The four Arabidopsis reduced wall acetylation genes are expressed in secondary wall-containing cells and required for the acetylation of xylan.

Xylan is one of the major polysaccharides in cellulosic biomass, and understanding the mechanisms underlying xylan biosynthesis will potentially help us design strategies to produce cellulosic biomass better suited for biofuel production. Although a number of genes have been shown to be essential for xylan biosynthesis, genes involved in the acetylation of xylan have not yet been identified. Here, we report the comprehensive genetic and functional studies of four Arabidopsis REDUCED WALL ACETYLATION (RWA) genes and demonstrate their involvement in the acetylation of xylan during secondary wall biosynthesis. It was found that the RWA genes were expressed in cells undergoing secondary wall thickening and their expression was regulated by SND1, a transcriptional master switch of secondary wall biosynthesis. The RWA proteins were shown to be localized in the Golgi, where xylan biosynthesis occurs. Analyses of a suite of single, double, triple and quadruple rwa mutants revealed a significant reduction in the secondary wall thickening and the stem mechanical strength in the quadruple rwa1/2/3/4 mutant but not in other mutants. Further chemical and structural analyses of xylan demonstrated that the rwa1/2/3/4 mutations resulted in a reduction in the amount of acetyl groups on xylan. In addition, the ratio of non-methylated to methylated glucuronic acid side chains was altered in the rwa1/2/3/4 mutant. Together, our results demonstrate that the four Arabidopsis RWA genes function redundantly in the acetylation of xylan during secondary wall biosynthesis.