拟南芥胞外超氧化物歧化酶（MnSOD2）调控花器官脱落的分子机制

Floral organ abscission is a key performance of plant senescence and response to environmental factors which take place at abscission zone. Previous study showed the lignification of secession cells is essential for abscission. The lignification occurs outside the cytoplasm and the H2O2 is necessary for such process. H2O2 is produced by the O2- catalyzed by extracellular SOD. However, there is no evidence to show the existence of extracellular SOD in plant. The pilot experiments showed that AtMSD2 is located in the apoplast and the MSD2 knock out mutant showed abscission delay. Here we will study the function of AtMSD2 in the process of floral organ abscission. On the one hand, the RP-HPLC and some transferred marker proteins will be used for supply some evidence of AtMSD2 is an apoplast protein. On the other hand, fluorescence staining, in vitro protein expression, qRT-PCR, and RNA-seq will be used for the detection of ROS and lignin accumulation in the abscission zone, the position of AtMSD2 in the abscission signal pathway. And finally, our results will show the function of AtMSD2 in the abscission zone. Our final goal is to supply some useful information for the model plant research and agricultural production.

花器官脱落是植物衰老及响应环境因素的重要表现。离开母体的器官离层细胞木质化是导致脱落的关键原因。木质化发生需源于胞外SOD催化生成的H2O2参与。是何种SOD在胞外行使此功能并影响木质素积累暂无定论。前期研究发现，拟南芥MnSOD2(AtMSD2)定位在离层细胞质外且msd2敲除突变体花脱落推迟。本项目以拟南芥AtMSD2为研究对象，对AtMSD2编码胞外SOD并参与花器官脱落开展研究：通过转荧光蛋白、RP-HPLC/MS等技术，从细胞和蛋白水平确定AtMSD2编码胞外SOD，为木质化发生中ROS来源寻找证据。通过化学染色、qRT-PCR、RNA-seq等技术，研究AtMSD2参与离区ROS产生、积累及木质化过程，并探究脱落信号通路中AtMSD2的调控分子。本项目将从AtMSD2编码胞外SOD影响离区木质化发生的角度，进一步完善植物花器官脱落发生的分子机制，为该领域研究提供一定实验依据。

花器官脱落是植物衰老及响应环境因素的重要表现，花器官脱落异常严重影响植物正常生长发育及生物量变化。花器官脱落的发生依赖脱落器官离层细胞的木质化发生。细胞壁木质化过程依赖质外体SOD催化来源于RBOHs的O2-生成H2O2。目前，究其是何种SOD在胞外行使此功能并影响木质素积累暂无定论。我们前期研究发现，拟南芥MnSOD2(AtMSD2)定位在离层细胞质外且MSD2缺失突变体花脱落异常。针对该问题，项目组从拟南芥胞外SOD的鉴定及其调控花器官脱落的机制两方面开展研究。利用氨基酸序列分析发现MSD2是不同于已发现的植物SOD；通过非变性凝胶电泳、Pull-down实验、蛋白免疫印迹、药理学实验发现MSD2具有SOD活性，与MSD1活性类似，其活性不受KCN和H2O2的抑制；通过蛋白高级构象比对，发现MSD2的活性中心与MSD1类似，MSD1Y63与MSD2Y68在空间上重合。蛋白硝基化实验证实MSD2Y68硝基化影响了MSD2的活性；构建MSD2-mcherry和MSD2△SP-mCherry载体获得转基因植物，以叶表皮和根为材料，进行亚细胞定位检测及质壁分离实验证实包含有信号肽序列的MSD2定位在细胞质外体；启动子活性分析发现，MSD2在花器官离区特异性表达；转录组测序分析、药理学实验、组织化学染色、突变体杂交等实验发现，ABA和NO影响了MSD2活性进而影响了IDA-HAE信号通路，最终调节了拟南芥花器官脱落的发生。本项目首次在植物中证实MSD2是植物质外体SOD，并通过影响IDA-HAE信号通路调节了拟南芥花器官脱落发生过程。项目的完成为人们研究质外体ROS产生提供了重要实验依据，也为进一步完善植物花器官脱落发生的分子机制提供了新的研究思路。

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