调控植物质膜H+-ATPase小分子化合物的鉴定和质膜H+-ATPase功能分析

Plant plasma membrane (PM) H+-ATPase is known as a master enzyme, which converts chemical energy derived from hydrolysis of ATP into pH and electrical gradients across the plasma membrane. The combined electrochemical gradient constitutes a driving force for the transport of solutes and metabolites across the plasma membrane. In Arabidopsis thaliana, PM H+-ATPases are encoded by an 11 member gene family (AHA1 to AHA11). Due to expression overlap and functional redundancy, genetic analyses of individual isoforms are difficult. For example, single AHA1 or AHA2 knockout mutant has no obvious phenotype; however the double knockout is lethal. PM H+-ATPase activity is highly regulated and controlled at both transcriptional and post-translational levels by many factors including hormones, calcium, light, and environmental stresses like increased soil salinity. The PM H+-ATPase plays an important role in many physiological processes that include cell growth, intracellular pH, both biotic and abiotic stress responses, and stomatal regulation. However it is not well understood how the PM H+-ATPase directly involves in these regulations because of lack of an assay system for specifically activating or inhibiting PM H+-ATPase. In this study, we screen small molecules that directly bind and specifically regulate PM H+-ATPase from plant materials or small molecular libraries (ChemDiv, Chemical Diversity). Using a bio-guided isolation strategy, the fractions extracted from plant and affecting PM H+-ATPase activity are further purified by silica gel column chromatography, Sephadex LH-20, Thin Layer Chromatography (TLC), preparative reversed-phase HPLC, and spectroscopic methods (1D-NMR, 2D-NMR, MS, IR, UV, etc.). We have identified few small molecules either activating or inhibiting the PM H+-ATPase activity. We also screen a small molecular library (ChemDiv, Chemical Diversity) and have identified few molecules that bind and regulate the PM H+-ATPase activity. Using these small molecules, we will study how the PM H+-ATPase regulates plant growth, development and response to environmental changes, specifically how this enzyme regulates plant salt tolerance.

质膜H+-ATPase（质子泵）被称为植物生命过程中的主宰酶，它们不仅在物质的跨膜运输中，同时在其它过程中如信号转导起重要作用。由于不可取代和功能冗余的原因，其机理的研究存在巨大的挑战。我们在研究盐碱胁迫下植物如何调控细胞内离子平衡时发现：内源小分子化合物可以直接参与质子泵活性的调节。通过测定对质子泵活性的影响,逐级分离内源活性小分子组分，采用薄层色谱、硅胶柱色谱、凝胶柱色谱、反相高效液相色谱等分离手段，及现代谱学技术，并结合理化性质，我们鉴定到一些小分子化合物，并解析了化学结构。拟南芥突变体敲除小分子合成的关键酶改变了植物盐敏感性；一小分子能增强拟南芥和玉米的耐盐性。同时，我们通过一酵母系统，筛选化学小分子化合物库，得到几个特异抑制/激活质子泵活性的小分子。本申请将在前期工作的基础上，鉴定/优化这些小分子的结构、研究它们对质子泵活性调控的作用机理，利用它们研究质子泵的生物功能。

盐胁迫是限制农业生产的重要环境因素，质膜H+-ATPase在植物应对盐胁迫、保持植物体内离子平衡方面起着十分重要的作用。内源小分子是植物在代谢过程中产生的一类十分重要的代谢产物，它们是否参与质膜H+-ATPase活性的调节并不清楚。为了寻找参与调控质膜H+-ATPase活性的植物内源有机小分子物质, 我们首先建立了质膜H+-ATPase活性导向分离模型,利用现代天然产物化学分离技术从拟南芥根中得到激活或抑制质膜 H+-ATPase活性的组分, 并进一步利用现代波谱和质谱等技术, 对活性组分中的化合物进行结构解析鉴定。最终我们从激活组分中分离鉴定到化合物油酸 (C18:1), 亚油酸 (C18:2), 亚麻酸 (C18:3), 从抑制组分中分离鉴定到化合物磷脂酰肌醇PI。体外和体内实验证明它们可以调控质膜H+-ATPase的活性。进一步的生化结合实验表明, C18:1, C18:2, C18:3和PI都与质膜H+-ATPase AHA2的C末端直接的相互作用。此外发现,在盐胁迫下, 拟南芥PI通过代谢转变为PI-4-P, 一方面解除PI对质膜H+-ATPase活性的抑制作用, 释放其活性, 另一方面PI-4-P激活SOS1的活性, 增强植物的耐盐能力。通过研究，我们阐明了这些小分子化合物调控质膜H+-ATPase活性使植物适应盐胁迫的分子机制，发现了植物在盐碱胁迫下调控细胞内离子平衡的新机制。并且，我们通过正向筛选方法直接寻找到了调节特异酶活性的内源小分子化合物，在国际上鲜有报道，研究方法有借鉴作用。