Mutations in multiple XXT genes of Arabidopsis reveal the complexity of xyloglucan biosynthesis.

Olga A Zabotina,Yi-Hsiang Chou,David Cavalier,Michael G Hahn,Utku Avci,Kenneth Keegstra,Stefan Eberhard,Linda Danhof,Sivakumar Pattathil

doi:10.1104/pp.112.198119

Abstract

Xyloglucan is an important hemicellulosic polysaccharide in dicot primary cell walls. Most of the enzymes involved in xyloglucan synthesis have been identified. However, many important details of its synthesis in vivo remain unknown. The roles of three genes encoding xylosyltransferases participating in xyloglucan biosynthesis in Arabidopsis (Arabidopsis thaliana) were further investigated using reverse genetic, biochemical, and immunological approaches. New double mutants (xxt1 xxt5 and xxt2 xxt5) and a triple mutant (xxt1 xxt2 xxt5) were generated, characterized, and compared with three single mutants and the xxt1 xxt2 double mutant that had been isolated previously. Antibody-based glycome profiling was applied in combination with chemical and immunohistochemical analyses for these characterizations. From the combined data, we conclude that XXT1 and XXT2 are responsible for the bulk of the xylosylation of the glucan backbone, and at least one of these proteins must be present and active for xyloglucan to be made. XXT5 plays a significant but as yet uncharacterized role in this process. The glycome profiling data demonstrate that the lack of detectable xyloglucan does not cause significant compensatory changes in other polysaccharides, although changes in nonxyloglucan polysaccharide amounts cannot be ruled out. Structural rearrangements of the polysaccharide network appear responsible for maintaining wall integrity in the absence of xyloglucan, thereby allowing nearly normal plant growth in plants lacking xyloglucan. Finally, results from immunohistochemical studies, combined with known information about expression patterns of the three genes, suggest that different combinations of xylosyltransferases contribute differently to xyloglucan biosynthesis in the various cell types found in stems, roots, and hypocotyls.

Highlights

Xyloglucan is an important hemicellulosic polysaccharide in dicot primary cell walls
Results from Driselase-high-performance anion-exchange chromatography (HPAEC) (Fig. 3) and oligosaccharide mass profiling (OLIMP) analyses of the xxt1 xxt2 and xxt1 xxt2 xxt5 mutants demonstrated that these plants lacked detectable xyloglucan, consistent with earlier results with the xxt1 xxt2 mutant (Cavalier et al, 2008; Park and Cosgrove, 2012) and leading to the conclusion that deleting the XXT5 gene from this double mutant does not restore the synthesis of xyloglucan
There were significant differences in the amounts of IP released from total alcohol-insoluble residue (AIR) and both hemicellulosic fractions (1 N KOH and 4 N KOH) prepared from xxt5, xxt1 xxt5, and xxt2 xxt5 plants compared with the ecotype Columbia (Col-0) wild type and xxt1 and xxt2 mutants (P, 0.01; Fig. 3)

Summary

Introduction

Xyloglucan is an important hemicellulosic polysaccharide in dicot primary cell walls. Screening of ethyl methanesulfonate-mutagenized plants for aberrant cell wall formation led to the isolation of the Arabidopsis mur mutant (Reiter et al, 1997), and subsequently, MUR3 was determined to be a xyloglucan galactosyltransferase that adds Gal to the third xylosyl residue, forming XXLG from XXXG (Madson et al, 2003). Heterologous expression of two of the genes in this family (At3g62720 and At4g02500) demonstrated that the encoded proteins have xylosyltransferase activity and were named XXT1 and XXT2, respectively (Cavalier and Keegstra, 2006) These biochemical studies demonstrated that the xyloglucan xylosyltransferases XXT1 and XXT2 have the same substrateacceptor specificity (i.e. transferring Xyl from UDP-Xyl to cellohexaose) and are able to catalyze the substitution of two glycosidic residues in adjacent positions, thereby generating GGXXGG structures

Methods

Results

Conclusion