Dissecting the Molecular Mechanism of Pectin Synthesis in Arabidopsis

剖析拟南芥果胶合成的分子机制

• 批准号: 0646109
• 负责人: Debra Mohnen
• 金额: $ 73.09万
• 依托单位: University of Georgia Research Foundation Inc
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0646109&HistoricalAwards=false
• 关键词: Dissecting; Molecular; Mechanism; Pectin; Synthesis

Pectin is a structurally complex family of plant cell wall polysaccharides that have multiple functions in plant growth, development and plant defense against pathogens. Modifications of pectin structure due to mutations in genes encoding pectin biosynthetic or pectinolytic enzymes result in altered cell-cell adhesion, dwarfism, brittle leaves and reduced numbers of shoots and flowers, indicating multiple roles for pectin in plant growth and development. Pectin biosynthesis is predicted to require ~50 biosynthetic enzymes of the glycosyltransferase type, including four or more galacturonosyltransferases (GalATs). Galacturonic acid comprises ~70% of pectin and is present in the three pectic polysaccharides: homogalacturonan (HG), rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II).In previous studies, the researchers have identified a superfamily of 25 Arabidopsis thaliana genes encoding demonstrated and putative GalATs: 15 GAUT (GAlactUronosylTransferase) and 10 GATL (GAUT-Like) genes. Sequence alignment of the 25 GAUT1-related superfamily genes and phylogenetic analyses identified four clades, GAUT A, B, C and GATL. Cell wall analyses of homozygous Arabidopsis T-DNA insertion mutants of 13 GAUT genes show that most of the GAUT mutants have modified levels of galacturonic acid in their walls and glycosyl residue changes consistent with defects in HG, RG-I and/or RG-II synthesis. The specific function of the mutated GAUTs, however, is not known. The goal of the research is to determine the molecular function of three GAUT1-related family genes for which mutant plants show novel pectin-related phenotypes. The SPECIFC AIMS are to:(1) Biochemically characterize GAUT6, GAUT14 and GATL8 proteins. Polyclonal antibodies will be raised against the heterologously expressed proteins. The possibility that specific GAUT/GATLs exist in complexes will be tested using SDS-PAGE and immunoprecipitation in combination with Western blotting and LC-MS/MS to identify protein subunits. The enzymatic activities of complexes and individual subunits will be tested. (2) Analyze homozygous mutants to elucidate GAUT/GATL biological function. Mutation-associated specific changes in plant growth and development will be analyzed and wall structure will be determined by a combination of glycosyl residue and linkage analyses and by immunolabeling tissue sections using antibodies against specific wall carbohydrate epitopes. (3) Determine cell-type specific expression of each GAUT/GATL. EST, microarray, and PCR transcript analyses, together with GUS promoter fusion studies will be done to determine tissue and cellular location of the encoded proteins. If time allows, immunogold labeling using antibodies generated against recombinant or purified GAUTs/GATLs, or GFP-fusion protein expression will be carried out to define the subcellular location of the proteins within the cell and the Golgi complex. Broader Impacts. The outcome of this project is likely to facilitate the engineering of plants to produce pectins with modified structures and properties, and with improved agricultural, industrial and biomedical value. The project will involve training of future scientists by recruiting undergraduate and graduate students through the University of Georgia Summer Undergraduate Research Program (SURP) and SURP-Bridge program and by participating in undergraduate science national conferences to identify meritorious historically underrepresented graduate student candidates. The research experience of students participating in this project will provide them with a broad training in diverse genetic, biochemical, and cell biological techniques.

果胶是一类结构复杂的植物细胞壁多糖，在植物生长发育和抗病等方面具有多种功能。编码果胶合成酶或果胶分解酶的基因突变导致果胶结构的改变，导致细胞粘附改变、矮化、叶脆、芽和花数量减少，表明果胶在植物生长发育中的多重作用。果胶的生物合成预计需要约50种糖基转移酶类型的生物合成酶，包括四种或更多的半乳糖醛酸转移酶（galat）。半乳糖醛酸约占果胶的70%，存在于三种果胶多糖中：均半乳糖醛酸（HG）、鼠李糖半乳糖醛酸I （RG-I）和鼠李糖半乳糖醛酸II （RG-II）。在之前的研究中，研究人员已经确定了一个由25个拟南芥基因组成的超家族，编码已证实和推测的galat: 15个gaat （GAlactUronosylTransferase）和10个gaat （like gaat）基因。对25个与gaut1相关的超家族基因进行序列比对和系统发育分析，鉴定出4个支系，分别为gauta、B、C和GATL。对13个gat基因的纯合子拟南芥T-DNA插入突变体细胞壁分析表明，大多数gat突变体细胞壁中半乳糖醛酸的水平和糖基残基的变化与HG、RG-I和/或RG-II合成缺陷一致。然而，突变gat的具体功能尚不清楚。该研究的目的是确定突变植物表现出新的果胶相关表型的三个gaut1相关家族基因的分子功能。具体目的是：(1)生物化学表征GAUT6， GAUT14和gaut8蛋白。针对异源表达的蛋白将产生多克隆抗体。将使用SDS-PAGE和免疫沉淀结合Western blotting和LC-MS/MS检测复合物中存在特异性GAUT/ gatl的可能性，以鉴定蛋白质亚基。复合物和单个亚基的酶活性将被测试。(2)分析纯合突变体，阐明GAUT/GATL的生物学功能。将分析植物生长发育中与突变相关的特异性变化，并通过结合糖基残基和连锁分析以及使用针对特定壁碳水化合物表位的抗体对组织切片进行免疫标记来确定壁结构。(3)测定各GAUT/GATL的细胞类型特异性表达。EST、微阵列和PCR转录分析以及GUS启动子融合研究将用于确定编码蛋白的组织和细胞位置。如果时间允许，将使用针对重组或纯化的gats /GATLs产生的抗体或gfp融合蛋白表达进行免疫金标记，以确定蛋白质在细胞和高尔基复合体中的亚细胞位置。更广泛的影响。该项目的成果很可能促进植物工程，以生产具有改良结构和性能的果胶，并提高农业、工业和生物医学价值。该项目将包括通过佐治亚大学夏季本科生研究计划（SURP）和SURP- bridge计划招募本科生和研究生，并通过参加本科生科学全国会议来确定有价值的历史上代表性不足的研究生候选人，从而培养未来的科学家。参与该项目的学生的研究经验将为他们提供各种遗传、生化和细胞生物学技术的广泛培训。