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Journal of Experimental Botany, Vol. 52, No. 354, pp. 11±23, January 2001 REVIEW ARTICLE Enzymes of the biosynthesis of octadecanoid-derived signalling molecules Florian Schaller1 Lehrstuhl fuÈr Pflanzenphysiologie, Ruhr-UniversitaÈt Bochum, D-44780 Bochum, Germany Received 14 June 2000; Accepted 16 August 2000 Abstract It is known that octadecanoids, i.e. jasmonic acid and related compounds are involved in plant defence reactions against (1) microbial pathogens, (2) herbivores and (3) damage by UV-B or UV-C light as well as (4) senescence and (5) mechanotransduction. Jasmonic acid is likely to occur ubiquitously in the plant kingdom, and it has also been found in some fungi. The pathway of octadecanoid biosynthesis was elucidated in the early 80s by Vick and Zimmerman. This review summarizes recent progress in the identification and characterization of octadecanoid biosynthetic enzymes and in the understanding of the regulation of octadecanoid biosynthesis. Key words: Lipase, lipoxygenase, allene oxide synthase, allene oxide cyclase, 12-oxo-phytodienoic acid reductase. Introduction More than 100 years ago, Charles Darwin and Julius von Sachs postulated the existence of chemical messengers that are responsible for the regulation of plant development. The ®rst such messenger, the growth hormone indole-3-acetic acid, was identi®ed in the 1930s. Since then, the phytohormones have been and still are an area of active research, but also one of major ignorance. Some analogies to animal hormones have become apparent, since over recent years brassinosteroids and jasmonates, resembling in structure the animal steroids or prostaglandins, respectively, have been identi®ed as plant signalling compounds. In 1962, the methyl ester of jasmonic acid was isolated by Demole and coworkers as a major fragrance in the etheral oil of jasmine (Demole et al., 1962), and the free 1 Fax : q49 234 32 14187. E-mail: [email protected] ß Society for Experimental Biology 2001 acid was identi®ed at the same time in culture ®ltrates of the fungus Botryodiplodia theobromae (Aldridge et al., 1971). Jasmonic acid is known to be involved as a signalling compound in multiple aspects of plant responses to their biotic and abiotic environment. Today it is known that jasmonic acid, in planta levels of which range from 0.01±3.0 mg g 1 FW (Mueller et al., 1993; Mueller and Brodschelm, 1994), is only one of several bioactive compounds within the class of octadecanoids which regulate a broad spectrum of plant responses (for review see Sembdner and Parthier, 1993). The pathway of jasmonic acid (JA) biosynthesis is shown in Fig. 1. Biosynthesis is believed to start with the oxygenation of free a-linolenic acid (LA), which is converted to (9Z,11E,15Z,13S)-13-hydroperoxy-9,11, 15-octadecatrienoic acid (13(S)-HPOT) in a reaction catalysed by a 13-lipoxygenase (LOX). The presumed release of LA from membrane lipids through the action of a lipase may be triggered by local or systemic signals like oligogalacturonides, chitosan, systemin (NarvaÂezVaÂsquez et al., 1999; Farmer and Ryan, 1992; Mueller et al., 1993) or wounding (NarvaÂez-VaÂsquez et al., 1999; Conconi et al., 1996a). The 13(S)-hydroperoxide serves as a substrate for several enzymes like divinylether synthase, peroxygenase, hydroperoxide lyase, and hydroperoxide reductase (Fig. 1) (BleÂe and Joyard, 1996; MareÂchal et al., 1997) or allene oxide synthase (AOS) (Vick and Zimmerman, 1981, 1987). AOS converts 13(S)-HPOT to an unstable epoxide (12,13(S)-epoxy9(Z),11,15(Z)-octadecatrienoic acid, 12,13-EOT) which is cyclized by allene oxide cyclase (AOC) to the ®rst cyclic and biologically active compound of the pathway, 12-oxo-10,15(Z)-phytodienoic acid (OPDA). Reduction of the 10,11-double bond by a NADPH-dependent OPDA-reductase then yields 3-oxo-2(29(Z)-pentenyl)cyclopentane-1-octanoic acid (OPC-8 :0) which is believed to undergo three cycles of b-oxidation to yield the end 12 Schaller product of the pathway, i.e. JA. Evidence for this b-oxidation process has emerged from radiotracer experiments, which have allowed the detection of 3-oxo-2(29-pentenyl)cyclopentane hexanoic acid (OPC-6 :0) and 3-oxo-2-(29-pentenyl)cyclopentane butanoic acid (OPC4:0) as intermediates in the conversion of OPDA to JA (Vick and Zimmerman, 1983). While this biosynthetic pathway results in the production of JA with (3R,7S)con®guration (i.e. (q)-7-iso-JA) (Vick and Zimmerman, 1984), JA extracted from plant tissue is predominantly in the thermodynamically more favourable (3R,7R)trans-con®guration (i.e. ( )-JA) (Quinkert et al., 1982). The biosynthesis of JA appears to