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Etd-04012015-214419.Pdf (1.273 Mb ) Automated Template A: Created by James Nail 2011 V2.02 A study of abscisic acid regulated enzymes and histone binding proteins in plants By Udhghatri Kolli A Dissertation Submitted to the Faculty of Mississippi State University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Molecular Biology in the Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology Mississippi State, Mississippi August 2015 Copyright by Udhghatri Kolli 2015 A study of abscisic acid regulated enzymes and histone binding proteins in plants By Udhghatri Kolli Approved: ____________________________________ Jiaxu Li (Major Professor) ____________________________________ Din-Pow Ma (Committee Member) ____________________________________ Daniel G. Peterson (Committee Member) ____________________________________ Zhaohua Peng (Committee Member) ____________________________________ Scott Willard (Committee Member) ____________________________________ Kenneth Willeford (Graduate Coordinator) ____________________________________ George Hopper Dean College of Agriculture and Life Sciences Name: Udhghatri Kolli Date of Degree: August 14, 2015 Institution: Mississippi State University Major Field: Molecular Biology Major Professor: Jiaxu Li Title of Study: A study of abscisic acid regulated enzymes and histone binding proteins in plants Pages in Study: 111 Candidate for Degree of Doctor of Philosophy Plant hormone abscisic acid (ABA) plays a main role in coordinating various stress signals in plants. ABA regulates the expression of genes and activities of enzymes in response to various stress conditions. In the following studies we were able to study the ABA mediated regulation of enzymes in plants. Using in-gel activity analysis we identified that ABA regulates the activity of aspartate aminotransferase (AAT), an enzyme involved in nitrogen assimilation and carbohydrate metabolism. Our results indicate that phosphorylation of AAT by SnRK2.2 and 2.3 kinases results in down regulation of AAT2 and AAT3 isozyme activities in Arabidopsis. AAT was identified as a negative regulator of drought stress and aat mutant plants showed improved survival following drought conditions. Using in-gel staining method we were able to visualize sugar phosphatases like fructose 1-6 bisphosphatase family, sedoheptulase-1,7- bisphosphatase, inositol mono phosphatases; protein serine/threonine phosphatases, protein tyrosine phosphatases and studied their response to ABA and drought stress. Fructose-1-6 bisphosphatase family of phosphatases were identified to be induced by ABA in Arabidopsis and rice. N-acetylglucosamine (GlcNAc) is present on glycoproteins and as post translational modification (PTM) in cytoplasmic and nuclear proteins. N- acetylglucosamine is removed from target proteins by hexosaminidases. Little is known about the hexosaminidases in plants. Using in-gel activity analysis we were able to identify an ABA induced Beta-hexosaminidase with a neutral pH optimum in soybean. The nuclear DNA in chromatin is associated with basic proteins called histones. The N-terminal tails of histones contain different PTMs including methylation, phosphorylation, ubiquitination, acetylation, ADP-ribosylation and glycosylation. The histone lysine methylation can serve as a binding site or repel/disrupt the histone binding proteins. The effector/reader proteins specifically recognize the post translational modifications and responsible for the downstream process. Many histone methyl modification effector proteins have been characterized but very few proteins whose binding was disrupted by the presence of a PTM were identified. Using peptide pulldown analysis, far western analyses we identified a WD-40 domain containing histone binding (HB01) protein as direct interactor of unmodified histone. The presence of post translational modifications disrupts HB01 binding to histone H3. DEDICATION This dissertation is dedicated to the loving memory of mother, Padma Sasi Rekha Kolli. ii ACKNOWLEDGEMENTS I am grateful to my major professor Dr. Jiaxu Li for his constant encouragement and guidance. I would like to thank my committee members Dr. Din-Pow Ma, Dr. Daniel G. Peterson, Dr. Zhaohua Peng and Dr. Scott T. Willard for their suggestions and advice. I thank Dr. Ying Wai Lam of the Vermont Genetics Network Proteomics Facility at the University of Vermont for help with mass spec analysis I thank Dr. Jie Song, Dr. Babi Ramesh Nallamilli, Dr. Hana Mujahid and Dr. Yuxuan Hou, for their support, help and friendship. I would also like to thank all the students, faculty and staff of Biochemistry, Molecular Biology, Plant Pathology, and Entomology. I would like to thank my family and friends for their unrelenting support and encouragement. None of this would have been possible without their support and love. iii TABLE OF CONTENTS DEDICATION .................................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................... iii LIST OF TABLES ............................................................................................................ vii LIST OF FIGURES ......................................................................................................... viii CHAPTER I. INTRODUCTION .............................................................................................1 II. LITERATURE REVIEW ..................................................................................4 Arabidopsis thaliana ..........................................................................................4 Oryza sativa .......................................................................................................5 Abscisic acid ......................................................................................................6 ABA biosynthesis ........................................................................................6 Abscisic acid catabolism ..............................................................................9 ABA transporters .......................................................................................11 Core ABA signaling pathway ....................................................................12 ABA signal transduction ............................................................................16 ABA regulated gene expression...........................................................17 ABA induced proteins ..........................................................................22 ABA mediated stomatal closure ..........................................................22 Nitrogen assimilation and abscisic acid ...........................................................24 In-gel activity analysis .....................................................................................26 Histone post translational modifications ..........................................................28 Histone lysine methylation ........................................................................28 Histone acetylation .....................................................................................30 WD repeat proteins ....................................................................................30 Peptide pulldown assay ....................................................................................32 Identification of peptide-protein interactions by far western analysis .............33 III. IDENTIFICATION OF ABSCISIC ACID REGULATED ENZYMES IN PLANTS .........................................................................................35 Introduction ......................................................................................................35 Aspartate aminotransferase ..............................................................................36 iv Materials and Methods .....................................................................................38 Plant material and growth conditions ........................................................38 Treatments..................................................................................................39 ABA treatment .....................................................................................39 Drought treatment ................................................................................40 Salt treatment .......................................................................................40 Protein extraction and quantification .........................................................40 Native gel electrophoresis ..........................................................................41 Activity measurement of aspartate aminotransferase ................................41 In-gel activity analysis of aspartate aminotransferase .........................41 In-solution activity analysis of aspartate aminotransferase .................42 Statistical analysis ......................................................................................42 Results ..............................................................................................................42 ABA dependent regulation of aspartate aminotransferase .........................42 The activity of aspartate aminotransferase isoforms is regulated by drought stress .................................................................................44 The activity of aspartate aminotransferase isoforms in response
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