University of Kentucky UKnowledge Theses and Dissertations--Plant and Soil Sciences Plant and Soil Sciences 2016 POLYPHENOL CONTENT AND DIFFERENTIAL EXPRESSION OF FLAVONOID BIOSYNTHETIC PATHWAY GENES OF FRAGARIA SPP. WITH WHITE FRUIT Sutapa Roy University of Kentucky, [email protected] Digital Object Identifier: http://dx.doi.org/10.13023/ETD.2016.033 Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Roy, Sutapa, "POLYPHENOL CONTENT AND DIFFERENTIAL EXPRESSION OF FLAVONOID BIOSYNTHETIC PATHWAY GENES OF FRAGARIA SPP. WITH WHITE FRUIT" (2016). Theses and Dissertations--Plant and Soil Sciences. 72. https://uknowledge.uky.edu/pss_etds/72 This Doctoral Dissertation is brought to you for free and open access by the Plant and Soil Sciences at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Plant and Soil Sciences by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless an embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Sutapa Roy, Student Dr. Douglas D. Archbold, Major Professor Dr. Arthur G. Hunt, Director of Graduate Studies POLYPHENOL CONTENT AND DIFFERENTIAL EXPRESSION OF FLAVONOID BIOSYNTHETIC PATHWAY GENES OF FRAGARIA SPP. WITH WHITE FRUIT DISSERTATION A Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Agriculture at the University of Kentucky By Sutapa Roy Director: Dr. Douglas D. Archbold, Professor, Department of Horticulture Lexington, Kentucky 2016 Copyright © Sutapa Roy 2016 ABSTRACT OF DISSERTATION POLYPHENOL CONTENT AND DIFFERENTIAL EXPRESSION OF FLAVONOID BIOSYNTHETIC PATHWAY GENES OF FRAGARIA SPP. WITH WHITE FRUIT Strawberries are a rich source of polyphenols which contribute to berry color and plant disease resistance, and have been shown to lower the risk of many chronic when consumed. While a considerable body of work exists on the polyphenolic composition of commercial strawberry ( Fragaria x ananassa Duch.), less information is available concerning polyphenols in Fragaria vesca , or Alpine strawberry, considered a model system for the Rosaceae family of crop species. The study of natural and genetically-engineered F. vesca mutants with white fruit can provide unique insight into regulation of metabolic flux through the complex branched phenylpropanoid/flavonoid biosynthetic pathway. Thus, the identity and quantity of major phenolic-derived anthocyanins, flavonols, flavan-3-ols, hydroxycinnamic acids, and ellagic acid (EA)-derived compounds, of red-fruited versus white-fruited genotypes of F. vesca and F. x ananassa were compared by high performance liquid chromatography-mass spectrometry. Due to the unknown origin of all but one white- fruited mutant of F. vesca , it was assumed that each resulted from independent mutation events and would exhibit different flavonoid profiles. A total of 27 phenolic-derived compounds were identified. The white genotypes of both species had very low anthocyanin levels . Total content of free EA and its conjugated forms were generally higher in white than in red F. vesca, but were the opposite in F. x ananassa, more in red than in white berries . Differences in content of individual flavonoids and in group totals among the white F. vesca genotypes suggested that they may represent different mutations affecting flavonoid production. Polyphenol profiles of a red and a white cultivar of F. vesca during four fruit developmental stages were determined along with transcriptional analyses of key structural and regulatory genes of the phenylpropanoid/ flavonoid biosynthesis. The final concentration of polyphenolic groups in red versus white F. vesca was due to the differential expression patterns of key pathway genes, especially dihydroflavonol-4-reductase, anthocyanidin synthase, and UDP-glucose-flavonoid-3-O-glucosyltransferase. The efficacy of phenolic compounds were evaluated in an in vitro study for inhibiting growth of Colletotrichum spp. associated with anthracnose fruit rot of strawberry. Only trans - cinnamic, p-coumaric, and ferulic acid inhibited isolates of the pathogen. KEYWORDS: polyphenol, strawberry, flavonoid, anthocyanin, proanthocyanidin ___________________________________________________Sutapa Roy ____ Student Signature ___________________________________________________03 -11 -2016 ____ Date POLYPHENOL CONTENT AND DIFFERENTIAL EXPRESSION OF FLAVONOID BIOSYNTHETIC PATHWAY GENES OF FRAGARIA SPP. WITH WHITE FRUIT By Sutapa Roy Dr. Douglas D. Archbold _____________________________________ Director of Dissertation Dr. Arthur G. Hunt____________________________________________ Director of Graduate Studies 03-11-2016___________________________________________________ ACKNOWLEDGMENTS I wish to express my never-ending gratitude to my advisor Dr. Douglas Archbold for his encouragement, endless support and constructive criticism for all these years. I am thankful to him for providing me the opportunity to work in these projects and his highly valued guidance throughout the entire process to improve my approach to research and ways to improve this dissertation. His patience, care, motivation, dedication to work and philosophy of life not only helped me to progress professionally but also at personal level. I’m tremendously fortunate to have him as my advisor and would have never reached my goal without his support. I would like to thank my committee members Dr. Ling Yuan, Dr. Lisa Vaillancourt and Dr. Robert Geneve for their advice and support. Also I’d like to thank my outside examiner Dr. Czarena Crofcheck for being on my defense committee. I would like to extend my deep appreciation to Dr. Ling Yuan for his guidance in the transcriptional analysis, and Dr. Sanjay Singh, Dr. Sitakanta Pattanaik in his lab, for their helpful involvement in the experiments and suggestions. I also greatly appreciate Dr. Lisa Vaillancourt for her advice and help regarding the pathology work and Etta Nuckles in her lab for helping me with the well-plate toxicity assay and explaining me the concepts of the experiments. I would like to thank John May at the ERTL of University of Kentucky for his assistance and support in HPLC-MS analysis. Special thanks to Marta for being a supportive lab member and a very dear friend to me. I’m thankful to Afaf, she is like an elder sister to me. I’m grateful to all the faculty and staffs of Department of Horticulture. They have become a second family to me with special mention to all my wonderful friends, Meera, Rekha, Priyanka, Andrea, Pam, Chad, Monica, Jozsef, Xia (Summer), and Jarrod who have made my time here so memorable. Lastly I want to thank my parents Niharendu and Sulekha Ray who gave me life and more love and affection than I ever thought was possible. I am thankful to my sister, Sudipta Roy as one of my best friends in life. And obviously my husband, Soham Basu for everything he did for me, for giving me strength, encouragement and keeping faith in me. iii TABLE OF CONTENTS ACKNOWLEDGMENTS ............................................................................................................................. iii TABLE OF CONTENTS .............................................................................................................................. iv LIST OF TABLES ....................................................................................................................................... vii LIST OF FIGURES ...................................................................................................................................... ix Chapter 1: Introduction ............................................................................................................................ 1 Chapter 2: Literature Review .................................................................................................................. 3 2.1 Fragaria vesca as a model species for strawberry and other Rosaceae ................................. 3 2.2 Structure and