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During plant development or in response to stress a variety of signal molecules are generated, some of them derive from fatty acids (Browse, 2009a). For example, jasmonic acid (JA) and its precursor (9S,13S)-12-oxo-phytodienoic acid (cis-(+)-OPDA) derive from oxygenated polyunsaturated fatty acids that are collectively named oxylipins (Andreou et al., 2009). Their biosynthesis is initiated by formation of fatty acid hydroperoxides catalyzed mainly by lipoxygenases (LOXs) (Andreou & Feussner, 2009). Next allene oxide synthase (AOS) catalyses dehydration of fatty acid hydroperoxides to an unstable allene oxide, which may hydrolyse to a mixture of α-ketol, γ-ketol or racemic cyclopentenones. The allene oxide (9Z,13S,15Z)-12,13-epoxy-9,11,15-octadecatrienoic acid (12,13-EOT) is metabolized by an allene oxide cyclase (AOC) to an enantiomeric pure cyclopentenone, cis-(+)-OPDA (Fig. 1a). This AOS/AOC reaction on 13-hydroperoxy octadecatrienoic acid (13-HPOTE) represents the first specific step leading to JA synthesis. While these steps were localized in the plastid (Browse, 2009a), the later steps of JA biosynthesis take place in peroxisomes. They include the reduction of cis-(+)-OPDA by OPDA reductase 3 (OPR3), activation to the coenzyme A (CoA) ester and three cycles of β-oxidation (Browse, 2009a).
Figure 1. Allene oxide synthase (AOS)/allene oxide cyclase (AOC) pathways. (a) 13-Hydroperoxy octadecatrienoic acid (HPOTE) is converted by AOS to an allene oxide (12,13-EOT) which may either hydrolyse to ketols and a racemic cyclopentenone (OPDA) or the AOC reaction leads to specific formation of cis-(+)-OPDA. (b) 12-Hydro(pero)xy eicosapentenoic acid (HPETE) is converted by AOS to an allene oxide (11,12-EETE) which is either hydrolysed to ketols and a racemic cyclopentenone or AOC2 is capable of catalysing formation of 11-oxo prostatrienoic acid (OPTA).
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JA and cis-(+)-OPDA are important signalling molecules in the coordination of the plants response to stress such as wounding, pathogen attack or water deficit. Mutants deficient in cyclopentenone formation or JA signalling are impaired in pathogen resistance, whereas mutants with constitutively active JA signalling show increased resistance (Browse, 2009b). In addition, JA plays a role in the regulation of developmental processes (Browse, 2009a). Leaf senescence as well as inhibition of growth and germination is induced by application of the methyl ester of JA. In Arabidopsis thaliana JA is important for the correct release of pollen and elongation of filaments and JA-deficient or JA-insensitive plants are male sterile. However, similar tomato mutants are female sterile (Browse, 2009b).
Seed plants use mainly C18 fatty acids as precursors of oxylipins. In animals and algae such compounds may also derive from C20 and C22 fatty acids (Andreou et al., 2009). Similarly, the moss Physcomitrella patens can use arachidonic acid in addition to C18-fatty acids to form oxylipins (Wichard et al., 2005).
Genes encoding AOC have been isolated from various plant species. Hitherto all described AOCs are specific for the formation of two cyclopentenones: cis-(+)-OPDA or the roughanic acid-derived (7S,11S,13Z)-dinor-10-oxo-8,13-phytodienoic acid (dn-OPDA). Both are precursors of JA. So far, all characterized AOCs carry a putative plastidic transit peptide and are located in plastids (Browse, 2009a).
Here, we isolated two cDNAs encoding AOCs from this moss. Characterization of both AOCs included substrate specificity of the recombinant enzymes and formation of a novel cyclopentenone derived from 12-hydroperoxy arachidonic acid (12-HPETE). Finally we could not detect any JA and we show that single knockout of an AOC-encoding gene already leads to reduced fertility, malformed spore capsules and to aberrant sporogenesis.
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Numerous studies describe the occurrence and function of cyclopentanones and cyclopentenones as important group of oxylipins in flowering plants (Browse, 2009a,b) but knowledge on occurrence and function of oxylipins in nonflowering plants is still scarce (Andreou et al., 2009). Therefore, we aimed to analyse the formation of cyclopentanones and cyclopentenones by analysing recombinant AOCs and putative function(s) of these AOC products via targeted knockout mutants of P. patens. This moss serves as a model system for nonflowering plants (Charron & Quatrano, 2009). Recently, we found that it is able to metabolize arachidonic acid to form oxylipins (Wichard et al., 2005). One major hydro(pero)xide that was formed endogenously in P. patens is 12-H(P)ETE (Fig. S4) which serves as precursor of volatiles such as octenols, octenals and nonenals (Stumpe et al., 2006a). These volatiles are formed by at least two LOX enzymes with an unusual lyase activity or a classical plant-type hydroperoxide lyase-derived pathway (Wichard et al., 2005; Anterola et al., 2009).
