Journal of Chemical Ecology, VoL 20, No. 6, 1994

METABOLIC COSTS OF TERPENOID ACCUMULATION IN HIGHER PLANTS

JONATHAN

GERSHENZON

Institute of Biological Chemistry Washington State Universi~. Pullman, Washington 99164-6340 (Received October 12, 1993; accepted January 25, 1994)

Abstract--The net value of any plant trait can be assessed by measuring the costs and benefits associated with that trait. While the other contributors to this issue examine the possible benefits of terpenoids to plants, this article explores the metabolic costs of terpenoid accumulation in plants in the light of recent advances in terpenoid biochemistry. Terpenoids are more expensive to manufacture per gram than most other primary and secondary metabolites due to their extensive chemical reduction. The enzyme costs of making terpenoids are also high since terpenoid biosynthetic enzymes are apparently not shared with other metabolic pathways. In fact, plant cells may even possess more than one set of enzymes for catalyzing the basic steps of terpenoid formation. Terpenoids are usually sequestered in complex, multicellular secretory structures, and so storage costs for these substances are also likely to be substantial. However, not all of the processes involved in terpenoid accumulation require large investments of resources. For instance, the maintenance of terpenoid pools is probably inexpensive because there is no evidence that substantial quantifies of terpenes are lost as a result of metabolic turnover, volatilization, or leaching. Moreover, plants may reduce their net terpenoid costs by employing individual compounds in more than one role or by catabolizing substances that are no longer needed, although it is still unclear if such practices are widespread. These findings (and other facets of terpenoid biochemistry and physiology) are discussed in relation m the assumptions and predictions of several current theories of plant defense, including the carbonnutrient balance hypothesis, the growth-differentiation balance hypothesis, and the resource availability hypothesis.

Key Words--Terpenoid biosynthesis, terpenoid storage, secretory structures, metabolic turnover, volatilization, catabolism, carbon-nutrient balance hypothesis, growth-differentiation balance hypothesis, resource availability hypothesis. 1281 00980331/94/06(~ 128 ! $07.00t0 © 1994 Plenum Publishing Cor~oration

1282

GERSHENZON INTRODUCTION

As the largest group of organic substances in the plant kingdom, terpenoids play a multitude of important physiological and ecological roles in higher plants. Terpenoid primary metabolites serve as hormones, membrane components, photoprotective pigments, and membrane-bound sugar carriers in glycoprotein and polysaccharide biosynthesis (Gershenzon and Croteau, 1993). Terpenoid secondary metabolites have been implicated in defense against herbivores and pathogens, allelopathic interactions, nutrient cycling, and attraction of pollinators, dispersers, and entomophages, as detailed by the other contributors to this issue. However, from an ecological perspective, any enumeration of the benefits of terpenoids to plants should be balanced by a consideration of their cost. Indeed, several lines of evidence indicate that the accumulation of terpenoids may entail substantial costs in terms of energy and nutrients. First of all, terpenoids are sometimes present in plants at very high concentrations, for example, 10-13% of dry weight in the leaves of Eucalyptus dives (Morrow and Fox, 1980) and 9-16% of dry weight in the juvenile twig internodes ofBetula resinifera (Reichardt et al., 1984). Second, terpenoids are usually stored in complex secretory structures, such as resin ducts, glandular trichomes, or laticifers (Fahn, 1979). Third, certain terpenoids have been reported to undergo rapid metabolic turnover in plants (Seigler and Price, 1976), implying that continual synthesis may be necessary to maintain a fixed concentration. Fourth, inverse correlations sometimes exist between terpenoid accumulation and growth (Adzet et al., 1992; Hanover, 1966; Mathur et al., 1988). In Cymbopogon winterianus, for instance, a strong negative relationship was demonstrated between monoterpene content and aboveground biomass among a group of over 200 individuals (Mathur et al., 1988). Finally, there are indications that terpenoid defense compounds are reduced when there is less need for them. On the island of Puerto Rico, for example, where a certain specialist seed predator found on the Central American mainland is absent, the tropical legume Hymenaea courbaril has lower amounts of terpenoid resins in its seed pods than do conspecifics growing on the mainland (Janzen, 1975). Thus, costs are widely believed to influence the amounts of terpenes and other secondary metabolites found in plants and may at least partially explain terpenoid distribution patterns among species, individuals, and organs (Chew and Rodman, 1979; Coley et aL, 1985; Fagerstrom, 1989; Gulmon and Mooney, 1986; Krischik and Denno, 1983; Rhoades, 1979). The costs of terpenoid accumulation can be evaluated in an evolutionary context in terms of reproductive fitness or in a metabolic context in terms of resources, such as fixed carbon or nutrients. While fitness is a more relevant currency for assessing ultimate costs and benefits (Simms and Rausher, 1987), it is difficult to measure under natural conditions (Chapin, 1989). Costs expressed in terms of resources, sometimes referred to as metabolic costs, direct costs

