高温诱导黄瓜叶片叶绿素酶促降解的分子机制

Chlorophyll (Chl) is the crucial pigment molecule in light-harvesting and photosynthesis-performing, which plays an extremely important role in the growth and production of plants. However, Chl contents in plants leaves was decreased by heat stress, as a result of growth inhibition and yield loss. Our previous results have proved that the transcriptional regulation was the primary way for degradation of Chl in cucumber (Cucumis sativus L.) seedling leaves at heat stress, but the detailed molecular mechanisms still remain unknown largely. The key genes function in the pathway of Chl enzymatic degradation in cucumber seedling leaves was analyzed by the physiological and biochemical methods and genetic transformation under heat stress, and the full-length promoter of key sites genes was cloned, the functional cis-elements within promoter sequences responded to heat stress was identified by the 5’ sequence deletion analysis. Moreover, the potential up-stream transcription factors interacted between cis-elements was chosen and identified by the Yeast one-hybrid assays. Finally, regulation of transcription factors to the key genes in Chl degradation was analyzed, and the possible molecular pathway of Chl enzymatic degradation in cucumber seedling leaves under heat stress was constructed, and its molecular mechanisms was elucidated. This work would provide a theoretical basis for cucumber cultivation to heat stress.

叶绿素（Chl）作为植物捕获光能、行使光合作用的关键色素分子，对植物生长及产量形成发挥着极其重要的作用。高温胁迫下，植物Chl含量降低，严重影响了植物生长发育和产量形成。前期研究结果证实，转录调控是高温下黄瓜（Cucumis sativus L.）叶片叶绿素酶促降解调控的主要方式，但其分子机制尚不清楚。本研究从Chl酶促降解途径入手，通过生理生化分析和遗传转化，揭示高温诱导黄瓜叶片叶绿素酶促降解的关键作用位点基因的功能；克隆关键位点基因启动子全长，通过5′端序列缺失方法，鉴定该启动子序列中响应高温的功能作用元件；运用酵母单杂交技术筛选鉴定与该功能作用元件互作的上游转录因子，解析转录因子对叶绿素降解关键作用位点基因的调控作用，构建高温诱导黄瓜叶绿素酶促降解的分子路径，从转录调控的角度阐明其分子机制，为黄瓜抗热栽培提供理论依据。

高温是制约植物生长发育的主要非生物胁迫之一。高温导致黄瓜（Cucumis sativus L.）幼苗生长受到严重抑制，生理代谢紊乱，叶绿素降解，光合速率降低。本研究从Chl酶促降解途径入手，构建高温诱导黄瓜叶片叶绿素酶促降解的分子路径，从转录调控的角度阐明其分子机制。主要结果如下：. 1. 高温抑制黄瓜植株生长，叶绿体结构被破坏，叶绿素降解加速，叶绿素降解酶基因CsPPH、CsPAO、CsHCAR显著上调，PPH、PAO酶活显著升高，表明高温诱导黄瓜叶片叶绿素降解，且CsPPH、CsPAO、CsHCAR在高温诱导的叶绿素酶促降解中起重要作用。. 2. CsHCAR在衰老叶片中表达量最高且响应高温、干旱、黑暗逆境和GA3、Me-JA、ABA、SA激素诱导。黑暗条件下，过表达CsHCAR烟草株系的生物量、叶绿素含量、Fv/Fm、LHCII捕光天线蛋白含量和叶片活性氧（ROS）积累量均显著降低，表明CsHCAR影响叶绿体光合蛋白的稳定性，正向调控植株衰老，并在维持叶绿体ROS平衡中发挥重要作用。. 3. ABI5在高温诱导的叶绿素降解中起核心调控作用。一方面，ABI5通过直接上调CsPPH、CsPAO的表达促进叶绿素降解；另一方面，ABI5通过与MYB44相互作用，抑制其转录活性，干扰其与CsPPH、CsPAO基因启动子的结合，减轻了MYB44对CsPPH、CsPAO基因的抑制作用，导致叶绿素降解速率加快。. 总之，本研究发现CsHCAR通过影响黄瓜叶绿体光合蛋白的稳定性正向调控叶片衰老，阐明了高温胁迫下CsMYB44、CsABI5通过调控黄瓜CsPPH、CsPAO促进叶绿素降解的分子机制，构建了高温诱导黄瓜叶绿素酶促降解的分子模型，为黄瓜抗热栽培提供了理论依据。