Another important enzyme in oxylipin metabolism is AOC which catalyses the formation of cyclopentenones. In this step the enantiomeric structure is established which occurs in the naturally occurring jasmonates. The available EST library from P. patens harboured two sequences with similarity to AOCs. This allowed us to isolate full-length cDNAs. Both cloned AOCs did not contain a predictable transit peptide (Fig. S1), but the immunocytological approach showed location of both AOCs in chloroplasts (Figs 3 and S3). Proteins lacking a transit peptide may be imported via a transit peptide-independent sorting route, as observed for the AOS of barley (Maucher et al., 2000).
Both corresponding proteins of the full-length cDNAs showed AOC activity (Fig. 2). We tested fatty acid hydroperoxides in a coupled AOS/AOC assay and the typical reaction with 13-HPOTE leading to enantiomeric pure cis-(+)-OPDA was catalysed by both AOCs. Common features of substrates for AOC were identified by analysis of potato and corn AOC (Ziegler et al., 1999; Stumpe et al., 2006b). There, an epoxy group at position n-6,7 and a β,γ-double bound relative to the epoxy group at position n-3 was found to be essential. In case of C20 fatty acids only the allene oxide derived from 15-HPETE was tested as substrate of the corn AOC (Ziegler et al., 1999). Here, we used 12-HPETE as substrate which leads to formation of an allene oxide with an epoxy group at position n-9,10 and the β,γ-double bound at position n-6. The recombinant PpAOC1 and PpAOC2 formed the corresponding cyclopentenone (Fig. 2). Based on HPLC/MS and GC/EI-MS data as well as the UV spectrum and biosynthetic considerations we assign the structure as 11-OPTA (Figs 1 and S2). This cyclopentenone cannot be a precursor of JA because it has an octenyl instead of a pentenyl side-chain (Fig. 1). Interestingly, the formation of a similar eicosanoid was observed by incubation of arachidonic acid with extracts of corals such as Plexaura homomalla (Brash, 1989). Here, arachidonic acid was oxidized by an (8R)-LOX-AOS fusion protein first to (8R)-HPETE. This is further metabolized by the same fusion protein to an unstable allene oxide, which hydrolyses to ketols and undergoes nonenzymatic cyclization to racemic (5Z,14Z)-9-oxo-prosta-5,10,14-trienoic acid, called pre-calvulone A. This cyclopentenone was discussed as putative precursor of prostaglandins, calvulones and other marine eicosanoids (Andreou et al., 2009). However, the novel cyclopentenone 11-OPTA shares the same α,β-unsaturated carbonyl group in the cyclopentenone ring as cis-(+)-OPDA. It is important to note that in flowering plants cis-(+)-OPDA has gene regulatory activity which is independent of JA (Stintzi et al., 2001; Farmer et al., 2003; Taki et al., 2005; Müller et al., 2008). Therefore, we analysed the endogenous amounts of cyclopentenones in protonema and gametophores first. An average amount of cis-(+)-OPDA of c. 0.5 nmol g−1 FW was detected corresponding to observed levels in Arabidopsis leaves (Stintzi et al., 2001; Müller et al., 2008). The amount of the novel cyclopentenone, 11-OPTA, in P. patens was c. 0.1 nmol g−1 FW. Furthermore, it was not possible to detect any amount of JA or amino acid conjugates of cis-(+)-OPDA or JA. This is in agreement with similar observations by other groups (Browse, 2009a), but contrasts with a recent report on infected moss cultures (Oliver et al., 2009). However, in the latter report JA was detected by head-space analysis only and was not confirmed by additional GC/MS experiments. Therefore, one may assume that in contrast to flowering plants P. patens harbours only the plastid-localized part of the LOX pathway (Wasternack, 2007; Browse, 2009a). This is supported by the fact that seven out of nine putative LOX genes, were identified as plastidial 13-LOXs while the other two appeared to be pseudo genes (Anterola et al., 2009).
