The Arabidopsis thaliana Cyclic-Nucleotide-Dependent Response – a Quantitative Proteomic and Phosphoproteomic Analysis Dissertation by May Majed Alqurashi In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Science King Abdullah University of Science and Technology, Thuwal, Kingdom of Saudi Arabia November, 2013 2 The dissertation of May Majed Alqurashi is approved by the examination committee. Committee chair: Liming Xiong Committee member: Christoph Gehring Committee member: Samir Hamdan Committee member: Stefania Pasqualini Committee member: Timothy Ravasi Committee member: Zhiping Lai 3 © November, 2013 May Majed Alqurashi All Rights Reserved 4 ABSTRACT The Arabidopsis thaliana Cyclic Nucleotide-Dependent Response – a Quantitative Proteomic and Phosphoproteomic Analysis May Majed Alqurashi Protein phosphorylation governs many regulatory pathways and an increasing number of kinases, proteins that transfer phosphate groups, are in turn activated by cyclic nucleotides. One of the cyclic nucleotides, cyclic adenosine monophosphate (cAMP), has been shown to be a second messenger in abiotic and biotic stress responses. However, little is known about the precise role of cAMP in plants and in the down-stream activation of kinases, and hence cAMP-dependent phosphorylation. To increase our understanding of the role of cAMP, proteomic and phosphoproteomic profiles of Arabidopsis thaliana suspension culture cells were analyzed before and after treatment of cells with two different concentrations of 8-Bromo-cAMP (1 µM and 100 nM) and over a time-course of one hour. A comparative quantitative analysis was undertaken using two- dimensional gel electrophoresis and the Delta 2D software (DECODON) followed by protein spot identification by tandem mass spectrometry combined with Mascot and Scaffold. Differentially expressed proteins and regulated phosphoproteins were categorized according to their biological function using bioinformatics tools. The results revealed that the treatment with 1 µM and 100 nM 8-Bromo-cAMP was sufficient to induce specific concentration- and time-dependent changes at the proteome and phosphoproteome levels. In particular, different phosphorylation patterns were observed overtime preferentially affecting proteins in a number of functional categories, notably phosphatases, proteins that remove phosphate groups. This suggests that cAMP both transiently activates and deactivates proteins through specific phosphorylation events and provides new insight into biological mechanisms and functions at the systems level. 5 ACKNOWLEDGEMENTS ﻢﺴﺑ ﷲ ﺍﻟﺮﺣﻤﻦ ﺍﻟﺮﻭ ،ﻢﻴﺣﺍﻟﺼﻼﺓ ﻭﺍﻟﺴﻼﻡ ﻋﻠﻰ ﺃﺷﺮﻑ ﺍﻷﻧﺒﻴﺎء ﻭﺍﻟﻤﺮﺳﻠﻴﻦ ﺳﻴﺪﻧﺎ ﻣﺤﻤﺪ ﻭﻋﻠﻰ ﺁﻟﻪ ﻭﺻﺤﺒﻪ ﺃﺟﻤﻌﻴﻦﷲﷲ Over the past three years, I have received support and encouragement from a great number of individuals that I would like to extend my gratitude to. First, I would like to thank King Abdullah bin Abdulaziz for transforming his dream to a beautiful university I call home. My university is more than classes, labs and exams; it is the spark that helped igniting my passion for science and gave me plenty of reasons to love it even more, and for that I am forever a KAUSTian. I want to take this opportunity to express my sincere gratitude to my advisor, Prof. Christoph Gehring, for his mentorship, excellent guidance and the inspiration to be a better student, a better researcher, a better person. My appreciation is extended to Dr. Claudius Marondedze and Dr. Ludivine Thomas, whom without their dedication and patience in teaching me all I need to know about working in the field, I wouldn't be writing this right now! To everyone in our lab, who gave me an advice, a correction or a smile; thank you. My wonderful friends that supported me along the way and gave me a push towards being a better person, I wish I can list you all but you know your place in my heart. Most importantly, to the foundation of all my hopes and dreams, my success and inspiration, my beloved family, especially my parents, Mona and Majed, my lovely sister Maram and my two brothers Moayad and Motasem. Finally, I would like to dedicate my dissertation to my late grandfather, who since I was a little girl taught me the importance of science, and I know he would be so proud of me now. God bless his soul. 6 TABLE OF CONTENTS EXAMINATION COMMITTEE APPROVAL FORM ................. Error! Bookmark not defined. ABSTRACT .................................................................................................................... 4 ACKNOWLEDGEMENTS................................................................................................ 5 TABLE OF CONTENTS ................................................................................................... 6 LIST OF FIGURES .........................................................................................................12 LIST OF TABLES ...........................................................................................................14 CHAPTER 1: INTRODUCTION .......................................................................................15 1.1 Control of cellular processes .....................................................................................15 1.2 The perception and modulation of signals ...................................................................15 1.3 Linking environmental stimuli to cellular response in animal systems ............................16 1.4 Plant signaling and responses ....................................................................................21 1.5 The role of cAMP in plant signal transduction .............................................................23 1.6 Role of plant cGMP in signal transduction ..................................................................26 1.7 Adenylyl cyclases in plants .......................................................................................28 1.8 Role of phosphodiesterases in restoring the resting levels of cAMP ...............................31 1.9 The importance of phosphorylation in animals ............................................................32 1.10 Role of phosphorylation in plants .............................................................................34 CHAPTER 2: MATERIALS AND METHODS ...................................................................37 2.1 Reagents .................................................................................................................37 2.2 Plant material and growth conditions ..........................................................................37 2.3 Plant treatment and measurements .............................................................................38 2.4 Protein extraction .....................................................................................................38 2.5 Protein quantification ...............................................................................................39 2.6 One-dimensional (1D) gel electrophoresis ..................................................................40 2.7 Two-dimensional (2D) gel electrophoresis ..................................................................41 2.8 Pro-Q Diamond and SYPRO Ruby protein staining .....................................................42 2.9 Comparative analysis ...............................................................................................43 2.10 In-gel digestion and mass spectrometry.....................................................................43 2.11 Computational tools for the inferring of biological functions .......................................45 CHAPTER 3: RESULTS ..................................................................................................46 7 3.1 Protein concentration measurement in the samples .......................................................46 3.2 Two-dimensional (2D) gels analysis...........................................................................46 3.3 Identification of the protein spots after cAMP treatment and comparative analysis ...........50 3.3.1 Interesting observations from a general overview..................................................52 3.3.2 Up-regulated proteins in the early response to cAMP treatment. .............................54 3.3.3 Down-regulated proteins in the early response to cAMP treatment. .........................55 3.3.4 Up-regulated proteins in the late response to cAMP treatment. ...............................56 3.3.5 Down-regulated proteins in the late response to cAMP treatment. ...........................58 3.3.6 Comparison between treatments .........................................................................58 3.3.7 Co-expression analysis of the phosphatases ..........................................................62 3.3.8 Identification of phosphorylated sites in differentially expressed proteins. ...............63 3.4 Functional enrichment and gene ontology (GO) analysis ..............................................66 3.4.1 Biological processes enrichment analysis .............................................................67 3.4.2 Cellular compartment enrichment analysis ...........................................................72 3.4.3 Molecular function enrichment analysis ...............................................................73 3.4.4 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analysis 76 3.4.5 Phosphatases gene ontology, co-expression and promoter analysis ..........................78 3.4.6 Unknown proteins