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

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.

Figure 1.

Analysis of the xxt1 xxt2 xxt5 T-DNA insertion mutant. A to C, Gene models of XXT1, XXT2, and XXT5, respectively. Noncoding regions and introns are represented by black lines; coding regions are represented by gray boxes. The T-DNA insertion site for each gene is indicated along with the T-DNA border. LB, Left border; LP, left primer; RP, right primer; ?, unknown border. D, RT-PCR analysis of the wild type (Col-0; left panel) and the xxt1 xxt2 xxt5 T-DNA insertion mutant (right panel) using the gene-specific primers denoted in A to C that flank the T-DNA inserts. Further details can be found in Supplemental Data S1.

Figure 2.

Phenotype of roots from xxt mutant lines and wild-type seedlings. All seedlings were grown under the same conditions on agar plates for 7 d as described in “Materials and Methods.” Plates were oriented vertically in the growth chamber. The labels at bottom left of each image indicate the plant line shown. Root hairs in A, B, and C were similar. Root hairs boxed in D to H were magnified and placed in the same image to clearly show the root hair phenotype. Bar in A = 200 µm for all images, except the insets.

Figure 3.

Quantity of the Driselase-generated xyloglucan signature fragment, IP, estimated by HPAEC analysis. Results are expressed as relative amounts of IP obtained after digestion of total AIR, and soluble in 1 n KOH and 4 n KOH fractions prepared from mutant lines in comparison with the amount of IP obtained from the same fraction prepared from Col-0 wild-type plants, which was taken as 100%. One milligram of each sample was dissolved in 30 µL of acetate buffer containing approximately 0.5 units of Driselase and incubated for 18 h at 37°C. The total volume of each digest was analyzed by HPAEC. Analyses were performed on three independent sample preparations for each mutant and wild-type plant. Results were averaged and expressed as mean values (n = 3 biological replications) ± sd.

Figure 4.

OLIMP of XEG-digested 1 n KOH and 4 n KOH sequential extracts of the Col-0 wild type and xxt mutants. Each XGO is named according to the nomenclature described by Fry et al. (1993). The relative abundance of each XGO is presented as a mean value (n = 3 biological replications) ± sd.

Figure 5.

Glycome profiling of cell wall extracts prepared from Arabidopsis Col-0 and xxt mutants. Cell walls of each plant line were prepared from whole seedlings of Arabidopsis Col-0 wild type (WT) and all single and multiple xxt mutants grown in liquid culture. The walls were subjected to sequential extraction with α-amylase, CDTA, 1 n KOH, and 4 n KOH as described in “Materials and Methods.” The solubilized extracts were then screened against an array of plant glycan-directed monoclonal antibodies (Pattathil et al., 2010) using ELISAs. The panels at right (colored boxes) depict the groups of antibodies used, identified according to the polysaccharides predominantly recognized by each group (for additional details about the individual antibodies used, see Supplemental Table S2). The extent of antibody binding is represented as a colored heat map, with the brightest yellow depicting the strongest binding and black depicting no binding.

Figure 6.

Immunofluorescence labeling of transverse sections of roots taken from 6-d-old seedlings of wild-type Col-0 (WT) and all xxt mutant plants using selected xyloglucan-directed antibodies. Toluidine blue-stained sections of wild-type and mutant lines show the overall root morphology from left to right. The xyloglucan-directed antibodies used (CCRC-M1, CCRC-M58, CCRC-M88, and CCRC-M101) recognize distinct xyloglucan epitopes and are identified with labels in the wild-type images. Bars = 50 µm for all images.

Figure 6.

Immunofluorescence labeling of transverse sections of roots taken from 6-d-old seedlings of wild-type Col-0 (WT) and all xxt mutant plants using selected xyloglucan-directed antibodies. Toluidine blue-stained sections of wild-type and mutant lines show the overall root morphology from left to right. The xyloglucan-directed antibodies used (CCRC-M1, CCRC-M58, CCRC-M88, and CCRC-M101) recognize distinct xyloglucan epitopes and are identified with labels in the wild-type images. Bars = 50 µm for all images.

Figure 7.

Immunofluorescence labeling of transverse sections of hypocotyls from wild-type Col-0 (WT) and all xxt mutant plants using selected xyloglucan-directed antibodies. Toluidine blue-stained sections of wild-type and mutant lines show the overall hypocotyl morphology from left to right. The xyloglucan-directed antibodies used (CCRC-M1, CCRC-M58, CCRC-M88, and CCRC-M101) recognize distinct xyloglucan epitopes and are identified with labels in the wild-type images. Bars = 50 µm for all images.

Figure 7.

Immunofluorescence labeling of transverse sections of hypocotyls from wild-type Col-0 (WT) and all xxt mutant plants using selected xyloglucan-directed antibodies. Toluidine blue-stained sections of wild-type and mutant lines show the overall hypocotyl morphology from left to right. The xyloglucan-directed antibodies used (CCRC-M1, CCRC-M58, CCRC-M88, and CCRC-M101) recognize distinct xyloglucan epitopes and are identified with labels in the wild-type images. Bars = 50 µm for all images.

Figure 8.

Effect of pectate lyase treatment of hypocotyl sections on xyloglucan and homogalacturonan labeling patterns. With the exception of the JIM5 section, which was treated with CAPS buffer only as a control, sections were treated with pectate lyase (PL) in CAPS buffer (pH 10) for 2 h at room temperature followed by immunolabeling with the indicated antibodies. No differences in the immunolabeling patterns were observed with xyloglucan-directed antibodies compared with those shown in Figure 7. Homogalacturonan epitopes recognized by JIM5 were almost completely removed from the sections after pectate lyase treatment compared with the control labeling with JIM5. Bar = 50 µm for all images.

Figure 9.

Immunofluorescence labeling of transverse sections of basal parts of inflorescence stems taken from 6-week-old wild-type Col-0 (WT) and all xxt mutant plants using selected xyloglucan-directed antibodies. Toluidine blue-stained sections of wild-type and mutant lines show the overall stem morphology from left to right. The xyloglucan-directed antibodies used (CCRC-M1, CCRC-M58, CCRC-M88, and CCRC-M101) recognize distinct xyloglucan epitopes and are identified with labels in the wild-type images. Bars = 50 µm for all images.

Figure 9.

Immunofluorescence labeling of transverse sections of basal parts of inflorescence stems taken from 6-week-old wild-type Col-0 (WT) and all xxt mutant plants using selected xyloglucan-directed antibodies. Toluidine blue-stained sections of wild-type and mutant lines show the overall stem morphology from left to right. The xyloglucan-directed antibodies used (CCRC-M1, CCRC-M58, CCRC-M88, and CCRC-M101) recognize distinct xyloglucan epitopes and are identified with labels in the wild-type images. Bars = 50 µm for all images.

Figure 10.

Expression of XXT1, XXT2, GT3, GT4, and XXT5 in Arabidopsis organs. The AtGE Development series (Schmid et al., 2005) was queried for XXT1 (At3g62720), XXT2 (At4g02500), GT3 (At5g07720), GT4 (At1g18690), and XXT5 (At1g74380) expression using the AtGenExpress Visualization Tool (http://jsp.weigelworld.org/expviz/expviz.jsp). Gene expression in roots, hypocotyls, leaves (composed of the first two leaves to emerge), whole rosettes from 7-d-old seedlings, and stems from 21-d-old plants is shown.