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Role of Arabidopsis receptor for activated C-protein kinase 1 in plant growth, development and abscisic… guo, jianjun 2010

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ROLE OF ARABIDOPSIS RECEPTOR FOR ACTIVATED C-PROTEIN KINASE 1 IN PLANT GROWTH, DEVELOPMENT AND ABSCISIC ACID RESPONSES by Jianjun Guo  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Botany)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2010  © Jianjun Guo, 2010  ABSTRACT  In mammalian cells and yeast, RACK1 (Receptor for Activated C Protein Kinase 1) regulates various signaling pathways and cellular processes through its interaction with numerous signaling proteins. However, its functions in plants are poorly understood. My PhD project utilizes a combination of genetic, molecular, biochemical, bioinformatic, and cell biological approaches to study the function of RACK1 in plants using Arabidopsis as a model system. The first part of my study focused on the role of RACK1 genes in plant growth and development. The Arabidopsis genome contains three RACK1 genes, namely RACK1A, RACK1B and RACK1C. Using a genetic complementation approach, I discovered that three Arabidopsis RACK1 genes are functionally equivalent and positively regulate plant root and shoot growth and development. The second part of my study focused on the role of RACK1 genes in abscisic acid (ABA) responses. ABA primarily mediates plant responses to abiotic stress. It is one of the five classic plant hormones. Through physiological and molecular biological assays, I established that the three RACK1 genes function as negative regulators of ABA responses and that they are also involved in salt and drought stress responses. In searching for the molecular function of RACK1 in ABA responses, I first looked into the potential interaction between RACK1 and the heterotrimeric G-protein complex (another negative regulator of ABA responses). Both protein(s) (complex) are highly conserved between Arabidopsis, yeast and mammal and a physical interaction between them were found in nonplant systems. I discovered that Arabidopsis RACK1 and a heterotrimeric G-protein complex appeared to work additively in ABA responses. Moreover, there was no physical interaction detected between the Arabidopsis homologs of RACK1 and the subunits of G-protein complex. These data indicate that Arabidopsis RACK1 and heterotrimeric G-protein complex work in independent manner in regulating ABA responses, distinct from their counterparts in mammalian and yeast cells. I next looked into the potentially evolutionarily-conserved role of RACK1 in regulating protein translation as a candidate mechanism via which RACK1 could negatively influence ABA responses. I found five lines of evidence directly or indirectly supporting this hypothesis: all three Arabidopsis RACK1s complemented the growth defects of the yeast rack1/cpc2 mutant; ii  the rack1 mutation had an additive effect with anisomycin, an inhibitor of protein translation, on root growth; RACK1 physically interacted with Arabidopsis eukaryotic initiation factor 6 (eIF6), also known to regulate ribosome assembly and translation initiation in mammalian cells; rack1 mutants displayed impaired 60S ribosome subunit biogenesis and 80S functional ribosome assembly. In addition, ABA constantly inhibited the expression of RACK1 and eIF6. In summary, my PhD work has advanced our understanding of the versatile role of RACK1 genes in regulating several traits in plant growth and development as well as ABA/stress responses. I also found that Arabidopsis RACK1 and heterotrimeric G-protein complex, different from their counterparts in mammals and yeast, worked independently in regulating ABA responses. In addition, I established a role of Arabidopsis RACK1 in regulating protein translation, which was the first defined cellular process in which RACK1 is involved. At last, the data from my study indicates a role of RACK1 as a molecular link between ABA signaling and its effect on protein translation.  iii  TABLE OF CONTENTS ABSTRACT .................................................................................................................................. ii TABLE OF CONTENTS ............................................................................................................. iv LIST OF TABLES...................................................................................................................... viii LIST OF FIGURES ...................................................................................................................... ix LIST OF ABBREVIATIONS ...................................................................................................... xi ACKNOWLEDGEMENTS......................................................................................................... xii CO-AUTHORSHIP STATEMENT ........................................................................................... xiv CHAPTER 1 GENERAL INTRODUCTION ............................................................................. 1 1.1 Abscisic Acid Signaling ...................................................................................................... 2 1.1.1 The physiological function of ABA ............................................................................. 2 1.1.2 ABA function in other organisms................................................................................. 5 1.1.3 The ABA receptors ....................................................................................................... 5 1.2 RACK1, a Versatile Scaffold Protein in Plants? ................................................................. 7 1.2.1 Introduction .................................................................................................................. 7 1.2.2 Discovery of RACK1 as an intracellular receptor........................................................ 7 1.2.3 RACK1 is a WD-repeat-containing protein ................................................................. 8 1.2.4 RACK1 as a scaffold protein........................................................................................ 8 1.2.5 Discovery of RACK1 in plants................................................................................... 12 1.2.6 Functional characterization of RACK1 in plants........................................................ 16 1.2.7 RACK1 interacting partners in plants ........................................................................ 18 1.2.8 Concluding remarks.................................................................................................... 22 1.3 Thesis objectives................................................................................................................ 24 1.4 References ......................................................................................................................... 25 CHAPTER 2 Arabidopsis RACK1 Genes Regulate Rosette Leaf Production and Root Growth with Unequal Genetic Redundancy ............................................................................................ 35 2.1 Introduction ....................................................................................................................... 36 2.2 Materials and methods....................................................................................................... 38 2.2.1 Plant materials and growth conditions........................................................................ 38 2.2.2 Isolation of rack1b and rack1c T-DNA insertional mutants ...................................... 38 2.2.3 Generation of rack1a, rack1b and rack1c double and triple mutants......................... 38 2.2.4 Genetic complementation ........................................................................................... 39 2.2.5 RNA isolation, RT-PCR and quantitative real-time PCR analyses............................ 39 2.2.6 Rosette leaf production assay ..................................................................................... 40 2.2.7 Root growth assay ...................................................................................................... 41 2.3 Results ............................................................................................................................... 41 2.3.1 T-DNA insertional mutants of RACK1B and RACK1C.............................................. 41 2.3.2 Loss-of-function mutations in RACK1B and RACK1C enhance the growth defects in rosette leaf production of rack1a mutant............................................................................. 42 2.3.3 Loss-of-function mutations in RACK1B and RACK1C enhance the defects in root growth of rack1a mutant ..................................................................................................... 45 2.3.4 Genetic complementation of rack1a mutants by overexpressing RACK1B and RACK1C .............................................................................................................................. 46 2.3.5 Tissue/organ Expression of Arabidopsis RACK1 genes............................................. 49 2.3.6 Cross-regulation of RACK1 genes at the transcription level ...................................... 50 2.4 Discussion.......................................................................................................................... 51 2.3.1 RACK1 genes in plant growth and development ........................................................ 52 iv  2.3.2 Mechanism of unequal genetic redundancy of RACK1 genes.................................... 53 2.5 References ......................................................................................................................... 56 CHAPTER 3 Dissection of the Relationship between RACK1 and Heterotrimeric G-proteins in Arabidopsis ................................................................................................................................. 58 3.1 Introduction ....................................................................................................................... 59 3.2 Materials and methods....................................................................................................... 60 3.2.1 Plant materials and growth conditions........................................................................ 60 3.2.2 Generation of rack1a-1, gpa1-4, and agb1-2 double mutants.................................... 60 3.2.3 ABA inhibition of seed germination and early seedling development assays............ 61 3.2.4 RT-PCR and quantitative RT-PCR............................................................................. 61 3.2.6 Yeast two-hybrid assay............................................................................................... 62 3.2.7 Yeast three-hybrid assay............................................................................................. 63 3.2.8 Plant two-hybrid protein-protein interaction assay..................................................... 63 3.2.9 Co-immunoprecipitation (Co-IP) ............................................................................... 64 3.3 Results .............................................................................................................................. 64 3.3.1 Double mutants between rack1a and gpa1 or agb1 ................................................... 64 3.3.2 An enhanced effect on ABA hypersensitivity was observed between rack1a and gpa1 or agb1 mutants in the ABA inhibition of cotyledon greening and root growth................. 67 3.3.3 RACK1 and AGB1 may not physically interact with each other............................... 72 3.3.4 The regulation of RACK1 expression by G-proteins ................................................. 76 3.4 Discussion.......................................................................................................................... 79 3.5 References ......................................................................................................................... 81 CHAPTER 4 RACK1 is a Negative Regulator of ABA Responses in Arabidopsis ................ 85 4.1 Introduction ....................................................................................................................... 86 4.2 Materials and methods....................................................................................................... 87 4.2.1 Plant materials and growth conditions........................................................................ 87 4.2.2 Generation of RACK1A overexpression lines ........................................................... 87 4.2.3 Generation of PRACK1::GUS reporter lines.................................................................. 88 4.2.4 Generation of PRACK1::RACK1-GFP/CFP/YFP reporter lines ................................... 88 4.2.5 ABA inhibition of seed germination and cotyledon greening assays......................... 89 4.2.6 ABA inhibition of root growth assay ......................................................................... 89 4.2.7 Water loss assay.......................................................................................................... 89 4.2.8 Salt stress germination assay ...................................................................................... 89 4.2.9 RT-PCR and quantitative real-time RT-PCR ............................................................. 90 4.3 Results ............................................................................................................................... 92 4.3.1 RACK1 genes act redundantly to negatively regulate ABA responses during seed germination and early seedling development ...................................................................... 92 4.3.2 Overexpression of RACK1A conferred ABA hyposensitivity .................................... 96 4.3.3 Expression of RACK1 in imbibed, germinating and germinated seeds ...................... 97 4.3.4 ABA marker genes, RD29B and RAB18, were up-regulated in rack1a mutants...... 101 4.3.5 The transcription of three RACK1 genes was down-regulated by ABA .................. 103 4.3.6 rack1 mutants display reduced water loss ................................................................ 104 4.3.7 rack1 mutants display hypersensitivity to salt during seed germination.................. 105 4.3.8 RACK1 interaction network ..................................................................................... 107 4.4 Discussion........................................................................................................................ 114 4.5 References ....................................................................................................................... 118 CHAPTER 5 Involvement of Arabidopsis RACK1 in Protein Translation and Its Regulation by Abscisic Acid ............................................................................................................................ 123 v  5.1 Introduction ..................................................................................................................... 