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Selective chemical intervention in plant cell wall polysaccharide metabolism: consequences for cell expansion

植物细胞壁多糖代谢的选择性化学干预：对细胞扩张的影响

基本信息

批准号：
BB/E013651/1
负责人：
Stephen Fry
金额：
$ 48.83万
依托单位：
University of Edinburgh
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2007
资助国家：
英国
起止时间：
2007 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FE013651%2F1
关键词：
Selective chemical intervention plant cell

项目摘要

Growing plants have cell walls composed of polysaccharides called pectins, hemicelluloses and cellulose. This type of wall (the primary cell wall) is unique to plants, and indispensable for their growth and development / especially for 'cell expansion', an aspect of plant physiology utterly different from the growth processes of animals and microbes. The enormity of plant cell expansion can be illustrated by the fact that a mature oak tree contains fewer cells than one human liver! Plant growth (indisputably a vital process in biology and agriculture) thus depends on biochemical processes that occur in the primary cell wall. Much is known about the chemistry of individual cell-wall polysaccharides, but we still cannot ascribe specific biological functions to specific chemical components of the wall. Usually in this situation, a powerful experimental method for finding such functional relationships would involve genetics: the gene for a wall component would be knocked out and we would observe the biological fate of the plant ('what it died of'). However, this approach is difficult to apply to cell wall components because there are often dozens of genes all encoding a group of very similar cell wall-related enzymes. For example there are 33 different genes all specifying functionally similar versions of a particular cell wall enzyme, xyloglucan endotransglucosylase (XET). It is not practicable to knock out all the members of such a family of genes. The alternative approach, adopted here, is to use foreign chemical agents (xenobiotics) that block the action of all members of the corresponding family of proteins. If we could apply such a xenobiotic, to inhibit the activity of all 33 XETs, for example, we could critically test the hypothesis that XETs (as a group) play a vital role in cell expansion. This approach is termed selective chemical intervention in biological systems (SCIBS), or 'chemical genetics'. We will explore the effects on plant cell expansion of non-toxic xenobiotics that specifically inhibit a family of wall-related enzymes. To discover such inhibitors, we will need to test 100's to 1000's of xenobiotics for their ability to inhibit the cell-wall enzymes of interest. Testing the effects of ~2000 xenobiotics on the activity of ~30 enzyme-families clearly requires a simple, 'high-throughput screen' for each enzyme family. This lab has invented such a screen, termed a dot-blot assay, for one particular enzyme family (the XETs), and we will now devise comparable dot-blot assays for ~29 additional families of cell-wall enzymes as well as for a group of other growth-enhancing cell-wall proteins called expansins. The xenobiotics to be tested will be sourced from commercial collections of natural products, already-known enzyme inhibitors, and herbicides (those with unknown modes of action). We will also modify some of the more promising xenobiotics by attaching chemical groups to them which may enhance their specificity or effectiveness. With these resources, we will screen the xenobiotics for effects on wall-related enzymes. Active xenobiotics will be tested in detail so we can define how effective they are. how specific they are (i.e., whether they inhibit only one family of wall enzymes), and whether they are toxic to plant cells (e.g. as respiratory poisons). Suitable xenobiotics will be used to diminish the target enzyme's activity; we will monitor consequent effects on the plant cells' ability to grow and develop. The aim is to provide definitive new information on the specific biological significance of selected cell-wall components.

生长中的植物有由称为果胶、半纤维素和纤维素的多糖组成的细胞壁。这种类型的壁(初级细胞壁)是植物所特有的，对于它们的生长和发育是必不可少的，尤其是对细胞扩张来说，这是植物生理学的一个方面，与动物和微生物的生长过程完全不同。一棵成熟的橡树含有的细胞比一个人的肝脏还少，这就说明了植物细胞扩张的严重性！因此，植物生长(无可争议地是生物学和农业中的一个重要过程)依赖于发生在初级细胞壁中的生化过程。关于单个细胞壁多糖的化学成分已知很多，但我们仍然不能将特定的生物学功能归因于细胞壁的特定化学成分。通常在这种情况下，寻找这种功能关系的一个强有力的实验方法将涉及遗传学：壁组件的基因将被敲除，我们将观察植物的生物学命运(它死于什么)。然而，这种方法很难应用于细胞壁成分，因为通常有几十个基因都编码一组非常相似的细胞壁相关酶。例如，有33个不同的基因都规定了一种特定的细胞壁酶--木葡聚糖内转葡糖苷酶(XET)--的功能相似版本。要敲除这样一个基因家族的所有成员是不现实的。这里采用的另一种方法是使用外来化学物质(异源化合物)来阻止相应蛋白质家族的所有成员的活动。例如，如果我们能应用这种异源物质来抑制所有33个XET的活性，我们就可以批判性地检验XET(作为一个群体)在细胞扩张中发挥关键作用的假设。这种方法被称为生物系统中的选择性化学干预(SCIBS)，或“化学遗传学”。我们将探索无毒的外源物质对植物细胞扩张的影响，这些物质专门抑制一系列与壁相关的酶。为了发现这种抑制剂，我们需要测试100‘S到1000’S的外源化合物对感兴趣的细胞壁酶的抑制能力。测试~2000种外源生物对~30个酶家族活性的影响显然需要对每个酶家族进行简单的高通量筛选。这个实验室已经为一个特定的酶家族(XETs)发明了这样一种被称为斑点杂交分析的筛选方法，现在我们将为另外29个细胞壁酶家族以及一组名为Expansins的其他促进生长的细胞壁蛋白质设计类似的斑点杂交分析。将测试的外源生物将来自商业收集的天然产品、已知的酶抑制剂和除草剂(那些作用模式未知的产品)。我们还将通过在一些更有前景的外源化合物上附加化学基团来修饰它们，这可能会增强它们的特异性或有效性。有了这些资源，我们将筛选外源物质对壁相关酶的影响。活性外源生物将进行详细测试，这样我们就可以定义它们的有效性。它们有多具体(即，它们是否只抑制一种壁酶家族)，以及它们是否对植物细胞有毒(例如，作为呼吸道毒素)。适当的外源物质将被用来降低目标酶的活性；我们将监测由此对植物细胞生长和发育能力的影响。其目的是就选定的细胞壁成分的特定生物学意义提供明确的新信息。