P1: GVM Journal of Chemical Ecology [joec] pp452-joec-370876 April 20, 2002 11:25 Style file version Nov. 19th, 1999

Journal of Chemical Ecology, Vol. 28, No. 5, May 2002 (C 2002)

POSTGENOMIC CHEMICAL ECOLOGY: FROM GENETIC CODE TO ECOLOGICAL INTERACTIONS1

MAY R. BERENBAUM2,

2Department of Entomology, 320 Morrill Hall University of Illinois 505 S. Goodwin Urbana, Illinois 61801-3795

(Received June 26, 2001; accepted January 12, 2002)

Abstract—Environmental response genes are defined as those encoding pro- teins involved in interactions external to the organism, including interactions among organisms and between the organism and its abiotic environment. The general characteristics of environmental response genes include high diver- sity, proliferation by duplication events, rapid rates of evolution, and tissue- or temporal-specific expression. Thus, environmental response genes include those that encode proteins involved in the manufacture, binding, transport, and breakdown of semiochemicals. Postgenomic elucidation of the function of such genes requires an understanding of the chemical ecology of the organism and, in particular, of the “small molecules” that act as selective agents either by pro- moting survival or causing selective mortality. In this overview, the significance of several groups of environmental response genes is examined in the context of chemical ecology. Cytochrome P-450 monooxygenases provide a case in point; these enzymes are involved in the biosynthesis of furanocoumarins (furo- coumarins), toxic allelochemicals, in plants, as well as in their detoxification by lepidopterans. Biochemical innovations in insects and plants have historically been broadly defined in a coevolutionary context. Considerable insight can be gained by defining with greater precision components of those broad traits that contribute to diversification. Molecular approaches now allow chemical ecol- ogists to characterize specifically those biochemical innovations postulated to lead to adaptation and diversification in plant/insect interactions.

Key Words—Allelochemical, cytochrome P-450, environmental response gene, Helicoverpa, olfactory receptor, Papilio, substrate recognition site, xenobiotic response element, furanocoumarin, furocoumarin.

1 ISCE-Silverstein/Simeone Award 2000. E-mail: [email protected]

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INTRODUCTION

The millenium bug, or Y2K bug, is the computer glitch that was a function of the fact that, in the 1950s, IBM engineers, to save memory, used only a two- digit year field; the two-digit field was anticipated to wreak havoc after December 31, 1999 (Anonymous, 2000). Such havoc conspicuously failed to materialize. Thus, an insect perhaps more worthy of the designation Y2K bug is Drosophila melanogaster, the vinegar or pomace fly. In March 2000, sequencing of the en- tire genome of D. melanogaster was completed and published, marking the first complete arthropod genome to be sequenced (Adams et al., 2000). This accom- plishment may herald a new era within the field of chemical ecology. Since its inception, practitioners of the science of chemical ecology have been concerned mostly with small molecules. Admittedly, some reach impressive size; a case in point may be the extremely large-ring monocyclic polyazamacrolides produced in the defensive secretions of some coccinellid pupae. Ring sizes in some of these remarkable macrocycles can range from 24 to over 150 members (Schroeder et al., 2000), but substances with the cognomen “macromolecules” are only recently subjects for study by chemical ecologists. Protein serving as olfactory receptors and enzymes involved in allelochemical biosynthesis now routinely merit study, but even bigger molecules loom on the horizon. In the postgenome era, DNA should be an increasing focus of attention even for those with an affection for small, tractable molecules. One reason is that DNA itself is a target site for many toxic natural products. The universal occurrence of DNA makes it a likely evolutionary target for broad-spectrum defense com- pounds, and the evolution of DNA must reflect in part interspecific interactions among organisms. B-DNA possesses quite a number of nucleophilic reactive sites that interact with natural products (Yangand Wang, 1999). Furanocoumarins (furo- coumarins), e.g., benz-2-pyrone compounds produced by a handful of angiosperm plants primarily in the Apiaceae and Rutaceae, covalently bind to thymine and cross-link strands. Ecteinascidin from the marine tunicate Ecteinascidia turbinata binds covalently to guanine N-2; the fungal metabolite aflatoxin B1 attacks N-7 guanine. Duacarmycin, a Streptomyces product, and bleomycin/pepleomycin, from Streptomyces verticillus, induce DNA backbone cleavage. Daunorubicin, initially isolated from Streptomyces caeruleorubidus, and doxorubicin, produced by Strep- tomyces peucetius, are anthracycline drugs that can intercalate (as can triostin A and echinomycin, naturally occurring bisintercalator quinoxaline antibiotics from Streptomyces echinatus and related species); distamycin A, from Streptomyces distallicus, binds in the narrow minor groove associated with AT sequences in B-DNA, and actinomycin D from Streptomyces spp. binds to DNA duplexes and interferes with transcription. Perhaps a more important reason for chemical ecologists to incorporate DNA into their studies is that one of the principal goals of postgenomic biology is to P1: GVM Journal of Chemical Ecology [joec] pp452-joec-370876 April 20, 2002 11:25 Style file version Nov. 19th, 1999

