Absence of branches from xylan in Arabidopsis gux mutants reveals potential for simplification of lignocellulosic biomass

Abstract

As one of the most abundant polysaccharides on Earth, xylan will provide more than a third of the sugars for lignocellulosic biofuel production when using grass or hardwood feedstocks. Xylan is characterized by a linear β(1,4)-linked backbone of xylosyl residues substituted by glucuronic acid, 4-O-methylglucuronic acid or arabinose, depending on plant species and cell types. The biological role of these decorations is unclear, but they have a major influence on the properties of the polysaccharide. Despite the recent isolation of several mutants with reduced backbone, the mechanisms of xylan synthesis and substitution are unclear. We identified two Golgi-localized putative glycosyltransferases, GlucUronic acid substitution of Xylan (GUX)-1 and GUX2 that are required for the addition of both glucuronic acid and 4-O-methylglucuronic acid branches to xylan in Arabidopsis stem cell walls. The gux1 gux2 double mutants show loss of xylan glucuronyltransferase activity and lack almost all detectable xylan substitution. Unexpectedly, they show no change in xylan backbone quantity, indicating that backbone synthesis and substitution can be uncoupled. Although the stems are weakened, the xylem vessels are not collapsed, and the plants grow to normal size. The xylan in these plants shows improved extractability from the cell wall, is composed of a single monosaccharide, and requires fewer enzymes for complete hydrolysis. These findings have implications for our understanding of the synthesis and function of xylan in plants. The results also demonstrate the potential for manipulating and simplifying the structure of xylan to improve the properties of lignocellulose for bioenergy and other uses.

Fig. 1.

Identification of candidate secondary cell wall GTs. (A) Coexpression clustering of all putative GT genes from the CAZy database, using transcriptomics data from the AtGenExpress expression atlas. Organ expression levels are shown above (green) or below (red) the average for the gene. White boxed section with branch labeled (*) is enlarged in B. (C) Subcellular localization of GUX1 and GUX2. GFP-tagged GUX1 and GUX2 and Golgi marker GONST1-YFP were transiently expressed in tobacco leaves and examined by laser-scanning confocal microscopy.

Fig. 2.

Xylan structure and quantity in stem of WT and gux mutant plants. (A) AIR from WT, gux1, gux2, and gux1 gux2 stems was digested with xylanase NpXyn11A and analyzed by PACE. (B) Quantification of Xyl residues in the xylan backbone substituted with [Me]GlcA by PACE as described in A. (C) Quantification of the xylan backbone by PACE as described in A. (D) Monosaccharide analysis of WT and gux1 gux2 stem. AIR was hydrolyzed to constituent monosaccharide sugars using TFA and analyzed by HPAEC-PAD. (Inset) Adjusted scale for GlcA. (E) MALDI-TOF MS analysis of xylan structure. Oligosaccharides produced by NpXyn11A digestion of stem AIR were deuteropermethylated. Note the different intensity scales in E. All data are from at least three independent biological experiments. (Error bars represent SD.) *Significantly different from WT, P < 0.05, Student t test.

Fig. 3.

GuxT activity in WT and gux1 gux2 stem microsomes. (A) Microsomes were incubated with fluorescently labeled acceptor [AMAC-(Xyl)₆] and UDP-GlcA. Resulting charged assay product was separated by electrophoresis. (B) GuxT assay product characterization. Assay product from WT microsomes was digested with NpXynllA, β-xylosidase, or β-xylosidase and α-glucuronidase together, and the product analyzed by electrophoresis.

Fig. 4.

Properties of WT and gux1 gux2 xylan. (A) WT and gux1 gux2 AIR was sequentially extracted using CDTA, Na₂CO₃, 1 M KOH, and 4 M KOH. Resulting fractions were analyzed by PACE using NpXyn11A. (B) AIR sequentially extracted as in A, hydrolyzed to monosaccharide sugars using methanolic HCl, and quantified using GC. Total sugars quantified in 1 M KOH soluble and insoluble fractions (of which the majority are xylan) are shown (complete data in Fig. S7). (C) Enzymatic hydrolysis of xylan to Xyl. WT, gux1 gux2, irx9, and irx14 AIR were hydrolyzed with NpXyn11A and β-xylosidase and quantified using PACE. Released Xyl monosaccharide is expressed as percent weight of enzyme-hydrolyzed xylan. Error bars represent SD (n = 3).