Phytochemicals: Differentiation and function

Vol 29,No 6, pp 1715-1724,1990 PrInted,n Great Brltaln

0031 9422/90$300+000 0 1990Pergamon Press plc

Phyfochemmy,

REVIEW ARTICLE NUMBER 53 PHYTOCHEMICALS:

DIFFERENTIATION

AND FUNCTION

OTTO R. GOTTLIEB

Instltuto de Quimlca, Universldade de SHoPaula, 05508 Srio Paula, SP, Brazil (Recewed 28 June 1989) Key Word Index-Primary metabohtes; general metabolites, special metabohtes; phytochemlcal dwwons; structural types, alternatlve expresslon, evolutionary canalization, oxldatlve evolution; turnover rates; protection

devices.

Abstract-In order to elucidate the causes of phytochemical differentlatlon, the alternative expressions of general vs special metabolic divisions, herbaceous vs woody metabolic classes, subclasses and structural types, as well as the selection and orientation of protection devices of these types m the formation of natural products, were submitted to stepwise analysis. Internal factors were shown to dominate the biosynthetic driving forces at all these metabolic ranks. Environmental factors, such as herbivory, constitute additional driving forces at the highest rank, accentuating the expression of special metabolism, and at the lowest rank, where the regospecific localization of protection devices on structural types directs compound diversity.

INTRODUCTION It 1s invariably assumed that the characteristics of an organism-from the morphological to the molecularhave been selected for their functional advantage Cl]. Nevertheless, the quest for the factors mvolved m the selection of phytochemlcals has proved to be extraordinarily elusive. The presently most widely respected concept envisages, according to Janzen, that “natural selection serves as a mechanism by which a population of herbivores may call forth de nouo the evolution of a biosynthetic pathway producing compounds toxic to the herbivore” [2]. In another opinion, expressed by Muller “associated animals are possessed of no mechanism by means of which they can call forth de nouo the evolution of a specific metabolic mechanism m plants. If, however, a plant species has several alternative and simultaneous metabolic pathways already m operation, producmg varying quantities of the numerous by-products characteristic of plants, selective pressure might well increase the proportion of one of these Thus the toxicity to animals of these metabolic wastes, no matter how important eventually, is subsequent and secondary to their elimination from protoplasm” [3]. However, as recalled by Haslam, “many biologists find metabolic inefficiency, as implied in Muller’s point of view of co-evolution difficult to concede” [l]. “It is a precept of modern chemical ecology that energy is not likely to be ‘wasted’ in the production of secondary metabolites unless there 1ssome compensating adaptive advantage to the organism in question” [4], These theories are based on circumstantial evidence and from here on the only rational step forward must imply the mtroduction of experimental evidence. This has so far been tested by considering the bioactivity of secondary metabohtes. We [S] have noted a trend to-

wards increasing toxicity of alkaloids associated with evolutionary advancement within the angiosperms. Evidence of this sort would seem, at least a priorr, to favour Janzen’s point of view. In contrast, Haslam [l] reported that vascalagin and castalagin predominate in oak leaves in spite of the fact that the astringency of the precursor of these two tannins, namely penta-O-galloyl-D-glucose, is considerably higher and considered this evidence to be in favour with Muller’s view of plant defence. However, it seems unlikely that evidence based on bioactivity will ever provide an answer to the question. With respect to natural products, is their biosynthesis triggered by their bioactivity (m Janzen’s sense) or is then bioactivity controlled by their biosynthesis (in Muiler’s sense)? An escape from such a circular argument should, at least theoretically, be possible by a step-by-step attribution of internal vs environmental driving forces to metabolic differentiation.

METABOLICDIFFERENTIATION The basic metabolism of autotrophic plants combines photosynthesis with respiration, leading from CO1 via the sugars of the Calvin cycle, pyruvic acid and acetic acid either to the fatty acids of the Lynen spiral (a reversible process) or to the simpie aliphatic acids of the Krebs cycle and thence back to CO,. Connected by mostly reversible pathways to the basic metabolites are some essential intermediates such as the Krebs-cycle-derived aliphatic ammo acids, the purines and pyrimidmes, the acetic acidderived mevalonic acid, the sugar-derived glycerol and the sugar-plus-pyruvic acid-derived shiklmlc acid, the latter functioning as a precursor to the aromatic amino acids.

