Expression and characterization of galacturonosyltransferase-6 (GAUT6) of the galacturonosyltranserferase-1 (GAUT1)-related gene family of Arabidopsis thaliana
Caffall, Kerry Hosmer
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Pectin is an important component of the plant primary wall that contributes to wall structural integrity, cell adhesion and modulation of wall expansibility. The function of pectic polysaccharides also extends to the elicitation of plant defense response-related signal transduction pathways. The pectic polysaccharides are defined by the presence of .-1,4-linked galacturonic acid residues in their backbone structures. The current knowledge of pectin structure, function and biosynthesis is being elucidated in the post-genomic era, however, the complexity of pectic polysaccharide structure, the difficulty of isolating membrane bound glycosyltransferases and the tolerance of plants to changes in wall structure, continue to hinder rapid progress towards the elucidation of the pectin biosynthetic process and machinery. Indeed, many hurdles were overcome by the identification of the Arabidopsis thaliana GALACTURONOSYLTRANSFERASE-1 (GAUT1), a protein that catalyzes the transfer of GalA from UDP-GalA onto oligogalacturonides, an activity consistent with pectin homogalacturonan galacturonosyltransferase activity. The identification of GAUT1 has led to the identification of 14 additional proteins that make up the Arabidopsis GAUT1-related gene family. Here it has been shown that most members of the Arabidopsis GAUT1-related gene family have conserved orthologs in Populus trichocarpa (black cotton wood; a woody dicot) and Oryza sativa (rice; a grassy monocot). In a survey of GAUT gene mutant cell walls, it was found that at least five other GAUT genes, GAUT 6, 8, 9, 10, and 11, are putative GalATs that function in pectin biosynthesis, but that at least three members, GAUT 12, 13 and 14, are putative GalATs involved in xylan synthesis. GAUT6 was selected for further analysis based on the gaut6 mutant wall compositional phenotype and by the relatedness of GAUT6 to GAUT1. A fractionation of gaut6 walls, compared to WT, was carried out and showed that gaut6 walls appeared to be altered in the synthesis of a pectic component indicative of homogalacturonan or rhamnogalacturonan-I. Additionally, GAUT6 expressed in Nicotiana benthamiana and Escherichia coli produced protein that had detectable HG:GalAT activity, proving that GAUT6 1414is able to transfer [C]GalA from UDP-[C]GalA onto oligogalacturonides, consistent with a function as an HG:GalAT and with a role in pectin biosynthesis.