:Miittl 2!. .. 1.... .{11 .M REY}: . 5. Juana»! . I . 1324. , ,\!_a.¢¥ is}. 5.. hi}. 1?: 14.9». An fit. . 435.. ; 5 :2 3.4.3.3? a? .l I. 1,) n. 7. 11.53:: I’V. V.,.V._..§.,#£$§-§flfir 9.. 7 . . .. , :1 . , . .n. .mtsmnuv .r ‘ ‘ .. fig .WW.§H¢§ hi“, . . , , . : A , ‘ , . , A. , @3319“. . v :1 . 2 3w. ,. sear-3 Michigan State . 1\:Avnu¥\ l lln . UlllVUlOlly This is to certify that the thesis entitled VOLATILE BIOSYNTHESIS DURING RIPENING OF ‘JONAGOLD' APPLE FRUIT: ASSOCIATION OF GENE EXPRESSION WITH AROMA VOLATILES presented by Nobuko Sugimoto has been accepted towards fulfillment of the requirements for the MS. degree in Horticulture Kfl‘“ Li I s;__\n rupw m (Ma’jor Professor’s Signature’ 5"- l " O 7 Date MSU is an affirmative-action, equal-opportunity employer PLACE IN RETURN Box to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE IWV222W9 072409 2/05 c:/C|RC/DateDue.indd.p.15 -———— VOLATILE BIOSYNTHESIS DURING RIPENING OF ‘JONAGOLD’ APPLE FRUIT: ASSOCIATION OF GENE EXPRESSION WITH AROMA VOLATILES By Nobuko Sugimoto A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Horticulture 2007 ABSTRACT VOLATILE BIOSYNTHESIS DURING RIPENING OF ‘JONAGOLD’ APPLE FRUIT: ASSOCIATION OF GENE EXPRESSION WITH AROMA VOLATILES By Nobuko Sugimoto Changes in the volatiles produced by ‘Jonagold’ apple (Ma/us x domestica Borkh) during ripening and senescence were related to changes in gene expression using a cDNA—based microarray containing over 10,000 gene fragments. Patterns for aroma biosynthesis, internal ethylene content, respiration, skin color, starch, and texture were typical for climacteric fruit. Volatile compounds and 002 increased after a rapid increase in ethylene production. Straight-chain esters hexyl acetate and butyl acetate and branched-chain esters 2-methylbutyl acetate and hexyl 2-methylbutanoate were found to be the major esters detected by GC/MS. Long chain esters predominated during the early stages of ripening and short chain esters increased later in proportion. Generally, the alcohols increased at an earlier development stage than the esters for which they acted as substrates. Esters are formed by combining alcohol with CoA derivative of fatty acid by the action of alcohol acyltransferase (AAT). Patterns in gene expression reflecting the rise and fall in ester formation were found in some putative genes for amino acid metabolism (branched-chain aminotransferase and branched-chain d-keto acid decarboxylase), fatty acid metabolism, and ester formation. The pathway for branched-chain ester biosynthesis is discussed. 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. In private, his family warmly welcomed me and made me a part of his family. I would also like to thank the members of my committee Drs. Steve van Nocker, Christoph Benning, and Eran Pichersky for their advice and guidance. 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, Ning Jiang, and Grumet laboratory members for their help and advice in molecular biology. 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 US. TABLE OF CONTENTS LIST OF TABLES ................................................................................................. v LIST OF FIGURES ............................................................................................ xiii CHAPTER I INTRODUCTION .................................................................................................. 1 References ................................................................................................. 4 CHAPTER II LITERATURE REVIEW ........................................................................................ 5 Flavor ......................................................................................................... 6 Apple aroma ............................................................................................... 8 Aroma physiology ..................................................................................... 10 Cultural practices affecting aroma ................................................. 11 Timing of harvest ........................................................................... 15 Ester precursor formation ......................................................................... 15 Catabolic pathways ....................................................................... 16 Synthetic pathways ........................................................................ 24 Ester synthesis ......................................................................................... 27 Hypothesis ............................................................................................... 30 References ............................................................................................... 33 CHAPTER III CHARACTERIZATION OF VOLATILE ESTER BIOSYNTHESIS BY ‘JONAGOLD’ APPLES DURING THE RIPENING PROCESS ............................ 54 Introduction .............................................................................................. 55 Materials and Methods ............................................................................. 57 Results ..................................................................................................... 61 Discussion ................................................................................................ 64 Conclusion ............................................................................................... 70 References ............................................................................................... 72 CHAPTER lV GENE EXPRESSION ASSOCIATED WITH BRANCHED-CHAIN ESTER FORMATION IN ‘JONAGOLD’ APPLE FRUIT ................................................. 114 Introduction ............................................................................................ 1 15 Materials and Methods ........................................................................... 118 Results ................................................................................................... 126 Discussion .............................................................................................. 129 Conclusion ............................................................................................. 136 References ............................................................................................. 137 APPENDIX ........................................................................................................ 154 LIST OF TABLES CHAPTER 2 Table 1. Representative esters identified in apples with sensory description. Only esters that had an odor description are listed. Esters included acetates, propanoates, 2-methylpropanoates, butanoates, 2-methylbutanoates, pentanoates, and hexanoates. ................................................................ 47 CHAPTER 3 Table 2. Matrix of esters detected organized by acid and alcohol precursors for apple fruit. * indicates that the acid and alcohol combinations are reciprocal. Numbers indicate the maximum GC/MS response (TIC) detected during preclimacteric to postclimacteric stages of fruit ripening in millions. ................................................................................................... 77 CHAPTER 4 Table 3. Constraint table defining expression limits for microarray elements to identify candidate gene fragments for sequencing. Constraints were developed to identify changes in expression associated with eight distinct developmental stages (Days 0, 11, 25, 32, 39, 49, 60, and 70) during ripening for ‘Jonagold’ apple fruit. Expression levels are relative to Day 0 (in I092 scale). Categories are based on the pattern of expression and numbers denote the day following which, pattern changes were detected; ‘low’ indicates expression patterns that declined and ‘high’ indicates expression patterns that increased; ‘middle’ indicates no change in pattern from Day 0 through Day 70; ‘peak’ indicates a transient increase in gene expression on the indicated day; and “double peak’ indicates an expression pattern with peaks on Days 25 and 39. ............................... 142 Table 4. Gene with accession number and primer list for semi-quantitative RT- PCR. Apple cluster numbers are from Tree Fruit Technology genomic analysis tool apple database v.3.0 (http://genomicsmsu.edu/fruitdb/analyses/apple.shtml). Accession and GI number indicates the longest EST from apple clusters. Genes include for which PCR was not successful. Single number in ‘cycles needed’ indicates that the same cycle was performed with both biological replications, rep. 1 and 2. Two numbers indicate that different cycles were performed between biological replications for optimum result. .............. 143 APPENDIX Table 5. Original data for Figure 10 (Chapter 3). Data are internal ethylene content, C02 production, and the GC/MS response total ion count (TIC) for all aroma volatiles. Each value is the average of four replications. .. 155 Table 6. Original data for Figure 11 (Chapter 3). Force (N) for bending/tensile failure and compressive failure during ripening and senescence of ‘Jonagold’ apple fruit. ............................................................................ 156 Table 7. Original data for Figure 12 (Chapter 3). Data are volatile compounds, GC retention time (second), and classes (esters, alcohols, and aldehydes) identified from ‘Jonagold’ apple fruit during ripening and senescence. .157 Table 8. Original data for Figure 13A and 14A (Chapter 3). Data are the GC/MS response (total ion count, TIC) of alcohols produced by ‘Jonagold' apple fruit during ripening and senescence. The GC/MS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TIC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. .................................................................................... 158 Table 9. Original data for Figure 138 and 148 (Chapter 3). Data are the percentages that each alcohol class comprises of all alcohols detected on each date using the respective GC/MS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no alcohols of these classes were detected. ............. 159 Table 10. Original data for Figure 14A (Chapter 3). Data are the GC/MS response (total ion count) of esters arranged by their alkyl (alcohol- derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. ...................................... 160 Table 11. Original data for Figure 148 (Chapter 3). Data are the percentages that each ester class comprises of all esters detected on each date using the respective GC/MS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Esters are arranged by the source of the alkyl (alcohol-derived) moiety. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no esters of these classes were detected. ................................... 161 Table 12. Original data for Figure 150 (Chapter 3). Data are the GC/MS response (total ion count, TIC) of ethanol esters arranged by their alkanoate (acid-derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. The GC/MS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TIC value is the result of dividing the ion count for the vi unique ion by the ion fraction. Each value is the average of four replications. ........................................................................................... 162 Table 13. Original data for Figure 15E (Chapter 3). Data are the percentages that each ester class comprises of all ethanol esters detected on each date using the respective GC/MS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no esters of these classes were detected. ................................... 163 Table 14. Original data for Figure 16C (Chapter 3). Data are the GC/MS response (total ion count, TIC) of propanol esters arranged by their alkanoate (acid-derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. The GC/MS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TlC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. ........................................................................................... 164 Table 15. Original data for Figure 16E (Chapter 3). Data are the percentages that each ester class comprises of all propanol esters detected on each date using the respective GC/MS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no esters of these classes were detected. ................................... 165 Table 16. Original data for Figure 170 (Chapter 3). Data are the GC/MS response (total ion count, TIC) of butanol esters arranged by their alkanoate (acid-derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. The GC/MS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TIC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. ........................................................................................... 166 Table 17. Original data for Figure 17E (Chapter 3). Data are the percentages that each ester class comprises of all butanol esters detected on each date using the respective GC/MS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no esters of these classes were detected. ................................... 168 Table 18. Original data for Figure 18B (Chapter 3). Data are the GC/MS response (total ion count, TIC) of pentanol esters arranged by their alkanoate (acid-derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. The GC/MS response is calculated from data for a vii single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TIC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. ........................................................................................... 169 Table 19. Original data for Figure 18C (Chapter 3). Data are the percentages that each ester class comprises of all pentanol esters detected on each date using the respective GC/MS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no esters of these classes were detected. ................................... 170 Table 20. Original data for Figure 190 (Chapter 3). Data are the GC/MS response (total ion count, TIC) of hexanol esters arranged by their alkanoate (acid-derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. The GC/MS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TIC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. ........................................................................................... 171 Table 21. Original data for Figure 19E (Chapter 3). Data are the percentages that each ester class comprises of all hexanol esters detected on each date using the respective GC/MS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no esters of these classes were detected. ................................... 172 Table 22. Original data for Figure 20C (Chapter 3). Data are the GC/MS response (total ion count, TIC) of 2-methylbutanol esters arranged by their alkanoate (acid-derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. The GC/MS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TIC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. ........................................................................................... 173 Table 23. Original data for Figure 20E (Chapter 3). Data are the percentages that each ester class comprises of all 2-methylbutanol esters detected on each date using the respective GC/MS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no esters of these classes were detected. ................ 174 Table 24. Original data for Figure 21A (Chapter 3). Data are the GC/MS response (total ion count) of esters arranged by their alkanoate (acid- viii derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. ...................................... 175 Table 25. Original data for Figure 21 B (Chapter 3). Data are the percentages that each ester class comprises of all esters detected on each date using the respective GC/MS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Esters are arranged by the source of the alkanoate (acid-derived) moiety. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no esters of these classes were detected. ................................... 176 Table 26. Original data for Figure 228 (Chapter 3). Data are the GC/MS response (total ion count, TIC) of acetate esters arranged by their alkyl (alcohol-derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. The GC/MS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TIC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. ........................................................................................... 177 Table 27. Original data for Figure 22C (Chapter 3). Data are the percentages that each ester class comprises of all acetate esters detected on each date using the respective GC/MS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no esters of these classes were detected. ................................... 178 Table 28. Original data for Figure 23B (Chapter 3). Data are the GC/MS response (total ion count, TIC) of propanoate esters arranged by their alkyl (alcohol-derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. The GC/MS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TIC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. ........................................................................................... 179 Table 29. Original data for Figure 23C (Chapter 3). Data are the percentages that each ester class comprises of all propanoate esters detected on each date using the respective GC/MS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no esters of these classes were detected. ................................... 180 Table 30. Original data for Figure 24B (Chapter 3). Data are the GC/MS response (total ion count, TIC) of butanoate esters arranged by their alkyl (alcohol-derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. The GC/MS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TIC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. ........................................................................................... 181 Table 31. Original data for Figure 240 (Chapter 3). Data are the percentages that each ester class comprises of all butanoate esters detected on each date using the respective GC/MS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no esters of these classes were detected. ................................... 182 Table 32. Original data for Figure 25B (Chapter 3). Data are the GC/MS response (total ion count, TIC) of hexanoate esters arranged by their alkyl (alcohol-derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. The GC/MS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TIC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. . .......................................................................................... 183 Table 33. Original data for Figure 250 (Chapter 3). Data are the percentages that each ester class comprises of all hexanoate esters detected on each date using the respective GC/MS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no esters of these classes were detected. ................................... 184 Table 34. Original data for Figure 26B (Chapter 3). Data are the GC/MS response (total ion count, TIC) of octanoate esters arranged by their alkyl (alcohol-derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. The GC/MS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TIC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. ........................................................................................... 185 Table 35. Original data for Figure 26C (Chapter 3). Data are the percentages that each ester class comprises of all octanoate esters detected on each date using the respective GC/MS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined. since no esters of these classes were detected. ................................... 186 Table 36. Original data for Figure 273 (Chapter 3). Data are the GC/MS response (total ion count, TIC) of 2-methylbutanoate esters arranged by their alkyl (alcohol-derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. The GC/MS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TIC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. ........................................................................................... 1 87 Table 37. Original data for Figure 27C (Chapter 3). Data are the percentages that each ester class comprises of all 2-methylbutanoate esters detected on each date using the respective GC/MS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no esters of these classes were detected. ................ 188 Table 38. Original data for Figure 31 (Chapter 4). Data are internal ethylene content, 002 production, and the GC/MS response total ion count (TIC) for all aroma volatiles. Each value is the average of four replications. .. 189 Table 39. Original data for Figure 32A and 320 (Chapter 4). Data are the GC/MS response (total ion count, TIC) of 2-methylbutanol esters arranged by their alkanoate (acid-derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. The GC/MS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TIC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. ........................................................................................... 190 Table 40. Original data for Figure 32A and 320 (Chapter 4). Data are the GC/MS response (total ion count, TIC) of 2-methylbutanoate esters arranged by their alkyl (alcohol-derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. The GC/MS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TIC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. ........................................................................................... 191 Table 41. Original data for Figure 32B (Chapter 4). Data are the GC/MS response (total ion count, TIC) of 2—methylbutanol and 2-methylbutanal produced by ‘Jonagold’ apple fruit during ripening and senescence. The GC/MS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TlC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. ........................ 192 xi Table 42. Original data for Figure 32E (Chapter 4). Data are the percentages that each ester class comprises of all 2-methylbutanoate esters detected on each date using the respective GC/MS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no esters of these classes were detected. ................ 193 Table 43. Original data for Figure 33 (Chapter 4). Data are the relative luminosity (I092) of microarray elements compared to Day 0 for branched-chain aminotransferase (BCAT), pyruvate decarboxylase (PDC), and 2- isopropylmalate synthase during ripening and senescence of ‘Jonagold’ apple fruit. ............................................................................................. 194 Table 44. Original data for Figure 34 (Chapter 4). Data are luminosity of electrophoresis agarose gel spots relative to the maximum value for each gene from semi-quantitative RT-PCR products. The spot measurements are for branched-chain aminotransferase (BCAT) genes during ripening and senescence of ‘Jonagold’ apple fruit. All data were normalized relative to control gene 18s rRNA luminosity. Each value is the average of two replications. Figures below are original images from which luminosity data were extracted. ...................................................................................... 195 Table 45. Original data for Figure 35 (Chapter 4). Data are luminosity of electrophoresis agarose gel spots relative to the maximum value for each gene from semi-quantitative RT-PCR products. The spot measurements are for pyruvate decarboxylase (PDC) genes during ripening and senescence of ‘Jonagold’ apple fruit. All data were normalized relative to control gene 18s rRNA luminosity. Each value is the average of two replications. Figures below are original images from which luminosity data were extracted. ...................................................................................... 197 Table 46. Original data for Figure 36 (Chapter 4). Data are luminosity of electrophoresis agarose gel spots relative to the maximum value for each gene from semi-quantitative RT-PCR products. The spot measurements are for 2-isopropylmalate synthase gene during ripening and senescence of ‘Jonagold’ apple fruit. All data were normalized relative to control gene 18$ rRNA luminosity. Each value is the average of two replications. Figures below are original images from which luminosity data were extracted. .............................................................................................. 198 xii LIST OF FIGURES CHAPTER 2 Figure 1. Pathways having potential to supply alcohol and acyI-CoA substrates for ester formation. .................................................................................. 48 Figure 2. Pathway for catabolism of fatty acids via B-oxidation. In plants, [3- oxidation takes place in peroxisomes. Free fatty acid Cn acyl-CoA are reduced by two carbons during each cycle of B-oxidation by four steps. 1. Dehydrogenation by acyI-CoA oxidase, 2. addition of water by 2-trans- enoyl-CoA hydratase, 3. dehydrogenation by L-3-hydroxyacyl-CoA dehydrogenase, and 4. cleavage of acetyI-CoA by 3-ketoacyl-CoA thiolase to produce Cn-2 acyI-CoA. Stars indicate the carbon position during each reaction. Hydrogens in the carbon-hydrogen bonds are not shown. ....... 49 Figure 3. Pathway for catabolism of lipids and fatty acids via lipoxygenase. In plants, Iipoxygenase activity are found to be in chloroplasts (Hatanaka, 1993). Linoleic (18:2) or Iinolenic (18:3) acids are formed by lipase. Lipoxygenase peroxidizes linoleic and linolenic acid to 13-hydroperoxy linoleic acid (13-HPOD) and 13-hydroperoxy linolenic acid (13-HPOT), respectively. Hydroperoxide lyase cleaves 13-HPOD and 13-HPOT into hexanal and cis-3-hexenal, respectively. Hexanal and cis-3-hexenal is reduced by alcohol dehydrogenase to hexanol and cis-3-hexenol respectively. Hydrogens in the carbon-hydrogen bonds are not shown. .50 Figure 4. Putative (dashed lines) and demonstrated (solid lines) pathways involved in branched-chain ester biosynthesis. * Indicates that gene is found in bacteria, but not in plants. Hydrogens in the carbon-hydrogen bonds are not shown. Stars and circles indicate the carbon position during each reaction. .......................................................................................... 51 Figure 5. Pathway for fatty acid biosynthesis through two-carbon chain elongation in plants. AcetyI-ACP (primer) and malonyl-ACP (chain extender) is condensed by 3-ketoacyl-ACP synthase Ill. The next 3 steps are reduction by 3-ketoacyl-ACP reductase, dehydration by 3-hydroxyacyl- ACP dehydrase, and reduction by enoyl-ACP reductase. The cycle repeats to condense with malonyI-ACP until the chain length is 16-18, in general. The final step is terminated by acyl-ACP thioesterase by hydrolysis. Stars indicate the carbon position during each reaction. Hydrogens in the carbon-hydrogen bonds are not shown. ...................... 52 Figure 6. Pathway for fatty acid biosynthesis through single-carbon chain elongation (d-keto acid elongation, dKAE) in plants. Pyruvate (primer) and acetyl-CoA (chain extender) is condensed by 2-isopropylmalate synthase. xiii The next 2 steps are isomerization and dehydration by isopropylmalate dehydratase, and decarboxylation by 3-isopropylmalate dehydrogenase. The cycle repeats to condense with acetyI—CoA or terminated to produce acyI-CoA. Stars indicate the carbon position during each reaction. Hydrogens in the carbon-hydrogen bonds are not shown. ...................... 53 CHAPTER 3 Figure 7. Pathways having potential to supply alcohol and acyl-CoA substrates for ester formation. .................................................................................. 78 Figure 8. Tensile failure was measured on bars of cortex tissue using a 3-point bending rig. The tissue bars were square in cross-section (9 mm x 9 mm) and approximately 7cm in length and were cut parallel to the axis of the fruit. The force was applied perpendicular to the fruit axis and the maximum force encountered during the test was recorded. .................... 79 Figure 9. Compressive failure was tested on cylinders of cortex tissue placed on the flat plate and compressed by a descending flat-faced probe. The cylinders were made with an internal diameter of 16.5 mm, trimmed to a length of 2 cm, and taken from tissue just beneath the skin. The cylinder was removed from the equator of the fruit, normal to the fruit axis. The force was applied perpendicular to the fruit axis and the maximum force encountered during the test was recorded. ............................................. 80 Figure 10. Ontogeny of total volatiles, ethylene and respiration (002 production) during ripening and senescence of ‘Jonagold’ apples. The apple fruit were examined from Sept 2, 2004 (Day 0) to Nov. 23, 2004 (Day 81). Fruits were harvested every 3-4 days from the field until Oct. 7, 2004 (Day 35), and thereafter maintained at room temperature (2111°C). Each symbol represents the average of four replications for total volatiles and respiration, and ten replications for ethylene. Vertical bars represent mean i SD. ....................................................................................................... 81 Figure 11. Ontogeny of total volatiles and textural changes during ripening and senescence of ‘Jonagold’ apples. Tensile failure/bending force was to measure ‘crispness’ and compressive force for ‘softening’. The apple fruit was examined from Sept 2, 2004 (Day 0) to Nov. 23, 2004 (Day 81). Fruits were harvested every 3-4 days from the field until Oct. 7, 2004 (Day 35), and thereafter maintained at room temperature (21i1°C). The samples were taken opposite sides of each fruit and each symbol represents four fruit replications. ...................................................................................... 