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ascertain gene function, and the function of many genes can be comprehended only within the context of chemical ecology, in particular, the function of those genes that can be considered “environmental response genes.” Environmental response genes can be defined as those encoding proteins involved in interactions external to the organism, including interactions among organisms and between the organ- ism and its abiotic environment. General characteristics of environmental response genes, particularly those involved in mediating interactions among organisms and thus allowing for reciprocal evolutionary responses, include (1) very high diversity, (2) proliferation by duplication events, (3) rapid rates of evolution, (4) occurrence in gene clusters, and (5) tissue- or temporal-specific expression. All of these char- acteristics are consistent with responses to evolutionary pressure emanating from a highly changeable external environment. Among environmental response genes, then, are those encoding proteins in- volved in the manufacture, binding, transport, and breakdown of semiochemicals, the principal medium of communication for many species between the organism and its environment. Examples of environmental response genes are numerous. Chemical signals are characterized by producers and recipients, and each has its own set of genes (Table 1). These genes may have genome-level effects that are very different from, for example, the so-called “housekeeping genes.” By the same token, the term in common use to contrast with housekeeping genes, i.e., “luxury genes” (e.g., Vinogradov, 1997), sells short the ecological and evolutionary contri- butions of genes allowing organisms to cope with environmental stress. The lack of correlation between genome size and number of genes (e.g., Caenorhabditis elegans at 97 Mb and 19,000 genes and Drosophila melanogaster with 180 Mb and 13,000 genes; http://www.ornl.gov/hgmis/faq/compgen.html) has led to the suggestion that evolution proceeds principally by altering patterns of gene ex- pression, rather than by increasing the protein inventory. Environmental response genes may be an exception to the trend; new functions may be acquired by prolif- eration of new proteins, not just by changes in regulation and expression. Parallel evolution in both gene function and expression pattern may be the key to diver- sification in environmental response genes. In this overview, the significance of

TABLE 1. SEMIOCHEMICALS AND ASSOCIATED ENVIRONMENTAL RESPONSE GENES

Semiochemical Producer Receiver

Pheromone Biosynthetic genes Pheromone-binding protein genes Odorant receptor genes Pheromone-degrading enzymes Allomone Biosynthetic genes Detoxification genes Transport protein genes Transport protein genes Kairomones Biosynthetic genes Odorant receptor genes Taste receptor genes P1: GVM Journal of Chemical Ecology [joec] pp452-joec-370876 April 20, 2002 11:25 Style file version Nov. 19th, 1999

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several groups of environmental response genes is examined in the context of chemical ecology. The “chemical senses” by which organisms perceive information about their environments encompass olfaction, or perception of chemical stimuli in gaseous phase, and gustation (and other forms of contact chemoreception), perception of chemical stimuli as solutes in liquid media. Irrespective of organism, chemical signaling (not only externally but also internally) shares several features. Most, if not all, chemical signaling systems include a receptor that reacts with a signal molecule; many also involve a signal transducer and amplifier. Perireceptor phe- nomena associated with movement of signal molecules (binding, transport, and degradation) also share many conserved features across a wide range of taxa. Many of the same classes of molecules serve these functions across a variety of taxa.