involve three different compartments (Fig. 4): the conversion of LA to OPDA is localized in the chloroplasts (Vick and Zimmerman, 1987; Song et al., 1993; Laudert et al., 1997), while the reduction of racemic OPDA to OPC-8 :0 (OPR1 and OPR2) occurs in the cytoplasm (Schaller and Weiler, 1997a, b). Finally, the postulated steps of b-oxidation, i.e. conversion of OPC-8 :0 to JA, are believed to occur in peroxisomes (Gerhardt, 1983; Vick and Zimmerman, 1984). Like JA, OPDA may accumulate to substantial amounts in plant tissue (Stelmach et al., 1998). In contrast, OPC-8 :0 occurs only in trace amounts. The release of a-linolenic acid By analogy to mammalian eicosanoid biosynthesis, a phospholipase A (PLA) activity has been postulated to be involved in JA biosynthesis (Figs 1, 3). An increase of PLA activity could be detected in tobacco cells 2 h after elicitation (Roy et al., 1995) and in soybean after treatment with extracts of a pathogenic fungus (Chandra et al., 1996). A rapid release of LA preceding the accumulation of JA upon elicitation (Mueller et al., 1993) or wounding (Conconi et al., 1996a) supported evidence for a regulated lipase (Fig. 4) which releases LA for octadecanoid biosynthesis from membrane lipids (NarvaÂez-VaÂsquez et al., 1999). Considering the localization of the early enzymes of JA biosynthesis, i.e. LOX, AOS and AOC in the chloroplasts, it is not the amount of LA within whole cells (which should be generally suf®cient for JA biosynthesis and even more so after induction by stress; Conconi et al., 1996a; Moreau and Preisig, 1993), but the amount of LA in the chloroplasts that needs to be determined. Since biosynthesis of OPDA occurs in the chloroplasts, the major sources of LA for JA biosynthesis may be monogalactosyl diacylglycerol and digalactosyl diacylglycerol as previously suggested (Simpson and Gardner, 1995). Galactosyl lipids are predominantly located in chloroplasts and are degraded after wounding (Conconi et al., 1996a) or mechanical stimulation (Weiler et al., 1993). Lipoxygenase Lipoxygenases (LOXs) are non-haem iron-containing dioxygenases that catalyse the oxygenation of fatty acids to their corresponding hydroperoxy derivatives (Figs 1, 3) and are widely distributed in animals, in man and higher plants (Siedow, 1991). Plant LOX proteins consist of a single polypeptide chain with a molecular mass of about 94±104 kDa. They have been correlated with plant growth and development, maturation and senescence (for review see Siedow, 1991), with responses to pathogen attack (Rance et al., 1998; VeÂroneÂsi et al., 1996) and wounding (Farmer and Ryan, 1992; Saravitz and Siedow, 1995, 1996) and the biosynthesis of signalling molecules, such as JA and OPDA (Fig. 3) (Creelman and Mullet, 1997; Vick and Zimmerman, 1987). Higher plants contain multiple LOXs (Fig. 2) comprising at least eight identi®ed isoforms in soybean (for review see Brash, 1999). It has been shown that many plant LOXs are soluble, cytoplasmatic enzymes (Siedow, 1991; Hildebrand, 1989; Gardner et al., 1991). Nevertheless, the presence of LOXs has been demonstrated in other cellular compartments, such as the stroma of chloroplasts (Douillard and Bergeron, 1981), lipid bodies (Feussner and Kindl, 1994; Feussner and KuÈhn, 1995), vacuoles (Tranbarger et al., 1991), and mitochondrial fractions (Braidot et al., 1993). A stroma-localized plastidial LOX (LOX2) appears to be responsible for the wound-induced biosynthesis of JA in Arabidopsis (Bell et al., 1995). Charge modi®cations of soluble LOXs, however, may result in their association with membranes (Droillard et al., 1993). Alternatively, soluble LOXs may be transferred via vesicles to target membranes where they attack polyunsaturated fatty acids of glycerolipids or free fatty acids liberated by membrane-bound phospholipases (Fornaroli et al., 1999). Since linolenic acid dissolves poorly in aqueous media at typical physiological pH values of 7.0±7.4 it is not likely to be available as a substrate for soluble LOXs (Brash, 1999). Therefore, membrane lipids attacked by membrane-associated LOXs are likely to be the initial substrate of octadecanoid biosynthesis (Siedow, 1991). Allene oxide synthase According to the biosynthetic pathway of Vick and Zimmerman, jasmonates originate from LA via the reaction of a 13-LOX. The resulting product (9Z,11E, 15Z,13S)-13-hydroperoxy-9,11,15-octadecatrienoic acid (13(S)-HPOT) serves as a substrate for several enzymes (BleÂe, 1998) like hydroperoxide lyase, hydroperoxide reductase, divinylether synthase, and peroxygenase (Fig. 1). The dehydration of 13(S)-HPOT to the unstable epoxide (9Z,11E,15Z,13S,12R-12,13-epoxy-9,11,15-octadecatrienoic