82 Figure 12. Representative gas chromatograph of the headspace of ‘Jonagold’ apples at the respiratory climacteric on Day 42. Predominant esters were butyl acetate, 2-methylbutyl acetate, hexyl acetate, and hexyl 2- xiv methylbutanoate. A total of 31 esters, '3 aldehydes, and 5 alcohols were detectable at this point in development. .................................................. 83 Figure 13. Patterns of alcohol emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81 ). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of ethanol, propanol, butanol, hexanol, and 2-methylbutanol. B. Alcohol proportions (% of total alcohols). Each symbol represents the average of four replications. ...................................................................................... 85 Figure 14. Patterns of alcohol ester emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81 ). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of propanol, propyl esters, butanol, butyl esters, hexanol, hexyl esters, 2- methylbutanol, and 2-methylbutyl esters. B. Alcohol and alcohol ester proportions (% of total alcohols or alcohol esters). Each symbol represents the average of four replications. .............................................................. 87 Figure 15. Patterns of ethanol ester emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of total ethanol esters and ontogeny of ethylene. B. GC/MS response (TIC) of total ethanol. C. GC/MS response (TIC) of ethyl acetate, ethyl propanoate, ethyl butanoate, and ethyl 2-methylbutanoate. D. Ethanol proportion (°/o of total alcohols). E. Ethanol ester proportions (% of total ethanol esters). Each symbol represents the average of four replications. Vertical bars represent mean 1: SD. ........................................................ 89 Figure 16. Patterns of propanol ester emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81). The fruits were collected from the . field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of total propanol esters and ontogeny of ethylene. B. GC/MS response (TIC) of total propanol. C. GC/MS response (TIC) of propyl acetate, propyl propanoate, propyl butanoate, propyl 2-methylbutanoate, propyl hexanoate, and propyl octanoate. D. Propanol proportions (% of total alcohols). E. Propanol ester proportions (% of total propanol esters). Each symbol represents the average of four replications. Vertical bars represent mean 1: SD. ............................................................................................. 91 XV Figure 17. Patterns of butanol ester emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81 ). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of total butanol esters and ontogeny of ethylene. B. GC/MS response (TIC) of total butanol. C. GC/MS response (TIC) of butyl acetate, butyl propanoate, butyl butanoate, butyl 2-methylbutanoate, butyl pentanoate, butyl hexanoate, butyl heptanoate. and butyl octanoate. D. Butanol proportions (% of total alcohols). E. Butanol ester proportions (% of total butanol esters). Each symbol represents the average of four replications. Vertical bars represent mean 1 SD. ........................................................ 93 Figure 18. Patterns of pentanol ester emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81 ). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of total pentanol esters and ontogeny of ethylene. B. GC/MS response (TIC) of pentyl acetate, pentyl propanoate, pentyl 2-methylbutanoate, and pentyl hexanoate. C. Pentanol ester proportions (% of total pentanol esters). Each symbol represents the average of four replications. Vertical bars represent mean i SD. ..................................................................... 95 Figure 19. Patterns of hexanol ester emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of total hexanol esters and ontogeny of ethylene. B. GC/MS response (TIC) of total hexanol. C. GC/MS response (TIC) of hexyl acetate, hexyl propanoate, hexyl butanoate, hexyl 2-methylbutanoate, hexyl hexanoate, and hexyl octanoate. D. Hexanol proportions (% of total alcohols). E. Hexanol ester proportions (% of total hexanol esters). Each symbol represents the average of four replications. Vertical bars represent mean 1 SD. .......................................................................................................... 97 Figure 20. Patterns of 2—methylbutanol ester emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of total 2-methylbutanol esters and ontogeny of ethylene. B. GC/MS response (TIC) of total 2-methylbutanol. C. GC/MS response (TIC) of 2- methylbutyl acetate and 2-methylbutyl butanoate. D. 2—Methylbutanol xvi proportions (% of total alcohols). E. 2-Methylbutanol ester proportions (% of total 2-methylbutanol esters). Each symbol represents the average of four replications. Vertical bars represent mean :t SD. ............................. 99 Figure 21. Patterns of acid ester emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of acetates, propanoates, butanoates, 2-methylbutanoates, hexanoates, and octanoates. 8. Acid ester proportions (% of total acid esters). Each symbol represents the average of four replications. .............................. 101 Figure 22. Patterns of acetate ester emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of total acetate esters and ontogeny of ethylene. B. GC/MS response (TIC) of ethyl acetate, propyl acetate, butyl acetate, pentyl acetate, hexyl acetate, 2-methylpropyl acetate, and 2-methylbutyl acetate. C. Acetate ester proportions (°/o of total acetate esters). Each symbol represents the average of four replications. Vertical bars represent mean 1: SD. ......... 103 Figure 23. Patterns of propanoate ester emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81 ). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of total propanoate esters and ontogeny of ethylene. B. GC/MS response (TIC) of ethyl propanoate, propyl propanoate, butyl propanoate, pentyl propanoate, and hexyl propanoate. C. Propanoate ester proportions (% of total propanoate esters). Each symbol represents the average of four replications. Vertical bars represent mean :I: SD. ........................... 105 Figure 24. Patterns of butanoate ester emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81 ). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of total butanoate esters and ontogeny of ethylene. B. GC/MS response (TIC) of ethyl butanoate, propyl butanoate, butyl butanoate, 2- methylbutyl butanoate, and hexyl butanoate. C. Butanoate ester proportions (% of total butanoate esters). Each symbol represents the average of four replications. Vertical bars represent mean 1 SD. ......... 107 xvii Figure 25. Patterns of hexanoate ester emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81 ). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of total hexanoate esters and ontogeny of ethylene. B. GC/MS response (TIC) of propyl hexanoate, butyl hexanoate, pentyl hexanoate, and hexyl hexanoate. C. Hexanoate ester proportions (% of total hexanoate esters). Each symbol represents the average of four replications. Vertical bars represent mean i SD. .................................. 109 Figure 26. Patterns of octanoate ester emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81 ). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of total octanoate esters and ontogeny of ethylene. B. GC/MS response (TIC) of propyl octanoate, butyl octanoate, and hexyl octanoate. C. Octanoate ester proportions (% of total octanoate esters). Each symbol represents the average of four replications. Vertical bars represent mean :I: SD. ........................................................................................................ 1 1 1 Figure 27. Patterns of 2-methylbutanoate ester emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81 ). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of total 2-methylbutanoate esters and ontogeny of ethylene. B. GC/MS response (TIC) of ethyl 2-methylbutanoate, propyl 2- methylbutanoate, butyl 2-methylbutanoate, pentyl 2-methylbutanoate, and hexyl 2-methylbutanoate. C. 2-Methylbutanoate ester proportions (% of total 2-methylbutanoate esters). Each symbol represents the average of four replications. Vertical bars represent mean :I: SD. ........................... 113 CHAPTER 4 Figure 28. Pathways having potential to supply alcohol and acyl-CoA substrates for ester formation. ................................................................................ 144 Figure 29. Putative (dashed lines) and demonstrated (solid lines) pathways involved in branched-chain ester biosynthesis. * Indicates that gene is found in bacteria, but not in plants. Hydrogens in the carbon-hydrogen bonds are not shown. Stars and circles indicate the carbon position during each reaction. ........................................................................................ 145 xviii Figure 30. Experimental design for microarray analysis. Arrows indicate comparison made. Numbers indicate the days for the eight stages of development. Evaluated sixteen arrays were used with two biological samples and a dye swap to reduce variation. The tail of the arrow indicates RNA probe labeled with cyanine 3; the head of the arrow indicates RNA probe labeled with cyanine 5. ........................................ 146 Figure 31. Internal ethylene concentration, total volatiles (TIC), and 002 production in pre-climacteric through post-climacteric ‘Jonagold’ apples. Eight stages (Days 0, 11, 25, 32, 39, 49, 60, and 70) were selected for genomic analysis based on physiological stages during ripening. Stage 1 (Day 0), early climacteric; stage 2 (Day 11), late preclimacteric and onset of trace ester biosynthesis; stage 3 (Day 25), onset of the autocatalytic ethylene and rapid Increase of ester biosynthesis; stage 4 (Day 32), half- maximal ester biosynthesis and engagement of the respiratory climacteric; stage 5 (Day 39), near maximal ester biosynthesis, peak in respiratory activity, and onset of rapid tissue softening; stage 6 (Day 49), end of maximal biosynthesis, the conclusion of the respiratory climacteric, and completion of tissue softening; stage 7 (Day 60), midpoint in the decline in ester biosynthesis, maximal ethylene production, and onset of senescent; stage 8 (Day 70), postclimacteric minimum in ester production and high fruit senescent. ...................................................................................... 147 Figure 32. Patterns of 2-methylbutanol and 2-methylbutanoate ester emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of total 2-methylbutanol and 2- methylbutanoate esters and ontogeny of ethylene. B. GC/MS response (TIC) of 2-methylbutanol and 2-methylbutanal. C. GC/MS response (TIC) of 2-methylbutyl acetate and 2-methylbutyl butanoate. D. GC/MS response (TIC) of ethyl 2-methylbutanoate, propyl 2-methylbutanoate, butyl 2-methylbutanoate, pentyl 2-methylbutanoate, and hexyl 2- methylbutanoate. E. 2-Methylbutanoate ester proportions (% of total 2— methylbutanoate esters). Each symbol represents the average of four replications. Vertical bars represent mean :t SD. .................................. 149 Figure 33. Gene expression based on microarray data (in logZ scale). For genes potentially involved in branched-chain ester formation including pyruvate decarboxylase (PDC), branched-chain aminotransferase (BCAT), and 2- isopropylmalate synthase gene expression relative to Day 0. ............... 150 Figure 34. Gene expression of putative branched-chain aminotransferase (BCAT) for ‘Jonagold’ apple fruit ripened at room temperature performed by semi-quantitative RT-PCR. The value is based on spot density relative xix to maximum value. 185 rRNA was used as a control. All data are normalized relative to control gene spot density. The control gene spot density ranged between 0.78-0.98. Each symbol represents the average of two replications. The average pooled standard deviation is 0.15. ..... 151 Figure 35. Gene expression of putative pyruvate decarboxylase (PDC) for ‘Jonagold’ apple fruit ripened at room temperature performed by semi- quantitative RT-PCR. The value is based on spot density relative to maximum value. 183 rRNA was used as a control. All data are normalized relative to control gene spot density. The control gene spot density ranged between 078—098. Each symbol represents the average of two replications. The average pooled standard deviation is 0.10. ................ 152 Figure 36. Gene expression of putative 2-isopropylmalate synthase for ‘Jonagold’ apple fruit ripened at room temperature performed by semi-quantitative RT-PCR. The value is based on spot density relative to maximum value. 18s rRNA was used as a control. All data are normalized relative to control gene spot density. The control gene spot density ranged between 0.78- 0.98. Symbol represents the average of two replications. The pooled standard deviation is 0.06. .................................................................... 153 XX CHAPTER I INTRODUCTION Scent is one of the features of fruits that make them attractive to animals. Scent attraction to herbivores is a significant evolutionary response to a lack of mobility to employ animals for seed dispersal (Levey, 2004). Fruit maximize aroma production during ripening when seeds are mature. In fruits, the major compounds responsible for aroma are esters, aldehydes, acids, and terpenes. Most of the scents humans perceive as sweet are derived from esters. In apples and apple products, the volatile profile is known to contain over 200 compounds (Dimick and Hoskin, 1983). During apple ripening, the volatile composition changes over time; quantitative and qualitative changes in volatiles stimulate the olfactory system of humans and mammals to indicate the presence of ripe fruit (Stoddart, 1980). In human olfaction, some compounds can be detected at parts-per-trillion level, but higher levels are needed for many compounds, depending upon their odor threshold. The degree to which a volatile impacts aroma is a function of the degree to which its concentration exceeds the odor threshold. In ‘Delicious’ apples, the concentration range for major esters is typically between 0001-0060 parts-per-million (ppm) (vlv) and the character impact compound ethyl 2- methylbutanoate can be detected as low as 0.0001 ppm (Flath et al., 1967). Cultural practices (e.g. long-term controlled atmosphere (CA) storage) of preserving fruit quality significantly diminish aroma and the recovery is not reversible for most apple varieties (Ferenczi et al., 2006; Plotto et al., 1999). Despite consumer value placed in flavor, aroma research has been somewhat of a low priority. In other apple exporting countries, the focus on flavor is growing and is considered vital to the maintenance of strong marketing strategies. The diverse aroma volatiles in apple are known to be produced by more than one biosynthetic pathway. But the actual pathways employed and their regulation are not well understood. Lack of progress in this area is in part a result of the difficulty associated with obtaining plant material, given the seasonality of the crop and the dynamic processes associated with ripening. Further, exploration of these pathways using forward or reverse genetic techniques is extremely difficult given the 5 to 10-year reproductive cycle of apple. Additionally, use of model organisms like Arabidopsis thaliana provides little insight, since Arabidopsis does not autonomously produce the same esters that are synthesized in apples. The aim of this research is to: first, characterize aroma profiles and individual compounds during apple fruit ripening by establishing their temporal association with changes in fruit texture, skin color, starch content, sugar content, ethylene content, and respiratory activity; second, to relate changes in the expression of genes associated with putative ester biosynthetic pathways with patterns of ester production and other features of the ripening process. We expect that this information will help improve our understanding of the physiology and biochemistry of ester formation in apples. REFERENCES Dimick, PS. and J.C. Hoskin. 1983. Review of apple flavor-state of the art. CRC Crit. Rev. Food Sci. Nutr. 18(4):387-409. Ferenczi, A., J. Song, M. Tian, K. Vlachonasios, D. Dilley, and RM. Beaudry. 2006. Volatile ester suppression and recovery following 1- methylcyclopropene application to apple fruit. J. Amer. Soc. Hort. Sci. 131(5):691-701. Flath, R.A., D.R. Black, D.G. Guadagni, W.H. McFadden, and TH. Schultz. 1967. Identification and organoleptic evaluation of compounds in Delicious apple essence. J. Agric. Food Chem. 15(1);29-35. Levey, DJ. 2004. The evolutionary ecology of ethanol production and alcoholism. Integr. Comp. Biol. 44(4):284-289. Plotto, A., MR. McDaniel, and JP. Mattheis. 1999. Characterization of 'Gala' apple aroma and flavor: differences between controlled atmosphere and air storage. J. Amer. Soc. Hort. Sci. 124(4):416-423. Stoddart, OM. 1980. The ecology of vertebrate olfaction. Chapman & Hall, London. CHAPTER II LITERATURE REVIEW Flavor Humans use five senses to enjoy food: sight, odor, taste, touch, and hearing. Using these senses, we classify food quality attributes into three sensory properties: appearance, flavor, and texture (Fisher and Scott, 1997). Consumers select fruits and vegetables based on these three properties and flavor is considered to be the most important factor (Péneau et al., 2006). Despite this preference for flavor, breeding of horticultural commodities has focused mostly on appearance and texture to aid in marketing and improve handling. The result has been that flavor is somewhat neglected. Flavor consists of odor and taste. The taste component is due to the interaction of chemicals released from the food with the tongue. The odor component is due to the interaction of volatiles with the nose. The mixture of aroma notes and other taste factors determine characteristic flavors. For example, when Marsh et al. (2006) added sugar to kiwi fruit pulps, tasters perceived kiwi as more banana-like. When they added acid, kiwi was perceived less banana-like and had an increased lemon flavor. Four categories of taste are prominent in fruits; sweetness, sourness, bitterness, and sometimes astringency. In fruits and vegetables, the perception of sweetness is primarily due to sugars and sugar alcohols, sourness due to organic acids, bitterness and astringency are due to alkaoloids and phenolics (Baldwin, 2004). Ripe fruit on average contain about 10-15°/o sugar by weight. The sugars are sometimes converted from stored starch as in apple, banana, and mango (McGee, 2004). Common sugars include sucrose, glucose and/or fructose, and sorbitol (Baldwin, 2002). Total acid content ranges from about 5% for the lemon to 0.1 % for the persimmon (Biale, 1964), and several forms of acids are found in fruits including ascorbic, citric, malic, quinic, tartaric, and oxalic. These organic acids are normally stored in the plant vacuoles. Ascorbic is described as soft, aspirin-like, astringent, and lemon-like; citric as sharp, fresh, and lemony; malic as lemony, tangy, bitter, sharp, and green apple-like; and quinic as chalky, aspirin-like, and fizzy (Marsh et al., 2006). Phenolic compounds range from low molecular weight, which tends to be bitter, to high molecular weight, which tends to be astringent (Drewnowski and Gomez-Carneros, 2000). One of the most important phenolic groups, flavonoids, includes flavonones, flavonols, flavones, isoflavones, flavans (catechins), and anthocyanins, and are found in many foods and beverages such as in green tea, red wine, soybean, grapefruit, and orange juice (Drewnowski and Gomez-Carneros, 2000). When phenolic compounds bind to proteins in saliva, it significantly reduces the lubricating qualities of saliva and decreases its viscosity, leading to an increase in friction, which we perceive as astringency (Prinz and Lucas, 2000). There are three classes of aroma volatiles based on how the volatiles are generated. Thermally-generated aroma compounds require heating or cooking, disruption-dependent aroma requires the mixing of cellular contents normally held separate by various organelles, and autonomous aromas are produced without external intervention from ripening fruits and vegetables (Beaudry, 2000). Volatiles that add to flavor have various notes. For example, alcohols and aldehydes are perceived as green-grassy, esters as fruity and floral, phenolic compounds as spicy, furanones as nutty, pyrazines as earthy, Iactones as creamy, and sulfur compounds as onion-like (McGee, 2004). Some odor compounds may add character-impact compounds, either desired or off-odor (Fan and Thayer, 2002), or may enhance the intensity of other flavors. Sweet flavor perception is enhanced in apple fruits by 2-methylbutyl acetate (Young et al,1996) Apple aroma Apple is a climacteric fruit. As such it experiences a strong increase in ethylene production during ripening, which drives an increase in 00; production. Volatile compounds with odor-activity increase concurrently with or follow the ethylene increase (Ferenczi, 2003; Mattheis et al., 1991b) (Table 1). However, not all volatiles can be perceived as odorants and contribute to aroma. Further, while many of the volatiles are spontaneously produced from the intact fruit, several important volatiles are formed by oxidation, hydrolysis, or other enzymatic reactions as the fruit is crushed during mastication and exposed to oxygen (Drawert, 1975). When the apple is consumed, the mixture of spontaneous and induced compounds creates characteristic flavors. In fresh and processed apples, more than 200 volatile compounds have been isolated; they consist mainly of alcohols, aldehydes, hydrocarbons, acids, and esters (Dimick and Hoskin, 1983). Of these volatiles, esters are the primary compounds that influence aroma and normally account for 80-95% of the total volatile emission (Paillard, 1990). Most esters contain two- to eight-carbon alkyl (alcohol-derived) or alkanoate (acid-derived) groups on either side of the oxygen atom. These alkyl or alkanoate groups can be straight or branched. For instance, hexyl acetate and butyl acetate are classified as straight-chain esters, and 2- methylbutyl acetate is a branched-chain ester. These three are the major esters considered to confer a characteristic apple aroma when the apple is ripening (Fellman et al., 2000). In general, they are perceived as fruity and floral (Plotto et al., 2000). The aldehydes, hexanal, cis-3-hexenal, and trans-2-hexenal are produced in abundance upon tissue disruption (as during mastication) by enzymatic oxidation and are responsible for green notes (Paillard, 1990) which are also important to the apple aroma. A low concentration of 4- methoxyallylbenzene (4-allylanisole) provides a ‘spicy’ or ‘anise—like’ aroma in some varieties and is thought to add a characteristic note (Williams et al., 1977). Other important aroma volatiles include 3-penten-2-ol (apple-like aroma) in ‘Starkspur Golden’ fruit (Vanoli et al., 1995) and B-damascenone (fruity odor) in ‘Cox Orange’ and ‘Elstar’ (Fuhrmann and Grosch, 2002). As fruit maturity shifts from preclimacteric to postclimacteric, the amount of individual compounds emitted by the fruit changes over time, altering the aroma profiles (Mattheis et al., 1991b). For example, in ‘Redchief Delicious’, longer carbon chains tend to predominate in esters produced at earlier ripening stages and shorter chain esters increase as fruit become overripe (Ferenczi, 2003). Unsaturated esters, which are normally in very low abundance in intact fruit, are formed when apple cells are crushed (Schreier et al, 1978). In processed apples, hexanal and trans- 2-hexenal increase due to enzyme activation by heat and alter the aroma composition while others decrease by inactivation of enzymes (Su and Wiley, 1998). Different cultivars emit different compounds (Kakiuchi et al., 1986). Cultivars can be classified by the type of esters they produce such as acetates in ‘Calville blanc’ and ‘Golden Delicious’, butanoates in ‘Bell de Boskoop’ and ‘Canada blanc’, propanoates in ‘Richared’ and ‘Reinette du Mans’, and ethyl esters in ‘Starking’ (Paillard, 1990). Apple physiology Ethylene is one of the plant hormones that regulate plant growth and development. From a postharvest perspective, it has a role in regulating fruit ripening. There are two systems to describe ethylene production (Biale, 1964; McMurchie et al., 1972; Pratt and Goeschl, 1969). System 1 is considered to be present in all fruit and plant tissues and is characterized as having feedback inhibition. System 2 is an autocatalytic system that is active in the climacteric fruit that stimulates ethylene production in response to ethylene presence. As climacteric fruits start to ripen, system 2 is activated, operating simultaneously with system 1. The onset of ethylene production causes physiological changes in apples: respiration starts to increase, flesh softens, and volatile compounds are emitted. In some cultivars, chlorophyll is degraded and anthocyanins accumulate. The internal ethylene content that initiates the climacteric rise in mango can be as little as 0.04 uL-L", but most climacteric crops require 0.1 pL-L'1 or more (Biale, 1964; McGlasson, 1970). Suppression of the onset of system 2 ethylene is 10 necessary to prolong storability substantially. Therefore, many studies have investigated inhibition of ethylene synthesis to delay ripening for long-term storage and export. The ethylene pathway was established by Yang and Hoffman (1984). Ethylene is formed from s-adenosyI-methionine (SAM) by SAM synthetase which is derived from methionine. SAM is converted to 1-aminocyclopropane-1- carboxylic acid (ACC) by ACC synthase (ACS), which is then oxidized by ACC oxidase (AC0), to ethylene. Differential expression of ACS and ACC gene family members result in the transition from auto-inhibitory to autocatalytic ethylene production (Barry et al., 2000; Lelievre et al, 1997). Inhibition of ethylene production can be accomplished by these two enzymes/genes. When ACS or ACO were silenced in fruit, firmness, sugar, and acid composition were maintained and shelf-life was increased; however, the total volatile esters were suppressed by 65-70% inhibition (Dandekar et al., 2004; Flores et al., 2002). In apple, MdACO1 and MdACS1 have been positioned on a molecular marker linkage map for breeders to develop new cultivars that maintain good shelf life (Costa et al., 2005). To an extent, continuous ethylene action and a high rate of ethylene production are required for full ester biosynthesis in apple (Fan et al., 1998). Cultural practices affecting aroma The impact of CA storage and the ethylene action inhibitor 1- hTM methylcyclopropene (1-MCP, SmartF res ) use on ripening and quality have been extensively studied. While these technologies permit preservation of fruit 11 quality for a fairly long time, aroma production is diminished and the rate and extent of recovery is reduced upon transfer to air (Ferenczi et al., 2006; Plotto et al., 1999; Tough and Hewett, 2001; Yahia, 1991). CA Storage. In 2005, about 84 % of the US. cold-stored apple crop was held in CA storage (USDA-NASS, 2005). CA storage has increased the marketing season of apples to year-round. It has an advantage in suppressing decay without necessitating the use of fungicides. It also delays ripening, maintains firmness, skin color, and acidity by inhibiting ethylene synthesis (Mattheis et al., 1998). However, improper management of storage condition can damage the crop significantly during storage. For example, low temperature, low 02, and high C02 may cause fermentation (Mattheis et al., 1991a) or internal disorders (Argenta et al., 2002); low temperature, low 02, and increased duration of storage greatly reduce aroma production (Brackmann et al., 1993; Echeverria et al., 2004a; Mattheis et al., 1991a; 1998; Plotto et al., 1999). On the other hand, Lavilla et al. (1999) reported that CA storage of “Granny Smith’ apple fruit did not diminish aroma production. Under ultra-low oxygen (ULO) conditions, ethanol and aldehydes may accumulate to varying degrees depending on cultivar, causing off-flavors (Dixon and Hewett, 2000a). Plotto et al. (1999) reported that sourness and astringency were significantly higher in ULO-stored fruit than in apples held in air storage in which fruitiness and floral scent were maintained. Also, the accumulation of ethanol or methanol by ULO conditions may alter the ester composition. Because of the abundance of these alcohols in ULO-stored fruit, the methanol- or ethanol- 12 derived esters increase while butyl and hexyl esters decrease (Argenta et al., 2004; Mattheis et al., 1991a). Moreover, ULO strongly suppresses straight-chain esters while branched-chain esters are not similarly suppressed by low 02, but they are suppressed by high 002 (Brackmann et al., 1993). Similarly, Fellman et al. (1993) observed an increase in 2-methylbutyl acetate after ‘Rome’ apples were removed from low 02 CA (1% C02) storage. Under the ULO condition, the suppression of ethylene action is probably the primary cause of reduced aroma volatile synthesis. Further, respiration may be suppressed, reducing carbon and energy available for ester biosynthesis (Rudell et al., 2002). It is also plausible that 02-dependent processes in ester biosynthesis may be limited under these conditions. When ‘Gala’ apple was stored in CA for 120 days and examined after 7 days at 20°C, ester emission was greater. in fruits treated with ambient air 1-3 days per week for 8 hours and returned to 1 kPa 02 compared to static 1 kPa 02 (Mattheis et al., 1998). These experiments suggest a potential for hypoxia to assist in acclimation and recovery of aroma after a long period of storage. Chemical treatments. Ethylene can be suppressed by chemical treatments in two ways: by inhibiting ethylene biosynthesis or ethylene action. One chemical tool is preharvest treatment with aminoethoxyvinylglycine (AVG), which inhibits ACS and can delay fruit maturity and ripening by reducing ethylene production. AVG application and additional CA storage benefit fruit quality (Brackmann and Waclawovsky, 2001 ). 