胡杨耐盐性调控的eATP信号途径研究

Extracellular ATP (eATP) is a crucial requirement for regulating plant growth,development and defence response. However, the contribution of eATP signaling to plant salt tolerance is unknown. eATP signaling in Populus euphratica programmed cell death (PCD) has been previously postulated in our lab. In this study, we attempt to characterize ATP signaling cascade and the molecular networks involved in plant response to salinity. The aim is to elucidate the mechanism of salinity tolerance in woody plants as P. euphratica is typically a salt-resistant tree species. We planned to investigate the regulation of salt-induced eATP in salinity tolerance of P. euphratica, including (1) how eATP mediates ionic and ROS (reactive oxygen species) homeostasis in salinized cells through second messengers, such as H2O2, NO and cytosolic Ca2+ ([Ca2+]cyt); (2) the contribution of Apyrase (nucleoside triphosphate-diphosphohydrolase) to the control of ATP concentrations in the extracellular matrix and the relevance to salt tolerance. The establishment of ATP signaling pathway in plant salt tolerance is helpful to discover the specific salt-resistance mechanism existing in tree species that differs from model herbaceous plants. Our work may not only extend fundamental biological understanding of trees, but also benefits salt-resistant gene modifications of economically important woody species in the future.

胞外ATP (eATP)作为信号分子，在植物生长发育以及防御反应中发挥重要调控作用，但eATP在植物耐盐性中的作用还属未知。本项目在前期胡杨细胞eATP-PCD(细胞程序化死亡)信号网络的基础上,拟探讨eATP对胡杨耐盐性的调控作用。胡杨为典型的耐盐树木，探讨胡杨细胞对盐胁迫响应的eATP信号途径，有助于深入阐明树木的耐盐机理。项目重点研究 (1) 盐诱导的eATP如何通过H2O2、NO和[Ca2+]cyt信号调控胞内的离子平衡和活性氧平衡；(2)胞外ATP水解酶- - Apyrase在盐胁迫下对eATP浓度的调控模式，及其对胡杨细胞耐盐性的影响。通过探究eATP对胡杨细胞耐盐性的调控机制，建立胡杨的eATP信号途径，不但能深入揭示耐盐树木特有的且区别于模式植物的耐盐机理，研究结果还对经济型林木的抗逆性遗传改良具有指导意义。

胡杨广泛分布于我国西北干旱、盐碱地区，具有极强的适应性，如耐盐、抗旱以及忍受极端温度等。本项目在前期研究的基础上，重点探讨胡杨耐盐性调控的胞外ATP（eATP）信号途径。研究结果表明，eATP能够在盐胁迫下调控不同耐盐性杨树细胞K+/Na+平衡。ATP (50uM) 能够增强盐胁迫下胡杨、群众杨细胞的Na+/H+交换，特别是在盐敏感杨树中效果更加显著。然而，ATP诱导的Na+外流和H+内流可以被阿米洛利（Na+/H+逆向转运体抑制剂）和原钒酸钠（质膜H+-ATPase抑制剂）所抑制，这表明ATP促进的Na+外排是由质膜（PM）上活化的Na+/H+逆向转运引起的。NaCl可导致两种杨树细胞K+的外流，且盐敏感的群众杨外流更加明显。K+通道抑制剂TEA能显著抑制盐诱导的K+外流，表明盐诱导K+外流是由去极化激活的外向整流型K+通道和非选择性阳离子通道介导的。ATP有助于杨树细胞尤其是盐敏感的群众杨维持盐胁迫下的K+平衡。这是由于ATP处理的杨树细胞质膜上质子泵被活化，从而抑制了经由去极化激活的K+通道介导的K+外流。由于Apyrase能水解盐诱导的胞外ATP以阻止其诱发细胞程序化死亡，因此，本项目还重点探讨了胡杨Apyrase (PeAPY1和PeAPY2) 调控植物耐盐性的生理与分子机理。以野生型拟南芥、拟南芥apyrase突变体 (Atapy1和Atapy2)、胡杨PeAPY1和PeAPY2过表达株系为材料，在盐胁迫下研究了各种基因型对盐胁迫响应的差异，从生理、生化及分子生物学角度分析了PeAPY1和PeAPY2对植物耐盐性的调控作用。表型分析结果显示，PeAPY1和PeAPY2过表达拟南芥株系的耐盐性增强。PeAPY1和PeAPY2过表达株系在盐胁迫下，能够提高抗氧化酶(SOD、CAT、APX) 的活性，维持体内ROS (活性氧) 平衡，减少体内MDA (丙二醛) 的积累，从而缓解了氧化胁迫。另外，PeAPY1和PeAPY2过表达株系在盐胁迫下，减少了Na+的摄入，并维持K+的吸收，从而维持体内的K+/Na+平衡，增强耐盐性。再者，PeAPY1和PeAPY2过表达株系的ABA响应基因受盐胁迫诱导表达量上调，表明PeAPY1和PeAPY2可能参与了盐胁迫下ABA的信号转导过程。此外，本项目还解析了胡杨Apyrase 调控植物抗旱和低温耐受的分子机制。