acid) by allene oxide synthase (AOS, Enzymes of octadecanoid biosynthesis 13 oligogalcturonides wounding O COOH chitosan m em br an e systemin 13-keto-9(Z),11(E),15(Z)octadecatrienoic acid Lipase OH COOH COOH 13-OH-9(Z),11(E),15(Z)octadecatrienoic acid Lipoxygenase linolenic acid Lipoxygenase Hydroperoxidereductase O OH OOH COOH COOH COOH O etheroleic acid 13(S)-HPOT Divinylethersynthase 15,16-epoxy-13-OH-9(Z), 11(E)-octadecadienoic acid Peroxygenase AOS Hydroperoxidelyase HO O COOH O n-hexenal COOH COOH 12,13-EOT AOC O OH COOH COOH O OHC 12-oxo-9(Z)dodecenoic acid OPDA OPR COOH O COOH Isomerization Faktor COOH OHC 12-oxo-10(E)dodecenoic acid OPC-8:0 -oxidation COOH COOH HOOC traumatic acid O jasmonic acid Fig. 1. a-Linolenic acid: a central substrate for the production of defence-related metabolites with biological activity. AOS: allene oxide synthase; AOC: allene oxide cyclase; OPR: 12-oxo-phytodienoic acid reductase; 13(S)-HPOT: (9Z,11E,15Z,13S)-13-hydroperoxy-9,11,15-octadecatrienoic acid; 12,13-EOT: (9Z,11E,15Z,13S,12R-12,13-epoxy-9,11,15-octadecatrienoic acid; OPDA: 12-oxo-10,15(Z)-octadecatrienoic acid; OPC-8:0: 3-oxo-2(29(Z)pentenyl)-cyclopentane-1-octanoic acid. formerly hydroperoxide isomerase and hydroperoxide dehydrase) (Vick and Zimmerman, 1981, 1987) represents the ®rst speci®c step in octadecanoid biosynthesis. The unstable allene oxide does either spontaneously hydrolyse to a mixture of a- and c-ketols (80% and 10%, respectively) and racemic cis-12-oxophytodienoic acid (OPDA, 10%) or, in a concerted action with allene oxide cyclase (AOC), it cyclizes to enantiomerically pure 9S,13S-OPDA (i.e. cis(q)-OPDA) (Hamberg and Fahlstadius, 1990; Laudert et al., 1997), the substrate for jasmonic acid biosynthesis (Fig. 1). AOS was ®rst puri®ed and subsequently cloned from ¯ax seeds (Song and Brash, 1991; Song et al., 1993), guayule (Pan et al., 1995), Arabidopsis (Laudert et al., 1996), tomato (Sivasankar et al., 2000; Howe et al., 2000), and barley (Maucher et al., 2000) and was also puri®ed from corn (Utsunomiya et al., 2000). AOS is a cytochrome P450 of the CYP74 family (Laudert et al., 1996; Song et al., 1993; Pan et al., 1995) which is independent of oxygen, NADPH and P450 reductases for enzymatic activity and is characterized by a lack of transmembrane segments and low af®nities for CO. According to 14 Schaller Fig. 2. Phylogenetic tree of plant lipoxygenases. The amino acid sequences were compiled into the phylogenetic tree using `clustalW' and `TreeView'. Arabidopsis thaliana, LOX 2 accession JQ2391; Arabidopsis thaliana, LOX accession JQ2267; Cucumis sativus, LOX (lipid body) accession X92890; Cucumis sativus, LOX accession U36339; Glycine max, LOX 1 accession AAA33986; Glycine max, LOX 2 accession J03211; Glycine max, LOX 3 accession X13302; Glycine max, LOX 4 (VSP 94) accession P38417; Glycine max, LOX 5 (vlxB) accession U50075; Glycine max, LOX 7 accession U36191; Glycine max, LOX accession X56139; Glycine max, LOX vlxC accession U26457; Hordeum vulgare, LOX A accession L35931; Hordeum vulgare, LOX B accession AAB60715; Lens culinaris, LOX 1 accession P38414; Lycopersicon esculentum, LOX (loxC, chloroplast) accession U37839; Lycopersicon esculentum, LOX (chloroplast) accession AAB65767; Lycopersicon esculentum, LOX A accession P38415; Lycopersicon esculentum, LOX B accession P38416; Nicotiana tabacum, LOX accession S57964; Oryza sativa, LOX 2 accession A53054; Oryza sativa, LOX 2 accession S23454; Phaseolus vulgaris, LOX accession S22153; Pisum sativum, LOX (cytoplasmic, seed) accession X07807; Pisum sativum, LOX (seed) accession CAA34906; Pisum sativum, LOX G accession X76124; Solanum tuberosum, LOX 2 accession U60201; Solanum tuberosum, LOX 3 accession U60202; Solanum tuberosum, LOX accession U24232; Solanum tuberosum, LOX H1 accession X96405; Solanum tuberosum, LOX H3 accession X96406; Solanum tubersosum, LOX 1 accession U60200; Vicia faba, LOX 1 accession CAA97845. differences in the haem-binding domain which is distinct from that of most other P450 enzymes, the CYP74 family is subdivided in two classes. The CYP74A subfamily contains all AOSs, whereas the pepper hydroperoxide lyase and the tomato hydroperoxide lyase are the sole member of the CYP74B subfamily thus far identi®ed (Matsui et al., 1996; Howe et al., 2000). AOS activity has been localized in the chloroplasts of spinach leaves (Vick and Zimmerman, 1987) and barley (Maucher et al., 2000).The AOS cDNAs from ¯ax (Song et al., 1993), tomato (Sivasankar et al., 2000; Howe et al., 2000) and Arabidopsis (Laudert et al., 1996) encode transit peptides characteristic for chloroplast targeting. Similar transit peptides could