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.

Les deux systèmes nodulaires symbiotiques les plus importants au niveau agronomique et environnemental sont, d'une part, les symbioses Rhizobium-légumineuses qui concernent environ 14 000 espèces, et d'autre part, les symbioses entre les plantes actinorhiziennes (environ 200 espèces) et l'actinomycète du sol Frankia. La plupart des plantes actinorhiziennes sont capables de fixer des quantités d'azote comparable à celles des Légumineuses ; ce sont généralement des plantes pionnières capables de coloniser des environnements pauvres en éléments minéraux. Elles représentent donc un atout écologique important. Si la symbiose Rhizobium-légumineuse est très étudiée, les mécanismes moléculaires à l'origine de la formation des nodules actinorhiziens restent actuellement peu connus. Ainsi, chez les Légumineuses, les flavonoïdes sont des molécules-clefs du processus de nodulation, alors que chez les plantes actinorhiziennes, l'implication des flavonoïdes dans la nodulation reste imprécise. L'objectif de cette thèse était de comprendre l'implication des flavonoïdes au cours de l'interaction symbiotique entre l'arbre actinorhizien tropical Casuarina glauca et son symbiote Frankia. L'analyse d'une base de données d'unigènes couplée à celle de données d'expression de puces à ADN a permis l'identification de huit genes de C. glauca impliqués dans la voie de biosynthèse des flavonoïdes. L'étude de leur expression dans les racines par PCR quantitative au cours d'une cinétique d'infection de C. glauca par Frankia a montré que les transcrits de la chalcone isomerase et de l'isoflavone reductase s'accumulaient très tôt après l'inoculation, suggérant ainsi une implication des isoflavonoïdes dans la symbiose actinorhizienne. Nous avons alors utilisé une stratégie d'ARN interférent pour réduire l'expression du gène de la chalcone synthase, la première enzyme de la voie de biosynthèse des flavonoïdes. La réduction de l'expression du gène de la chalcone synthase a provoqué une réduction significative du taux de flavonoïdes dans les racines ainsi qu'une très forte diminution du taux de nodulation chez les plantes transformées. Une restauration du taux de nodulation a pu être obtenu en présence de naringenin, une molécule centrale de la voie de biosynthèse des flavonoïdes.Nos résultats apportent donc, pour la première fois, une évidence directe de l'implication forte des flavonoïdes au cours de la nodulation des plantes actinorhiziennes. / Nitrogen-fixing root nodulation, confined to four plant orders, encompasses more than 14,000 Leguminosae species, and approximately 200 actinorhizal species forming symbioses with rhizobia and Frankia bacterial species, respectively. Most actinorhizal plants are capable of high rates of nitrogen fixation comparable to the nitrogen fixing symbiosis between legumes and Rhizobium. As a consequence, these plants are able to grow in poor and disturbed soils and are important elements in plant community worldwide. The basic knowledge of the symbiotic interaction between Frankia and actinorhizal plants is still poorly understood, although it offers striking differences with the Rhizobium-legume symbiosis. In the symbiosis between legumes and Rhizobium, flavonoids are key molecules for nodulation. In actinorhizal plants, the involvement of flavonoids in symbiosis is poorly understood, but because of the similarities of the infection process between some actinorhizal plants and legumes, flavonoids were proposed to act as plant signals for the bacteria Frankia. The objective of this thesis was to investigate the involvement of flavonoids during the actinorhizal nodulation process resulting from the interaction between the tropical tree Casuarina glauca and the actinomycete Frankia.Eight C. glauca genes involved in flavonoid biosynthesis were identified from a unigene database and their expression patterns were monitored by quantitative real-time PCR during the nodulation time course. Our results showed that chalcone isomerase and isoflavone reductase transcripts accumulated preferentially early after inoculation with Frankia, suggesting thus for the first time that isoflavonoids are implicated in actinorhizal nodulation. To go deeper in the understanding of the role of these molecules in actinorhizal symbiosis, we used RNA interference strategy to silence chalcone synthase, the enzyme that catalyzes the first committed step of the flavonoid pathway. Knockdown of chalcone synthase expression led to a strong reduction of specific flavonoids levels and resulted in a severely impaired nodulation. Nodule formation could be rescued by supplementation of plants with naringenin, which is an upstream intermediate in flavonoid biosynthesis. Our results provide, for the first time, direct evidence of a strong implication of flavonoids during actinorhizal nodulation.

Ultraviolet –B (UV-B) radiation is an abiotic stress that has significant effects on plant growth, development, and gene regulation. Due to the depletion of the stratospheric ozone layer over the past several decades, the amount of UV-B light that is reaching the earth’s surface has significantly increased. As a result, research over the past few decades on the effects of UV-B light on plant growth, development, and the mechanisms that regulate a plant’s protection and survival against UV-B light has grown greatly. Brachypodium distachyon is a relatively new model system and one that has not been extensively studied. The aim of this study was to determine the UV-B dose time required to elicit a significant increase in phenolic content, while subsequently assessing protein production to qualitatively implicate whether or not the experimental dosage of UV-B administered was initiating a UV-B specific or non-specific response. In addition, this research annotated the genes that encode the protein sequences for UVR8 and CHS proteins to see if B. distachyon possessed the necessary proteins to undergo a UV-B specific response similar to that of Arabidopsis. The results of the study show that in response to artificial UV-B light, the dose time of UV-B required to elicit a significant increase in total phenolic content is 2 hours. The data also shows an increase in total protein content after 4 hours of UV-B exposure. In addition to the metabolic data, computational analysis of chalcone synthase (CHS) and UV-RESISTANCE LOCUS 8 (UVR8) revealed that there are seven genes in B. distachyon that encode the protein transcripts for CHS and CHS-like proteins, and two genes that code for UVR8 proteins. The results of this study suggest that the UV-B dose regimen used in this study may be initiating the non-specific UV-B signaling pathway. In addition, the presence of UVR8 and CHS protein sequences suggest that B. distachyon has the capacity to work through the UV-B specific signaling pathway.

The organization of metabolism is an essential feature of cellular biochemistry. Metabolism does not occur as a linear assembly of freely diffusing enzymes, but as a complex web in which multiple interactions are possible. Because of the crowded environment of the cell, there must be structured and ordered mechanisms that control metabolic pathways. The following work will examine two metabolic pathways, one that is ubiquitous among living organisms and another that is entirely unique to plants, and examine the organization of each in an attempt to further define mechanisms that are fundamental features of metabolic control. One study offers some of the first characterizations of genes involved in [Fe-S] cluster assembly in Arabidopsis. The other explores the mechanisms that control localization of an enzyme that is part of the well-characterized flavonoid biosynthetic pathway. These two distinct pathways serve as unique models for genetic and biochemical studies that contribute to our overall understanding of plant metabolism. / Ph. D.