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.
