12-Oxophytodienoate reductases (OPRs) belong to a family of flavin-dependent oxidoreductases. With two new tomato isoforms reported here, three OPRs have now been characterized in both tomato and Arabidopsis. Only one of these isoforms (OPR3) participates directly in the octadecanoid pathway for jasmonic acid biosynthesis, as only OPR3 reduces the 9S,13S-stereoisomer of 12-oxophytodienoic acid, the biological precursor of jasmonic acid. The subcellular localization of OPRs was analyzed in tomato and Arabidopsis. The OPR3 protein and activity were consistently found in peroxisomes where they co-localize with the enzymes of beta-oxidation which catalyze the final steps in the formation of jasmonic acid. The octadecanoid pathway is thus confined to plastids and peroxisomes and, in contrast to previous assumptions, does not involve the cytosolic compartment. The expression of tomato (Lycopersicon esculentum,Le) OPR3 was analyzed in the context of defense-related genes using a microarray comprising 233 cDNA probes. LeOPR3 was found to be up-regulated after wounding with induction kinetics resembling those of other octadecanoid pathway enzymes. In contrast to the induction of genes for wound response proteins (e.g. proteinase inhibitors), the accumulation of octadecanoid pathway transcripts was found to be more rapid and transient in wounded leaves, but hardly detectable in unwounded, systemic leaves. Consistent with the expression data, OPDA and JA were found to accumulate locally but not systemically in the leaves of wounded tomato plants. The transcriptional activation of the octadecanoid pathway and the accumulation of JA to high levels are, thus not required for the activation of defense gene expression in systemic tissues.

12-Oxophytodienoate reductase (OPR) 3, a homologue of old yellow enzyme (OYE), catalyzes the reduction of 9S,13S-12-oxophytodienoate to the corresponding cyclopentanone, which is subsequently converted to the plant hormone jasmonic acid (JA). JA and JA derivatives, as well as 12-oxophytodienoate and related cyclopentenones, are known to regulate gene expression in plant development and defense. Together with other oxygenated fatty acid derivatives, they form the oxylipin signature in plants, which resembles the pool of prostaglandins in animals. Here, we report the crystal structure of OPR3 from tomato and of two OPR3 mutants. Although the catalytic residues of OPR3 and related OYEs are highly conserved, several characteristic differences can be discerned in the substrate-binding regions, explaining the remarkable substrate stereoselectivity of OPR isozymes. Interestingly, OPR3 crystallized as an extraordinary self-inhibited dimer. Mutagenesis studies and biochemical analysis confirmed a weak dimerization of OPR3 in vitro, which correlated with a loss of enzymatic activity. Based on structural data of OPR3, a putative mechanism for a strong and reversible dimerization of OPR3 in vivo that involves phosphorylation of OPR3 is suggested. This mechanism could contribute to the shaping of the oxylipin signature, which is critical for fine-tuning gene expression in plants.

OPDA likely plays signaling roles in plant defense that do not depend on its further conversion to the phytohormone jasmonic acid. To elucidate the role of OPDA in tomato plant defense, we have silenced the 12-oxophytodienoate reductase 3 (OPR3) gene. Two independent transgenic tomato lines (SiOPR3-1 and SiOPR3-2) showed significantly reduced OPR3 expression upon infection with the necrotrophic pathogen Botrytis cinerea. Moreover, SiOPR3 plants are more susceptible to this pathogen, and this susceptibility is accompanied by a significant decrease in the OPDA levels and an almost abolished JA-Ile production. OPR3 silencing also leads to a major reduction in the expression of other genes of the jasmonic acid (JA) synthesis and signaling pathways after infection. These results confirm that in tomato plants, as in Arabidopsis, OPR3 determines OPDA availability for JA biosynthesis. In addition, we show that an intact JA biosynthetic pathway is required for proper callose deposition since its pathogen-induced accumulation is reduced in SiOPR3 plants. Interestingly, OPDA, but not JA, treatment restored basal resistance to B. cinerea and induced callose deposition in SiOPR3-1 and SiOPR3-2 transgenic plants. These results provide clear evidence that OPDA by itself plays a major role in basal defense of tomato plants against this necrotrophic pathogen. This article is protected by copyright. All rights reserved.