The next enzyme in the biosynthetic pathway from cis-(+)-OPDA to JA is the OPDA reductase. Arabidopsis harbours at least five genes encoding proteins with activity and different substrate specificities but only OPR3 converts that enantiomeric form of OPDA, which is the precursor in JA biosynthesis (Breithaupt et al., 2006). Owing to undetectable levels of JA and its amino acid conjugates such as JA-Ile, JA-Leu and JA-Phe as well as cis-(+)-OPDA-Ile in P. patens, it is likely that the enzyme metabolizing cis-(+)-OPDA is missing or not active, although sequences were found with similarity to OPDA reductase genes (Li et al., 2009).
In respect to the JA deficiency and cis-(+)-OPDA accumulation P. patens is similar to the opr3 mutant of A. thaliana, which is a molecular tool to analyse cis-(+)-OPDA-specific gene activity and development (Stintzi et al., 2001; Browse, 2009a). However, it is a matter of debate how cis-(+)-OPDA perception takes place. In the case of JA perception, the F-box protein COI1 was recently identified as a JA receptor (Yan et al., 2009) which interacts with repressors of JA-responsive gene expression, the JAZ proteins, if (+)-7-iso-jasmonoyl-l-isoleucine is bound (Browse, 2009a; Fonseca et al., 2009). Consequently, JAZ proteins are directed to proteasomal degradation. JA or cis-(+)-OPDA are inactive in binding to COI1. As yet, there is no mechanistic explanation for cis-(+)-OPDA perception (Browse, 2009a), and a COI1-independent perception may be probable (Böttcher & Pollmann, 2009).
As P. patens is JA-deficient, but able to accumulate cis-(+)-OPDA, we investigated possible functions of cyclopentenones via targeted knockout mutants of PpAOC1 and PpAOC2. All our attempts to obtain double knockout mosses failed, revealing that both enzymes have overlapping functions in protoplast regeneration and that depletion of both enzymes in one P. patens plant may be lethal. The recombinant enzymes showed different substrate specificities as PpAOC2 is capable to form 11-OPTA in addition to formation of cis-(+)-OPDA (Fig. 2). This is reflected in unchanged amounts of cis-(+)-OPDA in all lines analysed, at least in the gametophytic tissues protonema and gametophores, but in an increase of 11-OPTA in ΔPpAOC1 line 15 (Fig. S5). Under standard growth conditions both gametophytic tissues were WT-like in the mutants. Seed plants affected in cis-(+)-OPDA and JA biosynthesis or JA perception show differences from WT often only under specific stress situations, such as pathogen attack or at certain developmental stages (Wasternack, 2007); for example, JA-deficient and JA-insensitive mutants of Arabidopsis are male sterile and show diminished resistance to herbivores and pathogens (Browse, 2009a). By contrast, the tomato mutant jai1 affected in the COI1 gene is female sterile (Li et al., 2004). Such differences in action of JA in reproductive tissues even in two dicot species prompted us to inspect sporophyte formation in P. patens ΔAOC mutants. This analysis revealed that targeted disruption of single members of the two PpAOC genes resulted in reduced fertility and in defective sporogenesis. Further, both mutants developed capsules (sporophytes) that did not release mature meiospores. The sporogenesis of the knockout mosses was interrupted in the post-meiotic stage of haploid tetrads. This leads to the conclusion that PpAOC1 and PpAOC2 are required for fertilization, for spore maturation and for a subsequent dehiscing of the capsules. The phenotype may be caused by a locally lowered cis-(+)-OPDA content or a yet unidentified metabolite derived there from, since our complementation experiments with cis-(+)-OPDA failed and the cis-(+)-OPDA amount was not reduced in mutant protonema or in gametophores. As the gametangia development of the PpAOC1 and PpAOC2 mutants is comparable to WT we assume that the fertilization process itself is hampered leading to the reduced amounts of spore capsules in the mutants. As P. patens is a monoecious moss (Reski, 1998), we were unable to assess if this was caused by specific problems in the male or in the female part of the fertilization process.
The mutant phenotypes observed here differ from those obtained after knockout of another nuclear-encoded but plastid-localized metabolic enzyme, sulphite reductase (Wiedemann et al., 2010), arguing in favour of a specific requirement of AOCs in moss fertilization and sporophyte development. The phenotypes of the knockout mosses described here, suggests that a role of oxylipins in reproductive development of plants is evolutionary conserved but is specified differentially in different branches of the plant kingdom, as has previously found for auxin (Ludwig-Müller et al., 2009; Paponov et al., 2009; Sun et al., 2010) and for gibberellin signalling (Vandenbussche et al., 2007).