METABOLIC COSTS OF TERPENOIDS

1283

(Gulmon and Mooney, 1986; Zangerl and Bazzaz, 1992), or allocation costs (Simms, 1992), may be easier to quantify and should help to identify the major physiological processes contributing to cost (Lerdau, 1993). This paper surveys the metabolic costs of terpenoid accumulation in higher plants in the light of recent progress in terpenoid biochemistry. The approach taken is, of necessity, quite speculative, since very little research has been directed specifically towards establishing the costs of terpenoid accumulation. However, within the last few years substantial advances have been made in our general understanding of terpenoid metabolism, yielding a wealth of new information on the pathways and enzymes of biosynthesis, and the sequestration, turnover, and catabolism of various terpenoids. These findings afford fresh insights into various aspects of the costs of terpenoid accumulation. In this review, I examine the costs of terpenoid biosynthesis, storage, and maintenance, and outline a number of mechanisms by which terpenoid costs may be reduced. Several current theories of plant defense are also discussed in the context of what is known about terpenoid costs. BIOSYNTHETIC COSTS

Costs of Substrates and Cofactors Terpenoids are formed from the fusion of five-carbon units having a branched, isopentanoid skeleton. Each unit is constructed from three molecules of acetyl-coenzyme A (acetyl-CoA) via the mevalonic acid pathway, a process that utilizes three molecules of ATP and two molecules of NADPH (Gershenzon and Croteau, 1993; Goodwin and Mercer, 1983). Following the initial condensation of five-carbon units, terpenoids undergo many types of cyclizations, couplings, and rearrangements to generate the basic representatives of each skeletal class. These products are then subject to a variety of ATP- or NADPH-mediated transformations, including oxidations, reductions, and conjugations, that eventually give rise to the many thousands of different terpenoid metabolites found in plants. Thus, terpenoid biosynthesis requires ample supplies of the substrate acetyl-CoA and the cofactors ATP and NADPH as raw materials. Nearly 20 years ago, a young Dutch ecologist named F.W.T. Penning de Vries, developed a straightforward approach to calculating the costs of the raw materials involved in the synthesis of plant metabolites, based on procedures for estimating growth yield and efficiency in microorganisms (Penning de Vries et al., 1974). In these methods, which were later refined by Mooney and coworkers (Chiariello et al., 1989; Gulmon and Mooney, 1986; Merino et al., 1984; Williams et al., 1987), cost is determined by computing the amount of glucose required to provide all the substrates and cofactors consumed in biosynthesis. Glucose or another simple carbohydrate seems a suitable unit of currency for

