SOURCE-SINK RELATIONSHIP DURING PAPAYA FRUIT DEVELOPMENT AND RIPENING A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERISTY OF HAWAIM IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN HORTICULTURE MAY 1999 BY Lili Zhou Dissertation Committee: Robert E. Pauli, Chairperson Kent Kobayashi Roy K. Nishimoto Chung-Shih Tang David A. Christopher We certify that we have read this dissertation and that, in our opinion, it is satisfactory in scope and quality as a dissertation for the degree of Doctor of Philosophy in Horticulture. DISSERTATION COMMITTEE Chairperson ACKNOWLEDGEMENTS My sincere thanks give to Dr. Robert E. Pauli, my major advisor, for his guidance, support, and encouragement throughout my Ph.D. program and to Dr. David Christopher, for his supervision and help on molecular techniques and allowing me to work in his lab, for the last two years. I am grateful to other committee members. Dr. Kent Kobayashi, Dr. Rey K. Nishimoto, Dr. Chung-Shih Tang for their valuable comments, input, and encouragement during my research. I also appreciate Dr. Chia’s kind help during my comprehensive examination. I express my special thanks to UH Poamoho Experimental Station, Doles Food Fresh Company and Mr. Nozawa at north shore private farm for management of the papaya field and for the provision of experiment fruit. Thanks also to Dr. Richard Manshardt for the use of his papaya seed and to Dr. Sturm for kindly providing carrot invertase antiserium. I deeply appreciate my lab colleagues; Dr. Nancy Chen, Mrs. Gail Uruu, Mr. Ted Goo, Mr. Achie Reyes’ technical help and friendship throughout my study. I would especially like to thank Dr. Penny Dudley, Ms. Yanxin Shen, Mr. Xinli Li, Mr. Chingcheng Chen, Mrs, Maria Q. Reyes, Ms. Theeranu Chantrachit, Mr. Paul Hoffer, Dr. Jingwei Dai, Mr. Cecilia kato. Dr. Hualiang, Dr. Hongmai Ma for their help and company. In addition, I am grateful for all help I had received from Department of Horticulture and Plant Molecular Physiology, especially to Dr. Harrienton’s lab. Dr. John Stiles’ lab, Dr. Yamamoto’s lab and Ms. Doris Victor, Mrs. Horiuchi, Lynne, and Takahashi Susan. Finally, I thank my parents, brother and sister, for their understanding, encouragement and support over my study at the university. To my husband and son, thank you for your enduring love while I was away from home earning my Ph.D. degree in the University of Hawaii. Ill ABSTRACT The source sink relationship during papaya fruit development and ripening was investigated. The source size and sink strength were modified by single or continual defoliation, and fruit thinning, respectively. The relationship between fruit growth, respiration, sugar accumulation and the activity of sucrose phosphate synthase (SPS), sucrose synthase (SS), and acid invertase were determined in fruit from 14 days after anthesis (DAA) to 140 DAA (harvest maturity) and in response to defoliation and fruit removal. A putative complete invertase gene and a SS gene fragment were isolated and characterized from nearly mature green papaya fruit. Single defoliation significantly reduced new flower and fruit set, and ripe fruit total soluble solids (TSS) but did not reduce fruit production, average fruit mass, percentage fruit flesh and seed, seed mass ratio and seed dry mass during a six weeks period. Continual defoliation in addition reduced fruit size, sugar and invertase enzyme activity and fruit production. The responses of defoliation and fruit thinning varied between different cultivars, weather conditions, defoliation time, degree and method. The pattern of gene expression during fruit development was compared with invertase extracted enzyme activity in the presence and absence of sodium chloride (NaCI) and by western blot analysis. The papaya invertase sequence had an open reading frame that encoded a polypeptide chain of 582 residues and calculated molecular weight of 65, 684 Da. The protein was highly homologous to known plant cell wall invertase and 67% identical at the amino acid level to carrot cell wall invertase. The cloned 720 bp SS fragment was highly homologous to A. glutinosa (X92378) and SS genes from other species. Invertase gene was expressed at a higher level during late fruit development stage than in young fruit and other tissues of papaya plant. SS gene expression was higher in young fruit and petiole tissues than in other tissues. The data demonstrated that SS enzyme was a major enzyme in fruit sink establishment and maintenance. Apoplastic invertase had an important function in phloem unloading during papaya fruit sugar accumulation and the activity was regulated at both transcriptional and translational levels. IV TABLE OF CONTENTS ACKNOWLEDGEMENT............................................................... iii ABSTRACT ........................................................................ iv LIST OF TABLES................................................................ x LIST OF FIGURES............................................................... xi LIST OF ABBREVIATIONS....................................................... xiii CHAPTER 1 INTRODUCTION...................................................... 1 CHAPTER 2 LITERATURE REVIEW ................................................ 3 2.1 Papaya fruit development and ripening............................. 3 2.1.1 Introduction....................................................... 3 2.1.1.1 Botany and fruit morphology............................... 3 2.1.1.2 Importance.................................................... 4 2.1.2 Fruit development and ripening................................ 4 2.1.2.1 Fruit set and growth pattern.............................. 4 2.1.2.2 Respiration and ethylene................................... 5 2.1.2. 3 Color and texture........................................... 5 2.1.2. 4 Sugar and volatiles........................................ 6 2.2 Preharvest factors affecting quality on papaya and other fruits 7 2.3 Source-sink relationship during fruit development.............. 9 2.3.1 Carbohydrate metabolism in ripening fruit as affected by leaf a r e a .......................................................... 10 2.3.2 Defoliation and source-sink manipulation.................... 11 2.3.3 Sink strength and relative enzyme activities during fruit development........................................................ 14 2. 3. 3.1 Sucrose phosphate synthase................................. 16 2. 3. 3. 2 Sucrose synthase............................................ 18 2. 3. 3. 3 Invertase..................................................... 18 2. 3. 3. 4 Enzymes and sink strength.................................. 22 2.3.4 Sugar unloading pathway at sinks.............................. 23 2.3.5 Structure and function of invertase enzymes and genes among plant species.............................................. 25 2.4 Conclusion.............................................................. 29 CHAPTER 3 HYPOTHESES AND OBJECTIVES....................................... 30 3.1 Hypotheses.............................................................. 30 3.2 Objectives.............................................................. 30 CHAPTER 4 THE RELATIONSHIP OF PAPAYA FRUIT GROWTH, RESPIRATION, SUGAR ACCUMULATION AND THE ACTIVITY OF SPS, SS, INVERTASE ENZYME DURING FRUIT DEVELOPMENT AND RIPENING.................................. 31 A b s t r a c t ...................................................................... 31 4.1 Introduction............................................................ 32 4.2 Material and methods.................................................. 34 4.2.1 Plant material.................................................... 34 4.2.2 Chemicals.......................................................... 35 4.2.3 Observations....................................................... 35 4.2.4 Sugar assay........................................................ 35 4.2.5.Dry mass assay.................................................... 35 4.2.6 Fruit respiration rate.......................................... 36 4.2.7 Carbon import rate by fruit flesh............................. 36 4.2.8 Enzyme extraction................................................. 36 4.2.9 SS and SPS assay.................................................. 36 4.2.10 Invertase assay.................................................. 37 4.2.11 Data analysis.................................................... 37 4.3 Results.................................................................. 37 4.3.1 Color development................................................. 37 4.3.2 Fruit growth curve and respiration rate....................... 38 4.3.3 Fruit sugars....................................................... 43 4.3.4 SPS, SS, invertase enzyme activities.......................... 43 4.3.5 The relationship between fruit mass, and length, diameter and flesh thickness.............................................. 43 4.3.6 The relationship between fruit skin, flesh color and dry mass accumulation...................................... 44 VI 4.3.7 The relationship between flesh color and TSS in full ripe fruit................................................................ 44 4.3.8 Correlations between respiration, sugar content and enzyme activity............................................................ 44 4.3.9 Carbon import and enzyme activity in fruit flesh tissue.. 50 4.3.10 Sugar accumulation and enzyme activity in 'Kapoho' and 'UH801'