BIOCHEMICAL AND MOLECULAFL CHARACTEIUZATION OF a-FARNESENE BIOSYNTHESIS IN RELATION TO SUPERFICIAL SCALD DEVELOPMENT IN APPLE (Malus x domestka Borkh.) A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph by H. P. VASANTHA RUPASINGHE In partial fulfilment of requirements for the degree of Doctor of Philosophy January, 200 1 " H.P.V. Rupasinghe, 200 1 National Library Bibliothèque nationale IM of canada du Canada Acquisitions and Acquisitions et 8ibliographic Services services bibliographiques 395 Wellington Street 345. nie Wellington Ottawa ON K1A ON4 Ottawa ON K1A ON4 Canada Canada The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sen reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/nlm, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. ABSTRACT BIOCHEMICAL AND MOLECULAR CHGIRACTERIZATION OF a-FARNESENEBIOSYNTHESIS IN RELATION TO SUPERFICLAL SCALD DEVELOPMENT IN APPLE (Malus x domestica Borkh.) H.P. Vasantha Rupasinghe Advisors: University of Guelph, 200 1 Dr. Demis P. Murr Dr. Gopi Paliyath Metabolism of a-famesene, a sesquiterpene that accurnulates in apple skin during cold storage, has been implicated in the development of superficial scald in pome fniits. Biosynthesis of a-famesene occurs through the classical mevalonate pathway, and is formed directly fiom trans,trans-farnesyl pyrophosphate. This step is catalyzed by trans,~rans-a-faniesene synthase, an enzyme located mainly in hypodermaI and epidennaI cells of apple hit. The enzyme was purified seventy-fold fiom the cytosolic fraction, where activity was highest among sub-cellular fractions. The enzyme required a divalent rnetal (M~"or ~n'3for activity and exhibited allosteric kinetics: S(o.jlfor FPP was 84 j~rnol-~-'.The Hill coefficient (nH)indicated that the native protein was a trimer. Activity of a-famesene synthase was not evident in apple skin at harvest, but was induced by Iow temperature storage and preceded the accumulation of a-famesene. In contrat, high activity of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) was present in the skin at harvest, but declined during the first 8 weeks in storage and then remained unchanged. The inhibition of ethylene (CzH4) action in apple fruit by 1- methylcyclopropene (1-MCP) reveaIed that C2& was required for a-farnesene synthesis and the development of superficial scald. However, activity of a-fmesene synthase \vas not af5ected by Cz&. Since fiuit respiration was suppressed significantiy by 1-MCP, the reghation of a-famesene biosynthesis by Cz& may be through control of glycolysis; e-g., acetyl CoA availability limits isoprene synthesis and HMGR activity. A full-length (hmgl) and a fragment (hmg2) of two cDNA clones comprising the HMGR gene farnily were isolated f?om apple skin. The transcription product of hmgl cDNA has an open reading fiame of 1767 nucleotides and encodes a protein of 589 polypeptide residues of 62.7 kD. The presence of two highiy hydrophobic domains near the amino terminus, a unique feature of plant HMGR genes, was recognized. The two genes were expressed differentially in response to developmental stimuli; hrngl being expressed relatively constitutively, and hmgZ being highly sensitive to low temperatures and Cr& The synthesis of a-farnesene possibly occurs through a complex of sequential metabolic enzymes or "metabolon" located in the cytosol/ER boundary, where hmg2 also may be involved. To rny wzye Pri-vanka. rny son Kalana. and rny dazcghter Viraji who received less of my attention while this thesis was heing prepared 1 wish to express my gratitude and appreciation to my CO-advisorsDrs. Demis Murr and Gopi Paliyath for providing me the opportunity to study the postharvest biotechnology of fiuit. Appreciation also goes to Drs. Kckey Yada and Geza Hrazdena (Corne11 University) for serving as members of my advisory cornmittee and for their guidance and encouragement during the doctoral study. 1 gratehlly acknowledge Dr. Daryl Rowan (HortResearch, NZ), Dr, Barry McGIasson (University of Western Sydney, Australia), and Dr. Albert Purvis (University of Georgia) for sharing their experience. 1 thank Drs. Gordon Lange and Rodney Memll (Department of Chernistry and Biochernistry) and Dr. Brian Allen (Department of Mathematics and Statistics) of University of Guelph for advice in their areas of expertise. Special thanks to Dr. John Proctor for reviewing the manuscripts written for publication fiom this thesis. 1 am very thankful to Kurt Almquist for his support given during the gene cloning and expression studies. 1 appreciate the technical assistance of Dr. Valerie Robinson, Dr. Sandy Smith, and Robert Harrison for studies on 31~-~~~,DSC and freeze fracture, respectively, although the results of these works were not included in the thesis. 