Clemson University TigerPrints All Dissertations Dissertations 12-2008 FUNCTIONAL CHARACTERIZATION OF HEXOKINASE-LIKE 1 (HKL1) FROM ARABIDOPSIS THALIANA Abhijit Karve Clemson University, [email protected] Follow this and additional works at: https://tigerprints.clemson.edu/all_dissertations Part of the Genetics Commons Recommended Citation Karve, Abhijit, "FUNCTIONAL CHARACTERIZATION OF HEXOKINASE-LIKE 1 (HKL1) FROM ARABIDOPSIS THALIANA" (2008). All Dissertations. 387. https://tigerprints.clemson.edu/all_dissertations/387 This Dissertation is brought to you for free and open access by the Dissertations at TigerPrints. It has been accepted for inclusion in All Dissertations by an authorized administrator of TigerPrints. For more information, please contact [email protected]. FUNCTIONAL CHARACTERIZATION OF HEXOKINASE-LIKE 1 (HKL1) FROM ARABIDOPSIS THALIANA A Thesis Presented to the Graduate School of Clemson University In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Genetics by Abhijit Avinash Karve December 2008 Accepted by: Dr. Brandon Moore, Committee Chair Dr. William Vance Baird Dr. Albert Abbott Dr. Lesly Temesvari Dr. James Morris ABSTRACT Arabidopsis hexokinase1 (AtHXK1) is a moonlighting protein with roles in both glucose signaling and catalysis. In this study, we first cloned and characterized the six HXK related genes from Arabidopsis. Three of the six encoded proteins were shown not to phosphorylate hexoses and thus, are designated as hexokinase-like (HKL) proteins. Though they are only 50% identical to HXK1, the amino acid sequences of HKL1 and HKL2 both have well conserved glucose binding domains and other recognized structural elements. The possible basis for their lack of catalytic activity was further probed by site-directed mutagenesis and ultimately was attributed to a suite of amino acid substitutions. Gene expression studies showed that transcripts of HKL1 and HKL2 occur in most plant tissues, thus supporting the hypothesis that they have regulatory functions. The function of AtHKL1 was more closely examined using a reverse genetics approach. We identified a T-DNA knockout mutant for HKL1 and made HKL1 overexpression lines in different genetic backgrounds. Their phenotypes showed that HKL1 is a negative regulator of plant growth. Interestingly, many of the phenotypes required the presence of HXK1 protein. Both HKL1-GFP and HXK1-GFP are expressed at mitochondria and both were shown to interact with each other by coimmunoprecipitation assays. However, even though the HKL1 phenotypes included some dependence on glucose treatments, we conclude that HKL1 likely does not have a direct role in glucose signaling. Instead, we found from seedling signaling assays and a novel root hair phenotype that HKL1 mediates plant growth responses at least in part by ii promoting ethylene biosynthesis and/or signaling. Overall, these studies have helped to identify, characterize, and define the function of non-catalytic HXKs from Arabidopsis. iii DEDICATION This work is dedicated to my wife, Rucha Karve whose constant inspiration lead me to pursue graduate studies. This work would not have been possible without her constant love and support. Thanks for everything! iv ACKNOWLEDGMENTS I would like to thank my research advisor Dr. Brandon Moore for giving me an opportunity to pursue PhD in his lab and for guidance. This work would not have been possible without his expertise, encouragement and enthusiasm. I would like to thank my former advisory committee member, the late Dr. Shu- Hua Cheng, for her encouragement and help. She was an invaluable source of information, support and encouragement. I would like to thank my advisory committee members, Dr. Albert Abbott, Dr. Vance Baird, Dr. Lesly Temesvari and Dr. James Morris for their constructive ideas, helpful discussions, and valuable suggestions. Many thanks to former Moore lab members, Dr. Rajagopal Balasubramanian, Dr. David Weston and Ms. Helen Metters for their assistance, and a former summer undergraduate student Anna Pavlovskaya for the help with the promoter assays. I would like to thank Xiaoxia (Penny) Xia for her tremendous help and support in the lab. I would also like to thank my family and my in laws for their encouragement, love and support. I would like to thank my friends in Clemson, Abhijit, Nikhil, Shreenivas, Sourabh, Santosh, Sourabh (Jr.), Namrata, Mayank and Prashant for their help and support. Finally, I would like to thank my wife, Rucha Karve for her love and support and for patiently standing by me during this long endeavor. v TABLE OF CONTENTS Page TITLE PAGE ................................................................................................................... i ABSTRACT.................................................................................................................... ii DEDICATION ............................................................................................................... iv ACKNOWLEDGMENTS................................................................................................v LIST OF TABLES ....................................................................................................... viii LIST OF FIGURES........................................................................................................ ix CHAPTER I. LITERATURE REVIEW...............................................................................1 Introduction..............................................................................................1 Glucose sensing and signaling in yeast and mammals.............................2 Glucose sensing and signaling in plants...................................................5 Molecular mechanism of glucose regulation in plants...........................11 Cellular context of HXK dependent signaling.......................................13 Interaction of glucose signaling with other signaling pathways ............15 Hexokinases...........................................................................................21 Non catalytic HXKs...............................................................................24 References..............................................................................................27 II. EXPRESSION AND EVOLUTIONALRY FEATURES OF HEXOKINASE GENE FAMILY IN ARABIDOPSIS....................45 Abstract..................................................................................................45 Introduction............................................................................................47 Materials and methods…………………………………………………50 Results....................................................................................................56 Discussion……………………………………………………………...76 References……………………………………………………………...83 vi III. FUNCTION OF ARABIDOPSIS HEXOKINASE-LIKE 1 AS AN EFFECTOR OF PLANT GROWTH .......................................90 Abstract..................................................................................................90 Introduction............................................................................................92 Materials and Methods...........................................................................95 Results..................................................................................................100 Discussion............................................................................................115 References............................................................................................121 IV A ROLE OF HKL1 IN MEDIATING CROSS-TALK BETWEEN GLUCOSE AND ETHYLENE SIGNALING.....................................127 Abstract................................................................................................127 Introduction..........................................................................................129 Materials and methods.........................................................................132 Results..................................................................................................136 Discussion............................................................................................152 References............................................................................................158 IV SUMMARY ...............................................................................................163 APPENDICES..............................................................................................................169 A: Domains and conserved regions of AtHXKs with respect to ScHXK2...........................................................................................170 B: Sequence analysis of HXK family genes from A. lyrata ...........................172 C: Amino acid sequence alignment for predicted HXK family proteins from A. thaliana and A. lyrata ..............................................173 D: Effect of ABA on seed germination of Col, hkl1-1 and abi4-1 .................175 E: Copyrights permission for chapter 2..........................................................176 vii LIST OF TABLES Table Page 2.1 Selected properties predicted for HXK family proteins from A. thaliana ....................................................................................57 2.2 Comparisons of HXK amino acid sequences from A. thaliana and A. lyrata ........................................................................72 viii LIST OF FIGURES Figure Page 1.1 Arabidopsis seedling