Disrupting two Arabidopsis thaliana xylosyltransferase genes results in plants deficient in xyloglucan, a major primary cell wall component

Abstract

Xyloglucans are the main hemicellulosic polysaccharides found in the primary cell walls of dicots and nongraminaceous monocots, where they are thought to interact with cellulose to form a three-dimensional network that functions as the principal load-bearing structure of the primary cell wall. To determine whether two Arabidopsis thaliana genes that encode xylosyltransferases, XXT1 and XXT2, are involved in xyloglucan biosynthesis in vivo and to determine how the plant cell wall is affected by the lack of expression of XXT1, XXT2, or both, we isolated and characterized xxt1 and xxt2 single and xxt1 xxt2 double T-DNA insertion mutants. Although the xxt1 and xxt2 mutants did not have a gross morphological phenotype, they did have a slight decrease in xyloglucan content and showed slightly altered distribution patterns for xyloglucan epitopes. More interestingly, the xxt1 xxt2 double mutant had aberrant root hairs and lacked detectable xyloglucan. The reduction of xyloglucan in the xxt2 mutant and the lack of detectable xyloglucan in the xxt1 xxt2 double mutant resulted in significant changes in the mechanical properties of these plants. We conclude that XXT1 and XXT2 encode xylosyltransferases that are required for xyloglucan biosynthesis. Moreover, the lack of detectable xyloglucan in the xxt1 xxt2 double mutant challenges conventional models of the plant primary cell wall.

Figure 1.

Analysis of xxt1, xxt2, and xxt1 xxt2 T-DNA Insertion Mutants. (A) Gene models of XXT1 and XXT2. Noncoding regions and introns are represented by heavy black lines; coding regions are represented by gray rectangles. The black box with diagonal lines represents the predicted transmembrane domain encoded by each gene. The T-DNA insertion site for each gene is indicated. The position and orientation of each primer is also indicated. (B) RT-PCR analysis of wild-type (Columbia [Col-0]) and T-DNA insertion mutants. Total RNA was isolated from 7-d-old wild-type, xxt1, xxt2, and xxt1 xxt2 mutant seedlings and digested twice with DNase. Wild-type and T-DNA insertion lines were assayed for the presence of XXT1 and XXT2 transcripts with RT-PCR for 35 cycles with gene-specific primers (see Supplemental Table 2 online). The experiment was conducted on three different pools of 7-d-old etiolated seedlings from each line, and the typical result of an ethidium bromide–stained agarose gel is presented.

Figure 2.

The xxt1 xxt2 T-DNA Insertion Mutant Has a Root Hair Phenotype. (A) to (D) Seven-day-old seedlings of wild-type, xxt1, xxt2, and xxt1 xxt2 grown on vertical agar plates. Bar = 1 mm. (E) Wild-type root hairs. Bar = 200 μm. (F) xxt1 xxt2 root hairs. Bar = 200 μm. (G) Representative example of a wild-type root hair located just above the zone of elongation. Bar = 50 μm. (H) Representative example of an xxt1 xxt2 double mutant root hair located just above the zone of elongation. Bar = 25 μm. (I) and (J) Root hairs of xxt1 xxt2 double mutants (T2 generation) complemented with either 35S_pro:XXT1 (I) or 35S_pro:XXT2 (J). Bars = 200 μm.

Figure 3.

OLIMP of XyG Oligosaccharides Released by XyG-Specific Endoglucanase. (A) OLIMP of XyG oligosaccharides released from AIR preparations of 4-d-old wild-type (Col-0), xxt1, and xxt2 etiolated seedlings digested with XEG. The relative abundance of XyG oligosaccharide is presented as mean value (n = 6 hypocotyls) ± sd. The m/z of each XyG oligosaccharide is presented in parentheses. All Ac, total relative amount of acetylated XyG oligosaccharides; All F, total relative amount of fucosylated XyG oligosaccharides; All L, total relative amount of galactosylated XyG oligosaccharides. (B) and (C) HPAEC chromatograms of a crude cell wall preparation of 7-d-old wild-type (B) and xxt1 xxt2 double mutant (C) etiolated seedlings digested with XEG. Each XyG oligosaccharide is named according to a XyG nomenclature detailed by Fry et al. (1993), where the XyG molecule is described from the nonreducing end to the reducing end with a single letter that denotes a specific glucosyl residue substitution pattern. An unsubstituted β-glucan backbone d-Glcp is assigned “G,” an α-d-Xylp-(1→6)-β-d-Glcp substitution pattern is assigned “X,” a β-d-Galp-(1→2)-α-d-Xylp-(1→6)-β-d-Glcp substitution pattern is assigned “L,” and an α-l-Fucp-(1→2)-β-d-Galp-(1→2)-α-d-Xylp-(1→6)-β-d-Glcp substitution pattern is assigned “F.” PAD, pulsed amperometric detector.

Figure 4.

Immunofluorescent Labeling of Wild-Type and Mutant Roots with XyG-Directed Antibodies. Immunofluorescent labeling of 250-nm transverse sections taken from ∼5 mm above the root apex of 4-d-old wild-type (Col-0), xxt1, xxt2, and xxt1 xxt2 seedlings. The antibodies used were directed against different epitopes of XyG and are described in Methods. (A) to (E) Col-0 root cross-sections labeled with CCRC-M1 (A), CCRC-M39 (B), CCRC-M58 (C), CCRC-M87 (D), and CCRC-M89 (E). (F) to (J) xxt1 single mutant root cross-sections labeled with CCRC-M1 (F), CCRC-M39 (G), CCRC-M58 (H), CCRC-M87 (I), and CCRC-M89 (J). (K) to (O) xxt2 single mutant root cross-sections labeled with CCRC-M1 (K), CCRC-M39 (L), CCRC-M58 (M), CCRC-M87 (N), and CCRC-M89 (O). (P) to (T) xxt1 xxt2 double mutant root cross-sections labeled with CCRC-M1 (P), CCRC-M39 (Q), CCRC-M58 (R), CCRC-M87 (S), and CCRC-M89 (T).

Figure 5.

The xxt1 xxt2 T-DNA Insertion Mutant has a 90% Decrease in 2-Xylose. (A) A schematic of the primary fragmentation patterns and corresponding m/z of 2-Xyl and 4-Xyl PMAA derivatives. (B) Electron-impact mass spectra of the 2-,4-Xyl peak from wild-type (Col-0) and xxt1 xxt2 PMAA derivatives of crude cell wall preparations. Arrows denote the diagnostic fragmentation ions for 2-Xyl (solid arrows) and 4-Xyl (dashed arrows).

Figure 6.

The xxt1 xxt2 Double T-DNA Insertion Mutant Lacked Driselase-Susceptible XyG. HPAEC analysis of Driselase-hydrolyzed AIR from wild-type (Col-0) (A) and xxt1 xxt2 (B) etiolated seedlings. GGMX, peak comprised of galactose, glucose, mannose, and xylose; X₂, xylobiose.

Figure 7.

xxt2 and xxt1 xxt2 T-DNA Insertion Mutants Have a Reduction in Stiffness and Ultimate Stress. Mechanical properties of 4-d-old etiolated hypocotyls of wild-type (Col-0; n = 217), xxt1 (n = 96), xxt2 (n = 51), and xxt1 xxt2 (n = 59) T-DNA insertion mutants. Stiffness (A); ultimate stress (B). Asterisks denote statistically significant difference with respect to the wild type (P < 0.001). Error bars ± sd.