not be found, however, in the AOS from rubber particles of guayule (Pan et al., 1995) and in the two isoforms of the barley AOS (Maucher et al., 2000). Catalytic activity While AOS from ¯ax and Arabidopsis will convert the 13(S)-hydroperoxides of linoleic and linolenic acids to the respective allene oxides (Song and Brash, 1991; Laudert et al., 1996) at comparable rates, the Km for 13(S)- octadecadienoic acid of barley AOS1 is approximately 5-fold higher compared to that for 13(S)-octadecatrienoic acid. The 15(S)-hydroperoxides of C20 fatty acids as well as the 9(S)-hydroperoxides of linoleic and linolenic acid are also substrates of barley AOS (Maucher et al., 2000). The physiological relevance of the latter activity, however, remains obscure. Physiology Wounding, OPDA and JA induce the expression of both ¯ax and Arabidopsis AOS (Harms et al., 1998; Laudert and Weiler, 1998) (Fig. 4). Arabidopsis AOS is also induced by ethylene, an indication that these two phytohormones might interact in some way (O'Donnell et al., 1996; Laudert and Weiler, 1998). Salicylic acid, an activator of pathogenesis-related gene expression, induces Arabidopsis AOS but represses ¯ax AOS, indicating different regulation of AOS gene expression among plant species (Harms et al., 1998; Laudert and Weiler, 1998). Overexpression of ¯ax AOS in transgenic potato plants led to a 6±12-fold increase in the endogenous JA levels, but despite the fact that the transgenic plants had `resting' Enzymes of octadecanoid biosynthesis 15 Fig. 3. Plant lipoxygenases metabolize a-linolenic acid to produce signalling compounds, induce defence mechanisms and produce energy. JA levels similar to those found in wounded wild-type plants, pin2 genes were not constitutively expressed (Harms et al., 1995). In contrast, the overexpression of Arabidopsis AOS in Arabidopsis and tobacco did not alter basal levels of JA, but in these transgenic plants, peak jasmonate levels were 2±3-fold higher after wounding as compared to wounded, untransformed plants and the transgenic plants reached the maximum JA-level signi®cantly earlier than wounded control plants. These ®ndings suggest that overexpression of AOS might be a way of controlling defence dynamics at least in Arabidopsis and tobacco (Laudert et al., 2000). Considering the results of both Harms et al. and Laudert et al., it could be argued that it is not the absolute, constitutive level of octadecanoids that determines the induction of defence mechanisms, but the relative increase of signalling molecules after, for example, wounding (Harms et al., 1998; Laudert and Weiler, 1998). The ®ndings that the expression of the AOS gene is upregulated by JA and OPDA as well as their methyl esters (Laudert and Weiler, 1998) demonstrate the autoinduction of the ®rst speci®c enzyme of the jasmonate pathway by its end products. Treatments which do not lead to endogenous accumulation of jasmonates in barley (e.g. SA, ABA, NaCl, and pathogen infection) failed to give upregulation of AOS expression (Maucher et al., 2000). Fig. 4. The biosynthesis of jasmonic acid takes place in three different compartments and is highly regulated. LA: a-linolenic acid; LOX: lipoxygenase; AOS: allene oxide synthase; AOC: allene oxide cyclase; OPR: 12-oxo-phytodienoic acid reductase; 13(S)-HPOT: (9Z,11E, 15Z, 13S)-13-hydroperoxy-9,11,15-octadecatrienoic acid; 12,13-EOT: (9Z, 11E,15Z,13S,12R-12,13-epoxy-9,11,15-octadecatrienoic acid; OPDA: 12oxo-10,15(Z)-octadecatrienoic acid; OPC-8:0: 3-oxo-2(29(Z)-pentenyl)cyclopentane-1-octanoic acid; JA, jasmonic acid; SA, salicylic acid; BR, brassinolide; Cor, coronatine. Regulation of the Arabidopsis AOS gene In order to gain a better understanding of the mechanisms involved in the control of AOS gene expression, the AOS gene of Arabidopsis was isolated and characterized (Kubigsteltig et al., 1999). The promoter region contains cis-elements such as CAAT boxes and ACGT elements (Foster et al., 1994) found in many regulated genes. These elements are almost identical to a stress response element described previously (Goldsbrough et al., 1993) as being salicylate-reponsive and a putative ethylene-reponse element (Montgomery et al., 1993; Itzhaki et al., 1994), respectively. In young transgenic plants (up to 21 d (tobacco) and 11 d (Arabidopsis) post-germination) expressing the pAOS :: uidA reporter gene, no GUS 16 Schaller activity was observed. In older plants, however, increasing GUS activity could be detected and was correlated with increasing leaf age. Generally, GUS activity was higher in Arabidopsis compared to tobacco. Furthermore, Arabidopsis pAOS::uidA reporter gene plants showed expression of the AOS