低温光照胁迫对红芸豆类囊体膜脂代谢及光系统结构与功能的影响

Chilling stress combined with light intensities result in reduction in crop production and become an important factor to limit the yield and quality of vegetables. To study the changes of thylakoid membrane fatty acid composition and the structure and function of photosystem protein complexes under chilling with different illumination stress is helpful to understand the plant adaptation mechanism to adverse environment. The leaves of red kidney bean (Phaseolus vulgaris) will be as experimental materials to determine the changes of antioxidant system and ultrastructure of chloroplast,also fatty acid composition changes in thylakoid membrane. Spectroscopy will be used to analyze the changes of pigments and proteins conformation of thylakoid membrane. The structural and functional changes of PSII and PSI protein complex will be studied by Blue native gel, SDS-PAGE and Western blot and clark electrode to investigate the extent of photoinhibition of PSII and PSI, including the protein content changes of LHCII,CP43,CP47,LHCI, D1, PasA/B combined with photosynthesis electron transport activity and chlorophyll fluorescence. The correlation between membrane fatty acids composition with PSII photoinhibition, the interaction between PSII and PSI during chilling-light stress and the course of recovery will be also examined. The study aims to elucidate the key factors of chilling with illumination stress resistance and provide theoretical basis for agricultural production and vegetable cultivation.

低温和光照协同会导致作物减产，成为限制蔬菜产量和质量的重要因素。研究低温光照下类囊体膜脂防酸组成与光系统复合物结构和功能的变化，有助于深入理解植物对低温光逆境的适应机理。本项目以红芸豆叶片为材料，对低温不同光强处理下叶片抗氧化系统、叶绿体超微结构、类囊体膜脂组分变化进行分析；采用波谱学技术测定类囊体膜色素和蛋白构象变化；蓝绿温和胶电泳结合SDS-PAGE和Western blot技术研究PSII和PSI色素蛋白复合体结构及捕光天线蛋白LHCII,CP43,CP47,LHCI和反应中心蛋白D1,PasA/B含量变化，结合电子传递活性和叶绿素荧光参数变化探索PSII和PSI光抑制程度。阐明低温光照胁迫下类囊体膜脂组分变化与PSII光抑制的相关性以及光抑制恢复过程中PSII和PSI之间的相互作用，揭示植物抗低温光逆境的关键因素，为作物生产和蔬菜高产栽培提供理论依据。

项目研究了低温光胁迫红芸豆叶片解剖特征、活性氧代谢、光合气体交换、叶绿素荧光、类囊体膜脂肪酸、色素吸光过程、类囊体膜蛋白复合物结构和功能变化以及MAPK基因家族成员鉴定及对胁迫的响应。结果表明：低温光胁迫导致ROS过量积累，SOD、APX活性受低温影响严重，POD、CAT和AsA协同抗氧化。随胁迫时间延长，Chla、Chlb和Chl (a+b)的含量降低，Chla/Chlb 升高；Pn、Gs和Tr降低，Ci升高，气孔开张比降低，数目增多。气孔因素和非气孔因素是低温光胁迫下光合速率下降原因。随胁迫时间延长，Fv/Fm、Fv'/Fm'、qP、ФPSII和ETR降低，NPQ升高。类囊体膜MGDG、DGDG和SQDG中的亚麻酸（C18：3）含量显著降低，棕榈酸(C16:0)含量显著升高，PG中的棕榈酸 (C16:0)和反式十六碳-烯酸[C16:1（3t）]含量降低，亚麻酸（C18：3）和亚油酸（C18：2）含量升高。类囊体膜总饱和脂肪酸(SFA)含量升高，多不饱和脂肪酸（PUFA）含量以及膜脂不饱和度（U/S）显著降低。PUFA含量与Fv/Fm的相关性达86.2 %，较高含量的PUFA增加了类囊体膜稳定性，减轻光抑制。低温弱光胁迫下离体类囊体膜对红光区吸收增加，而低温高光下对红和蓝光区的吸收先略有增加后降低。PSII超级复合体逐渐解聚，PSII双体降解，单体增加，LHCII变化不明显，PSI复合体发生部分降解。随胁迫延长，PSII和PSI复合体蛋白亚基降解，D1、PsaA/B，CP43、CP47和LHCII蛋白含量降低。低温光胁迫下总链、全链、PSII和PSI电子传递活性降低，PSI降低程度更显著。PSI是低温光抑制的首要位点，PSI光抑制会限制PSII受体侧的电子传递，加重PSII光抑制。PSI的低温光抗性是决定植物对低温光逆境抗性高低的关键因素之一。在红芸豆全基因组范围鉴定到PvMAPKs基因家族成员22个，qRT-PCR分析筛选出3个响应低温光胁迫差异显著基因。红芸豆通过多个调节机制来响应低温光照胁迫，如启动抗氧化反应；增加类囊体膜中多不饱和脂肪酸含量；调节吸收红和蓝光区的色素分子参与光能吸收；增加热耗散淬灭激发能；增加LHCII中Chla分子间吸和传能以减少过多光能对反应中心损伤。研究结果为蔬菜抗低温光照胁迫种质筛选培育和高效栽培提供理论依据。