1 aIso owe many thanks to the laboratories of Drs. Barry Shelp and Judy Strommer for allowing me to use their facilities. 1 also thank Brad Cooney (Laboratory Services) for the timely semice provided on primer synthesis and DNA sequencing. 1 wish to thank Dr. John Cline (Department of Plant Agriculture, Simcoe) and Ken Wilson (OMAFRA, Thornbury) for their help in collecting apple hit which were used in this study. 1 must also thank Len Wiley for his help harvesting and maintaining the storage of apple, and Ramany Paliyath, Valsala Kailidumbil, Bernie Watts, and Rodger Tschanz for technical help. 1 gratefully acknowledge the frnancid assistance provided for this research by the Ontario Apple Marketing Commission; the Natural Sciences and Engineering Research Council of Canada (NSERC); the Ontario Ministry of Agriculture, Food and Rural Af%airs (OMAFRA): Agriculture and Agi-Food Canada (AAFC); and the CanAdapt Program of the Agriculturai Adaptation Council (AAC). TABLE OF CONTENTS CHAPTER 1 General Introduction Superficial scald Factors determining the susceptibility to scald development Control of superficial scald 2 -3.1 Diphenylamine @PA) 1-3-2 DPA is under scrutiny 1.3 -3 Attempts to find a DPA alternative 1 -3-4 Manipulation of ethylene 1-3 -5 Controlled atmosphere storage 1.3 -6 Heat treatments 1-3 -7 Prediction Biochemical theories of scaid development 1-4.1 ceFarnesene theory Biosynthesis of isoprenoids 1-51 HMGR as the key regulatory enzyme 1-5.2 GAP/pyruvate pathway 1-5.3 Sesquiterpene cyclases and synthases Oxidative stress and membrane degradation 1 -6.1 Lipid peroxidation 1.6.2 Antioxidants and antioxidant enzymes 1.6.3 Alternative oxidase and stress Research hypothesis and objectives Outline of the thesis CHAPTER IX Biosynthesis of a-Farnesene and its Relation to Superficial Scaid Development in 'Delicious' Apples 3.1 Abstract 2.2 Introduction 2.3 Materials and Methods 2.4 Results and Discussion 2.5 Literature Cited CHAPTER III Sesquiterpene a-Famesene Synthase: Partial Purification, Characterization, and Activity in Relation to Superficial Scald Deveiopment in Apples 3-1 Abstract 3 -2' Introduction 3-3 Materials and Methods 3 -4 Results 3 -5 Discussion 3 -6 Literature Cited CHAPTER IV Regulation of a-Famesene Synthesis by Ethylene Study 1 Suppression of a-Famesene Synthesis in 'Delicious' Ap ple by Arninoethoxyvinylglycine (AVG) and 1-Methylcyclopropene (1-MCP) 4A. 1 Abstract 4A.2 Introduction 4A.3 Materials and Methods 4A.4 Results and Discussion 4A.5 Literature Cited Study 2 Inhibitory Effect of 1-MCP on Ripening and Superficial Scald Deveiopment in 'Mclntosh' and 'Delicious' Apples 4B. 1 Abstract 4B.2 Introduction 4B.3 Materiais and Methods 4B.4 Results and Discussion 4B.5 Literature Cited CHAPTER V Cloning and Expression of hmgl and /.mg2 cDNA Clones Encoding 3-Hydroxy-3-Methyiglutaryl Coenzyme A Reductase (HMGR) and its Activity in Relation to cc-Farnesene Synthesis in Apple 5.1 Abstract 5 -2 Introduction 5.3 Materials and Methods 5.4 Results and Discussion 5 -5 Literature Cited CHAPTER VI GENERAL DISCUSSION 179 LITERATURE CITED IN GENERAL INTRODUCTION AND GENERAL DISCUSSION LIST OF TABLES CHAPTER II 2.1 Incorporation of tram,trans-[l --'~]-farnes~l pyrophosphate into a- 47 famesene and farnesol by different tissue segments of 'Delicious' apple fruit. CHAPTER III 3.1 Distribution of a-farnesene synthase activity arnong different sub- cellular fiactions of skin tissue of 'Deiicious' apple hit. Partial purification of a-famesene synthase from skin tissue of 78 -Delicious' apple bit. Content of hexane-extractable a-faniesene, and its catabolites in the 85 skin tissue of 'Delicious' apple in relation to severity of scaid, DPA and CA treatment. CHAPTER IV production and in vivo a-famesene synthase activity of 'Delicious' apples following treatment with AVG and 1-MCP. Sumary of the statistical analysis of Fi,ve 4.2.1. Flesh fimess of 'McIntosh' and 'Delicious' apples treated with 1pL.L' 1-MCP a day afier harvest at 20 OC for 18 h or not treated and stored 60 days (removal 1) and 120 days (removal 2) in air at O - 1 OC and 90 - 95 % relative humidity. cc-Farnesene and conjugated triene alcohol (CTOL) contents in skin tissue of 'Mclntosh' and 'Delicious' apples treated with 1-MCP a day after harvest and stored in air at O - 1 OC with 90 - 95 % relative humidity for 120 days. Incidence of superficial scald in 'McIntosh' and 'Delicious' apples treated with 1-MCP and stored 120 days in air at O - 1 OC with 90 - 93 % relative humidity. Correlation (rZ) of mean Cz& production at removal tirne 1 with six postharvest measurements in 'Mclntosh' and 'Delicious' apples treated or untreated with 1-MCP.