BRANCHED-CHAIN ESTER BIOSYNTHESIS IN RIPENING APPLE FRUIT By Nobuko Sugimoto A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Horticulture 2011 ABSTRACT BRANCHED-CHAIN ESTER BIOSYNTHESIS IN RIPENING APPLE FRUIT By Nobuko Sugimoto In apple fruit, aroma is an essential element of organoleptic quality and it can suffer in response to a number of pre- and post-harvest cultural treatments. Of the several classes of odor-active compounds, esters are the most important, but little is known regarding pathways of biosynthesis. This research presents evidence for a ‘new’ pathway for ester biosynthesis in apple that uses the starting products pyruvate and acetyl-CoA for the synthesis of precursors to branched-chain (BC) and certain short, straight-chain (SC) esters. The initial step in the pathway involves the formation of citramalic acid from pyruvate and acetyl-CoA by citramalate synthase (CIM). Citramalic acid then provides for the formation of α-keto-β-methylvalerate and its tramsaminated product isoleucine via α-ketobutyrate, and also for the BC ester precursors 2-methylbutanol or 2- methylbutanoate. The hypothesized pathway also provides for the formation of 3-, 4-, and 5- carbon fatty acids via the process of single-carbon elongation of α-keto acids, which are metabolized to short-chain fatty acids. These short-chain fatty acids are proposed to contribute to SC ester formation. Analysis of ripening fruit revealed that citramalic acid increased about 120- fold as ester production increased during ripening. At the same time, the content of isoleucine increased more than 20-fold, while other amino acids remained steady or declined. Apple disc feeding studies documented the incorporation of 13C-labeled acetate into citramalic acid and isoleucine and into esters derived from 2-methylbutanoate, 2-methylbutanol, propanoate, and butanoate, supporting the hypothesized pathway. Furthermore, two novel genes were identified from apple, the sequence of which suggests that they are members of the 2-isopropylmalate synthase (IPMS) gene family. Purified His-tag protein from these genes was found to form citramalate and 2-ethylmalate from the α-keto acids pyruvate and α-ketobutyrate, respectively, when acetyl-CoA was added. Substrate specificity for α-keto acids in decreasing order was α- ketobutyrate, pyruvate, and α-ketovalerate and is characteristic of CIM. Therefore, the two genes (MdCIM1 and MdCIM2) are apparently alleles coding for CIM, which initiates carbon flux into esters via the ‘citramalate pathway’. The sequence of MdCIM1 differed from that of MdCIM2 by two amino acids, yet MdCIM2 was essentially non-functional, possessing only a fraction of the activity of MdCIM1. The hypothesized pathway constitutes a conceptual shift in the regulation of ester biosynthesis in that it implies synthetic, rather than catabolic pathways are responsible for ester precursor supply. iii ACKNOWLEDGMENTS I would like to thank my advisor Dr. Randolph M. Beaudry for his support, training, encouragement, and knowledge giving to me throughout my studies in postharvest physiology. He was more than just an advisor; taught me the excitement of science and gave me the opportunity to meet with other scientists to exchange the ideas, and gave me a freedom to pursue my curiosity in research. I would also like to thank the members of my committee Drs. Robert Last, A. Daniel Jones, and Eran Pichersky for their advice and guidance. They were the best committee members for my PhD study. Special appreciation to Drs. Imad Ajjawi in Robert Last lab, Banita Tamot in Dr. Benning lab, and Kyaw Aung in Jianping Hu lab for their help, advice, and training in molecular biology and biochemistry. I enjoyed joining our study group; Jeongwoon Kim, Chin-Mei Lee, Christine Shyu, and Kyaw Aung to broaden my knowledge. I enjoyed working with current and past members of the postharvest laboratory and also enjoyed the friendship with the viticulture/enology group on the same floor. I also want to thank Drs. van Nocker, and Ning Jiang laboratory members for their help and advice. Many thanks to other faculty, staff, and friends who supported me during my program. I am grateful for Dr. Dennis Murr / University of Guelph for supporting and giving me advice. Finally, I would like to sincerely appreciate my parents and my sister’s warm support and love during my stay in the U.S. iv TABLE OF CONTENTS LIST OF TABLES ....................................................................................................................... vii LIST OF FIGURES ........................................................................................................................x CHAPTER I INTRODUCTION ...........................................................................................................................1 REFERENCES ................................................................................................................................6 CHAPTER II LITERATURE REVIEW ..............................................................................................................10 Aroma diversity .................................................................................................................11 Ester formation...................................................................................................................12 Proposed catabolic pathways .............................................................................................15 Synthetic pathways............................................................................................................19 REFERENCES ..............................................................................................................................33 CHAPTER III CHANGES IN FREE AMINO ACID CONTENT IN ‘JONAGOLD’ APPLE FRUIT AS RELATED TO BRANCHED-CHAIN ESTER PRODUCTION, RIPENING, AND SENESCENCE ..............................................................................................................................48 Introduction........................................................................................................................49 Materials and Methods.......................................................................................................52 Results................................................................................................................................59 Discussion..........................................................................................................................62 Conclusion .........................................................................................................................68 REFERENCES ..............................................................................................................................88 CHAPTER lV BIOSYNTHESIS OF ISOLEUCINE VIA CITRAMALATE PATHWAY: CHARACTERIZING CITRAMALATE SYNTHASE AS THE FIRST COMMITTED STEP IN A NEW BIOSYNTHETIC PATHWAY FOR ESTER SYNTHESIS IN RIPENING APPLE FRUIT ......96 Introduction........................................................................................................................97 Materials and Methods.....................................................................................................103 Results..............................................................................................................................121 Discussion........................................................................................................................127 Conclusion .......................................................................................................................134 REFERENCES ............................................................................................................................166 APPENDIX CHARACTERIZING THE VOLATILE PROFILE AND STORABILITY OF APPLE FRUIT FROM THE GENEVA MALUS CORE COLLECTION ............................................................176 Introduction......................................................................................................................177 Materials and Methods.....................................................................................................180 v Results and discussion.....................................................................................................184 Conclusion .......................................................................................................................192 REFERENCES ............................................................................................................................223 vi LIST OF TABLES CHAPTER ll Table 1. Reported existence of citramalate pathway in several strains of bacteria supported by feeding studies and with characterized citramalate synthase gene name. (n.d. indicates not determined; n.a. indicates not available)............................................................................28 CHAPTER lll Table 2. Putative branched-chain aminotransferase (BCAT), pyruvate decarboxylase (PDC), threonine deaminase (TD) genes and the 18S ribosomal RNA (18s rRNA) gene with accession number, GenBank number, forward and reverse primer sequence (5'→3'), and expected PCR gene fragment size (bp) for semi-quantitative RT-PCR. With the exception of BCAT3, accession numbers represent one of the ESTs used in the assembly of contigs that contained the region between