Identification of xylan arabinosyl transferases and their role in determining xylan structural and cross-linking properties within grass cell walls

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The cell walls of grasses differ substantially from those of dicots, particularly in the hemicellulosic component xylan. Xylan is the most abundant polysaccharide after cellulose, often accounting for 25% of biomass; in grasses, unlike dicots, it is frequently heavily substituted with arabinose. Some of these arabinose are ester-linked to ferulic acid which can undergo oxidative coupling to provide covalent crosslinks believed to be key to digestibility of grass cell walls. We predicted that glycosyl transferase (GT) family 61 genes were involved in arabinoxylan (AX) synthesis and have recently demonstrated that some GT61 wheat and rice genes are responsible for the addition of 3-linked Ara to AX (Anders et al., 2012, "Glycosyl transferases in family 61 mediate arabinofuranosyl transfer onto xylan in grasses", PNAS). Here we propose to build on our lead in understanding xylan arabinosylation and explore the consequences for both non-starch polysaccharide (dietary fibre) in wheat flour and digestibility in grass biomass. We will determine which genes are responsible for different arabinose linkages present in grass xylans and test our hypothesis that GT61 genes are directly responsible for AX feruloylation. Arabinose addition is predicted to increase solubility, whereas AX feruloylation will decrease solubility and decrease digestibility. We will examine these predicted effects of modifying xylan arabinosyl transferase (XAT) gene activity in two grass systems; wheat starchy endosperm (which gives rise to white flour) and the stems of the model grass Brachypodium distachyon. We already know which GT61 genes are espressed in these systems and will supress or overexpress them and will determine effects on cell walls using a variety of approaches including 2D-NMR. We will also determine the effects of XAT gene suppression on soluble fibre and viscosity in flour from wheat transgenics and on digestibility in Brachypodium transgenics.

Academic: Identification of genes (XATs) responsible for the different forms of xylan arabinosyl substitution will have major scientific impact as they are key to evolutionary divergence of grass cell walls from those of dicots. In wheat grain, arabinosyl substitution determines the solubility of arabinoxylan (AX), the major non-starch polysaccharide. More generally, the feruloylation of grass cell walls occurs via the arabinosyl substitution of xylan in grasses. Xylan feruloylation does not occur in dicots but it allows cross-linking between xylan chains and from polysaccharide to lignin. Cell wall feruloylation has been implicated in many traits of grasses, including low digestibility of biomass, pest and pathogen resistance. Demonstration of the genes involved in feruloylation of grass cell walls would be a breakthorugh in plant science and merit publication in the highest impact journals. The proposed work will also provide invaluable genetic resources to the research community where these genes are modified in transgenic wheat, Brachypodium and Arabidopsis plants.

Industrial: (1) Improved wheat varieties tailored for different end-uses. We have already identified that decreasing TaXAT1 activity will decrease viscosity of wheat grain extracts, improving grain properties for non-food uses. We have filed a patent on this and are planning a follow-on proposal to develop elite wheat variety with this trait in partnership wih a commercial breeder who has already agreed to do the introgression and crossing. Identification of the other XAT genes in this proposal will allow a complete picture of how to determine solubility of grain AX; e.g. to incease its soluble dietary fibre for human use. (2) Digestibility. Identification of XAT with a specific role in xylan feruloylation would make it a major target for increasing the digestibility of grass biomass, particularly if such an effect can be demonstrated in Brachypodium transgenic lines. This would provide a novel route for improving the digestibility of pasture species for ruminants and for improving efficiency of biofuel prodcution from grass lignocellulosic biomass. (3) Biorefining. If, as we predict, XAT genes are integral to the control of feruloylation and this is the principle means by which lignin is attached to polysaccharide in grass biomass, it may be possible to develop new varieties where separation of these components is easier. Since this is a major part of the cost of deriving products from this material, this would make biorefining more economically attractive.

Societal: The proposed research should lead to new technologies which improve competitiveness of UK industry. Increased separation of UK wheat varieties for food and non-food uses is desirable as the beneficial quality traits are so different and the proposed reseearch will help to bring this about. Identification of genes responsible for dietary fibre in wheat foods should facilitate the breeding of varieties for healthier wheat products. If XAT genes are identified as a route to increased digestibility in grasses this would open the way to improved varietes of pasture species for improved ruminant digestion, and improved varieties of crops (e.g. wheat, barley, Miscanthus) where the non-food parts are intended for bioethanol production. Therefore the research could lead to novel methods of increasing the efficiency of processes using sustainable substitutes for fossil carbon.