Reduced Wall Acetylation proteins play vital and distinct roles in cell wall O-acetylation in Arabidopsis

Abstract

The Reduced Wall Acetylation (RWA) proteins are involved in cell wall acetylation in plants. Previously, we described a single mutant, rwa2, which has about 20% lower level of O-acetylation in leaf cell walls and no obvious growth or developmental phenotype. In this study, we generated double, triple, and quadruple loss-of-function mutants of all four members of the RWA family in Arabidopsis (Arabidopsis thaliana). In contrast to rwa2, the triple and quadruple rwa mutants display severe growth phenotypes revealing the importance of wall acetylation for plant growth and development. The quadruple rwa mutant can be completely complemented with the RWA2 protein expressed under 35S promoter, indicating the functional redundancy of the RWA proteins. Nevertheless, the degree of acetylation of xylan, (gluco)mannan, and xyloglucan as well as overall cell wall acetylation is affected differently in different combinations of triple mutants, suggesting their diversity in substrate preference. The overall degree of wall acetylation in the rwa quadruple mutant was reduced by 63% compared with the wild type, and histochemical analysis of the rwa quadruple mutant stem indicates defects in cell differentiation of cell types with secondary cell walls.

Figure 1.

Growth phenotype of 18-d-old rwa triple and quadruple mutants grown under a regime of 12-h (right) and 16-h (left) photoperiod. The wild type and three of the triple mutants grew to larger size in long day. However, the rwa2 rwa3 rwa4 mutant and the rwa1 rwa2 rwa3 rwa4 (quadruple) mutants were light stressed and showed more extreme dwarfism under long-day than short-day conditions.

Figure 2.

The rwa quadruple mutant presents extreme dwarfism and improper flowering. A, Mature plants of the wild type (left) and the quadruple mutant (right; B). C, E, and F, The wild type. D and G, rwa1 rwa2 rwa3 rwa4. C to E, Unopened buds. F and G, Flowers. E, Dissection of the wild-type bud shown in C to show that, unlike in the quadruple mutant, pollen grains were already dehisced at this stage of flower development.

Figure 3.

rwa mutants exhibited shoot and root growth deficiency. Shoot growth was measured for 6-week-old triple mutants along with the wild type and rwa2 rwa4 double mutant, the only double mutant with reduced growth. A and B, Diameter of leaf rosette. C, Length of inflorescence stem. A, Short day. B and C, Long day. D, The root growth of the same genotype was measured between 2 and 7 d after germination (n = 10–25). Asterisks indicate significant difference from the wild type (Student’s t test, P < 0.05). Error bars indicate se (n = 4–19).

Figure 4.

The quadruple rwa mutant shows an absence of interfascicular fibers and has abnormal xylem cells. Representative transverse stem sections of the mature wild type and quadruple rwa mutant stained with toluidine blue. A and D, Wild type. B, C, and E, rwa1 rwa2 rwa3 rwa4. C, A magnified micrograph of B that highlights a vascular bundle. The interfascicular fibers (marked by “if” in the wild type; A) is absent in the quadruple mutant (equivalent region to “if” is indicated by a star in C). D and E, Xylem cells located in the most inner region of xylem. The arrow in E highlights a secondary cell wall thickening. Bars = 50 µm (A–C) and 10 µm (D and E).

Figure 5.

Cell wall acetylation is depleted in double, triple, and quadruple rwa mutants. The amount of released acetic acid upon saponification of AIR was measured. A, AIR prepared from green stems of 6-week-old double mutants (n = 3). B, AIR prepared from green stems of 6-week-old triple mutants (n = 2; statistic analysis performed assuming same sd as for the wild type in A). C, AIR prepared from 6-week-old rosette leaves of the wild type and quadruple mutant (n = 3). Asterisks above each bar indicate statistically significant difference compared with the wild type (Student’s t test, P < 0.05). Error bars indicate se.

Figure 6.

35S:RWA2 complements phenotype of quadruple mutant. A, Mature wild-type (WT) plants (left), quadruple mutant rwa1 rwa2 rwa3 rwa4 (center), and quadruple mutant transformed with 35S:RWA2 (right). B, The level of acetylation in 6-week-old rosette leaves from the wild type and quadruple mutant transformed with 35S:RWA2. Values are mean ± se (n = 4).

Figure 7.

XyG acetylation is disproportionally decreased in the rwa2 rwa3 rwa4 mutant. XyG-specific acetylation was measured by OLIMP in etiolated seedlings (A) and senesced dry stem (C). Total acetylation level of the same senesced stem samples was determined to compare with XyG acetylation (B). Asterisks above each bar indicate statistically significant difference when compared with the wild type (Student’s t test, P < 0.05). Error bars indicate se (A, n = 10; B and C, n = 5).

Figure 8.

Acidic XOS liberated from Arabidopsis AIR samples by GH10 endoxylanase hydrolysis. A, Principle component analysis scores scatterplot showing the separation of the wild type, rwa1 rwa2, rwa2 rwa3, rwa3 rwa4, and rwa1 rwa3 rwa4. B, Loading plot for Principal Component 1 (PC1; 67% variance) showing mass peaks separating rwa3 rwa4 and rwa1 rwa3 rwa4 (positive) from the wild type and other double mutants (negative). C, Peak intensities for MeGlcA (1→2)-linked xylotriose with varying acetylation level. Error bars represent sd with three to four biological replicates. Asterisk indicates P value < 0.05 (Student’s t test). Xyl, (1→4)-Linked β-d-Xyl; Ac, acetyl residue. Numeric symbol indicates number of Xyl or acetyl residues. [See online article for color version of this figure.]