The Arabidopsis Family GT43 Glycosyltransferases Form Two Functionally Nonredundant Groups Essential for the Elongation of Glucuronoxylan Backbone    

Abstract

There exist four members of family GT43 glycosyltransferases in the Arabidopsis (Arabidopsis thaliana) genome, and mutations of two of them, IRX9 and IRX14, have previously been shown to cause a defect in glucuronoxylan (GX) biosynthesis. However, it is currently unknown whether IRX9 and IRX14 perform the same biochemical function and whether the other two GT43 members are also involved in GX biosynthesis. In this report, we performed comprehensive genetic analysis of the functional roles of the four Arabidopsis GT43 members in GX biosynthesis. The I9H (IRX9 homolog) and I14H (IRX14 homolog) genes were shown to be specifically expressed in cells undergoing secondary wall thickening, and their encoded proteins were targeted to the Golgi, where GX is synthesized. Overexpression of I9H but not IRX14 or I14H rescued the GX defects conferred by the irx9 mutation, whereas overexpression of I14H but not IRX9 or I9H complemented the GX defects caused by the irx14 mutation. Double mutant analyses revealed that I9H functioned redundantly with IRX9 and that I14H was redundant with IRX14 in their functions. In addition, double mutations of IRX9 and IRX14 were shown to cause a loss of secondary wall thickening in fibers and a much more severe reduction in GX amount than their single mutants. Together, these results provide genetic evidence demonstrating that all four Arabidopsis GT43 members are involved in GX biosynthesis and suggest that they form two functionally nonredundant groups essential for the normal elongation of GX backbone.

Identification of a disaccharide side chain 2-O-α-D-galactopyranosyl-α-D-glucuronic acid inArabidopsisxylan

“…The 2D 1 H- 13 C HSQC spectrum of Arabidopsis xylan showed a cross peak of H-1 and C-1 signals at 5.42 and 98 ppm (Fig. 2) corresponding to the resonances of Gal-MeGlcA disaccharide substituents, which is the same as that observed in E. globulus xylan.…”

“…In summary, our results from the 2D H- 13 C NMR spectroscopy and the MALDI-TOF-MS provide unequivocal evidence demonstrating that Arabidopsis xylan contains Gal-GlcA disaccharide side chains in addition to substitutions with GlcA, MeGlcA and acetyl groups (Fig. 4).…”

“…The MALDI-TOF-MS data showing the presence of ions corresponding to (Gal-GlcA)-substituted xylooligomers not only is consistent with the 2D NMR data but also demonstrate that the disaccharide side chain in Arabidopsis xylan is mainly composed Figure 2. two-dimensional 1 h- 13 C hSQC nmr spectrum of acetylated xylan from wildtype Arabidopsis stems. acetylated xylan was extracted with DmSo 28 and digested with β-endoxylanase m6 (megazyme) to generate xylooligosaccharides, 16 which were subsequently subject to structural analysis using nmr spectroscopy.…”

“…As a typical dicot xylan, Arabidopsis xylan consists of the xylosyl backbone, the reducing end tetrasaccharide sequence, and substitutions of xylosyl residues with GlcA/MeGlcA residues and acetyl groups. 5,10 Genetic and biochemical studies of xylan biosynthesis in Arabidopsis have revealed that the elongation of the xylosyl backbone requires glycosyltransferases from both GT43 (IRX9/I9H and IRX14/I14H) and GT47 (IRX10/IRX10L) families, 5,[11][12][13][14][15][16] the biosynthesis of the reducing end tetrasaccharide sequence involves glycosyltransferases from GT8 (IRX8 and PARVUS) and GT47 (FRA8/F8H) families, 5,6,11,[17][18][19] the substitutions by GlcA residues is mediated by 3 GT8 glycosyltransferases (GUX1/2/3), 20,21 and the methylation of GlcA residues is catalyzed by 3 DUF579 , and proposed that ESK1 and its homologs are putative acetyltransferases responsible for xylan acetylation. it was noticed that the 1 h- 13 C nmr spectroscopy and found that the signal peak at 5.42 ppm in the 1 h nmr spectrum was attributed to Glca residues substituted at O-2 with α-D-galactose (Gal), indicating the presence of Gal-Glca disaccharide side chains in Arabidopsis xylan.…”

Quantifying Bio-Engineering: The Importance of Biophysics in Biofuel Research

“…This lowered mechanical strength was attributed to differences in cellulose microfibril orientation in the walls of fra1. Irregular xylem8 (irx8), irx9, and irx14 mutations of Arabidopsis thaliana resulting in defects in glucuronoxylan biosynthesis (Keppler and Showalter 2010;Pena et al 2007;Lee et al 2010). These defects lead to decreased mechanical strength of the plant stems.…”