13 Another important chemical treatment is 1-MCP. 1-MCP is approved in the US for commercial use on edible crops and is marketed as SmartFreshTM (AgroFresh, lnc., Rohm and Haas). Food use registration for 1-MCP has been obtained in several countries. Use continues to expand, in terms of number of countries and variety of crops (Watkins and Miller, 2005). Research suggests that 1-MCP is very effective in maintaining apple quality by blocking ethylene receptors and reducing physiological disorders during storage (Fan et al., 1999). However, the effectiveness of 1-MCP depends on cultivar and storage conditions (Watkins et al., 2000), concentration (Lurie et al., 2002), temperature and duration (DeEll et al., 2002), growing region, fruit maturity, and time from harvest to treatment (Blankenship and Dole, 2003). It also has a negative effect on disease control, thought to be imposed through an inhibition iof the synthesis of phenolics in strawberry (Jiang et al., 2001). The significant decrease in volatile production is specific to climacteric fruit crops, dependent upon ethylene for normal ripening (Botondi et al., 2003; Defilippi et al., 2004; Ferenczi et al., 2006; Golding et al., 1999). Lurie et al. (2002) observed that 1-MCP-treated apples retained more alcohols, aldehydes, and [3- damascenone. In bananas, application of 1-MCP after an initiation of ripening caused an increase in branched-chain alcohols, altering volatile composition (Golding et al., 1999). Botondi et al. (2003) reported that there is no 1-MCP effect on apricot color, but 1-MCP did cause a reduction in lactone synthesis and rise in terpenols. It is possible that when ethylene action is excessively limited, there 14 can be a shortage of energy for maintenance reactions required for normal cellular activity, leading to undesirable reactions (Mir et al., 1999). Timing of harvest Timing of harvest can alter aroma production, composition, and recovery after storage (Echeverria et al., 2004a, 2004b). Early-picking of apples, harvested more than four weeks ahead of the optimal harvest date, delays the onset of volatile production and the amount is lower. If the fruit is harvested within two weeks of the optimal harvest date, the aroma production is normal after few days (Song and Bangerth, 1996). The fruits harvested at the climacteric stage produced more volatiles after removal of the fruit from storage than fruits harvested prior to the climacteric (Brackmann et al., 1993). Vanoli et al. (1995) found the change in volatile composition by harvest date that the optimum harvested apple had a best aroma composition, low content of butanoates and alcohols and high content of acetate esters. Ester precursor formation Apple skin is covered by a layer of wax and cutin (lvanov and Dodova- Anghelova, 1974). When Dimick and Hoskin (1983) removed the oily wax coating on the skin, ester production was not limited and suggested that the ester source is from the skin or cortex. Guadagni et al. (1971) observed the greatest ester production was from peels of apples suggesting aroma biosynthesis mainly takes place in the skin rather than the flesh. Rudell et al. (2002) observed that the apple skin tissue displayed a greater capacity to synthesize pentanol esters than 15 carpellary tissue (between the carpels and the core line) and hypanthial tissue (between the skin and the core line) when incubated with 1-pentanol. Esters have an alcohol-derived (alkyl) group and an acid-derived (alkanoate) group. The alkyl and alkanoate groups can be straight-chain or branched-chain. The immediate precursors are an alcohol and a CoA thioester of a fatty acid (Figure 1). Alcohols are formed from aldehydes by alcohol dehydrogenase (ADH). Alkyl group precursors normally range from 1 to 6 carbons in length and the alkanoate group precursors range from 2 to 8 carbons (Paillard, 1990). Carbon entry into these pathways has been variously proposed to depend on catabolic and biosynthetic pathways (e.g. B-oxidation, lipoxygenase, amino acid metabolism, two- and single-carbon fatty acid synthesis). Although some experimental results support a role for catabolism (Sanz et al., 1997), sufficient evidence does not exist to disregard biosynthetic pathways. Once formed, the alcohols and acyl-CoAs are combined by alcohol acyltransferase (AAT) to form esters. Catabolic pathways According to one hypothesis, straight-chain ester precursors are proposed to be derived from catabolism of fatty acids via the B-oxidation (Figure 2) and lipoxygenase pathways (Figure 3) (Dixon and Hewett, 2000b; Fellman et al., 2000; Sanz et al., 1997; Yahia, 1994). Branched-chain ester precursors may be supplied from branched-chain amino acid (BCAA) metabolism (Figure 4) (Perez et al., 2002; Tressl and Drawert, 1973). 16 B-oxidation. In plants, B-oxidation takes place in peroxisomes rather than in mitochondria as in mammals. Peroxisomes are classified according to their physiological function: glyoxisomes, leaf peroxisomes, and unspecialized peroxisomes (Hayashi, 2000). Leaf peroxisomes, are specialized organelles for photorespiration, glyoxisomes are primarily dedicated to fatty acid breakdown (as in germinating seeds), and unspecialized peroxisomes remain undefined (Hayashi, 2000). The function of peroxisomes and glyoxisomes has some degree of plasticity; transformation from peroxisome to glyoxisome can be observed and vice versa during specific developmental stages (Hayashi and Nishimura, 2003). For example, the glyoxisomes first appear in a germinating seedling and, when the seedling begins photosynthesis, the glyoxisome is functionally transformed to a peroxisome (Hayashi and Nishimura, 2003). The major role of B-oxidation in the glyoxisome is to convert fatty acids into carbon skeletons that can be metabolized into sugars, amino acids, and nucleotides, and to provide energy (Graham and Eastmond, 2002). The initial step of the B-oxidation pathway is the activation of free fatty acids with a high-energy thioester linkage with acyl-coenzyme A by acyI-CoA synthetase (EC 6.2.1.3). The activated acyI-CoAs are reduced by two carbons during each cycle of B-oxidation, which is comprised of four steps. 1) dehydrogenation, 2) addition of water, 3) dehydrogenation, and 4) cleavage of acetyl-CoA (Graham and Eastmond, 2002) (Figure 2). The first dehydrogenation step is catalyzed by acyI-CoA oxidase (ACX, EC 1.3.3.6). AcyI-CoA is converted to 2-trans-enoyl-CoA requiring FAD as a cofactor, passing electrons to Oz and 17 forming H202, which is further converted to H20 and 02 by catalase. There are multiple ACX isozymes with differences in substrate chain-length specificities (De Bellis et al., 1999, 2000; Hooks et al., 1996, 1999; Kirsch et al., 1986; Rylott et al., 2003). Chain-length specificities are categorized as: long- (>C14), medium- (C8- C14), and short-chain (C4-08). Several peroxisomal ACX isozymes have been isolated and characterized in pumpkin (DeBellis et al., 1999, 2000) and cucumber (Kirsch et al., 1986). In Arabidopsis, several genes in the ACX family have been isolated and characterized (Hooks et al., 1999; Rylott et al., 2003). The second and third steps in B-oxidation are catalyzed by a multifunctional protein containing both enzyme activities: 2-trans-enoyI-CoA hydratase (EC 4.2.1.17), hydrating 2-trans-enoyI-CoA to 3-hydroxyacyl-CoA, and L-3- hydroxyacyl—CoA dehydrogenase (EC 1.1.1.35) requiring NAD as a cofactor and oxidizing 3-hydroxyacyl-CoA to 3-ketoacyl-CoA, respectively. The final step is catalyzed by 3-ketoacyl-CoA thiolase (EC 2.3.1.16), which uses CoASH to cleave two carbons from 3-ketoacyl-CoA to form acetyl-CoA and an acyl-CoA that is two carbons shorter than in the previous cycle. The shortened acyl-CoA product cycles again through B-oxidation until completely reduced to acetyl-CoA. The major difference between mammalian mitochondrial and peroxisomal/glyoxisomal B-oxidation is that the first step in mammals is catalyzed by acyl-CoA dehydrogenase (ACDH, EC 1.3.99.3) instead of acyI-CoA oxidase (ACX) (Eaton et al., 1996; Graham and Eastmond, 2002). During the first step of dehydrogenation with ACDH, flavin adenine dinucleotide (FAD) passes electrons to the mitochondrial respiratory chain instead of to 02 and forming H202. Similar 18 to ACX, ACDH isozymes have different chain-length specificities of acyl-CoAs (Eaton et al., 1996). Chain-length specificities are categorized as: short— (C4-6), medium- (C4-12), long- (08-12), and very long-chain (012-24). In plants, the complete degradation of fatty acids takes place in the peroxisome/glyoxisome and the function of mitochondrial B-oxidation is unknown (Hayashi, 2000). In contrast, mammals have a functional difference between two organelles in metabolizing fatty acids. Mammalian B-oxidation takes place primarily in mitochondria, but very long chain fatty acids such as hexacosanoic acid (C26) and less common fatty acids are oxidized in the peroxisome (Eaton et al., 1996). Interestingly, based on the phylogenetic tree of ACX, plant glyoxisomal short-chain ACX has a high similarity with mammalian mitochondrial ACDH, whereas plant glyoxisomal long-chain ACX has a high similarity with mammalian peroxisomal ACX (Hayashi, 2000). Several 3-ketoacyl-CoA thiolase genes have been isolated and characterized in Arabidopsis (Germain et al., 2001). One of the 3-ketoacyl-CoA thiolases has substrate specificity based on chain-length. However, it is not clear whether chain-length specificity in Arabidopsis is imposed via ACX or the final step via 3—ketoacyI—CoA thiolase. No studies on the expression of B-oxidation genes have been reported for ester-producing plant organs. Lipoxygenase pathway. The pathway of lipoxygenase involves several enzymes (Figure 3). These enzymes are bound to the thylakoid membrane of chloroplasts of green leaves (Hatanaka, 1993). Linoleic or Iinolenic acids are usually the most abundant fatty acids in plants. These fatty acids are released 19 from phospholipids by lipase or hydrolase (Wang, 2001). The free fatty acids are oxidized by lipoxygenase (LOX, EC 1.13.11.12), requiring oxygen; LOX acts on C18 fatty acids to produce 9- or 13- hydroperoxy derivatives. Different forms of LOX have been analyzed in cotyledons and fruits, and in leaves of barley, spinach, and Arabidopsis (Feussner and Wasternack, 2002). The specificity of LOX determines the tendency toward C6 or 09 aldehyde synthesis. In apples, LOX is highly specific in peroxidizing linoleic acid to 13-hydroperoxy derivatives (Kim and Grosch, 1979), which would facilitate synthesis of hexanal. The second step involves the enzyme hydroperoxide lyase (HPL). HPL has been characterized as a special class of cytochrome P450 enzyme (Noordermeer et al., 2001) that cleaves the 13-hydroperoxy derivatives, 13-hydroperoxy linoleic acid (13-HPOD) and 13-hydroperoxy linolenic acid (13-HPOT) into hexanal and Z-3—hexenal, respectively. 13-HPOT can also be metabolized to C5 compounds such as 2-Z-pentenol or 1-penten-3-ol by a secondary activity of lipoxygenase under anaerobic conditions (Salch et al., 1995). The aldehyde products of HPL are reduced to corresponding alcohols (e.g. hexanol and Z-3-hexenol) by alcohol dehydrogenase (ADH). Salas et al. (2005 and 2006) studied silencing HPL and LOX genes in potato and Arabidopsis leaves. HPL knock-out plants had similar levels of C6 compounds but had 4-fold higher in 05 compounds, and LOX- silenced plants had severely decreased volatile production. The authors concluded that both LOX and HPL activities are required for volatile production. Finally, alcohols such as those produced by the LOX pathway can act as substrates for ester formation. 20 Branched-chain amino acid degradation. The pathways for branched- chain amino acid (BCAA) metabolism have been extensively studied in yeast and bacteria, and are an important in flavor development of microbial fermentations (Dickinson et al., 1997, 1998, 2000; Smit et al., 2005). The first step in BCAA catabolism is the removal of the amino group via branched-chain aminotransferase (BCAT, EC 2.6.1.42), which requires pyridoxal phosphate as a coenzyme (Figure 4). BCAT can transaminate all three BCAAs or may have a preference for a specific BCAA (Yvon et al., 2000). It is still not clear whether BCAT is regulated by an unique mechanism in plants, but in bacteria, nutritional factors such as carbohydrate and nitrogen source regulate aminotransferase activity (Chambellon and Yvon, 2003). In examples from non-ester producing plants, BCAA degradation is thought to occur in response to low carbohydrate availability (Graham and Eastmond, 2002). In a recent study with Arabidopsis thaliana, a total of six or possibly seven BCAT genes localized in mitochondria, plastids, and cytosol were cloned. The mitochondrial AtBCAT-1 is thought to contribute to the degradation of all three BCAA (Diebold et al., 2002; Schuster and Binder, 2005). Although BCAT enzymes catalyze a reversible reaction, the degradation is believed to contribute to aroma formation by bacteria since the mutant line of BCATgenes reduced the formation of branched-chain aldehydes and corresponding alcohols (Rijnen et al., 2003). The branched-chain d-keto acid is metabolized via two pathways to supply immediate precursors to branched-chain esters, either to branched-chain acyl- CoAs (acid-forming pathway) or to branched-chain alcohols (alcohol-forming 21 pathway). Mammals only have the capacity for catabolism in formation of branched-chain acyI-CoAs; a defect in branched-chain q-keto acid dehydrogenase in this pathway causes a disorder known as a maple syrup urine disease (Platell et al., 2000). In bacteria, metabolism of branched-chain a-keto acid differs among strains, favoring either the acid-forming or alcohol-fonning pathway, or utilizing both at the same time (Helinck et al., 2004). In the acid-forming pathway, a branched-chain d-keto acid is dehydrogenated to branched-chain acyl-CoAs by branched-chain d-keto acid dehydrogenase (BCKDH, EC 1.2.4.4), a multifunctional enzyme composed of three subunits (d-keto acid dehydrogenase, dihydrolipoyl acyltransferase, and dihydrolipoyl dehydrogenase) and structurally similar to pyruvate dehydrogenase (PDH) complex (Mooney et al., 2002). PDH catalyzes the oxidative decarboxylation of pyruvate to yield acetyI-CoA and NADH, and has two forms: plastidial and mitochondrial (Tovar—Méndez et al., 2003). In plants, BCKDH has been shown to take place both in peroxisomes (Gerblin and Gerhardt, 1988) and mitochondria (Taylor et al., 2004). Unlike BCAT, the activity of BCKDH is highly regulated through the mechanism of phosphorylation (inactivation) and dephosphorylation (activation) in mammals (Harper et al., 1984). However, regulation by phosphorylation has been difficult to prove in plants (Mooney et al., 2002). In mammals and bacteria, the branched-chain acyl-CoA (2-methylbutyl- 00A, for example, in figure 4) is further metabolized into acetyl-CoA and propionyI-CoA to supply for energy production (pathway not shown) (Harper et al., 1984; Massey et al., 1976). The same pathway is proposed in Arabidopsis, but, 22 some of the enzymes are still waiting to be characterized to prove the pathway (Taylor et al., 2004). If the proposed pathway is active in plants, propionyl-CoA _ can serve as precursor of esters. In the alcohol-forming pathway, the branched-chain a-keto acid is decarboxylated to branched—chain aldehyde by branched-chain d-keto acid decarboxylase (BCKDC, EC 4.1.1.72) with the release of CO2. This product is further dehydrogenated to branched-chain alcohol by alcohol dehydrogenase (ADH, EC 1.1.1.1) (Wyllie et al., 1996). In bacteria, BCKDC activity is capable of forming an alcohol from branched-chain a-keto acid and is proposed to be the rate controlling step; however, it is present in only few strains of bacteria (Smit et al., 2004). Similarly, in yeast, pyruvate decarboxylase (PDC, EC 4.1.1.1), an important enzyme that cleaves pyruvate to acetaldehyde during alcoholic fermentation, has been reported to catalyze the decarboxylation of branched- chain d-keto acids (Yoshimoto et al., 2001), but may not be an essential step for forming branched-chain alcohol (ter Schure et al., 1998). Several genes (PDC1, PDC5, PDCG, YDL0800) have been reported to be responsible for decarboxylation of branched-chain a-keto acids to branched-chain aldehydes and branched-chain alcohols (Dickinson et al., 1997, 1998, 2000; Yoshimoto et al., 2001). Dickinson et al. (1997, 1998, 2000) suggests that the catabolic pathways of three BCAAs are accomplished in different ways, a single YDL080C gene (PDC-like gene) is likely responsible for leucine catabolism and any one of the isozymes of PDC is responsible for valine and isoleucine catabolism. 23 Yoshimoto et al. (2001) adds that a PDC1 gene at least partially contributes to the formation of 3-methylbutanol in yeast. In fruits, three PDC genes have been isolated from strawberries and one from grape berries (Moyano et al., 2004; Or et al., 2000). Also, PDC activities have been measured during maturation of ‘Fuji’ apples (Echeverria et al., 2004c). Since PDC plays a significant role in the conversion of pyruvate to acetaldehyde, which is further reduced to ethanol by ADH, the main purpose of these studies was to explore the mechanism of ethanol production under anaerobic conditions and the formation of ethanol-derived esters such as ethyl esters or acetate esters. However, they did not evaluate PDC potential for BCAA catabolism. Thus, PDC activity in BCAA catabolism in fruits is unknown. Tieman et al. (2006) recently found that 2-phenylethanol, an important flavor and insect attractant in tomato and rose, is synthesized from phenylalanine by participation of an aromatic amino acid decarboxylase. This report suggested that decarboxylase activity might contribute to aroma formation in ester-producing fruits. To date, the involvement of PDC in forming branched—chain esters has not been addressed in the literature, to our knowledge. Synthetic pathways There are two pathways for fatty acid biosynthesis, either by two-carbon (2-C FAB) or one-carbon (1-C FAB) elongation. For two-carbon elongation, fatty acid synthase (EC 2.3.1.85) plays a major role with an acyl carrier protein to synthesize long chain fatty acids for membranes, storage lipids, and waxes (Ohlrogge and Jaworski, 1997). For one-carbon elongation, an d-ketoacid 24 elongation (aKAE) route is utilized, which may impact primary and secondary metabolic pathways including the tricarboxylic acid cycle, leucine biosynthesis, formation of sugar-ester acyl acids, and short-chain alcohols of yeast (Kroumova and Wagner, 2003). Two-carbon elongation has been extensively studied for its role in lipid biosynthesis, but, very limited information is available regarding one- carbon elongation. In certain strains of bacteria, isoleucine biosynthesis may utilize 1-C FAB pathway (Charon et al., 1974; Xu et al., 2004). Branched-chain amino acid biosynthesis. Generally, isoleucine is synthesized from threonine in plants and bacteria. However, radioactive carbon- labeling studies indicate that most of the isoleucine is synthesized by a pathway independent of threonine in the Leptospira bacteria (Charon et al., 1974). Branched-chain amino acids are formed from branched-chain a-keto acids (Figure 4). These branched-chain d-keto acids, a-keto-B—methylvalerate, a-keto- isovalerate, and d-ketoisocaproate are substrates in the aminotransferase reaction synthesizing the BCAAs, isoleucine, valine, and leucine, respectively. The immediate precursor to isoleucine is the branched-chain a-keto acid d-keto- B-methylvalerate. This compound can potentially serve to synthesize 2- methylbutyl and 2-methylbutanoate esters without the formation of isoleucine. Two-carbon elongation. 2-C FAB takes place in the plastids (Figure 5). Two enzyme activities are required, acetyl-CoA carboxylase (ACCase, EC 6.4.1.2) and fatty acid synthase (FAS, EC 2.3.1.85). ACCase provides the starting material of malonyl-CoA from acetyl-CoA, yielding C02. FAS refers to several enzymes required to form fatty-acids ranging in length from 12-22 25 carbons (Broun et al., 1999; Thelen and Ohlrogge, 2002). The predicted products found in most plants are 16- and 18-carbon fatty acids. Fatty acid synthesis occurs in four steps (Ohlrogge and Jaworski, 1997) (Figure 5), 1) condensation, 2) reduction, 3) dehydration, and 4) reduction. First, malonyl-CoA is transacylated to malonyl-ACP (chain extender), requiring acyl- carrier protein (ACP) as a cofactor, and condenses with acetyl-CoA (primer) via the enzyme 3-ketoacyl-ACP synthase Ill (KAS III, EC 2.3.1.41). Steps 2-4 are catalyzed by 3-ketoacyl-ACP reductase (EC 1.1.1.100), 3-hydroxyacyl-ACP dehydrase (EC 4.2.1.17), and enoyI-ACP reductase (EC 1.3.1.9), respectively, forming butyryl-ACP after step 4. The cycle repeats to condense with malonyl- ACP with an enzyme 3-ketoacyl—ACP synthase l (KAS l) and KAS II. KAS exhibits substrate specificity: KAS l prefers the shorter chain (04-14)- ACP and KAS II for longer chain (>C14)-ACP (Shimakata and Stumpf, 1982). The fatty acid normally elongates to 16 to 18 carbons in length until acyl-ACP ' thioesterase (EC 3.1.2.14) terminates fatty acid synthesis by hydrolysis. The terminated fatty acid either leaves the plastid to the ER for further elongation or desaturation or for storage as a lipid. The acyl-ACP thioesterase has two types: one is relatively specific for 18:1-ACP (FatA) and the other for short-chain saturated acyl-ACP (FatB) (Hawkins and Kridl, 1998; Jones et al., 1995). When the 12:0-ACP thioesterase gene from seed of California bay trees was expressed in developing seed of Arabidopsis, the transgenic plant produced large amounts of Iaurate (12:0) (Voelker et al., 1992). If acyl-ACP thioesterase can cleave fatty acids to produce shorter length C4-C8 acids, they have a potential to produce 26 precursors suitable for ester formation. However, to-date, no short- to medium- chain acyl—ACP thioesterase has been reported in fruit. One-carbon elongation. The 1-C FAB pathway is rather specialized and is not found in all plants. This pathway consists of 3-4 steps, starting with condensation of acetyl-CoA (chain extender) and pyruvate (primer) by the enzyme 2-isopropylmalate synthase (EC. 4.1.3.12) (Figure 6). The next step involves isomerization, dehydration, and decarboxylation by isopropylmalate dehydratase (EC 4.2.1.33), and 3-isopropylmalate dehydrogenase (EC 1.1.1.85), to produce d-keto butyrate, extended by one-carbon relative to the primer, pyruvate (Kroumova and Wagner, 2003). Further elongation by one carbon is accomplished by successive rounds of acetyl-CoA addition and CO2 elimination to make long odd- and even-numbered d-keto acids. Finally, d-keto acid dehydrogenase 2-oxovalerate dehydrogenase (often called 2-oxoacid dehydrogenase, EC 1.2.1.25) decarboxylates and acetylates to form an acyl-CoA with the same carbon number relative to the starting o-keto acid (Kroumova and Wagner, 2003). These acyl-CoAs can enter directly into the AAT reaction, forming straight—chain esters. Interestingly, d-keto butyrate produced in this pathway may also serve as a substrate for reactions leading to the formation of a-keto-B-methylvalerate and subsequent isoleucine biosynthesis (Figure 4). This possibility has not been demonstrated in plants to my knowledge. Ester synthesis The final step of ester formation is the combination of alcohol and acyl- CoA by alcohol acyltransferase (AAT, EC 2.3.1.84) (Figure 1). 0f the various 27 enzymes implicated in ester formation, only AAT has been characterized to any meaningful extent. The AATgene has been identified in apple, strawberry, melon, and banana (Aharoni et al., 2000; Harada et al., 1985; Jayanty et al., 2002; Souleyre et al., 2005; YahyaOui et al., 2002). Several isozymes of AAT have been studied and were determined to utilize a broad range of precursors although they do exhibit marked substrate preferences. The AAT reaction velocity is affected by carbon chain length, and architecture (e.g. straight— or branched-chain) of the acyl-CoAs, or alcohol substrates (Aharoni et al., 2000; Olias et al., 2002; Ueda et al., 1992; Yahyaoui et al., 2002). Substrate preference also differs by fruit species and, within a species, even between cultivars (Holland et al., 2005), and can not be predicted based on the sequence similarity among various members of the AAT family (Beekwilder et al., 2004). In apples, MpAA T1 (Souleyre et al., 2005) and MpAA T2 (Li et al., 2006) have been isolated. MpAA T1 is characterized and found to be expressed in several different organs. MpAAT1 prefers to produce hexyl esters of C3, C6, and C8 acyl-CoAs, but with acetate esters substrate preference depends on precursor alcohol concentration. In strawberry, AAT has substrate preference in the order of hexyl>butyl>amyl>isoamyl when acetyI-CoA is the acyl donor, and acetate>butanoate>propanoate when butanol is the alcohol donor (Pérez et al., 1993). Four genes CM-AA T1, CM-AA T2, CM-AA T3, and CM-AA T4 have been characterized in melon (El-Sharkawy et al., 2005; Yahyaoui et al., 2002). CM- AAT2 has no detectable activity; CM-AAT1 is capable of producing a wide range of esters but has a higher activity with hexanol relative to butanol in the 28 production of hexyl and butyl esters (Yahyaoui et al., 2002). CM-AAT1 can also take branched-chain alcohols, but the activity rate depends on the position of the methyl group; it has higher activity for 2-methylbutanol than for 3-methylbutanol. CM-AAT3 accepts a wide range of substrates with strong preference in benzyl acetate and CM—AAT4 exclusively forms acetates with strong preference for cinnamoyl acetate (El-Sharkawy et al., 2005). Such substrate specificity is likely to have an effect on individual ester concentration and which types of esters are produced at various stages of ripening and senescence. It is suggested that substrate supply is a major determinant rather than AAT enzyme activity for regulating the quantitative and qualitative composition of the aroma profile (Echeverria et al., 2004c; Wyllie and Fellman, 2000). In banana, Jayanty et al. (2002) observed AAT expression and the presence of AAT enzyme activity well before the onset of fruit ripening and ester biosynthesis, suggesting that the precursors are lacking, and therefore, the control must lie within the ester precursor biosynthetic pathway. However, Jayanty et al. (2002) also hypothesized that as aroma biosynthesis engages, the increase in AAT activity and gene expression exerts a major influence in overall ester formation. Also, since AAT cannot discriminate between 2-methylbutyl and 3-methylbutyl precursors (Wyllie et al., 1996), the fact that apple mostly produces 2-methylbutyl esters suggests control of biosynthesis must lie at the stage of ester precursor supply, rather than at the level of AAT (Wyllie and Fellman, 2000). An increase in AAT activity during ripening was observed for apple (Defilippi et al., 2005) and 29 melon (Shalit et al., 2001) suggesting a significant, but not limiting role for AAT in ester production. Hypothesis The involvement of B-oxidation, the lipoxygenase pathway, and BCAA metabolism for ester formation is suggested by various substrate feeding studies. In ‘Cox’s Orange Pippin’ apple, feeding of methyl hexanoate and methyl octanoate enhanced butanoate ester formation (Bartley et al., 1985) and feeding pentanoic acid increased propanoate esters in ‘Golden Delicious’ apples (De Pooter et al., 1983). In a deuterium-labeling study, feeding 018:0 and 18:1 fatty acids produced straight-chain C6-C8 alkanoate esters, hexanoic acids produced C4 alkyl and alkanoate esters, and linoleic acids only produced hexyl and hexanoate esters in apple (Rowan et al., 1999). On the other hand, labeled palmitic acid (C16) did not convert into volatile constituents in banana (Tressl and Drawert, 1973). These studies suggest that if LOX is active at the same time as B-oxidation, both pathways could act together to provide the large amount of C6 and the trace amount of unsaturated precursors found in esters. Labeled-Leucine produced 3-methylbutyl and 3-methylbutanoate esters, valine produced 2-methylpropyl and 2-methylpropanoate esters, and isoleucine produced 2-methylbutyl and 2-methylbutanoate esters in strawberry, apple, and banana (Perez et al., 2002; Rowan et al., 1996, 1998; Tressl and Drawert, 1973). In apples, esters are produced having branched-chain alkyl and branched-chain alkanoate groups that are likely from isoleucine and valine metabolism (Ferenczi, 2003). Additional feeding studies demonstrate that the catabolism of BCAAs can 30 supply the needed products for branched-chain esters (Wyllie et al., 1996; 2000). Although leucine is abundantly found in ripening apple fruit (Burroughs, 1970; Hansen, 1970), there are little-to—no esters produced from leucine. Paradoxically during apple fruit ripening, isoleucine accumulates, but the other BCAAs do not (Nie et al., 2005). According to conclusions derived from feeding studies, the increase in 2-methylbutyl ester is a result of increased catabolism of isoleucine. However, this suggestion runs counter to the observation that isoleucine accumulates. lsoleucine accumulation data are more suggestive of enhanced isoleucine synthesis and/or a reduction in its catabolism. It is possible that increased isoleucine synthesis is accompanied by an increase in the pool of the isoleucine precursor a-keto-B-methylvalerate, d-keto-B-methylvalerate may directly feed into the synthesis of 2-methylbutanol and 2-methylbutanoate, without forming isoleucine. 