The Flavonoid Biosynthetic Pathway of Arabidopsis thaliana

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Chapter 2 Proteomic Analysis of the Flavonoid Biosynthetic Machinery of Arabidopsis thaliana

SUMMARY

This thesis describes an effort to integrate mass spectrometry with a previouslyestablished affinity chromatography method to extend the analysis of protein complexes associated with the flavonoid biosynthetic pathway of Arabidopsis thaliana (Burbulis and Winkel-Shirley, 1999). For the affinity chromatography approach described in this chapter, the flavonoid enzyme, chalcone isomerase (CHI), was expressed in E. coli as a fusion to the carboxy terminus of thioredoxin (TRX). TRX was also expressed alone for use in control experiments. The recombinant proteins were used to prepare an affinity column using Affi-Gel 10 resin, to which soluble protein extracted from 4-day-old seedlings of Landsburg and the CHI null mutant,tt5(86) was applied. The resulting samples were then used to test the feasibility of using a proteomics approach to identify the full array of proteins recovered by affinity chromatography,as explained in Chapter 3. Keywords: Protein-protein interactions, Enzyme complexes, Affinity chromatography, Flavonoid Pathway

INTRODUCTION

The flavonoid biosynthetic pathway is a well-studied metabolic system that exists exclusively in plants. Products of the pathway are secondary metabolites, derived from phenylalanine and acetyl CoA (Stafford, 1990), that perform a diverse set of functions. Flavonoids are known for their roles as the major red, blue, and purple pigmentation that are used to attract pollinators and seed dispersers (Weiss, 1991). The products also provide UV protection (Li et al., 1993), and microbial defense (Stafford, 1990). Animals, including humans, consume large quantities of flavonoids in fruits and vegetables. The antioxidant and anticancer properties of flavonoids are advantageous for animal and human health (Harborne and Williams, 2000).It is believed that many metabolic pathways exist as networks of interacting enzymes that
enhance the efficiency of metabolism in diverse ways. For example, the formation of enzyme complexes helps to separate anabolic and catabolic reactions (Hrazdina and Jensen, 1992). This organization allows the accumulation of high local substrate concentrations and the channeling of intermediates (Winkel, 2004). The Winkel laboratory uses the flavonoid biosynthetic pathway of Arabidopsis thaliana as a model to understand the assembly of enzyme complexes. Interaction among several flavonoid enzymes, demonstrated by yeast two-hybrid, affinity chromatography, and coimmunoprecipitation, showed that the enzymes of the pathway interact with each other in highly specific manner (Burbulis and Winkel-Shirley, 1999). Also, two key enzymes of the pathway, chalcone synthase (CHS) and CHI, co-localize in both the cytoplasm and nuclei of specific cells at the root tip and in leaves (Saslowsky et al., 2005). In the proposed research, an effort was made to elucidate the organization of the flavonoid pathway using affinity directed mass spectrometry (Figure 2.1). One of the flavonoid enzymes, CHI, was used as a bait to attract interacting proteins from crude extract prepared from 4-day-old wild type Landsburg and mutant tt5(86) seedlings, a CHI gene mutant. An effort was then made to identify the captured proteins using an ESI LC-MS/MS method, as described in Chapter 3.

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Materials and Methods

Recombinant Protein Expression

Recombinant protein was expressed following a previously-published protocol (Burbulis and Winkel-Shirley, 1999). Two key flavonoid enzymes, CHS and CHI, were expressed as fusions to the carboxyl terminus of thioredoxin (TRX) using the pCD1 vector (Dana et al.,submitted). TRX was also expressed from the pCD1 vector. The plasmids were introduced in E.coli strain BL21(DE3) pLysS (Novagen). Protein expression was induced by the addition of dioxane-free isopropyl-β-D-thiogalactoside (Fisher Scientific, New Jersey) to a final concentration of 0.5 mM (1 ml of 0.5 M per liter of culture), followed by incubation at room temperature, 200 rpm for 4 h. The cells were collected by centrifugation at 5000 RPM in a JA10 rotor at 4°C for 15 min and then were stored at -20°C. The cell pellets were resuspended in 33 ml of lysis buffer (50 mM Hepes, pH 8.0, 150 mM NaCl, 10% glycerol, 1% Tween-20, 10 mM 2-mercaptoethanol) per liter of culture, and then sonicated (6 × 10 sec) on ice. Pellets were collected by centrifugation at 14,000 RPM in a JA-17 rotor at 4°C for 40 min.

ACKNOWLEDGMENTS 
TABLE OF CONTENTS
LIST OF FIGURES 
LIST OF TABLES
Chapter 1: Introduction 
Multienzyme Complexes 
The Flavonoid Biosynthetic Pathway of Arabidopsis thaliana 
Mass Spectrometry
Protein Profiling by Mass Spectrometry
Protein-Protein Interactions and Proteomics 
Proposed Study 
Acknowledgments
Chapter 2: Proteomic Analysis of the Flavonoid Biosynthetic Machinery of Arabidopsis thaliana
Summary
Introduction
Materials and Methods
Results
Discussion
Acknowledgment
Chapter 3: Mass Spectrometric Analysis of Recombinant Proteins, Affinity Eluted Samples,and Soluble Plant Extracts From Wild Type Landsburg Ecotype and Mutant tt5(86)
Arabidopsis thaliana
Summary
Introduction
Materials and Methods
Results
Discussion
Acknowledgments
Chapter 4: Conclusions 
Conclusions
References
Appendix A: Results from proteomic analysis of Landsburg seedlings
VITA

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