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The Role of NADP + Order Number 9411942 The role of NADP+-malic enzyme in tomato fruit Finger, Fernando Luiz, Ph.D. The Ohio State University, 1993 Copyright ©1993 by Finger, Fernando Luiz. All rights reserved. U M I 300 N. Zeeb Rd. Ann Arbor, M I 48106 THE ROLE OF NADP+-MALIC ENZYME IN TOMATO FRUIT DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Fernando Lufz Finger, B.S., M.S. ***** The Ohio State University 1993 Dissertation Committee: Approved by M. Knee L. M. Lagrimini [HLi t L ____ Advisor M. S. Biggs Department of Horticulture J. J. Finer Copyright by Fernando Lufz Finger 1993 To My Parents ii ACKNOWLEDGMENTS I would like to express my sincere appreciation to my advisor Dr. Michael Knee for his guidance and interest in this research. I would also like to thank Dr. Mark Lagrimini for sharing his expertise in molecular biology techniques. Thanks go to Drs. Scott Biggs and John Finer for their comments and suggestions. I would like to thank the following Brazilian institutions, Universidade Federal de Vigosa, CAPES and FAPEMIG for their financial support. Special thanks go to my colleague Dr. Raimundo Santos Barros for his friendship and encouragement. I am very grateful to Dr. Avtar Handa (Purdue University) for providing the tomato fruit lambda ZAP If library and Dr. Timothy Nelson (Yale University) for the corn malic enzyme cDNA clone. I would like to thank my fellow graduate students, in special, Joe Curran and Karen Klotz for their friendship. This research was in part supported by a grant from The Ohio State Graduate Student Alumni Research Award. VITA July 8, 1958 Born - Caxias do Sul, Brazil 1982 B.S., Universidade Federal de Pelotas, Brazil 1986 M.S., Universidade Federal de Vigosa, Brazil 1986-Present Assistent Professor of Universidade Federal de Vigosa, Brazil PUBLICATIONS Finger F. L. (1985). Effects of water loss on postharvest physiology of green pepper (Capsicum annuum L.) and (Musa acuminata Colla) fruits. M.S. Thesis, Universidade Federal de Vicosa, Brazil. Knee M. and Finger F. L. (1992). NADP+-Malic enzyme and organic acid levels in developing tomato fruits. J. Amer. Soc. Hort. Sci. 117: 799-801. FIELDS OF STUDY Major Field: Horticulture Postharvest physiology of fruits and vegetables TABLE OF CONTENTS ACKNOWLEGMENTS ................................................................................. iii VITA ................................................................................................................ iv LIST OF TABLES ........................................................................................ vii LIST OF FIGURES....................................................................................... viii GENERAL INTRODUCTION........................................................................ 1 CHAPTER I. NAD P+-MALIC ENZYME AND ORGANIC ACID METABOLISM IN DEVELOPING TOMATO FRUITS.................................................... 14 Introduction ............................................................................ 14 Materials and M ethods ......................................................... 17 Results and Discussion ....................................................... 21 Conclusions .......................................................................... 40 List of References ................................................................ 42 II. PURIFICATION AND INTRACELLULAR LOCATION OF TOMATO FRUIT NADP+-MALIC ENZYME ..................................................... 44 Introduction ............................................................................ 44 Materials and M ethods ......................................................... 47 Results and Discussion ....................................................... 61 Conclusions .......................................................................... 88 List of References ................................................................ 90 v III. ISOLATION AND CHARACTERIZATION OF A cDNA CLONE FOR NADP+-MALIC ENZYME FROM TOMATO FR U IT........................ 94 Introduction ............................................................................ 94 Materials and M ethods ......................................................... 97 Results and Discussion ....................................................... 113 Conclusions .......................................................................... 135 List of References ................................................................ 137 SUMMARY AND CONCLUSIONS.............................................................. 140 LIST OF REFERENCES.............................................................................. 