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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.

Flavonoid biosynthesis is an important secondary metabolic pathway in higher plants with a range of vital functions in plants and animals. This pathway has been developed as a model system for the study of multi-enzyme complexes. The goal of the work presented here was to structurally characterize a series of loss-of-function chalcone synthase (CHS) alleles and to define the molecular basis of the interaction between CHS and the second enzyme of flavonoid biosynthesis, chalcone isomerase (CHI). CHS proteins encoded by five previously characterized alleles were characterized by homology modeling in an effort to explain the alterations in function, stability, and dimerization exhibited by these variants. Four of the encoded proteins have a single amino acid substitution and the fifth is a truncated protein resulting from a frameshift. Models for each of these proteins were generated in silico and analyzed after molecular dynamics simulations. This analysis suggested reasons for changes in catalytic ability and stability for three of the five CHS variants. To characterize the molecular basis of the CHS-CHI interaction, a model was developed using X-ray crystallography, small-angle neutron scattering (SANS), in silico docking, molecular dynamics simulations, and yeast 2-hybrid analyses. These enzymes appear to be interacting in a manner that could facilitate the flow of intermediates from one active site to another. These experiments also identified a series of amino acids that appear to be involved in the interaction, which are currently undergoing alteration and analysis using a yeast 2-hybrid assay to verify the authenticity of the model. The data presented herein could be used in future engineering experiments to alter pathway flux to control the levels or types of flavonoid endproducts, resulting in more nutritious plants or flowers with novel pigments. These experiments advance the study of the structure of multi-enzyme complexes, an area that currently contains little information. As well, this is the first known use of SANS for the investigation of the architecture of metabolons. The techniques described herein could easily be applied to other systems in an effort to better understand the organization of multi-enzyme complexes and the implications of these assemblies on metabolic regulation. / Ph. D.

Polyclonal antibodies against the first two enzymes of the Arabidopsis thaliana flavonoid biosynthetic pathway were developed using conventional and phage antibody technology. cDNAs from Arabidopsis coding regions of chalcone synthase (CHS) and chalcone isomerase (CHI) were sub-cloned in frame into a bacterial expression vector as fusions with glutathione Stransferase (GST) using standard directional cloning techniques. Analysis of crude extracts of Escherichia coli containing GST .. CHS or GST .. CHI fusion protein indicated that the cells expressed equivalent amounts per volume of culture. CHS and CHI were purified to near homogeneity, yielding approximately 100 micrograms of GST .. CHS and 1 milligram of GST-CHI per liter of culture. The purified fusion proteins were injected into chickens and polyclonal lgY·s were purified from egg yolk Accumulation of CHS and CHI, as well as products of the pathway, were compared during the first eight days of Arabidopsis development. CHS and CHI are sequentially induced and reach maximal accumulation levels by day 5. Anthocyanidin levels are offset by one reaching maximal levels at day 6. The fusion proteins were also used to screen a phage-display library for Fabl fragments that recognize CHS and CHI epitopes. Preliminary data indicated that enrichment of phage displaying antibodies against CHS and CHI was successful. Phage-derived antibodies against CHS and CHI provide valuable tools for future experiments addressing Western blot analysis, immunolocalization experiments, and disruption of the flavonoid biosynthetic pathway by introduction of the corresponding genes into transgenic Arabidopsis plants. / Master of Science