1-C FAB can utilize several primers other than pyruvate to form various types of compounds such as glucosinolates and sugar esters (Kroumova and Wagner, 2003). Although there are some limitations by plant species, 1-C FAB is capable of making various chain length of fatty acids, generally 3-12 carbons; straight, branched, odd or even, short- or medium-chain length (up to C7 straight chain in petunia) (Kandra et al., 1990; Kroumova et al., 1994; Kroumova and Wagner, 2003; Oku and Kaneda, 1988). If 1-C FAB pathway is utilized in the fruit ester biosynthesis, it may give an explanation of forming esters with odd-number carbon chains (C3, 05, and C7) as well as even-number esters. 31 Extensive feeding studies demonstrate catabolic pathways have the potential to meet the needs for ester biosynthesis; however, genes/enzymes in these pathways are not well characterized in ester-forming fruits. In case of biosynthetic processes in ester formation, no studies have been evaluated the possible role in ester formation. Only the final step of ester formation, AAT gene expression and activity has been studied. 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Rapid reduction in aroma volatiles of 'Pacific Rose’ apples in controlled atmospheres. Acta Hort. 553:219-223. Tovar-Méndez, A., J.A. Miernyk, and DD. Randall. 2003. Regulation of pyruvate dehydrogenase complex activity in plant cells. Eur. J. Biochem. 270(6): 1 043-1 049. 44 Tressl, R. and F. Drawert. 1973. Biogenesis of banana volatiles. J. Agric. Food Chem. 21 (4):560-565. Ueda, Y., A. Tsuda, J.H. Bai, N. Fujishita, and K. Chachin. 1992. Characteristic pattern of aroma ester formation from banana, melon, and strawberry with reference to the substrate specificity of ester synthetase and alcohol contents in pulp. J. Jpn. Soc. Food Sci. Technol. 39:183-187. Ulrich, D., E. Hoberg, A. Rapp, and S. Kecke. 1997. Analysis of strawberry flavour - discrimination of aroma types by quantification of volatile compounds. Z. Lebensm. Unters. Forsch. A. 205(3):218-223. USDA-National Agricultural Statistics Service. Cold Storage 2005 Summary. USDA, Vanoli, M., C. Visai, and A. Rizzolo. 1995. 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Characterization and role of the branched-chain aminotransferase (BcaT) isolated from Lactococcus lactis subsp. cremon‘s NCDO 763. Appl. Environ. Microbiol. 66(2):571-577. 46 Esters Odor characters Reference Ethyl acetate pleasant, ethereal-fruity, brandy-like Dimick and Hoskin, 1983 Ethyl acetate Fruity, solvent like Paillard, 1990 very diffusive, ethereal-fruity, pungent, Butyl acetate pear odor Dimick and Hoskin, 1983 Butyl acetate slight wiskey Advanced Biotech. Inc. Butyl acetate nail polish, gala Plotto et al., 2000 Pentyl acetate apple-like, sweet Rizzolo et al., 1989 Pentyl acetate banana oil, fruity pineapple Paillard, 1990 hexyl acetate Sweet fruity, slightly floral Dimick and Hoskin, 1983 hexyl acetate green, fruity Salas et al., 2006 hexyl acetate Gala, ripe, pear Plotto et al., 2000 Heptyl acetate fruity, fatty-green, slight floral odor Dimick and Hoskin, 1983 2-methylbutyl acetate solvent, gala Plotto et al., 2000 2-methylbutyl acetate apple, pear, banana Advanced Biotech. Inc. Z-3-hexenyl acetate banana, green Salas et al., 2006 Ethyl propanoate ethereal, fruity-rum like odor Dimick and Hoskin, 1983 Propyl propanoate fruity Plotto et al., 2000 Butyl propanoate fruity, apple Plotto et al., 2000 Hexyl propanoate apple Plotto et al., 2000 Ethyl 2-methylpropanoate diffusive, sweet-ethereal, fruity odor Dimick and Hoskin, 1983 Ethyl butanoate . fruity, ester-like, sweet Ulrich et al., 1997 Ethyl butanoate fruity, banana, pineapple Paillard, 1990 powerful, ethereal-fruity odor, banana, Ethyl butanoate pinapple Dimick and Hoskin, 1983 Propyl butanoate pineapple, apricot Paillard, 1990 Butyl butanoate rotten apple, cheesy Plotto et al., 2000 Butyl butanoate pear, pineapple Paillard, 1990 Hexyl butanoate green apple Plotto et al., 2000 Methyl 2-methylbutanoate sweet fruity Plotto et al., 2000 powerful diffusive, green-fruity, pungent Ethyl 2-methylbutanoate odor Dimick and Hoskin, 1983 Ethyl 2-methylbutanoate sweet strawberry Plotto et al., 2000 Ethyl 2-methylbutanoate apple-like, green , fruity Paillard, 1990 Propyl 2-methylbutanoate very sweet, strawberry Plotto et al., 2000 Butyl 2-methylbutanoate fruity, apple Plotto et al., 2000 Hexyl 2-methylbutanoate apple, grapefruit Plotto et al., 2000 Hexyl 2-methylbutanoate powerful, fresh-green fruity odor Dimick and Hoskin, 1983 Butyl pentanoate apple, raspberry Paillard, 1990 Methyl hexanoate fruity Paillard, 1990 Ethyl hexanoate fruity, fresh, sweet Paillard, 1990 Butyl hexanoate green apple Plotto et al., 2000 Butyl hexanoate pineapple Paillard, 1990 Hexyl hexanoate sweet floral Advanced Biotech. Inc. Table 1. Representative esters identified in apples with sensory description. Only esters that had an odor description are listed. Esters included acetates, propanoates, 2-methylpropanoates, butanoates, 2-methylbutanoates, pentanoates, and hexanoates. 47 .coszLe Loywm Lou. mmflgmoam om ucm _ocoo_m 233w 9 EEQOQ 9.3m: m>m>>£mn_ e 939“. macaw » _C<o< + Ea 2829.8 6.3 £9: In 6:82 cozmmcofi cozmmcofi m I I AIIIIII c0380 03... coemo 0:0 wEON OHOXLO wwm_>xo€moon_ s ,2 82:82 3385526522.“. 0 m4 E05? co=mn_xo-n 9 . n_O<-_>Hmo< 9m>:._>n_ _Ewflm>w owmcmgxoa: _ wEom OEE< Emcofiococfim 228 as“. m_mo£c>wo_m 29mm B-Oxidation pathway R-C-C-Cfi-S-COA Cn acyl-CoA 0 H202 FAD X Acyl-CoA oxidase 02 FADH2 R-C=C-(&-S—COA 2-trans-enoyl-CoA O H20 2-trans-enoyl-CoA hydratase OH R-C-C-(R-S-COA 3-hydroxyacyl-CoA O NAD+ L-3-HydroxyacyI-CoA dehyd rogenase NADH R-c-é-c-s-CoA 3-ketoacyl-CoA I3-ketoacyl-CoA thiolase CoASH H20>\*C-C&-S-C0A acetyl-CoA V O R-C-S-COA Cn-2 acyl-CoA Figure 2. Pathway for catabolism of fatty acids via B-oxidation. In plants, [3- oxidation takes place in peroxisomes. Free fatty acid Cn acyl-CoA are reduced by two carbons during each cycle of B-oxidation by four steps. 1. Dehydrogenation by acyl-CoA oxidase, 2. addition of water by 2-trans-enoyl-CoA hydratase, 3. dehydrogenation by L-3-hydroxyacyI-CoA dehydrogenase, and 4. cleavage of acetyl-CoA by 3-ketoacyl-CoA thiolase to produce Cn-2 acyl-CoA. Stars indicate the carbon position during each reaction. Hydrogens in the carbon-hydrogen bonds are not shown. 49 Diacylglycerolipids Lipoxygenase pathway ,. A- ' Lipase I Linoleic acid (18:2) Linolenic acid (18:3) C-C—C-C-C-C=g-C-C=(93-R-COOH C-C-C=C-C-C=Cz-C-C=C-R-COOH Ikieexyggnéee , (.EQXH l OOH OOH C-C—C-C-C-C-1C%=C-C=(93-R-COOH C—C-C=C-C-C-g=C-C=C93-R-COOH 13-hydroperoxy linoleic acid (13-HPOD) 13-hydroperoxy linolenic acid (13-HPOT) WW“ lfi-Iydroperoxide lyase (HPLfll LL95» Pentenols O O c—c-c-c-c-c' c-c-c=c-c-5 Hexanal cis-3-Hexenal lADH; l 9H : l ,. C-C-C—C-C-C C-C-C=C-C-C 1-Hexanol cis-3-Hexenol Hexyl esters Hexenyl esters? Figure 3. Pathway for catabolism of lipids and fatty acids via lipoxygenase. In plants, lipoxygenase activity are found to be in chloroplasts (Hatanaka, 1993). Linoleic (18:2) or Iinolenic (18:3) acids are formed by lipase. Lipoxygenase peroxidizes linoleic and linolenic acid to 13-hydroperoxy linoleic acid (13-HPOD) and 13-hydroperoxy linolenic acid (13-HPOT), respectively. Hydroperoxide lyase cleaves 13-HPOD and 13-HPOT into hexanal and cis-3-hexenal, respectively. Hexanal and cis-3-hexenal is reduced by alcohol dehydrogenase to hexanol and cis-3-hexenol respectively. Hydrogens in the carbon-hydrogen bonds are not shown. 50 sausagemza 10-030000 IIY 22$ 0:03.3550 .:0=000: :000 0:00 :0500: :0900 05 0. 228 28:93:53-0 000.05 028.0 0:0 2000 026:0 00: 0:0 00:00 500.03-50.00 05 :_ 0:00.902: 9:03 :_ .0: 50 0:00.00: :_ 0:00: 0. 0:00. :05 09020:. .. 0.0055005 m0m0:0m0:0>:00 6:023 n__0 > > .0000 :_0:0-00:0:05 :_ 0023:. 00.3.5020 0..00 0- 0 <00- m. NWo-o <00. .3. 50.20 902.500 30:: 0:00 000:6:0E00 0. New ~00... 0. 0:0 60:: 00:000: 0505.: .0 059“. 000.080.0000 0800.00.00 OE :00 0.0090070 0.3 .. m .12 000:0_0>_>£0E-m.0.0v.-0 IOOO-O 00 O O A V 1000- O- 00 D 0 05000.00. a 0%. M000:000:0=0:_E0 :03 O O - 0a... 1 $0000.38 0.00 0833.3 5 3.04.2 $0000000:-0L0E00_ 0.00 08.032003 Iao00::om_-m 90.05.3908. 00.08.3058.- N nzu 0.0.0.0., 93..a ICOO-00.0 0.023 0.00..0 ~00 IOOO- n.0- O O am- F 0000 .0000L n_.0 6 IQ N 10 F ... Ann-1000-00-00 AIIAIAI 1000. 0-0-0 A.- . 1000000 A--. zoom-Wad A1". . 109.0 1000 .. w In. 0.0029.- -0 (Coum-O-O "“50 m A ‘XA/ IWMUIH m0m0£E000 05:005.; 0 i 4 N00 30.28... 0 O\ __ __ :0_Qo._n_ IO Fatty acid biosynthesis (2C elongation) II II II AcetyI-CoA C-C-S-COA + HO-C—C-g-S-ACP Malonyl-ACP /i 3-ketoacyl-ACP synthase Ill C02 + CoA-SH II II C—C-C—g—S-ACP acetoacetyl-ACP NADPH, H+ .. ._...- _._._ >lL3-ketoacyl-ACP reductase NADP+ 9“ Cu) C-C-C-g-S-ACP 3-Hydr0xybutyryI-ACP H 0 /i 3-hydroxyacyl-ACP dehydrase Cu) C-g=C-g-S-ACP trans-2-buten0yl-ACP NADPH, H+ >1 Enoyl-ACP reductase NADP+ O ll Butyryl-ACP C-C-C-g-S-ACP + MalonyI-ACP r3-ketoacyl-ACP synthase I I OB—ketoacyI-ACP synthase II J t 0 ll R-C-C-C-C-S-ACP H20 >1 AcyI-ACP thioesterase ACP O t 0 II R-C-C-C-C-OH Fatty acid Figure 5. Pathway for fatty acid biosynthesis through two-carbon chain elongation in plants. Acetyl-ACP (primer) and malonyI-ACP (chain extender) is condensed by 3-ketoacyl-ACP synthase Ill. The next 3 steps are reduction by 3- ketoacyl-ACP reductase, dehydration by 3-hydroxyacyl-ACP dehydrase, and reduction by enoyl-ACP reductase. The cycle repeats to condense with malonyl- ACP until the chain length is 16-18, in general. The final step is terminated by acyl-ACP thioesterase by hydrolysis. Stars indicate the carbon position during each reaction. Hydrogens in the carbon-hydrogen bonds are not shown. 52 or-keto acid elongation (aKAE) Pyruvate Acetyl-CoA c-c-COOH + *cf’c-s-CoA O 1 2-isopropylmalate synthase COOH I 0 c-cfb-COOH OH 2 l Isopropylmalate dehydratase COOH I :- 0 C-C-C-COOH OH 3 3-lsopropylmalate dehydrogenase CO2 0 Propionyl-CoA CO2 t V * ’3’ . . C-C-CIE-S-COA 4» -- C-C-E-COOH a-Keto butyrrc acrd O a-ketoacid DH 0 ¥ Repeat steps 1-3 ButyryI-CoA 002 t 0 V t r} . . C-C-C-C-S-CoA <- .- C-C-C-C-COOH a-Ketovalencacud C") a-ketoacid DH C") * Repeat steps 1-3 Kroumova and Wager, 2003 Figure 6. Pathway for fatty acid biosynthesis through single-carbon chain elongation (d-keto acid elongation, oKAE) in plants. Pyruvate (primer) and acetyl- CoA (chain extender) is condensed by 2-isopropylmalate synthase. The next 2 steps are isomerization and dehydration by isopropylmalate dehydratase, and decarboxylation by 3-isopropylmalate dehydrogenase. The cycle repeats to condense with acetyl-CoA or terminated to produce acyl-CoA. Stars indicate the carbon position during each reaction. Hydrogens in the carbon-hydrogen bonds are not shown. 53 CHAPTER III CHARACTERIZATION OF VOLATILE ESTER BIOSYNTHESIS BY ‘JONAGOLD’ APPLES DURING THE RIPENING PROCESS 54 INTRODUCTION Aroma is a flavor component with an important role in apple fruit quality. Of the aroma active compounds, esters are the predominant compounds produced by horticultural crops such as apple, banana, melon, and strawberry. The ratio and type of esters vary by fruit species and even among cultivars within species (Kakiuchi et al., 1986). Some aroma compounds increase intensity of ‘sweet flavor’ and can affect the perception of sweetness in fruits (Young et al., 1996) while others add additional flavor notes (Williams et al., 1977). Characteristic fruit volatiles are produced via three routes: from autonomous production, from heat during cooking, and from tissue disruption 2 (Beaudry, 2000). Some compounds are produced by more than one method, but to a significant extent these pathways provide distinct volatile aroma profiles. Fresh apples and prepared apple products (e.g. fresh apple slices, apple sauce, cooked apple products, apple cider) emit over 200 volatile compounds (Dimick and Hoskin, 1983). Fresh apples autonomously produce an abundance of hexyl acetate, butyl acetate, and 2-methylbutyl acetate during ripening. These esters are considered to confer typical apple aroma characteristics (Paillard, 1990). Despite the existence of ‘typical’ apple notes, apple cultivars have differing volatile profiles (Fellman et al., 1993; Guadagni et al., 1971; Kakiuchi et al., 1986; Mattheis et al., 1998; Song and Bangerth, 1996). For example, ‘Golden Delicious’ mostly produces acetate esters, ’Starking’ produces a large amount of propanoate esters, and ‘Richared’ and ‘Canada blanc’ produce elevated levels of butanoate esters (Paillard, 1990). As the ripening stage changes from 55 preclimacteric t0 postclimacteric, the amounts of individual compounds also change over time, altering the ester composition (Ferenczi, 2003; Mattheis et al., 1991b) Esters in apples are comprised of an alkyl (alcohol-derived) group and an alkanoate (acid-derived) group. The alkanoate group is derived from a CoA thioester of a short- to medium-chain length fatty acid. The acyl-CoA and the alcohol are joined by an enzyme called alcohol acyltransferase (AAT) to form the ester (Figure 7). The carbon chain length of alcohol substrates for AAT normally ranges between 2-6 carbons whereas acyl-CoA substrates typically have 2-8 carbons (Ferenczi, 2003). Several hypotheses have been suggested to propose pathways by which ester precursors are formed. Straight-chain ester precursors may be formed from fatty acids by a combination of B—oxidation and lipoxygenase activities (Drawert, 1975; Sanz et al., 1997; Yahia, 1994). Branched-chain ester precursors may be formed from the metabolism of branched-chain amino acids (BCAA) (Pérez et al., 2002; Rowan et al., 1996, 1998; Tressl and Drawert, 1973). In apple, the primary branched-chain esters have 2-methylbutyl alkanoate and/0r alkyl groups. These branched-chain groups are likely derived from isoleucine metabolism. A small amount of 2-methylpropyl esters are also found in apple, apparent products of valine metabolism. However, 3-methylbutyl esters which would be expected from leucine metabolism are typically absent. 3-Methylbutyl esters are abundantly produced in banana and provide this fruit its characteristic aroma (Jayanty et al., 2002). 56 Apple ripening is regulated by ethylene and ester biosynthesis begins as ethylene production commences (Biale, 1964; McGlasson, 1970). In addition to aroma volatile formation, ethylene induces several physiological changes including anthocyanin accumulation, chlorophyll loss, a rise in respiration, and cell wall degradation. These changes were used to measure the ripening stages of the fruit. The objective of this research was to investigate the pattern of ester biosynthesis as it relates temporally to physiological changes of ‘Jonagold’ apple during ripening and senescence. This study served to establish a physiological foundation for further genomic characterization. MATERIALS AND METHODS In order to temporally link the physiological changes of ‘Jonagold’ apple to the pattern of ester biosynthesis during ripening and senescence, ethylene, C02 production, texture, color, °Brix, firmness, and starch index were used to characterize the ripening stages of the fruit during an 81-day period. Data collection began well before the onset of ripening and continued until fruit were fully senescent. Plant Material ‘Jonagold’ apples were harvested from the Michigan State University Clarksville Horticultural Experiment Station, Clarksville, MI. From September 2, 2004 (Day 0) until ripening was fully engaged on October 7, 2004 (Day 35), fruits were collected for examination every three to four days from the field. On each occasion, fruit were held overnight in the laboratory to equilibrate to laboratory 57 temperature (211:1 °C) and covered with ventilated, black, 4-mil thick plastic bags to avoid desiccation and responses to intermittent laboratory light before analysis. All fruits (approximately 200) remaining on the tree were harvested and transported to the laboratory at once on October 7, 2004 (harvest date, Day 35) after it was apparent that ripening was underway. This was done to avoid damage in the field due to freezing and fruit drop. Thereafter, fruit were maintained at room temperature (211:1 °C) and covered with plastic bags as described previously. The fruit were examined every three to four days from harvest (October 7, 2004) until the conclusion of the study on November 23, 2004 (Day 81). On each evaluation date, 20 apples were randomly chosen and the internal ethylene content of each was measured. Of these, the fourteen fruit having an internal ethylene content nearest the median were selected for further analysis. The four fruit having ethylene levels closest to the median were used for analysis of CO2 production, ester emission, and textural properties. The remaining ten fruit were used to obtain skin color (percentage of redness), background, starch, °Brix measurements, and gene expression (Chapter IV). Volatile analysis To collect volatiles, apples were held at 20°C in a 1-L Teflon container (Savillex Corporation, Minnetonka, MN) fitted with two gas-sampling ports, each sealed with a Teflon-lined half-hole septum (Supelco Co., Bellefonte, PA). Headspace volatiles were sampled after 20 minutes using a 1-cm long solid- phase micro extraction (SPME) fiber (65 pm PDMS-DVB, Supelco Co., 58 Bellefonte, PA). SPME sorption time was 3 min. The exposed fibers were immediately transferred to a gas chromatograph (GC) (HP-6890, Hewlett Packard 00., Wilmington, DE) injection port (230°C) and desorbed for 2 min. Desorbed volatiles were trapped on-column using a liquid nitrogen cryofocussing trap. Separation of volatiles was by capillary column (SupelcoWax-10, Supelco, Bellefonte, PA, 29 m x 0.2 mm id, 0.2 pm coating film). The temperature of the G0 was programmed from 40 to 240°C at a rate of 50°C/min; the flow rate of the helium carrier gas was 1 mL/min and the G0 was operated in splitless mode. Detection was by time-of-flight mass spectrometry (Pegasus ll, LECO Corp., St. Joseph, MI) (GC/MS) according to the method of Song et al. (1997). Identification and quantification of compounds were by comparison of the mass spectrum and MS response (total ion count, TIC), respectively, with those of authenticated reference standards and spectra in the National Institute for Standard and Technology (NIST) mass spectra library (Search Version 1.5). Measurement of respiration To measure respiration, CO2 accumulation in the 1-L Teflon container was measured on 0.1-mL gas samples withdrawn from the sampling port using an insulin-type plastic syringe. CO2 was sampled at the same time volatiles were measured, following the 20-minute holding period. The gas sample was injected into an infrared gas analyzer (model 225-MK3; Analytical Development Co., Hoddesdon, England) operated in a flow-through mode with N2 as the carrier gas and a flow rate of 100 mL-min". The CO2 concentration was calculated relative to 59 a certified standard (Matheson Gas Products Inc., Montgomeryville, PA) containing 0.979 pL-L'1 ethylene, 4.85% CO2, and 1.95% 02 balanced with N2. Measurement of ethylene production The internal ethylene content of apple fruit was determined by withdrawing a 1-mL gas sample from the interior of the apples and subjecting the gas sample to gas chromatographic analysis (Carle Series 400 AGC; Hach Company, Loveland, 00) as previously described (Mir et al., 2001). The GC was fitted with a 6-m-Iong, 2-mm-i.d. stainless-steel column packed with activated alumina and was equipped with a flame ionization detector. The ethylene detection limit was approximately 0.005 pL-L'I. Ethylene concentrations were calculated relative to the certified standard noted previously. Texture analysis Texture was analyzed as the force (N) required to bring about tissue failure under both compressive and tensile strain using a texture analyzer (TA.XT2i, Texture Technologies, Scarsdale, NY). The signal was analyzed by commercial software (Texture Expert Exceed, version 2.60, Texture Technologies, Scarsdale, NY) (Figure 8). Tensile failure was measured on bars of cortex tissue cut from apple fruit using a 3-point bending rig (TA-92, 88.9 mm x 101.6 mm, the distance between the two supports was 28.57 mm). The tissue bars were square in cross-section (9 mm x 9 mm) and approximately 7cm in length and made using a F rench-fry maker (GPO-2549, Progressive International Corp., Kent, WA). To make the bars, apple fruit were trimmed to fit into the device and placed so that the tissue bars were cut parallel to the axis of the fruit. 60 The bars of tissue were placed on the bending rig such that the force applied was perpendicular to the fruit axis. The descending probe advanced at a rate of 2 mm/second and the maximum force encountered was recorded. Compressive failure was tested on cylinders of cortex tissue taken from portions of the fruits not used for tensile failure samples. The cylinders were made using a cork borer with an internal diameter of 16.5 mm. The cylinder was removed from the equator of the fruit, normal to the fruit axis. The skin was removed and the core end was trimmed away to yield a cylinder 2 cm in length. Compressive failure of the tissue was tested by placing the cylinder between two flat plates on the texture analyzer (Figure 9). The probe speed was 2 mm/second. When the probe touched the fruit, force (N), distance (mm), and speed (s) were recorded. The instrument continued to apply force until the probe had traveled 8 mm from the point initial contact with the fruit. The maximum force encountered was recorded. RESULTS Skin color (percent of redness) increased from 22% on Day 0 to over 95 % by Day 39 (data not shown). Background color (green=5, yellow=1) had a reciprocal pattern to red color development, beginning at 5 (green) on Day 0 and gradually decreasing to 1 (yellow) on Day 81 (data not shown). Starch conversion to sugars, as measured by the starch index (1 - 8) started at 2 on Day 0 and continued to increase, reaching a maximum of 8 on Day 32 (data not shown). The pattern for soluble solids was similar to that of starch conversion; 61 the initial soluble solid was 12 °Brix on Day 0 and reached its maximum of 16 °Brix on Day 39 (data not shown). Ethylene production remained low until Day 18 (Figure 10). Day 21 was considered to be the onset of the ethylene climacteric when the level rose above 0.2 pL-L". Esters were first detected at very low levels as early as Day 14, a week before the onset of a sustained increase in ethylene. Immediately after the ethylene increase, a rapid and large increase in ester biosynthesis began. On Day 32, ester biosynthesis was approximately half-maximal and the respiratory climacteric was engaged. On Day 39, ester biosynthesis approached its maximum, respiratory activity peaked, being twice as great as on Day 32, and rapid tissue softening began (Figure 11). The most abundant esters on Day 42, when the highest total ester response was recorded, were hexyl acetate, 2- methylbutyl acetate, butyl acetate, and hexyl 2-methylbutanoate; the complexity of the ester profile was at its maximum at this point (Figure 12). Near-maximal ester biosynthesis continued until Day 49 when the respiratory climacteric reached its conclusion and tissue softening was completed. On Day 60, the internal ethylene content reached its maximum (approximately 690 pL-L'I) and the decline in ester biosynthesis was approximately at its midpoint. By Day 70, fruit were highly senescent and esters reached a postclimacteric minimum. Data collection ceased on Day 81. A total of 39 volatile compounds, including 31 esters, 5 alcohols, and 3 aldehydes were detected and evaluated from early pre- climacteric through late post-climacteric stages of development (Table 2). Alcohol patterns 62 Alcohols were detected only after the onset of the ethylene climacteric (Day 21). Alcohols detected included ethanol, propanol, butanol, hexanol, and 2- methylbutanol (Figure 13). 2-Methylpropanol, pentanol, heptanol, and octanol were not detected. Hexanol was at its maximum relative abundance in the early ripening stages and butanol, propanol, and ethanol increased in the later developmental stages. Ethanol did not appear until Day 53 and contributed only a small amount relative to other alcohols in total pr0portion. Butanol appeared on Day 25, and predominated in total proportion from Day 42 until senescence. 2- Methylbutanol was detected as early as Day 21 and had a high peak on Day 39, increasing 5- to 7-fold immediately after the final harvest date (October 7, 2004, Day 35), and declined sharply thereafter. Ester patterns based on the alkyl moiety Abundant esters included those with butyl, hexyl, 2-methylbutyl, and propyl alkyl groups; pentyl and ethyl esters were less abundant and heptyl and octyl esters were not detected (Table 2). The emission of esters of the above alcohols increased sharply after the onset of the ethylene climacteric (Figure 14). The patterns for the esters reflected the patterns of their corresponding alcohols (Figures 13-20); hexyl esters were most abundant in the early ripening stages and butyl, propyl, and ethyl esters rose in the later stages (Figures 14-19). Total ion count (TIC) response was greatest for acetate esters (Figures 15-20). Butyl esters were the most diverse with the butyl alkyl group being produced in combination with 2, 3, 4, 5, 6, 7, and 8 carbon straight-chain alkanoate groups 63 and 2-methylbutanoate (Table 2). Several pentyl esters were detected but no pentanol was found. Ester patterns based on the alkanoate moiety Esters detected classed by the alkanoate moiety included: acetate, propanoate, butanoate, 2-methylbutanoate, hexanoate, and octanoate esters (Table 2). All esters rapidly increased after the onset of the ethylene climacteric (Figures 21-27). However, only acetate and propanoate esters were maintained at high levels during senescence, while butanoate, hexanoate, octanoate, and 2- methylbutanoate esters decreased after the climacteric peak on Day 42. Acetate esters had the greatest diversity of alkyl groups (Table 2). For each of the alkanoate ester classes, hexanol-derived alkyl groups predominated early in ripening and consistently declined in later development stages relative to other ester classes. Pentanoate and heptanoate esters were only produced with butanol-derived alkyl groups. No 2-methylpr0panoate esters were detected. DISCUSSION The essentially concurrent events of autocatalytic ethylene production and ester synthesis in ‘Jonagold’ was similar to that previously described for ‘Bisbee Delicious’ (Mattheis et al., 1991b), ‘Golden Delicious’ (Song and Bangerth, 1996), and ‘Redchief Delicious’ apples (Ferenczi, 2003). This is consistent with previous studies demonstrating that ester production requires ethylene action (Defilippi et al., 2004; Ferenczi et al., 2006). Once climacteric ethylene production was 64 engaged, the internal ethylene content did not decrease during senescence as previously observed in ‘Redchief Delicious’ apple (Ferenczi, 2003). The predominant emission of acetate esters by ‘Jonagold’ fruit can permit this cultivar to be categorized as an ‘acetate-producing’ type (Paillard, 1990). The four major esters: hexyl acetate, 2-methylbutyl acetate, butyl acetate, and hexyl 2-methylbutanoate found in abundance at the climacteric peak (Day 42) in this study were similarly abundant at the climacteric peak for ‘Redchief Delicious’ apple (Ferenczi, 2003), although the proportion of these four major esters differed between the two cultivars. The high amount of butyl and hexyl esters are a characteristic of the ‘Jonagold’ aroma profile (Dixon and Hewett, 2000). Ethanol-derived esters such as ethyl acetate, ethyl butanoate, and ethyl 2- methylbutanoate, which contribute significantly to ‘Delicious’ or ‘Bisbee Delicious’ apple aroma (Guadagni et al., 1971; Mattheis et al., 1991a, 1991b), were only found during postclimacteric stage and the production was quite low relative to other esters. 