Characterization of Arabidopsis Mutants Altered in Fucose- Containing Cell Wall Polysaccharides

为了进一步了解植物细胞壁的合成、结构和功能，采用直接生化筛选方法分离了细胞壁多糖组成改变的拟南芥突变体。已经确定了三个不同的位点，它们要么导致细胞壁材料（mur1）中L-聚焦的完全缺失，要么导致这种单糖的数量减少约50%（细胞系mur2和mur3）。为了充分利用这些突变系的有用性，有必要了解导致突变表型的生化或调节损伤，并获得受影响的基因。拟议的研究将使用生化工具来定义导致观察到的细胞壁组成变化的变化。将进行实验以确定突变系是否在前体可利用性方面存在缺陷，或者它们是否在糖基转移的某些方面存在缺陷。这些问题将通过量化GDP- focus （focusyltransferase的底物）的数量和确定含有细胞壁多糖的focus的结构变化来解决。这些研究将通过分析focusyltransferase的活性和重新合成GDP-L- focus的酶的活性来补充。所有选择的突变将相对于分子标记进行映射，作为通过基于图谱的程序获得受影响基因的先决条件。mur1突变似乎特别适合进行遗传分析，因为已经有大量独立的等位基因可用，并且可以使用简单的免疫筛选程序构建高分辨率的遗传图谱。因此，将尝试从标记文库或通过基于图谱的程序克隆MUR1基因。该基因的可用性将使人们对高等植物中糖核苷酸相互转换途径的调控有一些深入的了解，并允许对其他植物（包括经济上重要的物种）的细胞壁组成进行修饰。高等植物的细胞壁在植物生长发育过程中起着举足轻重的作用，决定着植物各部分的物理性质。例如水果的柔软度，纺织纤维的强度，饲料的消化率，以及纸张生产过程中木材的加工特性。此外，植物细胞壁是植物与环境之间的界面，在植物抵御害虫方面发挥着重要作用。最后，植物细胞壁材料占陆地新合成生物量的大部分，是还原碳最重要的可再生来源，可作为化学工业和燃料生产的原料。因此，了解植物细胞壁材料的合成机制，获得控制植物细胞壁材料特性的基因是十分必要的。这最终将为利用生物技术工具以从经济和/或生态角度来看理想的方式改造植物细胞壁开辟机会。＊＊＊

9405739 Reiter In an effort to further understand the synthesis, structure and function of the plant cell wall, a direct biochemical screening procedure has been used to isolate mutants of Arabidopsis with alterations in their cell wall polysaccharide composition. Three different loci have been identified that either lead to the complete absence of L- fucose in cell wall material (mur1) or to an approximately 50% reduction in the amount of this monosaccharide (lines mur2 and mur3). To fully exploit the usefulness of these mutant lines, it is necessary to understand the biochemical or regulatory lesions leading to the mutant phenotypes, and to get access to the affected genes. The proposed research will use biochemical tools to define the changes leading to the observed alterations in cell wall composition. Experiments will be carried out to determine whether the mutant lines show defects in precursor availability, or whether they are deficient in some aspects of glycosyl transfer. These questions will be addressed by quantifying the amount of GDP- fucose (the substrate for fucosyltransferase) and by determining structural changes in fucose containing cell wall polysaccharides. The studies will be complemented by assaying fucosyltransferase activities , and the activity of enzymes in the de novo synthesis of GDP-L-fucose. All selected mutations will be mapped relative to molecular markers as a prerequisite to get access to the affected genes by map-based procedures. The mur1 mutation appears to be particularly amenable to genetic analysis because a large number of independent alleles is already available, and an easy immunological screening procedure can be used to construct a genetic map of high resolution. Accordingly, attempts will be made to clone the MUR1 gene either from tagged libraries or by map -based procedures. The availability of this gene would allow some insights into the regulation of sugar nucleotide interconversion pathways in hi gher plants, and permit the modification of cell wall composition in other plants including economically important species. %%% The cell walls of higher plants play pivotal roles during plant growth and development, and determine the physical properties of all parts of the plant. Examples include the softness of fruits, the strength of textile fibers, the digestibility of forage, and the processing characteristics of wood during paper production. Furthermore, plant cell walls present the interface between the plants and their environment and play important roles in the defense of plants against pests. Finally, plant cell wall material accounts for most of the newly synthesized terrestrial biomass, making it the most important renewable source of reduced carbon, which can be used as raw material for the chemical industry and for fuel production. Accordingly, it is highly desirable to understand the mechanism by which plant cell wall material is synthesized, and to get access to the genes controlling the properties of plant cell wall material. This will ultimately open up opportunities to use tools of biotechnology to modify plant cell walls in ways that are desirable from an economically and/or ecological point of view. ***