1284

GERSHENZON

such calculations, since carbohydrates are the usual storage and transport forms of fixed carbon in plants and can be readily respired to generate ATP and NADPH. Using this methodology, I have calculated the substrate and cofactor costs for the construction of a variety of plant terpenoids based on the latest available biosynthetic information. The results are presented in Table 1 as the quantity of glucose in grams needed to manufacture a gram of each terpene. In preparing this list, I attempted to choose only substances whose formation is fairly well understood, since the validity of these computations obviously depends on the accuracy of our biosynthetic knowledge. Considerable progress has recently been made in the study of terpenoid metabolism in plants (Cane, 1990; Croteau, 1987; Gershenzon and Croteau, 1993; Towers and Stafford, 1990). Nevertheless, for several classes of terpenes, it was difficult to find representatives whose biosynthetic pathway had been completely worked out, and so, for some of the compounds selected, inferences regarding intermediates and cofactor requirements had to be made based on analogies to better-studied compounds. The calculated substrate and cofactor costs in Table 1 range from 3.54 g glucose/g for ct-pinene and several other compounds to 1.99 g glucose/g for stevioside, a diterpene glycoside (Figure 1). Cost varies inversely with the degree of oxygenation, with highly oxygenated terpenes being much cheaper to make than terpenes with little or no oxygenation. For instance, among the monoterpenes, the highly oxygenated iridoid glycosides aucubin (2.39 g glucose/g, C15H2209) and antirrhinoside (2.32, CI5H22010) are much less expensive to construct than the nonoxygenated olefins c~-pinene, limonene, and myrcene (all 3.54, Clont6 ). Thus, on a weight basis, it may be cheaper for a plant to accumulate greater amounts of oxygenated terpenoids than nonoxygenated terpenoids, as long as the costs of catalyzing the additional enzymatic transformations are not excessively high. Of course, from a functional point of view, oxygenated and nonoxygenated terpenoids may have substantially different biological properties and are not necessarily interchangeable (Langenheim, this issue). The overall costs of providing substrates and cofactors for terpenoid biosynthesis depend not only on the particular types of compounds produced, but also on the final concentration of terpenoids attained in plant tissue (Gulmon and Mooney, 1986). For example, among diterpenes, stevioside has a lower calculated cost per gram than abietic acid (1.99 g glucose/g vs. 3.26), so 1 g of stevioside is considerably cheaper to make than 1 g of abietic acid. However, the actual stevioside concentration in Stevia rebaudiana leaves is 3-8% of dry weight (Metivier and Viana, 1979), which is much greater than the 0.1% concentration of abietic acid found in the needles of Lar/x laricina (Ohigashi et al., 1981). Therefore, on a tissue basis, the cost of stevioside production in S. rebaudiana (60-160 mg glucose/g leaf) is about 20-50 times as high as that of abietic acid in L. laricina (3.3 mg glucose/g needles). Clearly, the outlay of

TABLE 1. SUBSTRATEAND COFACTOR COSTS FOR BIOSYNTHESIS OF SOME PLANT TERPENOIDSa Compound (and corresponding number in Figure 1)

Molecular formula

Monoterpenes a-Pinene (5)

C10Ht6

3.54

Limonene (3)

CioH~6

3.54

Myrcene (1) Menthone (4) Linalool (2) Camphor (6)

CIoH~6 CioHtsO CLoHtsO CfoHt60

3.54 3.37 3.12 3.10

CjsH2209 CisH22Oto

2.39 2.32

Jensen, 1991; Damtoff et al., 1993b Jensen, 1991; Damtoft et al., 1993a

CI~H2,, CI5H240 CisHx40 CtsH2~O CisH260

3.54 3.43 3.35 3.34 3.25

Dehal and Cmteau, 1988 Threlfall and Whitehead, 1991

C2oH32 C20H320 C:0H~O CmH3oOz

3.54 3.35 3.32 3.26

C3sH6oOI8

1.99

CaoHsoO C~-I4sO3 C321-1~Os C3sH~O8

3.46 3.34 2.87 2.72

Goodwin and Mercer, 1983 Tabata et al., 1993 Balliano et al., 1983

(C~Hs)~

3.54

Cornish, 1992; Patterson-Jones et al., 1990

Iridoid glycosides Aucubin (7) Antirrhinoside (8) Sesquiterpenes Caryophyllene (9) Capsidiol (13) Caryophyllene oxide (10) Germacmne (11) Patchoulol (12) Diterpenes Casbene (14) Pachydictyol A (15) Manool (16) Abietic acid (17) Stevioside (18) Triterpenes /3-Amyrin (19) Bryonolic acid (20) Cucurbitacin B (21) Papyriferic acid (22) Polyterpenes Rubber (23)

