小麦抗条锈过程中叶片黄化机制的研究

Wheat is one of the most important staples for human being. Wheat production, however, is seriously affected by fungal pathogens, e.g. strip rust, which leads to million tons of yield loss every year. The WHEAT KINASE START1 (WKS1, Yr36) gene confers a high-temperature adult-plant resistance to strip rust, and leads to the accumulation of hydrogen peroxide and an accelerated yellowing process. Hydrogen peroxide was regarded as an inducer of cell death and chlorosis but it was also predicted to be insufficient to confer all the changes by some scientists. Therefore it is still a mystery that whether there are other regulatory mechanisms underlying chlorosis. To reveal possible key elements in wheat chlorosis process, the applicant isolated the WKS1 protein complex from wheat and detected PsbO, an extrinsic component to stabilize the thylakoid-bound photosystem (PS) II complex and enhance oxygen releasing activity. PsbO is an ideal candidate to reveal the possible regulation of chlorosis controlled by WKS1. The applicant proposed to verify the binding between WKS1 and PsbO, reveal the subcellular localization of their binding site, detect the possible phosphorylation and discover the phosphorylation sites, demonstrate the effect of phosphorylation on biological functions of PsbO, and finally investigate the chlorosis process in PsbO transgenic, EMS mutant and transient suppressed plants. This work is supposed to reveal a novel regulation of chlorosis and open a new window to increase wheat resistance to fungal pathogens.

小麦是人类重要粮食作物，但其生产受到病虫害的严重威胁。WKS1（Yr36）基因是一个广谱抗条锈基因，可以加速病菌感染周围叶片黄化进程。叶片黄化在抗病、抗逆过程中广为发生，前人推测该过程受到过氧化氢的调控，但是否存在其他调控机制尚不清楚。为了研究小麦抗病中叶片黄化的内在机制，申请人从转基因小麦中分离了WKS1的蛋白复合体，发现了光合系统II的外膜成员PsbO。PsbO可以增加光系统II蛋白复合体稳定性，提高放氧能力。申请人拟以WKS1与PsbO的潜在调控关系为突破口，研究WKS1是否可以直接结合PsbO，观察两个蛋白的亚细胞结合位点；随后，检测WKS1对于PsbO蛋白的磷酸化，测定磷酸化位点，检测模拟磷酸化对PsbO蛋白功能的影响；然后利用转基因小麦、EMS突变体或者瞬时干扰研究PsbO在叶片黄化过程中的作用。本工作将揭示小麦广谱抗病的内在机制，为提高小麦抗病能力提供新靶基因和研究方向。

条锈病在全世界主要小麦产区都广为发生，具有发病面积广、频率高、致病菌株突变快和造成损失巨大的特点，对小麦生产造成了巨大威胁。局部坏死是小麦抗条锈病的主要手段，而活性氧（ROS）与细胞死亡和局部坏死密切相关。申请人前期发现了广谱抗条锈病基因WKS1通过磷酸化抑制tAPX造成ROS积累的机制，但是抗条锈病过程中的ROS从何而来尚不可知。小麦申请人在此项目的支持下，探索了小麦抗条锈病过程中活性氧产生的机制。申请人发现高温成株抗病基因WKS1可以结合并磷酸化光系统II外周蛋白PsbO，磷酸化降低PsbO结合光系统II的能力，进入迅速降解途径，失去PsbO的“裸露PsII”接收光能以后，释放的不再是氧气，而是超氧阴离子。申请人进一步获得了psbo-A1突变体和RNAi转基因小麦，发现它们抗条锈病的能力显著提高。通过该项目的支持，申请人发现了小麦抗条锈病过程中过氧化氢“开源”的机制，丰富了小麦抗条锈病的理论，相关工作发表在植物研究领域的权威杂志《Molecular Plant》。该项目支持了多位研究生的培养，其中金霄和王初阳分别获得了复旦大学硕士和博士国家奖学金，金霄同学获评复旦大学优秀毕业生，论文第一作者王帅同学获得了国家奖学金和上海市优秀毕业生等荣誉称号。