124 5.2 Materials and methods..................................................................................................... 125 5.2.1 Plant materials and growth conditions...................................................................... 125 5.2.2 DNA microarray assay ............................................................................................. 125 5.2.3 Yeast complementation assay................................................................................... 127 5.2.4 Isolation of eif6a and eif6b T-DNA insertional mutants .......................................... 127 5.2.5 Yeast two-hybrid assay............................................................................................. 128 5.2.6 Bi-molecular Fluorescence Complementation (BiFC) assay in Arabidopsis mesophyll protoplasts.......................................................................................................................... 128 5.2.7 Root growth assay with anisomycin ......................................................................... 129 5.2.8 Analysis of embryo development ............................................................................. 129 5.2.9 Ribosome profiling assay ......................................................................................... 129 5.2.10 Gene expression analysis........................................................................................ 130 5.3 Results ............................................................................................................................. 131 5.3.1 A group of genes co-regulated by ABA and rack1 mutation ................................... 131 5.3.2 Co-expression analysis of RACK1 genes.................................................................. 133 5.3.3 Arabidopsis RACK1 complements the S. cerevisiae cpc2/rack1 mutant ................. 134 5.3.4 RACK1 physically interacts with eukaryotic initiation factor 6 (eIF6) ................... 136 5.3.5 eIF6 homologs in Arabidopsis ................................................................................. 138 5.3.6 rack1 mutants are hypersensitive to anisomycin, an inhibitor of protein translation143 5.3.7 RACK1 in functional 80S ribosomal subunit assembly and 60S ribosome biogenesis ........................................................................................................................................... 145 5.3.8 ABA inhibits global protein translation.................................................................... 145 5.3.9 ABA regulates the expression of both RACK1 and eIF6 genes ............................... 147 5.4 Discussion........................................................................................................................ 148 5.4.1 Arabidopsis RACK1 genes are functional equivalent to S. cerevisiae CPC2........... 149 5.4.2 RACK1 is involved in the 60 ribosome subunit biogenesis and 80S functional ribosome assembly in Arabidopsis .................................................................................... 150 5.4.3 ABA inhibits translation initiation ........................................................................... 151 5.5 References ....................................................................................................................... 153 CHAPTER 6 Conclusions and General Discussion ................................................................ 158 6.1 Conclusions ..................................................................................................................... 159 6.1.1 Three Arabidopsis RACK1s influence plant growth and ABA responses in a manner of unequal genetic redundancy (Chapter 2 and Chapter 4) ............................................... 159 6.1.2 RACK1s function as negative regulators of ABA responses (Chapter 4)................ 160 6.1.3 Arabidopsis RACK1 and heterotrimeric G-protein complex interact in a mechanism that is distinct from their counterparts in mammalian cells and in yeast (Chapter 3) ....... 161 6.1.4 Arabidopsis RACK1s are involved in protein translation regulation and might serve as a molecular link between ABA signaling and its inhibitory effect on global protein translation (Chapter 5) ....................................................................................................... 163 6.2 General discussion and future directions......................................................................... 165 6.2.1 What is the role of RACK1 in ABA responses?....................................................... 165 6.2.2 What is the role of RACK1 in protein translation? .................................................. 167 6.2.3 Is RACK1 one of the molecular links between stress signaling, ABA signaling and their effect on protein translation?..................................................................................... 167 6.2.4 Other studies that could be done to better understand the function of RACK1 ....... 168 6.4 References ....................................................................................................................... 170 APPENDIX 1A. Genes that are co-up-regulated by rack1 mutation and ABA treatment........ 173 vi  APPENDIX 1B. Genes that are co-down-regulated by rack1 mutation and ABA treatment ... 185 APPENDIX 2. RACK1-coexpressed genes .............................................................................. 205  vii  LIST OF TABLES  Table 1.1 RACK1 protein and its homolog's interacting partner and their plant homologs ..................................................................................................................................................... 10 Table 4. 1 Primers used in this study ....................................................................................... 91 Table 4.2 RACK1A-interacting proteins identified by using the BAR Arabidopsis Interactions Viewer. ................................................................................................................ 109 Table 4.3 RACK1C-interacting proteins identified by using the BAR Arabidopsis Interactions Viewer. ................................................................................................................ 111  viii  LIST OF FIGURES  Figure 1.1 ABA signaling regulation of seed germination and early seedling development. ....... 3 Figure 1.2 ABA signaling regulation of stomatal movement........................................................ 4 Figure 1.3 The N-J phylogenetic tree of RACK1 orthologs in plants. ........................................ 13 Figure 1.4 Protein sequence alignment between plant RACK1 orthologs and mammalian RACK1. ....................................................................................................................................... 13 Figure 1.5 Protein sequence alignment between RACK1 proteins in Arabidopsis and rice. ...... 15 Figure 1.6 Loss-of-function rack1a mutant in Arabidopsis. ....................................................... 17 Figure 1.7 Protein sequence alignment between eIF6 proteins in Arabidopsis and human. ....... 19 Figure 1.8 Protein sequence alignment between protein phosphatase 2A-A subunits in Arabidopsis and human. .............................................................................................................. 19 Figure 1.9 Protein sequence alignment between protein phosphatase 2A-C subunits in Arabidopsis and human. .............................................................................................................. 20 Figure 1.10 Protein sequence alignment between Arabidopsis Gβ, AGB1 (NCBI accession number: NP_195172.1) and bovine Gβ, GNB1 (NCBI accession number: P62871). ................ 20 Figure 1.11 Illustration of genomic duplication of RACK1B and RACK1C genes. .................... 23 Figure 2.1 Multiple amino acid sequence alignment of RACK1 in plants and in humans. ........ 37 Figure 2.2 T-DNA insertional mutants of RACK1B and RACK1C. ........................................... 43 Figure 2.3 Loss of function mutations in RACK1B and RACK1C enhance the rosette leaf phenotype of rack1a mutants. ..................................................................................................... 44 Figure 2.4 Loss of function mutations in RACK1B and RACK1C enhance the root phenotype of rack1a mutants............................................................................................................................. 46 Figure 2.5 The complementation of rack1a mutants by overexpression of RACK1 genes......... 48 Figure 2.6 The expression of RACK1A, RACK1B and RACK1C genes. ..................................... 49 Figure 2.7 The expression of RACK1 genes in rack1a, rack1b and rack1c single and double mutants......................................................................................................................................... 51 Figure 2.8 The model of unequal genetic redundancy of RACK1 genes in regulating plant development................................................................................................................................. 55 Figure 3.2 ABA hypersensitivity of loss-of-function alleles of RACK1A, GPA1 and AGB1. .... 68 Figure 3.3 ABA hypersensitivity of agb1 rack1a and gpa1 rack1a double mutants in the ABA inhibition of cotyledon greening assay. ....................................................................................... 70 Figure 3.4 ABA hypersensitivity of agb1 rack1a and gpa1 rack1a double mutants in the ABA inhibition of root growth assay. ................................................................................................... 71 Figure 3.5 Test of the physical interaction between RACK1 and AGB1 using yeast two-hybrid assay............................................................................................................................................. 73 Figure 3.6 Test of the interaction between RACK1A and AGB1 using yeast three-hybrid assay, plant two-hybrid assay, and co-immunoprecipitation assay........................................................ 75 Figure 3.7 Expression analysis of RACK1A in the young seedlings of G-protein mutants. ...... 77 Figure 3.8 Expression analysis of RACK1A in the mature seeds of gpa1 mutants. ................... 78 Figure 4.1 ABA sensitivity of rack1 single and double mutants in seed germination assay. ..... 93 Figure 4.2 ABA sensitivity of rack1 double mutants in cotyledon greening assay. ................... 95 Figure 4.3 ABA sensitivity of rack1 double mutants in root growth assay. ............................... 96 Figure 4.4 Overexpression of RACK1A conferred ABA hyposensitivity.................................... 97 Figure 4.5 ABA sensitivity of RACK1A overexpression lines in seed germination assay. ......... 98 ix  Figure 4.7 Analysis of RACK1 protein expression in imbibed, germinating and germinated seeds using PRACK1::RACK1-GFP/CFP/YFP reporter lines. ..................................................... 101 Figure 4.8 Expression of ABA marker genes, RD29B and RAB18, in rack1 mutants. ............. 102 Figure 4.9 Regulation of the transcription of RACK1 by ABA................................................. 103 Figure 4.10 Water loss assay of rack1 mutants. ........................................................................ 104 Figure 4.11 Water loss assay of RACK1A overexpression lines. .............................................. 105 Figure 4.12 Salt stress sensitivity of rack1 mutants during seed germination. ......................... 106 Figure 4.13 Salt stress sensitivity of RACK1A overexpression lines during seed germination. 107 Figure 4.14 Quantitative RT-PCR analysis of the expression of selected putative RACK1 interactors in response to ABA.................................................................................................. 113 Figure 5.1 Gene ontology distribution of the genes that are differentially regulated in rack1a rack1b mutants........................................................................................................................... 132 Figure 5.2 A Venn diagram shows the number of genes that are co-regulated by 50 µM ABA treatment and by rack1 mutation. .............................................................................................. 133 Figure 5.3 RACK1 co-expression analysis................................................................................. 134 Figure 5.4 Complementation assay for failed pseudohyphal growth in the diploid S. cerevisiae cpc2 mutant, using three Arabidopsis RACK1 genes. ............................................................... 135 Figure 5.5 Physical interaction between RACK1 and eIF6 detected in yeast two-hybrid assays and in the BiFC system.............................................................................................................. 137 Figure 5.6 Subcellular localization of RACK1 and eIF6. ......................................................... 138 Figure 5.7 Arabidopsis eIF6 homologs. .................................................................................... 140 Figure 5.8 Expression of Arabidopsis eIF6 homologs. ............................................................. 141 Figure 5.9 eif6 mutant alleles..................................................................................................... 142 Figure 5.10 The synergistic effect of anisomycin treatment and rack1 mutation on Arabidopsis seedling root growth. ................................................................................................................. 144 Figure 5.11 Ribosome profiling of rack1a rack1b mutant and ABA-treated Arabidopsis seedlings. ................................................................................................................................... 146 Figure 5.12 The regulation of RACK1 and eIF6 expression by ABA. ...................................... 148 Figure 6.1 A schematic presentation of the role of RACK1 in stress/ABA responses and protein translation regulation. ................................................................................................................ 165 Figure 6.2 ABA signaling regulation of seed germination and early seedling development and the putative role of RACK1 in facilitating ABA responses. ..................................................... 