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Roles of Wrky Proteins in Mediating the Crosstalk of Hormone Signaling Pathways: an Approach Integrating Bioinformatics and Experimental Biology

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UNLV Retrospective Theses & Dissertations 1-1-2006 Roles of Wrky proteins in mediating the crosstalk of hormone signaling pathways: An approach integrating bioinformatics and experimental biology Zhen Xie University of Nevada, Las Vegas Follow this and additional works at: https://digitalscholarship.unlv.edu/rtds Repository Citation Xie, Zhen, "Roles of Wrky proteins in mediating the crosstalk of hormone signaling pathways: An approach integrating bioinformatics and experimental biology" (2006). UNLV Retrospective Theses & Dissertations. 2711. http://dx.doi.org/10.25669/0cw1-sipg This Dissertation is protected by copyright and/or related rights. It has been brought to you by Digital Scholarship@UNLV with permission from the rights-holder(s). You are free to use this Dissertation in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/or on the work itself. This Dissertation has been accepted for inclusion in UNLV Retrospective Theses & Dissertations by an authorized administrator of Digital Scholarship@UNLV. For more information, please contact [email protected]. ROLES OF WRKY PROTEINS IN MEDIATING THE CROSSTALK OF HORMONE SIGNALING PATHWAYS: AN APPROACH INTEGRATING BIOINFORMATICS AND EXPERIMENTAL BIOLOGY by Zhen Xie Bachelor of Sciences Shandong Agricultural University 1998 Master of Sciences Shandong Agricultural University 2001 A dissertation submitted in partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biological Sciences Department of Biological Sciences College of Sciences Graduate College University of Nevada, Las Vegas December 2006 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 3256298 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. UMI UMI Microform 3256298 Copyright 2007 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Dissertation Approval The Graduate College University of Nevada, Las Vegas September 26______, 20 06 The Dissertation prepared by Zhen X ie Entitled Roles of WRKY Proteins in Mediating the Crosstalk of Hormone Signaling Pathways: an Approach Integrating Bioinformatics and Experimental Biology _____________________________________________________ is approved in partial fulfillment of the requirements for the degree of Doctor of Philosophy ___________________________________ Examination Committee Chair duate College Examination Committee Member __ ExaminatioifCommittee Member Gmduate College Faculty Reprêsèfïtative Examin^Mm Committee Member 11 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT Roles of WRKY Proteins in Mediating the Crosstalk of Hormone Signaling Pathways: an Approach Integrating Bioinformatics and Experimental Biology by Zhen Xie Dr. Jeffery Qingxi Shen, Examination Committee Chair Associate Professor of Biological Sciences University of Nevada, Las Vegas The goal of my research is to understand the molecular mechanism by which hormones control seed germination. Gibberellins (GA) promote, while abscisic acid (ABA) and salicylic acid (SA) inhibit seed germination. Key moleeules in these signaling pathways include receptors, secondary messengers, protein kinases and phosphatases, and transcription factors. My study focuses on how WRKY transcription factors modulate the expression of an a-amylase gene {Amy32b), which is up-regulated by GA, but down- regulated by ABA and SA in the aleurone eells of germinating seeds. Chapter 2 started with the annotation and phylogenetic analyses of the WRKY gene superfamily, followed by functional studies of WRKY proteins in mediating ABA responses. Eighty-one WRKY genes were identified in the riee genome through computational analyses. Phylogenetie analyses based on WRKY domain sequences suggest that extensive duplications and losses of the WRKY domain occurred during evolution of this gene family. Transient expression studies suggest that among four WRKY genes that are ABA-inducible in aleurone cells, OsWRKY72 and OsWRKY77 111 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. function as transcription activators while OsWRKY24 and OsWRKY45 are repressors of the ABA-inducible HVA22 gene. Chapter 3 presents cellular and biochemical data to support a novel model that two transcription repressors OsWRKYSl and OsWRKYVl mediate the crosstalk of GA and ABA signaling. Both genes are ABA-inducible, but GA-repressible in the embryos and aleurone cells of germinating rice seeds. The interaction of OsWRKY51 and OsWRKYVl synergistically represses the GA-induced Amy32b expression, likely by functionally interfering with the GA-inducible transactivator, GAMYB. The mechanism underlying the crosstalk of SA, ABA and GA is reported in Chapter 4. Similar to ABA, SA blocks seed germination likely through repressing the expression of a-amylase genes such as Amy32b. Over-expression of the SA-inducible and GA- repressible HvWRKY38 in aleurone eells bloeks GA-induced Amy32b expression. Therefore, HvWRKY38 might mediate the crosstalk of SA, ABA and GA signaling in regulating a-amylase expression and seed germination. My research demonstrates the high efficiency of the approach integrating bioinformaties and experimental biology in addressing the cell-biological signaling network. The finding derived from this research helps advance towards aehieving our long-term goal: to improve the yield and quality of rice and other eereals. IV Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS ABSTRACT..................................................................................................................................ni LIST OF FIGURES....................................................................................................................vii LIST OF TABLES....................................................................................................................... ix ABBREVIATIONS...................................................................................................................... x ACKNOWLEDGMENTS........................................... xiii CHAPTER 1 GENERAL INTRODUCTION..........................................................................1 Gibberellic acid ...................................................................................................................... 4 The crosstalk of gibberellin and abscisic acid ...................................................................9 The crosstalk of gibberellin and salicylic acid ................................................................. 12 WRKY transcription factors ...............................................................................................13 The transient expression assay using barley aleurone cells ........................................... 14 The scope of the dissertation ...............................................................................................16 References............................................................................................................................. 17 CHAPTER 2 ANNOTATIONS AND FUNCTIONAL ANALYSES OF THE RICE PFRKTGENE SUPERFAMILY REVEAL POSITIVE AND NEGATIVE REGULATORS OF ABSCISIC ACID SIGNALING IN ALEURONE CELLS........................................................27 Abstract.................................................................................................................................27 Introduction.......................................................................................................................... 28 Results................................................................................................................................... 31 Discussion.............................................................................................................................52 Materials and methods ........................................................................................................ 59 Acknowledgements ............................................................................................................. 63 References.............................................................................................................................64 CHAPTER 3 E9TERACTI0NS OF TWO ABSCISIC-ACID INDUCED WRKY GENES IN REPRESSING GIBBERELLIN SIGNALING IN ALEURONE CELLS.......................................................................................71 Summary ......................................................................................................................
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