2-Methylbutanoate esters, which have a fruity and sweet aroma (Plotto et al., 2000), increased during the climacteric peak. In addition to the esters described, 4-methoxyallylbenzene, which contributes a spicy aroma to apples (Williams et al., 1977) was also detected in the ‘Jonagold’ (data not shown). Other common apple odorants including 3-penten-2-ol (apple-like aroma) found in ‘Starkspur Golden' fruit (Vanoli et al., 1995), or B-damascenone (fruity odor) found in ‘Cox Orange’ and ‘Elstar’ (F uhrmann and Grosch, 2002), were not detected in ’Jonagold’. Despite the abundance of alcohol precursors, free acids were not detected or were tentatively detected below threshold levels, 65 which is in contrast to data for ‘Redchief Delicious’ apple in which acetic acid, propanoic acid, 2-methylbutanoic acid, butanoic acid, and hexanoic acid were found (Ferenczi, 2003). ‘Jonagold’ apple is the result of a cross between ‘Jonathan’ and ‘Golden Delicious’ (Gianfranceschi et al., 1998). The ratios and abundance of butyl, 2- methylbutyl, and hexyl acetate esters in the ‘Jonagold’ may be a function of genetic descendance from the ‘Jonathan’ variety, which produced profiles similar to ‘Jonagold’ in butyl, 2-methylbutyl, and hexyl acetate esters (Kakiuchi et al., 1986) As noted previously, the final step for ester formation is catalyzed by AAT. Substrate preferences for AAT isozymes have been characterized (Olias et al., 2002; Ueda et al., 1992). Souleyre et al. (2005) cloned MpAAT1 from ‘Gala’ apple and determined the substrate preference for the transcribed protein. The rate of acetate ester formation by MpAAT1 depended on substrate alcohol concentration; at low alcohol substrate concentrations, the preference order was 2-methylbutanol>hexanol>butanol. At high alcohol substrate concentrations, the preference order was hexanol>2-methylbutanol>butanol. In the current study, for the acetate esters in ‘Jonagold’, the abundance of the various alcohol classes seemed to reflect the abundance and availability of corresponding alcohols. As a result, it was not clear whether MpAAT1 exerted any influence on the profile due to its substrate preferences. Despite the abundance of 2-methylbutanol, only 2- methylbutyl acetate and 2-methylbutyl butanoate esters were produced. In contrast, 2-methylbutanoate esters were formed with five different alkyl groups 66 from ethyl, propyl, butyl, pentyl, and hexyl alcohol. Interestingly, no 2-methylbutyl 2-methylbutanoate was detected, despite the high levels of 2-methylbutyl and 2- methylbutanoate esters. Furthermore, the production (TIC) of hexyl esters was greater than 2-methylbutyl esters even though the amount of hexanol detected was less than 2-methylbutanol. In apples, only MpAA T1 (Souleyre et al., 2005) and MpAA T2 (Li et al., 2006) have been isolated, but other AAT genes exist. In the Tree Fruit Technology genomic analysis tool apple database version 4.0 (http://genomicsmsu.edu/fruitdb/analyses/apple.shtml), there are 13 clusters tentatively identified as AAT. The observed diversity of ester formation in the present study might be explained by the involvement of several AAT isozymes that differ in substrate preferences (Holland et al., 2005; Ueda et al., 1992). The presence of butyl, hexyl, and 2-methylbutyl esters at low levels before the onset of ethylene production and the lack of detectable quantities of corresponding alcohols or acids suggest that isozymes of AAT were functional before the onset of the ethylene climacteric, but that precursors were low or limiting (Jayanty et al., 2002). This is in contrast to the suggestion that the AAT gene is fully under ethylene control (Defilippi et al., 2005a). Preclimacteric AAT activity has also been demonstrated for ‘Redchief Delicious’ apple (Ferenczi et al., 2006). Similarly in melon, the expression of CM-AA T1 gene was not completely suppressed under the ethylene-suppressed antisense ACC oxidase melon fruit and fruit treated with 1-MCP (an inhibitor of ethylene action), although the expression was severely reduced in both lines (Yahyaoui et al., 2002). 67 Collectively, the data suggests that AAT activity may be highly regulated developmentally, having both constitutive and ethylene-driven components. The rapid increase in branched-chain esters at the climacteric peak is indicative of elevated enzyme activity in the pathways for the formation of branched-chain ester precursors (Defilippi et al., 2005b; Nie et al., 2005). d-Keto- B-methylvalerate, which is a product of isoleucine degradation and a precursor in isoleucine formation, is believed to produce either 2-methylbuyI-C0A by branched-chain a-ketoacid dehydrogenase (BCKDH) or 2-methylbutanal by branched-chain a-ketoacid decarboxylase (BCKDC). 2-Methylbutanal is likely further converted to 2-methylbutanol by alcohol dehydrogenase (Wyllie et al., 1996). The fact that ‘Jonagold’ can produce both 2-methylbutanoate and 2- methylbutanol esters suggests that the apple utilizes BCKDH and BCKDC pathways where the branched-chain d-keto acid is either converted to acids or alcohols, respectively. This is in contrast to mammals, which can only catabolize branched-chain o-keto acid to form branched-chain acyl-CoAs (Platell et al., 2000). Lipoxygenase activity may supply the saturated C6 alcohol, hexanol, for ester biosynthesis (Rowan et al., 1999). This supposition is supported by the finding that lipoxygenase activity in apple is highly specific in peroxidizing linoleic acid to 13-hydroperoxy derivatives (Kim and Grosch, 1979). B-Oxidation can theoretically form short- to medium-chain C4, C6, C8, and C10 fatty acids from long-chain fatty acids. However, the longest chain esters detected were octanoate esters. Feeding studies suggest that both B-oxidation and 68 lipoxygenase pathway may play a major role in the abundance of even-chain number esters (Rowan et al., 1999). However, increase in the odd-number C3 alkyl and alkanoate groups in the later ripening stages is not supportive of the involvement of either lipoxygenase or B-oxidation, which do not easily allow an explanation for their biosynthesis. Further, the trends of decreasing C6 alkyl groups and increasing C3 alkyl and alkanoate groups, may be indicative of the involvement of an as-yet undescribed pathway. While 05 alcohols may be supplied from lipoxygenase, they have only been observed under anaerobic conditions (Salch et al., 1995). As for C7 esters, neither pathway explains their production easily without invoking o-oxidation. Since ester biosynthesis can not be readily explained by only fatty acid break down, it is possible that the biosynthetic pathways are involved. Likely, the complex profile of esters produced by ripening apple fruit is the product of both synthetic and catabolic reactions. The recently characterized fatty acid synthetic pathway involving single carbon elongation (Kroumova and Wagner, 2003) may, for instance, be responsible for synthesizing odd-number carbon ester substrates and may also supplement even-number carbon substrates. This pathway may also impact the branched-chain esters, especially those thought to be derived from isoleucine. One of the products of the single- carbon fatty acid synthetic pathway is a-keto butyrate, a precursor in the formation of o-keto-B-methylvalerate and, ultimately, isoleucine. The majority of the feeding studies suggest that ester biosynthesis is from fatty acid and amino acid catabolism pathways. Interestingly, however, no 69 published studies have been found exploring the idea of synthetic or anabolic precursor formation. So far, few of the genes or proteins thought to supply the substrates of alcohols and acyl-CoAs in these pathways have been characterized relative to ester biosynthesis in fruit. Only the final step of ester formation involving AAT has been studied at protein and molecular levels in ester- producing fruits. CONCLUSION Physiological changes during fruit ripening were similar to those found for previous studies of apple. The abundance in butyl acetate, 2-methylbutyl acetate, hexyl acetate, and hexyl 2-methylbutanoate were characteristic for ‘Jonagold’ during the climacteric peak. Ethyl ester production did not significantly contribute to the volatile profile of this variety. Esters possessing long-chain alkyl and alkanoate moieties were most prevalent relative to those with shorter-chain groups at earlier ripening stages. Developmentally-dependent changes in alcohol abundance were similar to those of their corresponding esters suggesting that alcohol precursor availability exerts a major influence in ester biosynthesis. Presence of esters before the onset of ethylene suggests that the AAT is active before ripening begins, but precursors are limiting. Not all the reciprocal combinations of alcohols and acids were found in ‘Jonagold’, suggesting that several AAT isozymes with differing substrate preferences may be active. 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Takamura, and H.W. Gardner. 1995. Characterization of a C-5,13-cleaving enzyme of 13(S)-hydroperoxide of linolenic acid by soybean seed. Plant Physiol. 108(3):1211-1218. Sanz, C., J.M. Olias, and AG. Pérez. 1997. Aroma biochemistry of fruits and vegetables, p. 125-155. In: F.A. Tomas-Barberan and R.J. Robins (eds). Phytochemistry of fruit and vegetables. Oxford University Press, New York. Song, J. and F. Bangerth. 1996. The effect of harvest date on aroma compound production from 'Golden Delicious' apple fruit and relationship to respiration and ethylene production. Postharvest Biol. Technol. 8(4):259- 269. Song, J., B.D. Gardner, J.F. Holland, and RM. Beaudry. 1997. Rapid analysis of volatile flavor compounds in apple fruit using SPME and GC/time-of-flight mass spectrometry. J. Agric. Food Chem. 45(5):1801-1807. Souleyre, E.J.F., D.R. Greenwood, E.N. Friel, S. Karunairetnam, and RD. Newcomb. 2005. An alcohol acyl transferase from apple (cv. Royal Gala), MpAAT1, produces esters involved in apple fruit flavor. FEBS J. 272(12):3132-3144. Tressl, R. and F. Drawert. 1973. Biogenesis of banana volatiles. J. Agric. Food Chem. 21(4):560-565. Ueda, Y., A. Tsuda, J.H. Bai, N. Fujishita, and K. Chachin. 1992. Characteristic pattern of aroma ester formation from banana, melon, and strawberry with reference to the substrate specificity of ester synthetase and alcohol contents in pulp. J. Jpn. Soc. Food Sci. Technol. 39:183-187. Vanoli, M., C. Visai, and A. Rizzolo. 1995. The influence of harvest date on the volatile composition of 'Starkspur Golden' apples. Postharvest Biol. Technol. 6(3-4):225-234. Williams, AA, 00. Tucknott, and M.J. Lewis. 1977. 4-methoxyallylbenzene: an important aroma component of apples. J. Sci. Food Agric. 28(2):185-190. Wyllie, S.G., D.N. Leach, H.N. Nonhebel, and l. Lusunzi. 1996. Biochemical pathways for the formation of esters in ripening fruit, p. 52-57. In: A.J. Taylor and DS. Mottram (eds). Flavour science, recent developments. Royal Society of Chemistry, Cambridge, UK. Yahia, EM. 1994. Apple flavor. Hort. Rev. 16:197-234. 75 Yahyaoui, F.E., C. Wongs-Aree, A. Latché, R. Hackett, D. Grierson, and J.C. Pech. 2002. Molecular and biochemical characteristics of a gene encoding an alcohol acyl-transferase involved in the generation of aroma volatile esters during melon ripening. Eur. J. Biochem. 269(9):2359-2366. Young, H., J.M. Gilbert, S.H. Murray, and RD. Ball. 1996. Causal effects of aroma compounds on Royal Gala apple flavours. J. Sci. Food Agric. 71 (3):329-336. 76 _ .m:0:..E :_ 05:03. :2: :0 00005 0:0:00E:0:00: 0: 000508.005 9.50 00:00:00 A05 00:800.. 0200 £38000: 0:: 0:00.05 0.0::52 0005.00. 00 0:0.:0:.:E00 5:80 0:0 0.00 0:: :05 00:00.05 - :2: 05:0 :0: 0.005005 .3020 0:0 0.00 3: 00N.:0m:0 00:00:00 90:00 :0 x305. .N 030-: I- I. 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Compressive failure was tested on cylinders of cortex tissue placed on the flat plate and compressed by a descending flat-faced probe. The cylinders were made with an internal diameter of 16.5 mm, trimmed to a length of 2 cm, and taken from tissue just beneath the skin. The cylinder was removed from the equator of the fruit, normal to the fruit axis. The force was applied perpendicular to the fruit axis and the maximum force encountered during the test was recorded. 80 3.0e+9 - Total volatiles (TIC) 2.0e+9 - 1.0e+9 1 + Total volatiles —O— Ethylene —A— C02 ---——.-—-— Harvest date a' — 10000 ~ 100 - 1000 80 r: .C it o: A ‘é 1003‘ ~ 60 o: 2. E, V w 8 10 5 ”0'8 3.. :3 s 8 IJJ h. 1 —20 o. N O O 0.1 ~ 0 - 0.01 Figure 10. Ontogeny of total volatiles, ethylene and respiration (CO; production) during ripening and senescence of ‘Jonagold’ apples. The apple fruit were examined from Sept 2, 2004 (Day 0) to Nov. 23, 2004 (Day 81). Fruits were harvested every 3-4 days from the field until Oct. 7, 2004 (Day 35), and thereafter maintained at room temperature (21:I:1°C). Each symbol represents the average of four replications for total volatiles and respiration, and ten replications for ethylene. Vertical bars represent mean 1 SD. 81 15 + Total volatiles P335409 2 ‘ A A Bending a, 10 ‘ ‘ A i 0 Compression E t‘tatt o u- ; ‘ t: .2: 5 c g -2.05+09 .g 0 O 3 I- 'D A o rm . . . _ 9 £120: 0. 3 0 a = e . a 03 g 5 3 30.1 . Q -1.05+09 o d? ‘ > ..>. « 3 (D 1 O 3 404 J g! t. g‘ ‘ l- l- .i . a , . 2 g . o o I ' U T Fr I V U ' T T I' I j r1 I V T V r ffi 1b0IOE+oo . . . , 0 1O 20 30 t 40 50 60 70 80 90 Day Harvest date Figure 11. Ontogeny of total volatiles and textural changes during ripening and senescence of ‘Jonagold’ apples. Tensile failure/bending force was to measure ‘crispness’ and compressive force for ‘softening’. The apple fruit was examined from Sept 2, 2004 (Day 0) to Nov. 23, 2004 (Day 81). Fruits were harvested every 3-4 days from the field until Oct. 7, 2004 (Day 35), and thereafter maintained at room temperature (211:1°C). The samples were taken opposite sides of each fruit and each symbol represents four fruit replications. 82 3 s o 3 b 8 2 2 .8 2 3 2.4E+07- g E i g g ‘5 i’ is d _ E E a «<1 .-. A 2.05+07- m g 2 . =3 l: m 1.6E-I-07- m C . o % 12E+07- 2 o . 8 ,0 8.09067 3 . m E 4.0E+06q 0.0E+OO- . - . L i. 1‘20 140 160 180 200 220 240 260 280 300 Seconds Figure 12. Representative gas chromatograph of the headspace of ‘Jonagold’ apples at the respiratory climacteric on Day 42. Predominant esters were butyl acetate, 2-methylbutyl acetate, hexyl acetate, and hexyl 2—methylbutanoate. A total of 31 esters, 3 aldehydes, and 5 alcohols were detectable at this point in development. 83 Figure 13. Patterns of alcohol emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of ethanol, propanol, butanol, hexanol, and 2-methylbutanol. 8. Alcohol proportions (% of total alcohols). Each symbol represents the average of four replications. 84 8.0e+7 -<>- Ethanol A --D-- Propanol —O— Butanol 8 -A- Hexanol F 6.0e-I-7 - + 2-Mbutanol C .2 fl 3 ; - '8 4.0e+7 - Harvest date ———> . . h I 3 o . . . . ’ . o O .C 2.; 2.0e+7 - z < . I I A A A 0.0 m-‘II-‘3'Eooooooooooo 1,7 100 .0 —0— Ethanol B :0: —D— Propanol 2 -O— Butanol g 80 1 -A— Hexanol E + 2—Mbutanol 2 A “5 60 - a". . rn A I 0 o o . C o . . o .2 40- . - t' O h a 20 ‘i ' A ‘ I I I .2 5 I A ‘ “A o i ' ‘ ‘2', 0 1 . '9‘!‘ 90900099999 0 1O 20 30 4O 50 60 70 80 90 Day Figure 13. 85 Figure 14. Patterns of alcohol ester emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of propanol, propyl esters, butanol, butyl esters, hexanol, hexyl esters, 2-methylbutanol, and 2-methylbutyl esters. 8. Alcohol and alcohol ester proportions (% of total alcohols or alcohol esters). Each symbol represents the average of four replications. 86 8.0e+8 —I— Propanol .\ A ° 0 A g —D— Propyl esters ‘ A 3;; + Butanol . g —0— Butyl esters '8 6.0e+8 - -L- Hexanol ° ‘ o ’ h —A— Hexyl esters 0 o. + 2-Mbutanol , ' ' .23.. -<>— 2-Mbutyl esters ‘ o m ; . A 2 a 4.0e+8 - Harvest date -—9§ ° 0 - ‘ i g t g N '0 J C 2.0e+8 (U '5 .C 8 2 0.0 """"" 100 g A -l— Propanol B 0 g -D— Propyl esters :E ’6 —O— Butanol O 0 so . A -O— Butyl esters @- 3 + Hexanol n- .8 -A— Hexyl esters - o .9 Ta 60 ~ '0 h- A A 1’ ° . o e «c O O A 2 ‘5 ‘° N 2 '2 3 - ° ‘_' 503 20 J .2 a Q- 3 ° ' ' — \° < 2.. o a r O 10 20 Figure 14. 87 90 Figure 15. Patterns of ethanol ester emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of total ethanol esters and ontogeny of ethylene. B. GC/MS response (TIC) of total ethanol. C. GC/MS response (TIC) of ethyl acetate, ethyl propanoate, ethyl butanoate, and ethyl 2- methylbutanoate. D. Ethanol proportion (% of total alcohols). E. Ethanol ester proportions (% of total ethanol esters). Each symbol represents the average of four replications. Vertical bars represent mean :I: SD. 88 Total ethanol esters (11C) Ethanol esters (110) Ethanol ester proportions (%) 2.0e+7 1.5047 1 1 .Oe+7 -‘ 5.00+6 d 0.0 + Total Ethanol esters —O— Ethylene Harvest date —>: 10000 - 1000 ~100 F10 0.1 ‘ 0.01 3.0e+5 1 .59+5 . -i-— Ethanol 8.09+6 4 4.00+6 - 0.0 100 -O- Ethyl acet -D— Ethyl prop -V— Ethyl but —0— Ethyl 2—Mbut O I E O O I l I I 6 9‘ I l I o ’ I . . .~ (MO 00 o o o o 0 0,9,3. 1‘3. 3"::$ .v__v y__v_v_v__v V 80+ sol 40~ 201 o... + Ethanol 100 80 - 60 i 40 . 20~ -<>- Ethyl acet -CJ- Ethyl prop -V- Ethyl but —0— Ethyl 2-Mbut Figure 15. 89 Ethylene (pL/L) Figure 16. Patterns of propanol ester emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81 ). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of total propanol esters and ontogeny of ethylene. B. GC/MS response (TIC) of total propanol. C. GC/MS response (TIC) of propyl acetate, propyl propanoate, propyl butanoate, propyl 2- methylbutanoate, propyl hexanoate, and propyl octanoate. D. Propanol proportions (% of total alcohols). E. Propanol ester proportions (% of total propanol esters). Each symbol represents the average of four replications. Vertical bars represent mean i SD. 90 4.09+8 + Total Propanol esters ; -O— Ethylene ' 3.094-8 J 2.0e+8 ‘ 1 .Oe+8 ' r 1000 r 100 0.0 Total Propanol esters (TIC) 0.1 L 0.01 —-b— Propanol 8 E g -<)— Propyl aoet ’ a -O— Propyl prop : o 3 —V— Propyl but ; . . 9 . .0. 1.59+8 '1 —O— Propyl 2—Mbut o c -o— Propyl hex f ’ a + Propyl oct 3 o 9 1.0e+8 4 O. I 5.09+7 J . I ' ' I - :.:oov.°o.o . 5 ’Nxm"""" 0.0 ' “ ‘ ‘ 100 80 1 —+— Propanol 60 1 40 4 20 . 103 r i -<>— Propyl acet -Ci— Propyl prop 3° 1 —v— Propyl but —0— Propyl 2-Mbut + Propyl hex 60 "l + Propyloct Propanol ester proportions (%) 40a 20« __'-\ _ 34.-g... _ _ o . . A“ ‘3'}:7” o 10 20 Figure 16. 91 10000 Ethylene (pJJL) Figure 17. Patterns of butanol ester emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of total butanol esters and ontogeny of ethylene. B. GC/MS response (TIC) of total butanol. C. GC/MS response (TIC) of butyl acetate, butyl propanoate, butyl butanoate, butyl 2- methylbutanoate, butyl pentanoate, butyl hexanoate, butyl heptanoate, and butyl octanoate. D. Butanol proportions (% of total alcohols). E. Butanol ester proportions (°/o of total butanol esters). Each symbol represents the average of four replications. Vertical bars represent mean :t SD. 92 Ethylene (nun A 8.0e+8 10000 g + Total Butanol esters v 6.0e+8 4 -O— Ethylene r 1000 2 3 — 100 8 4.0e+8 « 8 Harvest date ——>‘E b 10 g 2.0e+8 - - 1 .D g 0.0 r I j I r 0.1 I- L 0.01 6.09-0-7 * 4 09+? + Butanol A 2.0e+7 E o 0 V 420e+8 . 2 -<>- Butyl acet 5 . 3 -C}- Butyl prop 3 o 3 -v— Butyl but — o _ 3-09+8 ~ —0— Butyl 2-Mbut ; ’ ’ 0 g -I— Butyl pent § 9 ° ’ g + Butyl hex E . . o + Butyl hept 5 . + . . m 2 0° 8 + Butyloct : I - . - I l I ° ' ' I 1 0e 8 O k I I I I . 4» ~ - ' o i // v V :t .' \0' ° 0 3\ [=5 .‘ V 9 Iv\ . ' . 0.0 _.“ A; I; a 100 . 30 ‘ + Butanol 3 60 - E s 40- : E 20 1 . o I 1 I . I I V U U .9 100 A t -O- Butyl acet ’ 8. 80 _ -Ci- Butyl prop 2 -V- Butyl but 0. -O— Butyl 2-Mbut ‘- + Butyl pent ; 3 6° ‘ -o— Butyl hex ' : . . , 3 + Butylhept , . O ’ 9 -°- 40 ‘ + Butyl oct , ’ ’ ’ r: 2 . 5 i a 20 lfi: III-I'I'll -4 . . 1.5! s 0 t t O 10 20 30 400 50 60 70 80 90 ay Figure 17. 93 Figure 18. Patterns of pentanol ester emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of total pentanol esters and ontogeny of ethylene. B. GC/MS response (TIC) of pentyl acetate, pentyl propanoate, pentyl 2-methylbutanoate, and pentyl hexanoate. C. Pentanol ester proportions (% of total pentanol esters). Each symbol represents the average of four replications. Vertical bars represent mean i SD. 94 2.0e+8 1.5e+8 * 1 .0e+8 ~ 5.0e+7 d Total pentanol esters (TIC) 0.0 + Total Pentanol esters —O— Ethylene Harvest date ———> - 1000 F 100 ~10 r1 0.1 - 0.01 1 .Oe-I-B 8.0e+7 - 6.0e+7 - 4.0e+7 - 2.0e+7 ~ Pentanol esters (TIC) P o -<>- Pentyl acet —l:l-— Pentyl prop —O— Penyl 2-Mbut -O— Pentyl hex 100 80‘ 60- 40‘ 20- Pentanol ester proportions (%) -<>- Pentyl acet -D- Pentyl prop -O— PentyIZ—Mbut + Pentyl hex Figure 18. 95 A o 0.. ‘ T t B O .0 O O I I I I I C 1 0000 Ethylene (leL) Figure 19. Patterns of hexanol ester emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of total hexanol esters and ontogeny of ethylene. B. GC/MS response (TIC) of total hexanol. C. GC/MS response (TIC) of hexyl acetate, hexyl propanoate, hexyl butanoate, hexyl 2- methylbutanoate, hexyl hexanoate, and hexyl octanoate. D. Hexanol proportions (% of total alcohols). E. Hexanol ester proportions (% of total hexanol esters). Each symbol represents the average of four replications. Vertical bars represent mean :I: SD. 96 8.09+8 6.0e+8 4.0e+8 2.00+8 0.0 Total hexanol esters (TIC) 2.0e+7 1 .Oe+7 3.0e+8 Hexanol esters (TIC) 2.094-8 1.0e+8 20 80 60 40 Hexanol ester proportions (%) 20 .1 I -O-— Total Hexanol esters -O— Ethylene Harvest date ———> - 1000 0.1 - 0.01 -I— Hexanol q -<>- Hexyl aoet -D— Hexyl prop -V- Hexyl but -0— Hexyl 2-Mbut + Hexyl hex + Hexyl oct .4 + Hexanol V I U 1 U .1 1 d -‘ -<>— Hexyl acet -l:I— Hexyl prop —v— Hexyl but —0— Hexyl 2-Mbut -O— Hexyl hex + Hexyl oct Figure 19. 97 1 0000 Ethylene (pJJL) Figure 20. Patterns of 2-methylbutanol ester emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of total 2-methylbutanol esters and ontogeny of ethylene. B. GC/MS response (TIC) of total 2-methylbutanol. C. GC/MS response (TIC) of 2—methylbutyl acetate and 2- methylbutyl butanoate. D. 2-Methylbutanol proportions (% of total alcohols). E. 2-Methylbutanol ester proportions (% of total 2-methylbutanol esters). Each symbol represents the average of four replications. Vertical bars represent mean 1 SD. 98 + 2-Mbutanol 8 E E .9 3 -<>— 2-Mbutyl acet .5 -v- 2-Mbutyl but : 4.0e+8 4 g 3.0e+8 - 5 g 2.0e+8 - o'l 1.0e+8 ~ 0.0 100 °\° 80 - V 60 . 2 40 ‘ .3 20 - 0 8 100 g. -<>— 2-Mbutyl acet —v— 2—Mbutyl but 5 30 .l *3 -°- 60 * E 40 . .o 2‘ g 20 4 N 0 r w—vwW—n—v-v—v—v—v—v—w—w—l o 10 20 30 4o 50 60 Day Figure 20. 99 90 Ethylene (ulJL) E 5.0e+8 ; 10000 3 -0— Total 2-Mbutanol esters? 8 —O- Ethylene . . . r- 1000 '5 i _. C 0 o [- 100 g A 2 5e+8 _ Harvest date ——> n U ' i - 3. l: : 1° .r.: V = o ‘2' " l1 . . . O . «l o 0 . . AW o 1 E o y y y. T I V I I O ; I- 0 5 L 0.01 Figure 21. Patterns of acid ester emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of acetates, propanoates, butanoates, 2-methylbutanoates, hexanoates, and octanoates. B. Acid ester proportions (% of total acid esters). Each symbol represents the average of four replications. 100 1 .Ze+9 1 .0e+9 4 8.0e+8 n 6.0e+8 ‘ 4.0e+8 . Acid ester production (TIC) 2.0e+8 - '100- O 00 O O L l (% of total acid esters) 3 Acid ester proportions N c l -<>— Acetates -D- Propanoates + Butanoates + 2-Mbutanoates + Hexanoates + Octanoates Harvest date ——> -<>— Acetates -Cl— Propanoates -v- Butanoates + 2-Mbutanoates -I— Hexanoates + Octanoates O Figure 21. 101 Figure 22. Patterns of acetate ester emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81 ). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of total acetate esters and ontogeny of ethylene. B. GC/MS response (TIC) of ethyl acetate, propyl acetate, butyl acetate, pentyl acetate, hexyl acetate, 2-methylpropyl acetate, and 2- methylbutyl acetate. C. Acetate ester proportions (% of total acetate esters). Each symbol represents the average of four replications. Vertical bars represent mean 1: SD. 102 ””9 10000 --0— Total Acetate esters A -O— Ethylene 8 . . F 1000 O .1. . . E 8.0e+8 - _ \I‘ g Harvest date —> ' 100 g . 3 ~ 10 5 4.0e+8 ~ rlr F 1 _ o o o ‘3 Al' I- . o 0.0 . - 1 T 1 V T —[ 0‘1 ~7 w -l*- 0' - 0.01 5.0e+8 -<>- Ethyl acet -D— Propyl acet q -V- Butyl acet A 4'03” -O— Pentylacet ' g + Hexyl acet ' v + 2—Mpropyl acet v 2 3.0e+8 - + 2-Mbutylacet V 3 . f 3 / . + J 3 2 De 8 t < 1.0e+8 4 / V / ., .. ~. 0-0 T . . . 4:...6.009909029292:2020 100 7 A -O— Ethyl acet °\° -C}- Propyl aoet v . -v- Butyl acet g 80 -O— Pentyl aoet .9 -I— Hexyl aoet t + 2-Mpropyl acet 8- 6° " -O— 2-Mbutyl aoet 2 Q h ‘3 ‘° ‘ g 20 - 8 < . . . O . ...-...-a-’\ x“ ._fl 0 T ‘ - - -- ‘.€\/-V-V_V-V-V.V-VSV§V-V=V 0 10 20 30 40 50 60 70 80 90 Day Figure 22. 103 Ethylene (pIJL) Figure 23. Patterns of propanoate ester emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81 ). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of total propanoate esters and ontogeny of ethylene. B. GC/MS response (TIC) of ethyl propanoate, propyl propanoate, butyl propanoate, pentyl propanoate, and hexyl propanoate. C. Propanoate ester proportions (% of total propanoate esters). Each symbol represents the average of four replications. Vertical bars represent mean 1: SD. 104 4.0e+8 : + Total Propanoate esters - 1000 F 100 r10 A -O- Ethylene 0 E 3.0e+8 - E 3 8 2.0e+8 - - .3 Harvest date —> C 3. 1.0e+8 - 2 D. ‘3 '— 0.1 - 0.01 2.0e+8 -<>- Ethyl prop -CI- Propyl prop a ~v— amyl prop . . . : 1.5e+8 . -O— Pentyl prop ' V -I— Hexyl prop V v 2 v 3 3 1.0e+8 ~ § C 3, 5.0e+7 J 2 0. 0.0 100 °\° -<>- Ethyl prop ‘6 -Cl— Propyl prop I: 80 - -V— BUT)!l prop .9 -o— Pentyl prop g. —I-— Hexyl prop 60 ~ 9 D. 3 {g 40 . 3 8 20 4 i g o . . Figure 23. 105 1 0000 Ethylene (pUL) Figure 24. Patterns of butanoate ester emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of total butanoate esters and ontogeny of ethylene. B. GC/MS response (TIC) of ethyl butanoate, propyl butanoate, butyl butanoate, 2-methylbutyl butanoate, and hexyl butanoate. C. Butanoate ester proportions (% of total butanoate esters). Each symbol represents the average of four replications. Vertical bars represent mean 3: SD. 106 10000 —0— Total Butanoate esters A —O- Ethylene ‘ U 2.0e+8 - . - 1000 E 9 . o . . ‘ o‘ g ' ’ - 100 g Harvest date —> N 1.0e+8 ‘ 1 _ 1o 8 _ . *3 t , a O O . ..I. g 0 o ' ' I”! i o 1 .v v -_ ‘.' 3 ' L 0.01 1.2e+8 : -<>— Ethyl but 3 ' -Cl- Propyl but 3 A -V- Butyl but v 0 -O— 2-Mbutyl but .‘ F + Hexyl but 3 ' v V 8.0e+7 ‘ : 2 f3 3 , v 8 4.0e-l-7 ~ E g v m -. ' I 0.0 A 100 °\° -<>- Ethyl but V -D— Propyl but 2 80 _ "V- Butyl but 3 -o— 2-Mbutyl but ‘5 + Hexyl but 60 e .1 v v v v V G v v ' v v v V L g 40 - 3 . 8 204 -I.'.:‘I'"- I C ‘3 . - ' m o U I ~‘.‘:.: « . . ‘ 0 10 20 30 40 50 60 70 80 90 Day Figure 24. 107 Ethylene (pJJL) Figure 25. Patterns of hexanoate ester emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of total hexanoate esters and ontogeny of ethylene. B. GC/MS response (TIC) of propyl hexanoate, butyl hexanoate, pentyl hexanoate, and hexyl hexanoate. C. Hexanoate ester proportions (% of total hexanoate esters). Each symbol represents the average of four replications. Vertical bars represent mean :I: SD. 108 1 .2e+8 -0— Total Hexanoate esters —O— Ethylene * 8.0e+7 - Harvest date ——>3 4.0e+7 I 0 .-/ - 1000 r 100 F10 0.1 17 Total hexanoate esters (TIC) “ '4 1 r 0.01 -Cl-— Propyl hex 8.0e+7 " —v.— Buty| hex -O— Pentyl hex (E: -I- Hexyl hex 6.0e+7 . I! t 2 4.0617 1 5 g 2.0e+7 - 0.0 A 100 °\° -Cl- Propyl hex " -v— Butyl hex 2 80 4 —O— Pentyl hex .% -I— Hexyl hex Q h g 40 . fi 8 20 . t! X £ 0 . . Figure 25. 109 1 0000 Ethylene (pL/L) Figure 26. Patterns of octanoate ester emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81 ). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of total octanoate esters and ontogeny of ethylene. B. GC/MS response (TIC) of propyl octanoate, butyl octanoate, and hexyl octanoate. C. Octanoate ester proportions (% of total octanoate esters). Each symbol represents the average of four replications. Vertical bars represent mean i SD. 110 r 1000 r 100 -10 0.1 - 0.01 5.0e+6 ‘ -0— Total Octanoate esters: A .0. Ethylene :. U E E s Harvest date —>: 3 2.5e+6 ( ,- 3 N O c ‘3 E O 0 o I- 0.0 r '- ..l- .' 5.0e-I-6 —El— Propyl oct -V- Butyl oct 8 4.0e+6 ~ -I— Hexyloct E g 3.0e+6 ~ g 2.0e+6 « E 8 1.0e-l-6 ~ 0.0 A 100 E: -D- Propyl oct 2 -v— Butyl oct O 30 ‘ -I— Hexyloct IE 60 - 2 D. h g .0. 3 3 20 - c S 8 o , . Figure 26. 111 10000 Ethylene (pLJL) Figure 27. Patterns of 2-methylbutanoate ester emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81 ). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of total 2-methylbutanoate esters and ontogeny of ethylene. B. GC/MS response (TIC) of ethyl 2-methylbutanoate, propyl 2-methylbutanoate, butyl 2-methylbutanoate, pentyl 2-methylbutanoate, and hexyl 2-methylbutanoate. C. 2-Methylbutanoate ester proportions (°/o of total 2-methylbutanoate esters). Each symbol represents the average of four replications. Vertical bars represent mean :1: SD. 112 5.0e+8 2.5e+8 - 0.0 Total 2-methylbutanoate esters (TIC) + Total 2-Mbutanoate esters —O— Ethylene E 3.0e+8 2.0e+8 * 1 .Oe+8 ‘ 2-Methylbutanoate esters (TIC) -<>- Ethyl 2-Mbut -Cl— Propyl 2—Mbut -V- Butyl 2—Mbut —(>- Pentyl 2-Mbut + Hexyl 2-Mbut -<>— Ethyl 2-Mbut -Cl— Propyl 2-Mbut -V- Butyl Z-Mbut -O— Pentyl 2-Mbut + Hexyl Z-Mbut ‘ =1- 0_ ‘ 0.. 3‘0‘3—3 o I- 80~ .3 8 ,. \° 13:" 604 O'é’ =0 g3 40- £0. >0 5 in 20~ I N o Figure27. 1o 20 30 4o 50 Day 113 09990000,. 60 70 80 A - 1000 - 100 - 1o - 1 0.1 - 0.01 B c 90 10000 Ethylene (pL/L) CHAPTER IV GENE EXPRESSION ASSOCIATED WITH BRANCHED-CHAIN ESTER FORMATION IN ‘JONAGOLD’ APPLE FRUIT Nobuko Sugimoto, Zhenyong Wang, Sungchung Park, Schuyler Korban, Steve van Nocker, Randolph Beaudry 114 INTRODUCTION Esters, produced during ripening by many horticultural crops such as apple, banana, melon, and strawberry, contribute importantly to aroma. In apples, the esters hexyl acetate, butyl acetate, 2-methylbutyl acetate are abundantly produced and considered to confer typical apple aroma characteristics; however, the volatile profile is highly complex and differs with each cultivar (Kakiuchi et al., 1986) Esters in apples are largely composed of either straight-chain or branched-chain alkyl (alcohol-derived) and alkanoate (acid-derived) groups (Figures 28 and 29). The alcohol and acid (actually acyl—CoA thioesters of fatty acids) precursors are joined by an enzyme called alcohol acyltransferase (AAT) to form the ester end product (Aharoni et al., 2000; Jayanty et al., 2002; Souleyre et al., 2005; Yahyaoui et al., 2002). Straight-chain ester precursors may be formed from fatty acid degradation via B-oxidation or lipoxygenase system (Sanz et al., 1997) or from fatty acid biosynthesis via single- or two—carbon fatty acid elongation synthetic pathways (Kroumova and Wagner, 2003; Thelen and Ohlrogge, 2002). Branched-chain ester precursors may be derived from branched-chain amino acid (BCAA) degradation (Rowan et al., 1996; Tressl and Drawert, 1973) or by diversion of branched-chain o-keto acid precursors to BCAAs. The single-carbon fatty acid synthesis pathway, if active in apple, may also produce o-keto butyrate which is necessary for the biosynthesis of o-keto-B- methylvalerate, the immediate precursor to isoleucine. While it can be demonstrated that apple fruit can make esters by metabolizing various 115 exogenous substrates involving pathways such as lipoxygenase, B—oxidation, and amino acid degradation, isotopic evidence is not conclusive that these pathways operate in vivo in the same way. 2-Methylbutyl and 2-methylbutanoate esters are abundantly produced by ‘Jonagold’ apples (Mir et al., 1999; Chapter 3). The 2-methylbutyl alkyl and alkanoate moieties are likely derived from o-keto-B-methylvalerate. In isoleucine formation, o-keto-B-methylvalerate is transaminated by branched-chain aminotransferase (BCAT) to isoleucine (Figure 29). This reversible reaction is also the first step in isoleucine degradation. In apple, o-keto-B-methylvalerate can be formed from added isoleucine, suggesting that isoleucine degradation may be necessary for 2-methylbutyl and 2-methylbutanoate ester formation in apple (Rowan et al., 1996). Alternatively, o-keto—B-methylvalerate used in ester biosynthesis may be diverted from the pathway of isoleucine biosynthesis, which increases during apple ripening (Nie et al., 2005; Singh and Shaner, 1995). In the biosynthesis of 2-methylbutyl and 2-methylbutanoate esters, o—keto- B-methylvalerate can be dehydrogenated by branched—chain a-ketoacid dehydrogenase (BCKDH) to 2-methylbutyI-CoA or decarboxylated by branched- chain d-ketoacid decarboxylase (a.k.a. branched-chain 2-ketoacid decarboxylase or pyruvate decarboxylase, PDC) to 2-methylbutanal (Wyllie et al., 1996). The dehydrogenase pathway is considered as a major route for BCAA catabolism in most organisms, whereas the PDC pathway has been extensively studied only in yeast and bacteria (Dickinson et al., 1997, 1998, 2000; Smit et al., 2004). 