169  x  LIST OF ABBREVIATIONS ABA ABRC ATP cDNA BAR BiFC BLAST CaMV35S cDNA CFP CYFP/NYFP DIC DNA eIF6 EV GD GFP GUS hr kb IP LB mRNA MS OFP1 ORF PAGE PCR qRT-PCR RACK1 RNA RNAi RT-PCR SC SD S.E. TAIR T-DNA WT X-gluc Y2H YFP  abscisic acid Arabidopsis biological resource center adenosine triphosphate complementary DNA Bio-Array Resource for Arabidopsis Functional Genomics Bi-molecular fluorescence complementation Basic Local Alignment Search Tool Cauliflower mosaic virus 35S (constitutive) promoter Complementary DNA (DNA reverse transcribed from messenger RNA Cyan fluorescent protein Carbon-terminus/nitrogen-terminus of yellow fluorescent protein Differential interference contrast deoxyribonucleic acid eukaryotic initiation factor 6 empty vector Gal4 DNA binding domain green fluorescent protein β-glucuronidase hours Kilo bases immunoprecipitation Luria-Bertani bacterial growth medium formula Messenger RNA Murashige and Skoog Ovate family protein 1 Open reading frame polyacrylamide gel electrophoresis polymerase chain reaction Quantitative reverse transcription-polymerase chain reaction Receptor for activated protein C kinase 1 ribonucleic acid RNA interference reverse transcription-polymerase chain reaction synthetic complete Standard deviation Standard error The Arabidopsis Information Resources transfer DNA wild type 5-bromo-4-chloro-3-indolyl-β-D-glucuronide yeast two hybrid yellow fluorescent protein  xi  ACKNOWLEDGEMENTS  I want to first express my gratefulness to my parents, who are always supporting me and encouraging me selflessly. I owe them too much to ever be able to pay them back. I want to thank my younger sister who has been taking care of our parents with her full heart, and never complained about my inability to help her. She granted me peace in my mind for these 7 years of my study in Canada. I want to thank my wife, Kelly, who has always been there for me and accompanied me through lots of difficult times in the path, who has inspired me to work harder in pursuit of a life that we have dreamed and designed together. I want to say thanks to Dr. Jin-Gui Chen, my PhD supervisor. I thank him for the great passion and incredible patience that he put into training me and helping me build up my confidence in academic research. He has always been available, always been amazingly efficient and always challenging me to the next level. I also owe him too much to be able to pay back. I want to thank my committee members, Dr. Brian Ellis, Dr. George Haughn, and Dr. Lacey Samuels, who gave me enormous help and valuable advice on my thesis research and my career choice, who also serve as great role models for me to observe and learn from on a daily basis. Specifically, I want to show my gratitude to Dr. Brian Ellis, who took on much more responsibility than he needed to on my PhD training. I’ve learned so much from attending Ellis’ lab’s PBG (Plant Biology Group) meeting, a place for open discussion of pretty much any topic in plant biology. I want to thank Chen lab members who gave me various help during my PhD program: Dr. Shucai Wang, Qingning Zeng (Dr. to be), Junbi Wang (Dr. to be), Dr. Yajun Gao, Dr. Ying Chang, and Jia Cheng. I want to say special thanks to Dr. Shucai Wang, who is the most generous researcher I’ve ever seen. He gave me much precious advice on designing and conducting my day-to-day experiments and made my research life much easier. I want to thank many fellow graduate students and postdocs who gave me friendship, support and help at times when I needed them the most. They are: Hardy Hall, Dr. Jinsuk Lee, Dr. Sarah McKim, Dr. Jie Le, Dr. Jun Chen, Dr. Eiko Kawamura, Dr. Minako Kaneda, Dr. Eryang Li, Dr. Songhua Zhu, Dr. Yunkun Dang, Jun Huang, Allan DeBono, Eric Johnson, Dr. Xianzhong Wu, Dr. Fenling Li xii  and many more whose names did not show up here. I also should not forget those undergraduate students who did a lot dirty work without any complaint and with whom I had a lot of fun time in lab: Phoebe Lee, Candy Luk, Linda Lin, Cameron Grisdale, Sarah Mak, and Asal Ghazal. I wish them the best for the career and life that they’re pursuing.  xiii  CO-AUTHORSHIP STATEMENT  Below is a list of papers that have been published or submitted for publication as a result of this work and a summary of the contribution made by the candidate.  Guo J, Wang S, Valerius O, Hall H, Zeng Q, Ellis B and Chen JG. Involvement of Arabidopsis RACK1 in protein translation and its regulation by abscisic acid. Under revision for Plant Physiology. Chen JG and the candidate designed the experiments. The candidate conducted majority of the research work and wrote the manuscript. Chen JG revised the manuscript. Wang S assisted in protoplast assay. Valerius O supplied new material and offered technical comments. Hall H conducted data analysis for DNA microarray experiment. Ellis B revised the manuscript and offered insightful discussion. Other co-authors assisted the candidate in some of the experiments.  Guo J, Wang, J, Xi L, Huang WD, Liang J, and Chen JG (2009a) RACK1 is a negative regulator of ABA responses in Arabidopsis. Journal of Experimental Botany 60:3819-3833. Chen JG and the candidate designed the experiments. The candidate conducted majority of the research work and wrote the manuscript. Chen JG revised the manuscript. Other co-authors assisted the candidate in some of the experiments.  Guo J, Wang S, Wang J, Huang WD, Liang J, and Chen JG (2009b) Dissection of the relationship between RACK1 and heterotrimeric G-proteins in Arabidopsis. Plant & Cell Physiology 50:1681-1694. Chen JG and the candidate designed the experiments. Chen JG prepared most of the genetic materials used in this study and contributed to Figure 1 and Figure 2. The candidate conducted the rest of the research work and wrote the manuscript. Chen JG revised the manuscript. Other co-authors assisted the candidate in some of the experiments.  xiv  Guo J, Chen JG (2008) RACK1 genes regulate plant development with unequal genetic redundancy in Arabidopsis. BMC Plant Biology 8: 108 Chen JG and the candidate designed the experiments. The candidate conducted all of the experiments and wrote the manuscript. Chen JG revised the manuscript.  Guo J, Liang J, Chen JG (2007) RACK1: a versatile scaffold protein in plants? International Journal of Plant Developmental Biology 1: 95-105. The candidate outlined the concepts, collected the literature and wrote the manuscript and did most of the data analysis and prepared the Figures. Chen JG revised the manuscript. Liang J contributed to Figure 3 and offered some useful discussions.  xv  CHAPTER 1  GENERAL INTRODUCTION  1  1.1 Abscisic Acid Signaling Plant hormone abscisic acid (ABA) regulates diverse aspects of plant growth, development and stress responses, including seed germination, plant vegetative growth, leaf stomatal movement, and abiotic and biotic stress responses. As numerous reviews about every aspect of ABA biosynthesis, metabolism, and signaling events are available (Finkelstein et al., 2002; Finkelstein and Gibson, 2002; Nambara and Marion-Poll, 2005; Finkelstein et al., 2008), I will briefly summarize some basic physiological functions of ABA in plants in this subchapter. I will also discuss some of the recent development on the ABA receptors identification and the ABA signaling in non-plant organisms.  1.1.1 The physiological function of ABA  ABA generally has inhibitory effect on plant growth. Exogenous application of ABA inhibits seed germination and early seedling development. The inhibitory effect of ABA on seed germination is best understood in cereal grains in which ABA and GAs (Gibberellins) have antagonistic function in mobilizing starch reserve in the endosperm. Starch mobilization is catalyzed by α-amylase, an enzyme synthesized in aleurone and secreted into the endosperm, whose expression is promoted by GA and inhibited by ABA (Lovegrove and Hooley, 2000). In contrast, the mechanism of the inhibitory effect of ABA on seed germination in Arabidopsis is poorly understood despite the fact that many ABA signaling molecules were discovered from mutants with altered seed germination response to ABA (Figure 1.1, Seo et al., 2009). Similarly, there has been little study of the mechanism of the inhibitory effect of ABA on early seedling development. One study pointed out that this effect might be mainly due to ABA’s inhibitory effect on plant cell division by promoting the expression of a cyclin kinase inhibitor, ICK1 (Hong et al., 1998). Another important role of ABA is promotion of stomatal closure and inhibition of stomatal opening. The elevation of ABA concentration, sensed and transduced via various signaling molecules within the guard cells (Figure 1.2), leads to the elevation of cytosolic Ca2+, which in turn activates the slow-anion channels and inactivates the inward-rectifying K+ channel. The activation of these two channels leads to the depolarization of plasma membrane, which  2  Figure 1.1 ABA signaling regulation of seed germination and early seedling development. The pointed-end arrow indicates a directional signal transduction route that involves direct biochemical interaction (such as physical binding, phosphorylation etc.) or extrapolated through epistasis analysis. The blunt-end arrow indicates an inhibitory effect. ABI, ABA insensitive ; PP2Cs, protein phosphatase 2Cs; PYR, pyrabatin resistance; SnRK, sucrose non-fermenting kinase; ABF, ABA-responsive elements (ABRE) binding factor; ICK1, inhibitor of cyclindependent kinase 1; GTG, GPCR-type G protein; CHLH, H subunit of the magnesiumprotoporphyrin-IX chelatase; Rop, plant-specific subfamily of RHO GTPase; CPK, calciumdependent protein kinase; GCR, putative G-protein coupled receptor; GPA1, G-protein alpha subunit 1; AGB1, G-protein beta subunit 1.  3  Figure 1.2 ABA signaling regulation of stomatal movement. The pointed-end arrow indicates a directional signal transduction route that involves direct biochemical interaction (such as physical binding, phosphorylation etc.) or extrapolated through epistatic study. The blunt-end arrow indicates an inhibitory effect. ABI, ABA insensitive ; PP2Cs, protein phosphatase 2Cs; PYR, pyrabatin resistance; SnRK, sucrose non-fermenting kinase; ABF, ABA-responsive elements (ABRE) binding factors; ICK1, inhibitor of cyclin-dependent kinase 1; GTG, GPCRtype G protein; CHLH, H subunit of the magnesium-protoporphyrin-IX chelatase; Rop, plantspecific subfamily of RHO GTPase; CPK, calcium-dependent protein kinase; GCR, putative Gprotein coupled receptor; GPA1, G-protein alpha subunit 1; AGB1, G-protein beta subunit 1; OST1, open stomata 1; PP2A, protein phosphatase 2A; SNARE, soluble N-ethylmaleimidesensitive factor attachment protein receptors; PLD, phospholipids D; PA, phosphatidic acid, ROS, reactive oxygen species; GCA, growth controlled by abscisic acid; cADPR, cyclic ADPribose; cGMP, cyclic guanosine monophosphate; NAPDH, nicotinamide adenine dinucleotide phosphate; MPK, (mitogen-activated protein kinase); S1P, sphingosin-1-phosphate; PI-PLC, phosphatidylinositol-specific phospholipase C; AtRac1, Arabidopis Ras-like small GTPase 1.  4  then activates the outward K+ channel. The leaking out of anions and K+ causes loss of turgor in guard cells and hence stomatal closure (Schroeder et al., 2001; Ward et al., 2009). Furthermore, ABA has been long recognized to be important in abiotic stress adaptation. Multiple stresses have been shown to increase cellular ABA levels. This is believed to be mainly achieved by promoting ABA biosynthesis to exceed its degradation rate (Kushiro et al., 2004). Meanwhile, increasing evidence supports a regulatory role for ABA in responding to biotic stresses (Ton et al., 2009).  1.1.2 ABA function in other organisms  It has been recently found that endogenous ABA synthesis also occur in lower Metazoa and in human granulocytes (Zocchi et al., 2003; Puce et al., 2004; Bruzzone et al., 2007). In sponges, ABA functions as an intracellular messenger downstream of a thermosensor (Zocchi et al., 2003). In the hydroid, Eudendrium reacemosum, ABA is activated by light and plays a signaling role in light-induced regeneration. In humans, ABA was identified in granulocytes as an endogenous pro-inflammatory cytokine, which acts as an endogenous stimulator of insulin (Bruzzone et al., 2008), and is involved in the development of atherosclerosis (Magnone et al., 2009). Interestingly, in all of these different species, ABA signals through activation of ADPribosyl cyclase, promoting the production of the secondary messenger, cyclic ADP-ribose (cADPR), and thereby increasing intracellular Ca2+ concentration (Zocchi et al., 2003; Puce et al., 2004; Bruzzone et al., 2007). Although no obvious homolog of ADP-ribosyl cyclase is present in plant genomes, cADPR itself has been established as a key player in ABA signaling transduction in plants (Wu et al., 1997), indicating that an evolutionary conserved ABA/cADPR signaling cascade may exist.  1.1.3 The ABA receptors  Unlike the case for many other plant hormones, a traditional forward genetic approach has failed to identify an ABA receptor. This led to the assumption that either ABA receptors belong to a gene family with redundant functions, or that mutations in the ABA receptors are lethal. The 5  identification of multiple ABA receptors by other strategies later supported both assumptions. Through a biochemical assay looking for ABA binding proteins, an H subunit of Mg-chelatase (CHLH) was shown to specifically bind ABA with high affinity (Shen et al., 2006). The CHLH protein is a key component in both chlorophyll biosynthesis and plastid-to-nucleus signaling, and is knock-out lethal. It has long been known that at least one ABA receptor resides on the plasma membrane (Anderson et al., 1994), and two research groups identified different G-protein coupled receptors (GPCRs) as candidate ABA receptors associated with the plasma membrane (Liu et al., 2007; Pandey et al., 2009). GCR2 (G-protein coupled receptor 2) was reported to have seven predicted trans-membrane domains and the ability to interact with the sole Gα subunit in Arabidopsis, GPA1, which are both characteristics of mammalian GPCR (Liu et al., 2007). However, gcr2 mutants displayed mild or no response to ABA (Gao et al., 2007; Guo et al., 2008), and other studies questioned the prediction of the seven transmembrane domains (Johnston et al., 2007; Chen and Ellis, 2008; Illingworth et al., 2008) as well as the ABAbinding property of GCR2 (Risk et al., 2009). Therefore, whether GCR2 really works as an ABA receptor remains unclear. More recently, a novel GPCR gene, GTG (GPCR-type G proteins), was identified as an ABA receptor and reported to mediate ABA signaling via its interaction with heterotrimeric G-proteins (Pandey et al., 2009). Despite the identification of the above ABA receptors, none of these receptors have a direct relationship with any of the five classical ABA signaling molecules, ABI1, ABI2, ABI3, ABI4 and ABI5, which were identified from forward genetic screening as mutants with ABA insensitivity. More recently, two research groups using very different approaches identified the same 14-member gene family, PYR (Pyrabatin Resistance)/ RCAR (regulatory component of ABA receptor), as encoding an ABA receptors (Ma et al., 2009; Park et al., 2009). With the identification of this ABA receptor family and characterization of the structure of PYR, a linear ABA signaling transduction pathway, PYR/RCAR-PP2C-SnRK2, that regulates downstream gene expression is now becoming clear (Fujii et al., 2009; Melcher et al., 2009; Miyazono et al., 2009; Nishimura et al., 2009; Santiago et al., 2009; Sheard and Zheng, 2009).  6  1.2 RACK1, a Versatile Scaffold Protein in Plants? 