UDP-GLUCOSE:SINAPIC ACID GLUCOSYLTRANSFERASE IN BRASSICACEAE by SHAWN X. WANG B. Sc., The Shanghai Agricultural College, 1983 M. Sc., The University of Saskatchewan, 1992 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF PLANT SCIENCE We accept this thesis as conforming to therequiredstandard THE UNIVERSITY OF BRITISH COLUMBIA October, 1996 © Shawn X. Wang, 1996 In presenting this thesis in partial fulfilment , of the requirements: for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada Date tfew '??7 DE-6 (2/88) ABSTRACT Sinapine is a bitter phenolic choline ester that is ubiquitous in crucifer seeds. It occupies about 1-4% of the dry matter of rapeseed. Elimination of sinapine from the seeds would improve the flavor, palatability and nutritional properties of rapeseed meal for its utilization as supplemental protein source for animal feed. UDP-glucose.sinapic acid glucosyltransferase (SGT; EC 2.4.1.120) is a key enzyme involved in biosynthesis of both sinapine in seed and sinapoylmalate in vegetative tissues of many members of Brassicaceae. Both esters are strongly UV-absorbing but their physiological functions in plants are unknown. By a series of column chromatography techniques, SGT has been purified from 60-h-old seedlings of Brassica napus and characterized. SGT is a monomeric polypeptide with a molecular weight of 42 kDa and a pi of pH 5. The subcellular location of SGT appears to be the cytosol. SGT activity is not inducible by heat shock or UV radiation stresses. Its general characteristics are similar to those of SGT from Raphanus sativus, as well as a number of other UDP-glucose-dependent glucosyltransferases. The Km (UDp.giUCOse) was 0.24 mM and Km (sinapic acid) was 0.16 mM. SGT also catalyzes the reverse reaction in vitro, using UDP and sinapoylglucose to form UDP-glucose. No cofactors are required for SGT activity, but reducing reagents and glycerol are required to stabilize the enzyme. The enzyme is strongly inhibited by p- hydroxyl-mercuribenzoic acid, UDP, TDP, Zn++, Cu++ and Hg++. Kinetic properties and substrate affinity data suggest that the catalytic mechanism of SGT is best described by a "random bi-bi" model. An analysis of developmental profiles showed that SGT was expressed in all growth stages of B. napus plants, but was most active during the early ii germination and seed development stages, particularly in cotyledons, juvenile leaves and young shoots. Partial amino acid sequence data were obtained from tryptic digests of the putative SGT protein. These sequences showed a very high degree of similarity to the HSP/HSC70 protein family. iii TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iv LIST OF TABLES . x LIST OF FIGURES xii ABBREVIATIONS xvi ACKNOWLEDGMENTS xviii Chapter I GENERAL INTRODUCTION 1 1. Sinapoyl esters and phenolics in plants 2 2. The central role of the phenylpropanoid pathway 4 3. Glucosylation and UDP-glucose-dependent glucosyltransferases 5 4. Biological significance of SGT 8 5. Economic significance of SGT 10 Chapter II TAXONOMIC DISTRIBUTION OF SGT IN BRASSICA CEAE 14 INTRODUCTION 15 OBJECTIVE 16 MATERIALS 16 1. Plant materials 16 2. Chemicals 19 3. Equipment 19 METHODS 19 1. Determination of sinapine by HPLC 19 1.1. Sample preparation 19 1.2. HPLC analysis 20 1.3. Calculation of sinapine content • 20 2. SGT assay for dry seeds and seedlings 21 2.1. Seedling growth 21 2.2. Protein sample preparation 21 2.3. Determination of protein concentration 22 2.4. Determination of SGT activity 23 2.5. Calculation of SGT activity 23 iv RESULTS 25 1. Sinapine accumulation 25 2. SGT activity 25 DISCUSSION 29 Chapter III THE DIVERSITY OF SINAPIC ACID ESTERS, ENZYMOLOGY AND DEVELOPMENTAL EXPRESSION OF SGT IN B. NAPUS AND S. ALBA 33 INTRODUCTION 34 OBJECTIVES 39 MATERIALS 39 1. Plant materials 39 2. Chemicals 39 METHODS 40 1. Plant growth conditions and sampling 40 2. HPLC analysis of methanol-soluble phenolics 41 2.1. Sample preparation 41 2.2. HPLC analysis 41 2.3. Qualitative and quantitative determination of sinapic acid derivatives 42 3. Enzymatic assays of SE, SGT, SCT, SMT and 4CL .... 