116 In the final enzymatic step, AAT can combine 2-methylbutyl-COA and/or 2- methylbutanol with various alcohols and acyl-CoAs, respectively, to create a wide variety of esters. The substrate specificity of AAT is believed to markedly impact the ester profile (Aharoni et al., 2000; Olias et al., 2002; Souleyre et al., 2005; Ueda et al., 1992; Yahyaoui et al., 2002). Although AAT likely plays a major role in establishing the ester profile, control of ester synthesis probably lies at the level of ester precursor formation (Wyllie and Fellman, 2000). For branched- chain esters, control may lie in the BCAA catabolism pathway, either BCAT, BCKDH and/or PDC. However, little is known about activity and regulation of BCAT and PDC in higher plants. To our knowledge, no studies on ester synthesis in fruit have characterized the expression of genes associated with these enzymes. Given that the biochemistry of ester formation is so poorly understood, a genomic analysis may aid in the identification of highly regulated genes in the ester formation pathways. The relationship between ester-production and gene expression has been studied in apple, strawberry, and pear fruit using expressed sequence tag (EST) analysis (Park et al., 2006) and microarrays made with ESTs (Aharoni et al., 2000; Fonseca et al., 2004). Fonseca et al. (2004) found that when the expression of the ethylene synthesis gene, ACC oxidase, increased, gene expression for a short-chain type alcohol dehydrogenase, pyruvate decarboxylase, and 3-ketoacyl-CoA thiolase (B-oxidation) increased at the same time in ripening pear fruit. Aharoni et al. (2000) found that AATgene expression underwent a 16-fold increase in strawberry between the green and 117 red fruit stages; they observed an increase in volatile esters as fruit turned from pink to red. Aharoni et al. (2000) also observed an increase in PDC gene expression during fruit development and explored the relationship between ethyl ester formation and PDC gene expression. Park et al., (2006) observed that expression of a lipoxygenase gene increased as total volatiles increased during apple ripening. However, the relationship between lipoxygenase gene expression and the production of C6 esters, which are believed to be derived from fatty acid metabolism via the lipoxygenase pathway, was not evaluated. In order to improve our understanding of the pathways involved in ester formation, we developed an apple microarray for genomic analysis. The microarray was a composite of presumed ester formation-related ESTs, available at the time of printing and 10,000 unknown, unsequenced gene fragments obtained from a library representing genes expressed at all stages of apple fruit development. The objective of this experiment was to establish a correlative link between changes in gene expression and changes in branched-chain ester biosynthesis in ripening apple. We hypothesized that branched-chain ester formation was from BCAA catabolism and expected the expression of genes associated with BCAA catabolism such as BCAT and PDC to be upregulated during the increase in branched-chain ester production. MATERIALS AND METHODS Plant Material 118 ‘Jonagold’ apples were harvested from the Michigan State University Clarksville Horticultural Experiment Station, Clarksville, Ml. From September 2, 2004 (Day 0) until ripening was fully engaged on October 7, 2004 (Day 35), fruits were collected for examination every three to four days from the field. On each occasion, fruit were held overnight in the laboratory to equilibrate to laboratory temperature (211:1 °C) and covered with ventilated, black, 4-mil thick plastic bags to avoid desiccation and responses to intermittent laboratory light before analysis. All fruits (approximately 200) remaining on the tree were harvested and transported to the laboratory at once on October 7, 2004 (harvest date, Day 35) after it was apparent that ripening was underway. This was done to avoid damage in the field due to freezing and fruit drop. Thereafter, fruit were maintained at room temperature (211:1 °C) and covered with plastic bags as described previously. The fruit were examined every three to four days from harvest (October 7, 2004) until the conclusion of the study on November 23, 2004 (Day 81). On each evaluation date, 20 apples were randomly chosen and the internal ethylene content of each was measured. Of these, the fourteen fruit having an internal ethylene content nearest the median were selected for further analysis. The four fruit having ethylene levels closest to the median were used for analysis of C02 production, ester emission, and textural properties. From the remaining ten fruit, the skin and a small amount of cortex were removed and immediately frozen in liquid nitrogen and stored at -80°C until extraction of RNA. 119 Two replicates were created from the ten apples; each replicate consisted of four to five apples. Aroma Analysis To collect volatiles, apples were held at 20°C in a ‘I-L Teflon container (Savillex Corporation, Minnetonka, MN) fitted with two gas-sampling ports, each sealed with a Teflon-lined half-hole septum (Supelco Co., Bellefonte, PA). Headspace volatiles were sampled after 20 minutes using a 1-cm long solid- phase micro extraction (SPME) fiber (65 pm PDMS-DVB, Supelco Co., Bellefonte, PA). SPME sorption time was 3 min. The exposed fibers were immediately transferred to a gas chromatograph (GC) (HP-6890, Hewlett Packard Co., Wilmington, DE) injection port (230°C) and desorbed for 2 min. Desorbed volatiles were trapped on-column using a liquid nitrogen cryofocussing trap. Separation of volatiles was by capillary column (SupelcoWax-10, Supelco, Bellefonte, PA, 29 m x 0.2 mm id, 0.2 pm coating film). The temperature of the GC was programmed from 40 to 240°C at a rate of 50°C/min; the flow rate of the helium carrier gas was 1 mL/min and the GC was operated in splitless mode. Detection was by time-of-flight mass spectrometry (Pegasus II, LECO Corp., St. Joseph, MI) (GC/MS) according to the method of Song et al. (1997). Identification and quantification of compounds were by comparison of the mass spectrum and MS response (total ion count, TIC), respectively, with those of authenticated reference standards and spectra in the National Institute for Standard and Technology (NIST) mass spectra library (Search Version 1.5). Isolation of RNA 120 In brief, eight developmental stages were selected for analysis of expressed genes based on physiological changes during ripening. These stages are: stage 1 (Day 0), early climacteric; stage 2 (Day 11), late preclimacteric and onset of trace ester biosynthesis; stage 3 (Day 25), onset of the autocatalytic ethylene and rapid increase of ester biosynthesis; stage 4 (Day 32), half-maximal ester biosynthesis and engagement of the respiratory climacteric; stage 5 (Day 39), near maximal ester biosynthesis, peak in respiratory activity, and onset of rapid tissue softening (Chapter 3, Figure 11); stage 6 (Day 49), end of maximal ester biosynthesis, the conclusion of the respiratory climacteric, and completion of tissue softening; stage 7 (Day 60), midpoint in the decline in ester biosynthesis, maximal ethylene production, and onset of senescence; and stage 8 (Day 70), postclimacteric minimum in ester production and extensive fruit senescence. Total RNA was isolated from the ‘Jonagold’ apple skin and 2-3 mm of underlying cortex tissue by hot borate/phenol extraction followed by LiCI precipitation (Lopez-Gomez and Gémez-Lim, 1992). Microarray Printing Microarray slides were created by the Genomics Technology Support Facility (GTSF) of the Genomics Core in Michigan State University, East Lansing, MI. Approximately 10,000 unsequenced cDNA gene fragments were generated from the lambda phage cDNA library from ‘Mutsu’ apple fruit described by Gao et al. (2005) using a mass excision kit and protocols as described by the manufacturer (ZAP-cDNA synthesis kit, Stratagene, LaJolla, CA). In addition to the unsequenced cDNA fragments, an additional 116 nucleotide gene fragments 121 were arrayed. These 116 genes were selected based on their putative identity using the Tree Fruit Technology genomic analysis tool apple database version 2.0 (http://genomics.msu.edu/fruitdb/analyses/apple.shtml). Putative gene identities included aminotransferase, alcohol acyltransferase, alcohol dehydrogenase, acyl-ACP thioesterase, fatty acid hydroperoxide lyase, lipoxygenase, 3-ketoacyI-CoA thiolase, acyl-CoA oxidase, enoyl-CoA hydratase, and omega-3 fatty acid desaturase, allene oxide synthase, mannitol dehydrogenase, mannitol transporter, NADP-dependent D-sorbitol—6-phosphate dehydrogenase, sorbitol dehydrogenase, and sorbitol transporter. Additional human and bacterial control genes were also placed on the array. These genes were used as controls for non-specific binding and to normalize for background signal noise when analyzing microarray data. Microarray design and labeling The experimental design was a one-way classification with a single multi- level treatment factor (stage of development) having eight levels. The comparison arrangement was mixed and consisted of loops and indirect comparisons (Figure 30) (Churchill, 2002; Yang and Speed, 2002). There were two biological and two technical replicates with dye swaps. The microarray protocol used for labeling, hybridization, and washing has been described by Hegde et al. (2000). Reverse transcription to make cDNA was performed using total RNA (20 pg) and an oligo(dT) 20-mer primer (4 pg). Products were labeled with fluorescent dyes: cyanine 3 (cy3) and cyanine 5 (cy5). Labeled cDNA was mixed, vacuum dried, and re-suspended in 14 pL of 122 hybridization buffer (50% formamide, 5x SSC, and 0.1% SDS) with COT1-DNA (20 pg) and Poly(A)-DNA (2 pg) to block nonspecific hybridization. The cy3 and cy5 probes were combined and placed on a microarray slide for hybridization and incubated for 16—20 hours at 42°C using a water bath. After washing and drying, the slides were scanned (Affymetrix 428 Array Scanner, Santa Clara, CA) at 635 nm for cy5 and 532 nm for cy3. Images were analyzed for feature and background intensities using GenePix Pro 3.0 (Molecular Devices, Union City, CA). Statistical analysis Raw data were submitted to linear models for microarrays Graphical User Interface (LimmaGUl) library modules for the R statistical package (http://bioinf.wehi.edu.au/limmaGUl/index.html) software (Wettenhall and Smyth, 2004). Data was analyzed with minimum background correction and normalized within array only using the global loess method. The least squares method was used for the linear model fit according to the Benjamini & Hochberg method to control the false discovery rate. The logz differential expression ratio (M) value among the eight developmental stages was used to measure degree of expression changes relative to Day 0, which was considered as a reference. The probability of differences in expression was calculated. Gene fragments were selected for sequencing based on the statistical probability (P<0.00025) that expression changes were non-random. Selection was also accomplished using a constraint table developed to capture additional large expression changes associated with specific physiological events, but not statistically different from 123 Day 0 (greater than 60% of these had P<0.01) (Table 3). Sequencing was successful on approximately 85% of selected cDNA fragments. Gene identity was tentatively established by first screening for the presence of vector sequence. Following low quality sequence trimming, acceptable sequences were clustered and consensus sequences were obtained using stackPACK (httpzllgenomics.msu.edu/stackpack/tool). Identity was tentatively assigned by BLAST analysis against the NCBI non-redundant protein and Arabidopsis thaliana protein database. Approximately 80% genes were identified for their identity from total of 758 genes BLASTed. Semi-quantitative reverse transcription polymerase chain reaction (RT- PCR) analysis PCR was used to verify microarray data and to compare expression of all available members for putative pyruvate decarboxylase (PDC), and branched- chain aminotransferase (BCAT) gene families and for the 2-isopropylmalate synthase gene. All genes evaluated using semi-quantitative RT-PCR, apple cluster number, primers, the expected size of the PCR-product, optimum cycle number, and optimum temperature for primer binding to gene are listed (Table 4). PCR for two PDC genes and one BCATgene were unsuccessful. No products were evident for PDC6 and BCA T11 after two trials using different primers and PDC7 was too short to design appropriate primers. Two biological replicates were used for PCR analyses for each developmental stage used in the microarray analysis. cDNA synthesis and PCR reactions were performed according to the manufacturer (lnvitrogen, Carlsbad, 124 CA) directions. Before creating cDNA, total RNA was treated with DNase using RNase-free DNase set kit according to the manufacturer (Qiagen Inc, Valencia, CA). One pg of DNase-treated total RNA was reverse transcribed using oligo(dT)12-13 primer and SuperScript II as described by the manufacturer (lnvitrogen, Carlsbad, CA). The cDNA (1.0 pL) was used as a template in a 50 pL PCR reaction containing 10 pM of forward and reverse gene-specific primers designed using Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). The PCR reaction was performed as described in the following steps: 1) 5 min at 95°C, 2) 30 s at 95°C, 3) 30 s at 55-59°C, 4) 30 s at 72°C, repeating 18-35 cycles from steps 2-4, and final elongation 5 min at 72°C. The amplified PCR products were separated by electrophoresis on a 1.5% (w/v) agarose gel, visualized with ethidium bromide, and photographed. Relative light density of the bands was quantified by a digital imaging system (EagleEye Il, Stratagene, La Jolla, CA). To identify the Optimum cycle, the gene products amplified by PCR had to be visible on the gel electrophoresis and be able to quantifiable by light density measurement without saturation. The number of PCR cycles needed ranged from 26 to 35 (Table 4). A single number indicates that the same cycle was performed with both biological replications. Two numbers indicate that different number cycles were performed for each replicate. PCR products were cleaned by QlAquick PCR purification kit (Qiagen inc., Valencia, CA) and sequenced at the GTSF facility to verify identity. All the PCR generated sequences had to be 97-100% identical to the reported apple sequence in the apple sequence database (version 3.0). 125 Semi-quantitative RT-PCR data analysis A partial sequence of the 18S ribosomal RNA gene (gi85717895) was used as an internal control for PCR analyses. Expression data for all genes (PDC, BCAT, and 2-isopropylmalate synthase) were normalized based on the 18S spot density. The spot density for the 188 ribosomal RNA gene varied approximately i10% across the eight developmental stages (data not shown). The values for PCR are calculated as the spot density relative to the maximum value obtained for each gene. RESULTS Branched-chain esters Total ester production increased coincident with increased ethylene accumulation in the fruit and continued well beyond the end of the respiratory climacteric (Figure 31). The pattern for branched-chain esters was generally similar to that for total esters, but had a higher, sharper initial peak (Figure 32). The GC/MS response for all esters derived from 2-methylbutanoate (ethyl, propyl, butyl, pentyl, and hexyl 2-methylbutanoate) and from 2-methylbutanol (2- methylbutyl acetate and 2-methylbutyl butanoate) peaked on Day 39 and declined thereafter (Figure 32). 2-Methylbutanol esters had a lower diversity, but approximately the same abundance as 2-methylbutanoate esters based on total ion count (TIC). The most abundant 2-methylbutanoic acid-derived branched- chain esters were hexyl and butyl 2-methylbutanoate, and the most abundant alcohol—derived branched-chain ester was 2-methylbutyl acetate. 2-Methylbutanol 126 and 2-methylbutanal production patterns were similar to those for 2-methylbutyl esters, rapidly increasing during the climacteric to a peak on Day 39 and declining rapidly thereafter, then undergoing a slow increase as ripening and senescence continued. Free 2-methylbutanoic acid was not detected. Gene expression related to branched-chain amino acid metabolism Of the 116 nucleotide gene fragments printed on the microarray slide, a branched-chain aminotransferase [BCAT, MDC410280 (BCA T10)] was the only gene that was related with branched-chain amino acid (BCAA) metabolism. Based on the BLAST analysis of the 758 unknown microarray nucleotide fragments, only three genes were related to BCAA metabolism. Two pyruvate decarboxylase (PDC) clusters and one 2-is0propylmalate synthase singleton (gi7387848) were identified. The two PDC clusters were judged to be similar to a single apple gene (MDC015210, PDC1) found in the Tree Fruit Technology genomic analysis tool apple database (version 3.0, httpzllgenomics.msu.edu/fruitdb/analyses/apple.shtml). There were no sequence similarities found in the apple sequence database for 2-isopropylmalate synthase. The apple sequence database contained a total of 11 putative BCAT and 7 putative PDC genes. Microarray expression analysis Relative expression of BCA T10 changed relatively little, but peaked simultaneously with all branched-chain esters (Figure 33). The change in expression relative to Day 0 was not significant (P=0.36), but the overall range in expression between the peak and lowest expression levels was approximately a 127 12-fold change. Expression remained low until onset of ethylene accumulation on Day 25, maximized on Day 39 at 3.9x initial level, and decreased rapidly after Day 49 to a low of 0.3x on Day 70. PDC1 relative expression remained very low until onset of ethylene accumulation on Day 25, but changed significantly thereafter (P<0.005). PDC1 expression rapidly increased as volatiles began to increase, reached maximum of about 5.7x initial levels on Day 49, and decreased slightly during senescence. 2-Isopropylmalate synthase relative expression increased rapidly after Day 11 coincident with increasing ethylene and branched-chain ester production, reaching maximum 34-fold change on Day 39 and remained high during senescence. Of the three genes characterized using the microarray, BCA T10 had an expression pattern that was most similar to the pattern of branched-chain ester production. However, PDC1 and 2-isopropylmalate synthase initial increases closely corresponded with the onset of branched-chain ester biosynthesis and, reflecting the pattern of production several of the branched-chain esters, continued to be highly expressed during the later stage of fruit development. Semi-quantitative RT-PCR analysis PCR product spot densities for BCA T10, PDC1, and 2-isopropylmalate synthase yielded patterns that were similar to the microarray data, validating the microarray analyses (Figures 3436). Of the ten BCAT genes in addition to BCA T10, nine were found to be expressed in the fruit (Table 4). Four BCAT genes [MDC021750 (BCA T1), MDCOZB270 (BCA 72), MDC238050 (BCA T6), 128 MDC405820 (BCA T9)] were expressed similarly to BCA T10 during branched- chain ester production (Figure 34A), five genes [MDC160840 (BCA T3), MDC192460 (BCA T4), MDCZ38040 (BCA T5), MDC301230 (BCAT7), MDC385660 (BCA T8)] had relatively stable expression until approximately Day 25, then declined slightly as ripening and senescence progressed (Figure 348). Even though the expression of some BCATgenes followed the same pattern as branched-chain ester production, their relative expression change was not large. Of the six PDC genes in addition to PDC1, five were detected as being expressed in the fruit (Table 4). Expression patterns for the five genes differed, however only one (PDC1) increased (Figure 35). MDCZOG810 (PDC4) rapidly increased on Day 32 and declined thereafter. MDC433930 (PDC5) was stable throughout ripening and slightly increased during senescence. MDC133700 (PDCZ) and MDC206800 (PDC3) had the highest expression before the climacteric peak and gradually declined afterwards. With exception of PDC5, most of the PDC genes had a relatively high expression compared to the BCAT genes based on PCR cycle numbers (Table 4). DISCUSSION Ester synthesis Autocatalytic ethylene production, which we considered to occur above an internal ethylene content of 0.2 pL-L", had a pattern relative to ester synthesis similar to that previously described for ‘Bisbee Delicious’ (Mattheis et al., 1991b), ‘Golden Delicious’ (Song and Bangerth, 1996), and ‘Redchief Delicious’ apples 129 (Ferenczi, 2003). This is consistent with previous studies demonstrating that the bulk of ester production requires ethylene action (Defilippi et al., 2004; Ferenczi etaI,2006) The abundantly produced 2-methylbutyl acetate is also produced in large amounts by ‘Bisbee Delicious’ (Mattheis et al., 1991a, 1991b), ‘Redchief Delicious’ (Ferrenczi, 2003), ‘Rome’ (Fellman et al., 1993), and ‘Golden Delicious’ (Song and Bangerth, 1996) fruit. The ‘Delicious’ variety also produces ethyl 2-methylbutanoate in abundance, however, very little of this compound was produced by ‘Jonagold’, accounting for only 0.4% of total ion count (TIC) for all branched-chain esters on average. The high production of hexyl 2- methylbutanoate in ‘Jonagold’ is also found in ‘Redchief Delicious’ (Ferrenczi, 2003). On the other hand, ‘Annurca’ apple produces little or no branched-chain esters (Lo Scalzo et al., 2001). The maintenance of a high rate of production of 2-methylbutyl esters throughout ripening and senescence suggests a consistent production of 2-methylbutanol, which, in fact was detected. The sharp increase, followed by sustained production of 2-methylbutanol is suggestive of a sharp increase and subsequent sustained PDC activity. Gene expression The similarity in the expression pattern of putative BCAT genes with branched-chain ester production suggests expression activity may influence isoleucine metabolism and subsequent ester formation. lsoleucine metabolism is known to change during apple fruit ripening. A significant increase in isoleucine concentration takes place during apple ripening (Nie et al., 2005), indicating 130 biosynthesis of isoleucine outpaces its catabolism. Deuterium-labeled feeding studies by Rowan et al. (1996) demonstrated that isoleucine catabolism can contribute to ester biosynthesis during apple fruit ripening. It is also possible, however, that increased synthesis of isoleucine is accompanied by increased availability of o-keto-B-methylvalerate, the precursor to isoleucine. a-Keto-B- methylvalerate rather than isoleucine may serve as the source of substrate for decarboxylation reactions leading to the formation of 2-methylbutanoate and 2- methylbutanol. In a recent study with Arabidopsis thaliana, a total of six or possibly seven BCATgenes have been cloned. BCAT proteins are found localized in different tissues and possess differing activities. AtBCAT—1 is localized in mitochondria, and has the capacity to initiate degradation of leucine, isoleucine, and valine in all tissues, AtBCAT-2, -3 and -5 are located in plastids, expressed at rather low levels, and, with exception of AtBCAT-3, transcribed in all tissues. AtBCAT-4 and -6 are cytoplasmically located, expressed in tissues associated with transport function and in meristematic tissues (Diebold et al., 2002; Schuster and Binder, 2005). Of these, mitochondrial AtBCAT—1 is suspected to be important in BCAA catabolism. None of the apple BCATgenes characterized in this study had a similarity with the mitochondrial AtBCA T-1, but were more similar to plastid-localized AtBCA T-2 [BCA T2 (MDC028270)], AtB CA T-3 [B CA T5 (MDC238040), BCA T6 (MDC238050), BCA T8 (MDC385660), and BCA T10 (MDC410280)], and AtBCA T-5 [BCA T9 (MDC405820)]. The enzymatic functions of all BCAT proteins in Malus sp. are currently unknown. 131 In bacteria, nutritional factors such as carbohydrate and nitrogen source regulate aromatic aminotransferase and branched-chain aminotransferase gene expression (Chambellon and Yvon, 2003). In higher plants, BCAA degradation is thought to occur due to a limitation in carbon supply (Graham and Eastmond, 2002). It is possible that increase in BCATgene expression changes in apple were in response to a limitation in the supply of carbon being delivered to the fruit after being detached from the tree. On the other hand, in apple, there is a plentiful supply of energy-rich carbohydrates stored in the fruit arguing against this possibility of starvation-induced BCATexpression. Branched-chain o-ketoacid decarboxylases, or PDCs, are poorly studied in higher plants. Failure to detect PDC6 gene expression in fruit suggests that it may lack a function in fruit and is therefore not expressed. Little information is found characterizing PDC genes relative to ester formation. In yeast, which produces important esters in some food products and beverages, five genes have been reported to be responsible for decarboxylation of branched-chain o- keto acids to branched-chain aldehydes (Dickinson et al., 1997, 1998, 2000; Yoshimoto et al., 2001). Dickinson et al. (1997, 1998, 2000) suggests that the catabolic pathways of three BCAAs are accomplished in different ways, a single YDL080c gene (PDC-like gene) is likely responsible for leucine catabolism and any one of the isozymes of PDC can enable valine and isoleucine degradation. It is possible that some apple PDCs may have a similar capacity to metabolize branched-chain or-keto acids. The predominant production of 2-methylbutyl and 132 2-methylbutanoate esters by ‘Jonagold’ apple fruit suggests a specificity of PDC activity for d-keto-B-methylvalerate, a component of isoleucine metabolism. In fruits, three PDC genes were isolated from strawberries and one from grape berries (Moyano et al., 2004; Or et al., 2000). Also, PDC activities were measured during maturation of ‘Fuji’ apples (Echeverria et al., 2004). The main purpose of these studies was to relate PDC activity and expression to ethanol production under anaerobic conditions or to the formation of ethanol-derived esters such as ethyl esters, but not for BCAA metabolism. In ‘Jonagold’, the pattern of ethyl ester formation did not appear to reflect the pattern of expression of any of the PDC genes, they were found only at low levels and tended to increase only during later developmental stages. The TIC for total ethyl ester production only accounted for 1% of the TIC for total alcohol esters and the TIC for ethanol accounted for only 0.2% of the TIC for all alcohols. The lack of a correlation between ethanol ester production and expression for any of the five PDC genes argues against a causative relationship between the expression of these genes and ethanol metabolism. In tomato fruits, Tieman et al. (2006) recently found that 2-phenylethanol, an important flavor and insect attractant in tomato and rose, is synthesized from phenylalanine by an aromatic amino acid decarboxylase. Tieman et al. (2006) also proposed the pathway that phenylalanine conversion to phenethylamine without producing phenylpyruvate as in yeast and then to 2-phenylacetaldehyde and to 2-phenylethanol. Theoretically, this report suggests that a decarboxylase may directly convert free branched-amino acid to 2-methylbutylamine for 133 example, from isoleucine and then to 2-methylbutanal. The involvement of PDC in BCAA catabolism in ester synthesizing fruits awaits characterization. 2-lsopropylmalate synthase is normally not considered to be involved in isoleucine formation or degradation. It is engaged in the first step of leucine biosynthesis, transferring an acetyl group from acetyl-CoA to o-ketoisovalerate to form isopropylmalate. Given that Nie et al. (2005) observed high accumulation of isoleucine, but not leucine during ripening of apple, a rationale for the marked increase in 2-isopropylmalate synthase expression is not obvious. However, it is possible that the 2-isopropylmalate synthase protein may have a different activity; 2-isopropylmalate synthase may also be involved in fatty acid biosynthesis (Charon et al., 1974; Kroumova et al., 1994; Kroumova and Wagner, 2003) The single-carbon fatty acid elongation pathway for fatty acid synthesis may contribute to o-keto-B-methylvalerate formation via q-keto butyrate and may also contribute to propionyl-CoA formation from this same metabolite (Figure 29). If so, 2-isopropylmalate synthase may be involved in supplying precursors of both straight— and branched-chain esters. The increase in branched-chain and propanol and propanoate esters at the climacteric peak (Chapter 3, Figures 14 and 21) and their maintenance at high levels during senesce supports a role for engagement of the single-carbon fatty acid elongation pathway in ester biosynthesis. Nevertheless, the single-carbon fatty acid elongation pathway needs to be more fully evaluated. Apart from the current study, we have not been able to find any studies linking this pathway to volatile ester biosynthesis. 