Functional genomics of intracellular membrane trafficking in Arabidopsis thaliana

细胞膜内运输途径连接真核生物膜系统的细胞器。在植物中，这些途径在蛋白质、脂质和多糖的生物合成和积累中发挥着重要作用，这些蛋白质、脂质和多糖为世界提供了食物、衣服和住所。除此之外，它们也被认为是植物细胞发育和细胞适应环境胁迫的更广泛方面的核心。我研究的总体目标是了解植物基因如何控制细胞膜内运输，因为植物发育过程中细胞形态的变化和细胞对环境胁迫的反应。小的拉布gtp酶是关键的膜运输调节因子。在拟南芥中，已经鉴定出57个推定的Rabs，并将其划分为18个结构亚类，但只有少数Rabs在功能上得到了研究。根据我们最近对rabb - d2和rabb - e1亚类的表征，我们建议研究rabb - a1亚类在膜运输、细胞信号传导、细胞形态发生和应激适应中的功能。我们将使用显性负突变方法结合植物细胞中共表达的荧光标记物的体内成像来测试rab - a1在膜运输中的作用。我们还将确定每个Rab-A1成员的组织特异性和亚细胞定位，并生成用于功能丧失研究的敲除突变体。构建基于分裂泛素的cDNA文库，筛选Rab-A1和Rab-E1相互作用物，以剖析rab介导的信号通路。此外，我们还计划描述和克隆三种新的膜运输突变体，这些突变体是在基于ems诱变群体中分泌GFP的细胞内积累的遗传筛选中鉴定出来的。我们的工作将1)提供植物作为多细胞生物是如何发育的分子和细胞细节，2)为植物如何应对当前环境提供新的见解，3)阐明有价值的蛋白质、脂质和多糖是如何在植物膜系统中积累的。这些知识可用于开发有益于农业和生物技术的技术。

The intracellular membrane trafficking pathways connect the organelles of the eukaryotic endomembrane system.  In plants, these pathways play important roles in the biosynthesis and accumulation of proteins, lipids and polysaccharides that literally feed, clothe and house the world. Beyond this, they have also been recognized as central to wider aspects of plant cell development and cellular adaptation to environmental stresses.       The overall goal of my research is to understand how plant genes control intracellular membrane trafficking as cell morphology changes during plant development and in cellular responses to environmental stresses. Small Rab GTPases are key membrane trafficking regulators. In Arabidopsis, 57 putative Rabs are identified and clarified into 18 structural subclasses, yet only few have been functionally studied. Following our recent characterization of the Rab-D2 and Rab-E1 subclasses, we propose to investigate the function of the Rab-A1 subclass in membrane trafficking, cell signaling, cell morphogenesis, and stress adapation. We will test the roles of Rab-A1s in membrane trafficking using a dominant negative mutant approach combined with in vivo imaging of fluorescent markers co-expressed in plant cells. We will also determine the tissue specificity and subcellular localization of each Rab-A1 members, and generate knockout mutants for loss-of-function studies. A split ubiquitin-based cDNA library will be constructed and screened for Rab-A1 and Rab-E1 interacters in order to dissect Rab-mediated signaling pathways. In addition, we also plan on characterizing and cloning three novel membrane trafficking mutants identified in a genetic screen based on the intracellular accumulation of a secretory GFP in an EMS-mutagenized population.       Our work will 1) give molecular and cellular details on how plants, as multicellular organisms are developed, 2) provide fresh insight into how plants deal with prevailing environment, and 3) shed light on how valuable proteins, lipids, and polysaccharides are accumulated in the plant endomembrane system. This knowledge can be used to develop technologies of benefit to agriculture and biotechnology.