Modification of the degree of 4-O-methylation of secondary wall glucuronoxylan

• Specific expression of GXMs in secondary wall forming cells.
• A complete loss of GlcA methylation in xylan of the gxm1/2/3 triple mutant.
• Elevation of glucuronoxylan methyltransferase activity in GXM overexpressors.
• Increased GlcA methylation in xylan of GXM overexpressors.

Plant secondary walls are the major constituent of plant biomass targeted for second-generation biofuel production. Therefore, a thorough understanding of how secondary walls are constructed is critical for a better utilization of plant biomass for biofuel production. One of the major components in secondary walls is xylan, which is composed of a linear chain of β-1,4-linked xylosyl residues. In Arabidopsis, about 10% of xylosyl residues in xylan are substituted with glucuronic acid (GlcA), of which 60% are methylated at O-4. By contrast, all of the GlcA substituents in Populus xylan are methylated at O-4. It is not known how the degree of GlcA methylation in xylan is controlled. In this report, we demonstrated that simultaneous T-DNA knockout mutations of the three glucuronoxylan methyltransferase (GXM) genes, GXM1, GXM2, and GXM3/GXMT1, which are specifically expressed in secondary wall-forming cells, led to a complete loss of GlcA methylation in xylan in Arabidopsis stems. Overexpression of GXM2 and GXM3 in wild-type Arabidopsis resulted in an up to 5-fold increase in glucuronoxylan methyltransferase activity and as a result, up to 90% of the GlcA side chains in xylan were methylated as opposed to 60% seen in the wild type. The increased degree of GlcA methylation in xylan had no discernable effects on cell wall sugar composition and lignin monomer composition. These results reveal that the activities of GXM1, GXM2 and GXM3 are responsible for all of the GlcA methylation in xylan in Arabidopsis stems and that the degree of GlcA methylation in xylan can be modified by altered expression of GXMs.