1  1.2.1 Introduction  RACK1 was originally identified as a receptor for activated protein kinase C (PKC) (Ron et al. 1994). Now RACK1 has been recognized as a scaffold protein that has versatile roles in diverse signal transduction pathways. There are several excellent review articles that highlight the structure and function of RACK1 in mammals and yeasts (Chen et al. 2004b; McCahill et al. 2002; Sklan et al. 2006). Here we mainly focus on the study of RACK1 in plants, particularly in the model plant Arabidopsis. For details of RACK1 in other systems, readers are referred to the abovementioned review articles and the references therein. We begin with a brief review of the discovery and functional properties of RACK1 in mammalian cells. 1.2.2 Discovery of RACK1 as an intracellular receptor RACK1 gene was first cloned from a chicken liver cDNA library as a gene that is closely linked to the major histocompatibility complex loci (Guillemot et al. 1989). A few years later, Ron et al. (1994) isolated the RACK1 gene from a rat brain cDNA expression library and proved that RACK1 protein fulfilled all the criteria for a receptor for activated PKC established by MochlyRosen (Mochly-Rosen et al. 1991a, 1991b). Binding of RACK1 to the activated form of PKC anchors the latter to a sub-cellular location where its substrates are present (Ron et al. 1995). RACK1 was found to interact with both the “conventional (calcium-dependent)” and the “novel (calcium-independent)” PKC isoforms (Besson et al. 2002) with the conventional PKCβII isoform being the preferred binding partner (Stebbins and Mochly-Rosen 2001).  1  . A version of this section has been published.  Guo J, Liang J, Chen JG (2007) RACK1: a versatile scaffold protein in plants? International Journal of Plant Developmental Biology 1: 95-105  7  1.2.3 RACK1 is a WD-repeat-containing protein RACK1 is composed of seven tryptophan-aspartic acid-domain (WD40) repeats. Each WDrepeat spans approximately 40-60 amino acids. The WD domains start with a glycine-histidine (GH) dipeptide 11 to 24 residues from the N terminus and end with a tryptophan-aspartic acid (WD) dipeptide at the C terminus (Smith et al. 1999), though neither GH nor WD is absolutely conserved. A summary of some of the substitute amino acids at GH and WD positions of WDrepeat proteins is available at http://BMERC-www.bu.edu/wdrepeat (Smith et al. 1999). The first WD-repeat-containing protein whose crystal structure was determined was the heterotrimeric G protein β subunit (G) in which WD40 repeats form a seven-bladed β propeller structure (Wall et al. 1995; Sondek et al. 1996). Each WD domain contains the first three strands of one blade and the last strand in the next blade; the last WD domain comprised of the first three strands of the last blade and the last strand of the first blade to form a circular propeller structure. Other WD40-repeat-containing proteins whose crystal structures were resolved all form β propeller, indicating that the β propeller may be the predominant fold for this protein family (Robinson et al. 2001; Orlicky et al. 2003; Voegtli et al. 2003; Madrona and Wilson 2004). 1.2.4 RACK1 as a scaffold protein A scaffold protein is a protein whose main function is to bring other proteins together for them to interact. Besides its role as an intracellular receptor for activated PKC, RACK1 is capable of interacting with numerous other signaling molecules and modulating their cellular functions. As shown in Table 1.1, most RACK1-interactors identified in mammals and fungi/yeasts are signaling proteins, such as receptors, kinases, phosphatases, transcription factors, and GTPases. These RACK1-interacting proteins include membrane- anchored proteins, cytosolic proteins, and nuclear proteins. Through the interaction with these partners, RACK1 play regulatory roles in diverse developmental processes and physiological responses, such as cell cycle control, cell movement and growth, immune responsiveness, and neural responses (McCahill et al. 2002; Sklan et al. 2006). Scaffolding property of RACK1 entitles it to integrate inputs from distinct signaling pathways. There are many examples that RACK1 functions as a scaffold protein to bring other proteins together to facilitate the interaction between them. For example, STAT1 (Signal  8  Transducers and Activators of Transcription 1) is associated with one of its receptors, type I interferon (IFN), via RACK1 (Usacheva et al. 2001). Disruption of the interaction between RACK1 and IFNα receptor abolishes IFNα-induced tyrosine phosphorylation of STAT1. One way for RACK1 to scaffold two or more proteins together is through its ability to bind multiple proteins simultaneously. In consistent with this scenario, RACK1 has several independent protein binding sites (McCahill et al. 2002). On the other hand, two interacting proteins could have a same binding site on RACK1. RACK1 scaffolds their interaction by forming a homo dimer to bring them together (Thornton et al. 2004).  9  Table 1.1 RACK1 protein and its homolog's interacting partner and their plant homologs Reference  Arabidopsis homolog  Mochly-Rosen et al. (1991a) Disatnik et al. (1994)  None  I. Human proteins C2 domain proteins PKC PLCγ1 Synaptotagmin p65 protein PH domain proteins Dynamin1 β-Spectrin oxysterol binding protein  Ras-guanine nucleotide releasing factor (GRF) SH2 domain proteins Src tyrosin kinase protein tyrosine kinase, Fyn p120GAP (Ras GTPaseactivating protein) WD repeat domain protein Gβ1γ1 and transducin hetero-trimer Gα1β1γ1 FAN, an adapter protein factor associated with neutral sphingomyelinase activation RACK1  Other proteins PDE4D5 p73α pRB p63α p19 (H-RasIDX) alternative splicing variant of c-H-ras Type I interferon receptor β long subunit (IFNαRβL) and STAT1 eIF6 translation initiation factor  Mochly-Rosen et al. (1992) Rodriguez et al. (1999) Rodriguez et al. (1999) Rodriguez et al. (1999)  Rodriguez et al. (1999)  All plant PI-PLC isoforms appear to belong to δ-type of mammalian PIPLCs, but lacking a PH domain (Mueller-Roeber B and Pical C, 2002). However, C2 domains are conserved among all the PLC isozymes. At5g11100 and At1g05500 shared highest homology to p65, in the C2 domain region, each share 27% identity (44% similarity) and 26% identity (46% similarity) (Craxon M, 2004, BMC genomics) None None At1g13170, At2g31020, At2g31030, At4g08180, At4g12460 and At4g22540; share above 30% identity (>42% similarity) with their human homolog; based on skirpan AL 2006, there are 12 oxysterol binding protein in Arabidopsis and expressed in various tissues (skirpan AL 2006). None  Chang et al. (1998); Chang et al. (2001) Yaka et al. (2002)  None  Koehler and Moran (2001)  At5g05710 share 22% identity and 51% similarity to the PH domain of the query protein  Dell et al. (2002)  bovine AGB1 and Arabidopsis AGB1 (At4g34460) shared 43% identity and 61% similarity At2g45540 and At1g58230, both are WD-40 repeat family protein / beige-related; they share 35% identity( 51% similarity) and 33% identity (52% similarity)  Tcherkasowa et al. (2002)  None  Thornton, C. et al. 2004 Yarwood et al. (1999) Ozaki et al. (2003) Ozaki et al. (2003) Fomenkov et al. (2004) Guil et al. (2003) Usacheva et al. (2001); Croze et al. (2000) Ceci et al. (2003)  None. None. At3g12280, retinoblastoma-related protein share 22% identity (40% similarity) to pBR None None. None At3g55620, shares 72% identities and 84% similarity with human IEF6 (p27BBP); At2g39820 (NP_181512) shares 58% identity, 77% similarity with human IEF6 (p27BBP)  10  Table 1.1 (continued) RACK1 protein and its homolog's interacting partner and their plant homologs PTPmu  Hellberg et al. (2002); Mourton et al. (2001)  Integrin  Buensuceso et al. (2001); Liliental and Chang (1998) Hermanto et al. (2002)  Insulin-like growth factor I (IGF-IR) P0 (MPZ) myelin protein Plectin (cytoskeletal linker protein) Angiotensin II receptorassociated protein (Agtrap) Beta chain of IL-5/IL3/GM-CSF receptor inositol 1,4,5trisphosphate receptors NR2B subunit of the NMDA receptor Na(+)/H(+) exchange regulatory factor (NHERF1), a binding partner of CFTR Androgen receptor (AR) Dopamine transporter (DAT) (GABA) gammaaminobutyric acid type A receptor β1 subunit acetylcholinesterase variant AChE-R Tyrosine kinase 2  (At1g71860, (PTP1) shares 32% identities and 52% similarity to the aa 913-1153 of human PTPmu; aa 915–1178 of human PTPmu was used to interact with RACK1 None None.  Xu et al. (2001) Osmanagic-Myers and Wiche (2004) Wang et al. (2002)  None None  Geijsen et al. (1999)  None  Patterson et al. (2004)  No homolog. (Nagata T 2004)  Yaka et al. (2002)  None  Liedtke et al. (2002, 2004)  None  Rigas et al. (2003) Lee et al. (2004)  None None  Brandon et al. (1999)  None  Perry et al. (2004)  None  Haro et al. (2004)  Lots of homologs to the tyrosine kinase domains which binds to RACK1  (RanI) Pat I kinase  Mcleod et al. (2000)  Scp160p  Baum et al. (2004)  No structure homolog (Mcleod et al. 2000), lots of homologs to the tyrosine kinase domain At1g33680, single-strand RNA binding protein, share 27% identity (44% similarity) to the KH-I domain of Scp160p None None  None  II yeast proteins  Msa2/Nrd1 Jeong et al. (2004) Pck2 Won et al. (2001) III. Viral and bacterial proteins HIV-1 Nef protein synthesized early after infection. Crucial for high viral loads and pathogenesis Epstein-Barr BLZF1 Epstein-Barr A73 Influenza A M1 Adenoviral E1A Mumps virus protein V Human papillomavirus E2 protein Helicobacter pylori VacA cytotoxin  Gallina et al. (2001)  Baumann et al. (2000) Smith et al. (2000) Reinhardt and Wolff (2000) Sang et al. (2001) Kubota et al. (2002) Boner and Morgan (2002) Hennig et al. (2001)  11  1.2.5 Discovery of RACK1 in plants RACK1 is highly conserved across several kingdoms of eukaryotic organisms including animals, plants, and fungi. Although not recognized as such, the first plant RACK1 gene was cloned from tobacco BY-2 cells as an auxin (2,4-dichlorophenoxyacetic acid, 2,4-D) inducible gene, arcA (Ishida et al. 1993, 1996). Subsequently, a cDNA clone that encodes a protein that is highly similar to arcA was cloned from the greening leaves of rice (Iwasaki et al. 1995). Since then, RACK1 orthologs have been cloned in other plant species including alfalfa (McKhann et al. 1997), rape (Kwak et al. 1997), Arabidopsis (Vahlkamp and Palme 1997), and tomato (Kiyosue and Ryan 1999). RACK1 was also found in the green algae (Schloss 1990). The completely sequenced Arabidopsis genome encodes three RACK1 proteins, designated as RACK1A, RACK1B, and RACK1C (Chen et al. 2006). All three RACK1 proteins belong to the WD-repeat superfamily which contains 237 proteins in Arabidopsis (van Nocker and Ludwig 2003). Using a bovine G whose crystal structure has been resolved (Lambright et al. 1996; Sondek et al. 1996) as a template, RACK1A was modeled as a 7-bladed β propeller structure (Chen et al. 2006) with each blade comprised of four anti-parallel β sheets as that found in bovine Gβ (Sondek et al. 1996). The BLASTP search using Arabidopsis RACK1A protein (NCBI accession number: NP_173248) as a template revealed 15 RACK1 homologs in plants with full length proteins (Figure 1.3, Figure 1.4). Interestingly, besides Arabidopsis, other plant species also contain more than one copy of RACK1 genes, in contrast to only one copy of RACK1 gene in other nonplant organisms. For example, rice contains two RACK1 homologs, RWD1 and RWD2, which are approximately 80% similar to Arabidopsis RACK1 proteins at the amino acid level (Figure 1.5). All plant RACK1 proteins share over 65% identity and 80% similarity each other at the amino acid level when aligned with Blast 2 Sequences (Tatusova and Madden 1999).  12  Figure 1.3 The N-J phylogenetic tree of RACK1 orthologs in plants. Fifteen full-length proteins from 10 species were included. Each protein was labelled with an NCBI accession number or protein name followed by a common species name (in parentheses). The N-J phylogenetic tree was generated by GenomeNet CLUSTALW Server (http://clustalw.genome.jp/). Figure 1.4 Protein sequence alignment between plant RACK1 orthologs and mammalian RACK1. The NCBI accession number or protein name is indicated at the beginning of each sequence. Proteins analyzed include RACK1A (Arabidopsis, NCBI accession number NP_173248), RACK1B (Arabidopsis, NCBI accession number NP_175296), RACK1C (Arabidopsis, NCBI accession number NP_188441), Q39336 (rapeseed), O24076 (alfalfa), Q39836 (soybean), BAA76896.1 (tomato), BAA76895.1 (tomato), CAA96528.1 (leadwortleaved tobacco), P93340 (leadwort-leaved tobacco), ABB86277.1 (potato), ABB02625.1 (potato), P49026 (tobacco), RWD1 (Japanese rice), RWD2 (Japanese rice), P25387 (green algae), and human RACK1 (NCBI accession number P25388). The positions for conserved GH and WD dipeptides are indicated by “##” and “**” respectively on the top of the sequences. The conserved PKC binding domains are indicated by blocks on the top of the sequences. Within each sequence, identical amino acids among RACK1 proteins are shown as dots, and similar amino acids are shaded by grey color. Gaps are shown as dashed lines. All sequence alignments in this chapter were generated by the ClustalW multiple alignment of BioEdit Sequence Alignment Editor (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). The positions for WD repeat domains were obtained from the SMART database (http://smart.embl-heidelberg.de).  13  14  Figure 1.5 Protein sequence alignment between RACK1 proteins in Arabidopsis and rice. The NCBI accession numbers for rice RACK1 proteins, RWD1 and RWD2, are NP_001043910.1 and NP_001056254.1. The highest variation in sequence between Arabidopsis and rice RACK1 proteins are underlined. The RACK1 homolog in Brassica napus shared the higher homology to Arabidopsis RACK1A (96% identity and 98% similarity) than to RACK1B and RACK1C (87% identity and 94% similarity). All plant RACK1 proteins are approximately 75% similar to mammalian RACK1 at the amino acid level (Figure 1.4). The number and position of GH and WD core sequences as well as the PKC binding sites are largely conserved in plant RACK1 proteins (Figure 1.4) Despite the highly conserved amino acid sequences of RACK1 proteins in different organisms, the region between the 6th and 7th conserved GH-WD core sequences displays high variation (Figure 1.4). This region is the greatest variable region in all WD-repeat proteins and is 15  predicted to be exposed at the surface of the 7-bladed propeller structure of the WD-repeat proteins, thus presumably determining their binding properties (Smith et al. 1999). Consistent with this scenario, the majority of RACK1-interacting partners bind the 6th and 7th WD repeats of RACK1 protein (McCahill et al. 2002). Interestingly, two RACK1 proteins in rice, RWD1 and RWD2, the only monocot plant in our list, have distinctive sequences between the 2nd and 3rd conserved GH-WD core sequences but a shorter sequence between the 6th and 7th GH-WD core sequences when compared with RACK1 proteins in Arabidopsis and other dicot plants (Figure 1.4, Figure 1.5). These distinctive sequence patterns suggest that there may exist some binding partners for RACK1 protein that differ between rice and Arabidopsis. 