145 vi LIST OF TABLES TABLE PAGE 1.1 NADP+-malic enzyme activity, malic and citric acid contents in various tissues of mature-green 'Ohio 7814’ tomato fruits 37 2.1. Purification of NADP+-malic enzyme from mature-green tomato fruit pericarp ........................................................................................ 62 2.2. Activity of NADP+-glyceraldehyde-3-phosphate dehydrogenase in intact and 0.1% Triton X-100 lysed chloroplast fraction of immature-green tomato fruit pericarp, tomato leaves and corn leaves ................................................................................................... 78 2.3. Total and specific activities of ribulose-1,5- bisphosphate carboxylcise in the Sephadex G-25 protein fraction of the supernatant and 0.1% Triton X-100 lysed chloroplast fraction from immature-green tomato fruit, tomato leaves and corn leaves ................................................................................................... 79 2.4. Total and specific activities of NADP+-malic enzyme in the Sephadex G-25 protein fraction of the supernatant and 0.1% Triton X-100 lysed chloroplast fraction from immature-green tomato fruit, tomato leaves and corn leaves .................................... 81 3.1. Comparison of nucleotide sequence between ME-4 cDNA and the sequence of malic enzyme cDNAs from other sources. ... 130 LIST OF FIGURES FIGURE PAGE 1.1. Transverse section of a tomato fruit showing the various tissues ............................................................................................... 18 1.2. Changes in specific activity of NADP+-malic enzyme in the outer pericarp of Cherry tomato fruits through ripening ........................ 23 1.3. Changes in specific activity of NADP+-malic enzyme in the inner tissues of Cherry tomato fruits through ripening .......................... 24 1.4. Changes in malic acid concentration during Cherry tomato fruit ripening ............................................................................................. 27 1.5. Fruit growth in fresh weight and soluble protein changes in the outer pericarp and inner tissues of ’Ohio 7814’ tomatoes 29 1.6. Changes during fruit development for the outer pericarp of 'Ohio 7814' tomato in specific activity of NADP+-maiic enzyme 30 1.7. Changes during fruit development for inner tissues of 'Ohio 7814’ tomato specific activity of NADP+-malic enzyme ........................... 31 1.8. Changes during fruit development for outer pericarp of ’Ohio 7814’ tomato, in specific activity of NADP+-ma!ic enzyme, malic acid and citric acid contents ........................................................... 34 1.9. Changes during fruit development for inner tissues of 'Ohio 7814’ tomato, in specific activity of NADP+-malic enzyme, malic acid and citric acid contents ................................................................... 35 2.1. Purification of NADP+-malic enzyme from tomato fruit on DEAE- Cellulose column .............................................................................. 63 2.2. Purification of NADP+-malic enzyme from tomato fruit on gel filtration column ................................................................................. 65 2.3. Estimation of the molecular weight of native NADP+-malic enzyme (ME) from tomato fruit by Sephacryl S-300 chromatography................................................................................ 66 2.4. Purification of tomato fruit NADP+-malic enzyme on pseudo­ affinity chromatography Cibacron Bluecolumn .............................. 68 2.5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% separating gel) of fractions eluted from Cibacron Blue 3GA Agarose column ................................................................................ 70 2.6. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8% separating gel) of a fraction eluted from Cibacron Blue 3GA Agarose column ................................................................................ 72 2.7. Western blots following 8% sodium dodecyl sulfate- polyacrylamide gel electrophoresis ................................................. 74 2.8. Immunoinhibition of tomato fruit NADP+-malic enzyme activity in a Sephadex G-25 protein fraction ................................................... 76 2.9. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8% separating gel) of total and chloroplastidic protein fraction from tomato fruit, tomato leaves and corn leaves .................................. 83 2.10. Western blot following 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total and chloroplastidic protein fraction from tomato fruit, tomato leaves and corn leaves ........................... 84 2.11. Western blot following 8% sodium dodecyl
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