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

生长中的植物细胞壁由称为果胶、半纤维素和纤维素的多糖组成。这种类型的壁（初生细胞壁）是植物所独有的，并且对于它们的生长和发育/特别是对于“细胞扩张”是不可或缺的，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。一棵成熟的橡树所含的细胞比一个人的肝脏还少，这一事实可以说明植物细胞扩张的巨大性。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）取决于初生细胞壁中发生的生化过程。关于单个细胞壁多糖的化学性质已经知道很多，但是我们仍然不能将特定的生物功能归因于细胞壁的特定化学成分。通常在这种情况下，找到这种功能关系的有力实验方法将涉及遗传学：壁成分的基因将被敲除，我们将观察植物的生物命运（“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种不同的基因，它们都指定了一种功能相似的特定细胞壁酶，木葡聚糖内切转葡糖基酶（XET）。把这样一个基因家族的所有成员都敲除是不切实际的。这里采用的替代方法是使用外来化学试剂（外源性物质），阻止相应蛋白质家族的所有成员的作用。例如，如果我们能够应用这样的生物外源性物质来抑制所有33种XET的活性，我们就可以批判性地检验XET（作为一个群体）在细胞扩增中发挥至关重要作用的假设。这种方法被称为生物系统选择性化学干预（SCIBS）或“化学遗传学”。我们将探讨非毒性外源性物质对植物细胞扩增的影响，这些外源性物质特异性地抑制一个与细胞壁相关的酶家族。为了发现这样的抑制剂，我们需要测试100到1000的异生物素抑制感兴趣的细胞壁酶的能力。测试约2000种外源性物质对约30种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”。这个实验室已经发明了这样一种筛选方法，称为斑点印迹法，用于一个特定的酶家族（Xcadin），我们现在将为大约29个额外的细胞壁酶家族以及一组其他生长增强细胞壁蛋白（称为expansins）设计类似的斑点印迹法。待测试的外源性物质将来自天然产物、已知酶抑制剂和除草剂（作用模式未知的那些）的商业收集。我们还将通过将化学基团连接到它们上来修饰一些更有前途的外源性物质，这可能会增强它们的特异性或有效性。有了这些资源，我们将筛选外源性物质对细胞壁相关酶的影响。将详细测试活性异生物质，以便我们可以定义它们的有效性。它们有多具体（即，它们是否仅抑制一个壁酶家族），以及它们是否对植物细胞有毒（例如作为呼吸毒物）。合适的外源性物质将被用来降低目标酶的活性;我们将监测对植物细胞生长和发育能力的影响。其目的是提供明确的新信息的特定生物学意义的选定细胞壁组件。

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.