1.2.6 Functional characterization of RACK1 in plants It has been proposed that the biological function of RACK1 was established prior to the separation of plant and animal kingdoms approximately 600 million to 1 billion years ago (Neer et al. 1994). In model mammals and in humans, RACK1 was found to be expressed ubiquitously in different tissues and organs, including brain, liver and spleen, consistent with its general scaffolding function (Chou et al. 1999). Because RACK1 was originally identified as an auxin-inducible gene in tobacco BY-2 suspension cells in a differential screen for genes involved in auxin-mediated cell division (Ishida et al. 1993), RACK1 was proposed to have a role in auxin-mediated cell division. The induction of RACK1 (ArcA) transcription in the BY-2 cells was specific to auxin, since other plant hormones, such as abscisic acid, gibberellic acid, ethylene, and cytokinin (benzylaminopurine, BAP) had no such effect. Interestingly, the transcription of Msgb1, a RACK1 ortholog in alfalfa, was induced in root by BAP, but not by 2, 4-D (McKhann et al. 1997). Kwak et al. (1997) observed that when the RACK1 ortholog in Brassica napus L. was injected into Xenopus laevis oocytes, the insulin-induced maturation of oocytes, a PKCmediated pathway, was inhibited, mimicking the effect of rat RACK1 (Smith and MochlyRosen 1992). This result suggests that plant RACK1 has a conserved function by acting as an intracellular receptor for activated PKC, though no PKC ortholog has been identified in plants. Perennes et al. (1999) found that RACK1 was induced by ultraviolet (UV) irrdation in tobacco BY-2 cells and that this induction could be blocked by salicylic acid (SA) treatment. Because UV irradiation and SA acted as agonists to arrest BY-2 cells at cell cycle entry, and 16  RACK1 transcription was correlated with the point of at cell cycle entry, it was hypothesized that RACK1 might be involved in UV and SA mediated-cell cycle arrest (Perennes et al. 1999). Komatsu et al. (2005) used a proteomic approach to analyze protein expression profiles in the embryos of the rice d1 mutant, a loss of function mutant of the heterotrimeric G protein  subunit (G). RACK1 was found to be one of the seven proteins whose expression is downregulated in d1 mutant. Accumulation of RACK1 protein was induced by abscisic acid (ABA) in imbibed seeds of wild-type, but not in d1 mutant. Based on these results, the authors proposed that the expression of RACK1 is regulated by G and that RACK1 may play important roles in rice embryogenesis and germination (Komatsu et al. 2005). Chen et al. (2006) provided direct genetic evidence for the function of RACK1 in plants by characterizing the loss-of-function mutants of RACK1 in Arabidopsis. Like mammalian RACK1, Arabidopsis RACK1 genes are expressed ubiquitously. Knocking out one of the three Arabidopsis RACK1 genes, RACK1A, conferred defects in multiple developmental processes and resulted in pleiotropic phenotypes (Figure 1.6), including shorter hypocotyls in etiolated seedlings and epinastic cotyledons in light-grown seedling. When grown under short day conditions, rack1a mutants are late flowering and the rate of rosette leaf production is reduced by approximately 40%. Furthermore, rack1a mutants display altered sensitivities to several plant hormones, including hyposensitivity to gibberellic acid and brassinolide in seed germination, hyposensitivity to auxin in adventitious and lateral root formation, and hypersensitivity to abscisic acid in seed germination and early seedling development (Chen et al..  Figure 1.6 Loss-of-function rack1a mutant in Arabidopsis. Wild-type Columbia-0 (Col, left) and rack1a-1 mutant (right) were photographed 52 days after being grown under short-day conditions (8/16 h photoperiod). 17  2006). The pleiotropic phenotype of rack1a mutants is consistent with a general scaffolding function of RACK1 1.2.7 RACK1 interacting partners in plants Although RACK1 has been shown to interact with numerous proteins with diverse functions in mammals, little is known about RACK1-interactors in plants. Recently Chang et al. (2005) used a proteomic approach to demonstrate that RACK1 proteins are associated with the 40S subunit of cytosolic ribosome in Arabidopsis. Giavalisco et al. (2005) reported that RACK1 proteins comigrate with the 80S ribosome in two-dimensional gel electrophoresis. These two independent studies provided the first biochemical evidence that at least some parts of RACK1’s function are conserved in plants, because RACK1 proteins are associated with ribosomes in both mammals and yeasts (Link et al. 1999; Ceci et al. 2003; Shor et al. 2003; Nilsson et al. 2004; Sengupta et al. 2004). Interestingly, among approximately 60 RACK1-interacting proteins identified thus far in mammals and yeasts, only a few of them have significant homologs in plants (Table 1.1). On the basis of BLAST search analysis of the NCBI Arabidopsis protein database using each mammalian or fungi/yeast RACK1-interacting protein as a template, we found that mammalian RACK1-interacting proteins eIF6, protein phosphatase 2A, 14-3-3β, and G have highest homologies in Arabidopsis (Figure 1.7, Figure 1.8, Figure 1.9, Figure 1.10). In mammalian cells, eIF6 translation initiation factor binds free 60S ribosome subunit and keeps the 40S and 60S subunits from assembling into a functional 80S ribosome. RACK1 functions as a physical linker to bring activated PKC and its substrate eIF6 together and lead to the phosphorylation of eIF6 by PKC; the phosphorylated eIF6 could eventually dissociate from the 60S subunit and allow joining of the two ribosomal subunits (Ceci et al. 2003). The RACK1 homolog in yeast, Asc1p/Cpc, is also associated with the ribosome (Shor et al. 2003) and co-precipitated with eIF6 complex (Volta et al. 2005). Mutation in Asc1p led to an impaired 80S formation and a reduced efficiency of translation (Chantrel et al. 1998). There are two eIF6 homologs (At3g55620 and At2g39820) in Arabidopsis. Mammalian eIF6 has 72% identity and 84% similarity with At3g55620, and 58% identity and 77% similarity with At2g39820 at the amino acid level (Figure 1.7). RACK1 proteins have been found to be associated with the small ribosomal subunit in Arabidopsis (Chang et al. 2005) and in algae (Manuell et al. 2005); a role of RACK1 in the regulation of translation may be evolutionarily conserved across kingdoms. Further 18  studies are required to examine a direction interaction between RACK1 and eIF6 proteins in plant cells.  Figure 1.7 Protein sequence alignment between eIF6 proteins in Arabidopsis and human. The NCBI accession numbers for Arabidopsis eIF6 proteins, eIF6A and eIF6B, are AAP75806.1 (At3g55620) and NP_181512.1 (At2g39820). The NCBI accession number for human eIF6 is AAK39426.  Figure 1.8 Protein sequence alignment between protein phosphatase 2A-A subunits in Arabidopsis and human. The NCBI accession numbers for Arabidopsis PP2A A subunits, PP2AA1/RCN1, PP2AA2, and PP2AA3, are Q38845 (At1g25490), AAP37715.1 (At3g25800), and NP_001031035.1 (At1g13320), respectively. The NCBI accession number for human PP2A A subunit α isoform, PPP2R1A, is N30153. 19  Figure 1.9 Protein sequence alignment between protein phosphatase 2A-C subunits in Arabidopsis and human. The NCBI accession numbers for Arabidopsis PP2A C subunits, PP2AC1 to PP2AC5, are Q07098 (At1g10430), Q07099 (At1g59830), Q07100 (At2g42500), P48578 (At3g58500), and ABF85773.1 (At1g69960). The NCBI accession number for human PP2A C subunit α isoform, PPP2CA, is NP_002706.1.  Figure 1.10 Protein sequence alignment between Arabidopsis Gβ, AGB1 (NCBI accession number: NP_195172.1) and bovine Gβ, GNB1 (NCBI accession number: P62871). Boxed are the RACK1-interacting domains mapped by Dell et al. (2002). The second promising candidate interactor of RACK1 in plants is the protein phosphatase 2A (PP2A), an intracellular serine/threonine protein phosphatase. In mammalian cells, PP2A holoenzymes exist as either a heterodimer consisting of a 36-KDa catalytic subunit (C subunit) and a 65-kDa regulatory A subunit, or as a heterotrimer consisting of this heterodimer and one of the regulatory B subunits (Xu et al. 2006). While the regulatory A subunit is required for scaffolding the PP2A holoenzyme heterotrimer, the regulatory B subunit 20  regulates the subcellular location and specificity of the enzyme (Sheng 2003). RACK1 was found to interact with the heterodimer of PP2A comprising of the regulatory A subunit and the catalytic C subunit (Kiely et al. 2006). The Arabidopsis genome contains three PP2A subunits, 17 B subunit, and five C subunits, which can be theoretically assembled into 255 heterotrimeric PP2A isoforms (Zhou et al. 2004). The three Arabidopsis PP2A A subunit isoforms, PP2AA1, PP2AA2, and PP2AA3, exhibit approximately 56% identity with the human homolog at the amino acid level (Figure 1.8). The five Arabidopsis PP2A C subunit isoforms, PP2AC1 to PP2AC5, exhibit about 80% identity with the human C subunit α isoform (Figure 1.9). Genetic study of Arabidopsis PP2A A subunit mutant, rcn1 (root curl in naphthylphthalamic acid 1), and B subunit mutant, ton2 (tonneau 2), revealed that PP2A and its regulatory subunits are crucial for plant development and hormonal signaling (DeLong 2006). It would be interesting to determine if RACK1 can interact with PP2A A and C subunits in plants. The 14-3-3β protein was identified as a RACK1 interactor in mouse cells (Chu et al. 2005). 14-3-3 proteins are highly conserved across the eukaryotic organism. Their general function is to specifically bind to phosphorylated proteins and change their activity, stability or localization (Ferl 2004). The discovery of the function of 14-3-3 reinforces the long-time held concept that phosphorylation/dephosphorylation of the target protein is sufficient to change its activity. There are 15 14-3-3 genes in the Arabidopsis genome and at least 13 of them are expressed. Arabidopsis 14-3-3 proteins share over 50% identity at the amino acid level each other, and can be divided into two major evolutionary groups, ε group and non-ε group, based on the phylogenetic analysis (Sehnke et al. 2002). Results from tissue and subcellular localization, genetic study, and biochemical binding assays suggested the presence of both overlapping and distinct functions among plant 14-3-3 proteins (Sehnke et al. 2002). Several important roles of 14-3-3 proteins have been revealed in plants. For example, binding of phosphorylated nitrate reductase (NR) to 14-3-3 proteins and divalent cations inactivates the enzyme activity of NR (Huber et al. 1996). 14-3-3s and Mg2+ can also bind the H+-ATPase and stimulate its pump activity (Malerba and Bianchetti 2001). The mouse 14-3-3β which interacts with RACK1 is more closely related to the non-ε group 14-3-3 proteins (over 59% identity at the amino acid level). However, the interaction between 14-3-3 proteins and RACK1 remains to be determined in plant cells. Another promising candidate interactor of RACK1 in plants is the Gβ subunit. Heterotrimeric G proteins are evolutionarily conserved in all eukaryotes (Temple and Jones 2007). Arabidopsis G, AGB1, shares 43% identity and 61% similarity with bovine Gβ protein 21  at the amino acid level (Figure 1.10). In mammalian cells, binding of RACK1 to Gβγ results in a specific inhibition of Gβγ-mediated activation of PLCβ2 and adenylyl cyclase II whereas has no effect on other functions of Gβγ (Dell et al. 2002; Chen et al. 2004a). Dell et al. (2002) mapped the Gβ-interacting region on RACK1 to the 207 amino acids at the N-terminus of RACK1. The amino acid sequence that is most divergent between mammalian RACK1 and Arabidopsis RACK1 proteins (the amino acid 280-300 region, Figure 1.4) is not required for its interaction with Gβ (Dell et al. 2002). Chen et al. (2005) mapped the RACK1-interacting regions on Gβ and identified five amino acid segments that are important for the interaction (Chen et al. 2005). These amino acid segments of Gβ are approximately 38% to 90% identical to those in Arabidopsis G (Figure 1.10). Plant heterotrimeric G proteins play important roles in multiple developmental processes, hormonal responsivenesses, and stress responses (Perfus-Barbeoch et al. 2004). As mentioned above, RACK1 protein was down-regulated in the embryos of rice G mutant (Komatsu et al. 2005), raising the possibility that the interaction between RACK1 and the heterotrimeric G proteins might be conserved in plants. Such an interaction deserves further investigation. Palmer et al. (2006) reported that a RACK1 homolog in the fungus Cryptococcus neoformans, Gib2, can function as an atypical heterotrimeric Gβ subunit and interact with one of the three Gα homologs, Gpa1, and two Gγ homologs, Gpg1 and Gpg2 (Palmer et al. 2006). However, in mammalian cells, RACK1 only interacts with Gβ1γ1 heterodimer and Gα1β1γ1 heterotrimer, but not with Gα subunit alone (Dell et al. 2002). Similarly, RACK1A does not interact with the sole Gα in Arabidopsis in a yeast split-ubiquitin assay (Chen et al. 2006).  1.2.8 Concluding remarks The study of RACK1 in plants is still at its fetal stage. Despite a long list of RACK1-interacting proteins in mammals, most of these interactors do not have significant homologs in plants. Therefore it remains mysterious if RACK1 could also function as a scaffold protein in plant cells. The identification and characterization of the whole spectrum of RACK1’s physical interacting partners in plants will be of great importance to unravel the molecular mechanism of the action of RACK1 in plants. If the scaffolding function of RACK1 is indeed conserved in plants, we would expect to identify a wide range of RACK1-interacting proteins. Future studies should also focus on the determination of the physiological pathways in which RACK1 plays a 22  regulatory role and the signals which regulate others via the scaffolding properties of RACK1. In addition, because most plant species have more than one RACK1 gene whereas there is only one in other non-plant organisms, it is essential to clarify a potential functional redundancy among these genes. At least in Arabidopsis, two of three RACK1 genes, RACK1B and RACK1C, were a consequence of chromosomal segment duplication (Figure 1.11). RACK1B and RACK1C sit in the duplicated chromosomal segments corresponding to the most recent polyploidy in the Arabidopsis genome which occurs sometime between 24 and 40 million years ago before the split of the Arabidopsis and Brassica lineages (Blanc et al. 2003). Because the formation of homodimer is one of the mechanisms through which RACK1 scaffolds other proteins’ interaction in mammalian cells, the unique feature of multiple RACK1 proteins in plant cells offers extra opportunities for forming heterodimeric complex among RACK1 proteins. Three Arabidopsis RACK1 proteins have variations in the major protein-interacting regions (Figure 1.4, Figure 1.5), implying that each RACK1 protein may have diverse binding partners. These diverse proteins could bind different RACK1 proteins and interact with each other through the platforms of homodimeric and heterodimeric complexes formed by RACK1 proteins. Such homodimeric and heterodimeric complex may help achieve a maximal scaffolding function of RACK1 in plant cells.  