1715

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R. GnnLlEB

Basic and Intermediary (jomtly designated primary) metabohsm forms an integrated system, a first metabohc dlvlslon, which extends branches mto two further broad dlvlslons of metabolites, a general one and a special (usually called secondary) one General metabolism transforms primary metabohtes into macromolecules, particularly ammo acids mto proteins, purmes and pyrlmldmes plus sugars mto nucleic acids, sugars into polysaccharides and glycerol plus fatty acids mto hplds Special metabolism can be separated into two subdlvlsions. a herbaceous one and a woody one The former comprises classes of compounds of simple blosynthetlc ortgm such as ahphatlc ammo acid-derived alkaloids (e g pyrrohzldines and quinohzidines), non-protein ammo acids and ohgopeptldes, mevalomc acid-derived terpenolds, acetic acid-derived polyketides, sugar-derived ohgosaccharides, as well as classes of mlxed blosynthetlc origin such as the tropane alkaloids, which are derived from ahphatic ammo acids and acetic acid The woody subdivlslon comprises shlklmlc acid-derived hgnolds and benzyhsoqumolme alkaloids, as well as classes of mixed blosynthetlc origin such as the indole alkaloids, which are formed from shiklmlc acid plus mevalomc acid and the flavonolds, synthesized from shlklmlc acid and acetlc aad. Each of these classes of special metabohsm may comprose a series of subclasses of progressively lower rank. For instance the class flavonolds incorporates three subclasses of rank 1: flavonolds, lsoflavonolds and neoflavonolds, and for instance the subclass of rank 1 lsoflavonolds mcorporates several subclasses of rank 2: isoflavanones, Isoflavones, rotenolds, pterocarpans, coumestans, 3-aryl-4-hydroxycoumarins, isoflavans, etc. Particular structural features of the compounds classified mto the subclass of lowest rank define structural types With flavonolds, these features concern oxygenation patterns eg the subclass lsoflavones comprises the 7,4’dlhydroxy-, 5,7,3’,4’-tetrahydroxyand other types. Finally differential substltutlon (glycosylatlon, methylatlon, prenylatlon etc) ofeach of the structural types defines the mdlvldual natural products. As occurs m the case of the macromolecules of primary metabohsm (which lead to the macromolecules of general metabolism), the macromolecules of special metabolism also may combine mto macromolecules (of special metabohsm) Thus, flavonolds lead to condensed tannins, sugars plus shlklmate-derived gallic acid lead to hydrolysable tannins, hgnolds lead to hgnins, terpenoids lead to latlces and resins, hydroxy fatty acids plus hgnolds lead to cutms and suberms, fatty acids plus fatty alcohols lead to waxes Thus, by analogy with the hlerarchlcal classification of hvmg orgamsms mto kingdoms, dlvlslons, classes, subclasses, orders, famlhes, genera, species, populations, speclmens, a classlficatlon of phytochemlcals leads to the recogmtlon of dlvdons, subdlvlslons, classes, subclasses, structural types and mdivldual compounds. In both cases the classificatory units sit on a dendrogram with a progressively larger number of branches Equally m both cases no functional roles are implied in the characterization of any of the classificatory units. With relief one can stop sophlstlcatmg about primary roles for secondary compounds, e.g. the effect on growth of mdoleacetlc acid [6], or about secondary roles for primary compounds, e g the attraction for pollinators of fructose [7]. If consideration of function IS necessary, this can be done independently on another plane of mformatlon

