Biosynthesis of the phytohormone jasmonoyl-isoleucine (JA-Ile) requires reduction of the JA precursor 12-oxo-phytodienoic acid (OPDA) by OPDA reductase 3 (OPR3). Previous analyses of the opr3-1 Arabidopsis mutant suggested an OPDA signaling role independent of JA-Ile and its receptor COI1; however, this hypothesis has been challenged because opr3-1 is a conditional allele not completely impaired in JA-Ile biosynthesis. To clarify the role of OPR3 and OPDA in JA-independent defenses, we isolated and characterized a loss-of-function opr3-3 allele. Strikingly, opr3-3 plants remained resistant to necrotrophic pathogens and insect feeding, and activated COI1-dependent JA-mediated gene expression. Analysis of OPDA derivatives identified 4,5-didehydro-JA in wounded wild-type and opr3-3 plants. OPR2 was found to reduce 4,5-didehydro-JA to JA, explaining the accumulation of JA-Ile and activation of JA-Ile-responses in opr3-3 mutants. Our results demonstrate that in the absence of OPR3, OPDA enters the β-oxidation pathway to produce 4,5-ddh-JA as a direct precursor of JA and JA-Ile, thus identifying an OPR3-independent pathway for JA biosynthesis.

植物激素茉莉基异亮氨酸（JA-Ile）的生物合成需要通过OPDA还原酶3（OPR3）还原JA前体12-氧代-植物二烯酸（OPDA）。先前对opr3-1拟南芥突变体的分析表明，OPDA信号传导作用独立于JA-Ile及其受体COI1。然而，该假设受到质疑，因为opr3-1是JA-Ile生物合成中未完全受损的条件等位基因。为了阐明OPR3和OPDA在不依赖JA的防御中的作用，我们分离并鉴定了功能丧失的opr3-3等位基因。令人惊讶的是，opr3-3植物对坏死性病原体和昆虫摄食仍然具有抗性，并激活了依赖COI1的JA介导的基因表达。OPDA衍生物的分析确定了受伤的野生型和opr3-3植物中的4,5-didehydro-JA 。发现OPR2将4,5-didehydro-JA还原为JA，解释了opr3-3突变体中JA-Ile的积累和JA-Ile反应的激活。我们的结果表明，在不存在OPR3的情况下，OPDA进入β-氧化途径，产生4,5-ddh-JA作为JA和JA-Ile的直接前体，从而确定了JA生物合成的不依赖OPR3的途径。