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. 2012 Jan;158(1):465-75.
doi: 10.1104/pp.111.189779. Epub 2011 Nov 22.

Changes in cell wall biomechanical properties in the xyloglucan-deficient xxt1/xxt2 mutant of Arabidopsis

Yong Bum Park  1 , Daniel J Cosgrove
Affiliations

Changes in cell wall biomechanical properties in the xyloglucan-deficient xxt1/xxt2 mutant of Arabidopsis

Yong Bum Park et al. Plant Physiol. 2012 Jan.

Abstract

The main load-bearing network in the primary cell wall of most land plants is commonly depicted as a scaffold of cellulose microfibrils tethered by xyloglucans. However, a xyloglucan-deficient mutant (xylosyltransferase1/xylosyltransferase2 [xxt1/xxt2]) was recently developed that was smaller than the wild type but otherwise nearly normal in its development, casting doubt on xyloglucan's role in wall structure. To assess xyloglucan function in the Arabidopsis (Arabidopsis thaliana) wall, we compared the behavior of petiole cell walls from xxt1/xxt2 and wild-type plants using creep, stress relaxation, and stress/strain assays, in combination with reagents that cut or solubilize specific components of the wall matrix. Stress/strain assays showed xxt1/xxt2 walls to be more extensible than wild-type walls (supporting a reinforcing role for xyloglucan) but less extensible in creep and stress relaxation processes mediated by α-expansin. Fusicoccin-induced "acid growth" was likewise reduced in xxt1/xxt2 petioles. The results show that xyloglucan is important for wall loosening by α-expansin, and the smaller size of the xxt1/xxt2 mutant may stem from the reduced effectiveness of α-expansins in the absence of xyloglucan. Loosening agents that act on xylans and pectins elicited greater extension in creep assays of xxt1/xxt2 cell walls compared with wild-type walls, consistent with a larger mechanical role for these matrix polymers in the absence of xyloglucan. Our results illustrate the need for multiple biomechanical assays to evaluate wall properties and indicate that the common depiction of a cellulose-xyloglucan network as the major load-bearing structure is in need of revision.

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Figures

Figure 1.
Figure 1.
Size of wild-type and xxt1/xxt2 plants. A, Representative photographs of wild-type (WT) and xxt1/xxxt2 rosettes at 28 d old. B, Blade areas of fifth to eighth leaves (not counting cotyledons) of wild-type and xxt1/xxxt2 plants. C, Petiole and leaf blade lengths of wild-type and xxt1/xxxt2 plants. Error bars represent se. * Statistically different from the wild type at P < 0.05 by Student’s two-tailed t test (n = 16).
Figure 2.
Figure 2.
No detectable xyloglucan in 28-d-old xxt1/xxt2 plants. A and B, MALDI-TOF analysis (A) and HPAEC-PAD analysis (B) of XGOs resulting from XEG digestion of alkali-soluble cell wall fractions from wild-type (WT) and xxt1/xxt2 rosettes. These experiments were carried out twice with similar results. C, Immunoblot assays to detect XyG from six replicate petioles (with or without XEG digestion), detected with three antibodies (CCRC-M1, CCRC-M39, and CCRC-M87). D, Immunoblot assays with CCRC-M39, comparing XyG detection in xxt1/xxt2 petioles with a dilution series of extracts from wild-type petioles (six replicates each).
Figure 3.
Figure 3.
Elastic and plastic compliances of wild-type (WT) and xxt1/xxt2 petiole walls. Error bars represent se. * Statistically different from the wild type at P < 0.05 by Student’s two-tailed t test (13 ≤ n ≤ 15).
Figure 4.
Figure 4.
Acid-induced and α-expansin-induced wall extension of wild-type (WT) and xxt1/xxt2 walls. A, Extension of native walls in response to acidic buffer (pH 4.5). Extension of wild-type walls in pH 6.8 buffer is also shown. Each curve is the average of 10 responses. B, Extension of heat-inactivated wild-type and xxt1/xxt2 walls in pH 4.5 buffer upon the addition of cucumber α-expansin. These curves are averages of eight to 14 individual responses. The wild-type control without the addition of α-expansin is also shown. Arrows indicate when the incubation buffer was switched (A) and α-expansin was added (B). In both A and B, the total extension (30–150 min) of wild-type walls was significantly greater than that of xxt1/xxt2 walls at P < 0.015 by Student’s one-tailed t test. The initial length of samples was 5 mm.
Figure 5.
Figure 5.
Wall stress relaxation spectra of wild-type (WT) and xxt1/xxt2 walls. Each line represents the average of 12 measurements, and shaded regions indicate statistical significance between wild-type and xxt1/xxt2 samples (P < 0.05 by Student’s two-tailed t test; n = 12). Force was measured as gram force.
Figure 6.
Figure 6.
Fusicoccin (FC)-induced growth of wild-type (WT) and xxt1/xxt2 petioles. White circles with the solid line (wild type) and white squares with the broken line (xxt1/xxt2) indicate buffer controls. Error bars represent se. * Statistically significant difference between wild-type and xxt1/xxt2 values of fusicoccin-treated petioles at P < 0.05 by Student’s two-tailed t test (9 ≤ n ≤ 12).
Figure 7.
Figure 7.
Wall extension (creep) assays for wild-type (WT) and xxt1/xxt2 petioles. Change in length (Δ extension; μm) was measured for 2 h (1 h for PGase) after applying each protein or agent and subtracted from the value for buffer controls (wild type = 20.8, xxt1/xxt2 = 18.0). A, Wall extension assays of the wild type and xxt1/xxt2 in response to xylan-loosening proteins. Error bars represent se (10 ≤ n ≤ 18). B, Wall creep responses of the wild type and xxt1/xxt2 induced by pectin-loosening agents. Error bars represent se (10 ≤ n ≤ 12). * Statistically greater response in xxt1/xxt2 (P < 0.05 by one-tailed t test).
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