Analysis of Multiple Cellulases in Plant Cell Wall Processes

植物细胞壁的修饰与几乎所有的植物发育过程相协调。细胞壁的变化被认为是由一系列酶介导的，这些酶可以破坏构成细胞壁的结构聚合物中的连接，特别是葡萄糖分子之间的连接。用于修饰纤维素的酶，通常被称为纤维素酶，就是这个家族的成员。在拟南芥中，纤维素酶家族是广泛的（超过12个成员），由结构上不同类别的基因编码。在该家族中，有位于质膜的成员，被认为是在细胞壁的最内层起作用，还有分泌的成员，被认为是在细胞壁的任何层起作用，包括最外层。这两个结构基团具有相似的催化特征，并且与细菌纤维素酶家族9具有同源性。这个项目有一个基本的假设，即锚定在质膜上的纤维素酶在细胞壁的生物合成过程中起作用，而那些在细胞外的酶在细胞壁分解代谢过程中起特定的作用，这些过程的活性范围从局部和局部到大规模和灾难性的。这一活动范围分别与细胞生长和细胞死亡等不同功能有关。对于所有拟南芥纤维素酶基因，我们对它们在体内的真正底物、作用方式以及它们对纤维素或其他β -1-4葡聚糖的作用程度一无所知。为了更好地理解多种纤维素酶在植物发展的作用,提出了遗传和分子研究调查如何调制geneexpression和酶水平变化期间differentcellulase函数三个结构不同的拟南芥cellulasegenes:一个假定的membrane-anchored纤维素酶Cel6.1.1, putativesecreted, auxin-stimulated纤维素酶Cel5.1.1,和一个假定的abscissioncellulase Cel115L。这一比较具有重要意义，因为人们认为，在生长和扩张过程中发生的主要细胞壁变化发生在细胞壁的最内层。预计参与生长的纤维素酶在生长过程中上调，在细胞壁拆卸过程中下调，而参与细胞壁拆卸的纤维素酶在生长过程中反之亦然。本研究采用的实验系统是拟南芥下胚轴生长，其中纤维素酶在同一组织的不同发育阶段表现出不同的功能。这些功能包括纵向和径向生长、细胞分离和细胞死亡。下胚轴生长将在野生型植物、在横向或纵向生长中有缺陷但在纤维素酶基因中没有缺陷的形态突变体和预计在特定纤维素酶基因中有缺陷的T-DNA插入突变体（敲除突变体）之间进行比较。敲除突变可能会改变植物的形状或其他特征，与突变基因的假设功能一致，并将为纤维素酶的功能提供强有力的证据。

The plant cell wall is modified in coordination with almost allplant developmental processes. Changes in the cell wall are thought to bemediated by a family of enzymes that break down links in the structuralpolymers making up the cell wall, in particular links between glucosemolecules. The enzymes expected to modify cellulose, commonly referred to ascellulases, are members of this family. In Arabidopsis the family of cellulases is extensive (more thantwelve members) and is encoded by structurally different classes of genes.Within the family there are members located at the plasma membrane that arepresumed to act at the innermost layers of the cell wall, and members thatare secreted and are presumed to act at any stratum within the cell wall,including the outermost layer. Both structural groups have similarcatalytic signatures and homology to Family 9 of bacterial cellulases. This project has the underlying hypothesis that cellulases anchoredin the plasma membrane play a role in cell wall biosynthetic processes whilethose that are extracellular play specific roles in cell wall catabolicprocesses that range in activity from partial and localized, to massive andcatastrophic. This range in activity is linked to contrasting functionslike cell growth and cell death, respectively. For all Arabidopsiscellulase genes nothing is known about their true in vivo substrate, mode ofaction and to what extent they can act on cellulose or other beta-1-4 glucans. To better understand the role of cellulases in plant development,genetic and molecular studies are proposed to investigate how geneexpression and enzyme levels are modulated during shifts between differentcellulase functions for three structurally distinct Arabidopsis cellulasegenes: a putative membrane-anchored cellulase Cel6.1.1, a putativesecreted, auxin-stimulated cellulase Cel5.1.1, and a putative abscissioncellulase Cel115L. The comparison is significant since it is believed thatthe major cell wall changes occurring in processes of growth and expansiontake place in the innermost layer of the cell wall. It is anticipated thatcellulases involved in growth will be up-regulated during growth anddown-regulated during cell wall disassembly, and vice versa for cellulasesinvolved in cell wall disassembly The experimental system to be used for these studies is Arabidopsishypocotyl growth in which contrasting functions of cellulase are manifestedin different developmental stages of the same tissue. These functionsinclude longitudinal and radial growth, cell separation and cell death.Hypocotyl growth will be compared among wild-type plants, morphologicalmutants that are defective in lateral or longitudinal growth but are notdefective in cellulase genes and T-DNA insertional mutants (knock-outmutants) that are predicted to be defective in a particular cellulase gene.Knock-out mutations may change the shape or other characteristics of theplant consistent with the mutated genes hypothesized function and willprovide strong evidence for cellulase function.