Figure 1.11 Illustration of genomic duplication of RACK1B and RACK1C genes. RACK1B (At1g48630) and RACK1C (At3g18130) sit in the genomic duplication block # 0103319703610. The graph was generated from http://wolfe.gen.tcd.ie/athal/index.html (Blanc et al. 2003).  23  1.3 Thesis objectives Although numerous studies have been done in mammalian cell and in yeast on the molecular function of RACK1 and a versatile and essential role for RACK1 has been revealed in these organisms, little is known about its function in plants. The overall objective of my thesis was to advance our understanding of the function of RACK1 in plants, using Arabidopsis as a model. A combination of molecular biological, genetic, cell biological and biochemical approaches were employed in this study. Due to the fact that RACK1 is very likely involved in multiple signaling pathways, like its mammalian counterpart, my thesis focuses on the following specific objectives:  1) To determine the relationship between the three Arabidopsis RACK1 homologous genes. 2) To define the role of Arabidopsis RACK1 genes in plant growth and development. 3) To define the role of Arabidopsis RACK1 genes in ABA responses. 4) To test the genetic and physical interaction between RACK1 and heterotrimeric Gproteins as a candidate mechanism for the role of RACK1 in ABA responses. 5) To explore the role of RACK1 in protein translation as a candidate mechanism for its function in mediating ABA responses.  24  1.4 References Anderson BE, Ward JM, Schroeder JI (1994) Evidence for an extracellular reception site for abscisic acid in commelina guard cells. 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BMC Plant Biology 8: 108 35  2.1 Introduction  Receptor for activated C kinase 1 (RACK1) is a protein containing seven tryptophanaspartic acid-domain (WD40) repeat, and was originally identified as an anchoring protein for protein kinase C (PKC) in mammals, shuttling the activated enzyme to different subcellular sites (Mochly-Rosen et al., 1991; Ron et al., 1994). Structurally, RACK1 is similar to the heterotrimeric G-protein  subunit (G) which has a seven-bladed propeller structure with one WD40 unit constituting each blade (reviewed by McCahill et al. 2002 and Sklan et al. 2006). The WD40 unit is known to be involved in protein-protein interactions. Increasing evidence suggests that mammalian RACK1 physically interacts with many other proteins and works as scaffold protein that facilitates other proteins’ interactions. Through the interaction with these partners, RACK1 plays regulatory roles in diverse developmental and physiological responses, including cell cycle control, cell movement and growth, immune response and neural responses in mammals (McCahill et al. 2002; Sklan et al. 2006). Therefore, RACK1 is now viewed as a versatile scaffold protein, serving as a nexus for multiple signal transduction pathways. Although not recognized as such, the first plant RACK1 gene was cloned from tobacco BY-2 cells as an auxin (2,4-dichlorophenoxyacetic acid, 2,4-D) inducible gene, arcA (Ishida et al. 1993). Subsequently, the amino acid sequence homologs of RACK1 were found in all plant species examined (reviewed by Guo et al., 2007). Earlier studies based on gene expression and induction analysis implied that plant RACK1 may have a role in hormone-mediated cell division (Ishida et al., 1993; McKhann et al., 1997), UV and salicylic acid responses (Perennes et al., 1999). In rice, RACK1, named RWD (Iwasaki et al., 1995), was found to be one of the seven proteins whose expressions were down-regulated in d1 mutant, a loss-of-function allele of rice heterotrimeric G-protein  subunit (Komatsu et al., 2005). Further, rice RACK1 protein was induced by abscisic acid (ABA) in imbibed wild-type seeds, but not in d1 mutant seeds. It was proposed that RACK1 may play a role in rice embryogenesis and germination (Komatsu et al. 2005). RACK1 proteins in Arabidopsis were found to be associated with the subunits of ribosomes (Chang et al., 2005; Giavalisco et al., 2005). However, a scaffolding function of RACK1 proteins in plants has not been established. Analysis of RACK1 proteins in plants and in non-plant organisms revealed an important feature of plant RACK1 proteins: some plants have more than one RACK1 genes, in contrast to the single copy of RACK1 gene in non-plant organisms. For example, the sequenced genomes of 36  rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana) contain two and three RACK1 homologous genes, respectively (Figure 2.1). The three RACK1 proteins encoded by the Arabidopsis genome were designated as RACK1A, RACK1B and RACK1C, respectively (Chen et al. 2006). Previously, we provided evidence that RACK1A mediates multiple hormone responses and developmental processes (Chen et al., 2006). However, two major questions remained: (1) what are the functions of the other two Arabidopsis RACK1 genes, RACK1B and RACK1C? and (2) what is the relationship between Arabidopsis RACK1 genes? In this study, we took genetic and molecular approaches to answer these two questions. We demonstrate that although RACK1B and RACK1C genes are likely dispensable, they still contribute significantly to the RACK1A-regulated developmental processes in Arabidopsis. We provide evidence that the difference in the gene expression level and the cross-regulation are likely the molecular determinants of unequal genetic redundancy of RACK1 genes in regulating plant growth and development.  Figure 2.1 Multiple amino acid sequence alignment of RACK1 in plants and in humans. The amino acid sequences were aligned by CLUSTALW multiple alignment of BioEdit Sequence Alignment Editor http://www.mbio.ncsu.edu/BioEdit/bioedit.html. Amino acids that are identical or similar are shaded with black or gray, respectively. Gaps are shown as dashed lines. The proteins aligned are (name of species and accession number in parentheses): RACK1A_At (Arabidopsis thaliana, NP_173248), RACK1B_At (Arabidopsis thaliana, NP_175296), RACK1C_At (Arabidopsis thaliana, NP_188441), RWD1_Os (Oryza sativa, NP_001043910), RWD2_Os (Oryza sativa, NP_001056254), RACK1_Pt (Populus trichocarpa, ABK92879), RACK1 _Vv (Vitis vinifera, CAN61810), and RACK1_Hs (Homo sapiens, NP_006089). The positions of GH and WD dipeptides in each WD40 repeat are indicated by triangles and asterisks, respectively, on the top of residues. The positions for WD repeat domains were obtained from the SMART database http://smart.embl-heidelberg.de.  37  2.2 Materials and methods  2.2.1 Plant materials and growth conditions All mutants are in the Arabidopsis Columbia (Col-0) ecotype background. The rack1a-1 and rack1a-2 mutants have been reported previously (Chen et al., 2006). Plants were grown in 5  5 cm pots containing moistened 1:3 mixture of Sunshine Mix #1 (Sun Gro Horticulture Canada Ltd., Seba beach, Alberta, Canada) and Metro-Mix 220 (W.R. Grace & Co., Ontario, Canada) with 10/14 h (short-day conditions) or 14/10 h (long-day conditions) photoperiod at approximately 120 mol m-2 s-1 at 23C.  2.2.2 Isolation of rack1b and rack1c T-DNA insertional mutants The T-DNA insertion mutants of RACK1B (At1g48630), rack1b-1 (SALK_117422) and rack1b-2 (SALK_145920), and the T-DNA insertion mutants of RACK1C (At3g18130), rack1c-1 (SAIL_199_A04) and rack1c-2 (SALK_017913), were identified from the SALK TDNA Express database (http://signal.salk.edu/cgi-bin/tdnaexpress). For the SALK T-DNA insertional mutants (Alonso et al., 2003), the insertion was confirmed by PCR using RACK1Bspecific primers (5’-TCTCGACCTCAAACCCTG-3’ and 5’GAGAAGACTTTAGAGTCGATGGA-3’) or RACK1C-specific primers (5’ATCTCTCGCTCTGTTACGC-3’ and 5’-ACAATACTGACGCAGTCTGG-3’) and a T-DNA left border-specific primer JMLB1 (5’-GGCAATCAGCTGTTGCCCGTCTCACTGGTG-3’). For the SAIL T-DNA insertion mutants (Sessions et al., 2002), a different T-DNA left borderspecific primer, GarlicLB3 (5’-TAGCATCTGAATTTCATAACCAATCTCGATACAC-3’), was used. The absence of full-length transcript of RACK1B or RACK1C in these alleles was examined by RT-PCR.  2.2.3 Generation of rack1a, rack1b and rack1c double and triple mutants Double mutants between rack1a-1 and rack1b-2 or rack1c-1 were generated by crossing rack1b-2 or rack1c-1 into rack1a-1 single mutant and isolated in the F2 progeny by PCR genotyping. Similarly, double mutants between rack1b-2 and rack1c-1 were generated by 38  crossing rack1c-1 into rack1b-2 single mutant and isolated in the F2 progeny by PCR genotyping. For simplicity, the rack1a rack1b, rack1a rack1c and rack1b rack1c double mutant nomenclatures in this report refer specifically to the rack1a-1 rack1b-2, rack1a-1 rack1c-1 and rack1b-2 rack1c-1 mutants, respectively. Triple mutant among rack1a-1, rack1b-2 and rack1c-1 was generated by crossing rack1b-2 rack1c-1 into rack1a-1 rack1b-2 double mutants. Because rack1a-1 rack1b-2 rack1c-1 triple mutants cannot survive in soil to maturity, they are maintained in plants homozygous for the rack1b-2 and rack1c-1 loci and heterozygous for the rack1a-1 locus. The triple mutant was confirmed by PCR genotyping.  2.2.4 Genetic complementation The full-length open-reading frames of RACK1A (At1g18080), RACK1B and RACK1C were amplified from a cDNA library made from seedlings grown in light for 10d, cloned into the pENTR/D-TOPO vector (Invitrogen Canada Inc., Burlington, Ontario, Canada), and then subcloned into Gateway plant transformation destination binary vector pB2GW7 (Karimi et al., 2002) by LR recombination reactions. In these constructs, the expression of RACK1A, RACK1B or RACK1C was driven by the 35S promoter of the Cauliflower mosaic virus. Binary vectors were transformed into rack1a-1 or rack1a-2 mutants by Agrobacterium-mediated transformation (Clough and Bent, 1998). At least 16 independent transgenic lines were selected from each transformation, and two to four representative lines were used for further studies. The expression of transgene was examined by RT-PCR.  2.2.5 RNA isolation, RT-PCR and quantitative real-time PCR analyses For tissue/organ expression pattern analysis, total RNA was isolated from different parts of seedlings or mature plants, using the TRIzol reagent (Invitrogen). cDNA was synthesized from 1 g total RNA by oligo(dT)20-primed reverse transcription, using THERMOSCRIPT RT (Invitrogen). RACK1A-specific primers (5’-GGCATCTCCAGACACCGAAA-3’ and 5’GCAGAGAGCAACGACAGC-3’), RACK1B-specific primers (5’TCTCGACCTCAAACCCTG-3’ and 5’-GAGAAGACTTTAGAGTCGATGGA-3’), and RACK1C-specific primers (5’-ATCTCTCGCTCTGTTACGC-3’ and 5’39  ACAATACTGACGCAGTCTGG-3’) were used to amplify the transcripts of these three genes, respectively. The expression of ACTIN2 (amplified by primers 5’GTTGGGATGAACCAGAAGGA-3’ and 5’-GAACCACCGATCCAGACACT-3’) was used as control in PCR reactions. For the examination of the transcript level of RACK1A, RACK1B and RACK1C in the T-DNA insertional mutants or in the transgenic lines, 10d-old, light-grown seedlings were used for total RNA isolation. For the quantitative analysis of RACK1A, RACK1B and RACK1C transcript levels in the different tissues/organs of wild-type Col plants or in the rack1a-1, rack1b-2 and rack1c-1 single and double mutants, real-time PCR was used. RACK1A-specific real-time PCR primers (5’CTGAGGCTGAAAAGGCTGACAACAG-3’ and 5’CTAGTAACGACCAATACCCCAAACTC-3’), RACK1B-specific real-time PCR primers (5’GGTTCTACTGGAATCGGAAACAAGACC-3’ and 5’CTAGTAACGACCAATACCCCAGACCC-3’), and RACK1C-specific real-time PCR primers (5’-GCAGAGAAGAATGAAGGTGGTGT-3’ and 5’CTAGTAACGACCAATACCCCAGACCC-3’) were used. The expression of ACTIN2 (amplified by real-time PCR primers 5’-CCAGAAGGATGCATATGTTGGTGA-3’and 5’GAGGAGCCTCGGTAAGAAGA-3’) was used to normalize the expression of each gene. The quantitative real-time PCR was performed using the MJ MiniOpticon real-time PCR system (Bio-Rad Laboratories (Canada) Ltd., Mississauga, Ontario, Canada) and IQ SYBR Green Supermix (Bio-Rad Laboratories (Canada) Ltd.).  2.2.6 Rosette leaf production assay The number of rosette leaves was collected from wild-type Col and mutant plants grown under 10/14 h photoperiod with light intensity of approximately 120 mol m-2 s-1 and temperature was controlled at 23C. At least four plants from each genotype were used. The rate of rosette leaf production was expressed as the number of rosette leaves divided by the age of plant.  40  2.2.7 Root growth assay Seedlings were grown on MS/G plates consisting of ½ Murashige & Skoog (MS) basal medium supplemented with vitamins (plantmedia, Dublin, Ohio), 1 % (w/v) sucrose and 0.6 % (w/v) phytoagar (plantmedia), with pH adjusted to 5.7 with 1N KOH. The plates were placed under 14/10 h photoperiod with approximately 120 mol m-2 s-1 at 23C with a vertical orientation for monitoring root growth. The length of primary and the number of lateral roots were collected from at least 15 seedlings each genotype.  2.3 Results  2.3.1 T-DNA insertional mutants of RACK1B and RACK1C  The Arabidopsis genome contains three RACK1 homologous genes, designated as RACK1A, RACK1B and RACK1C, respectively (Chen et al., 2006). Previously, we showed that loss-of-function mutations in one member of this small gene family, RACK1A, resulted in altered hormone responses and multiple defects in plant growth and development (Chen et al., 2006). In order to examine the function of the other two RACK1 genes, RACK1B and RACK1C, we took a reverse genetic approach to seek and characterize the loss-of-function alleles for RACK1B and RACK1C. By searching the Salk Institute sequence-indexed insertion mutant collection (http://signal.salk.edu/cgi-bin/tdnaexpress), we obtained two independent T-DNA insertional alleles for each RACK1 gene. All alleles are in the Columbia (Col-0) ecotypic background. We designated the two mutant alleles for RACK1B as rack1b-1 and rack1b-2, respectively. In rack1b-1 allele, the T-DNA was inserted in the second exon of RACK1B gene, and in the rack1b-2 allele, the T-DNA was inserted in the first intron (Figure 2.2A). RT-PCR analysis indicated that the full-length transcript of RACK1B was absent in both alleles (Figure 2.2B), implying that they are likely loss-of-function alleles. Unlike rack1a mutants, rack1b mutants do not display any apparent developmental defects (Figure 2.2C). We designated the two mutant alleles for RACK1C as rack1c-1 and rack1c-2, respectively (Figure 2.2D). In rack1c-1 allele, the T-DNA was inserted in the second exon of RACK1C gene, and in the rack1c-2 allele, the T-DNA was inserted in the 5’-UTR region. RT-PCR analysis indicated that the full-length transcript of RACK1C was absent in both alleles (Figure 2.2E), implying that they 41  are likely loss-of-function alleles. Similar to rack1b mutants but unlike rack1a mutants, rack1c mutants do not display any obvious defects in plant growth and development (Figure 2.2C).  