METABOLIC

FLNCTION

The analysis of internal vs envlronmental driving forces towards metabolic differentlatlon must start at the bifurcation leading from primary metabohsm either to general or to special metabolism (Fig 1) Primary metabolism effects the complete turnover of material Nearly all the oxygen produced by photosynthesls m green plants and algae is cycled through the atmosphere and used up m that other fundamental actlvlty of hfe, respiration, m a relatively short space of time This complementary process thus cannot, by Itself, lead to the accumulation of phytochemlcals unless at least one of two phenomena occurs: the fast ehmmatlon of orgamcs from the equlhbrmm and/or the dlmmutlon of their rate of decomposltlon back to CO,. Elimmatlon from the equlhbrmm occurs by dehydrative polymerization and leads to the production of the polymeric structures of general metabohtes It should not be imagined that either the reductions leadmg from CO, to monomers or the dehydrations leading on to polymers are simple, perhaps even spontaneous, processes Indeed m the aerobic and aqueous medmm of the orgamsm precisely the opposite is the case In order to proceed at all, such reactlons need powerful activation regulated by the ratios of NADH/NAD+, ATP/ADP and acetyl CoA/ HCoA or their analogues. If the ratios of these different pairs of compounds IS high (e g. 10 1) then the cell 1s fully ‘charged’ with energy rich compounds (1 e NADH, ATP and thloesters) that can be used to carry out energyconsummg processes [S]. It 1s easy to foresee that shortage of some basic requirements, such as orgamsm-specliic quantities of phosphate, mtrate etc and levels of hght, temperature etc should Interfere with this complex machmery conslstmg of a large number of addltional components. If in consequence those ratios become low (e.g. one or less) then the cell has been depleted of the compounds it needs to carry out energy-consuming processes [S]. At this instant the concentration of UTP, GTP, CTP, ATP+UTP+GTP+CTP and dATP + dGTP + dCTP + dTTP bemg relatively low the dehydratlve polymerization to polysaccharldes, proteins, IIplds, RNA and DNA will slow down At the same time the concentration of NAD+ IS relatively high and the oxldative degradation of acetic acid via the Krebs cycle to CO, IS enhanced

GiAL.

co2

metaboliles

m~cromoiecules

General

Special macromolecules

macromolecules

Fig. 1 SchematIc representanon of carbon flow through bolic C

pools in plants condensation,

Major reactlon types d dehydration, o

r

metareduction, oxldatlon

Phytochemlcals

drfferentlatron and functron

1717

Aromattc annno actds ( Phe, Tyr )

I

Flavanones

Cmnamrc acrds

II 1

-

* Fiavonols

Chalcones

Flavones

I I Benzotc

I 2.3 - Drhydroxyflavanones

acidd

Fig 2. Drrect (vra /?-oxtdation) and mdnect (vra some flavonord types) turnover of cmnamic acids (spud Bar-z and Hose1 [ll])

Dtminutton of the rate of decomposition of the intermediates, i.e resistance to thts loss of carbon may constitute the driving force towards them scavenging, by condensation reactions, into more difficultly degradable, and hence necessarily complex, btzarre ‘natural products’.

This driving force for the generation of natural products is conststent with the concept of their turnover, i.e. progressive oxidative transformations which frequently lead back to primary metabolites [9]. For instance, cmnamic acids, intermediates of the primary shtkimate pathway, are degraded by very few steps, successively /Ioxidatton (to benzoic acids), Woodward fisston (to simple aliphattc acids) and oxidation (to CO,) [lo], unless obliged to pass through a long series of intermediates after protective condensation wtth acetic acid This concept 1s implicit m Barz and Hosel’s metabohc grid depicting anabolic and catabolic pathways of some flavonoids (Fig. 2) [1 11. Analogous diagrams can be drawn to explain the scavenging of amino acids (after oxidative transamination to oxoacids) by mtra- or intermolecular nitrogen, via Schiff bases, into complex alkaloids (Fig. 3); of acetic acid into polyketides; of mevalonic acid into terpenoids of homogentisic acid into compounds of Aromatic am,no acids ( f%

TY~ )

Arylpyruv~c

I

aads

L

1 Arylacets actds /e”m,c sods ~

Schlff bases -

Benzyltetrahydmlsoqulnollnes 1

Altphattc acads

CO2 Frg. 3 Direct (via oxrdatrve transaminatron, oxtdattve decarboxylatron and Woodward fission) and mdnect (via benzyhsoqumolme alkalotd types) turnover of phenylalanme and tyrosme

mixed biosynthetic origin (Fig. 4). A highly schematic dragram for the turnover of oligomeric products is shown

