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Conversion of Essential Amino Acids Into Aroma Volatiles in Developing Melon Fruit

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Conversion of Essential Amino Acids Into Aroma Volatiles in Developing Melon Fruit Conversion of Essential Amino Acids into Aroma Volatiles in Developing Melon Fruit Thesis submitted in partial fulfillment of the requirements for the degree of “DOCTOR OF PHILOSOPHY” by Itay Gonda Submitted to the Senate of Ben-Gurion University of the Negev July 2014 Beer-Sheva Conversion of Essential Amino Acids into Aroma Volatiles in Developing Melon Fruit Thesis submitted in partial fulfillment of the requirements for the degree of “DOCTOR OF PHILOSOPHY” by Itay Gonda Submitted to the Senate of Ben-Gurion University of the Negev Approved by the advisors: Aaron Fait ________________ Date:_______19/01/15 ____ Efraim Lewinsohn ________________ Date:_____21/01/15______ Approved by the Dean of the Kreitman School of Advanced Graduate Studies July 2014 Beer-Sheva 1 This work was carried out under the supervision of Dr. Aaron Fait In The Jacob Blaustein Institutes for Desert Research The French Associates Institute for Agriculture and Biotechnology of Drylands and Dr. Efraim Lewinsohn Agricultural Research Organization Institute of Plant Sciences, Newe Ya’ar Research Center The following publications resulted from this thesis: Gonda, I., Lev, S., Bar, E., Sikron, N., Portnoy, V., Davidovich-Rikanati, R., Burger, J., Schaffer, A., Tadmor, Y., Giovannonni, J.J., Huang, M., Fei, Z., Katzir, N., Fait, A. and Lewinsoh E. (2013) Catabolism of L-methionine in the formation of sulfur and other volatiles in melon (Cucumis melo L.) fruit. Plant J. 61, 458-472. Gonda, I., Burger, Y., Schaffer, A.A., Ibdah, M., Tadmor, Y., Katzir, N., Fait, A. and Lewinsohn, E. (2015) Biosynthesis and perception of melon aroma. In Havkin- Frenkel, D., Dudai, N. and Belanger, F.C. Eds. Biotechnology in Flavor Production. Blackwell Publishing Ltd., Oxford, UK. In press. 2 Thanks First of all, I want to thank Dr. Efraim Lewinsohn, who was my main mentor in this work. He always encouraged to try and test new ideas and never stopped me from preforming new experiments, no matter what. He supported my work and enthusiasm by all means, and always was ready to hear my ideas as well as their criticisms. There are not enough words to describe my gratitude and thankful feelings for him. Many thanks to Dr. Aaron Fait who was my mentor from Ben-Gurion University. Aaron always helped me when I needed him, both scientifically and academically. He knew when to push and when to let go and gave me many good advises and ideas, and I am thankfulness for that. Also, he solved any issues I had towards the university quickly and satisfactory. Special thanks to Dr. Nurit Katzir from Newe-Ya’ar that many of the work described here was carried out in tight collaboration with her and with the Genetics lab and lab members, Vitaly Portnoy, Galil Tzuri and Rotem Harel-Beja. A big thanks to Shery Lev and Dr. Navot Galpaz also at the Genetics lab that helped me both physically and mentally with much of the work I did, especially with the RIL population that was also part of their own projects. I would like also to thank all the members of Efraims’ lab at present and in the past. Especially to Einat Bar and Rachel Davidovich-Rikanati who thought me everything I know about lab work, and also to Raz Krizevski, Karin Rand, Ilan Butnick and Asaf Levy that were always ready to help and gave good ideas and advices. Thanks to all the cucurbits department people who were always pleased to help me with the plants in the field and gave good suggestions when I need them most. In particular to Dr. Yossi Burger and Dr. Kobi Tadmor, as well as to Ayala Meir, Fabian Baumkualler and Uzi Sa’ar. Final thanks to all my friends in Newe-Ya’ar, Zohar Ben-Simhon, Hanoch Glessner, Ari Feder, Noam Chayut and many others that were already mentioned above who made my time there pleasant and valuable. 3 Research-Student's Affidavit when Submitting the Doctoral Thesis for Judgment I_____Itay gonda________, whose signature appears below, hereby declare that (Please mark the appropriate statements): _√_ I have written this Thesis by myself, except for the help and guidance offered by my Thesis Advisors. _√_ The scientific materials included in this Thesis are products of my own research, culled from the period during which I was a research student. ___ This Thesis incorporates research materials produced in cooperation with others, excluding the technical help commonly received during experimental work. Therefore, I am attaching another affidavit stating the contributions made by myself and the other participants in this research, which has been approved by them and submitted with their approval. Date: ______22-01-2015_______________ Student's name: _____________________Itay Gonda ______ Signature:___________ itay______________ g. ___ 4 Contents Summary ........................................................................................................................ 8 Symbols and abbreviations .......................................................................................... 10 Units ............................................................................................................................. 13 List of figures and tables .............................................................................................. 14 Literature survey .......................................................................................................... 17 Flavor and aroma ................................................................................................... 17 The melon .............................................................................................................. 18 Melon aroma ......................................................................................................... 19 Specific characteristics of the melon aroma .......................................................... 20 Melon aroma – biosynthesis and genetic aspects .................................................. 21 Amino-acid-derived aroma compounds – general aspects .................................... 22 The biosynthesis of amino-acid-derived aroma compounds in plants .................. 23 Specific aspects of L-methionine degradation ...................................................... 25 Thioesters – occurrence and biosynthesis ............................................................. 26 The degradation of L-phenylalanine into (E)-cinnamaldehyde and other aroma volatiles ...................................................................................................... 27 Working hypotheses & research objectives ................................................................. 30 Experimental procedures ............................................................................................. 31 Chemicals .............................................................................................................. 31 Plant material......................................................................................................... 31 Incubation experiments and volatile analyses ....................................................... 31 Gas-chromatography mass-spectrometry volatile analyses .................................. 32 Gas-chromatography mass-spectrometry analysis of amino acids ....................... 35 Liquid-chromatography mass-spectrometry analysis of amino acids ................... 36 Preparation of cell-free extracts from melon flesh tissues .................................... 36 MGL enzymatic assays ......................................................................................... 37 L-methionine aminotransferase radiolabeled assay .............................................. 38 L-methionine aminotransferase GC-MS assay ..................................................... 38 L-methionine aminotransferase LC-MS assay ...................................................... 39 Methanethiol acetyl transferase (ThAT) enzymatic assay .................................... 39 (E)-cinnamaldehyde coupled enzymatic assays .................................................... 39 Cloning of CmMGL, CmMetAT and CmThAT1, and their heterologous expression in E. coli .............................................................................................. 40 Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) analyses of the recombinant proteins .................................................................... 40 Co-expression of CmThAT1 and CmMGL and their heterologous expression in E. coli ............................................................................................................... 41 5 Cloning of CmCNL and heterologous expression in E. coli ................................. 41 (E)-cinnamic acid:coenzyme A ligase enzymatic assay ....................................... 42 cDNA preparation and quantitative real-time PCR analysis ................................. 43 RNA-seq gene expression analysis ....................................................................... 43 Bioinformatic and statistical analyses ................................................................... 44 Results .......................................................................................................................... 45 L-methionine-derived aroma volatiles in melon fruits .......................................... 45 Melon cultivars contained different levels of sulfur aroma volatiles .................... 45 Feeding melon cubes with stable-isotope-labeled L-methionine resulted in enriched volatile aroma compounds .....................................................................
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