2.3.2 Loss-of-function mutations in RACK1B and RACK1C enhance the defects in rosette leaf production of rack1a mutant  Because loss-of-function alleles of RACK1B and RACK1C did not display significant defects in plant growth and development whereas loss-of-function alleles of RACK1A displayed multiple defects, we wanted to test if mutations in RACK1B or RACK1C can enhance the defects of rack1a mutants in growth and development. Therefore, we generated rack1a-1 rack1b-2 and rack1a-1 rack1c-1 double mutants. One of the most dramatic phenotypes observed in rack1a single mutants was the reduced number of rosette leaves (Chen et al., 2006). Therefore, we grew single and double mutants together with wild-type (Col) under identical, short-day conditions with 10/14h photoperiod, counted the number of rosette leaves in double mutants and compared it with Col and rack1a-1 single mutant. We found that while rack1b-2 and rack1c-1 single mutants produced wild-type number of rosette leaves, both rack1b-2 and rack1c-1 significantly enhanced the phenotype of reduced number of rosette leaves of rack1a-1 single mutants (Figure 2.3A and B). When plants were grown under 10/14h photoperiod for 48 days, wild-type produced approximately 30 rosette leaves, whereas rack1a-1 single mutant produced 22 rosette leaves. Under these conditions, rack1a-1 rack1b-2 and rack1a-1 rack1c-1 double mutants only produced about 16 and 19 rosette leaves, respectively (Figure 2.3B). The rate of rosette leaf production was reduced approximately 27% and 14%, respectively, in rack1a-1 rack1b-2 and rack1a-1 rack1c-1 double mutants, compared with rack1a-1 single mutant (Figure 2.3C). We also examined the rosette size by measuring the diameter of rosette of each genotype. Similar to that of number of rosette leaves, the diameter of rosette was significantly reduced in rack1a-1 single mutant, compared with wild-type plants, and such reduction was further enhanced in  42  Figure 2.2 T-DNA insertional mutants of RACK1B and RACK1C. A. A diagram to illustrate the T-DNA insertion sites in rack1b-1 and rack1b-2 mutants. Gray boxes represent coding regions and white boxes represent 5’-UTR and 3’-UTR regions. The T-DNA inserts are not drawn to scale. LB, T-DNA left border. B. RT-PCR analysis of RACK1B transcript in rack1b mutants. Total RNA was isolated from 10d-old, light-grown seedlings. RACK1B-specific primers that amplify the full-length transcript of RACK1B in wild-type (Col) were used. RTPCR was performed with 30 cycles. The expression of ACTIN2 was used as control. C. The rosette morphology of rack1b and rack1c mutants. Shown are plants grown 48 days under 10/14 h (L/D) photoperiod. D. A diagram to illustrate the T-DNA insertion sites in rack1c-1 and rack1c-2 mutants. E. RT-PCR analysis of RACK1C transcript in rack1c mutants. RACK1Cspecific primers that amplify the full-length transcript of RACK1C in Col were used. RT-PCR was performed with 35 cycles. The expression of ACTIN2 was used as control. rack1a-1 rack1b-2 and rack1a-1 rack1c-1 double mutants (Figure 2.3D). However, no synergistic effect was observed between rack1b-2 and rack1c-1 mutations. Statistically, rack1b2 rack1c-1 double mutants had wild-type traits of these phenotypes (Figure 2.3A, B). Subsequently, we generated rack1a-1 rack1b-2 rack1c-1 triple mutant. Very few triple mutants could survive in soil. For those survived, they were extremely slow in growth and  43  Figure 2.3 Loss of function mutations in RACK1B and RACK1C enhance the rosette leaf phenotype of rack1a mutants. A. The phenotype of rack1 mutants. Shown are plants grown for 48 days under 10/14 h photoperiod. Scale bars, 2 cm. B. The number of rosette leaves of rack1 mutants. C. The rate of rosette leaf production of rack1 mutants. The rate of rosette leaf production is expressed as the number of rosette leaves divided by the age of plants. D. The size of rosette of rack1 mutants. The number of rosette leaves, the rate of rosette leaf production and the size of rosette were measured from plants grown for 48 days under 10/14 h photoperiod. Shown are the averages of at least four plants ± S.E. The same experiment was repeated twice with similar trends and the data from one experiment were presented. *, significant difference from Col, P<0.05. #, significant difference from rack1a single mutant. **, significant difference from rack1a-1 rack1b-2 double mutant.  44  development, and produced fewest rosette leaves and smallest rosette size among all genotypes examined (Figure 2.3A-D). Not surprisingly, the rate of rosette leaf production in the triple mutant is the slowest among all genotypes (Figure 2.3C). Because rack1a-1 rack1b-2 rack1c-1 triple mutants could not survive to maturity to produce seeds, these triple mutants were maintained in plants homozygous for the rack1b-2 and rack1c-1 loci and heterozygous for the rack1a-1 locus. Because rack1b-2 rack1c-1 double mutants had wild-type morphology whereas rack1a-1 rack1b-2 rack1c-1 had extreme pleiotropic phenotype, rack1a-1 rack1b-2 rack1c-1 triple mutants can be readily picked up from the segregating progeny of plants homozygous for the rack1b-2 and rack1c-1 loci and heterozygous for the rack1a-1 locus.  2.3.3 Loss-of-function mutations in RACK1B and RACK1C enhance the defects in the growth and development of rack1a mutant roots  Genetic analysis indicated that loss-of-function mutations in RACK1A affect the production of rosette leaves and the size of rosette, and that the effect of rack1a-1 mutation can be enhanced by the rack1b-2 or rack1c-1 mutation or both (Figure 2.3). We wanted to extend our analysis to non-aerial organs by examining the impact of these mutations on roots. We measured the length of primary root and counted the number of lateral root and used them as parameters of root growth and root architecture. We found that the length of primary root of rack1a-1 mutant was slightly shorter than that of wild-type whereas rack1b-2 and rack1c-1 mutants had wild-type length of primary root (Figure 2.4A). The length of primary root was further shortened in rack1a-1 rack1b-2 and rack1a-1 rack1c-1 double mutants, compared with that in rack1a-1 single mutant (Figure 2.4A), indicating that rack1b-2 and rack1c-1 mutations can also enhance the effect of rack1a-1 mutation on primary root growth. Similar to the observation in primary root, rack1a-1 mutant produced fewer lateral roots than wild-type whereas rack1b-2 and rack1c1 mutants had wild-type number of lateral roots (Figure 2.4B). As expected, rack1b-2 and rack1c-1 mutations enhanced the lateral root phenotype of rack1a-1 mutant (Figure 2.4B). Among all genotypes examined, the rack1a-1 rack1b-2 rack1c-1 triple mutant produced the shortest root and did not produce any lateral root under our assay conditions (Figure 2.4A and B).  45  Figure 2.4 Loss of function mutations in RACK1B and RACK1C enhance the root phenotype of rack1a mutants. A. The length of primary root of rack1 mutants. B. The number of lateral roots of rack1 mutants. The length of primary root and the number of lateral roots were measured from 10d-old, light-grown seedlings (under 14/10 h photoperiod). Shown are the averages of at least 15 seedlings ± S.E. *, significant difference from Col, P<0.05. #, significant difference from rack1a single mutant. **, significant difference from rack1a-1 rack1b-2 double mutant.  2.3.4 Genetic complementation of rack1a mutants by overexpressing RACK1B and RACK1C  Genetic analyses indicated that there is unequal genetic redundancy among three Arabidopsis RACK1 genes in regulating rosette leaf production and root growth and development, and that RACK1A is likely a non-dispensable gene in this small gene family. Although RACK1B and RACK1C are likely dispensable, they still contribute significantly to the overall activity of RACK1 genes in regulating plant growth, as revealed by the analysis of double and triple mutants. We wanted to further explore the mechanism of the unequal genetic 46  redundancy of RACK1 genes. Firstly, since RACK1B and RACK1C are highly similar (about 90% identity) to RACK1A at the amino acid level (Figure 2.1), we wanted to test if RACK1B and RACK1C are in principal functionally equivalent to RACK1A. We reasoned that if RACK1B and RACK1C are indeed functionally equivalent to RACK1A, one would expect that overexpression of RACK1B or RACK1C complements the growth and developmental defects of rack1a mutants. Therefore, we generated transgenic lines overexpressing RACK1B or RACK1C in the rack1a mutant background using the CaMV 35S promoter. As a control, we generated transgenic plants overexpressing RACK1A in rack1a mutant background. At least two independent transgenic lines were analyzed for each transformation. Overexpression of the transgene in these lines was confirmed by RT-PCR analysis (Figure 2.5A). We examined the same parameters described above, namely the number of rosette leaves, the length of primary root and the number of lateral roots, in the transgenic lines overexpressing RACK1 genes, and compared them with those in rack1a mutants. As expected, overexpression of RACK1A fully complemented the mutant phenotype of rack1a mutant (Figure 2.5B, C, D). Similarly, we found that the overexpression of RACK1B or RACK1C fully restored rack1a mutant to wild-type morphology, evident by the wild-type number of rosette leaves, wild-type length of primary root and wild-type number of lateral roots in transgenic lines (Figure 2. 5B, C, D).  47  Figure 2.5 The complementation of rack1a mutants by overexpression of RACK1 genes. (A) RT-PCR analysis of the expression of RACK1 genes in transgenic lines. The transgenic lines 2-7, 6-2, 8-3 and 25-3 are RACK1A overexpressors in rack1a-2 mutants. The transgenic lines 4-5 and 28-2 are RACK1B overexpressors in rack1a-1 mutants. The transgenic lines 4-3, 5-3, 8-3 and 96 are RACK1C overexpressors in rack1a-1 mutants. RT-PCR was performed at 28 cycles. The expression of ACTIN2 was used as a control. (B) The number of rosette leaves in transgenic plants overexpressing individual RACK1 gene in rack1a mutant background. The number of rosette leaves was collected from plants grown for 37 d under 14/10 h photoperiod. Shown are the averages of number of rosette leaves from at least four plants ± S.E. (C) The length of primary root in transgenic plants overexpressing individual RACK1 gene in rack1a mutant background. The length of primary roots was measured from seedlings grown for 10 d under 14/10 h photoperiod. (D) The number of lateral roots in transgenic plants overexpressing individual RACK1 gene in rack1a mutant background. The number of lateral roots was counted from seedlings grown for 11 d under 14/10 h photoperiod. Shown in (C) and (D) are the averages of at least 20 seedlings ± S.E. *, significant difference from Col, P < 0.05. 48  2.3.5 Tissue/organ Expression of Arabidopsis RACK1 genes Because constitutive expression of RACK1B or RACK1C could efficiently complement rack1a mutant’s defects at plant growth and development, these results suggested that RACK1B and RACK1C are in principal functionally equivalent to RACK1A, and implied that the unequal genetic redundancy of RACK1 genes is likely due to the difference in their expression patterns or expression levels. To test this possibility, we examined the expression patterns of RACK1A, RACK1B and RACK1C in various tissues and organs of young seedlings and mature plants by RT-PCR. We found that all three Arabidopsis RACK1 genes were expressed ubiquitously (Figure 2.6A). These results are largely consistent with the results of analysis of RACK1 gene  Figure 2.6 The expression of RACK1A, RACK1B and RACK1C genes. (A) RT-PCR analysis of the expression of RACK1 genes in various tissues and organs of young seedlings and mature plants. RT-PCR was performed at 30 cycles. The expression of ACTIN2 was used as a control. (B) Quantitative real-time PCR analysis of the transcript levels of RACK1 genes. The transcript level of each RACK1 gene was normalized against the transcript level of ACTIN2 in each sample. The relative transcript levels of RACK1 genes were compared to that of RACK1C in the roots of 4 d-old, light-grown seedlings (set as 1). Shown are the averages of three replicates ± S.D. 49  promoter:-glucuronidase (GUS) transcriptional reporter lines (Chen et al., 2006). However, by using RT-PCR, we noticed that in any given tissues or organs examined, the transcript level of three RACK1 genes were different, with a general trend of RACK1A>RACK1B>RACK1C (Figure 2.6A). Such a trend was also previously observed in 10d-old, light-grown whole seedlings (Chen et al., 2006). In order to quantify the difference in transcript level of RACK1A, RACK1B and RACK1C genes, we used quantitative real-time PCR to more accurately compare the transcript level of three RACK1 genes in different tissues and organs of wild-type Col plants. We selected the samples of shoots and roots of 4d- and 7d-old light-grown seedlings and rosette leaves and roots of mature plants for quantitative real-time PCR analysis. We found that consistent with the result of in-gel RT-PCR analysis, the transcript level of RACK1C was the lowest and that of RACK1A was the highest among three RACK1 genes, with a trend of RACK1A>RACK1B>RACK1C in all samples examined (Figure 2.6B). For example, the transcript level of RACK1A is about 5-fold higher than that of RACK1C in the roots of 4d-old, light-grown seedlings. In this sample, the transcript level of RACK1B is approximately 2-fold higher than that of RACK1C. 2.3.6 Cross-regulation of RACK1 genes at the transcription level  The analysis of the expression patterns and transcript level of three RACK1 genes in various tissues and organs supported the view that the unequal genetic redundancy of RACK1 genes is likely due to the difference in the gene expression level. However, other possibilities may also exist. For example, as reviewed by Briggs et al. (2006), cross-regulation is another mechanism that attributes to the unequal genetic redundancy of some homologous genes. Because RACK1A, RACK1B and RACK1C are approximately 90% identical to each other at the amino acid level, we were unable to obtain antibodies that can specifically recognize each RACK1 protein. Therefore, in this study, we examined the impact of loss-of-function mutations of each RACK1 gene on the transcription of the other two RACK1 genes. Further, we examined the impact of combination of loss-of-function mutations of two RACK1 genes on the transcription of the other RACK1 gene. Specifically, we examined the transcript level of RACK1A in rack1b and rack1c single and double mutants, the transcript level of RACK1B in rack1a and rack1c single and double mutants, and the transcript level of RACK1C in rack1a and rack1b single and double mutants, and compared with their transcript levels in wild-type. For 50  this analysis, we used the 4.5d-old, light-grown whole seedlings. We found that the transcript level of any given RACK1 gene was reduced in the loss-of-function mutations in each or both of the other two RACK1 genes (Figure 2.7). Among three RACK1 genes, the transcript level of RACK1C was most dramatically affected by the loss-of-function mutations in the other two RACK1 genes (Figure 2.7).  