gene in pollen, which is consistent with the observation that jasmonates are required for male fertility in Arabidopsis, as both the coronatinand jasmonate-insensitive mutant coi1 (Feys et al., 1994) and the triple mutant fad3-2 fad7-2 fad8, incapable of producing normal levels of JA, are male-sterile (McConn and Browse, 1996). Furthermore, jasmonates have been shown to occur in pollen (Yamane et al., 1982; Miersch et al., 1998). After wounding, both local and systemic activation of the AOS promoter could be detected in Arabidopsis and tobacco. Two hours after wounding, GUS activity was observed in the main veins of Arabidopsis and tobacco leaves as well as the petioles and 24 h after wounding the whole shoot had reacted uniformly. In contrast to the systemic induction after wounding, after the application of jasmonates, such as OPDA, and JA as well as the octadecanoid analogue, coronatin, only a local induction of AOS promoter activity was observed in the treated leaves. JA anduor OPDA are thus unlikely to serve as systemic inducers of defence reactions in Arabidopsis (Kubigsteltig et al., 1999). Rather, octadecanoids act locally at the site of application, that is, they behave like short distance messengers in planta. Deletion analyses of the Arabidopsis AOS promoter localized a putative cis-element for wound inducibility to a domain of only 50 base pairs. The experiments to clarify if this element is both necessary and suf®cient to confer wound-inducibility are in progress (I Kubigsteltig, personal communication). Allene oxide cyclase In 1981, Vick and Zimmerman (Vick and Zimmerman, 1981) postulated the existence of a hydroperoxide cyclase for the conversion of 13(S)-HPOT into 12-oxo-phytodienoic acid and the allene oxide, 12,13(S)-epoxylinolenic acid, has been shown to serve as the immediate precursor of OPDA in plants (Fig. 1; Hamberg and Hughes, 1988; Baertschi et al., 1988; Crombie and Morgan, 1988). In 1990, an allene oxide cyclase (AOC) was identi®ed as a soluble enzyme in corn with a molecular mass of about 45 kDa (Hamberg and Fahlstadius, 1990). Recently, AOC was puri®ed from maize kernels (Ziegler et al., 1997), cloned from tomato (Ziegler et al., 2000) and the substrate speci®city of the enzyme has been analysed (Ziegler et al., 1999). Catalytic activity Allene oxide cyclase (AOC, EC 5.3.99.6) catalyses the cyclization of the unstable allene oxide 12,13(S)- epoxy-9(Z),11,15(Z)-octadecatrienoic acid (12,13-EOT) yielding 12-oxo-10,15(Z)-phytodienoic acid (OPDA) the ®rst octadecanoid bearing the characteristic cyclopentenone structure (Figs 1, 4). Potato AOC in combination with recombinant Arabidopsis AOS expressed in E.coli resulted in the production, in vitro, of highly asymmetrical cis-OPDA consisting nearly exclusively of the 9(S),13(S)-form (i.e.the cis(q)-enantiomer) (Laudert et al., 1997), which is the enantiomer occurring in plant tissues (Stelmach et al., 1998; Laudert et al., 1997) and the precursor of JA biosynthesis. The maize enzyme is a dimer of 47 kDa apparent molecular mass which accepts 12,13(S)-epoxy-9(Z),11,15(Z)-octadecatrienoic acid, but not 12,13(S)-epoxy-9(Z),11-octadecadienoic acid, as a substrate (Ziegler et al., 1997, 1999). This is in contrast to AOS which produces both allene oxides from the respective substrates, 13(S)-hydroperoxylinolenic acid or 13(S)-hydroperoxylinoleic acid. Thus, the speci®city in the octadecanoid biosynthetic pathway results from AOC, rather than LOX or AOS. The extremely short half-life of allene oxides (t1u2 less than 30 s in water), the optical purity of natural OPDA, and the absence of a- and c-ketols in plant tissues in vivo (Weiler et al., 1998), suggest a tight coupling of the AOS and AOC reactions possibly in a synthase-cyclase complex. 12-oxo-phytodienoic acid reductase Vick and Zimmerman (1984), analysing the metabolism of 18O-labelled OPDA in several plant tissues, identi®ed 3-oxo-2-(29(Z)-pentenyl)-cyclopentane-1-octanoic acid (OPC-8 :0) as one of the labelled metabolites. The enzyme catalysing the reduction of the CC double bond of the conjugated enone moiety (Fig. 1) was called 12-oxophytodienoic acid reductase (OPR). OPR was identi®ed as a protein of a molecular mass of about 54 kDa, with a KM of 15 mM for its substrate OPDA and a preference for NADPH as the reducing agent. Molecular cloning of the reductase from Arabidopsis (Schaller and Weiler, 1997b) revealed a close relationship to Otto Warburg's Old Yellow Enzyme (OYE), a ¯avoprotein enone reductase (Warburg and Christian, 1932, 1933), to morphinone reductase of Pseudomonas putida, an enzyme which reduces morphinone to hydromorphone or codeinone to hydrocodone (French and