Role of UDP-rhamnose and UDP-galacturonic acid biosynthetic genes in pectic synthesis

这项拟议的研究验证了这样一个假设，即核苷酸-糖底物的合成数量和位置在果胶合成的调节中发挥作用。果胶是一类复杂的植物细胞壁多糖家族，参与细胞间黏附、植物防御、植物发育和细胞间通讯。长期目标是阐明果胶的生物学作用以及参与其合成和组装的基因。在这里，我们将研究果胶合成的两个直接前体UDP-半乳糖醛酸(UDP-GALA)和UDP-鼠李糖(UDP-Rha)的调节和合成。所有果胶的脊骨中都含有半乳糖醛酸，而RG-I脊椎中同时含有GALA和RHA。RG-II的侧链上还含有Rha和Gala。将UDP-葡萄糖转化为UDP-Rha的重组拟南芥酶和将UDP-GLCA转化为UDP-GALA的重组差向异构酶已被功能克隆，并用PI进行了生化特征鉴定。在拟南芥中已鉴定出两个基因家族，包括三个UDP-Rha合成酶(URhaS)和六个UDP-Glca异构体(UGlcAEp)。UGlcAEp亚型被预测分为三个不同的类别(A、B和C)，以确定它们的亚细胞驻留。UDP-RhaSyn成员包含一个不同亚型的连接域，了解这些UDP-糖是如何供应给不同高尔基池中的各种果胶生物合成酶的，对于了解果胶是如何合成的至关重要。例如，UGlcAEp的不同亚型是否定位于不同的内膜，目前尚不清楚。与脊椎动物不同，植物产生大量的核苷酸-糖同工酶。有证据表明，植物产生多种NDP-糖合成酶亚型，以提供合成复杂多糖所需的各种活性糖。为了阐明UDP-GLCA差向异构酶和UDP-RHA合成酶亚型的生物学作用，提出了以下研究目标：(1)确定UGlcAEp和URHAS亚型的酶性质。每种异构体的完整生化特征将确定同种异构体特有的酶特性。(2)分析T-DNA突变体的壁结构，以确定UGlcAEp或URhaS亚型中是否有任何一种参与了特定果胶聚合物的合成。(3)鉴定天然UGlcAEp(或GFP融合)的亚细胞位置，并确定天然URHA(或GFP融合)是否与膜相关。针对不同亚型的抗体将被用来通过电子显微镜来确定不同的亚型是否定位于独特的内膜。针对UDP-RhaSyn亚型(或针对GFP-融合)的抗体将解决酶是可溶的还是膜结合的问题。这两家私人投资机构都坚定地致力于培养本科生、研究生和博士后，目前他们的实验室中有6名少数民族研究人员。PIS将使用佐治亚大学暑期本科生研究计划(SURP)和Sup-Bridge计划来招收未来的少数族裔学生。这项研究资助的学生将接受细胞壁生物化学、细胞生物学和分子生物学方面的培训，这是有效理解细胞壁生物学所需的罕见专业知识的组合。智力优势-拟议的研究检验了果胶的合成受特定核苷酸-糖合成酶亚型表达调控的假设，并将确定核苷酸-糖合成酶亚型是否局限于不同的高尔基池。突变分析有望深入了解果胶的生物学作用，并为果胶生物合成糖基转移酶提供新的底物。

The proposed research tests the hypothesis that the amount and location of synthesis of nucleotide-sugar substrates plays a role in the regulation of pectin synthesis. Pectin is a family of plant complex cell wall polysaccharides involved in cell-cell adhesion, plant defense, plant development and cell-cell communication. The long-term goal is to elucidate the biological roles of pectins and the genes involved in their synthesis and assembly. Here the regulation and synthesis of two immediate precursors of pectin synthesis, UDP-galacturonic acid (UDP-GalA) and UDP-rhamnose (UDP-Rha), will be studied. All pectins contain galacturonic acid in their backbones and the RG-I backbone contains both GalA and Rha. RG-II also has Rha and GalA in its side chains. Recombinant Arabidopsis enzymes that convert UDP-glucose to UDP-Rha and recombinant epimerases that convert UDP-GlcA to UDP-GalA have been functionally cloned and biochemically characterized by the PIs. Two gene families containing three UDP-Rha synthases (URhaS) and six UDP-GlcA epimerase (UGlcAEp) isoforms have been identified in Arabidopsis. The UGlcAEp isoforms are divided into three distinct classes (A, B, and C) predicted to determine their subcellular residency. The UDP-RhaSyn members contain a linker domain that differs among the isoforms.An understanding of how these UDP-sugars are supplied to the various pectin biosynthetic enzymes in different Golgi cisternae is crucial to understand how pectins are synthesized. For example, it is not known if different isoforms of UGlcAEp are localized to different endomembranes. Unlike vertebrates, plants produce numerous nucleotide-sugar isozymes. There is evidence that plants produce multiple NDP-sugar synthase isoforms to provide the diverse activated sugars required for complex polysaccharide synthesis. To elucidate the biological roles of UDP-GlcA epimerase and UDP-Rha synthase isoforms the following research goals are proposed: (1) Determine the enzymatic properties of the UGlcAEp and URhaS isoforms. Complete biochemical characterization of each isoform will identify isoform-specific enzyme properties. (2) Analyze the wall structure of T-DNA mutants to determine if any of the UGlcAEp or URhaS isoforms are involved in the synthesis of a particular pectic polymer. (3) Characterize the subcellular location of the native UGlcAEp's (or of GFP fusions) and determine if the native URhaS's (or GFP fusions) are associated with membranes. Antibodies raised toward distinct isoforms will be used to establish by electron-microscopy whether different isoforms are localized to unique endomembranes. Antibodies specific to UDP-RhaSyn isoforms (or to GFP-fusions) will address whether the enzymes are soluble or membrane associated.The broader impact of the proposed research. Both PIs have a strong commitment to educating undergraduates, graduate students, and postdocs and currently have 6 minority researchers in their labs. The PIs will use the University of Georgia Summer Undergraduate Research Program (SURP) and the SURP-Bridge program to recruit future minority students. The students funded by this research will receive training in cell wall biochemistry, cell biology and molecular biology; a rare combination of expertises that are required to effectively understand wall biology. Intellectual merit- The proposed research tests the hypothesis that the synthesis of pectin is regulated by the expression of specific nucleotide-sugar synthase isoforms and will establish whether the nucleotide-sugar synthase isoforms are localized to different Golgi cisternae. The mutant analyses are expected to provide insight into the biological roles of pectin and provide novel substrates for pectin biosynthetic glycosyltransferases.