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

草种对全球农业至关重要，包括三种最具生产力的粮食作物（水稻、小麦和玉米）以及反刍动物的牧草和芒属等生物能源作物。细胞壁占植物生物量的大部分，是国际研究工作的重点，特别是为了提高它们的消化率以有效转化为液体生物燃料，提高放牧动物的消化效率，并增加它们作为食物中的膳食纤维的有益作用，如小麦粉。禾本科植物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。木聚糖是纤维素之后最丰富的多糖（含有连接糖的分子），通常占生物质的25%;在草中，它具有大量的糖阿拉伯糖连接到主链上。我们最近已经证明，称为GT 61的基因家族中的一些小麦和水稻基因负责将阿拉伯糖添加到木聚糖中（Anders等人，2012，“Glycosyl transferases in family 61 mediate arabinofuranosyl transfer onto xylan in grasses”，PNAS，109：989-993.）在这里，我们建议建立在我们的领导在理解GT 61基因功能和木聚糖阿拉伯糖基化和探索小麦籽粒中的非淀粉多糖（膳食纤维）和草生物质的消化率的后果。通过将草基因添加到缺乏木聚糖上的阿拉伯糖和附着于该阿拉伯糖的阿魏酸的草特异性特征的系统中，我们可以确定哪些GT 61基因负责草木聚糖中存在的不同类型的阿拉伯糖添加;至关重要的是，我们还可以测试我们的假设，即GT 61基因直接负责阿魏酸添加到木聚糖中。这对于草细胞壁的消化率是至关重要的，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连接，从而产生交联。（木质素是细胞壁的防水成分，它抑制纤维素和木聚糖分子的消化，防止这些分子中存在的糖的释放。在小麦籽粒中，主要的非淀粉多糖组分是来自细胞壁的阿拉伯木聚糖（AX），并且其溶解性对于不同的最终用途是重要的。对于人类食物，AX是小麦中的主要膳食纤维，不溶性和可溶性形式赋予不同的健康益处。对于小麦谷粒的非食品用途，可溶性AX是不期望的组分。因此，小麦粒AX的溶解度是许多应用的感兴趣的参数，并且它将由阿拉伯糖添加的量和性质决定;预计更多的阿拉伯糖将增加溶解度，而阿魏酸介导的交叉，这些预测将在所有GT 61基因的活性都将被改变的转基因小麦植物中进行测试。木聚糖上的更多阿魏酸也有望减少由于与木质素的交联更大，因此提高了草生物质的消化率。我们将在一种名为二穗短柄草（Brachypodium distachyon）的草中研究这一点（这是一种方便的草作物生物量模型）。我们已经知道在短柄草中活跃的GT 61基因的数量，并将使用转基因技术特异性地抑制它们。在短柄草转基因植物中，我们将表征细胞壁并测试消化率的预测增加。阳性结果将使相关的GT 61基因成为改善用于生物燃料和反刍动物营养的草生物量的主要靶标。

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.