The role of the plant-specific ALTERED XYLOGLUCAN9 protein in Arabidopsis cell wall polysaccharide O-acetylation

Abstract

A mutation in the ALTERED XYLOGLUCAN9 (AXY9) gene was found to be causative for the decreased xyloglucan acetylation phenotype of the axy9.1 mutant, which was identified in a forward genetic screen for Arabidopsis (Arabidopsis thaliana) mutants. The axy9.1 mutant also exhibits decreased O-acetylation of xylan, implying that the AXY9 protein has a broad role in polysaccharide acetylation. An axy9 insertional mutant exhibits severe growth defects and collapsed xylem, demonstrating the importance of wall polysaccharide O-acetylation for normal plant growth and development. Localization and topological experiments indicate that the active site of the AXY9 protein resides within the Golgi lumen. The AXY9 protein appears to be a component of the plant cell wall polysaccharide acetylation pathway, which also includes the REDUCED WALL ACETYLATION and TRICHOME BIREFRINGENCE-LIKE proteins. The AXY9 protein is distinct from the TRICHOME BIREFRINGENCE-LIKE proteins, reported to be polysaccharide acetyltransferases, but does share homology with them and other acetyltransferases, suggesting that the AXY9 protein may act to produce an acetylated intermediate that is part of the O-acetylation pathway.

Figure 1.

XyG oligosaccharide profiles of cotyledons from 12-d-old plants. The observed oligosaccharides are labeled using the nomenclature described by Fry et al. (1993). Ac, Acetyl; m/z, mass-to-charge ratio; OE, overexpression.

Figure 2.

Growth phenotype of axy9.2. Two plants from each genotype are shown for Col-0, axy9.1, axy9.2, and AXY9 overexpressed (OE) in the axy9.2 background. A, Seven-day-old etiolated seedlings. B to D, Soil-grown plants at 12 d (B), 21 d (C), and 36 d (D).

Figure 3.

Stem cross sections. A, Stem cross sections of the four genotypes stained with toluidine blue. B, Higher magnification of axy9.2 xylem tissue. Arrows indicate some of the large, open xylem vessels.

Figure 4.

Total wall-bound acetyl ester content for stem (A) and leaf tissue (B). Ac, Acetate; OE, overexpression. Error bars indicate sd (n = 3). Asterisks indicate statistically significant differences from Col-0 (Student’s t test, P < 0.05).

Figure 5.

A and B, Total acetic ester content of a pectin extract (A) and the remaining pellet (B) of etiolated seedling material. C and D, The uronic acid content was measured for the pectin extract (C), and the molar ratio of acetic acid to uronic acid was calculated (D). Ac, Acetate; OE, overexpression. Error bars indicate sd (n = 3). Asterisks indicate statistically significant differences from Col-0 (Student’s t test, P < 0.05).

Figure 6.

Subcellular localization of AXY9. A fusion construct of AXY9-GFP was transiently expressed in N. benthamiana with or without a Golgi marker (N-terminal fragment of a mannosidase from soybean [Glycine max] fused to mCherry; Nelson et al., 2007). Leaves of the infiltrated plants were examined by confocal microscopy.

Figure 7.

Protein topology of AXY9. A, Split YFP protein fusions, AXY9-YFP_N and AXY9-YFP_C, were coexpressed with the complementary half of the YFP expressed inside (TMD-YFP_N/C) or outside (YFP_N/C-TMD) of the Golgi in N. benthamiana and examined using confocal microscopy. B, Protease protection assay using microsomes prepared from N. benthamiana transiently expressing the AXY9-GFP fusion protein. A western blot using an anti-GFP antibody was used to detect the fusion peptide after digestion with (+) or without (−) protease and detergent.

Figure 8.

A hypothetical model of plant wall polysaccharide acetylation. The RWA proteins have been hypothesized to act as transporters for an acetyl donor molecule, possibly acetyl-CoA. The TBL proteins have been reported to likely represent polysaccharide acetyltransferases, but their donor substrate is unknown. The AXY9 protein, with its active site in the Golgi lumen, likely acts downstream of the RWA proteins but upstream of the polysaccharide acetyltransferases, as disruption of this gene affects multiple polysaccharides. As a putative acetyltransferase, the AXY9 protein may act to produce a hitherto unknown acetyl donor substrate used by some or all of the TBL polysaccharide acetyltransferases.