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There are at least two models describing how the enzymes of metabolic pathways are arranged in living cells. The first is a stochastic model, where enzymes are freely-diffusing in the aqueous environment of the cell, and the second, the metabolon model, has pathway enzymes organized as enzyme complexes. Both are valid scientific hypotheses in that they make predictions that can be tested regarding pathway regulation, localization, and function. The goal of the work presented here was to test the metabolon model using the flavonoid biosynthetic pathway in Arabidopsis, which has been hypothesized to exist as a metabolic enzyme complex. Five novel mutants of the gene encoding the first enzyme of flavonoid biosynthesis, chalcone synthase (CHS), were characterized in an effort to develop tools for investigating the organization of flavonoid metabolism in Arabidopsis. A variety of mutant CHS genotypes were identified in this allelic series, including ones that displayed both null and temperature-sensitive phenotypes, based on endproduct analysis. Characterization of protein and RNA levels indicated that the stability of the CHS enzyme was reduced in some of the mutants as compared to wild type. In several of the alleles, homodimerization of CHS was also impaired. Effects of the mutations at the amino acid level were predicted from the three-dimensional crystal structure of the highly-homologous alfalfa CHS, which indicated substitutions at diverse sites on the enzyme, including ones that may disrupt folding and/or active site function. This allelic series should provide a useful genetic resource for ongoing studies of flavonoid enzyme structure, function, and subcellular organization. In an effort to determine the in planta location of the first two enzymes in flavonoid biosynthesis, CHS and chalcone isomerase (CHI), immunolocalization experiments were performed. Results indicate that CHS and CHI are abundant in epidermal and cortex cells of the root elongation zone and the root tip, consistent with the accumulation of flavonoid endproducts at these sites. At the subcellular level, both of these enzymes were found to localize to the endoplasmic reticulum (ER), consistent with the hypothesis that the enzymes of flavonoid biosynthesis are organized as a membrane-associated enzyme complex. Analysis of the tt7(88) mutant, which lacks the cytosolic domain of the putative 'anchor' P450 enzyme, flavonoid 3'-hydroxylase, showed an altered distribution of CHS and CHI as compared to wild type, however CHS and CHI were still found to be associated with ER. These results suggest that complex interactions occur within the flavonoid enzyme complex to mediate the subcellular distribution of its constituents. Also evident from these studies was the asymmetric distribution of CHS and CHI in cortex cells of the elongation zone, a finding that may provide clues about the physiological function of flavonoids in roots. Together, these immunolocalization data support the metabolon model for the organization of flavonoid biosynthesis in Arabidopsis. In an effort to develop tools to investigate the in vivo dynamics of flavonoid biosynthesis, fusion proteins between CHS or CHI and the reporter, green fluorescent protein (GFP), were produced. Transient transfection assays in epidermal cells from onion root bulbs and Arabidopsis seedlings indicated that the GFP component of the fusion constructs was functional, as determined via GFP fluorescence. To investigate the spatial and temporal dynamics of these fusion proteins in all cell types, Arabidopsis plants stably transformed with the CHI-GFP fusion constructs were generated. The analysis of these transgenic plants should provide information regarding the localization and dynamics of flavonoid biosynthesis in vivo, and thereby serve to offer new insights into the function and regulation of this important plant metabolic pathway. Overall, the research presented here represents a significant contribution toward understanding how subcellular organization may be important in regulating metabolism. / Ph. D.

Vascular tissues provide both the mechanical support to the plant body and the conducting cells for the transport of water, mineral solutes, hormones and other signaling molecules, amino acids, and sugars. To identify genes that may regulate vascular tissue-specific functions, we isolated xylem, phloem-cambium, and nonvascular tissues from the Arabidopsis root-hypocotyl, performed a genome-wide comparative analysis of tissue-specific transcripts using the 24K Affymetrix Arabidopsis ATH1 Genome Array (24K GeneChip), and identified potential genes that are required for xylem and phloem differentiation or tissue-specific functions. Based on this comparative analysis, two phloem-specific G2-like transcription factors, MYR1 and MYR2, and a xylem-specific NAC domain family member, XND1, were selected for further characterization. Under continuous light, myr2 plants flowered early, while myr1 plants did not differ significantly from wild type controls. However, double mutant myr1myr2 plants exhibited a novel phenotype characterized by elongated petioles, semi-erect leaf orientation, and suppression of lateral shoot outgrowth. These characteristics are reminiscent of yucca, a dominant Arabidopsis mutant with elevated levels of free auxin. Preliminary results indicated that like yucca, myr1myr2 plants were more resistant than wt plants to 5-mT, a toxic tryptophan analog, suggesting that MYR1 and MYR2 may be involved in regulating tryptophan-dependent auxin biosynthesis. Overexpression of any one of MYR1 isoforms resulted in a phenotype that in some cases resembled that observed in the double mutant, indicating that the regulation mediated by MYR1 and MYR2 may depend on formation of specific heterodimers consisting of isoforms of MYR1 and/or MYR2, and that the dimerization was susceptible to disruption both by overexpression and loss-of-function of MYR1/MYR2. Overexpression of XND1 resulted in the absence of TEs as determined from the absence of both secondary cell wall deposition and TE death. Using 3 tissue-specific promoter-GUS lines as genetic backgrounds, we demonstrated that overexpression of XND1 suppressed only TE-specific GUS expression but not phloem-specific GUS expression. Three T-DNA/transposon insertion lines, xnd1-1, -2, and -3, were identified. Under normal conditions, xnd1 did not exhibit significantly different growth and development compared to wild type plants. However, preliminary data indicated that xnd1 plants were ABA and cold hypersensitive. Yeast-two hybrid screening using the N-terminal portion of XND1 as bait identified a novel RING finger protein, At3g62970 that may function as the ubiquitin ligase (E3). These results suggested that XND1 functions as a negative regulator of xylem cell differentiation, and that the regulation mediated by XND1 may be integrated with the ubiquitin/26S proteasome pathway. / Ph. D.