Analysis of novel polar auxin transport pathway components in Arabidopsis

植物激素生长素在协调植物发育的各个方面起着关键作用，有助于作物生产力。这包括芽、根、花、种子、果实、茎的伸长、组织分化的发育。生长素作用模式的一个重要方面是其在发育组织内的极性运输，以建立浓度梯度，从而产生位置信息以允许基因表达模式的空间调节。生长素定向运输机制的最重要组成部分是家族性的生长素流出载体（PIN蛋白），其中一些定位于质膜的特定表面，以控制生长素的定向流出。细胞极性的快速变化涉及网格蛋白介导的内吞作用和PIN的再循环。生长素本身抑制这种循环，导致PIN蛋白在质膜上积累，从而促进其自身的流出。虽然内吞模型解释了PIN在细胞不同表面的动态再定位，但它不能机械地解释PIN蛋白在内质网（ER）中翻译后如何被递送到质膜。我们已经确定了一个新的突变体缺陷的极性生长素运输途径。涉及的基因是VAMP 714，被鉴定为功能获得和功能丧失突变体（Lindsey实验室，未发表）。生物信息学分析表明该基因是一个R-SNARE，有证据表明它定位于高尔基体囊泡，并与PIN蛋白共定位。vamp 714突变体不能正确地将PIN蛋白定位于质膜，并且显示出降低的极性生长素运输（Lindsey实验室，未发表）。因此，我们假设VAMP 714是必不可少的PIN蛋白质的质膜传递。本项目的目的是阐明拟南芥VAMP 714及其相关蛋白在将PIN蛋白传递到质膜中的重要作用。具体目标是：1）通过与亚细胞区室标记物共定位来表征VA MP 7家族蛋白在拟南芥细胞中的定位; 2）使用回收抑制剂（例如latrunculin B和Brefeldin A）来确定VA MP 7家族蛋白定位是否构成内吞回收途径的一部分; 3）详细表征VA MP 7家族突变体的分生组织表型。初步数据表明，VAMP 714是维持根分生组织中干细胞生态位所必需的; 4）在vamp 7家族突变体中定位VampPIN; 5）观察vamp 7家族突变体中基因转录的变化，重点是分析已知为a）分生组织中生长素调节的关键基因和B）分生组织身份和功能调节因子的关键基因。该项目提供新工作方式的培训，特别是先进的生物成像，生物信息学和转录组学。也有机会使用该项目的信息来进一步开发我们已经开发和发布的预测数学模型，该模型使我们能够理解生长素之间的串扰，其合成和运输（通过PIN蛋白），以及根分生组织中的其他基因和信号组分（例如摩尔等人2015新植物学家207（4）：1110-1122）。

The plant hormone auxin plays a key role in coordinating diverse aspects of plant development, contributing to crop productivity. This includes the development of the shoot, root, flowers, seeds, fruit, stem elongation, tissue differentiation. An important aspect of the mode of action of auxin is its polar transport within developing tissues, to establish concentration gradients that create positional information to allow spatial regulation of gene expression patterns. The most important components of the directional auxin transport mechanism are the familiy auxin efflux carriers (the PIN proteins), some of which localize to specific faces of the plasmamembrane, to control directional auxin efflux. Rapid changes in cell polarity involve clathrin-mediated endocytosis and recycling of PINs. Auxin itself inhibits this recycling, resulting in an accumulation of PIN proteins at the plasmamembrane, so promoting its own efflux. While the endocytic model accounts for the dynamic relocalization of PINs to different surfaces of the cell, it does not explain mechanistically how PIN proteins are delivered to the plasmamembrane following their translation in the endoplasmic reticulum (ER). We have identified a novel mutant defective in the polar auxin transport pathway. The gene involved is VAMP714, identified as both gain of function and loss of function mutants (Lindsey lab, unpublished). Bioinformatics analysis suggests the gene is an R-SNARE, and evidence suggests it is localised to the trans-Golgi vesicles, and co-localises with PIN proteins. vamp714 mutants fail to correctly localise PIN proteins to the plasmamembrane, and show reduced polar auxin transport (Lindsey lab, unpublished). We therefore hypothesise that VAMP714 is essential for the delivery of PIN proteins to the plasmamembrane. The aim of this project is to characterise the essential role of the Arabidopsis thaliana VAMP714 and related VAMP proteins in delivering PIN proteins to the plasmamembrane. Specific objectives are: 1) to characterise the localisation of VAMP7 family proteins in Arabidopsis cells, by co-localization with subcellular compartment markers; 2) to determine whether VAMP7 family protein localisation forms part of the endocytic recycling pathway, using recycling inhibitors such as latrunculin B and Brefeldin A; 3) to characterise in detail the meristem phenotype of vamp7 family mutants. Preliminary data suggest that VAMP714 is required for maintenance of the stem cell niche in the root meristem; 4) to characterise PIN localization in vamp7 family mutants; 5) to characterise changes in gene transcription in vamp7 family mutants with a focus on the analysis of key genes known to be a) auxin-regulated in the meristem and b) regulators of meristem identity and function. The project provides training in New Ways of Working, and specifically in advanced bioimaging, bioinformatics and transcriptomics. There is also the opportunity to use information from the project to develop further the predictive mathematical model we have developed and published that allows us to understand the crosstalk between auxin, its synthesis and transport (via PIN proteins), and other genes and signalling components in the root meristem (e.g. Moore et al. 2015 New Phytologist 207(4): 1110-1122).