134 However, there is solid data describing the involvement of the single-carbon fatty acid elongation pathway in sugar ester biosynthesis and excretion in several members of the Solanaceae family, including tobacco (Nicotiana tabacum), and petunia (Kroumova and Wagner, 2003). While no data are found verifying the function of the single-carbon fatty acid synthesis pathway in apple, the fact that 2-isopropylmalate synthase catalyzes the first step in this pathway, is suggestive that this gene plays a role in ester biosynthesis and/or other ripening processes not yet elucidated. On average, the RT-PCR cycle number needed for BCATfamily members tend to be larger than PDC or 2-isopropylmalate synthase genes. The lower number of PCR cycles required for quantification of PDC genes suggests transcript levels for these genes are several-fold higher than for BCAT genes. Further, the expression patterns for 2-isopropylmalate synthase and PDC exhibited a greater degree of change during ripening. The pattern of 2- methylbutanol, 2-methylbutanal, and 2-methylbutanol ester production remaining elevated or increasing slightly even in the senescence stage is similar to the expression pattern of PDC1. Since the expression patterns of PDCZ-5 declined sharply in the later ripening stage, we suggest that PDC1, detected on the microarray, may be involved in converting the isoleucine product/precursor d- keto-B-methylvalerate to 2-methylbutanal to form 2-methylbutyl esters in apples. Considering that 2—isopropylmalate synthase gene expression had a pattern similar to that for the production of branched-chain esters, we further suggest that this gene may have a role supplying q-keto butyrate for branched-chain o- 135 keto acid (d-keto-B-methylvaIerate) synthesis and ultimately contributing to branched-chain ester biosynthesis. CONCLUSION Several putative genes of branched-chain aminotransferase, pyruvate decarboxylase, and 2-isopropylmalate synthase were found to have an expression pattern that increase concurrently with branched-chain ester production. BCAA and 2-isopropylmalate synthase may be involved in supplying branched-chain o-keto acids. 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The tail of the arrow indicates RNA probe labeled with cyanine 3; the head of the arrow indicates RNA probe labeled with cyanine 5. 146 3.0e+9 - r 10000 ~ 100 —l— Total volatiles —O— Ethylene A —A— C02 0 - 1000 80 I: -_- 5 I; 2.Oe+9( 3 g 100 60 C) 2 3 E z =- v (u v ‘5 8 .5 > 10 9 40 a5 -"' 1.0e+91 > D s 8 L— l— 1 _ 20 0. N O 0 0.1 L 0 Day E 0.01 ©®®®©©®O Figure 31. Internal ethylene concentration, total volatiles (TIC), and C02 production in pre-climacteric through post-climacteric ‘Jonagold’ apples. Eight stages (Days 0, 11, 25, 32, 39, 49, 60, and 70) were selected for genomic analysis based on physiological stages during ripening. Stage 1 (Day 0), early climacteric; stage 2 (Day 11), late preclimacteric and onset of trace ester biosynthesis; stage 3 (Day 25), onset of the autocatalytic ethylene and rapid increase of ester biosynthesis; stage 4 (Day 32), half-maximal ester biosynthesis and engagement of the respiratory climacteric; stage 5 (Day 39), near maximal ester biosynthesis, peak in respiratory activity, and onset of rapid tissue softening; stage 6 (Day 49), end of maximal biosynthesis, the conclusion of the respiratory climacteric, and completion of tissue softening; stage 7 (Day 60), midpoint in the decline in ester biosynthesis, maximal ethylene production, and onset of senescent; stage 8 (Day 70), postclimacteric minimum in ester production and high fruit senescent. 147 Figure 32. Patterns of 2—methylbutanol and 2-methylbutanoate ester emissions during ripening and senescence of ‘Jonagold’ apple. The volatile profile was tracked from early September (Day 0) until late November (Day 81). The fruits were collected from the field until Oct. 7, 2004 (Day 35) and thereafter maintained at room temperature (indicated by dashed vertical line). A. GC/MS response (TIC) of total 2-methylbutanol and 2-methylbutanoate esters and ontogeny of ethylene. B. GC/MS response (TIC) of 2-methylbutanol and 2- methylbutanal. C. GC/MS response (TIC) of 2-methylbutyl acetate and 2- methylbutyl butanoate. D. GC/MS response (TIC) of ethyl 2-methylbutanoate, propyl 2-methylbutanoate, butyl 2-methylbutanoate, pentyl 2-methylbutanoate, and hexyl 2-methylbutanoate. E. 2-Methylbutanoate ester proportions (% of total 2-methylbutanoate esters). Each symbol represents the average of four replications. Vertical bars represent mean 1: SD. 148 10000 5.0e+8 149 L-I:l- Ethylene f c + Total 2-Mbutanolestersf ' ‘5 —0— Total 2-Mbutanoate estfie \ I . . * 100° .C S l ' I ' ' I j A ' I 9 0 ; .3 ’o‘ . 100 a. E E 2.Se+8 . Harvest date —9 g. V 0 2 i . ~ 10 2 C o ’ 0 N 0 o I 0 . . - I. H .- > .0 8 .. ' o . ° - 1 .C — I I l_-/ m g . A” '- 0 0 o?. . : . I r l r t I W 0.1 ' l u 0.01 — 8.0e+7 8.0e+5 _ g —-h— 2-Mbutanol g g 8 -a-— 2-Mbutanal 4.0e+7 ( - 4.0e+5 g a E .0 2. 2. N 0.0 0.0 N A 5.0e+8 0 —<>— 2-Mbutyl acetate E —v— 2-Mbutyl butanoate It! 3 E 2.59-0-8 ~ 0 C 3 3 E“ N 0.0 3.0e+8 a -<)- Ethyl 2-Mbutanoate D 3 -Cl— Propyl 2-Mbutanoate ; 0) -V- Butyl 2-Mbutanoate . 3 2.09,,8 . —o— Pentyl 2-Mbutanoate 5 E 8 -I— HexyIZ-Mbutanoate : E g 1.0e+8 ~ .9 g N 0.0"00 - 3 E g A 8 rI,\° 80 q V —O— Ethyl 2-Mbutanoate . g on 60 . -Cl— PropyIZ-Mbutanoate 8 g + Butyl2-Mbutanoate c "' 40 - —O— PentyIZ-Mbutanoate ; v. _ J, _ v v v v g E. -I— Hexyl2-Mbutanoate v ' I ' V ”‘1'- l I I - g e 20 ‘ v v " I I I Olin 0.. 332-s=gggoooqooooo O 10 20 30 40 50 60 70 80 90 Day Figure 32. (TIC) O) J O .- 51. Harvest date ——> '0 .9 0 4 " .2 A ‘5 2 2 3 . C ID 2 l ‘ I o N I l- g I g — A 2 4 ‘ ' ‘ g 0 t. I I ' I I I I ID 10 20 30 i 40 50 70 0 i: C : 0 EDay (D .2 ' —D— Pyruvate decarboxylase (PDC1) MDCO15210 —A- BC aminotransferase (BCAT10) MDC410280 + 2-isopropylmalate synthase gi7387848 Figure 33. Gene expression based on microarray data (in logz scale). For genes potentially involved in branched-chain ester formation including pyruvate decarboxylase (PDC), branched-chain aminotransferase (BCAT), and 2- isopropylmalate synthase gene expression relative to Day 0. 150 Harvest date ——>. A + BCAT1 + BCAT2 0.2 ‘ + BCAT 6 \7 + BCAT 9 - —v- BCAT10 -¢r— BCAT 3 —0— BCAT 4 0-2 ‘ -0— BCAT 5 -<>- BCAT 7 —0— BCAT 8 000 I T I l T I I U 0 10 20 30 40 50 60 70 Branched-chain aminotransferase (BCAT) relative spot density Day Figure 34. Gene expression of putative branched-chain aminotransferase (BCAT) for ‘Jonagold’ apple fruit ripened at room temperature performed by semi-quantitative RT-PCR. The value is based on spot density relative to maximum value. 185 rRNA was used as a control. All data are normalized relative to control gene spot density. The control gene spot density ranged between 0.78098. Each symbol represents the average of two replications. The average pooled standard deviation is 0.15. 151 1JZ‘ 1.0 \ 0.8 I, ’ 0.6 ..o'k" Harvest date ——>_ ' " «It 0.4 - 0.2- I 0.0 fi r Pyruvate decarboxylase (PDC) relative spot densrty 0 10 —v— PDC1 + PDC2 + PDC3 + PDC4 —-*- PDC5 20 30 50 60 70 Day Figure 35. Gene expression of putative pyruvate decarboxylase (PDC) for ‘Jonagold’ apple fruit ripened at room temperature performed by semi- quantitative RT-PCR. The value is based on spot density relative to maximum value. 183 rRNA was used as a control. All data are normalized relative to control gene spot density. The control gene spot density ranged between 0.78098. Each symbol represents the average of two replications. The average pooled standard deviation is 0.10. 152 1.2 _ Harvest date——> 8 m 1.0 ‘ a) 3 g 0.8 - {a 2 '5 8 0.6 . E m - d) 52.2 04. :2. 5 ° 0 2 E 0.2 ‘ I ; 0': f + 2-isopropylmalate synthase 0.0 I f l fi T I l T 0 10 20 3O 40 50 60 70 Figure 36. Gene expression of putative 2-isopropylmalate synthase for ‘Jonagold’ apple fruit ripened at room temperature performed by semi—quantitative RT—PCR. The value is based on spot density relative to maximum value. 18s rRNA was used as a control. All data are normalized relative to control gene spot density. The control gene spot density ranged between 078-098. Symbol represents the average of two replications. The pooled standard deviation is 0.06. 153 APPENDIX 154 Day Ethylene (pUL) SD C02 (mglkg‘h r) Total Volatiles (TIC) 0 0.0074 0.0119 24.61 1509500 5 0.1423 0.0422 18.74 15432000 7 0.0399 0.0288 30.66 1 162500 11 0.1326 0.0385 21.07 1057100 14 0.0225 0.0094 19.69 31861000 18 0.0602 0.0330 18.36 1 12090000 21 0.5149 0.3553 16.59 117370000 25 0.5182 0.2718 24.05 136640000 28 0.5538 0.1666 22.15 472390000 32 0.6956 0.2349 22.63 1 160300000 35 1.5031 0.7281 37.21 1329000000 39 3.0271 1 .8698 50.01 2758000000 42 109.536 46.980 44.50 2755600000 46 97. 825 54.030 30. 89 2997700000 49 179.507 49. 798 21 .99 2913700000 53 413.057 138.730 24.57 2239900000 56 533.818 119.016 21.55 2301200000 60 690.425 283.249 23.17 2083300000 63 485. 390 212.733 20.59 2148300000 67 371.956 94.518 19.07 1759200000 70 482.844 217.468 19.22 1727100000 74 448.329 210.926 19.62 1438100000 77 653.707 150. 345 19.88 1770300000 81 429.090 227.916 20.96 1427500000 Table 5. Original data for Figure 10 (Chapter 3). Data are internal ethylene content, C02 production, and the GC/MS response total ion count (TIC) for all aroma volatiles. Each value is the average of four replications. 155 Bending test (N) Day Replication 11 14 18 21 25 28 32 35 39 Rep 1-1 8.61 8.97 9.40 8.69 9.00 8.88 8.36 8.64 8.88 Rep 1-2 7.23 9.62 9.47 8.60 9.14 9.95 7.51 9.83 8.18 Rep 2-1 10.54 8.53 8.03 7.95 8.37 8.55 9.41 9.14 8.39 Rep 2-2 10.10 8.49 7.64 8.94 7.91 8.65 8.24 10.16 7.55 Rep 3-1 7.79 11.39 9.03 9.21 8.00 7.82 7.34 10.45 9.08 Rep 3-2 8.64 12.20 7.64 10.20 8.36 8.63 8.59 11.01 8.92 Rep 4-1 10.49 8.65 11.98 7.81 8.33 9.15 8.74 9.66 8.81 Rep 4-2 9.89 8.53 9.70 8.47 8.07 9.25 8.17 10.05 7.89 Day Replication 42 46 49 53 56 60 63 67 70 Rep 1-1 4.85 2.31 2.88 3.54 3.52 3.89 2.17 2.33 1.84 Rep 1-2 4.73 2.27 2.49 3.88 3.49 3.94 2.55 3.03 1.69 Rep 2-1 3.00 1.85 4.03 3.45 3.07 3.09 2.42 2.35 1.72 Rep 2-2 3.09 1.96 3.89 4.22 2.62 2.47 2.67 2.52 1.91 Rep 3—1 5.50 1.96 3.92 4.26 3.36 1.57 3.11 1.73 2.10 Rep 3-2 6.03 2.27 3.52 3.13 3.63 2.21 3.24 1.59 1.91 Rep 4-1 7.55 2.90 3.57 2.42 2.96 2.39 2.30 2.71 2.37 Rep 4-2 3.58 2.88 2.58 2.79 2.59 2.20 2.41 2.19 Compression test (N) Day Replication 5 7 1 1 14 18 21 25 28 32 35 Rep 1-1 120.39 101.09 93.15 89.89 83.29 77.13 87.71 91.15 78.24 83.32 Rep 1-2 110.81 117.58 88.07 97.40 90.82 89.49 84.39 74.07 76.20 86.52 Rep 2-1 111.12 88.02 94.87 89.59 84.29 91.55 83.53 70.38 61.48 89.14 Rep 2-2 99.21 99.68 96.48 102.98 80.40 79.38 83.50 71.70 73.71 79.37 Rep 3-1 104.91 108.98 93.59 107.06 88.50 80.01 84.62 63.98 81.65 85.21 Rep 3-2 97.62 94.55 94.99 86.60 81.64 83.54 79.43 94.75 76.94 86.63 Rep 4-1 90.60 94.62 91.04 90.88 86.26 90.44 87.53 84.02 70.74 73.66 Rep 4-2 96.38 109.41 96.31 80.11 77.65 91.72 82.46 69.68 91.87 83.85 Rep 4-3 94.82 Day Replication 39 42 46 49 53 56 60 63 67 70 Rep 1-1 83.76 44.73 39.46 32.60 44.64 40.81 58.82 35.57 39.05 40.00 Rep 1-2 70.04 41.85 40.46 44.23 32.40 45.50 50.98 31.77 32.86 29.85 Rep 2-1 67.58 43.73 32.10 45.87 50.43 47.99 48.66 42.15 31.60 37.09 Rep 2-2 78.06 39.12 20.66 52.92 48.60 42.11 42.30 36.32 35.95 32.27 Rep 3-1 79.77 51.14 30.55 42.98 41.22 29.09 32.73 35.94 17.91 29.81 Rep 3-2 78.26 58.97 30.17 36.15 38.84 30.79 37.88 46.62 22.25 30.79 Rep 4-1 75.36 47.54 45.65 34.36 41.54 47.95 33.30 38.83 30.33 39.55 Rep 4-2 44.73 56.93 41.84 36.10 32.21 43.14 36.50 33.13 33.35 30.75 Table 6. Original data for Figure 11 (Chapter 3). Force (N) for bending/tensile failure and compressive failure during ripening and senescence of ‘Jonagold’ apple fruit. 156 No. Compound RT (second) Class 1 Butanal 114.31 Aldehyde 2 Ethyl acetate 114.86 Ester 3 2-Methylbutanal 120.13 Aldehyde 4 Ethanol 121.68 Alcohol 5 Ethyl propanoate 127.09 Ester 6 Propyl acetate 130.21 Ester 7 2-Methylpropyl acetate 137.68 Ester 8 Propanol 141.03 Alcohol 9 Ethyl butanoate 142.62 Ester 10 Propylpropanoate 143.89 Ester 11 Ethyl 2-methylbutanoate 145.97 Ester 12 Butyl acetate 149.89 Ester 13 Hexanal 153.21 Aldehyde 14 2-Methylbutyl acetate 160.37 Ester 15 Propylbutanoate 161.59 Ester 16 Butanol 163.64 Alcohol 17 PropyI2-methylbutanoate 164.27 Ester 18 Butylpropanoate 165.29 Ester 19 Pentyl acetate 171.19 Ester 20 Pentylpropanoate 175.84 Ester 21 2-Methylbutanol 178.97 Alcohol 22 Butylbutanoate 182.70 Ester 23 Butyl2-methylbutanoate 185.82 Ester 24 2-Methylbutylbutanoate 192.09 Ester 25 Hexyl acetate 194.95 Ester 26 Propylhexanoate 203.79 Ester 27 Butylpentanoate 203.84 Ester 28 PentyIZ-methylbutanoate 207.07 Ester 29 Hexylpropanoate 209.40 Ester 30 Hexanol 210.10 Alcohol 31 Butylhexanoate 225.22 Ester 32 Hexylbutanoate 225.50 Ester 33 HexyI2-methylbutanoate 227.82 Ester 34 Pentylhexanoate 245.20 Ester 35 Butylheptanoate 245.40 Ester 36 Propyloctanoate 245.98 Ester 37 Hexylhexanoate 264.60 Ester 38 Butyloctanoate 264.96 Ester 39 Hexyloctanoate 300.86 Ester Table 7. Original data for Figure 12 (Chapter 3). Data are volatile compounds, GC retention time (second), and classes (esters, alcohols, and aldehydes) identified from ‘Jonagold’ apple fruit during ripening and senescence. 157 Day Ethanol Propanol Butanol Hexanol 2-Mbutanol 0 0 0 0 0 0 5 0 0 0 0 0 7 0 0 0 0 0 11 0 0 0 0 0 14 0 0 0 0 0 18 0 0 0 0 0 21 0 0 0 0 1277184 25 0 0 130985 0 1356999 28 0 65117 280817310988762 2444501 32 0 172094 7243258 16803031 10570357 35 0 216622 6393902 17336530 10402166 39 0 1545900 22210397 18375822 74834387 42 45755 1377952 22670479 16568597 15101710 46 0 5126017 37799365 14080643 17507796 49 0 6574075 30097459 12208675 16221393 53 39537 12422605 31316951 8795541 16073867 56 80978 14816826 31705307 11363277 19166305 60 142928 12947110 34101107 8701354 18636585 63 242565 12920652 33910922 12122987 25520562 67 171391 13122409 38082128 8224758 22429805 70 237862 15718028 44701783 10800553 31140692 74 178244 10428265 28421878 7438556 21044632 77 172917 10763077 31327303 10844211 24048526 81 244142 12196363 33790393 6137536 29826410 Ion 45 31 33 31 41 lon fraction 0.22358 0.55748 0.01690 0.09059 0.17844 Table 8. Original data for Figure 13A and 14A (Chapter 3). Data are the GCIMS response (total ion count, TIC) of alcohols produced by ‘Jonagold’ apple fruit during ripening and senescence. The GCIMS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TIC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. 158 Day Ethanol Propanol Butanol Hexanol 2-Mbutanol 0 nd nd nd nd nd 5 nd nd nd nd nd 7 nd nd nd nd nd 11 nd nd nd nd nd 14 nd nd nd nd nd 18 nd nd nd nd nd 21 0.00 0.00 0.00 0.00 100.00 25 0.00 0.00 8.80 0.00 91.20 28 0.00 0.40 17.22 67.39 14.99 32 0.00 0.49 20.82 48.30 30.38 35 0.00 0.63 18.61 50.47 30.28 39 0.00 1.32 18.99 15.71 63.98 42 0.08 2.47 40.65 29.71 27.08 46 0.00 6.88 50.73 18.90 23.50 49 0.00 10.10 46.23 18.75 24.92 53 0.06 18.10 45.62 12.81 23.41 56 0.10 19.21 41.10 14.73 24.85 60 0.19 17.37 45.76 11.68 25.01 63 0.29 15.25 40.03 14.31 30.12 67 0.21 16.00 46.42 10.03 27.34 70 0.23 15.32 43.57 10.53 30.35 74 0.26 15.45 42.10 11.02 31.17 77 0.22 13.95 40.60 14.05 31.17 81 0.30 14.84 41.11 7.47 36.29 Table 9. Original data for Figure 138 and 148 (Chapter 3). Data are the percentages that each alcohol class comprises of all alcohols detected on each date using the respective GCIMS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no alcohols of these classes were detected. 159 Ethanol Propanol Butanol 2-Mbutanol Pentanol Hexanol Day esters esters esters esters esters esters 0 0 0 0 0 0 0 5 0 0 0 0 0 0 7 0 0 0 0 0 0 1 1 0 0 0 0 0 0 14 0 0 73330 544232 0 58889 18 0 0 83154 66706 0 570601 21 0 0 4650657 4135506 1248775 9151795 25 0 0 7648340 4476358 3988414 14924860 28 0 486171 104050337 27454993 24440953 177021079 32 105421 1 3768227 214015744 107774833 45983035 367175606 35 957638 7505138 233873587 106544048 39889493 322774259 39 3383294 93199521 775917475 415832480 192039727 727724087 42 638924 61534735 617500508 217011577 105136342 745865025 46 2707736 195737296 775501480 239357798 97455215 700593932 49 3565715 245034720 759929280 241729378 89219198 614885143 53 5077692 286498097 561 181076 215081835 58239977 345668171 56 7602839 369272427 664983847 254520293 91627767 469876634 60 8603966 313026664 600304346 219037073 64556391 359405892 63 6916506 304853959 606827901 241751786 87970809 426317622 67 1 1045524 262074575 509882747 203489888 54227242 250314174 70 14128221 283759336 534265021 221951898 59521053 296472154 74 8806151 239687287 412288858 197089486 42315368 217490846 77 1 1560499 268848735 537759061 221358650 70492799 294728574 81 15217937 264940302 476333456 213434085 55658328 201483545 Total 101266855 3200227190 8397070205 3352642903 1184010886 6542502888 Table 10. Original data for Figure 14A (Chapter 3). Data are the GCIMS response (total ion count) of esters arranged by their alkyl (alcohol-derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. 160 Ethanol Propanol Butanol 2—Mbutanol Pentanol Hexanol Day esters esters esters esters esters esters 0 nd nd nd nd nd nd 5 nd nd nd nd nd nd 7 nd nd nd nd nd nd 11 nd nd nd nd nd nd 14 0.00 0.00 10.84 80.45 0.00 8.71 18 0.00 0.00 11.54 9.26 0.00 79.20 21 0.00 0.00 24.24 21.55 6.51 47.70 25 0.00 0.00 24.64 14.42 12.85 48.09 28 0.00 0.15 31.20 8.23 7.33 53.09 32 0.14 0.51 28.93 14.57 6.22 49.63 35 0.13 1.05 32.87 14.97 5.61 45.36 39 0.15 4.22 35.14 18.83 8.70 32.96 42 0.04 3.52 35.33 12.42 6.02 42.68 46 0.13 9.73 38.56 11.90 4.85 34.83 49 0.18 12.54 38.88 12.37 4.57 31.46 53 0.35 19.47 38.13 14.61 3.96 23.49 56 0.41 19.88 35.79 13.70 4.93 25.29 60 0.55 20.00 38.36 14.00 4.13 22.97 63 0.41 18.20 36.24 14.44 5.25 25.46 67 0.86 20.30 39.49 15.76 4.20 19.39 70 1.00 20.12 37.89 15.74 4.22 21.02 74 0.79 21.45 36.89 17.63 3.79 19.46 77 0.82 19.14 38.28 15.76 5.02 20.98 81 1.24 21.59 38.82 17.39 4.54 16.42 Table 11. Original data for Figure 14B (Chapter 3). Data are the percentages that each ester class comprises of all esters detected on each date using the respective GCIMS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Esters are arranged by the source of the alkyl (alcohol- derived) moiety. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no esters of these classes were detected. 161 Ethyl Ethyl Ethyl Ethyl Day acetate propanoate butanoate 2-Mbutanoate 0 0 0 0 0 5 0 0 0 0 7 0 0 0 0 11 0 O 0 0 14 O 0 0 0 18 0 0 0 0 21 0 0 0 0 25 0 0 0 0 28 0 0 0 0 32 757266 0 101763 195183 35 667936 0 241740 47962 39 1947659 420387 382040 633208 42 452915 0 86705 99303 46 1980734 329346 88585 309071 49 2496052 520588 87348 461727 53 3993076 749268 80848 254500 56 5506875 1296852 183483 615629 60 6117778 1685613 160476 640101 63 51 17759 1 169138 95136 534474 67 8312465 1695279 256405 781374 70 10836238 2004585 270795 1016602 74 6970138 1 176384 171887 487742 77 8130761 2039725 293869 1096144 81 10825273 2672355 400953 1319356 Ion 43 57 88 102 Ion fraction 0.54264 0.26430 0.10491 0.14837 Table 12. Original data for Figure 150 (Chapter 3). Data are the GCIMS response (total ion count, TlC) of ethanol esters arranged by their alkanoate (acid-derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. The GCIMS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TIC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. 162 Ethyl Ethyl Ethyl Ethyl Day acetate propanoate butanoate 2-Mbutanoate 0 nd nd nd nd 5 nd nd nd nd 7 nd nd nd nd 11 nd nd nd nd 14 nd nd nd nd 18 nd nd nd nd 21 nd nd nd nd 25 nd nd nd nd 28 nd nd nd nd 32 71.83 0.00 9.65 18.51 35 69.75 0.00 25.24 5.01 39 57.57 12.43 11.29 18.72 42 70.89 0.00 13.57 15.54 46 73.15 12.16 3.27 11.41 49 70.00 14.60 2.45 12.95 53 78.64 14.76 1.59 5.01 56 72.43 17.06 2.41 8.10 60 71.10 19.59 1.87 7.44 63 73.99 16.90 1.38 7.73 67 75.26 15.35 2.32 7.07 70 76.70 14.19 1.92 7.20 74 79.15 13.36 1.95 5.54 77 70.33 17.64 2.54 9.48 81 71.13 17.56 2.63 8.67 Table 13. Original data for Figure 15E (Chapter 3). Data are the percentages that each ester class comprises of all ethanol esters detected on each date using the respective GCIMS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no esters of these classes were detected. 163 Propyl Propyl Propyl Propyl Propyl Propyl Day acetate propanoate butanoate 2-Mbutanoate hexanoate octanoate 0 0 0 0 0 O 0 5 0 0 0 0 0 0 7 0 0 0 0 0 0 11 0 0 0 0 0 0 14 0 O 0 0 0 0 18 0 0 0 0 0 0 21 0 0 0 0 0 0 25 0 0 0 0 0 0 28 414457 0 71715 0 0 0 32 2048090 507334 633475 494961 84367 0 35 3413804 1485801 1 1 13596 1268839 223098 0 39 37608468 16857424 7388455 26119739 5196125 29310 42 27873726 8012876 5581 135 16001919 3975271 89808 46 114297757 34911242 12656492 26093516 7558456 219833 49 138006569 48247979 16984255 29639901 11916659 239356 53 163563464 59606493 20440790 28782475 13937652 167223 56 188014101 80665998 28531982 49177139 22626920 256286 60 168549933 75814458 22038081 36387441 10170995 65757 63 163483734 70828608 23633593 31639119 15221512 47393 67 154296474 54277559 17207960 28805571 7478334 8678 70 159947361 56294731 19451684 36552478 11489634 23448 74 140654281 44556421 15704732 28558255 10186096 27503 77 135337264 58784522 20012059 39151461 15461013 102416 81 136382064 57285036 20780573 35854075 14597304 41249 Ion 61 75 89 103 117 145 Ion fraction 0.15051 0.16752 0.11242 0.10392 0.12400 0.08257 Table 14. Original data for Figure 16C (Chapter 3). Data are the GCIMS response (total ion count, TIC) of propanol esters arranged by their alkanoate (acid-derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. The GCIMS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TIC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. 164 Propyl Propyl Propyl Propyl PrOpyl Propyl Day acetate propanoate butanoate 2-Mbutanoate hexanoate octanoate 0 nd nd nd nd nd nd 5 nd nd nd nd nd nd 7 nd nd nd nd nd nd 11 nd nd nd nd nd nd 14 nd nd nd nd nd nd 18 nd nd nd nd nd nd 21 nd nd nd nd nd nd 25 nd nd nd nd nd nd 28 85.25 0.00 14.75 0.00 0.00 0.00 32 54.35 13.46 16.81 13.14 2.24 0.00 35 45.49 19.80 14.84 16.91 2.97 0.00 39 40.35 18.09 7.93 28.03 5.58 0.03 42 45.30 13.02 9.07 26.00 6.46 0.15 46 58.39 17.84 6.47 13.33 3.86 0.11 49 56.32 19.69 6.93 12.10 4.86 0.10 53 57.09 20.81 7.13 10.05 4.86 0.06 56 50.91 21.84 7.73 13.32 6.13 0.07 60 53.85 24.22 7.04 11.62 3.25 0.02 63 53.63 23.23 7.75 10.38 4.99 0.02 67 58.88 20.71 6.57 10.99 2.85 0.00 70 56.37 19.84 6.85 12.88 4.05 0.01 74 58.68 18.59 6.55 11.91 4.25 0.01 77 50.34 21.87 7.44 14.56 5.75 0.04 81 51.48 21.62 7.84 13.53 5.51 0.02 Table 15. Original data for Figure 16E (Chapter 3). Data are the percentages that each ester class comprises of all propanol esters detected on each date using the respective GCIMS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no esters of these classes were detected. 165 Butyl Butyl Butyl Butyl Butyl Butyl Day acetate propanoate butanoate 2-Mbutanoate pentanoate hexanoate 0 0 0 0 0 0 0 5 0 0 0 0 0 0 7 O O 0 0 0 0 11 0 0 0 0 0 0 14 73330 0 0 0 0 0 18 83154 0 0 0 0 0 21 4650657 0 0 0 0 0 25 7126722 0 521618 0 0 0 28 78576048 7754501 10032725 3038240 23596 4550864 32 125374274 34876400 19166243 23413937 155087 10785473 35 110905433 55736708 22333337 30182603 333355 14155968 39 270136771 156898323 114983654 148234603 3333613 80063605 42 169785919 147746452 99078766 119929683 2296889 74021672 46 369037955 146293135 87564772 103640952 2193415 62475437 49 344772295 173527613 83716422 98535206 2521761 54547534 53 304316462 141107484 46585937 40169381 1587307 26459589 56 322329500 160173677 67004923 70416587 2788663 41200400 60 302019260 166314968 48951982 57906444 1503062 23169541 63 281995191 163363226 62876066 63177714 2547400 32418850 67 280877951 133389806 38654196 40706810 1001717 14965410 70 272861267 129597161 47697412 55536206 1534070 26739338 74 226101851 105133987 28974460 33423413 955526 17495363 77 251296583 134593058 54297453 62561190 2155057 32326845 81 235478823 118927742 46367945 49531429 1602393 24188531 Ion 61 75 89 103 85 117 lon fraction 0.06377 0.10197 0.12317 0.14318 0.12247 0.09374 Table 16. Original data for Figure 17C (Chapter 3). Data are the GCIMS response (total ion count, TIC) of butanol esters arranged by their alkanoate (acid-derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. The GCIMS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TIC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. 166 Butyl Butyl Day heptanoate octanoate 0 0 0 5 0 0 7 0 0 1 1 0 0 14 0 0 18 0 0 21 0 0 25 0 0 28 0 74365 32 0 244330 35 0 226181 39 402039 1864868 42 290288 4350839 46 562155 3733659 49 415686 1892763 53 1 86450 768466 56 355579 714518 60 142632 296456 63 168758 280696 67 94387 192470 70 121893 177673 74 70193 134066 77 201617 327258 81 83477 1531 17 Ion 1 13 145 Ion fraction 0.06613 0.08478 Table 16. Continued. 167 Butyl Butyl Butyl Butyl Butyl Butyl Butyl Butyl Day acetate propanoate butanoate 2-Mbut pentanoate hexanoate heptanoate octanoate 0 nd nd nd nd nd nd nd nd 5 nd nd nd nd nd nd nd nd 7 nd nd nd nd nd nd nd nd 11 nd nd nd nd nd nd nd nd 14 100.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 18 100.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 21 100.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 25 93.18 0.00 6.82 0.00 0.00 0.00 0.00 0.00 28 75.52 7.45 9.64 2.92 0.02 4.37 0.00 0.07 32 58.58 16.30 8.96 10.94 0.07 5.04 0.00 0.11 35 47.42 23.83 9.55 12.91 0.14 6.05 0.00 0.10 39 34.82 20.22 14.82 19.10 0.43 10.32 0.05 0.24 42 27.50 23.93 16.05 19.42 0.37 11.99 0.05 0.70 46 47.59 18.86 11.29 13.36 0.28 8.06 0.07 0.48 49 45.37 22.83 11.02 12.97 0.33 7.18 0.05 0.25 53 54.23 25.14 8.30 7.16 0.28 4.71 0.03 0.14 56 48.47 24.09 10.08 10.59 0.42 6.20 0.05 0.11 60 50.31 27.71 8.15 9.65 0.25 3.86 0.02 0.05 63 46.47 26.92 10.36 10.41 0.42 5.34 0.03 0.05 67 55.09 26.16 7.58 7.98 0.20 2.94 0.02 0.04 70 51.07 24.26 8.93 10.39 0.29 5.00 0.02 0.03 74 54.84 25.50 7.03 8.11 0.23 4.24 0.02 0.03 77 46.73 25.03 10.10 11.63 0.40 6.01 0.04 0.06 81 49.44 24.97 9.73 10.40 0.34 5.08 0.02 0.03 Table 17. Original data for Figure 17E (Chapter 3). Data are the percentages that each ester class comprises of all butanol esters detected on each date using the respective GCIMS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no esters of these classes were detected. 168 Pentyl Pentyl Pentyl Pentyl Day acetate propanoate 2-Mbutanoate hexanoate 0 0 0 0 0 5 0 0 0 0 7 O 0 0 0 11 0 0 0 0 14 0 0 0 0 18 0 0 0 0 21 1248775 0 0 0 25 3988414 0 0 0 28 24028772 0 412181 0 32 38817751 1776737 3593381 1795166 35 31414858 4204037 2772000 1498598 39 98396608 66886681 16874175 9882263 42 78811290 17431405 5237831 3655815 46 75671392 15422169 3025049 3336606 49 65864975 19871052 2126883 1356288 53 45228066 12080687 412261 518962 56 63259797 25744242 1331161 1292567 60 45953173 17671340 931878 0 63 54003345 31785957 1503927 677580 67 40336013 13435316 455913 0 70 42191914 16425812 903327 0 74 29308441 12605036 401891 0 77 43996128 24717999 1337721 440952 81 35918068 19125035 615226 0 Ion 61 53 103 117 Ion fraction 0.06723 0.00346 0.14220 0.01772 Table 18. Original data for Figure 188 (Chapter 3). Data are the GCIMS response (total ion count, TIC) of pentanol esters arranged by their alkanoate (acid-derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. The GCIMS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TIC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. 169 Pentyl Pentyl Pentyl Pentyl Day acetate propanoate 2-Mbutanoate hexanoate 0 nd nd nd nd 5 nd nd nd nd 7 nd nd nd nd 11 nd nd nd nd 14 nd nd nd nd 18 nd nd nd nd 21 100.00 0.00 0.00 0.00 25 100.00 0.00 0.00 0.00 28 98.31 0.00 1.69 0.00 32 84.42 3.86 7.81 3.90 35 78.75 10.54 6.95 3.76 39 51.24 34.83 8.79 5.15 42 74.96 16.58 4.98 3.48 46 77.65 15.82 3.10 3.42 49 73.82 22.27 2.38 1.52 53 77.66 20.74 0.71 0.89 56 69.04 28.10 1.45 1.41 60 71.18 27.37 1.44 0.00 63 61.39 36.13 1.71 0.77 67 74.38 24.78 0.84 0.00 70 70.89 27.60 1.52 0.00 74 69.26 29.79 0.95 0.00 77 62.41 35.06 1.90 0.63 81 64.53 34.36 1.11 0.00 Table 19. Original data for Figure 18C (Chapter 3). Data are the percentages that each ester class comprises of all pentanol esters detected on each date using the respective GCIMS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no esters of these classes were detected. 170 Hexyl Hexyl Hexyl Hexyl Hexyl Hexyl Day acetate propanoate butanoate 2-Mbutanoate hexanoate octanoate 0 0 0 0 0 0 0 5 0 0 0 0 0 0 7 0 0 0 0 0 0 11 0 0 0 0 0 0 14 58889 0 0 0 0 0 18 570601 0 0 0 0 0 21 9151795 0 0 0 0 0 25 14924860 0 0 0 0 0 28 131498506 5522563 12387526 23743315 3869168 0 32 183912774 22733102 17414263 137088190 6027278 0 35 152882255 30808824 19759306 113268595 6055280 0 39 294259645 84247680 72800548 256254115 20144312 17787 42 364627548 108166313 77847525 170940079 23837029 446530 46 332492806 108249568 74261456 158676067 26447777 466259 49 323247968 110095687 55248853 112904713 13221424 166498 53 225050323 53572738 22004134 36898464 8005050 137463 56 262989871 86296100 36881668 75432768 8276227 0 60 213873194 68823064 24181018 48854871 3673745 0 63 225978774 85414278 35304101 72644413 6976055 0 67 174267987 32684977 13288105 26193534 3879571 0 70 185370126 41161098 23144300 43018220 3778410 0 74 155662452 25983911 12408862 21276635 2158986 0 77 174419158 49696094 23484719 44263571 2865032 0 81 140823494 26606633 13631588 18648496 1773334 0 Ion 61 75 89 103 117 145 Ion fraction 0.06301 0.09639 0.08893 0.13061 0.09131 0.03399 Table 20. Original data for Figure 19C (Chapter 3). Data are the GCIMS response (total ion count, TIC) of hexanol esters arranged by their alkanoate (acid-derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. The GC/MS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TIC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. 