Bruce, 1995) and other OYErelated proteins (Fig. 5). OYE (EC 1.6.99.1) was initially isolated from brewer's bottom yeast and was the ®rst enzyme shown to possess a ¯avin cofactor (Warburg and Christian, 1932, 1933). Despite extensive biochemical and spectroscopic characterization, the physiological function of the enzyme has remained obscure. OYE has been described as a diaphorase catalysing the oxidation of NADPH in the presence of molecular oxgen, but a physiological oxidant has not been found (Massey and Schopfer, 1986). A number of arti®cial electron acceptors for Enzymes of octadecanoid biosynthesis 17 Fig. 5. Phylogenetic tree of the family of Old Yellow Enzymes. The amino acid sequences were compiled into the phylogenetic tree using `clustalW' and `TreeView'. Agrobacterium radiobacter, GTN-reductase accession CAA74280; Arabidopsis thaliana, OPR 1 accession AAC78440; Arabidopsis thaliana, OPR 2 accession AAC78441; Arabidopsis thaliana, OPR 3 accessions AAF67635, AAD38925, CAB66143, AAD19764; Arabidopsis thaliana, OPR-like protein accession AAC33200; Azotobacter vinelandii, 2-cyclohexen-1-one reductase accession BAA88211; Candida albicans, estrogen-binding protein accession P43084; Catharanthus roseus, OPR-like accession T09943; Enterobacter cloacae, pentaerythritol tetranitrate reductase accession AAB38683; Escherichia coli, N-ethylmaleimide reductase accession P77258; Kluyveromyces lactis, KYE 1 (OYE 1) accession P40952; Lithospermum erythrorhizon, LEDI-5a protein accession BAA83083; Lithospermum erythrorhizon, LEDI-5b protein accession BAA83084; Lycopersicon esculentum, OPR 1 accession CAB43506; Lycopersicon esculentum, OPR 2 accession AJ278331; Lycopersicon esculentum, OPR 3 accession AJ278332; Pseudomonas putida, morphinone reductase accession S64687; Saccharomyces cerevisiae, OYE 1 accession Q02899; Saccharomyces cerevisiae, OYE 2 accession NP_012049; Saccharomyces cerevisiae, OYE 3 accession NP_015154; Saccharomyces cerevisiae, OYE-like accession CAA65573; Schizosaccharomyces pombe, OYE A accession Q09670; Schizosaccharomyces pombe, OYE B accession Q09671; Vigna unguiculata, OPR-like accession T11580. reduced OYE has been identi®ed including the ole®nic double-bond of a,b-unsaturated ketones and aldehydes (Stott et al., 1993; Vaz et al., 1995). Several OYE homologues have been reported in prokaryotic and eukaryotic organisms (Stott et al., 1993; French and Bruce, 1994; Franklund et al., 1993; Miura et al., 1997; Buckman and Miller, 1998). The puri®cation (Schaller and Weiler, 1997a) and molecular cloning of a plant OPR (Schaller and Weiler, 1997b) led to the identi®cation of the ®rst member of the family of OYEs in higher plants. Up to now, two OPRs from Rock Harlequin (Corydalis sempervirens, Schaller and Weiler, 1997a; Schaller et al., 1998), three OPR isoforms from Arabidopsis (Schaller and Weiler, 1997b; Biesgen and Weiler, 1999; MuÈssig et al., 2000; Schaller et al., 2000) and three OPR isoforms from tomato (Straûner et al., 1999a; A Schaller, personal communication) have been described. A phylogenetic tree of a sequence alignment of the amino acid sequences of the three Arabidopsis and tomato OPRs as well as OYEs 1±3 from Saccharomyces and other plant OYE-related sequences is shown in (Fig. 5). Sequence similarities were observed over the entire lengths of the protein sequences, furthermore two of the three amino acids involved in substrate binding are conserved throughout the plant and the yeast sequences (tyr376 and his192), whereas asp195 is replaced by his in most of the plant sequences. Catalytic activity Looking at the phylogenetic tree of OYEs and their related enzymes and the OPRs (Fig. 5) it becomes obvious that enzymes of the OPR3-type from Arabidopsis and tomato are more closely related to the OYEs than OPR1 and OPR2. This re¯ects the enzymatic properties of the enzymes. While OPRI from Corydalis sempervirens (Schaller et al., 1997a), LeOPR1 from tomato (Straûner et al., 1999b) and OPR1 and OPR2 from Arabidopsis (Schaller et al., 2000) preferentially catalyse the reduction of cis( )-OPDA, the isomer which may result from a partial uncoupling of the AOSuAOC reactions, OPRII from Corydalis sempervirens (Schaller et al., 1998), LeOPR3 from tomato (A Schaller, F Schaller and P Macheroux, unpublished results) and OPR3 from Arabidopsis (Schaller et al., 2000) are the enzymes which 18 Schaller prefer cis(q)-OPDA, the naturally occurring precursor of JA biosynthesis, as their respective substrate. OPR3 rather than its relatives OPR1 and OPR2, is thus the enzyme involved in JA biosynthesis in Arabidopsis and tomato. The functions of OPR1 and OPR2 are not yet understood, but it seems