Plant Glycosyltransferases Beyond CAZy: A Perspective on DUF Families

Hemicelluloses are polysaccharides in plant cell walls that have beta-(1-->4)-linked backbones with an equatorial configuration. Hemicelluloses include xyloglucans, xylans, mannans and glucomannans, and beta-(1-->3,1-->4)-glucans. These types of hemicelluloses are present in the cell walls of all terrestrial plants, except for beta-(1-->3,1-->4)-glucans, which are restricted to Poales and a few other groups. The detailed structure of the hemicelluloses and their abundance vary widely between different species and cell types. The most important biological role of hemicelluloses is their contribution to strengthening the cell wall by interaction with cellulose and, in some walls, with lignin. These features are discussed in relation to widely accepted models of the primary wall. Hemicelluloses are synthesized by glycosyltransferases located in the Golgi membranes. Many glycosyltransferases needed for biosynthesis of xyloglucans and mannans are known. In contrast, the biosynthesis of xylans and beta-(1-->3,1-->4)-glucans remains very elusive, and recent studies have led to more questions than answers.

Forward genetic screens have led to the isolation of several genes involved in secondary cell wall formation. A variety of evidence, however, suggests that the list of genes identified is not exhaustive. To address this problem, microarray data have been generated from tissue undergoing secondary cell wall formation and used to identify genes that exhibit a similar expression pattern to the secondary cell wall-specific cellulose synthase genes IRREGULAR XYLEM1 (IRX1) and IRX3. Cross-referencing this analysis with publicly available microarray data resulted in the selection of 16 genes for reverse genetic analysis. Lines containing an insertion in seven of these genes exhibited a clear irx phenotype characteristic of a secondary cell wall defect. Only one line, containing an insertion in a member of the COBRA gene family, exhibited a large decrease in cellulose content. Five of the genes identified as being essential for secondary cell wall biosynthesis have not been previously characterized. These genes are likely to define entirely novel processes in secondary cell wall formation and illustrate the success of combining expression data with reverse genetics to address gene function.

Coexpression patterns of gene expression across many microarray data sets may reveal networks of genes involved in linked processes. To identify factors involved in cellulose biosynthesis, we used a regression method to analyze 408 publicly available Affymetrix Arabidopsis microarrays. Expression of genes previously implicated in cellulose synthesis, as well as several uncharacterized genes, was highly coregulated with expression of cellulose synthase (CESA) genes. Four candidate genes, which were coexpressed with CESA genes implicated in secondary cell wall synthesis, were investigated by mutant analysis. Two mutants exhibited irregular xylem phenotypes similar to those observed in mutants with defects in secondary cellulose synthesis and were designated irx8 and irx13. Thus, the general approach developed here is useful for identification of elements of multicomponent processes.