Analysis of novel polar auxin transport pathway components in Arabidopsis

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 The gene involved is VAMP714, identified as both gain of function and loss of function mutants (Lindsey lab, unpublished).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fail to correctly localise PIN proteins to the plasmamembrane, and show reduced polar auxin transport (Lindsey lab, unpublished).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).