GENES INVOLVED IN BINDING AND TRANSPORT OF SEMIOCHEMICALS

Taste Receptors. Molecular characterization of taste receptors has eluded investigators for a long time; molecular techniques provided a new and ultimately successful approach to this goal. Clyne et al. (2000) searched the Drosophila melanogaster genome to find a large family of putative odorant receptors, which they call the gustatory receptor family; the search algorithm identified proteins with characteristic structural properties (with sequence length around 380 amino acids, and regions encoding seven predicted transmembrane domains, suggestive of G- protein coupled receptors). At the time the study was done, 60% of the genome yielded 75 proteins in the family. Amino acid identity ranges from 34% to less than 10%, indicating tremendous sequence divergence. These gustatory receptors tend to be found in clusters; two large clusters contain four genes, whereas several other clusters contain two or three genes. Tissue specificity is high as well, in that 18 of the 19 characterized are expressed only in the labellum, the gustatory organ. Moreover, gene expression could not be detected in the labellum of a mutant, pox neuro, which lacks taste neurons. Subsequent completion of the genome sequence of D. melanogaster unveiled many other members of this group of candidate taste receptors to bring the total to at least 57 genes (http://sdb.bio.purdue.edu/fly/aignfam/odorcpt3.htm). Clyne et al. (2000) concluded that “The large size of this protein family likely reflects the diversity of compounds that flies can detect.” Alternatively, expression of indi- vidual genes is often restricted to subsets of gustatory neurons in developmentally different sense organs (Scott et al., 2001; Dunipace et al., 2001; Robertson, 2001). Given that so little is known of the chemical ecology of D. melanogaster in nature, identifying which compounds were likely selective agents in the evolution of this protein family will be a challenge. P1: GVM Journal of Chemical Ecology [joec] pp452-joec-370876 April 20, 2002 11:25 Style file version Nov. 19th, 1999

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Olfactory Receptors. Like gustatory receptors, olfactory receptors, charac- terized by multiple transmembrane domains, are extremely diverse within most genomes characterized to date. In the genome of the free-living nematode Caenorhabditis elegans, Robertson (1998) examined genes in the serpentine str (seven transmembrane receptor) family encoding seven-transmembrane G-protein coupled receptors related to ODR-10 diacetyl chemoreceptors (with a known func- tion in detecting environmental chemicals) and identified 197 members of this group. The family shares a close relationship with another family of serpentine receptors, srd (serpentine receptor class D), with approximately 55 genes. In a sub- sequent study, Robertson (2000) discovered another family of candidate serpen- tine chemoreceptors: the srh (serpentine receptor class H) family, which contains 214 genes and 90 pseudogenes. As much as 6% of the genome may encode over 500 chemoreceptors, constituting up to 4% of C. elegans proteins, an indication of the importance of chemical senses to this soil-dwelling organism. Phylogenetic analyses suggest frequent gene duplication events, e.g., as many as 85 duplica- tion events in family srh can be inferred to have occurred since C. briggsae and C. elegans differentiated. C. elegans is by no means unique in its reliance upon functional chemoreceptor genes; in mammals, olfactory receptors may number in excess of 1000, representing 1% of the gene complement. Pheromone-Binding Proteins. Odorant-binding proteins in a wide range of organisms serve to transport generally hydrophobic environmental odorants to in- ternal (olfactory) receptors. This is known to be a large family in D. melanogaster; Rubin et al. (2000) list 14 members of the family, and subsequent investigation has brought the number up to 39 (H. Robertson, personal communication). Among the odorant-binding proteins of insects are pheromone-binding proteins (PBP); these are involved in the protection and transport of pheromones from the external envi- ronment to internal receptors, and, like other members of the family, these proteins act generally as lipophilic ligand-transporting agents. In insects, they have been characterized in extracellular fluids bathing receptor cells in antennal hemolymph. Willett (2000) has conducted a study of pheromone-binding proteins within the genus Choristoneura in order to ascertain the forces driving diversification and differentiation in this group of proteins. Choristoneura is a genus of leaf-rolling caterpillars of economic importance, and a considerable amount of effort has been invested in characterizing the pheromones they produce. The putative ancestral pheromone is an acetate; the pheromone occurs as an alcohol in C. parallela and as an aldehyde in C. fumiferana. Willett (2000) was interested in finding evidence of selective impact on PBPs in association with changes in pheromone composition, specifically changes in the ratio of nonsynonymous to synonymous substitutions indicative of diversifying selection. Evidence of changes in PBPs corresponding to changes in pheromone composition was found in the case of C. fumiferana, with its novel aldehyde pheromone. However, other pheromone changes (e.g., chain P1: GVM Journal of Chemical Ecology [joec] pp452-joec-370876 April 20, 2002 11:25 Style file version Nov. 19th, 1999

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lengthening in C. muriana and a shift to an alcohol in C. parallela) are not re- flected in changes in PBP.

