Development and application of a suite of polysaccharide-degrading enzymes for analyzing plant cell walls

Abstract

To facilitate analysis of plant cell wall polysaccharide structure and composition, we cloned 74 genes encoding polysaccharide-degrading enzymes from Aspergillus nidulans, Aspergillus fumigatus, and Neurospora crassa and expressed the genes as secreted proteins with C-terminal Myc and 6x His tags. Most of the recombinant enzymes were active in enzyme assays, and optima for pH and temperature were established. A subset of the enzymes was used to fragment polysaccharides from the irregular xylem 9 (irx9) mutant of Arabidopsis. The analysis revealed a decrease in the abundance of xylan in the mutant, indicating that the IRX9 gene, which encodes a putative family 43 glycosyltransferase, is required for xylan synthesis.

Fig. 1.

Cell wall analysis of A. thaliana WT and irx9 stems. (A and B) Principal component plots for WT vs. mutant irx9 spectra. Spectra show a clear separation based on principal component (PC) 1. Spectra show differences in the carbohydrate fingerprint regions that correspond to the deformations of cellulosic and noncellulosic polymers. (C) Monosaccharide composition of WT and irx9 as determined by gas chromatography of alditol acetate derivatives of total polysaccharides from stems. (D) Capillary electrophoresis electropherograms of WT and irx9 after endo-xylanase AN1818.2 (Upper) and AN3613.2 (Lower) treatment. (E and F) Indirect immunofluorescence and differential interference contrast micrographs of LM11 antibody binding to transverse sections of WT (E) and irx9 (F).