Grass species are of huge importance to global agriculture and include the three most productive food crops (rice, wheat and maize) as well as pasture species for ruminants and bioenergy crops such as Miscanthus. Cell walls account for the majority of biomass in plants and are the focus of a large international effort in research, particularly to increase their digestibility for efficient conversion to liquid biofuels, to increase the efficicency of digestion by grazing animals, and to increase the beneficial role that they play as dietary fibre in foods such as wheat flour. The cell walls of grasses differ substantially from those of other plants, particularly in the hemicellulosic component xylan. Xylan is the most abundant polysaccharide (molecule containing linked sugars) after cellulose, often accounting for 25% of biomass; in grasses, it has a large quantities of the sugar arabinose attached to the backbone. We have recently demonstrated that some wheat and rice genes in a gene family called GT61 are responsible for the addition of arabinose to xylan (Anders et al., 2012, "Glycosyl transferases in family 61 mediate arabinofuranosyl transfer onto xylan in grasses", PNAS, 109: 989-993.)

Here we propose to build on our lead in understanding GT61 gene function and xylan arabinosylation and explore the consequences for both non-starch polysaccharide (dietary fibre) in wheat grain and digestibility in grass biomass. By adding grass genes to systems which lack the grass-specific feature of arabinose on xylan and ferulic acid which is attached to this arabinose, we can determine which GT61 genes are responsible for the different types of arabinose addition present in grass xylans; crucially we can also test our hypothesis that GT61 genes are directly responsible for addition of ferulic acid to xylan. This is critically important for digestibility of grass cell walls because ferulic acid on xylan can link with ferulic acid on other xylan molecules or with lignin giving cross-links. (Lignin is the water-repelling component of cell walls which inhibits digestion of the cellulose and xylan molecules, preventing release of the sugars present in these.)

In wheat grain, the major non-starch polysaccharide component is arabinoxylan (AX) from cell walls and the solubility of this is important for different end-uses. For human food, AX is the major dietary fibre within wheat and insoluble and soluble forms confer different health benefits. For non-food uses of wheat grain, soluble AX is an undesirable component. Therefore solubility of wheat grain AX is a parameter of interest for many applications and it will be determined by the amount and nature of arabinose addition; more arabinose is predicted to increase solubility, whereas ferulic-acid mediated cross-linking will decrease it. These predictions will be tested in GM wheat plants where the activity of all the GT61 genes will be altered.

More ferulic acid on xylan is also expected to decrease digestibility of grass biomass because of the greater cross-linking to lignin. We will examine this in a grass called Brachypodium distachyon (which serves as a convenient model for grass crop biomass). We already know the number of GT61 genes which are active in Brachypodium and will specifically suppress them using GM technology. In the Brachypodium GM plants, we will characterise the cell walls and test for the predicted increase in digestibility. A positive result would make the relevant GT61 gene(s) a major target for improvement of grass biomass for biofuel and ruminant nutrition.

Technical Abstract:
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.

Potential Impact:
Academic: Identification of genes (XATs) responsible for the different forms of xylan arabinosyl substittion 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; in particular, 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, sugar cane) 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.