BIOSYNTHETIC AND DETOXIFICATIVE PROTEINS

Over a quarter century of studies in chemical ecology have made clear the importance of semiochemical biosynthesis and detoxification to organisms across a wide range of taxa. Perhaps the best known group of environmental response genes are the cytochrome P-450s. Cytochrome P-450 monooxygenases (P-450s) comprise a large superfamily of heme-thiolate proteins that metabolize a wide range of both endogenous and exogenous hydrophobic compounds by incorpo- rating oxygen into a functionalized product. This generalized reaction, converting lipophilic substrates into hydrophilic products, means that these enzymes display great versatility; reactions catalyzed by P-450s include such oxidative transforma- tions as monooxygenations, NO-synthase type oxidations, dehydrogenations, and peroxidase-type oxidations and nonoxidative reactions such as dehydrase activ- ity, nitric acid reductase activity, and isomerization (Mansuy, 1998). Thus, these enzymes participate in a tremendous variety of transformations, including both biosynthetic and detoxification reactions. A standardized nomenclature based on levels of amino acid sequence identity is in use for this huge superfamily (Gonzalez and Gelboin, 1989). Sequences sharing over 40% identity are placed in a family (designated by a number); those sharing over 55% identity are placed in a separate subfamily (designated by a letter), and those sharing over 97% identity are recognized as allelic variants at a locus (designated by a number). The number of P-450s represented in a genome varies with the organism. They make up one of the largest superfamilies in plants; the recent genome se- quencing of Arabidopsis thaliana revealed over 280 P-450 genes (Paquette et al., 2000). In Drosophila melanogaster, 90 P-450s have been characterized, of which only seven are thought to be pseudogenes (Tijet et al., 2001a), and in C. elegans there are at least 60 P-450 genes (Gotoh, 1998). Gonzalez and Nebert (1990) sug- gested that P-450s began to diversify over 400 million years ago, concomitant with the colonization of terrestrial habitats by plants and herbivorous animals. The rapid diversification may have been a consequence of reciprocal selection pressures whereby plants biosynthetically evolve novel defense compounds, and insects (and other herbivores) overcame the erstwhile toxins with novel detox- ification pathways. Multiple gene duplication events allowed P-450s to acquire new functions in the presence of new environmental stress factors while retaining ancestral metabolic capabilities. Thus, P-450s may well be the consummate en- vironmental response genes, evolving and diversifying to a certain extent in the context of both interspecific and intraspecific interactions. P1: GVM Journal of Chemical Ecology [joec] pp452-joec-370876 April 20, 2002 11:25 Style file version Nov. 19th, 1999

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However, protein diversification is only one of several possible evolutionary mechanisms. P-450 expression is regulated in response to a wide range of factors, including developmental stage and exposure to both endogenous and exogenous substances (Tables 2 and 3). The majority of studies have focused on synthetic xenobiotics as inducers, whose efficacy was initially determined in studies of human P-450 (e.g., phenobarbital); their ecological significance is often difficult to interpret. The relative contributions of P-450 diversification and regulation of expression to the evolution of plant/animal interactions are unknown and will likely remain unknown until ecologically relevant inducers can be identified. Despite the enormous significance of ecological interaction in understanding P-450s, the ecological function and evolutionary history of most P-450s have yet to be determined. The vast majority of studies of P-450s involve humans, par- ticularly in the context of understanding drug metabolism and carcinogenesis (in that P-450s can act as activators of procarcinogens as well as detoxification en- zymes) (e.g., Guengerich, 1998). Although there is no question that such work is of great importance to human health, it does not provide a great deal of in- sight into the evolutionary processes that led to P-450 diversification; humans are uniquely effective at circumventing the action of natural selection and adapting the environment to themselves rather than adapting themselves to the environment (Berenbaum, 1999). In organisms other than vertebrates, the emphasis in P-450 studies is generally on human-produced xenobiotics as substrates. Until recently, in plants, efforts in characterizing function have focused on elucidating the role of P-450s in herbicide tolerance (Werck-Reichart et al., 2000); similarly, most work with insect P-450s has been in the context of characterizing genes associated with resistance to synthetic organic insecticides (Scott, 1999). The environmental context of P-450 evolution is perhaps best characterized in plants. Many P-450s have been characterized at the enzyme level and are known to participate in the biosynthesis of allelochemicals (Table 4, Fig. 1). As autotrophs, plants (with rare exceptions) are reliant upon biosynthesis, rather than sequestra- tion, for the acquisition of secondary compounds. Moreover, as sedentary organ- isms that form the base of most food webs, they are subject to intense selection pressure from herbivores and have few options other than chemical or morpho- logical defense for reducing the impact of herbivores (Berenbaum, 1995a). Thus, the allelochemical inventory of plants is staggering; over 40,000 compounds have been characterized. P-450-mediated transformations may well be involved in the biosynthesis of the majority of these compounds. Genome sequencing of plants with known secondary chemistry will facilitate elucidation of the function of many P-450 genes, as has already proved to be the case in A. thaliana; great strides have been made, e.g., in characterizing the genes governing glucosinolate biosynthesis (Table 4). Moreover, as in animals, P-450s play an important role in xenobiotic detoxification in plants; herbicides and other environmental toxins are metabolized by an array of P-450s (Table 4). P1: GVM Journal of Chemical Ecology [joec] pp452-joec-370876 April 20, 2002 11:25 Style file version Nov. 19th, 1999

