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

9405739为了进一步了解植物细胞壁的合成、结构和功能，利用直接生化筛选程序分离了细胞壁多糖组成发生变化的拟南芥突变体。已经确定了三个不同的基因座，它们要么导致细胞壁材料(Mur1)中完全没有L岩藻糖，要么导致这种单糖的数量减少约50%(株系Mur2和Mur3)。为了充分利用这些突变系的有效性，有必要了解导致突变表型的生化或调节性损伤，并获得受影响的基因。这项拟议的研究将使用生化工具来定义导致观察到的细胞壁成分变化的变化。将进行实验，以确定突变系是否在前体可获得性方面存在缺陷，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-岩藻糖(岩藻糖基转移酶的底物)的数量和确定含有岩藻糖的细胞壁多糖的结构变化来解决。这些研究将通过分析岩藻糖基转移酶的活性以及国内生产总值-L-岩藻糖从头合成中的酶的活性来补充。所有选定的突变都将相对于分子标记进行图谱绘制，作为通过基于图谱的程序获得受影响基因的先决条件。由于已经有大量独立的等位基因可用，而且一种简单的免疫学筛选程序可以用来构建高分辨率的遗传图谱，因此Mur1突变似乎特别适合进行遗传分析。因此，将尝试从标记的文库或通过基于图谱的程序来克隆MUR1基因。该基因的获得将使人们能够深入了解高密度植物中糖核苷酸相互转化途径的调控，并允许改变包括经济重要物种在内的其他植物的细胞壁组成。高等植物的细胞壁在植物的生长发育过程中起着举足轻重的作用，并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，以及造纸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，在植物抵御病虫害方面发挥着重要作用。最后，植物细胞壁材料占新合成的陆地生物质的大部分，使其成为减少碳的最重要的可再生来源，可用作化学工业和燃料生产的原料。因此，了解植物细胞壁材料合成的机制，并获得控制植物细胞壁材料性质的基因是非常有必要的。这最终将为利用生物技术工具以经济和/或生态角度所希望的方式改变植物细胞壁提供机会。***

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. ***