Xyloglucan biosynthesis: Identification and characterization of fucosyltransferase and cellulose synthase-like genes

Plant cell walls provide strength and shape to plant cells, and play a critical role in intra- and intercellular communications during plant growth and development. Xyloglucan is the major hemicellulose in the primary wall of dicots and non-graminaceous monocots. XyG is composed of a beta-(1,4)-glucan backbone that is usually decorated with alpha-(1,6)-xylose, beta-(1,2)-galactose and alpha-(1,2)-fucose residues. Until very recently, none of the genes encoding xyloglucan synthetic enzymes had been identified. I am therefore interested in identification of these genes and in exploration of their expression patterns during plant growth and development.; Utilizing sequence information obtained from a partially purified xyloglucan fucosyltransferase enzyme from pea epicotyls, the full-length cDNAs encoding Arabidopsis and pea XyG fucosyltransferase, AtFut1 and PsFut1, were identified. The biochemical function of AtFUT1 was confirmed by the immunoprecipitation of XyG fucosyltransferase activities from Arabidopsis microsomes using antibodies against E. col-expressed AtFUT1 proteins and, by the presence of XyG-specific fucosyltransferase activity in COS7 cells expressing the AtFut1 gene. The identity of the PsFUT1 was confirmed through sequence comparisons with the purified enzyme, its sequence similarity with AtFUT1, and its expression in COS7 cells. In young pea plants, the expression level of PsFut1 was found to be highly correlated with the internode elongation rate of etiolated pea seedlings.; Cellulose synthase-like proteins of Arabidopsis show 7% to 46% identity with cellulose synthases and, therefore, have been hypothesized to be involved in the biosynthesis of wall matrix polysaccharides such as XyG. Expression profiling of AtCsl genes was performed by examining EST libraries, MPSS libraries and microarray databases, but did not reveal any strong candidate genes for encoding the xyloglucan backbone glucan synthase.; Among all AtCsl genes, those of the D subfamily have the highest level of identity to AtCesA genes. To distinguish whether AtCslD proteins might be isoforms of AtCesA proteins or enzymes involved in the biosynthesis of matrix polysaccharides, a polyclonal antibody against an AtCslD2 peptide was generated and utilized to locate the proteins at Golgi membranes using cellular membrane fractionation techniques such as sucrose density gradient and two-phase partitioning. Therefore AtCslD genes are likely to encode enzymes involved in the matrix polysaccharide biosynthesis instead of CesA proteins.; In another attempt to identify functions for AtCslD genes, transgenic plants containing RNAi constructs targeting AtCslD transcripts were generated. These plants showed deformed cotyledons, were dwarfed as adult plants, and had shortened roots with more branches. However, the transcript suppression of AtCslD genes was only clearly seen in cotyledons, and no clear alteration in cell wall sugar composition was detected. Therefore, the exact biochemical functions of AtCslD proteins still could not be determined.