Arabidopsis 2010: RUI: Genetic and Physiological Characterization of Arabidopsis Plasma Membrane H+-ATPase Mutants

Western Washington University, Bellingham WA

Investigators

Abstract

The initial annotation of the complete sequence of Arabidopsis thaliana indicates that of the 11,601 distinct gene types found, only 35% are represented in the genome by one gene, while 37% are represented by gene families with five or more different members. For a majority of the larger gene families, the molecular function of the encoded proteins is known. However, the role of individual gene family members in specific physiological or development pathways is often uncertain. The focus of this project is to develop protocols to assist in describing the in planta function of individual gene family members in the AHA, Arabidopsis plasma membrane H+-ATPase, gene family. The AHA genes code for proteins that couple ATP hydrolysis to proton transport across the plasma membrane of plant cells. The proton-motive force produced by the ATPases then drives the transport of solutes via carriers and channels. In Arabidopsis, the proton pumps comprise a gene family with at least twelve gene isoforms. Expressed in different tissues, the pumps provide the underlying energy for a variety of fundamental plant processes such as cellular homeostasis, mineral and metabolite transport, cell growth and plant morphogenesis, and a variety of responses to the environment including stomatal control, tropic responses, and defense related mechanisms. As a first step towards determining the function of the AHA genes, we have screened over 80,000 Arabidopsis T-DNA insertion lines and have identified twenty-nine mutants. This collection of mutants includes ten of twelve AHA gene family members, with multiple alleles for many isoforms. Exhaustive screening of the current 80,000 lines and screening of recently available lines should make it possible to reach the goal of finding multiple alleles for each gene family member. To understand the role these genes individually and in the gene family, we will take the following approaches: - We will isolate, outcross and prepare aha mutant lines for analysis and distribution to the Arabidopsis Biological Resource Center, Columbus, OH 43210. - We will perform genetic crosses and create multiple insert lines (i.e., double, triple mutants, etc.) in order to uncover genetic redundancy in the gene family. - We will test for conditional phenotypes in the mutant AHA lines by comparing mutants to wild-type plants under conditions designed to reveal differences in development, responses to environmental stimuli, nutrient uptake, etc. - We will establish an on-line database of experimental conditions designed for a broad range of phenotypic analysis. Preliminary protocols will be available Year 1, first quarter. Detailed protocols and associated data will be added as it is generated. The availability of twenty-nine AHA gene-family mutant alleles provides the raw materials for substantial progress into the function of the AHA genes and gene family. The availability of this resource at a predominantly undergraduate institution provides a means of integrating genomic scale research with undergraduate training. The AHA genes can be referenced at http://www.ncbi.nlm.nih.gov:80/Database/index.html, via the accession numbers: At2g18960, AT4g30190, MJB24.16, T17F15.180, At2g24520, At2g07560, F27H5.120, T12K4.90, F23A5.1, F20D23.4, MRG21.9, AT4g11730.