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TABLE 2. TISSUE LOCALIZATION AND INDUCERS OF INSECT P-450 GENES IN INSECTSa

Isoform Species Tissues Inducers Ref.

CYP4C1 Blaberus discoidalis FB HTH CYP4D1 D. melanogaster NR CYP4D10 Drosophila mettleri NR SCA, PB Danielson et al. (1998) CYP4E1 D. melanogaster VNR CYP4L1,2 Manduca sexta MG, FB NR CYP4M1 M. sexta MG, FB C, PB, U CYP4M2 M. sexta MG, FB Not induced CYP4M3 M. sexta MG, FB C, T, U CYP6A1 M. domestica G, FB PB, PBO, ET CYP6A2 D. melanogaster FB, MG, MT PB CYP6A4, 5 M. domestica NR NR CYP6A8,9 D. melanogaster NR BA CYP6B1 Papilio polyxenes MG, FB, I X, BG, A Petersen et al. (2001) CYP6B2 Hellothis armigera MG, FB PO, PBO, P CYP6B3 P. polyxenes MG, FB X, BG, A Petersen et al. (2001) CYP6B4 P. glaucus MG X, A, B, I CYP6B7 H. armigera MG Pinene CYP6B8 H. zea MG, FB, RT X, CA, IC, PB X. Li et al. (2002) CYP6B9 H. zea MG X, CA, IC, PB X. Li et al. (2002) CYP6B11 P. canadensis MG X CYP6B12 P. glaucus MG X X. Li et al. (2001) CYP6B16 P. glaucus MG X X. Li et al. (2001) CYP6B17 P. glaucus MG X X. Li et al. (2001) CYP6B18 P. canadensis MG X X. Li et al. (2001) CYP6B20 P. glaucus MG X X. Li et al. (2001) CYP6B21 P. glaucus MG X X. Li et al. (2001) CYP6B24 P. glaucus MG X X. Li et al. (2001) CYP6B25 P. canadensis MG X X. Li et al. (2001) CYP6B26 P. canadensis MG X X. Li et al. (2001) CYP6B27 H. zea MG, FB X, CA, IC, PB X. Li et al. (2001) CYP6B28 H. zea MG, FB, RT, I X, CA, IC, PB X. Li et al. (2001) CYP6D1 M. domestica FB, PI, RT PB, PBO, C CYP9A1 H. virescens FB, G NR CYP9A2 M. sexta MG U, IC, PB, T, X Stevens et al. (2000) CYP9A4 M. sexta MG C, X Stevens et al. (2000) CYP9A5 M. sexta MG X Stevens et al. (2000) CYP18 D. melanogaster BW, G EC CYP28A1 D. mettleri NR PB, SA CYP28A2 D. mettleri NR PB, SA, SCA CYP28A33 D. nigrospiracula NR PB, SCA