in Fig. 5 Thus, as Bu‘Lock [12] has suggested, “secondary metabolism serves to maintain basic metabolism in ctrcumstances when its normal substrates, through deple+ton of nutrients, cannot be exploited for normal cellular growth and replication”. A complementary aspect of the concept refers to the tmphctt unity of prtmary and special metabolism. As Haslam has pointed out, “many secondary metabolites are formed, at least in part, by reaction sequences that are entirely analogous to those in the more ubiquitous pathways of primary metabolism. In some mstances a secondary biosynthetic pathway (and its associated enzymes) appears to have evolved directly from a prtmary pathway” [l]. This mechanism of metabolic alternation may work also m a plant under normal conditions. When NADH/ NAD+ and ATP/ADP ratios are correct, e.g. for frmting, nutrtents are canalized into fruits to promote production of general metabolites and hence their growth. At the same ttme nutrient-deficient conditions may ensue, e.g. in leaves and favour, in these organs, synthesis of spectal metabohtes. Analogously upon attack by herbivores or chemicals, the consequent diversion of energy and nutrients from other plant needs to reconstruction of the offended parts may again favour the synthesis of special metabohtes. Thus, although the prtmary function of natural products (i.e. secondary or better, as 1s here proposed, special metabolites) is Intrinsic, then productton, tf considered from the viewpomts of timing and quantity, nevertheless constitutes an important link m the mechanism of plant-herbivore interaction. The second and third ranks of decision concern the choices mittally among the subdivisions and next among the biosynthetic classes of special metabolites. Here preliminartly a distinctton must be made between herbaceous and woody chemistry. The former uses sugars, ammo acids, acettc acid and mevalonic acid as precursors for phytochemicals, while the latter uses addittonally or singly shtktmic acid (chiefly post-phenylalanine compounds)

1718

0

OH

R

GOTTLIEB

OH

Fa

I

0 0

0-O

++

OH

Y-

J

0

Fig 4 Direct (via Woodward

AromatIc precursors

fission) [lo] and proposed mchrect (via metabohtes [33]) turnover of homogentislc acid Fa = Farnesyl

Ahphatlc precursors

D

A E

B F

C G

Benzolc UT

acids

Sample -8 ahphatlc acids

+ a

Fig. 5 Direct and mdlrect (via special metabohsm) turnover of primary metabohtes SchematIc pathways are deplcted for classes ofnatural products. such as (A) benzyhsoqumohne alkaloids, (B) ffavonolds, (C) hgncnds, (D) qumohzldme alkaloids, (E) terpenolds, (F and G) polyketldes

isolated

from Otohn part~jiibl~u

Shlklmate derived (woody) chemistry was added to acetate derived (polyketlde and terpenold) chemistry with the early land flora and dlversrfied therefrom gradually, only to decline notlceably m the more modern anglosperms. Presumably hgnold chemrstry arrived at a chmax with the primitive flowering plants, attammg propenylphenols and allylphenols as the termmal metabohtes of the C, C,-sequences [S, 13. 141 From here on two alternatlve evolutionary pathways are followed One leads on by oxldatlve dlmerlzatlon of these phenols to neohgnans, which are common constituents of prlmrtlve angiospermous families Neolignans appear subject to oxldatlve degradation via simple compounds to CO, (Fig 6) The other pathway leads, by progressive shortening of the shlklmate route to cmnamyl alcohol derived hgnans and hgmns, to cmnamlc acid derived coumarms, to phenylalamne derived benzyhsoqumohne alkaloids, to anthramhc acid derived acrtdones, and at the end of the retrogression, the galhc acid derived gallo- and ellagltanruns which are common constituents of angiosperm famihes of intermediate evolutionary status Substantial abandonment of the shlklmate pathway leads to pre-

Phytochemlcals

dlfferentlatlon and function

1719

Fig. 6. Formulae of two neohgnans from Ocotea aclphylla [34], putative mtermedlates In the degradatlve pathway from the shlklmate-derived allylphenols and propenylphenols to simple ahphatlc acids and finally CO,

