钾缺乏诱导棉花根系质外体H2O2信号网络和氧化还原体系的变化及其调控

Root is the first organ of crops subjected to potassium deficiency stress. Apoplast is extracellular space, including cell wall and intercellular space, among which fluid is circulating. It is not only a barrier between cell and environments, but also bridge of sensing and transferring environmental signals. We verified that potassium deficiency induced H2O2 increase and oxidative stress, and that coronatine treatment further increased H2O2 content, but significantly alleviated the oxidative stress. Its molecular physiology mechanism is yet unclear. Therefore, we further study the potassium deficiency and “potassium deficiency + coronatine”, respectively, induced changes in (1) H2O2 content, redox homeostasis, proteomics, phosphoproteomics, and non-targeted and targeted metabolomics of apoplastic fluid and (2) H2O2 distribution in apoplast and (3) ClassⅢ peroxidases (PODs) of apoplastic fluid, cell wall ionically tightly binding protein and cell wall covalently binding proteins. Based on the above results, it will be clarified about (1) apoplastic proteins, phosphoproteins, PODs isoenzymes and metabolites in response to, respectively, potassium deficiency and “potassium + coronatine”, and (2) differences in H2O2 signaling network and redox system between potassium deficiency and “potassium + coronatine”.This study will supply references for application of root physiology improvement in cotton cultivating and molecular breeding , and for coronatine application against oxidative stress induced by abiotic stresses.

根系是作物遭受钾缺乏胁迫的首要器官。质外体是细胞膜外空间，既是细胞和外界环境的一道屏障，又是感受和传递外界信号的桥梁。我们证明钾缺乏诱导了棉花根系H2O2增加和氧化胁迫，而冠菌素处理进一步增加了H2O2含量但显著缓解了氧化胁迫。但其分子生理机制尚不清楚。本项目拟在研究钾缺乏和“钾缺乏+冠菌素”诱导的（1）质外体-汁液中H2O2含量、氧化还原稳态、蛋白质组、磷酸化蛋白质组和非靶向、靶向代谢组和（2）质外体H2O2分布以及（3）质外体-汁液中、细胞壁上离子键紧密结合和共价键结合的第三类过氧化物酶（PODs）的变化。根据研究结果，（1）明确棉花根系分别应答钾缺乏和“钾缺乏+冠菌素”的质外体蛋白质、磷酸化蛋白质、PODs同工酶和代谢物；（2）阐明钾缺乏和“钾缺乏+冠菌素”诱导的棉花根系质外体H2O2信号网络和氧化还原体系差异，挖掘反应氧化胁迫的生物标记。为根系改良在棉花栽培管理中应用提供参考。

根系是作物遭受钾缺乏胁迫的首要器官，钾吸收减少，导致地上部器官如叶片遭受钾缺乏胁迫。质外体是细胞膜外空间，既是细胞和外界环境的一道屏障，又是感受和传递外界信号的桥梁。分别建立了棉花根系和叶片质外体汁液分离的方法，并根据建立的方法分离了质外体汁液，从氧化还原平衡生理指标、代谢组和蛋白组角度解析了钾缺乏和冠菌素对棉花根系和叶片氧化还原平衡及H2O2产生和信号传导的调节，并从miRNA和mRNA水平解析了钾缺乏改变根系氧化还原平衡的调控机制。无论从生理生化测定还是代谢组水平显示，钾缺乏提高了根系质外体H2O2含量，降低了质外体还原性抗坏血酸和还原性谷胱甘肽的含量，增加了蛋白质羰基化程度，使得质外体偏向氧化状态。质外体蛋白质组结果显示，质外体草酸氧化酶和氨氧化酶在钾缺乏条件表达量显著增加，是质外体H2O2的主要来源；质外体抗坏血酸氧化酶增加，但是抗坏血酸过氧化物酶显著下降，导致单脱氢和双脱氢抗坏血酸含量增加，单脱氢抗坏血酸还原酶和细胞色素b561含量增加，负责把氧化胁迫或自由基信号传递到细胞内。在钾缺乏条件下，冠菌素处理使得质外体偏向还原状态，主要是增加了质外体的还原型谷胱甘肽和还原型烟酰胺腺嘌呤二核苷酸磷酸。与钾缺乏相对于高钾相比，钾缺乏条件下添加冠菌素相对于钾缺乏，在质外体次生代谢物方面有相反的调节模式，在质外体蛋白组方面表现为不同的调控模式。相对于高钾，钾缺乏诱导显著下调基因是显著上调基因的2.6倍，按GO注释定位到质外体的显著上调基因是下调基因的9.6倍，其中和氧化还原密切相关的第三类过氧化物酶基因上调是下调的12.8倍。钾缺乏诱导了miRNA如miR480、miR7508和miR319等显著变化，负调控其靶基因表达，进而调控了根系质外体空间的氧化还原平衡。