likely that they are not involved in JA biosynthesis. However, looking at the broad substrate speci®city of Le OPR1 and Arbidopsis OPR1 (Straûner et al., 1999b; F Schaller, unpublished results) it is tempting to speculate that other physiologically relevant compounds may be reduced by this enzyme. Yeast OYE forms charge transfer (CT) complexes with a variety of aromatic and heteroaromatic compounds carrying an ionizable hydroxyl group. Formation of the CT complex results in a long wavelength transition in the absorbance spectra. A positive correlation was observed between the absorbance maximum of the newly formed long wavelength transition and the Hammett paraconstant of the p-substituted phenolic compounds, which can be considered as a measure of the phenolate ionization potential (Massey et al., 1984). This ®nding and the pH dependence of binding suggest that in the CT complex, the bound phenolate and the oxidized ¯avin act as the electron donor and acceptor, respectively. It could be demonstrated that a series of phenolic ligands form CT complexes with LeOPR1 (Straûner et al., 1999b) and LeOPR3 as well as OPR3 from Arabidopsis (A Schaller, F Schaller and P Macheroux, unpublished results). This ®rst demonstration of such complexes in plant ¯avoproteins provides further biochemical evidence for the relationship between yeast OYEs and their plant homologues. It could be concluded from these experiments that the ¯avin moiety acts as the acceptor and the phenolic ligand as the donor in the LeOPR1 CT complex, as was concluded earlier for yeast OYE (Abramovitz and Massey, 1976). Work towards the elucidation of LeOPR structure by X-ray diffraction is in progress (A Schaller, personal communication) and will yield an even better understanding of the relationship between yeast and plant OYEs. Physiology The analysis of the regulation of OPR activity is of fundamental interest because JA is not the only biologically active compound of the jasmonate pathway. It could be demonstrated that the ®rst cyclic intermediate, OPDA, functions as an endogenous signal transducer in mechanotransduction (Stelmach et al., 1998; Weiler et al., 1993, 1994) and, furthermore, it could be demonstrated that OPDA may be more relevant in vivo than JA for signal transduction in elicited cell-suspension cultures from several plant species (Blechert et al., 1995; Parchmann et al., 1997). In tendrils of Bryonia dioica, there is a strong and transient increase in OPDA levels after mechanical stimulation while JA levels remain unaffected (Blechert et al., 1999). Thus the OPRs and OPR3 in particular may play a decisive role in octadecanoid signalling in that the enzyme appears to control the metabolite ¯ow from the biologically active C18compound OPDA to the C12-metabolite JA (Fig. 1). OPRs may thus regulate the level of OPDA independently from that of JA. Changes in the relative concentration of octadecanoid compounds (i.e. the oxylipin signature) rather than the concentration of any single compound, may be relevant for the activation of diverse physiological responses (Weber et al., 1997). The expression of OPRs was analysed on the mRNA and protein levels as well as in transgenic plants expressing the GUS reporter gene under the control of the Arabidopsis OPR promoters. Transient changes in steadystate OPR1 and OPR2 mRNA levels were observed in response to wounding, UV-C illumination and heatucold stress (Fig. 4). Likewise, the expression of GUS under the control of the OPR1 and OPR2 promoters was stimulated after touch- and wind-stimulation, as well as after wounding and UV-irradiation (Biesgen and Weiler, 1999). The very same conditions, which are known to stimulate octadecanoid signalling transduction (Farmer and Ryan, 1990; Falkenstein et al., 1991; Conconi et al., 1996b; Stelmach et al., 1998), also resulted in a rapid and transient increase of OPR3 mRNA (MuÈssig et al., 2000). The signi®cance of transcriptional activation of OPR genes remains unclear, however, since no induction was observed for OPR1 and 2 at the levels of protein or enzymatic activity (Biesgen and Weiler, 1999). b-Oxidation 12-(18O)-oxo-PDA, 10,11-dihydro-12-(18O)-oxo-PDA as well as the fully saturated 10,11,15,16-tetrahydro12-(18O)-oxo-PDA are converted to JA or 9,10-dihydroJA, respectively and, in addition, two labelled C16 and C14 intermediates with the carboxyl chain shortened by two and four carbons, respectively, have been isolated, suggesting that three cycles of b-oxidation are the terminal steps in JA biosynthesis (Figs 1, 4) (Vick and Zimmerman, 1983, 1984). The compartmentalization of these b-oxidation steps has not been investigated yet. Presumably, they