Rab蛋白LDS1调控保卫细胞脂滴大小和气孔功能的分子机制

Lipid droplets (LDs) are dynamic organelles and specialized for storage and retrieval of energy in the form of triacylglycerol which has been reported to be used to maintain functionality of stomata. LD size is an important factor that determines lipolysis efficiency, but the regulatory mechanism remains largely unknown. In a focused genetic screen, we uncovered an Arabidopsis recessive mutant lds1 (for lipid droplet size and stomata function 1) that showed aberrantly large LDs in guard cells and dysfunctional stomata as stomatal conductance (gs) induced by light and low [CO2] were significantly smaller in lds1 than that in WT plants. The inhibitive effect of lds1 mutation on stomatal opening was further confirmed as both light and fusicoccin failed to bring out a comparable increase in stomatal aperture as they did in WT. However, there is no appreciable difference in terms of both stomatal density and stomatal index between lds1 and WT plants. In addition, the capabilities of plants to closing their stomata when exposed to high [CO2] as well as to fighting against bacterial infection when inoculated with Pst DC3000 remain intact in lds1 mutant. More interestingly, in lds1 no rapid reduction of LD abundance in guard cells that is associated with and required for stomatal opening in WT plants was observed. Map based gene cloning, genetic complementation test and independent allele analysis together revealed that LDS1 gene encodes a member of Rab branch of the Ras superfamily of GTPases which are widely reported to act as membrane associated molecular switches. In addition, that homologous LDS1 from the early land plants and human being could rescue LD size and stomatal function in lds1 suggested gene function of LDS1 is conserved across evolution. Here, we propose LDS1 predominately expresses in guard cells and actively regulate the LD size and stomatal function. Loss function of LDS1 will cause aberrant large LDs which reduce the access of lipases to their TAG substrate and lead to defective lipolysis and hamper the extensive membrane cycling which both are required during stomatal opening. In this study, we will employ microscopic, molecular, biochemical, genetic, and physiological approaches to unravel the mechanism whereby LDS1 controls LD size and stomatal function. To this end, we will 1. examine the temporal-spatial expression pattern and subcellular location of LDS1 and its paralogue, observe whether LDS1 is preferentially expressed in guard cells and whether LDS1 protein translocate between LD, ER and Golgi body; 2. determine alterations to fatty acid levels/composition when LDS1 misexpressed in plants; 3. characterize the effectors/interacting partners of LDS1; 4. define LDS1 functions in plant response to environmental stresses. Knowledge gained during this study will provide essential insights into plant stomatal biology, Rab-mediated LD homeostasis, LD size control and the metabolic disease etiology in human, and may have implications for enhancing the neutral lipid content and composition of bio-energy crop plants.

脂滴参与调控细胞脂质稳态、物质转运和能量代谢平衡。我们发现，Rab蛋白家族成员LDS1突变导致保卫细胞中脂滴异常增大，抑制光照等诱导的气孔开放，但不影响气孔关闭、气孔密度、气孔指数以及植物对病原菌的抗性。导入拟南芥野生型LDS1基因及小立碗藓、卷柏和人的同源基因均可回复突变体表型。目前尚未见Rab蛋白特异调控保卫细胞脂滴代谢与气孔功能的相关报道。我们因此提出假说：LDS1特异调节保卫细胞脂滴大小控制气孔开放。本研究拟确定LDS1的时空表达特点、亚细胞定位及其结合GTP的能力，分析LDS1对脂滴代谢相关分子表达的影响，检测LDS1突变引起的脂质组分与含量变化，通过定点突变鉴定LDS1的活性位点，分离LDS1的效应蛋白和相互作用分子等，阐明LDS1调控脂滴稳态和气孔功能的分子机制。预期成果将加深对脂滴和气孔功能调控的理解，为农业抗逆育种、可再生生物能源研发和脂质代谢紊乱疾病治疗提供新思路。

气孔作为植物与外界环境进行气体交换的主要门户，其发育和运动受到遗传和环境因子的共同调节。脂滴广泛参与调控细胞脂质稳态、物质转运和能量代谢平衡。气孔保卫细胞中含有丰富的脂滴。之前的研究表明脂滴动员对于光照引起的气孔运动不可或缺。但是，截至目前，还没有脂滴直接参与气孔发育调控的研究报道。在本研究中，我们发现，Rab蛋白家族成员LDS1特异调节保卫细胞的脂滴大小。LDS1广泛表达于多个组织器官，且在气孔世系细胞中的表达强度存在明显的动态变化。干扰脂滴代谢的动态变化，可以显著抑制气孔的形态和功能建成。lds1突变导致保卫细胞中脂滴异常增大，并抑制气孔开放功能，但不影响气孔关闭、气孔密度和气孔指数以及植物对病原菌的抗性。导入拟南芥野生型LDS1基因及哺乳动物的同源基因均可回复突变体表型。进一步的研究发现，LDS1具有Rab蛋白类GTP酶活性。其参与调控气孔发育的功能依赖于鸟苷酸交换因子LDS1GEF和下游的效应蛋白SEIPIN2以及SEIPIN3。LDS1、LDS1GEF和SEIPIN2以及SEIPIN3突变均导致保卫细胞内脂滴大小异常，脂质组分和含量改变。但是双突变体seipin2seipin3的表型缺陷要显著轻于lds1突变体，表明还有其它效应蛋白参与LDS1的功能发挥过程，尚待进一步分析鉴定。综合起来，我们的研究首次发现了脂滴在气孔发育中具有不可或缺的作用。