a From Scott et al. (1998) unless otherwise indicated. Tissues: BW = body wall, FB = fatbody, I = integument, MG = midgut, G = gut, MT = Malpighian tubules, PI = proximal intestine, RT = reproductive tissue, V = viscera, NR = not reported. Inducers: A = angelicin, BA = barbital, B = bergapten, C = clofibrate, CA = chlorogenic acid, EC = ecdysone, ET = ethanol, HTH = hypertrehalosemic hormone, I = isopimpinellin, IC = indole-3-carbinol, N = nicotine, P = -pinene, PB = phenobarbital, PBO = piperonyl butoxide, PO = peppermint oil, SA = senita alka- loids, SCA = saguaro cactus alkaloids, T = tridecanone, U = undecanone, X = xanthotoxin. P1: GVM Journal of Chemical Ecology [joec] pp452-joec-370876 April 20, 2002 11:25 Style file version Nov. 19th, 1999

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TABLE 3. KNOWN FUNCTIONS OF INSECT P-450S

Isoform Species Substrates Ref.

CYP6A1 Musca domestica farnesyl, geranyl, neryl methyl Andersen et al. (1997) esters, aldrin, heptachlor CYP6A2 Drosophila aflatoxin, 7, 12 dimethyl Saner et al. (1996) melanogaster benz[a]anthracene 3-amino-1- Dunkov et al. (1997) methy1-5H-pyridoindole; aldrin, heptachlor, diazinon CYP6B1 Papilio polyxenes bergapten, xanthotoxin, psoralen, Ma et al. (1994) isopimpinellin CYP6B3 Papilio polyxenes xanthotoxin? J. Chen (unpublished results) CYP6B4 Papilio glaucus bergapten, xanthotoxin, Hung et al. (1997) isopimpinellin, imperatorin CYP6D1 Musca domestica benzo[a]pyrene, methoxyresorufin, Scott (1999) deltamethrin, cypermethrin, chlorpyrifos

The C. elegans genome has revealed more than 60 P-450 genes (Gotoh, 1998), with gene structures “strikingly divergent within each group.” The majority may deal with xenobiotic substrates—all of the P-450s characterized show relation- ships to mammalian CYP2, CYP3, and CYP4 subfamilies, which are involved in xenobiotic metabolism. Because the chemical ecology of C. elegans in nature is not particularly well characterized, elucidating the specific catalytic properties of these P450s will be challenging. Drosophila genome sequencing has yielded 90 P-450s (Tijet et al., 2001a) (http://ag.arizona.edu/p450/insectlist.html), the function of the vast majority of which is unknown. Although much attention has been focused on P-450 partici- pation in detoxification of such xenobiotics as insecticides, the majority of P-450s in Drosophila melanogaster may be involved in allowing this relatively gener- alized consumer of microbes and microbial fermentation products to deal with the wide range of microbial products, rather than plant allelochemicals, encoun- tered. Furthermore, Danielson et al. (1998) have documented P-450 involvement in detoxification of cactus alkaloids in cactophilic Drosophila species; CYP fam- ilies 4, 6, and 27 may be involved, as suggested by induction in the presence of potential cactus-derived substrates (Table 2). Other degradative functions of P-450s may involve endogenous substrates involved in intraspecific communication; in a small expressed sequence tag project with Manduca sexta, Robertson et al. (1999) reported the presence of three P-450s that are candidate odorant degrading enzymes. P-450s are also involved in the biosynthesis of other intraspecific chemical signals such as pheromones and cuticular hydrocarbons, and such hormones as ecdysone and juvenile hormone (Feyereisen, 1999). Thus, given the multitude of functions of P-450s in insects, P1: GVM Journal of Chemical Ecology [joec] pp452-joec-370876 April 20, 2002 11:25 Style file version Nov. 19th, 1999

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TABLE 4. PLANT P-450S WITH KNOWN FUNCTIONSa