Cost (g glucose/g)

References to its biosynthesisb Gambliel and Croteau, 1984; Lewinsohn et al., 1992 Alonso et al., 1992; Kjonaas and Croteau, 1983 Gambliel and Croteau, 1984 Croteau and Venkatachalam, 1986 Croteau and Gershenzon, 1994 Croteau and Shaskus, 1985; Dehal and Croteau, 1987

Cane, 1990 Cmteau et al., 1987a Dueber et al., 1978 West, 1981 Seaman et aL, 1990; Funk and Croteau, 1994 Graebe, 1987

"Costs were calculated as the quantity of glucose required to make all the starting materials, reactants, and cofactors necessary for biosynthesis using methods developed by Penning de Vries et al. (1974), Merino et al. (1984), and Williams et al. (1987). See Gershenzon (1994) for further details. The results are sometimes at variance with costs computed by other authors for the same compounds (C-ulmon and Mooney, 1986; Lambers and Rychter, 1989; Williams et al., 1987) due to the use of newer biosynthetic information in the present treatment and to slight variations in some of the assumptions made regarding cofactor formation. bGeneral references to terpenoid biosynthesis are: Gershenzon and Cmteau (1993), Porter and Sporgeon (1981), and Towers and Stafford (1990).

1286

GERSHENZON

(1) myrcene

(2) (-)-Iinalool

(3) (-)4imonene

(5) (+)-c¢-pinene

(6) (+)-camphor

(4) (+menthone

HO

HO

HO OH

c (7) aucubin

(9) caryophyllene

OGIc

(8) antirrhinoside

(10) caryophyllene oxide

FIG. 1. Structures of compounds listed in Table 1. resources to terpenoid biosynthesis is very strongly controlled by the actual concentration present in the plant. Another factor that could affect the raw material costs of terpenoid formation is the type of cell in which biosynthesis occurs. In computing the glucose requirements in Table 1, terpenoid biosynthesis was assumed to occur in heterotrophic cells with NADPH arising from glucose via the pentose phosphate pathway and ATP being derived from glycolysis and the citric acid cycle coupled to mitochondrial electron transport (Gershenzon, 1994). However, in photosynthetic cells, both of these cofactors can be produced in the chloroplast by lightdriven electron transport and transferred to other subceUular compartments by metabolic shuttles and membrane-bound translocators (Heldt and Flugge, 1987). Therefore, cofactor supply may be much less expensive in green tissue than in nongreen tissue (Chiariello et al., 1989; McDerrnitt and Loomis, 1981), and

1287

METABOLIC COSTS OF TERPENOIDS

(11)germacrone

(12) patchoulol

OH HO""_ ~ : ~ (14)casbene

(13)capsidiol

OH

(15) pachydictyolA

(16)(+)-manool

COOGIc

(17) (+}-abielicacid

(18)stevioside

FIG. 1. Continued hence terpenoids made in photosynthetic cells, such as carotenoids, phytol, or the diterpenes of Nicotiana tabacum (Keene and Wagner, 1985), could cost considerably less to manufacture than the estimates in Table 1 would indicate. In comparison to other classes of plant secondary metabolites, terpenoids generally have greater raw materials costs due to their high degree of chemical reduction. The average cost of all the terpenoids listed in Table 1 (3.18 g glucose/g) is greater than the average costs of both a representative group of plant phenolics (2.11) and a selected group of nitrogen-containing secondary metabolites (2.27), but similar to the average cost of a representative group of alkaloids (3.24) (Table 2). Terpenoids are also more expensive than nearly all types of primary metabolites, including carbohydrates, organic acids, amino acids, and nucleotides. However, fatty acids, which, like terpenoids, are biosynthesized from acetyl-CoA units and have a high level of chemical reduction, are equally costly. It is important to note that all of these comparisons of raw

