活性氧调控乙烯生成及线粒体功能活性的桂花衰老机制解析

Sweet Osmanthus (Osmanthus fragrans Lour.) is one of the top ten traditional flowers in China and loved by Chinese people due to the unique fragrance. However, the lifespan of individual flower is generally short, limiting both the ornamental and economic value. It has been reported that the petal senescence of ornamental flowers is related to the pollination-induced ethylene production. Whereas, our preliminary research has found that many fruitless cultivars of O. fragrans are also sensitive to ethylene, and the petal senescence is regulated by the non-pollination-induced endogenous ethylene production. However, the mechanism of ethylene biosynthesis during this process has not been clear. It has been found that reactive oxygen species (ROS) is an important signal factor in programmed cell death (PCD) that participated by mitochondria, and there is a feedback regulation between ROS and ethylene. Therefore, attached and detached flowers of O. fragrans, treated by free radicals and its specific scavenger superoxide dismutase, distilled water, ethylene and its inhibitor silver thiosulfate would be sampled, to investigate the influence of ROS on ethylene production which is measured by the content of 1-amino-cyclopropane carboxylic acid (ACC), activities and transcriptional changes of ACC synthase and oxidase; and on regulation of mitochondria activities which is measured by the mitochondrial ultra structure, membrane potential, activities of respiratory enzymes and release of cytochrome c. This research is aim to explore the regulatory mechanisms of ROS in PCD that participated by ethylene production and mitochondrial activity. Our research would offer new theoretical basis for postponing the senescence of woody flowers.

桂花(Osmanthus fragrans L.)是我国十大传统名花之一，花香甜润久远，深受人们喜爱。但因寿命短，闻香赏花和经济采收期受到限制。观赏植物花瓣衰老常与授粉诱导的乙烯跃变有关，而课题组研究发现许多不结实桂花品种也对乙烯敏感，非授粉诱导的内源乙烯跃变是其衰老的重要调控因子。然而，乙烯在该过程中的合成途径尚不清楚。研究表明，活性氧是线粒体途径细胞程序性死亡（PCD）的重要信号因子，且与乙烯之间存在反馈调节。本课题拟通过不同浓度超氧自由基及其清除剂、蒸馏水、乙烯及其抑制剂处理离体桂花，检测乙烯合成中间产物AAC含量、限速酶ACS和ACO活性及转录水平变化，以及反应线粒体功能活性的线粒体超微结构、膜电势、电子传递链呼吸酶、细胞色素c释放等指标的变化规律，探讨桂花花瓣衰老过程中活性氧介导的乙烯生成和线粒体功能失调的PCD发生和调控机制。该研究旨在为木本花卉衰老机理研究提供新的理论依据。

以“柳叶金桂”（Osmantuhs fragrans ‘Liuye Jingui’）为实验材料，使用蒸馏水（CK）、乙烯利（ETH）、乙烯作用抑制剂硫代硫酸银（STS）、维生素C（VC）、硫代硫酸钠（Na2S2O3）进行盘插处理，通过测定不同处理下的盘插寿命、珀酸脱氢酶（SDH）活性、细胞色素氧化酶（COX）活性、超氧阴离子自由基（O2-•）含量、氧化氢（H2O2）含量，从线粒体释放到细胞质中细胞色素c的含量、线粒体膜电位的变化，探讨乙烯对桂花衰老过程中线粒体活性的影响；通过测定ACO和ACS基因相对表达量变化、ACO和ACS酶活性的变化，探讨不同处理对乙烯合成的影响。结果表明，经过乙烯利（ETH）处理的样品盘插寿命缩短，SDH活性处于较低水平，COX活性降低时的降幅最大，超氧阴离子自由基和H2O2的含量增幅最大。与蒸馏水相比，其他四种溶液处理下桂花花瓣细胞色素c前两天的释放均显著提高；而第三天样品中乙烯利处理下细胞色素c释放显著降低，可能与此时花瓣中大分子物质蛋白质降解以及线粒体功能活性丧失有关。与蒸馏水相比，乙烯利、维生素C和硫代硫酸钠处理均不同程度提高了ACO基因的表达丰度，硫代硫酸银溶液处理则显著降低了其表达丰度；而对于ACS基因来说，乙烯利和维生素C均显著提高了ACS基基因的表达丰度，硫代硫酸银溶液和硫代硫酸钠处理则显著降低了ACS基因的表达丰度。由此推测，维生素C溶液虽然可以延缓桂花花瓣可见的衰老，但其延缓机制可能并非抑制内源乙烯合成。与蒸馏水相比，乙烯利和硫代硫酸钠处理不同天数的样品中检测到的荧光强度显著提高，而在硫代硫酸银和维生素C溶液处理下则显著降低。由此可以推测，桂花花瓣衰老过程中乙烯可能不断加剧呼吸速率，产生过量的活性氧自由基，破坏膜脂结构，使线粒体膜的完整性受到破坏，膜透性增大、离子泄漏，导致细胞的代谢紊乱，活性氧自由基进一步在机体内积累，并继续破坏线粒体的结构和功能，形成恶性循环，最终导致细胞死亡。