CYP number Function Reference

CYP51 obtusifoliol 14 a-demethylase CYP71A5 (3S)-cintronellol 10-hydroxylase CYP71A11 chlortoluron metabolism CYP71B7 7-ethoxycoumarin O-deethylase CYP71C DIBOA biosynthesis CYP71C3v2 triasulfuron 5-hydroxylase CYP71D9 flavonoid-6-hydroxylase Latunde-Dada et al. (2001) CYP71D13 limonene-3-hydroxylase Wust et al. (2001) CYP71D18 limonene-6-hydroxylase Wust et al. (2001) CYP71E1 p-hydroxymandelonitrile synthesis CYP72A1 secologanin synthase Irmler et al. (2000) CYP73 cinnamate 4-hydroxylase CYP74A alleneoxide synthase CYP74B fatty acid hydroperoxidelyase CYP74C fatty acid hydroperoxide lyase Tijet et al. (2001b) CYP75 flavonoid 30,50-hydroxylase CYP76 7-ethoxycoumarin O-deethylase CYP78A1 fatty acid hydroxylase CYP79A1 tyrosine N-hydroxylase CYP79A2 phenylalanine N-hydroxylase Wittstock and Halkier (2000) CYP79B2 tryptophan to indole-3-acetaldoxime Mikkelson et al. (2000) CYP79F1 dihomomethionine to Hansen et al. (2001) 5-methylthiopentanaldoxime CYP80A1 berbamunine synthase CYP80B1v7 (S)-N-methylcoclaurine 30-hydroxylase CYP80B1v2 fatty acid hydroxylase CYP81B1 fatty acid hydroxylase CYP81E1 flavonoid biosynthesis CYP83B1 indole-3-acetaldoxime hydroxylase Bak et al. (2001) CYP84 ferulate 5-hydroxylase CYP86A1 fatty acid w-hydroxylase CYP88A1 gibberellin biosynthesis CYP90A1 cathasterone 23-hydroxylase CYP90B brassinosteroid biosynthesis CYP90C1 steroid hydroxylase CYP93A1 dihydroxypterocarpan 6a-hydroxylase CYP93B1 (2S)-flavanone 2-hydroxylase CYP93B2 flavone synthase II Martens and Forkman (1999) CYP93C1 8-hydroxyisoflavanone synthase Latunde-Dada et al. (2001) CYP93C2 2-hydroxyisoflavanone synthase Akashi et al. (1999) CYP94A1 cutin monomers synthase CYP701A3 ent-kaurene oxidase CYP703A1 fatty acid hydroxylase

a From Ohkawa et al., 1999 unless otherwise indicated. P1: GVM Journal of Chemical Ecology [joec] pp452-joec-370876 April 20, 2002 11:25 Style file version Nov. 19th, 1999

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FIG. 1. Biosynthetic pathway leading to furanocoumarin production. Hollow arrows in- dicate steps known or suspected to be catalyzed by cytochrome P-450s. Angular fura- nocoumarin synthesis on left of figure, linear furanocoumarin synthesis on right of figure.

their diversity in at least the single insect genome characterized is not altogether surprising. Like other environmental response genes, P-450s appear to arise and diver- sify via gene duplication and acquisition of new functions. Some are involved in metabolism of endogenous substrates, such as hormones, but extreme diversifica- tion appears to be a hallmark of the xenobiotic-metabolizing P-450s. To understand P1: GVM Journal of Chemical Ecology [joec] pp452-joec-370876 April 20, 2002 11:25 Style file version Nov. 19th, 1999