are localized in the peroxisomes anduor glyoxysomes, since these are the only sites where b-oxidation is known to occur in plants (Gerhardt, 1983; Vick and Zimmerman, 1984). Alternatives should be considered, since b-oxidation of unsaturated fatty acids, such as oleic acid, terminates after only two cycles, leaving a D5,6-enoyl-CoA which cannot be b-oxidized further unless the double bond is reduced (Tserng and Jin, 1990). The in¯uence of a cyclohexenone ring attached to an acyl chain on the ef®ciency of the b-oxidation enzymes Enzymes of octadecanoid biosynthesis is unknown, nor has it been shown whether or not OPC8:0 can be converted to its coenzyme A thioester required to initiate b-oxidation. Likewise, the question of whether the transformation of the C18-compound OPDA to the C12-compound JA should be considered a biosynthetic, or rather, a catabolic reaction is still open. It has been reported that OPDA is active per se in the induction of benzophenanthridine alkaloids after elicitation of plant suspension cultures (Blechert et al., 1995). Therefore, these authors assumed that OPDA and OPC-8 :0 are primary signal transducers in the elicitation process, notwithstanding the biological activity of JA and its possible interaction with the same hypothetical receptor. Furthermore, OPDA is 50±100 times more active as compared to JA in eliciting tendril coiling in Bryonia dioica (Weiler et al., 1994). Thus, just like in mammalian eicosanoid metabolism where b-oxidation is a characteristic degradation route and usually associated with complete loss of the biological activity, it is possible that JA represents a degradation product of biologically more active C18-compounds, which is then further inactivated in the cell by conjugation (Sembdner et al., 1988). Discussionuperspectives Although all enzymes of JA-biosynthesis are present in low levels in uninduced plants, it is now certain that key steps in the pathway are under inductive control (Fig. 4). This induction may lead to an autoinduction of the biosynthetic pathway or may help to regulate the metabolite pools of the biologically active C18-compounds versus those of the C12-compounds. OPR operates at such a decisive point in the pathway. If one bears in mind that the OPDA content in plants varies by more than one order of magnitude between different species (Stelmach et al., 1998), it becomes obvious that (1) the 19 release of OPDA from its biosynthetic compartment, the chloroplast, anduor (2) the reduction of released OPDA by OPR may be rate-limiting steps in JA biosynthesis. Since the localization of OPR3 from Arabidopsis and tomato is not yet known the reduction of cis(q)-OPDA may be localized in the peroxisomes. The observation that OPR3 mRNA levels are upregulated by 24-epi-brassinolide (MuÈssig et al., 2000) suggests a functional relationship between octadecanoid and brassinolide signalling. Some further observations which point to this connection are: (1) brassinosteroids are present in pollen at very high concentrations, and jasmonic acid is required for pollen fertility (Xie et al., 1998) and (2)OPR3 gene expression by membrane damage after detergent treatment is impaired in the brassinosteroidde®cient mutant cbb1 (MuÈssig et al., 2000). Because of the fact that neither JA nor OPDA led to a systemic induction of the AOS promoter like, for example, wounding (Kubigsteltig et al., 1999), it seems clear that (1) these octadecanoids are not involved in systemic defence signalling (Fig. 6), rather they behave like local response regulators and (2) the systemic activation of the AOS promoter after wounding of a leaf or a root does involve other wound factor(s) released by these organs. On the other hand a crucial role for octadecanoids in herbivore defence could be demonstrated: (1) the LA-de®cient fad 3-2 fad 7-2 fad 8 mutant of Arabidopsis (McConn et al., 1997) lacks the ability to accumulate JA after wounding and is more susceptible to attacks of chewing insects than the wild-type, (2) the defenceless mutant (def 1) of tomato does not raise the JA-level after wounding, produces less proteinase inhibitor and is more susceptible to herbivore attack than the wild-type (Howe et al., 1996), (3) the JA-insensitive mutant (coi1) of Arabidopsis is more susceptible to herbivore attack than wild-type plants (Rojo et al., 1998). Therefore it may be possible that there exist different Fig. 6. There exist three wound-induced signalling pathways in higher plants (adapted from Weiler et al., 1999). 20 Schaller signal transduction pathways and defence mechanisms for herbivore attack and wounding, respectively. 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