The role and synthesis of β1,3-galactans in plant cell wall formation

The plant cell wall associated hydroxyproline rich glycoproteins (HRGPs) play an important role in cell wall formation, but the mechanisms are not well understood. The HRGP family consists of the heavily glycosylated arabinogalactan proteins (AGPs) and the moderately glycosylated extensins (EXTs) and proline rich proteins (PRPs). The HRGP glycans are functionally import and investigation of their synthesis provides a way to elucidate HRGP function. In my PhD thesis I studied enzymes with putative functions in AGP glycosylation and cell wall formation. I show that the two GLYCOSIDE HYDROLASE 43 (GH43) family enzymes of Arabidopsis thaliana are Golgi localized β-1,3-galactosidases involved in cell wall biosynthesis. The gh43null mutant displays root cell expansion defects when grown on media supplemented with glucose. This phenotype is accompanied by increased cell wall matrix association of AGPs based on β-Yariv quantification and comprehensive microarray polymer profiling of sequentially extracted cell walls. My findings suggest that the loss of exo-β-1,3- galactosidase activity changes the cell wall extensibility in roots, which may be explained by the involvement of the GH43s in AGP glycan biosynthesis. I also investigated two members of the Arabidopsis glycosyl transferase family 31 (GT31), named GALT7 and GALT8 here. galt7galt8 exhibits severe growth defects and 30% cellulose reduction in both primary and secondary cell walls. Cellulose biosynthesis rate in galt7galt8 is reduced, but this is not due to reduced CESA transcript or protein levels. Both GALT7 and GALT8 proteins are Golgi localized and a recombinant GALT8 shows galactosyltransferase activity. Quantification of membrane and cell wall associated proteins using mass spectrometry revealed that galt7galt8 has reduced levels of a subgroup of fasciclin-like AGPs (FLAs). I hypothesize that GALT7 and GALT8 are involved in glycosylating this subgroup of FLAs and that a defect in their glycosylation reduces the rate of cellulose biosynthesis. The role of HRGPs in wood and wood formation in trees is poorly understood. A total of 157 HRGPs are expressed during secondary growth and wood formation in Populus tremula. To facilitate functional studies of these HRGPs I analyzed their phylogeny and expression during wood formation as well as enzymes putatively involved in HRGP glycosylation. Many of the genes show tight spatio-temporal expression patterns, indicative of specific functions during wood formation. Immunolabeling of mature wood with AGP and EXT glycan specific antibodies unveiled cell type specificity of different epitopes. These data were accompanied by β-Yariv characterization of AGPs and immunoblotting of AGP/EXT glycan epitopes in phloem/cambium, developing wood and mature wood fractions. This revealed differential glycosylation between different wood tissues. Combined the data document the diverse changes in HRGP related processes during wood formation at both gene expression and HRGP glycan biosynthesis level, and associate specific HRGPs and GTs to specific developmental processes during wood formation