1288

GERSHENZON

..,,-"

.~OOH

(20)bryonoticacid

(19) ~-amyrin

HO o

HO

'

O" ~...,,

I.OH

o,,;'z°~ Ac

HOOCCH=CO0""~.,,

v

TM

(21)cuc:urbitacinB

v

(22)papydfericacid

~

OH

(23) rubber FIG. l. Continued

materials costs are based on the use of glucose as a standard currency. When the supply of nitrogen limits growth, nitrogen may be a more appropriate currency for evaluating costs (Chapin, 1989), and under such conditions nitrogencontaining metabolites could well become significantly more expensive to manufacture than terpenoids (Gulmon and Mooney, 1986).

Costs of Biosynthetic Enzymes In addition to substrates and cofactors, the formation of terpenoids requires specific enzymes to catalyze the reactions of the biosynthetic pathway. Enzyme costs will depend, first of all, on the total number of enzymes needed, a quantity that varies with the length of the pathway and the extent to which enzymes are shared among several pathways. Enzyme costs are also contingent upon the characteristics of individual enzymes, such as their molecular weight, amino acid composition, catalytic efficiency, and turnover rate.

1289

METABOLIC COSTS OF TERPENOIDS

TABLE 2. AVERAGE SUBSTRATE AND COFACTOR COSTS FOR TERPENOIDS AND VARIOUS OTHER CLASSES OF PLANT PRIMARY AND SECONDARY METABOLITESa

Cost (g glucose/g) Class

N

Mean

Range

Terpenoids Primary rnetabolites Fatty acids Amino acids Nucleotides Carbohydrates Organic acids Secondary metabolites Alkaloids Other nitrogen-containing compounds~ Phenolics

23

3.18

1.99-3.54

2 20 4 5 4

3.10 2.09 1.59 1.07 0.73

3.01-3.18 1.23-2.82 1.27-1.80 1.00-1.11 0.61-0.87

5

3.24

2.89-3.62

8 9

2.27 2.11

1.70-2.83 1.28-3.39

°Raw data for terpenoids are from Table 1. Raw data for other classes of compounds are from Gershenzon (1994). Each class includes representativesof a variety of different skeletal types. blncludes cyanogenicglycosides, glucosinolates,nonproteinamino acids, and proteinaseinhibitors.

Length of Pathway. Terpenoids exhibit great variability in the overall length of their biosynthetic pathways. Using the examples in Table 1, the number of enzymatic conversions involved in terpenoid formation (starting from acetylCoA) ranges from nine for the sesquiterpenes caryophyltene and patchoulol, the diterpene casbene, and several monoterpenes to 23 for the iridoid glycoside antirrhinoside. In general, substances with a lesser degree of oxygenation require fewer steps, which may offset their higher substrate and cofactor costs (Table 1). Unfortunately, not enough is known about the properties o f terpenoid biosynthetic enzymes to estimate the costs o f additional enzymatic steps in a meaningful way. The expense o f additional steps may be reduced if enzymes are not specific to one metabolic pathway but instead are shared among several pathways. Enzyme Sharing among Different Pathwaysof TerpenoidBiosynthesis. The formation o f all terpenoid metabolites begins with the seven basic reactions o f the mevalonate pathway, from acetyl-CoA to dimethylaUyl pyrophosphate (Gershenzon and Croteau, 1993). Hence, in theory a cell could employ a single set of mevalonate pathway enzymes for producing all of its terpenoid constituents. Nevertheless, recent research suggests that plant cells possess multiple sets of mevalonate pathway enzymes distributed among different subcellular compartments, although this is still a somewhat contentious issue (Bach, 1986; Gray,