42 3.1. Sample preparation for protein extraction 42 3.2. Determination of SE activity by HPLC 42 3.3. Determination of SGT activity by HPLC 43 3.4. Determination of SMT activity by HPLC 43 3.5. Determination of SCT activity by HPLC 44 3.6. Determination of 4CL activity by HPLC 44 RESULTS 45 1. Profiles of methanol-soluble phenolics in various tissues at different growth stages 45 1.1. Pattern of phenolic compound accumulation in young seedlings 45 1.2. Pattern of phenolic compound accumulation in various tissues of adult plants 49 1.2.1. B. napus cv. Westar 49 1.2.2. S. alba cv. Ochre 52 1.3. Profiles of phenolic compounds accumulated in developing seeds 55 V 2. Developmental pattern of expression of SE, SGT, SMT, SCT and 4CL 57 2.1. SE, SGT, SMT, SCT and 4CL activities in young seedlings 57 2.2. SE, SGT, SMT, SCT and 4CL activities in vegetative tissues of adult plant 60 2.3. SE, SGT, SMT, SCT and 4CL activities in fruit 63 3. Developmental expression of SGT in B. napus cv. Westar .66 DISCUSSION 70 Chapter IV ENZYMOLOGY OF UDP-GLUCOSE:SINAPIC ACID GLUCOSYLTRANSFERASE FROM B. NAPUS 81 INTRODUCTION 82 r OBJECTIVE 83 MATERIALS 87 1. Plant materials 87 2. Chemicals 87 METHODS 88 1. Determination of SGT activity 88 1.1. Multi-well plate method 88 1.2. HPLC method 88 1.3. Radioisotope method 89 1.4. Spectrophotometry method 90 2. Induction of SGT 92 2.1. Induction of SGT by sinapic acid 92 2.2. Induction of SGT by light 93 2.3. Induction of SGT by heat shock 94 3. Subcellular localization of SGT 94 4. Purification of SGT 96 5. Physical and chemical characterization of SGT 100 5.1. Determination of molecular weight 100 5.2. Determination of SGT pH stability and pH optimum 101 5.3. Determination of SGT thermal stability and temperature optimum 102 5.4. Requirement for metal ions 103 5.5. Requirement for reducing reagents 103 5.6. Determination of SGT substrate specificity 104 vi 5.7. Determination of the inhibitory effect of UDP-glucose analogues 105 5.8. Determination of effects of other inhibitors 105 5.9. Determination of SGT reversibility 106 5.9.1. From the forward reaction 106 5.9.2. From the reverse reaction 107 5.10. Investigation of affinity matrices binding for SGT 107 5.10.1. UDP-glucose-hydrazide membrane 107 5.10.2. UDP-glucuronic acid-agarose 109 5.10.3. Sinapic acid-Sepharose 110 5.11. Determination of SGT substrate kinetics Ill 6. Recovery of SGT activity from native PAGE 112 7. Determination of amino acid composition and sequence 113 8. Protein sequence analysis 114 9. Production of polyclonal antibodies 115 9.1. Production of polyclonal antibodies with native antigen 115 9.2. Production of polyclonal antibodies with denatured antigen 116 10. Immunodetection of SGT by Western blot 116 11. Immunoprecipitation of SGT activity 117 RESULTS 118 1. Induction of SGT 118 1.1. Substrate - sinapic acid effect on SGT 118 1.2. Light effect on SGT activity 121 1.3. Heat stress effect on SGT activity 121 2. Subcellular localization of SGT 121 3. Purification of SGT 126 4. Physical and chemical characterization of SGT 131 4.1. Molecular weight 131 4.2. pi, pH stability and pH optimum 131 4.3. Thermal stability and optimum temperature 133 4.4. Effect of metal ions 133 4.5. Effect of reducing reagents 139 4.6. Effect of other inhibitors 139 4.7. Substrate specificity 143 4.8. Inhibitory effect of substrate analogues 143 4.9. Reversibility of SGT 143 4.10. Affinity matrices , 147 4.11. Kinetic parameters 147 5. Amino acid composition and sequence 147 6. Protein sequence homology and comparison 152 vii 7. Immunological responses 152 DISCUSSION 160 1. Induction of SGT 160 2. Subcellular localization of SGT 162 3. Physical and chemical properties of SGT 164 4. Amino acid sequence homology and immunological responses 168 Chapter V ATTEMPTS TO IDENTIFY A GENE ENCODING SGT .... 172 INTRODUCTION 173 OBJECTIVE 175 MATERIALS 175 1. Plant materials 175 2. Chemicals 175 METHODS 176 1. Synthesis of BEK oligo-d(T)20 oligo adapter 176 2. Synthesis of SGT specific N-terminal primers 176 3. Isolation of total RNA from seedlings 177 4. Synthesis of single strand cDNA from total RNA by AMV-RT and M-MLV-RT 178 5. Amplification of hsp70/hsc70 and sgt by PCR 179 6. Agarose gel electrophoresis 180 7. Labeling hsplOlhsclQ probes with a-32P-dATP by random primer labeling system 180 7.1. DNA preparation 180 7.2. Random primer labeling 181 8. Northern blot analysis of seedling RNA 181 8.1. Electrophoresis of RNA 182 8.2. Capillary transfer with sodium citrate buffer 182 8.3. Pre-hybridization 183 8.4. Hybridization with probe 183 9. Southern blot analysis of PCR products 184 9.1. Electrophoresis of PCR products 184 9.2. Capillary transfer with 0.4 M NaOH 184 9.3.