Studies on signal transduction pathways of jasmonic acid in suspension-cultured rice cells

在这个项目中，我们试图分离和鉴定茉莉酸(JA)反应基因，以阐明茉莉酸在悬浮培养水稻细胞中的信号转导途径。此外，还合成了茉莉酸受体的分子探针。茉莉酸反应基因RRJ1和RRJ2分别编码水稻半胱氨酸γ裂解酶和丙酮酸脱羧酶。RRJ1可能参与含硫氨基酸的生物合成和代谢及相关的生化过程，RRJ2可能参与激发子诱导水稻细胞防御反应的能量代谢。我们成功地克隆了JA应答基因OsOPR1，它编码12-氧植物二烯酸还原酶(OPR)，是JA生物合成的关键酶之一。这是首次从单子叶植物中克隆OPR基因。由于OsOPR1优先催化(-)-顺式-OPDA的还原，而不是()-顺式-OPDA，因此作为JA的天然前体，OsOPR1可能参与了其他生理性…RERJ1是一个早期的茉莉酸反应基因，它编码一个具有基本区域/螺旋-环-螺旋基序的转录调控因子。通过对过量表达正、反义RERJ1基因水稻植株的表型分析，推测RERJ1基因可能参与了茉莉酸对水稻幼苗生长的抑制作用。RERJ1也可能参与包括防御反应在内的其他生理现象。对RERJ1的生物学功能的进一步分析正在进行中。JA在几丁质激发子诱导悬浮培养的水稻细胞产生二萜类植物保卫素的过程中发挥着关键的信号转导作用。我们从甲壳素诱导的悬浮培养水稻细胞中分离到一个编码二萜环化酶的基因，并证明该基因产物具有Et-Cassa-12，15-二烯合成酶的功能。这是首次分离到一个编码二萜环化酶的基因，该酶参与二萜碳氢化合物的生物合成，该二萜碳氢化合物可能是水稻中二萜类植物保卫素的前体。本文所克隆的Et-Cassa-12，15-二烯合成酶基因将是研究水稻二萜类植物抗毒素(-)-植物决明素生物合成调控机制的有用工具。我们合成了几种类型的JA衍生物，包括JA-氨基酸结合物、JA-生物素结合物、JA-地塞米松异二聚体和JA-荧光素结合物，作为JA结合蛋白的分子探针。由于这些化合物除JA-荧光素结合物外，在水稻幼苗实验、大豆PAL诱导实验和/或水稻木霉内酯A诱导实验中都表现出明显的JA活性，因此它们可能是分离和鉴定JA结合蛋白的有用探针。较少

In this project, we have tried to isolate and characterize jasmonic acid (JA)-responsive genes to elucidate the signal transduction pathways of JA in suspension-cultured rice cells. In addition, molecular probes for JA receptors were also synthesized.The JA-responsive genes RRJ1 and RRJ2 were found to encode a putative rice cystathionine γ-lyase and a pyruvate decarboxylase, respectively. RRJ1 is possibly involved in biosynthesis and/or metabolism of sulfur-containing amino acids and related biochemical processes, and RRJ2, in energy metabolism for elicitor-induced defense reactions in suspension-cultured rice cells.We succeeded in cloning of the JA-responsive gene OsOPR1 encoding 12-oxophytodienoic acid (OPDA) reductase (OPR) that is one of key biosynthetic enzymes of JA. This is the first cloning of an OPR gene from a monocot plant. Since OsOPR1 catalyze reduction of (-)-cis-OPDA preferentially over (+)-cis-OPDA, a natural precursor of JA, OsOPR1 might contribute to other physiologic … More al phenomena than JA biosynthesis.RERJ1 was isolated as an early JA-responsive gene which encodes a transcriptional regulator possessing a basic region/helix-loop-helix motif. Based on analysis of phenotypes of sense- or antisense-RERJ1 mRNA overexpressing transgenic rice plants, RERJ1 is suggested to be involved in JA-induced inhibition of shoot growth in rice. Involvement of RERJ1 in other physiological phenomena including defense response is also quite possible. Futher analysis of biological functions of RERJ1 is in progress.JA functions as a key signal transducer in a chitin elicitor-induced diterpenoid phytoalexin production in suspension-cultured rice cells. We isolated a cDNA encoding a diterpene cyclase from the chitin-elicited suspension-cultured rice cells, and demonstrated that the gene product functioned as ent-cassa-12, 15-diene synthase. This is the first report of isolation of a cDNA encoding a diterpene cyclase involved in biosynthesis of a diterpene hydrocarbon which is a putative precursor of diterpenoid phytoalexins in rice. ent-Cassa-12, 15-diene synthase cDNA isolated here will be a useful tool to investigate the regulatory mechanisms of biosynthesis of the rice diterpenoid phytoalexins (-)-phytocassanes during plant development and by change of environmental conditions.We synthesized several types of JA derivatives including JA-amino acid conjugates, JA-biotin conjugate, JA-dexamethasone heterodimer, and JA-fluoresceine conjugate as candidates for molecular probes for JA-binding proteins. Since these synthesized compounds except JA-fluoresceine conjugate exhibited significant JA-like activities in rice seedling assay, soybean PAL-inducing assay, and/or rice momilactone A-inducing assay, they might be useful probes to isolate and characterize JA-binding proteins. Less