171 Hexyl Hexyl Hexyl Hexyl Hexyl Hexyl Day acetate propanoate butanoate 2-Mbutanoate hexanoate octanoate 0 nd nd nd nd nd nd 5 nd nd nd nd nd nd 7 nd nd nd nd nd nd 11 nd nd nd nd nd nd 14 . nd nd nd nd nd nd 18 100.00 0.00 0.00 0.00 0.00 0.00 21 100.00 0.00 0.00 0.00 0.00 0.00 25 100.00 0.00 0.00 0.00 0.00 0.00 28 74.28 3.12 7.00 13.41 2.19 0.00 32 50.09 6.19 4.74 37.34 1.64 0.00 35 47.37 9.55 6.12 35.09 1.88 0.00 39 40.44 11.58 10.00 35.21 2.77 0.00 42 48.89 14.50 10.44 22.92 3.20 0.06 46 47.46 15.45 10.60 22.65 3.78 0.07 49 52.57 17.91 8.99 18.36 2.15 0.03 53 65.11 15.50 6.37 10.67 2.32 0.04 56 55.97 18.37 7.85 16.05 1.76 0.00 60 59.51 19.15 6.73 13.59 1.02 0.00 63 53.01 20.04 8.28 17.04 1.64 0.00 67 69.62 13.06 5.31 10.46 1.55 0.00 70 62.53 13.88 7.81 14.51 1.27 0.00 74 71.57 11.95 5.71 9.78 0.99 0.00 77 59.18 16.86 7.97 15.02 0.97 0.00 81 69.89 13.21 6.77 9.26 0.88 0.00 Table 21. Original data for Figure 19E (Chapter 3). Data are the percentages that each ester class comprises of all hexanol esters detected on each date using the respective GCIMS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no esters of these classes were detected. 172 2-Mbutyl 2-Mbutyl Day acetate butanoate 0 0 0 5 0 0 7 0 0 11 0 0 14 544232 0 18 66706 0 21 4135506 0 25 4476358 0 28 27454993 0 32 107605390 169444 35 106339430 204619 39 403136230 12696250 42 215097186 1914391 46 238299202 1058597 49 241304588 424791 53 214884501 197334 56 254053612 466681 60 218814342 222731 63 241371052 380734 67 203334247 155641 70 221685592 266306 74 196966986 122500 77 220904941 453709 81 213224107 209978 Ion 74 89 Ion fraction 0.02069 0.01 143 Table 22. Original data for Figure 200 (Chapter 3). Data are the GCIMS response (total ion count, TIC) of 2-methylbutanol esters arranged by their alkanoate (acid-derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. The GCIMS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TIC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. 173 2-Mbutyl 2-Mbutyl Day acetate butanoate 0 nd nd 5 nd nd 7 nd nd 11 nd nd 14 100.00 0.00 18 100.00 0.00 21 100.00 0.00 25 100.00 0.00 28 100.00 0.00 32 99.84 0.16 35 99.81 0.19 39 96.95 3.05 42 99.12 0.88 46 99.56 0.44 49 99.82 0.18 53 99.91 0.09 56 99.82 0.18 60 99.90 0.10 63 99.84 0.16 67 99.92 0.08 70 99.88 0.12 74 99.94 0.06 77 99.80 0.20 81 99.90 0.10 Table 23. Original data for Figure 20E (Chapter 3). Data are the percentages that each ester class comprises of all 2-methylbutanol esters detected on each date using the respective GCIMS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no esters of these classes were detected. 174 Acetate Propanoate Butanoate 2-Mbutanoate Hexanoate Octanoate Day esters esters esters esters esters esters 0 0 0 0 0 0 0 5 0 0 0 0 0 0 7 0 0 0 0 0 0 1 1 0 0 0 0 0 0 14 676452 0 0 0 0 0 18 720461 0 0 0 0 0 21 19230579 0 0 0 0 0 25 31001745 0 521618 0 0 0 28 265402885 13277064 22491965 27193736 8420031 74365 32 462973702 59893573 37485187 164785652 18692283 244330 35 413714315 92235371 43652598 147539999 21932943 226181 39 1 139999484 325310494 208250947 4481 15840 1 15286304 191 1966 42 866341985 281357046 184508522 312208815 105489788 4887176 46 1 152954103 305205461 175629902 291744655 99818276 4419750 49 1 136140920 352262919 156461668 243668431 81041906 2298617 53 979235446 2671 16669 89309043 106517081 48921254 1073152 56 1 124061950 354176869 133068737 196973284 733961 15 970803 60 979347835 330309443 95554288 144720735 37014281 362214 63 999597405 352561208 122289631 169499647 55293997 328089 67 886777792 235482938 69562306 96943201 26323315 201 148 70 924436609 245483388 90830496 137026833 42007382 201 122 74 772449834 189455739 57382441 84147936 29840445 161568 77 855603196 269831398 98541809 148410087 51093842 429674 81 797389466 224616800 81391037 105968582 40559169 194366 Total 13808056163 3898576380 1666932196 2825464515 855131329 17984521 Table 24. Original data for Figure 21A (Chapter 3). Data are the GCIMS response (total ion count) of esters arranged by their alkanoate (acid-derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. 175 Acetate Propanoate Butanoate 2-Mbutanoate Hexanoate Octanoate Day esters esters esters esters esters esters 0 nd nd nd nd nd nd 5 nd nd nd nd nd nd 7 nd nd nd nd nd nd 11 nd nd nd nd nd nd 14 100.00 0.00 0.00 0.00 0.00 0.00 18 100.00 0.00 0.00 0.00 0.00 0.00 21 100.00 0.00 0.00 0.00 0.00 0.00 25 98.35 0.00 1.65 0.00 0.00 0.00 28 78.79 3.94 6.68 8.07 2.50 0.02 32 62.22 8.05 5.04 22.15 2.51 0.03 35 57.52 12.82 6.07 20.51 3.05 0.03 39 50.92 14.53 9.30 20.02 5.15 0.09 42 49.37 16.03 10.51 17.79 6.01 0.28 46 56.80 15.04 8.65 14.37 4.92 0.22 49 57.62 17.86 7.93 12.36 4.11 0.12 53 65.62 17.90 5.99 7.14 3.28 0.07 56 59.71 18.81 7.07 10.46 3.90 0.05 60 61.70 20.81 6.02 9.12 2.33 0.02 63 58.81 20.74 7.20 9.97 3.25 0.02 67 67.42 17.90 5.29 7.37 2.00 0.02 70 64.20 17.05 6.31 9.52 2.92 0.01 74 68.15 16.72 5.06 7.42 2.63 0.01 77 60.09 18.95 6.92 10.42 3.59 0.03 81 63.79 17.97 6.51 8.48 3.24 0.02 Table 25. Original data for Figure 21 B (Chapter 3). Data are the percentages that each ester class comprises of all esters detected on each date using the respective GCIMS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Esters are arranged by the source of the alkanoate (acid- derived) moiety. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no esters of these classes were detected. 176 Day Ethyl acetate Pr0py| acetate Butyl acetate Pentyl acetate Hexyl acetate 2-Mpropyl acetate 2-Mbutyl acetate 11 14 18 21 25 28 32 35 39 42 46 49 53 56 60 63 67 70 74 77 81 000000000 757266 667936 1947659 452915 1980734 2496052 3993076 5506875 6117778 5117759 8312465 10836238 6970138 8130761 10825273 0 0 0 O 0 0 0 0 414457 2048090 3413804 37608468 27873726 114297757 138006569 163563464 188014101 168549933 163483734 154296474 159947361 140654281 135337264 136382064 0000 73330 83154 4650657 7126722 78576048 125374274 110905433 270136771 169785919 369037955 344772295 304316462 322329500 302019260 281995191 280877951 272861267 226101851 251296583 235478823 000000 1248775 3988414 24028772 38817751 31414858 98396608 78811290 75671392 65864975 45228066 63259797 45953173 54003345 40336013 42191914 29308441 43996128 35918068 0000 58889 570601 9151795 14924860 131498506 183912774 152882255 294259645 364627548 332492806 323247968 225050323 262989871 213873194 225978774 174267987 185370126 155662452 174419158 140823494 0 0 0 0 0 0 43846 485391 3430109 4458158 8090600 34514103 9693400 21174258 20448473 22199554 27908195 24020155 27647550 25352654 31544110 16785685 21518361 24737638 0000 544232 66706 4135506 4476358 27454993 107605390 106339430 403136230 215097186 238299202 241304588 214884501 254053612 218814342 241371052 203334247 221685592 196966986 220904941 213224107 Ion 43 61 61 61 61 56 74 Ion fraction 0.54264 0.15051 0.06377 0.06723 0.06301 011443 0.02069 Table 26. Original data for Figure 22B (Chapter 3). Data are the GCIMS response (total ion count, TIC) of acetate esters arranged by their alkyl (alcohol- derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. The GCIMS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TIC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. 177 Ethyl Propyl Butyl Pentyl Hexyl 2-Mpropyl 2-Mbutyl Day acetate acetate acetate acetate acetate acetate acetate 0 nd nd nd nd nd nd nd 5 nd nd nd nd nd nd nd 7 nd nd nd nd nd nd nd 11 nd nd nd nd nd nd nd 14 0.00 0.00 10.84 0.00 8.71 0.00 80.45 18 0.00 0.00 11.54 0.00 79.20 0.00 9.26 21 0.00 0.00 24.18 6.49 47.59 0.23 21.50 25 0.00 0.00 22.99 12.87 48.14 1.57 14.44 28 0.00 0.16 29.61 9.05 49.55 1.29 10.34 32 0.16 0.44 27.08 8.38 39.72 0.96 23.24 35 0.16 0.83 26.81 7.59 36.95 1.96 25.70 39 0.17 3.30 23.70 8.63 25.81 3.03 35.36 42 0.05 3.22 19.60 9.10 42.09 1.12 24.83 46 0.17 9.91 32.01 6.56 28.84 1.84 20.67 49 0.22 12.15 30.35 5.80 28.45 1.80 21.24 53 0.41 16.70 31.08 4.62 22.98 2.27 21.94 56 0.49 16.73 28.68 5.63 23.40 2.48 22.60 60 0.62 17.21 30.84 4.69 21.84 2.45 22.34 63 0.51 16.35 28.21 5.40 22.61 2.77 24.15 67 0.94 17.40 31.67 4.55 19.65 2.86 22.93 70 1.17 17.30 29.52 4.56 20.05 3.41 23.98 74 0.90 18.21 29.27 3.79 20.15 2.17 25.50 77 0.95 15.82 29.37 5.14 20.39 2.51 25.82 81 1.36 17.10 29.53 4.50 17.66 3.10 26.74 Table 27. Original data for Figure 220 (Chapter 3). Data are the percentages that each ester class comprises of all acetate esters detected on each date using the respective GCIMS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no esters of these classes were detected. 178 Ethyl Propyl Butyl Pentyl Hexyl Day propanoate propanoate propanoate propanoate propanoate 0 0 0 0 0 0 5 0 0 0 0 0 7 0 0 0 0 0 1 1 0 0 0 0 0 14 0 0 0 0 0 1 8 0 0 0 0 0 21 0 0 0 0 0 25 0 0 0 0 0 28 0 0 7754501 0 5522563 32 0 507334 34876400 1776737 22733102 35 0 1485801 55736708 4204037 30808824 39 420387 16857424 156898323 66886681 84247680 42 0 8012876 147746452 17431405 108166313 46 329346 3491 1242 146293135 15422169 108249568 49 520588 48247979 173527613 19871052 1 10095687 53 749268 59606493 141 107484 12080687 53572738 56 1296852 80665998 160173677 25744242 86296100 60 1685613 75814458 166314968 17671340 68823064 63 1169138 70828608 163363226 31785957 85414278 67 1695279 54277559 133389806 13435316 32684977 70 2004585 56294731 129597161 16425812 41161098 74 1 176384 44556421 105133987 12605036 2598391 1 77 2039725 58784522 134593058 24717999 49696094 81 2672355 57285036 1 18927742 19125035 26606633 Ion 57 75 75 53 75 Ion fraction 0.26430 0.16752 0.10197 0.00346 0.09639 Table 28. Original data for Figure 238 (Chapter 3). Data are the GCIMS response (total ion count, TIC) of propanoate esters arranged by their alkyl (alcohol-derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. The GCIMS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TIC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. 179 Ethyl Propyl Butyl Pentyl Hexyl Day propanoate propanoate propanoate ropanoate propanoate 0 nd nd nd nd nd 5 nd nd nd nd nd 7 nd nd nd nd nd 11 nd nd nd nd nd 14 nd nd nd nd nd 18 nd nd nd nd nd 21 nd nd nd nd nd 25 nd nd nd nd nd 28 0.00 0.00 58.41 0.00 41.59 32 0.00 0.85 58.23 2.97 37.96 35 0.00 1.61 60.43 4.56 33.40 39 0.13 5.18 48.23 20.56 25.90 42 0.00 2.85 52.51 6.20 38.44 46 0.11 11.44 47.93 5.05 35.47 49 0.15 13.70 49.26 5.64 31.25 53 0.28 22.31 52.83 4.52 20.06 56 0.37 22.78 45.22 7.27 24.37 60 0.51 22.95 50.35 5.35 20.84 63 0.33 20.09 46.34 9.02 24.23 67 0.72 23.05 56.65 5.71 13.88 70 0.82 22.93 52.79 6.69 16.77 74 0.62 23.52 55.49 6.65 13.72 77 0.76 21.79 49.88 9.16 18.42 81 1.19 25.50 52.95 8.51 11.85 Table 29. Original data for Figure 23C (Chapter 3). Data are the percentages that each ester class comprises of all propanoate esters detected on each date using the respective GCIMS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no esters of these classes were detected. 180 Ethyl Propyl Butyl 2-Mbutyl Hexyl Day butanoate butanoate butanoate butanoate butanoate 0 0 0 0 0 0 5 0 0 0 0 0 7 O 0 0 0 0 11 0 O 0 0 0 14 0 0 0 0 0 18 0 0 0 0 0 21 0 0 0 0 0 25 0 0 521618 0 0 28 0 71715 10032725 0 12387526 32 101763 633475 19166243 169444 17414263 35 241740 1113596 22333337 204619 19759306 39 382040 7388455 114983654 12696250 72800548 42 86705 5581135 99078766 1914391 77847525 46 88585 12656492 87564772 1058597 74261456 49 87348 16984255 83716422 424791 55248853 53 80848 20440790 46585937 197334 22004134 56 183483 28531982 67004923 466681 36881668 60 160476 22038081 48951982 222731 24181018 63 95136 23633593 62876066 380734 35304101 67 256405 17207960 38654196 155641 13288105 70 270795 19451684 47697412 266306 23144300 74 171887 15704732 28974460 122500 12408862 77 293869 20012059 54297453 453709 23484719 81 400953 20780573 46367945 209978 13631588 Ion 88 89 89 89 89 Ion fraction 0.10491 0.11242 0.12317 0.01143 0.08893 Table 30. Original data for Figure 24B (Chapter 3). Data are the GCIMS response (total ion count, TIC) of butanoate esters arranged by their alkyl (alcohol-derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. The GCIMS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TlC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. 181 Ethyl Propyl Butyl 2-Mbutyl Hexyl Day butanoate butanoate butanoate butanoate butanoate 0 nd nd nd nd nd 5 nd nd nd nd nd 7 nd nd nd nd nd 11 nd nd nd nd nd 14 nd nd nd nd nd 18 nd nd nd nd nd 21 nd nd nd nd nd 25 nd nd nd nd nd 28 0.00 0.32 44.61 0.00 55.08 32 0.27 1.69 51.13 0.45 46.46 35 0.55 2.55 51.16 0.47 45.26 39 0.18 3.55 55.21 6.10 34.96 42 0.05 3.02 53.70 1.04 42.19 46 0.05 7.21 49.86 0.60 42.28 49 0.06 10.86 53.51 0.27 35.31 53 0.09 22.89 52.16 0.22 24.64 56 0.14 21.44 50.35 0.35 27.72 60 0.17 23.06 51.23 0.23 25.31 63 0.08 19.33 51.42 0.31 28.87 67 0.37 24.74 55.57 0.22 19.10 70 0.30 21.42 52.51 0.29 25.48 74 0.30 27.37 50.49 0.21 21.62 77 0.30 20.31 55.10 0.46 23.83 81 0.49 25.53 56.97 0.26 16.75 Table 31. Original data for Figure 240 (Chapter 3). Data are the percentages that each ester class comprises of all butanoate esters detected on each date using the respective GCIMS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no esters of these classes were detected. 182 Propyl Butyl Pentyl Hexyl Day hexanoate Hexanoate hexanoate hexanoate 0 0 0 0 0 5 0 0 0 0 7 0 0 0 0 11 0 0 0 0 14 0 0 0 0 18 0 0 0 0 21 0 0 0 0 25 0 0 0 0 28 0 4550864 0 3869168 32 84367 10785473 1795166 6027278 35 223098 14155968 1498598 6055280 39 5196125 80063605 9882263 20144312 42 3975271 74021672 3655815 23837029 46 7558456 62475437 3336606 26447777 49 11916659 54547534 1356288 13221424 53 13937652 26459589 518962 8005050 56 22626920 41200400 1292567 8276227 60 10170995 23169541 0 3673745 63 15221512 32418850 677580 6976055 67 7478334 14965410 0 3879571 70 11489634 26739338 0 3778410 74 10186096 17495363 0 2158986 77 15461013 32 326845 440952 2865032 81 14597304 24188531 0 1773334 Ion 117 117 117 117 Ion fraction 0.12400 0.09374 0.01772 0.09131 Table 32. Original data for Figure 25B (Chapter 3). Data are the GCIMS response (total ion count, TIC) of hexanoate esters arranged by their alkyl (alcohol-derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. The GCIMS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TIC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. 183 Propyl Butyl Pentyl Hexyl Day hexanoate hexanoate hexanoate hexanoate 0 nd nd nd nd 5 nd nd nd nd 7 nd nd nd nd 11 nd nd nd nd 14 nd nd nd nd 18 nd nd nd nd 21 nd nd nd nd 25 nd nd nd nd 28 0.00 54.05 0.00 45.95 32 0.45 57.70 9.60 32.24 35 1.02 64.54 6.83 27.61 39 4.51 69.45 8.57 17.47 42 3.77 70.17 3.47 22.60 46 7.57 62.59 3.34 26.50 49 14.70 67.31 1.67 16.31 53 28.49 54.09 1.06 16.36 56 30.83 56.13 1.76 11.28 60 27.48 62.60 0.00 9.93 63 27.53 58.63 1.23 12.62 67 28.41 56.85 0.00 14.74 70 27.35 63.65 0.00 8.99 74 34.14 58.63 0.00 7.24 77 30.26 63.27 0.86 5.61 81 35.99 59.64 0.00 4.37 Table 33. Original data for Figure 250 (Chapter 3). Data are the percentages that each ester class comprises of all hexanoate esters detected on each date using the respective GCIMS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no esters of these classes were detected. 184 Propyl Butyl Hexyl Day octanoate octanoate octanoate 0 0 0 0 5 0 0 0 7 0 0 0 1 1 0 0 O 14 0 0 0 18 0 0 0 21 0 0 0 25 0 0 0 28 0 74365 0 32 0 244330 0 35 0 226181 0 39 29310 1864868 17787 42 89808 4350839 446530 46 219833 3733659 466259 49 239356 1892763 166498 53 167223 768466 137463 56 256286 714518 0 60 65757 296456 0 63 47393 280696 0 67 8678 192470 0 70 23448 177673 0 74 27503 134066 0 77 102416 327258 0 81 41249 1531 17 0 Ion 145 145 145 lon fraction 0.08257 0.08478 0.03399 Table 34. Original data for Figure 263 (Chapter 3). Data are the GCIMS response (total ion count, TIC) of octanoate esters arranged by their alkyl (alcohol-derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. The GCIMS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TIC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. 185 Propyl Butyl Hexyl Day octanoate octanoate octanoate 0 nd nd nd 5 nd nd nd 7 nd nd nd 11 nd nd nd 14 nd nd nd 18 nd nd nd 21 nd nd nd 25 nd nd nd 28 0.00 100.00 0.00 32 0.00 100.00 0.00 35 0.00 100.00 0.00 39 1.53 97.54 0.93 42 1.84 89.03 9.14 46 4.97 84.48 10.55 49 10.41 82.34 7.24 53 15.58 71.61 12.81 56 26.40 73.60 0.00 60 18.15 81.85 0.00 63 14.45 85.55 0.00 67 4.31 95.69 0.00 70 11.66 88.34 0.00 74 17.02 82.98 0.00 77 23.84 76.16 0.00 81 21.22 78.78 0.00 Table 35. Original data for Figure 260 (Chapter 3). Data are the percentages that each ester class comprises of all octanoate esters detected on each date using the respective GCIMS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no esters of these classes were detected. 186 Ethyl Propyl Butyl Pentyl Hexyl Day 2-Mbut 2-Mbut 2-Mbut 2-Mbut 2-Mbut 0 0 0 0 0 0 5 0 0 0 0 0 7 0 0 O 0 0 1 1 0 0 0 0 0 14 0 0 0 0 0 18 0 0 0 0 0 21 0 0 0 0 0 25 0 0 0 0 0 28 0 0 3038240 412181 23743315 32 195183 494961 23413937 3593381 137088190 35 47962 1268839 30182603 2772000 1 13268595 39 633208 26119739 148234603 16874175 256254115 42 99303 16001919 1 19929683 5237831 170940079 46 309071 26093516 103640952 3025049 158676067 49 461727 29639901 98535206 2126883 1 12904713 53 254500 28782475 40169381 412261 36898464 56 615629 49177139 70416587 1331161 75432768 60 640101 36387441 57906444 931878 48854871 63 534474 31639119 63177714 1503927 72644413 67 781374 28805571 40706810 455913 26193534 70 1016602 36552478 55536206 903327 43018220 74 487742 28558255 33423413 401891 21276635 77 1096144 39151461 62561190 1337721 44263571 81 1319356 35854075 49531429 615226 18648496 Ion 102 103 103 103 103 Ion fraction 0.14837 0.10392 0.14318 0.14220 0.13061 Table 36. Original data for Figure 27B (Chapter 3). Data are the GCIMS response (total ion count, TIC) of 2-methylbutanoate esters arranged by their alkyl (alcohol-derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. The GCIMS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TIC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. 187 Ethyl Propyl Butyl Pentyl Hexyl Day 2-Mbut 2-Mbut 2-Mbut 2-Mbut 2-Mbut 0 nd nd nd nd nd 5 nd nd nd nd nd 7 nd nd nd nd nd 11 nd nd nd nd nd 14 nd nd nd nd nd 18 nd nd nd nd nd 21 nd nd nd nd nd 25 nd nd nd nd nd 28 0.00 0.00 11.17 1.52 87.31 32 0.12 0.30 14.21 2.18 83.19 35 0.03 0.86 20.46 1.88 76.77 39 0.14 5.83 33.08 3.77 57.18 42 0.03 5.13 38.41 1.68 54.75 46 0.11 8.94 35.52 1.04 54.39 49 0.19 12.16 40.44 0.87 46.34 53 0.24 27.02 37.71 0.39 34.64 56 0.31 24.97 35.75 0.68 38.30 60 0.44 25.14 40.01 0.64 33.76 63 0.32 18.67 37.27 0.89 42.86 67 0.81 29.71 41.99 0.47 27.02 70 0.74 26.68 40.53 0.66 31.39 74 0.58 33.94 39.72 0.48 25.28 77 0.74 26.38 42.15 0.90 29.83 81 1.25 33.83 46.74 0.58 17.60 Table 37. Original data for Figure 270 (Chapter 3). Data are the percentages that each ester class comprises of all 2-methylbutanoate esters detected on each date using the respective GCIMS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no esters of these classes were detected. 188 Day Ethylene (uL/L) SD C02 (mg/kg’hr) Total Volatiles (TIC) 0 0.0074 0.0119 24.61 1509500 5 0.1423 0.0422 18.74 15432000 7 0.0399 0.0288 30.66 1 162500 11 0.1326 0.0385 21.07 1057100 14 0.0225 0.0094 19.69 31861000 18 0.0602 0.0330 18.36 112090000 21 0.5149 0.3553 16.59 117370000 25 0.5182 0.2718 24.05 136640000 28 0.5538 0.1666 22.15 472390000 32 0.6956 0.2349 22.63 1160300000 35 1 .5031 0.7281 37.21 1329000000 39 3.0271 1 .8698 50.01 2758000000 42 1 09.536 46.980 44.50 2755600000 46 97.825 54.030 . 30.89 2997700000 49 179.507 49.798 21 .99 2913700000 53 413.057 138.730 24.57 2239900000 56 533.818 119.016 21.55 2301200000 60 690.425 283.249 23.17 2083300000 63 485.390 212.733 20. 59 2148300000 67 371.956 94.518 19.07 1759200000 70 482.844 217.468 19.22 1727100000 74 448.329 210.926 19.62 1438100000 77 653.707 150.345 1988 1770300000 81 429.090 227.916 20.96 1427500000 Table 38. Original data for Figure 31 (Chapter 4). Data are internal ethylene content, C02 production, and the GCIMS response total ion count (TIC) for all aroma volatiles. Each value is the average of four replications. 189 Total 2-Mbutyl 2-Mbutyl 2-Mbutanol Day acetate butanoate esters 0 0 0 0 5 0 0 0 7 0 0 0 1 1 0 0 0 14 544232 0 544232 18 66706 0 66706 21 4135506 0 4135506 25 4476358 0 4476358 28 27454993 0 27454993 32 107605390 169444 107774833 35 106339430 204619 106544048 39 403136230 12696250 415832480 42 215097186 1914391 217011577 46 238299202 1058597 239357798 49 241304588 424791 241729378 53 214884501 197334 215081835 56 254053612 466681 254520293 60 218814342 222731 219037073 63 241371052 380734 241751786 67 203334247 155641 203489888 70 221685592 266306 221951898 74 196966986 122500 197089486 77 220904941 453709 221358650 81 213224107 209978 213434085 Ion 74 89 Ion fraction 0.02069 0.01143 Table 39. Original data for Figure 32A and 320 (Chapter 4). Data are the GCIMS response (total ion count, TIC) of 2-methylbutanol esters arranged by their alkanoate (acid-derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. The GCIMS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TIC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. 190 Table 40. Original data for Figure 32A and 32D (Chapter 4). Data are the GCIMS response (total ion count, TIC) of 2-methylbutanoate esters arranged by their alkyl (alcohol-derived) moiety during ripening and senescence of ‘Jonagold’ apple fruit. The GCIMS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TlC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. 191 Total Ethyl Propyl Butyl Pentyl Hexyl 2-Mbutanoate Day 2-Mbut 2-Mbut 2-Mbut 2-Mbut 2-Mbut esters 0 0 0 0 0 0 0 5 0 0 0 0 0 0 7 0 0 0 0 0 0 1 1 0 0 0 0 0 0 14 0 0 0 0 0 0 18 0 0 0 0 0 0 21 0 0 0 0 0 0 25 0 0 0 0 0 0 28 0 0 3038240 412181 23743315 27193736 32 195183 494961 23413937 3593381 137088190 164785652 35 47962 1268839 30182603 2772000 113268595 147539999 39 633208 26119739 148234603 16874175 256254115 448115840 42 99303 16001919 119929683 5237831 170940079 312208815 46 309071 26093516 103640952 3025049 158676067 291744655 49 461727 29639901 98535206 2126883 112904713 243668431 53 254500 28782475 40169381 412261 36898464 106517081 56 615629 49177139 70416587 1331161 75432768 196973284 60 640101 36387441 57906444 931878 48854871 144720735 63 534474 31639119 63177714 1503927 72644413 169499647 67 781374 28805571 40706810 455913 26193534 96943201 70 1016602 36552478 55536206 903327 43018220 137026833 74 487742 28558255 33423413 401891 21276635 84147936 77 1096144 39151461 62561190 1337721 44263571 148410087 81 1319356 35854075 49531429 615226 18648496 105968582 Ion 102 103 103 103 103 Ion fraction 0.14837 0.10392 0.14318 0.14220 0.13061 2-Methyl 2-Methyl Day butanol butanal 0 0 5 0 7 0 11 0 0 14 0 0 18 0 0 21 1277184 0 25 1356999 0 28 2444501 0 32 10570357 0 35 10402166 0 39 74834387 275777 42 15101710 0 46 17507796 132737 49 16221393 151713 53 16073867 226604 56 19166305 393190 60 18636585 247492 63 25520562 291262 67 22429805 503137 70 31140692 618697 74 21044632 365645 77 24048526 387025 81 29826410 456629 Ion 41 41 Ion fraction 0.17844 0.21027 Table 41. Original data for Figure 323 (Chapter 4). Data are the GCIMS response (total ion count, TIC) of 2-methylbutanol and 2-methylbutanal produced by ‘Jonagold’ apple fruit during ripening and senescence. The GCIMS response is calculated from data for a single unique ion; the fraction that the ion comprises of the total ion count is provided. Each TIC value is the result of dividing the ion count for the unique ion by the ion fraction. Each value is the average of four replications. 192 Ethyl Propyl Butyl Pentyl Hexyl Day 2-Mbut 2-Mbut 2-Mbut 2-Mbut 2-Mbut 0 nd nd nd nd nd 5 nd nd nd nd nd 7 nd nd nd nd nd 11 nd nd nd nd nd 14 nd nd nd nd nd 18 nd nd nd nd nd 21 nd nd nd nd nd 25 nd nd nd nd nd 28 0.00 0.00 11.17 1.52 87.31 32 0.12 0.30 14.21 2.18 83.19 35 0.03 0.86 20.46 1.88 76.77 39 0.14 5.83 33.08 3.77 57.18 42 0.03 5.13 38.41 1.68 54.75 46 0.11 8.94 35.52 1.04 54.39 49 0.19 12.16 40.44 0.87 46.34 53 0.24 27.02 37.71 0.39 34.64 56 0.31 24.97 35.75 0.68 38.30 60 0.44 25. 14 40.01 0.64 33.76 63 0.32 18.67 37.27 0.89 42.86 67 0.81 29.71 41.99 0.47 27.02 70 0.74 26.68 40.53 0.66 31.39 74 0.58 33.94 39.72 0.48 25.28 77 0.74 26.38 42.15 0.90 29.83 81 1.25 33.83 46.74 0.58 17.60 Table 42. Original data for Figure 32E (Chapter 4). Data are the percentages that each ester class comprises of all 2-methylbutanoate esters detected on each date using the respective GCIMS response (total ion count) during ripening and senescence of ‘Jonagold’ apple fruit. Each value is the average of four replications. The term ‘nd’ indicates percentages were not determined since no esters of these classes were detected. 193 Day BCAT10 PDC1 2"505‘32tfiya'smea'ate (MDC410280) (MDCO15210) ($7387848) 0 0.000 0.000 0.000 11 0.711 -0.172 0.020 25 0.399 -0137 2.605 32 0.835 0.408 4.172 39 1.961 1.282 5.078 49 0.440 2.205 4.880 50 -0.396 1.864 5.064 70 -1523 1.693 5.300 Table 43. Original data for Figure 33 (Chapter 4). Data are the relative luminosity (logz) of microarray elements compared to Day 0 for branched-chain aminotransferase (BCAT), pyruvate decarboxylase (PDC), and 2-isopropylmalate synthase during ripening and senescence of ‘Jonagold’ apple fruit. 194 BCAT1 BCAT2 BCAT3 BCAT4 BCAT5 MDC021750 MDC028270 MDC160840 MDC192460 MD0238040 Day Avg STDEV Avg STDEV Avg STDEV Avg STDEV Avg STDEV 0 0.748 0.008 0.964 0.051 0.808 0.051 0.944 0.079 1.000 0.000 11 0.751 0.094 0.768 0.095 0.817 0.131 0.761 0.268 0.835 0.196 25 0.860 0.108 0.778 0.011 0.935 0.092 0.776 0.197 0.855 0.179 32 1.000 0.000 0.834 0.026 0.986 0.011 0.822 0.251 0.802 0.022 39 0.918 0.010 0.964 0.051 0.890 0.037 0.706 0.241 0.695 0.078 49 0.835 0.035 0.593 0.066 0.920 0.113 0.608 0.268 0.650 0.139 60 0.703 0.121 0.435 0.404 0.754 0.148 0.504 0.017 0.655 0.055 70 0.660 0.084 0.252 0.219 0.655 0.043 0.356 0.048 0.717 0.204 Avg SD 0.074 0.170 0.091 0.198 0.133 BCAT6 BCAT7 BCAT8 BCAT9 BCAT1 0 18s rR NA MDC238050 MDC301230 MDC385660 MDC405820 MDC410280 giz85717895 Day Avg STDEV Avg STDEV Avg STDEV Avg STDEV A_yg STDEV Avg 0 0.750 0.094 0.691 0.437 1.000 0.000 0.771 0.257 0.588 0.142 86790 11 0.676 0.045 0.873 0.180 0.854 0.100 0.626 0.129 0.593 0.036 91298 25 0.805 0.013 0.876 0.076 0.901 0.056 0.794 0.154 0.723 0.024 89126 32 0.965 0.050 0.789 0.140 0.687 0.220 1.000 0.000 1.000 0.000 83377 39 0.859 0.146 0.753 0.160 0.735 0.119 0.792 0.132 0.675 0.116 87071 49 0.918 0.115 0.597 0.032 0.836 0.050 0.624 0.026 0.404 0.363 72491 60 0.746 0.277 0.633 0.086 0.650 0.190 0.498 0.136 0.354 0.477 74058 70 0.671 0.261 0.694 0.164 0.591 0.054 0.284 0.128 0.210 0.297 73491 Avg SD 0.155 0.197 0.121 0.141 0.246 Fooled average SD 0.153] Table 44. Original data for Figure 34 (Chapter 4). Data are luminosity of electrophoresis agarose gel spots relative to the maximum value for each gene from semi-quantitative RT-PCR products. The spot measurements are for branched-chain aminotransferase (BCAT) genes during ripening and senescence of ‘Jonagold’ apple fruit. All data were normalized relative to control gene 18s rRNA luminosity. Each value is the average of two replications. Figures below are original images from which luminosity data were extracted. 195 Rep 1 Rep 2 Day Day (BCAT1) M E53535)” ”$33334" m MDC192460 (BCAT4) MDC238040 __ (BCAT5) MDC238050 (BACT6) MDC301230 (BCAT7) MD0385660 (BCAT8) MDC405820 (BCAT9) m MDC410280 4.... -- w -' - (BCAT10) 18$ rRNA m Table 44. Continued. “ mil. m we“. “’1’"- mm‘n .— Min- M~" ‘ ’ ‘ 196 PDC1 PDC2 PDC3 PDC4 PDC5 18$ rRNA MDCO15210 MDC133700 MDC206800 MDC206810 MDC433930 gi285717895 Day Avg STDEV Avg STDEV Ayg STDEV Avg STDEV Avg STDEV Avg 0 0.026 0.037 0.846 0.137 0.923 0.108 0.578 0.087 0.716 0.070 86790 11 0.020 0.028 0.839 0.063 0.837 0.231 0.552 0.014 0.767 0.038 91298 25 0.260 0.005 0.937 0.070 0.951 0.065 0.542 0.125 0.811 0.038 89126 32 0.552 0.333 1.000 0.108 0.808 0.227 1.000 0.000 0.818 0.094 83377 39 0.679 0.086 0.924 0.079 0.531 0.083 0.745 0.040 0.834 0.037 87071 49 1.000 0.000 0.794 0.133 0.206 0.103 0.475 0.151 1.000 0.000 72491 60 0.890 0.040 0.593 0.181 0.071 0.049 0.213 0.097 0.817 0.012 74058 70 0.691 0.153 0.434 0.044 0.012 0.016 0.215 0.073 0.728 0.040 73491 Average SD 0.135 0.110 0.133 0.088 0.050 [Pooled average SD 10.103] Rep1 Rep2 Day Day 11 25 32 35 49 60 70 11 25 32 35 49 60 70 (PDC1) "' MDC133700 (PDC2) .4... .._. M ”00206800 (PDC3) ' "'5 M09068” ”$038333" (PDC4) 18$ rRNA qw- M 9119 cur-t9 ’21.?!- 9,84; ""1"". i" '1. '10-». "up... a” "a 01- «v. o Table 45. Original data for Figure 35 (Chapter 4). Data are luminosity of electrophoresis agarose gel spots relative to the maximum value for each gene from semi-quantitative RT-PCR products. The spot measurements are for pyruvate decarboxylase (PDC) genes during ripening and senescence of ‘Jonagold’ apple fruit. All data were normalized relative to control gene 18$ rRNA luminosity. Each value is the average of two replications. Figures below are original images from which luminosity data were extracted. 197 2-isopropylmalate 1 85 rRN A §Ynthase gi:85717895 gr7387848 Day Ag STDEV AyL 0 0.029 0.017 86790 11 0.062 0.010 91298 25 0.489 0.013 89126 32 0.738 0.076 83377 39 0.736 0.070 87071 49 1 .000 0.000 72491 60 0.884 0.109 74058 70 0.789 0.079 73491 Average SD 0.060 Rep1 Rep2 Day Day 01125 32 35 49 60 70 2-lsopropylmalate synthase 01125 32 35 49 60 70 185 rRNA Table 46. Original data for Figure 36 (Chapter 4). Data are luminosity of electrophoresis agarose gel spots relative to the maximum value for each gene from semi-quantitative RT-PCR products. The spot measurements are for 2- isopropylmalate synthase gene during ripening and senescence of ‘Jonagold’ apple fruit. All data were normalized relative to control gene 18s rRNA luminosity. Each value is the average of two replications. Figures below are original images from which luminosity data were extracted. 198 llilllillzlllllllljlfiiill11))inl