Dissecting the Molecular Mechanism of Pectin Synthesis in Arabidopsis

果胶是一类结构复杂的植物细胞壁多糖家族，在植物生长发育和植物抵御病原菌等方面具有多种功能。由于编码果胶生物合成或果胶分解酶的基因突变导致果胶结构的改变，导致细胞与细胞的黏附改变，矮化，叶片脆化，新梢和花朵减少，表明果胶在植物生长发育中具有多种作用。据预测，果胶的生物合成需要大约50种糖基转移酶类型的生物合成酶，其中包括四种或更多的半乳糖醛酸基转移酶(GalATs)。半乳糖醛酸约占果胶的70%，存在于三种果胶多糖中：高半乳糖醛酸(HG)、鼠李半乳糖醛酸I(RG-I)和鼠李半乳糖醛酸II(RG-II)。在先前的研究中，研究人员已经确定了一个由25个已证实和可能编码GalAT的超家族组成：15个Gaut(GAlactUronosylTransferase)和10个GATL(Gaut-like)基因。与GAUT1相关的25个超家族基因的序列比对和系统发育分析确定了4个分支，Gaut A、B、C和GATL。对13个Gaut基因的纯合子拟南芥T-DNA插入突变体的细胞壁分析表明，大多数Gaut突变体的细胞壁中的半乳糖醛酸水平发生了改变，糖基残基的变化与HG、RG-I和/或RG-II合成缺陷相一致。然而，突变的GAUT的具体功能尚不清楚。这项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。本研究的目的是：(1)对GAUT6、GAUT14和GATL8蛋白进行生化鉴定。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来鉴定蛋白质亚基，以确定复合体中存在特定Gaut/GATL的可能性。将测试复合体和单个亚基的酶活性。(2)对Gaut/GATL纯合突变株进行分析，阐明Gaut/GATL生物学功能。将分析与突变相关的植物生长和发育的特定变化，并将通过糖基残基和连锁分析的组合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)确定各Gaut/GATL的细胞类型特异性表达。将进行EST、微阵列和PCR转录分析，以及GUS启动子融合研究，以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GAUT/GATL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。该项目的成果可能会促进植物工程，以生产具有改良结构和特性的果胶，并具有更高的农业、工业和生物医学价值。该项目将通过乔治亚大学夏季本科生研究计划(SURP)和Sup-Bridge计划招收本科生和研究生，并参加本科科学全国会议，以确定优秀的历史上代表性不足的研究生候选人，从而培训未来的科学家。参与这个项目的学生的研究经验将为他们提供不同的遗传、生化和细胞生物学技术的广泛培训。

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.