Functional Identification of Genes for Pectin Biosynthetic Galacturonosyltransferases

University Of Georgia Research Foundation Inc, Athens GA

Investigators

Abstract

The enzyme activity of proteins encoded by putative pectin biosynthetic genes will be determined. Pectin is a family of structurally complex cell wall polysaccharides that has multiple roles in plant growth, development and disease resistance. The pectic polysaccharides homogalacturonan (HGA), rhamnogalacturonan-I (RG-I) and the substituted galacturonans rhamnogalacturonan II (RG-II) and xylogalacturonan (XGA) and apiogalacturonan all contain 1,4-linked alpha-D-galactosyluronic acid. Thus, pectin synthesis is catalyzed in part by galacturonosyltransferases that transfer galacturonic acid from UDP-GalA onto pectic oligosaccharide/polysaccharide acceptors. Arabidopsis putative galacturonosyltransferase (GalAT) genes were identified by trypsin cleavage of partially purified detergent-solubilized Arabidopsis GalAT containing protein fractions followed by liquid chromatography-tandem mass spectrometry to obtain amino acid sequences. Screening of the Arabidopsis gene/protein database with the derived amino acid sequences identified two putative GalAT proteins/genes based on the presence of predicted glycosyltransferase and membrane spanning domains. Heterologous expression of one of these genes (GALAT1) in a mammalian expression system in the presence of HGA yielded low levels of GalAT activity, suggesting the gene encodes an Arabidopsis thaliana UDP-galacturonic acid: HGA a-1,4-galacturonosyltransferase (GALAT1). Blast analysis of the Arabidopsis genome identified additional genes with high sequence similarity to GALTA1. At least 15 of these genes are proposed to be members of a putative GalAT gene family. The goals of the project are to clone selected members of the proposed GalAT gene family, heterologously express the cloned putative GalATs, and test the expressed proteins for GalAT activity. The developmental phenotype and wall pectin structure of plants mutated by T-DNA inserts in selected putative GalAT genes will also be determined. The long-term goals are to identify and characterize genes involved in pectin biosynthesis. The research will lead to the functional characterization of Arabidopsis genes that catalyze pectin synthesis. The identification of genes encoding pectin biosynthetic enzymes will allow the engineering of plants to produce pectins with modified structures and properties, pectins with improved agricultural value, and pectin-based neutraceutical and pharmacological products. The participation of historically underrepresented groups in the research will be achieved by recruiting undergraduate and graduate students through the University of Georgia Summer Undergraduate Research Program (SURP) (www.gradsch.uga.edu/rr/) and the new SURP-Bridge program, which draw in minority students from across Georgia. The latter program offers incoming graduate students from underrepresented groups research experience prior to graduate school in an effort to develop the students' research skills and increase their likelihood of success in graduate school.