拟南芥13-脂氧合酶调控气孔运动的作用机制

13-Lipoxgenases (13-LOX) catalyzes the first step of lipid oxidation leading to the production of oxylipins such as Jasmonic Acid (JA). Oxylipins are ubiquitous signal molecules involved in many biological processes. Our previous experiment indicated that 13-LOXs might be involved in stomatal movement regulation. There are four genes encoding 13-LOXs in Arabidopsis genome, two of which, LOX2/6, are expressed in guard cells. Moreover, LOX2 is related to JA and NO in the regulation of stomatal movement. In the project, biological technologies, such as Confocal and patch clamp, will be employed to study the functions of LOX2/6 in stomatal movement and the interacting relationship between LOX2/6 and Ca2+. Furthermore, the changes in plasma membrane Ca2+, K+ and Cl- currents in guard cells will be monitored to determine the channel types regulated by LOX2/6 in Arabidopsis. We will construct the Arabidopsis transgenic lines overexpressing genes LOX2/6 in double mutant background of COI1 and JAZ respectively, which are JA receptor and a negative factor in JA signaling. We will also measure the differences in response to JA and NO treatments among the mutants and the transgenic lines. Meanwhile, some key responsive genes in stomatal movement regulated by LOX2/6 will be identified by RNA-Seq and translatomes. All together, some essential components acting in signal transduction in Arabidopsis guard cells will be determined. The mechanisms by which LOX2/6 integrates multiple signals and regulates stomatal movement will be eventually elucidated.

13-脂氧合酶（13-LOX）催化茉莉酸（JA）等氧脂素合成的第一步反应，氧脂素作为信号分子广泛参与了诸多生物学过程。预研表明，13-LOX参与调控气孔运动，拟南芥四个13-LOX基因中LOX2/6在保卫细胞中表达，LOX2在气孔运动调节过程中与JA和一氧化氮（NO）相关。本项目拟运用Confocal和膜片钳等技术，研究LOX2/6与气孔运动及胞内Ca2+的作用关系；检测保卫细胞Ca2+、K+和Cl-通道电流变化，确定LOX2/6调控的通道类型。创制LOX2/6在茉莉酸受体COI1和JA信号转导负调节因子JAZ双突变体中过量表达的转基因材料，检测双突变体和转基因植物保卫细胞对JA、NO等处理的应答情况。同时利用RNA测序和翻译组学技术鉴定LOX2/6调节气孔运动的关键应答基因。确定LOX2/6调控的气孔保卫细胞信号转导组分，阐明LOX2/6通过整合多种信号分子调节气孔运动的机制。

13-脂氧合酶（13-LOX）催化茉莉酸（JA）等氧脂素合成的第一步反应，氧脂素作为信号分子广泛参与了诸多生物学过程。本项目以在保卫细胞表达的LOX2、LOX6基因为研究对象，对其功能缺失后突变体对不同刺激的响应及可能的作用机制开展研究。结果发现：LOX2、LOX6功能缺失后，突变体离体叶片失水较快，气孔开度较大，对外源ABA诱导的气孔关闭反应正常，但对外源SNP处理不敏感。共聚焦显微镜观察发现：SNP诱导的保卫细胞[Ca2+]cyt升高在突变体中被削弱。膜片钳技术分析发现：SNP对保卫细胞质膜内向K+通道活性的抑制效应被削弱。此外，我们发现：在光诱导气孔开放过程中，lox6-1突变体气孔开放得更快，并且这一现象是蓝光特异的。外源添加OPDA可以有效回补到野生型水平，而MJ则不能。表明LOX6可能作为负调节因子抑制光诱导的气孔开放过程。以lox6-1为代表，通过RNA seq比较分析了突变体和野生型材料保卫细胞在光照和黑暗条件下基因转录表达，构建了光暗转换前后差异表达基因的共表达模式。结合已有研究，选取包括OPDA高亲和结合蛋白编码基因在内的多个基因功能缺失突变体，通过气孔bioassay等实验初步鉴定到数个OPDA下游响应因子。该项目揭示了新的气孔保卫细胞运动调控途径，为揭示保卫细胞运动调控机制和培育抗旱节水作物提供了新的理论基础。