ponderance of acetate-derived phytochermcals which are common constituents of advanced anglospermous famiiles. The opposite evoiutlonary polarity can be ruled out because the biosynthetic classes iabeiied as primitive in the scheme (e.g. neohgnans, tyrosme-denved alkaloids and cyanogens) do sporadically occur m advanced famliles, whereas biosynthetic classes considered to be advanced (e.g. phenyiaianine-derived cyanogens, anthramiatederived alkaloids, poiyacetyienes, lridolds) never occur m primitive families Gaiio- and eilagltannins constitute a special case. Not only, as indicated above, are they absent from primitive angiosperms, but they are also usually absent from advanced ones m which the expression of shlklmate-derived phytochemlcais IS often superceded by the expression of acetate- and mevaionate-derived products Analogous evolutionary transitions from shlklmate-toacetate-derived compounds can be noted in angiosperms also at lower hierarchical levels. For instance, the relatively primitive famlhes of the superorder Magnoliiflorae contam a considerable number of structural types belonging to the shlkimate-derived neohgnan and benzyiisoquinohne classes [IS], whereas the relatively advanced Asterlflorae contain a rich variety of acetatederived poiyacetyienes and sesquiterpene iactones [S, 163. An analogous trend can also be observed within families Thus morphoiogicaI differentiation of the various tribes of Rutaceae IS accompanied by the stepwise replacement of benzyhsoquinoimes (wholly hgnold) by prenyiated quinoiones and coumarms (non-iignoid/hgnoid) and finally hmonolds (wholly non-hgnold) [17] Chemical evoiutlon m angiosperms, as portrayed above, IS a manifestation of evolutionary canalization, if we equate hgnold deveiopment in primitive members of’ this division with the “certain stage” mentioned m the teachings of Kubltzkl et al. “Plant evolution IS subJect to intrinsic constraints which may iead to evolutionary canalization The latter process IS due to the fact that a certain stage reached m the evoiutlonary history of an organism clearly restricts the posslbliities for further evolutionary change so that, m the extreme case, under the necessity for further evoiutlonary change, only one option remams. Clearly this option may be identical even for phyiogenetlcaily vastly separated groups” [18] The fourth rank of decision concerns the choice among the biosynthetic subclasses of special metabohtes This depends on available, le inherited, enzymatic control The expression of the genetic potential can be correlated with established phyiogenetic lineages. An iliustratlve hst of findings includes the presence of benzyiisoqumoime aikaiolds in the magnofiaiean famliies, ofiridoids in C’ornitforae, Gentlanifforae and’ Lamn-

florae and of poiyacetyienes plus sesqmterpene iactones in Araiiiflorae and Asteriflorae [19] Confidence of the scientific community in the relevance of such biosynthetic cnteria in plant classification has increased conslderabiy in the past decade to the point that ail recent systematic treatises include chemical data [20, 211 and some reclassifications have been based to a surprising extent on chemical cnterla [22]. Examples are the use of betalams to help delimit the order Caryophyliaies, of giucosinolates to dismantle the Rhoedaies and to associate the Papaveraceae with the Ranuncuiiflorae and of tlgiiane, daphnane and ingenane type dlterpenolds to transfer the Thymeieaceae from the Myrtiflorae to the Malvlflorae ~231. Such molecular homology IS implied to reveal common ancestry. Indeed chemical gradients for genera, families, orders, superorders and divisions 15, 191 would not be perceptible if analogy, 1.e. appearance of Identical blosynthetic classes of metabohtes in different lineages on a non-homologous basis and m adaptation to similar functions and/or environmental conditions, 1s not relatively rare. However, even if switches of chemical composition within a morphological lineage are observed, they are not random, but are usually characterized by the replacement of one metabolic class or subclass by one other class or subclass. Such switches, usually from common, wldespread chemistries to unusuai, distrlbutlonaiiy restricted chemlstnes, become specially noticeable with geographical isolation of the plant population, and m angiosperms can often be correlated with trends towards the abandonment of the shiklmate pathway and/or with economy in the use of nitrogen [17] In our work on the magnolialean families we noted the rich diversitication oftwo groups ofnaturalproducts, the neoiignans and benzyiisoquinoiines. From a survey of their distribution the predominance of one of these groups or their mutual exclusion m these famlhes becomes evident. Furthermore, apparently the presumed loss of benzyhsoquinoimes in various small, isolated families, such as the Hlmantandraceae and Caiycanthaceae, has triggered the evolution of still other biosynthetic classes of compounds m compensation [24]. Other examples of chemlcai dichotomy Include the mutually exclusive presence of quassinoids vs iimonolds in families of the order Rutaies [25], of neohgnans vs mtrana alkaloids in genera of the family Zygophyilaceae, of benzyhsoqumoiine alkaloids vs diterpene alkaloids m genera of the family Ranuncuiaceae [26], of neohgnans vs aryi- and styryi-a-pyrones m species of the genus Aruba [27], the general trend of stepwise replacement of benzyhsoqumofmes by simple and compfex anthramhc acidderived afkafoids and eventually by coumarms

120

0

R

GOTTLIEB

Phytochemicals

and/or hmonoids accompanying morphological dtfferentiatton of tribes of Rutaceae [17], and the trend to predominance of polyacetylenes vs sesquiterpene lactones in the tribe Hehantheae of the family Asteraceae