1290

GERSHENZON

1987; Kleinig, 1989; Schulze-Siebert and Schultz, 1987). Each compartment involved in terpenoid formation appears to produce a distinctive complement of terpenoid metabolites. Chloroplasts, for instance, synthesize carotenoids, tocopherols, and the phytol side chain of chlorophyll (Kleinig, 1989), while other types of plastids seem to be responsible for carrying out monoterpene and diterpene biosynthesis (Dudley et al., 1986; Gleizes et al., 1983). Elsewhere in the cell, the Golgi vesicles are thought to be the site ofplastoquinone and ubiquinone formation (Swiezewska et al., 1993), while the cytoplasm and endoplasmic reticulum together produce sesquiterpenes, triterpenes, and sterols (Berlingheri et al., 1988; Kleinig, 1989). Whether all the steps of terpenoid formation beginning with acetyl-CoA actually occur in each location is still uncertain. Nevertheless, plants clearly do not economize by using a single set of biosynthetic enzymes to make all the terpenoids they require. Perhaps it is more critical for them to partition the various branches of terpenoid biosynthesis among separate subcellular locations so that the formation of different end products can be independently regulated. Enzyme Sharing among Different Branches of Plant Metabolism. Enzyme sharing is also a possibility for certain enzymes of terpenoid biosynthesis that catalyze general reactions, such as the insertion of a hydroxyl group, the reduction of a carbon-carbon double bond, or the formation of glucoside linkage. Each of these reaction types could in theory be mediated by a single enzyme of low substrate specificity that participates in many different pathways, including those outside the realm of terpenoid metabolism. However, plants do not appear to reduce their biosynthetic costs in this manner. Most well-characterized enzymes of terpenoid biosynthesis that catalyze general types of reactions have high substrate specificities. For example, the hydroxylase that transforms (+)sabinene to (+)-cis-sabinol during the biosynthesis of the monoterpene thujone in Salvia officinalis (garden sage) is very selective for its substrate (Karp et al., 1987). Eleven monoterpenes that are structurally related to (+)-sabinene were tested as possible substrates for this enzyme, but none was hydroxylated at a detectable rate (Figure 2). Several other hydroxylases of monoterpene biosynthesis have been extensively characterized, and these also appear to utilize only a limited range of substrates (Karp et al., 1990). In addition to hydroxylases, other classes of terpenoid biosynthetic enzymes exhibit high degrees of substrate specificity as well, including dehydrogenases (Dehal and Crotean, 1987; Kjohaas et al., 1985), keto-reductases (Kjonaas et al., 1982), and glucosyltransferases (Kalinowska and Wojciechowski, 1988; Paczkowski and Wojciechowski, 1985; Zimowski, 1991, 1992). Thus, most enzymes involved in terpenoid formarion seem unlikely to participate in multiple metabolic pathways. Turnover Rate. Plant proteins are subject to continuous degradation in vivo, with the half-lives of specific enzymes ranging from less than an hour to several weeks (Vierstra, 1993). Rapid turnover is thought to benefit an organism by

1291

METABOLIC COSTS OF TERPENOIDS

HIGH S U B S T R A T E SPECIFICITY O F AN ENZYME OF M O N O T E R P E N E BIOSYNTHESIS

sabinene hydroxylase

%

(+ )-cis-sabinol

(+)-sabinene

Monoterpenes that are structurally-similar to sabinene, but are not accepted as substrates:

