拟南芥小G蛋白ROPα调控磷转运体PHT1;1和PHT1;4的功能研究

Phosphorus acts as an essential macroelement to plant growth and development. Given the general phosphate deficiency in cultivated soil and lack of phosphate fertilizer resources, it has become an important factor limiting crop production. Therefore, it is imperative to further explore the regulation mechanism of phosphate uptake for improving phosphate utilization efficiency in plant. ROPs are small GTP-binding proteins unique to plant, which are involved in regulating numerous cellular signal transduction processes. However, whether it could regulate phosphate signal pathway has not been reported. The applicant found that ropα mutant exhibited more tolerant to low phosphate and more sensitive to high phosphate stress, and a significant increase in phosphate accumulation in ropα mutant compared with the wild type. Moreover, ROPα interacted with the key phosphate uptake transporters PHT1;1 and PHT1;4 by multiple protein-protein interaction methods. We demonstrated for the first time here that a small GTP-binding protein could directly interact with transporter in plant. Based on these results, this project intends to further verify the interactions between ROPα and phosphate transporters through Co-IP assay, analyze the temporal and spatial responses of ROPα to phosphate stress by tissue expression and subcellular localization experiments, explore phosphate transporter protein activity by using isotope 32P uptake experiment and voltage-clamp technique, and reveal the ROPα function and molecular mechanism in regulation of phosphate transporters combined with genetic analysis, hoping to provide theoretical foundation for improving the signal regulation network in response to phosphate stress.

磷是植物生长发育所必需的大量营养元素，因耕地土壤普遍缺磷且磷肥资源匮乏，成为限制农作物生产的重要因素，因此深入研究植物对磷吸收的调控机制，对于提高植物磷素利用效率具有重要意义。ROPs是植物中特有的小G蛋白，参与调控众多的细胞信号转导过程，但其是否调控磷吸收却未有报道。申请人前期研究发现，拟南芥ropα突变体表现低磷耐受、高磷敏感的表型，且磷积累量显著升高。通过多种蛋白互作技术发现ROPα与主要负责磷吸收的转运体PHT1;1和PHT1;4互作，这也是首次在植物中发现小G蛋白与转运体直接互作。本项目拟以此为切入点，通过Co-IP等实验进一步鉴定ROPα与磷转运体互作，通过组织表达和亚细胞定位分析ROPα对磷胁迫的响应，利用32P吸收实验和电压钳技术探究ROPα对磷转运体活性调控的过程，结合遗传学材料分析深入揭示ROPα调控磷转运体的作用和分子机制，以期为作物磷高效利用的遗传改良奠定理论基础。

磷是植物生长发育所必需的大量元素之一，是核酸、蛋白质、磷脂类等物质的重要组成成分，在植物生长发育和逆境胁迫过程中发挥着重要作用。然而，土壤中的有效磷浓度往往很低，是限制农作物产量和品质的重要因素。因此，深入研究植物吸收利用磷的分子机制具有重要意义。Rho家族成员ROPs (Rho-related GTPase from plants) 是植物中特有的小G蛋白，在信号转导过程中具有不可替代的作用。ROPs有两种状态，一种是与GTP结合的激活态，另一种是与GDP结合的非激活态，通过激活态与非激活态的转变，调控植物众多生命过程。但ROPs家族成员是否参与植物磷信号转导途径却鲜有报道，其分子机制更是知之甚少。.该研究发现拟南芥rop6突变体对五价砷胁迫显著敏感，ROP6OE过表达株系对五价砷胁迫明显耐受，ROP6COM转基因回复株系能够回复突变体至野生型的表型。进一步研究表明，rop6突变体对低磷胁迫明显耐受，主根更长，花青素含量降低，磷吸收能力增强，磷含量显著升高。低磷胁迫处理条件下，ROP6蛋白活性降低并被26S蛋白酶体降解。通过蛋白-蛋白互作 (Y2H、BiFC、LCI和Pull-down) 方法证明，ROP6与拟南芥主要磷转运体PHT1;1和PHT1;4存在物理互作。电压钳实验发现，ROP6抑制PHT1;1和PHT1;4的磷转运活性。最后，遗传学上下游分析表明，ROP6位于PHT1;1和PHT1;4的上游。综上所述，该研究揭示了ROPs家族参与低磷胁迫的作用和分子机制，为农作物磷高效利用的遗传改良提供了新思路。