L-281. The fifth rank of decision concerns the formation of parttcular structural types within each biosynthetic class or subclass Thts is determined, since the origin of life on Earth, by a gradually increasmg level of oxtdation. An overview of the structure of flavonoids shows Cmethylatton to become increasingly common with evolutionary advancement within the pteridophytes [ZO] and characterizes prtmitive woody angiosperms (especially of the family Annonaceae) C-Prenylation, very rare in these groups, 1s common in advanced angiosperms [13]. Replacement of an aromatic hydrogen by methyl, and even more efficiently by prenyl, lowers the energy required for oxtdative attack on the phenol. Wtth or without the help of such a mechanism, a gradual mcrease of mean oxidatton level of the collection of metabohtes within biosynthetic classes occurs with evolution of the parent taxa. We have measured the phenomenon quantttatively for carotenotds in families of algae, lignms and flavonoids in divtsions of terrestrial plants, labdanes and other classes of diterpenotd in families of Dillenndae, Rostdae and Astertdae, irtdoids in the superorders Corniflorae, Loasiflorae, Gentianiflorae and Lamuflorae, polyacetylenes m famtlies of the Araliiflorae and Asteriflorae and of some other superorders, benzyhsoquinohne alkaloids m families of the superorders Magnoliiflorae and Ranunculiflorae [ 193 and mdole alkaloids m the families Apocynaceae, Loganiaceae and Rubiaceae [S]. The evolutionary importance of oxidation reactions is indeed to be expected. After reduction of inorganic to organic carbon the initial anabolic stages (all established during the biottc evolutton in an anoxic atmosphere) are based mainly on condensatton (and reduction) reacttons leading to most primary metabolites. The pathways beyond these precursors on to special metabohtes (the vast majority added durmg evolutton in an oxygen

containing atmosphere) requires oxidative steps. In the words of Haslam “oxidative reactions of one form or another play a large part m many of the pathways to secondary metabolism” [l]. We have demonstrated a positive correlatton to exist between oxidatton level and skeletal spectalization for the structural types of several biosynthettc classes of metabolites (e.g. Fig 7) [19]. Thus, as the oxidation level, the structural type of natural products must also be independent of herbivore pressure The sixth and seventh ranks of decision concern the transformation of structural types into parttcular natural products, respecttvely through the selection and the orientation of protective devices. Progresstve increase in the oxidation level of metabolites would lead to progressive ease of then breakdown. However, trends towards gradually higher turnover rates of secondary mtcromolecules have not been observed. On the contrary, compounds of high oxidation level are often isolated in high yield. The reason for this relative stability lies m the concomitant evolution of protective devices Abstraction of a phenolic hydrogen by glycostdatton or etherification, in this evoluttonary order, is certainly a common reactton. We have evaluated the phenomenon quantitatively for flavonoids in tribes of Asteraceae and suggested its posstble usefulness m the determmation of the evolutionary polarity of parallel chemical gradtents in plant lineages [16]. Increasing frequency of 0-methylatton and O,Omethylenatron also accompany increasing oxidation level of compounds and hence evolutton of plants We have demonstrated the positive correlatton of oxidation level and 0-methylation of flavonoids for spectes of the genus Lonchocarpus [29]. Of course 0-methylatton of a phenol does not prevent it bemg oxidized, it only dtmmishes the rate of the reaction Excessive stability of a compound would lead to the accumulation of large quantities of material, which may harm the producer btodynamtcally or energetically. In agreement with this concept, in nature de-0-methylation of phenols does not proceed by acid catalysts, but again by oxidation [9]. Stmtlarly methylenation of catechols and its reversal does not proceed by

Fig. 8 General sequence of react&on types leading to special metabohtes to a chalcone,

l), oxldatlon

(e.g. to an lsoflavone,

1721

differentiation and function

2) and reduction

condensation (e g to an lsoflavan,

(e.g. cinnamate + trlacetate 3, and to a pterocarparf, 4)

1722

0. R GOT-TLIEB

ketahzatton and hydrolyses, but again by oxidation of guaracyl derivatives and hydrrde abstractron from the methylene Fmally, oxtdattve sequences of natural products are

sporadrcally Interrupted by reductrve steps A case in pomt concerns the formatron of flavonotd types. Here reductions all comcrde with late brosynthettc steps, leading to what apparently are end products of sequences, but

it

R A3

A

OMe

R PI + P2

OMe

R

PI

+ Al

R PI + A2

OMe

R

Fig 9 Blogenesls of selected neohgnan types, from related species of Lauraceae and Plperaceae, ratlonahzed by the couphng of propenylphenol-(P) and allylphenol-(A) dewed radicals, respectrvely Pl, P2 and Al, A2, A3 [36]