(-)-a-thulene

(-)-Iimonone

terpinolene

(-)-tz-phellartdrene (-)-~-phellandrene

p'cymene

(x-terpinene

(-)-a-pinene

y-terpinene

(-)-~-pinene

geraniol

FIG. 2. Substrate specificity of a monoterpene biosynthetic enzyme. Sabinene hydroxylase converts (+)-sabinene to (+)-cis-sabinol, an intermediate in tht,:ione formation. This enzyme catalyzes a general type of reaction (allylic hydmxylation), but displays great selectivity for its natural substrate, (+)-sabinene. When the 11 structurally related monoterpenes shown were tested as possible substrates, none was hydroxylated at a rate approaching that of (+)-sabinene (Karl) et al., 1987). allowing its biochemical machinery to quickly adapt to changing environmental conditions (Hawkins, 1991; Vierstra, 1993). However, this process could significantly raise enzyme costs. Unfortunately, no information is yet available on the turnover rate of any enzyme of terpene biosynthesis, except 3-hydroxy-3methylglutaryl coenzyme A reductase (HMGR), a well-studied enzyme of the mevalonate pathway that has been shown to have a PEST amino acid sequence in its structure (Bach et al., 1991; Caelles et al., 1989; Chye et al., 1992; Monfar et al., 1990). Such a sequence, named for its high content of proline (P), glutamine (E), serine (S) and threonine (T) residues, is thought to target proteins for rapid degradation (Rogers et al., 1986). It would not be surprising if HMGR was actually found to have a higher turnover rate than most other plant proteins, since this enzyme has been postulated to catalyze an important, rate-controlling step in terpenoid biosynthesis (Chappell et al., 1991); Gershenzon and Croteau, 1990, 1993). The rapid turnover of regulatory proteins should

1292

GERSmSNZON

be especially important in facilitating prompt metabolic adjustment to changing conditions (Hawkins, 1991; Vierstra, 1993). Accurate measurements of enzyme turnover rates are essential for making realistic estimates of the cost of terpenoid biosynthesis in plants. However, in the absence of such information, it may be instructive to examine patterns of change in enzyme occurrence. If enzymes are only present in a plant for a short period of time, there will be less opportunity for them to undergo turnover, and their potential cost will be lower. We recently investigated the formation of monoterpenes in Mentha x piperita (peppermint) in relation to leaf development and found that monoterpene biosynthesis only occurs for a brief interval during the first two to three weeks of leaf ontogeny (J. Gershenzon and R. Croteau, unpublished results) (Figure 3A). When assays were carried out for the eight enzymes of the monoterpene pathway (Figure 4) between dimethylallyl pyrophosphate and menthone, the principal monoterpene in maturing peppermint leaves, it was discovered that these activities are uniformly high during the first few weeks of leaf development, but then decline to very low levels (Figures 3B and 3C). If it is assumed that changes in enzyme activity reflect changes in the amount of enzyme protein, the enzymes of monoterpene biosynthesis in M. × piperita are only present for a brief span of leaf development, thus minimizing the opportunity for turnover with its attendant costs of replacement. Many terpenoid compounds are not constitutively present in plants, but are produced only in response to herbivore or pathogen attack (Puritch and Nijholt, 1974; Takabayashi et al., 1991; Tallamy and Raupp, 1991). The enzymes involved in the biosynthesis of these induced substances are also found to occur in plants for only restricted periods of time. They are readily apparent following herbivory or infection, but are usually not detectable in undamaged plants (Croteau et al., 1987b; Dudley et al., 1986; Gijzen et al., 1992; Vogeli and Chappell, 1990). For instance, Abies grandis (grand fir) produces large quantities of monoterpenes after wounding that have a different composition than the monoterpenes present constitutively in this species. These compounds serve as a defense against fungi and bark beetles (Gershenzon and Croteau, 1991). The enzymatic machinery responsible for making these wound-induced monoterpenes is not active in uninjured trees, and was only discernible several days after wounding (Gijzen et al., 1992). Another example of induced terpenoid synthesis is seen in tobacco cell cultures, where the application of a fungal elicitor preparation triggers the formation of capsidiol, a sequiterpene phytoalexin. Detailed studies of the enzyme that catalyzes the first committed step of capsidiol biosynthesis, 5-epi-aristolochene synthase, showed that this protein is not detectable at all by assay or immunoblotting in unelicited cultures, but is only observed after elicitor treatment (Vogeli and Chappell, 1990). Thus, many enzymes of terpenoid biosynthesis appear to be only transiently present in plants,

1293

METABOLIC COSTS OF TERPENOIDS

1000

A ¢:: 0

750

e-~

500

V rate of monoterpene biosynthesis • monoterpene content

/ V\

12

/\

.-~~ Ore,=.

8 *"

E

K~-

'o -= 0

I

2

: 0I [ B - : .-__ •~ ' ~

i

/~

~

4

o

5

''7

gerry, ,yropho$

nihas

7

8

;