Institut de Biologie Moléculaire des Plantes, Unité Propre de Recherche 2357 CNRS, Université de Strasbourg, 12 rue du Général Zimmer, 67084 Strasbourg Cedex, France.
J Biol Chem. 2012 Feb 24;287(9):6296-306. doi: 10.1074/jbc.M111.316364. Epub 2012 Jan 3.
The jasmonate hormonal pathway regulates important defensive and developmental processes in plants. Jasmonoyl-isoleucine (JA-Ile) has been identified as a specific ligand binding the COI1-JAZ co-receptor to relieve repression of jasmonate responses. Two JA-Ile derivatives, 12OH-JA-Ile and 12COOH-JA-Ile, accumulate in wounded Arabidopsis leaves in a COI1- and JAR1-dependent manner and reflect catabolic turnover of the hormone. Here we report the biochemical and genetic characterization of two wound-inducible cytochromes P450, CYP94C1 and CYP94B3, that are involved in JA-Ile oxidation. Both enzymes expressed in yeast catalyze two successive oxidation steps of JA-Ile with distinct characteristics. CYP94B3 performed efficiently the initial hydroxylation of JA-Ile to 12OH-JA-Ile, with little conversion to 12COOH-JA-Ile, whereas CYP94C1 catalyzed preferentially carboxy-derivative formation. Metabolic analysis of loss- and gain-of-function plant lines were consistent with in vitro enzymatic properties. cyp94b3 mutants were largely impaired in 12OH-JA-Ile levels upon wounding and to a lesser extent in 12COOH-JA-Ile levels. In contrast, cyp94c1 plants showed wild-type 12OH-JA-Ile accumulation but lost about 60% 12COOH-JA-Ile. cyp94b3cyp94c1 double mutants hyperaccumulated JA-Ile with near abolition of 12COOH-JA-Ile. Distinct JA-Ile oxidation patterns in different plant genotypes were correlated with specific JA-responsive transcript profiles, indicating that JA-Ile oxidation status affects signaling. Interestingly, exaggerated JA-Ile levels were associated with JAZ repressor hyperinduction but did not enhance durably defense gene induction, revealing a novel negative feedback signaling loop. Finally, interfering with CYP94 gene expression affected root growth sensitivity to exogenous jasmonic acid. These results identify CYP94B3/C1-mediated oxidation as a major catabolic route for turning over the JA-Ile hormone.
茉莉酸激素途径调节植物中重要的防御和发育过程。已鉴定出茉莉酰异亮氨酸 (JA-Ile) 作为一种特定的配体，与 COI1-JAZ 共受体结合以解除茉莉酸反应的抑制。两种 JA-Ile 衍生物，12OH-JA-Ile 和 12COOH-JA-Ile，以 COI1 和 JAR1 依赖的方式在受伤的拟南芥叶片中积累，并反映了激素的分解代谢周转。在这里，我们报告了两个与伤口诱导的细胞色素 P450（CYP94C1 和 CYP94B3）相关的生化和遗传特征，这些细胞色素 P450 参与 JA-Ile 氧化。在酵母中表达的两种酶都催化 JA-Ile 的两个连续氧化步骤，具有不同的特征。CYP94B3 有效地进行 JA-Ile 的初始羟化作用生成 12OH-JA-Ile，很少转化为 12COOH-JA-Ile，而 CYP94C1 则优先催化羧基衍生物的形成。对丧失和获得功能的植物系进行的代谢分析与体外酶学特性一致。cyp94b3 突变体在受伤后 JA-Ile 水平大大降低，12COOH-JA-Ile 水平降低较少。相比之下，cyp94c1 植物表现出野生型 12OH-JA-Ile 积累，但失去了约 60%的 12COOH-JA-Ile。cyp94b3cyp94c1 双突变体 JA-Ile 超积累，几乎消除了 12COOH-JA-Ile。不同植物基因型中不同的 JA-Ile 氧化模式与特定的 JA 反应转录谱相关，表明 JA-Ile 氧化状态会影响信号转导。有趣的是，JA-Ile 水平的夸大与 JAZ 抑制剂的过度诱导有关，但不会持久地诱导防御基因的诱导，揭示了一种新的负反馈信号环。最后，干扰 CYP94 基因表达会影响根生长对外源茉莉酸的敏感性。这些结果表明，CYP94B3/C1 介导的氧化是 JA-Ile 激素代谢的主要分解途径。
