植物特有蛋白FS4调控细胞内吞作用和植物对硼元素吸收转运的分子机制研究

Tight control of plasma membrane (PM) protein homeostasis is crucial for plants to adjust the growth and development status and to adapt to the ever-changing environment. An important scientific question is how plants respond to environmental signals, and thereby selectively activate the endocytosis, recycling or degradation of PM proteins. In order to further investigate the regulatory mechanisms of endocytosis of plant PM proteins, we have used plant boron transporter BOR1-GFP as a PM marker protein to screen factors involved in endocytic degradation of plant PM proteins via a forward genetic approach. We have successfully obtained a novel regulatory factor FS4, which affects the endocytic degradation of BOR1-GFP. In this project, using a combination of molecular, cellular, biochemical and genetic approaches, we aim to study the expression profiles of FS4 gene and the dynamic subcellular localizations of FS4 protein; the functions of FS4 in the regulation of endocytosis, the multivesicular body (MVB) formation and the MVB-mediated PM protein sorting; the regulatory role of FS4 in the boron absorption and transport in plants. We will focus on characterizing the regulatory roles of FS4 in plant endocytic pathway as well as in plant boron absorption network, and will finally clarify the molecular mechanisms of FS4 functions in the regulation of endocytic degradation of plant PM proteins (especially BOR1) and in the modulation of plant boron absorption. This study will reveal the new regulatory mechanism of endocytic degradation of plant PM proteins, and will help us to further understand the regulatory network of uptake and transport of micronutrients in plants, and thereby provide the potential theoretical basis for crop breeding to improve the nutrient use efficiency.

细胞膜蛋白稳态的精确调控对于植物及时调整生长发育状态以应对复杂多变的外界环境至关重要。其中重要的科学问题是植物如何感应环境信号并选择性启动细胞膜蛋白的内吞、再循环或降解。为了深入探讨植物细胞膜蛋白内吞的调控机理，我们以植物硼元素通道蛋白BOR1-GFP为细胞膜标记蛋白，通过正向遗传筛选获得了影响细胞膜蛋白内吞降解的全新调控因子FS4。本项目拟从分子细胞、生化及遗传多个角度研究FS4基因表达模式和蛋白定位、FS4调控内吞作用和多泡体形成及多泡体介导的细胞膜蛋白分选的功能、FS4调控植物硼元素吸收转运的功能，重点研究FS4介导内吞作用及硼元素吸收转运的机理，最终阐明FS4调控植物细胞膜蛋白尤其是BOR1选择性内吞降解并影响植物硼元素吸收转运的分子机制。本研究将揭示植物细胞膜蛋白内吞降解的全新调控机制，并有助于我们深入理解植物对微量元素吸收转运的调控网络，为农作物的高效肥利用育种提供理论基础。

细胞膜蛋白稳态的精确调控对于植物及时调整生长发育状态以应对复杂多变的外界环境至关重要。其中重要的科学问题是植物如何感应环境信号并选择性启动细胞膜蛋白的内吞、再循环或降解。本项目以植物特有转运必需内体分选复合物（ESCRT）组分FYVE4/FS4及FREE1的功能研究为切入点，主要探究FYVE4调控多泡体形成以及多泡体介导的细胞膜蛋白内吞分选，最终调控植物非生物胁迫响应的功能和机制。本项目研究表明：1）FYVE4编码一个植物特有FYVE结构域蛋白，FYVE4与ESCRT-III复合物亚基SNF7互作进而影响SNF7以及VPS4到内含体膜上的募集；2）FYVE4调控多泡体形成及多泡体介导的细胞膜蛋白内吞分选，fyve4突变体中多泡体形成缺陷，泛素化膜蛋白分选受阻并在细胞中积累。遗传分析显示FYVE4与LIP5以及SNF7等ESCRT复合物组分协同调控多泡体形成及其介导的泛素化膜蛋白分选过程；3）fyve4突变体中硼转运蛋白BOR1内吞降解受阻，导致植物在高硼条件下吸收过多的硼元素，因而表现为对高硼胁迫超敏感；4）MLKs蛋白激酶家族磷酸化修饰FREE1蛋白，进而影响FREE1蛋白核质穿梭，最终调控植物脱落酸信号响应。以上核心研究结果分别于2021和2022年发表于国际主流期刊《New Phytologist》和《Plant Cell and Environment》。此外，本项目还探究了植物特有囊泡运输因子BLISTER调控内体介导的细胞膜蛋白和液泡蛋白分选转运的功能和机制，相关研究论文发表在国际主流期刊《PNAS》。本项目资助共发表SCI收录论文20篇、专著章节1篇，培养博士后1名，博士3名，硕士4名。本研究解析了FYVE4/FS4作为植物特有ESCRT复合物组分调控多泡体形成、泛素化膜蛋白内吞降解和植物高硼胁迫响应的分子机理，阐明了MLKs激酶调控FREE1蛋白磷酸化修饰和植物脱落酸信号响应的分子机制。这些研究结果完善了植物ESCRT途径的调控蛋白网络，并解析了ESCRT参与调控植物脱落酸信号及高硼胁迫响应的功能，为将来应用生物技术和分子辅助育种技术改良作物营养吸收效率和抗性提供了潜在理论依据。