Phytochemlcals

dlfferentiatlon

what indeed may be compounds reductively protected against facile oxldatlve breakdown. Such products (5 deoxyflavonolds, flavans, lsoflavans, pterocarpans) (Fig. 8) agam only occur in angiosperms [29, 301. Flavonoids in primitive pteridophytes and gymnosperms are endowed with a cmnamate derived B-ring which 1s hydroxylated at least at the para-position (as m aplgenin) or even at the para- and mefa-positions (as m luteolin). Introduction of trihydroxylatlon (concomitantly with protective O-methylatlon) surely constitutes an evolutionary advance However, in some special cases, concentrated m advanced families of both plant divisions, respectively In Ptendaceae, Dryopterldaceae and m Pmaceae, absence of B-ring hydroxylation also occurs, a reductive and possibly stabilizing trend [20] Reduction as a protection device must also operate in other classes of natural products, such as the terpenolds We have measured this effect quantitatively for sesquiterpene lactones m tribes of Asteraceae [313. Besides modulation of the turnover rates of natural products possessmg progressively higher oxidation levels, the evolution of protective devices entails another consequence: orientation of the transformations of the oxldatlon products m particular directions. The elaboration of the protective devices 1s under strict enzymatic control and thus functions regiospeclfically [ 15). Indeed according to Poulton, “methyltransferases possess a much narrower substrate specificity than previously imagined” [32]. Let us examine this questlon through its most conspicuous example. A caffelc acid derived moiety, either by itself m a C,.C,-denvative or as part of a hgnold, flavonoid or alkaloid etc. could a prrm suffer, by partial ethenficatlon, two types of protection, one forming 3-hydroxy-4-methoxyphenyl units and one forming 4-hydroxy-3-methoxyphenyl (guaiacyl) units. The former alternative would be expected to be favoured from the chemical, mechamstlc point of view, the ConJugated pamhydroxyl in caffeic acid being more reactive. Nevertheless it is feruhc acid which if favoured enzymatlcally [32]. The biological explanation 1s easy it 1s the free 4-hydroxyl which confers upon all metabolites a much greater potential for diverslficatlon. Compare in this respect for example the ferulic acid-derived comferyl alcohol, and its tremendous potential of forming oxidatlve coupling products involving the side chain p-carbon, with 3-hydroxy4-methoxycinnamyl alcohol exempt of oxidative side chain activation Indeed if several dnectlons of postprotectional transformations are mechanistically feasible they are all realized, even if mostly m different proportions (Fig. 9). Clearly the formation of a large vs a small number of structural vanants (i.e. the orientation of protective devices mto positions which result in intermediates of greater versatility of reaction) could a prum represent the adaptive factor we are looking for, the synthetic potential which herbivores elicit de nouo and which, through the relative plasticity of the molecular consequences it engenders, integrates the evolutionary counterpoint between plant and herbivore. But even at this lowest rank of decision, co-evolution of phytochemlcals and herbivores cannot be taken for granted. Natural products are not only toxic to the associated biota, they are also toxic to the producer itself Furthermore, toxlclttes of compounds vary widely and the production of a large number rather than of a few may constitute natural selection of the least harmful for the

and function

1723

plant (the intrinsic factor) which in a variable environment still guarantees reasonable deterrence against herbivory (the extrinsic factor).

CONCLUSION

The present essay suggests implicitly that a plant composed only of primary and general metabolites, even m a sterile environment, 1s a theoretical abstractlon Indeed, contrary to a century-old belief, the so-called secondary (here designated special) metabohtes are equaIly essential to plant hfe. True, they also adapt an organism to herbivore pressure, but their protective functions are accidental, rather than original or predestined. Acknowledgements-The author has benefited greatly from helpful crltlcism of Prof. Dr Klaus Kubitzkl (Umversltat Hamburg) and Prof. Dr Peter von Sengbusch (Umversltat Bielefeld) and IS Indebted to Conselho National de Pesqmsas Cientificas e Tecnolbglcas, Bra&a, for a research fellowslup.

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