苹果质膜型H+-ATPase应答非生物胁迫的分子机制与调控途径

Various abiotic stresses seriously threaten the maintainable development of fruit tree industry. Higher plants including fruit trees have evolved elaborately responsive mechanisms to adapt environmental stresses. Plasma membrane (PM) H+-ATPase is a crucial component of the complicated machinery for stress responses. It is unclear yet, however, concerning the molecular mechanism and regulatory pathway by which PM H+-ATPase responds to abiotic stresses. Our preliminary investigations demonstrated that the expression of PM H+-ATPase gene MdAHA15 is positively induced by multiple stresses, and that bHLH transcription factor MdbHLH104 binds to the promoter region of MdAHA15 gene. Furthermore, MdbHLH104 interacts with a scaffold protein MdBT4, while MdBT4 interacts with two ubiquitin E3 ligases MdUbiE3-1 and MdUbiE3-2, respectively. Therefore, it is reasonable to draw a hypothesis concerning that PM H+-ATPase responds to abiotic stresses via a putatively regulatory pathway, i.e. abiotic stresses→MdBT2(MdUbiE3-1 or MdUbiE3-2)→MdbHLH104→MdAHAs→PM H+-ATPase→stress responses. Based on this hypothesis, various techniques of molecular biology, genetics，biochemistry and physiology will be adopted in this study to further verify this pathway and elucidate the molecular mechanism underlying each step. First of all, several techniques, such as EMSA, ChIP-PCR, transient expression assays and so on, will be conducted to verify if MdbHLH104 really binds to the promoter region of MdAHA15 gene to regulate its expression. Meanwhile, it will be verified if MdBT2 interacts with MdbHLH104 protein to modulate its stability. Furthermore, the interaction between MdBT2 and MdUbiE3-1 or MdUbiE3-2 is to be verified, and the function of MdUbiE3-1 or MdUbiE3-2 in the modulation of MdBT2 in MdbHLH104 protein stability is to be characterized. Finally, the whole regulatory pathway will be integrated, and molecular mechanism will be refined.

非生物逆境胁迫严重威胁果树产业的可持续发展，质膜型(PM)H+-ATPase在非生物胁迫应答中具有重要地位，但分子机制和调控途径不清楚。根据我们的前期研究结果，推测出PM H+-ATPase应答非生物逆境的可能调控途径，即逆境胁迫→MdBT2→MdbHLH104→MdAHA15→PM H+-ATPase→应答。在此基础上，拟采用分子生物学和遗传学及生理生化技术验证该调控途径，并解析各步骤的分子机理。首先鉴定MdbHLH104对MdAHA15的转录激活，然后验证MdBT2与MdbHLH104的互作，并鉴定其对MdbHLH104蛋白的调控作用；验证MdBT2与MdUbiE3-1或MdUbiE3-2的互作，并鉴定二者是否参与MdBT2对MdbHLH104蛋白的调控；最后，通过鉴定MdBT2和MdbHLH104转基因材料的功能，进一步整合该调控途径，揭示其分子机制。

质膜型（Plasma membrane, PM）H+-ATPase作为质子泵，在质膜两侧形成跨膜质子驱动力，水解ATP驱动次级离子或溶质的主动运输，在植物的生长发育、逆境应答和营养吸收等方面具有至关重要的作用。本项目依据苹果基因组数据库和转录组数据鉴定了一个受缺铁诱导的bHLH转录因子基因MdbHLH104，通过分析MdbHLH104过表达苹果植株，发现MdbHLH104转基因株系表现出明显的抗缺铁能力，并且根际质子外泌和三价铁离子还原酶活性都增加。进一步研究发现，MdbHLH104蛋白能够直接结合到MdAHA8基因（编码PM H+-ATPases）的启动子上，激活MdAHA8基因的表达，从而正调控PM H+-ATPases的酶活性。以MdbHLH104为诱饵蛋白进行酵母双杂交筛选cDNA文库，得到一些与MdbHLH104互作的蛋白。酵母双杂交和Pull-Down分析的结果表明，MdbHLH104蛋白分别与MdbHLH105、MdbHLH115和MdbHLH121等其他bHLH蛋白相互作用，形成异源二聚体共同正调控下游基因MdAHA8的表达，分别共表达MdbHLH104与其互作蛋白明显增强转基因苹果愈伤组织中的H+-ATPase活性和铁含量。此外，还筛选到了2个BTB/POZ-TAZ蛋白（MdBT1和MdBT2）和1个SUMO蛋白（MdSIZ1）。酵母双杂交、Pull-Down和Co-IP（免疫共沉淀）分析试验表明，MdBT1/2和MdSIZ1都能够与MdbHLH104相互作用。Western杂交分析的结果表明MdBTs蛋白负调控MdbHLH104蛋白的丰度，MdSIZ1增强其蛋白稳定性。深入研究表明，MdBT2蛋白能够与MdCUL3互作形成CRL3BT2 E3泛素化连接酶复合体介导MdbHLH104蛋白的泛素化修饰，进而通过26S蛋白酶体途径降解；而MdSIZ1于缺铁条件下对MdbHLH104进行SUMO化修饰，进而抑制MdbHLH104的泛素化降解。因此，MdBT2或MdSIZ1蛋白通过MdbHLH104调控植物体内质子外泌和铁离子的吸收及动态平衡。