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the evolutionary diversification of P-450s, it is, therefore, important to identify the environmental chemicals (natural as well as synthetic) that act as selective agents promoting change. P-450s in Plant/Insect Interactions—Furanocoumarins as Exemplars. The coevolutionary interactions between flowering plants and herbivorous insects (col- lectively constituting over half of all terrestrial species) are thought to have gen- erated much of the terrestrial diversity on the planet. Ehrlich and Raven (1964) suggested that the evolution of biochemical novelty leads to speciation in an- giosperms and that subsequent biochemical innovations in detoxification lead to speciation in herbivores associated with those plants: “If recombination or muta- tion appeared in a population of insects that enabled individuals to feed on some previously protected plant group, selection could carry that line into a new adaptive zone. Here it would be free to diversify.” Generally, biochemical innovations in insects and plants have been broadly defined, e.g., the ability to feed on a particular plant, in the case of an insect, or the ability to biosynthesize a particular toxin, in the case of a plant. Rarely have specific adaptive traits been identified biochemically or genetically. Considerable insight can be gained by defining with greater preci- sion components of those broad traits that contribute to diversification. Molecular approaches now allow chemical ecologists to characterize those biochemical inno- vations that are postulated to lead to adaptation and diversification in plant/insect interactions. A case in point involves plants that contain furanocoumarins and the insects that eat them; for both insects and plants, P-450s are essential mediators of the interaction. Furanocoumarins owe their toxicity to their ability to bind covalently to DNAs and proteins in the presence of ultraviolet light; moreover, in the presence of molecular oxygen they can act as prooxidants that generate toxic oxygen species such as singlet oxygen and superoxide anion. Thus, furanocoumarins can interfere with cellular processes to cause toxicity in a wide variety of organisms, including insects (Berenbaum, 1991, 1995b). Over 200 furanocoumarins have been described from plants. Plant furanocoumarins occur in two structural configurations (Fig. 1): linear furanocoumarins (with the furan ring attached at the 6,7 positions of the benz-2-pyrone nucleus) can cross-link opposing strands in DNA helices as well as bind to proteins, whereas angular furanocoumarins (with the furan ring attached at the 7,8 positions of the benz-2-pyrone nucleus) bind to proteins but form only monoadducts with DNA. Linear furanocoumarins are known from approximately a dozen families, although they are most diverse and ubitquitous in Rutaceae and Apiaceae. Angular furanocoumarins are more narrowly distributed and are found only in a few genera in three plant families (Fabaceae, Rutaceae, Apiaceae); they are most frequently encountered in two tribes in the Apiaceae (Murray et al., 1982). The biosynthesis of both linear and angular furanocoumarins begins with L-phenylalanine and involves several cytochrome P-450 monooxygenases (Fig. 1). Three P-450s catalyze in sequence the biosynthesis of bergaptol from P1: GVM Journal of Chemical Ecology [joec] pp452-joec-370876 April 20, 2002 11:25 Style file version Nov. 19th, 1999

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demethylsuberosin (6-prenylated umbelliferone) via (+)-marmesin and psoralen (Stanjek et al., 1999; Matern et al., 1999). Although these P-450s have been char- acterized biochemically, they have not yet been defined at the molecular level. Furanocoumarin biosynthesis, however, is clearly inducible by multiple forms of environmental stress, including insect damage (Zangerl and Berenbaum, 1998; Berenbaum and Zangerl, 1999); in Apium graveolens, furanocoumarin biosynthe- sis is stimulated by jasmonic acid and its analogs (Stanjek et al., 1997). The pattern of furanocoumarin response to insect damage in Pastinaca sativa is different from that elicited by mechanical damage (Zangerl and Berenbaum, 1990), raising the possibility that, as in some other plant–insect interactions, insect-derived oral se- cretions may contain compounds that influence induction of plant biosynthesis (Frey et al., 2000). Few insects other than lepidopterans specialize on furanocoumarin-containing plants, and, within the Lepidoptera, specialization on these toxic plants is restricted to a handful of families (Berenbaum, 1983). Cytochrome P-450-mediated detox- ification of furanocoumarins is associated with specialization on these plants in the Papilionidae, the swallowtail butterflies. Within the family, furanocoumarin- feeding is restricted to the genus Papilio within the subfamily Papilioninae; over 75% of these species are associated with furanocoumarin-containing plant hosts. Within the genus, P-450-mediated metabolism of furanocoumarins corresponds to the frequency of ecological exposure. Constitutive levels of activity (associated with host plants unsupplemented with furanocoumarins) against xanthotoxin, a linear furanocoumarin, are high in Papilio cresphontes, a specialist restricted to linear furanocoumarin-containing Rutaceae, the putative ancestral host plant fam- ily of the group. Constitutive activity is high as well in P. polyxenes and P. bre- vicauda, two specialists associated with furanocoumarin-containing Apiaceae. In contrast, P-450-mediated metabolism of xanthotoxin is undetectable in P. troilus, a specialist on host plants in the Lauraceae, all of which lack furanocoumarins; furanocoumarin metabolic activity is also not inducible by supplemental dietary furanocoumarins (Cohen et al., 1992). The molecular basis of furanocoumarin resistance has been characterized at least in part in P.polyxenes. Transcripts of two cDNAs, CYP6B1v1 and CYP6B1v2, accumulate in response to supplemental furanocoumarins (Cohen et al., 1992); that these cDNAs encode furanocoumarin-metabolizing P-450s was demonstrated by baculovirus-mediated expression in two different lepidopteran cell lines