BIOSYNTHESIS OF BENZOATE-DERIVED BIPHENYL PHYTOALEXINS IN CELL CULTURES OF Pyrus pyrifolia

Abstract

Pear is an important fruit crop, known for its beautiful texture and high nutritional value. In comparison to European pears (Pyrus communis), Asian pears (Pyrus pyrifolia) exhibit higher ontogenic resistance, thereby they are less susceptible to the scab disease caused by the fungus Venturia pirina. To date, the scab-resistance mechanisms in pear are not well understood. The present study investigates the comparative basal metabolomics profile of immature and mature healthy leaves of P. pyrifolia and P. communis with aim to identify defense responsive metabolites in these two plant groups. Gas chromatography–mass spectrometry (GC-MS) coupled with multivariate analysis were applied to analyze the metabolite profiles in both the pear species between immature and mature leaves. Using non-targeted comparative metabolomics a total of 30 differentially accumulating metabolites were identified in both the pear species, out of which phenolic accumulation were significantly higher in P. pyrifolia leaves. Benzoic acid is an important plant natural product which serves as precursor for biosynthesis of an array of the other economically important secondary metabolites. In pears, benzoic acid serves as the precursor for biphenyl biosynthesis, which serves as defense metabolite protecting against pathogen infection. Till date, the biosynthesis of benzoic acid is poorly understood in pears. Therefore this thesis further aims at exploring the enzymatic route of benzoic acid formation in cell cultures of Asian pear (P. pyrifolia). Pear cell cultures responded to yeast-extract elicitor treatment by producing two biphenyls, the aucuparin and the noraucuparin. Cell free extract / partially purified proteins prepared from these elicited cell cultures were used to elucidate the enzymatic route of benzoic acid formation. A novel C2-chain cleavage enzyme, benzaldehyde synthase (BS), has been detected and characterized first time from these cell cultures. BS catalyzed the formation of benzaldehyde from cinnamic acid. The next step of the biosynthetic pathway was catalyzed by a NAD+-dependent benzaldehyde dehydrogenase (BD) which catalyzed the conversion of benzaldehyde into benzoic acid. The next step was detection and characterization of benzoate-CoA-ligase (BZL) activity catalyzing conversion of benzoic acid into benzoyl-CoA. Finally benzoyl-CoA enters into the biphenyl biosynthesis using biphenyl synthase (BIS) enzyme. The properties of BS, BD and BZL were biochemically characterized. Upon YE treatment BS, BD, BZL and BIS activities were coordinately induced. In conclusion, biosynthesis of benzoic acid in elicited cell cultures of P. pyrifolia was found to proceed via CoA-independent and non-β-oxidative route. Keywords: Asian pear; benzaldehyde synthase; benzaldehyde dehydrogenase; benzoate-CoA ligase; cell culture; metabolomics; Pyrus communis; Pyrus pyrifolia. vi Acknowledgements Thanks to Braham Swarup Almighty, the creator of universe and source of all knowledge for his divine sanction on me and allowing me to see immensity in his creatures. I take immense pleasure in expressing my deep sense of gratitude to my supervisor and mentor Dr. Debabrata Sircar for giving me the opportunity to join his research laboratory. His trust in me and constant supervision has been a constant source of positive energy for me. He has patiently listened and accordingly supervised and supported me throughout my Ph.D. His enthusiasm and profound concern for my success has made this journey a joyous one. It has been a great privilege and honour to work and study under his precious guidance. His sincerity, dynamism, vision and motivation deeply inspired me to learn new things. Along with academic support his affectionate behaviour and moral support also helped me through the rough road to finish my thesis. I express my wholehearted indebtedness to him. I would also like to express my profound gratitude to Prof. Partha Roy, Head of Department of Biotechnology for providing the basic infrastructural facilities in the department that allowed me to successfully complete my Ph.D. I’m thankful to him for giving me access to his laboratory instrumentation. His punctuality and sense of duty have been a source of motivation. His expert advices and jovial nature have encouraged me all along the path to success. Besides, I would like to thank the Department of Biotechnology, and Institute Instrumentation Centre (IIC), IIT Roorkee for instrument facilities to carry out the research work. I am also grateful to my SRC committee members: Dr. Bijan Choudhury, Dr. Paravindar Kumar and Dr. P. Gopinath; DRC chairman: Prof. Vikas Pruthi for their continuous advice and kind support. I also express my sincere gratitude towards Dr. Harsh Chauhan and Dr. Sri Ram Yadav for their kind and valuable suggestions and other faculty members of Department of Biotechnology during my PhD. My warmest thanks to my colleagues in my lab and in my department who made this study a successful experience. I especially thank Ms. Deepa Teotia for her timeless support and constructive ideas. I was lucky to work with a kind and helpful colleague Mr. Amol Sarkate. vii I am thankful to Ms. Kriti Juneja, Mr. Ashwani Kumar, Mr. Mukund Kumar, Ms.Varsha Tomar, Mr. Bhairavnath Waghmode and Ms. Komal Kushwaha for the pleasant working environment and companionship during laboratory works. I am also thankful to the office staff, Department of Biotechnology especially Mr. Veadpal Saini for their kind help and support, campus administration and workers team of my hostel, Azad Bhawan, their hard work made my stay comfortable in campus. A special note of thanks to my friends in IIT Roorkee namely Mr. Yogesh Sariya, Mr. Vijay Sharma, Mr. Ragvender Singh, Mr. Somesh Baneerjee, Mr. Swapneel Jaiswal, Mr. Sunil Sharma and Mr. Harvijay singh for their encouragement, support and giving unforgettable moments during my stay in IIT and in my life. I would also like to express my deep gratitude to Mrs. Rakhi Sircar and two little angels Ishika and Annie for their moral support, kind blessings and for letting me feel like home during my stay in IIT Roorkee. Finally, my sincere thanks to my Father, Mother and Brother for standing with me in every situation of my life. Special thanks to my Father for teaching and integrating spirituality in my life style and to teach me for being calm in every situation of life.