Xylan arabinosyl transferases: identification and characterisation of their role in determining properties of grass cell walls

禾草物种对全球农业至关重要，包括三种最多产的粮食作物（水稻、小麦和玉米），以及反刍动物的牧场物种和芒草等生物能源作物。细胞壁占植物生物量的大部分，是国际上大量研究的重点，特别是提高它们的消化率，以便有效地转化为液体生物燃料，提高食草动物的消化效率，并增加它们在小麦粉等食物中作为膳食纤维发挥的有益作用。草的细胞壁与其他植物的细胞壁有很大的不同，特别是在半纤维素成分木聚糖方面。木聚糖是仅次于纤维素的最丰富的多糖（含有连接糖的分子），通常占生物量的25%；在草中，它有大量的阿拉伯糖附着在主干上。我们最近证明了一个名为GT61的基因家族中的一些小麦和水稻基因负责将阿拉伯糖糖添加到木聚糖中（Anders等人，2012，“61家族的糖基转移酶介导阿拉伯糖烷基转移到草中的木聚糖上”，PNAS, 109: 989-993。）在此，我们建议在了解GT61基因功能和木聚糖阿拉伯糖基化的基础上，探索其对小麦籽粒中非淀粉多糖（膳食纤维）和草生物量消化率的影响。通过在缺乏阿拉伯糖对木聚糖和阿魏酸的草特异性的系统中添加草基因，我们可以确定哪些GT61基因负责草木聚糖中不同类型的阿拉伯糖的添加；重要的是，我们还可以验证我们的假设，即GT61基因直接负责将阿魏酸添加到木聚糖中。这对草细胞壁的消化率至关重要，因为木聚糖上的阿魏酸可以与其他木聚糖分子上的阿魏酸或与木质素产生交联。（木质素是细胞壁的阻水成分，它抑制纤维素和木聚糖分子的消化，阻止其中存在的糖的释放。）在小麦颗粒中，主要的非淀粉多糖成分是来自细胞壁的阿拉伯木聚糖（AX），其溶解度对不同的最终用途很重要。对于人类食物来说，AX是小麦中主要的膳食纤维，不溶性和可溶性形式具有不同的健康益处。对于小麦谷物的非食品用途，可溶性AX是不受欢迎的成分。因此，小麦籽粒AX的溶解度是许多应用中感兴趣的参数，它将由阿拉伯糖添加量和性质决定；预计更多的阿拉伯糖会增加溶解度，而阿魏酸介导的交联会降低溶解度。这些预测将在转基因小麦中得到验证，所有GT61基因的活性都将被改变。木聚糖上更多的阿魏酸也会降低草生物量的消化率，因为它与木质素的交联更大。我们将以一种叫做短柄草（Brachypodium distachyon）的草（它可以作为牧草生物量的方便模型）来检验这一点。我们已经知道在短柄草中有活性的GT61基因的数量，并将利用转基因技术特异性地抑制它们。在短柄草转基因植物中，我们将描述细胞壁的特征，并测试预测的消化率的增加。如果研究结果为阳性，那么GT61相关基因将成为改善草生物量用于生物燃料和反刍动物营养的主要靶点。

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.