Targeted Modification of Hemicellulose Biosynthesis and Identification of New Wall Biosynthesis Enzymes.

Senior personnel - Debra Mohnen (CCRC), Maor bar-Peled (CCRC), Michael Hahn (CCRC), Malcolm ONeill (CCRC), and William York (CCRC).

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Background - Glucuronoxylan and arabinoxylan are the quantitatively major hemicellulosic polysaccharides in Populus and switchgrass biomass, respectively (see Figure 1). These polysaccharides are often modified by acetylation and methylation and by the presence of phenolic esters.

The interactions of hemicellulose and cellulose, the modifications of hemicelluloses and the cross-linking of hemicellulose to lignin impact recalcitrance. Thus, we will use multiple strategies to manipulate genes involved in hemicellulose synthesis to decrease recalcitrance. We will target

          Glycosyltransferases involved in hemicellulose initiation and elongation

          Enzymes involved in the formation of nucleotide sugars

          Enzymes involved in hemicellulose modifications.

We have identified three Arabidopsis genes (FRA8, IRX8, and IRX9), which encode putative glycosyltransferases that are required for normal glucuronoxylan synthesis in secondary cell walls (Pena et al. 2007). Our studies suggest that FRA8 and IRX8 are involved in the synthesis of the glycosyl sequence:

           4-β-D-Xylp-(1,3)-α-L-Rhap-(1,2)-α-D-GalpA-(1,4)-D-Xylp

that is located at the reducing terminus of secondary cell wall glucuronoxylan and that IRX9 is involved in xylan elongation. We have also identified several Arabidopsis GAUT and GATL mutants whose cell wall changes are consistent with altered glucuronoxylan synthesis (Persson et al. 2007; Hosmer-Caffall and Mohnen, unpublished results; Zhou and Hahn, unpublished results).

We have identified homologs of these genes in Populus and switchgrass. Altering the expression of these genes is likely to alter the amounts and properties of hemicellulose and may lead to decreased recalcitrance.

An alternative strategy to manipulate hemicellulose synthesis is to control the expression of genes involved in the synthesis of NDP-sugars (see Figure 2). We have identified several genes required for the formation of UDP-Xyl, UDP-Araf, UDP-Glc, UDP-GalA, UDP-GlcA, and UDP-Rha (Harper and Bar-Peled, 2002; Gu and Bar-Peled, 2004; Watt et al., 2004; Pattathil et al., 2005; Konishi et al., 2006).

We have also identified genes that may encode O-methyl, O-acetyl, and feruloyl transferases (Bar-Peled, unpublished results).

Altering the availability of NDP-sugars or the expression of acyl transferases may affect cellulose and hemicellulose synthesis and thereby influence recalcitrance.

Selected gene constructs will be used by the Populus and switchgrass transformation groups to generate plants so that the affects of modified gene expression on recalcitrance can be determined. Changes in cell wall chemistry and recalcitrance will be determined by the Characterization and Modeling team (UGA, NREL, Ga Tech).

Walls with significant changes in recalcitrance will be structurally characterized in detail (see biomass characterization). Changes in cell wall morphology and the distribution of polysaccharides will be probed by immunolabeling tissue sections using monoclonal antibodies against specific cell wall-carbohydrate epitopes (see biomass characterization).

Identifying new enzymes involved in polysaccharide biosynthesis.

Our understanding of cell wall formation is limited by the lack of biochemical assays for identifying the hundreds of genes/enzymes involved in polysaccharide biosynthesis

Acceptor substrates are required to study the mechanisms of polysaccharide biosynthesis and to identify candidate gene-products involved in polysaccharide chain initiation, elongation, and termination.

Different strategies are required to assay enzymes that modify polysaccharides. Thus, we will develop diverse assays to identify and purify new cell wall biosynthesis proteins and protein-complexes. Once a new enzymes has been identified its corresponding gene will be cloned.

Glyco-acceptors will be generated for glycosyltransferases required for hemicellulose biosynthesis and for O-acetylation, O-methylation, and O-feruloylation activities.

Acceptors will be enzymatically and chemically generated from diverse polysaccharides. Acceptors will be purified and then fluorescently labeled at their reducing ends to facilitate sensitive detection.

Enzyme activity assays will be developed based on our extensive expertise with diverse glycosyltransferases. We have available all the enzymes required to synthesize UDP-Rha and UDP-Araf, and feruloyl CoA; UDP-Xyl, UDP-Arap,and UDP-GalA are available at NSF-funded CarboSource Services. UDP-GlcA, acetyl CoA and SAM are commercially available.

Cellulose is known to be synthesized by membrane-localized protein complexes. We have preliminary evidence that matrix polysaccharide synthesis also involves protein complexes (Mohnen and Bar-peled, unpublished results). To identify such complexes we will pull-down interacting proteins using affinity matrices and antibody-protein complexes (Sterling et al., 2006). Interacting proteins will be sequenced by mass spectrometry and the corresponding genes then identified.

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