盐胁迫下胡杨胞内Cl-稳态平衡的形成及其受H2O2信号调控的机理研究

Excess accumulation of Na+ and Cl- in the cytoplasma inhibits plant growth and development under salinity conditions. Thus, the studies on the mechanisms of cytoplasmic Na+ and Cl- detoxification are especially important for the understanding of plant salt tolerance. However, most studies are focus on the Na+ transport and detoxification while pay no attention to the process of cytosolic Cl- detoxification and this situation is more serious in woody plants. Populus euphratica exhibited a greater ability to regulate intracellular ion homeostasis under saline conditions and has been widely considered as a model plant to elucidate physiological and molecular mechanisms of salt tolerance in woody species. In this project, using a series of novel techniques, such as the patch clamp technique, the scanning ion-selective electrode technique, the fluorescence microscopic imaging techniques, the X-Ray microanalysis and real time PCR, we tend to investigate the mechanisms of cytosolic Cl- detoxification in salt-resistant Populus euphratica cells. Salt-sensitive Populus popularis cells were adopted as a comparison. We will investigate several contents in this project, including the characteristics of Cl- accumulating in the cytoplasma, activities of plasma membrane anion channels, activities of vacuolar Cl-/H+ antiport， gene expression related to the Cl- transport and the roles of H2O2 and plasma membrane H+-ATPase invovled in the process of cytosolic Cl- detoxification. This project put forwards a novel hypothesis that help to elucidating the mechanisms of intracellular Cl- homeostasis in Populus euphratica. This project is aslo important for the understanding of salt tolerance in woody plants.

盐胁迫下，盐离子（Na+和Cl-）在植物胞质中过度积累对植物生长具有严重毒害作用，因此，研究盐离子的胞质脱毒机制对理解植物抗盐性形成具有重要意义。然而，国内外学者大多专注于胞质Na+脱毒机制的研究，而较少关注Cl-的脱毒过程，尤其是木本植物。本课题拟以耐盐木本植物胡杨（具有极强的胞质盐离子脱毒能力）作为单细胞研究模型，以盐敏感的群众杨做为对照，应用膜片钳，非损伤微测技术，荧光成像技术，X射线微区分析和荧光定量PCR等先进手段，从细胞-亚细胞层次探讨胡杨细胞质Cl-脱毒机制。研究内容包括胞质和液泡Cl-积累特征，质膜阴离子通道活性，液泡膜Cl-/H+转运活性，Cl-平衡相关基因表达，H2O2信号和质膜H+-ATPase在Cl-脱毒中的作用等方面。本课题力求揭示盐胁迫下胡杨胞内Cl-稳态平衡形成的调控机制，这将为理解木本植物胞内Cl-脱毒提供新证据，也为研究植物细胞Cl-跨膜运输提供了新思路。

盐胁迫下, 盐离子(Na+和Cl-)过度积累对植物生长具有毒害作用。研究盐离子的脱毒机制对理解植物抗盐性形成具有重要意义。然而，国内外学者大多专注于研究Na+脱毒机制，极少关注盐胁迫下Cl-转运及脱毒机制，尤其是木本植物。本课题以耐盐木本植物胡杨（具有极强抗盐能力及离子平衡调控能力）作为单细胞研究模型，以盐敏感的群众杨细胞作为对照，采用非损伤微测技术，扫描电镜+能谱分析技术，荧光成像技术和荧光定量PCR等先进技术手段研究了盐胁迫下胡杨细胞与群众杨细胞Cl-转运和积累的特点，并揭示了H2O2信号在调控胡杨细胞Cl-平衡中的重要作用。. 研究结果发现：（1）与盐敏感的群众杨细胞相比，胡杨细胞在盐胁迫下能够在细胞质中积累相对较少的Cl-。非损伤微测实验结果揭示胡杨细胞这种有效调控细胞质Cl-积累的能力可能源于质膜Cl-外排系统和液泡Cl-/H+逆向转运系统的激活。（2）药理学实验证明质膜H+-ATPase活性在调控盐胁迫下胡杨细胞Cl-外排过程中具有重要作用。（3）与群众杨相比，胡杨细胞能够迅速响应盐胁迫，产生H2O2信号。药理学实验证明盐胁迫诱导H2O2信号对胡杨细胞Cl-脱毒具有正向调控作用。（4）盐胁迫下，胡杨细胞H2O2信号的产生调控细胞质钙信号系统（液泡钙释放途径），H2O2信号从而通过下游钙信号途径来实现对质膜Cl-外排和液泡膜Cl-/H+逆向转运相关基因表达的调控作用，最终实现细胞质Cl-稳态平衡。. 本项目已正式发表或接受发表SCI源刊论文9篇，培养或辅助培养硕士研究生4名（尚未答辩），较好地完成了既定目标。本课题的研究对今后深入探讨木本植物耐盐生理及分子机制奠定了良好的基础。