide

/ \ HO CH.,

HO / H3C~~ 'TO ~ ~ o ~ o

CH~ o- Senkirkine Jacobine N-oxide

Figure 6. In various Senecio spp. senecionine N-oxide is synthesized in roots as a common key-metabolite which in turn is structurally diversified into the species-specific PA-pattems by simple but specific reactions such as dehydrogenation, hydroxylation, epoxidation, acetylation etc.

secondary metabolism to cope with and adapt to a continuously changing environment. At least three important features become visible that form the mechanistic basis for diversification: (1) a high 'degree of chemical freedom' in secondary metabolism (freedom for structural modifications with almost no restrictions); (2) the existence of strictly regulated central pathways leading to complex key-metabolites well suited for chemical diversification; (3) recruitment and optimization of enzymes for specific but unlimited chemical diversification of the key-metabolites. Like diversity, intraspecific variation is inherent to secondary metabolism. For a number of secondary compounds, such as the

PAs, the main targets for variation are the peripheral enzymes responsible for generating species-specific patterns of secondary compounds. Due to the high degree of chemical freedom, the potential for variation of metabolite patterns caused by spontaneous mutations in structural or regulatory genes governing a given pathway is, in principle, inexhaustible. Whether or not a certain compound, or pattern of compounds, or the whole plant population producing it, will survive in nature, depends mainly on its capability and flexibility to withstand selection pressure in a competitive environment. We are still a long way from understanding how secondary compounds are synthesized and diver-

sifted, h o w these p r o c e s s e s are r egu la t ed and h o w they

are a d a p t e d to the n e e d s o f the e n v i r o n m e n t . Bes ides

b iochemis t ry , the p o w e r f u l tools o f m o l e c u l a r b io lo -

gy m a y a l low us to s tudy s t ruc tura l t rai ts on the gene

level , w h i c h m a k e s the ou t l ook p r o m i s i n g for fu ture

d e v e l o p m e n t s in th is field.

Acknowledgement

T h e r e s e a r c h in the a u t h o r ' s lab was suppo r t ed by

gran t s f r o m the ' D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t '

and the ' F o n d s de r C h e m i s c h e n I ndus t r i e ' .

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