Figure 2.7 The expression of RACK1 genes in rack1a, rack1b and rack1c single and double mutants. A. RT-PCR analysis of the expression of RACK1 genes in rack1a, rack1b and rack1c single and double mutants. RT-PCR was performed at 28 cycles. The expression of ACTIN2 was used as a control. B. Quantitative real-time PCR analysis of the transcript level of RACK1 genes in rack1a, rack1b and rack1c single and double mutants. The transcript level of RACK1 genes was normalized against the transcript level of ACTIN2 in each sample. The relative transcript level of RACK1 genes in mutant backgrounds was compared with that in wild-type (Col) (set as 1). Shown are the averages of three replicates ± S.D.  2.4 Discussion  RACK1 gene is evolutionarily conserved in diverse organisms. While all non-plant organisms contain a single RACK1 gene in their genomes, the Arabidopsis genome contains three RACK1 genes. We report here that three Arabidopsis RACK1 genes regulate rosette leaf 51  production and root growth and development with unequal genetic redundancy. We provided evidence that both the difference in gene expression level and the cross-regulation contribute to the unequal genetic redundancy of RACK1 genes in regulating plant growth and development.  2.3.1 RACK1 genes in plant growth and development  Although the research interest in RACK1 has grown exponentially since its discovery (Mochly-Rosen et al., 1991) and RACK1 is now viewed as a multi-functional, versatile scaffold protein in mammals and in yeasts (McCahill et al. 2002; Sklan et al. 2006), the function of RACK1 in plants remains poorly understood. We are just starting to have some hints about its potential functions in plants. Preliminary analysis suggested RACK1 may mediate multiple hormone responses and developmental processes in Arabidopsis (Chen et al., 2006). In this study, we focused on the two characteristic growth defects of rack1a mutants, namely the reduction in rosette leaf production and the reduction in root growth and lateral root formation, to study the function of RACK1B and RACK1C and the genetic relationship of RACK1 homologous genes in plant growth and development. We demonstrated that RACK1 genes are critical regulators of plant growth and development and are essential for plant survival. Simultaneous disruption of the function of all three RACK1 genes results in lethality. Thanks to the unequal genetic redundancy of RACK1 genes, we are still able to study the role of RACK1 genes in plant growth and development. The rack1a single mutants, rack1a rack1b and rack1a rack1c double mutants all display growth and developmental defects, but are viable. Therefore, these mutants can be treated as “weak alleles” of rack1 mutants. Now that we have identified RACK1 genes as critical regulators of plant growth and development and all “weak alleles” of rack1 mutants are available, future studies should focus on the elucidation of the molecular mechanism by which RACK1 genes regulate plant growth and development, including rosette leaf production and root growth and lateral root formation. Because rack1a mutants have also been shown to display altered responses to hormones (Chen et al., 2006; Guo et al., 2008), it remains unclear if the growth and developmental defects observed in rack1 mutants are due to the altered responses to multiple hormones and if there is also unequal genetic redundancy of RACK1 genes in mediating hormone responses. This is a fertile area that is worth further investigation.  52  2.3.2 Mechanism of unequal genetic redundancy of RACK1 genes  Genetic redundancy of homologous genes is thought to be due to gene duplication events during the evolution of the organism. Between homologous genes, genetic redundancy can be classified as full redundancy, partial redundancy, and unequal redundancy (Briggs et al., 2006). While full redundancy and partial redundancy have been documented in numerous cases, unequal genetic redundancy has just begun to be recognized as a common phenomenon of genetic relationship of homologous genes (Briggs et al., 2006). Unlike non-plant organisms whose genomes contain only a single RACK1 gene, some plant genomes contain more than one RACK1 genes (Figure 2.1). In particular, the Arabidopsis genome contains three RACK1 genes, which share the similar gene structure with two exons and one intron, and encode three highly similar proteins with approximately 90% identity at the amino acid level (Chen et al., 2006). However, the relationship of three Arabidopsis RACK1 homologous genes was unknown. Previously, we showed that loss-of-function mutation in one member of Arabidopsis RACK1A genes, RACK1A, conferred multiple defects in plant growth and development (Chen et al., 2006). Here we show that loss-of-function mutations in RACK1B or RACK1C do not confer apparent growth and developmental defects (Figure 2.2). These results suggested that RACK1B and RACK1C are likely dispensable in plant growth and development. However, we found that although rack1b and rack1c mutants displayed wild-type morphology, rack1b and rack1c can strongly enhance the growth and developmental defects of rack1a mutants (Figures 2.2 and 2.3). These results suggested that RACK1B and RACK1C still contribute significantly to the overall activity of RACK1 genes. Taken together, the behaviors and relationship of rack1 mutants satisfy the key criteria for RACK1 genes being unequally redundant homologous genes (Briggs et al., 2006). The unequal genetic redundancy is caused by many factors. Among them, the difference in gene expression pattern, expression level and cross-regulation of homologous genes have been recognized as major determinants (Briggs et al., 2006). The unequal genetic redundancy of some homologous genes is mainly due to the difference in expression pattern and/or expression level, which is believed to be caused by the difference in gene promoter activity. For example, as reviewed by Briggs et al. (2006), CAULIFLOWER (CAL) is closely related in sequence to APETALA1 (AP1), but AP1 and CAL regulate the formation of floral meristem in an unequally redundant manner because AP1 is expressed at much higher level than CAL throughout sepal 53  and petal development (Kempin et al., 1995). The unequal genetic redundancy of homologous genes can also be primarily due to the cross-regulation. For example, LONG HYPOCOTYL 5 (HY5) and its close homolog HY5 HOMOLOG (HYH), both of which are regulators of photomorphogenesis, are a pair of unequally redundant genes with similar expression patterns and levels (Holm et al., 2002; Briggs et al., 2006), but a normal protein expression and activity of HYH was dependent on the presence of a functional HY5 (Holm et al., 2002). In order to get insight into the mechanism of unequal genetic redundancy of three RACK1 genes, we examined each of these possibilities. Firstly, we showed that RACK1B and RACK1C are in principal functionally equivalent to RACK1A, because overexpression of either RACK1B or RACK1C under the constitutive CaMV 35S promoter fully complemented the growth and developmental defects of rack1a mutants (Figure 2.5). These results implied that the unequal genetic redundancy of RACK1 genes is likely due to the difference in gene expression pattern and/or expression level, rather than the difference in protein sequence or activity. To test this directly, we found that three RACK1 genes are widely expressed in various tissues and organs in young seedlings and in mature plants (Figure 2.6). However, RACK1 genes are expressed at different levels with a general trend of RACK1A>RACK1B>RACK1C in all tissues and organs examined (Figure 2.6). These results supported the view that the difference in gene expression level likely attributes to the unequal genetic redundancy of RACK1 genes in plant growth and development. We also tested the possibility of cross-regulation by examining the transcript level of each RACK1 gene in the loss-of-function alleles of either or both of the other two RACK1 genes. We found that the transcript level of any given RACK1 gene was reduced in the single or double mutants for the other two RACK1 genes (Figure 2.7). Therefore, both the difference in gene expression level and the cross-regulation contribute to the unequal genetic redundancy of RACK1 genes. Unlike HY5 and HYH, for which the expression of the duplicate gene (HYH) depends on the presence of the ancestral gene (HY5) (Holm et al., 2002), RACK1 homologous genes mutually depend on each other for reaching full expression, adding another level of complexity for the unequal genetic redundancy. The molecular basis of such mutual cross-regulation of RACK1 genes is unknown. Taken together, these results suggested that among three RACK1 homologous genes in Arabidopsis, RACK1A is likely the ancestral gene whereas RACK1B and RACK1C are duplicate genes because RACK1A appears to retain most of the function of RACK1 gene family. We propose that RACK1 genes regulate plant growth and development possibly in a continuous, quantitative manner. It is likely that a certain threshold of gene activity is required for the 54  RACK1 genes to have any influence on the plant growth and development (Figure 2.8). Because rack1b and rack1c single mutants do not exhibit any defects in plant growth and development whereas the rack1a rack1b and rack1a rack1c double mutants display enhanced phenotypes compared with the rack1a single mutant, it is likely that the residual activities of RACK1B and RACK1C are above this threshold (Figure 2.8). Therefore, although both RACK1B and RACK1C are likely dispensable, they still contribute significantly to the overall activity of RACK1 genes. We proposed that both the difference in gene expression level and the cross-regulation are likely the molecular determinants of unequal genetic redundancy of RACK1 genes in regulating plant growth and development.  Figure 2.8 The model of unequal genetic redundancy of RACK1 genes in regulating plant growth. The Arabidopsis genome contains three RACK1 homologous genes, designated as RACK1A, RACK1B and RACK1C, respectively, which encode three highly similar proteins. RACK1 genes regulate plant growth and development likely in a continuous quantitative manner. RACK1A is likely the ancestral gene whereas RACK1B and RACK1C are the duplicate genes, because RACK1A retains the most functions of RACK1 genes. The expression of RACK1 follows a general trend of RACK1A > RACK1B > RACK1C. A certain threshold of gene activity is likely required for the RACK1 genes to have any influence on plant growth, and the gene activity can be saturated once an excess of gene activity is reached. Because the loss-of-function mutations in RACK1B or RACK1C or both do not confer any defects in plant growth while enhancing the growth defects of rack1a mutants, the residual activities of RACK1B and RACK1C are likely above this threshold but below the point of saturation. RACK1 genes mutually regulate each other's transcription. Both the difference in gene expression and the cross-regulation are likely the molecular determinants of unequal genetic redundancy of RACK1 genes in regulating plant growth. The model is schematically based on the possible explanations for unequal genetic redundancy provided by Briggs et al. (2006).  55  2.5 References  Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen HM, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R, Gadrinab C, Heller C, Jeske A, Koesema E, Meyers CC, Parker H, Prednis L, Ansari Y, Choy N, Deen H, Geralt M, Hazari N, Hom E, Karnes M, Mulholland C, Ndubaku R, Schmidt I, Guzman P, AguilarHenonin L, Schmid M, Weigel D, Carter DE, Marchand T, Risseeuw E, Brogden D, Zeko A, Crosby WL, Berry CC, Ecker JR (2003) Genome-wide Insertional mutagenesis of Arabidopsis thaliana. Science 301: 653-657 Briggs GC, Osmont KS, Shindo C, Sibout R, Hardtke CS (2006) Unequal genetic redundancies in Arabidopsis - a neglected phenomenon? Trends Plant Sci 11: 492-498 Chang IF, Szick-Miranda K, Pan S, Bailey-Serres J (2005) Proteomic characterization of evolutionarily conserved and variable proteins of Arabidopsis cytosolic ribosomes. Plant Physiol 137: 848-862 Chen JG, Ullah H, Temple B, Liang J, Guo J, Alonso JM, Ecker JR, Jones AM (2006) RACK1 mediates multiple hormone responsiveness and developmental processes in Arabidopsis. J Exp Bot 57: 2697-2708 Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735-743 Giavalisco P, Wilson D, Kreitler T, Lehrach H, Klose J, Gobom J, Fucini P (2005) High heterogeneity within the ribosomal proteins of the Arabidopsis thaliana 80S ribosome. Plant Mol Biol 57: 577–591 Guo J, Liang J, Chen JG (2007) RACK1: a versatile scaffold protein in plants? Int J Plant Dev Biol 1: 95-105 Guo J, Wang S, Wang J, Huang WD, Liang J, and Chen JG (2009b) Dissection of the relationship between RACK1 and heterotrimeric G-proteins in Arabidopsis. Plant Cell Physiol 50:1681-1694. Holm M, Ma LG, Qu LJ, Deng XW (2002) Two interacting bZIP proteins are direct targets of COP1-mediated control of light-dependent gene expression in Arabidopsis. Genes Dev 16: 1247-1259 Ishida S, Takahashi Y, Nagata T (1993) Isolation of cDNA of an auxin-regulated gene encoding a G-protein -subunit-like protein from tobacco BY-2-cells. Proc Natl Acad Sci USA 90: 11152-11156 Iwasaki Y, Komano M, Ishikawa A, Sasaki T, Asahi T (1995) Molecular cloning and characterization of cDNA for a rice protein that contains seven repetitive segments of the Trp-Asp forty-amino-acid repeat (WD-40 repeat). Plant Cell Physiol 36: 505-510 Karimi M, Inze D, Depicker A (2002) GATEWAY((TM)) vectors for Agrobacteriummediated plant transformation. Trends Plant Sci 7: 193-195 Komatsu S, Abbasi F, Kobori E, Fujisawa Y, Kato H, Iwasaki Y (2005) Proteomic analysis of rice embryo: an approach for investigating G protein-regulated proteins. Proteomics 5: 3932-3941 56  Kempin SA, Savidge B, Yanofsky MF (1995) Molecular basis of the cauliflower phenotype in Arabidopsis. Science 267: 522-525 McCahill A, Warwicker J, Bolger GB, Houslay MD, Yarwood SJ (2002) The RACK1 scaffold protein: A dynamic cog in cell response mechanisms. Mol Pharmacol 62: 12611273 McKhann HI, Frugier F, Petrovics G, delaPena TC, Jurkevitch E, Brown S, Kondorosi E, Kondorosi A, Crespi M (1997) Cloning of a WD-repeat-containing gene from alfalfa (Medicago sativa): a role in hormone-mediated cell division? Plant Mol Biol 34: 771780 Mochly-Rosen D, Khaner H, Lopez J (1991) Identification of intracellular receptor proteins for activated protein kinase C. Proc Natl Acad Sci USA 88: 3997-4000 Perennes C, Glab N, Guglieni B, Doutriaux MP, Phan TH, Planchais S, Bergounioux C (1999) Is arcA3 a possible mediator in the signal transduction pathway during agonist cell cycle arrest by salicylic acid and UV irradiation? J Cell Sci 112: 1181-1190 Ron D, Chen CH, Caldwell J, Jamieson L, Orr E, MochlyRosen D (1994) Cloning of an intracellular receptor for protein kinase C – A homolog of the β-subunit of G-proteins. Proc Natl Acad Sci USA 91: 839-843 Sessions A, Burke E, Presting G, Aux G, McElver J, Patton D, Dietrich B, Ho P, Bacwaden J, Ko C, Clarke JD, Cotton D, Bullis D, Snell J, Miguel T, Hutchison D, Kimmerly B, Mitzel T, Katagiri F, Glazebrook J, Law M, Goff SA (2002) A high-throughput Arabidopsis reverse genetics system. Plant Cell 14: 2985-2994 Sklan EH, Podoly E, Soreq H (2006) RACK1 has the nerve to act: structure meets function in the nervous system. Prog Neurobiol 78: 117-134  57  CHAPTER 3  Dissection of the Relationship between RACK1 and Heterotrimeric G-proteins in Arabidopsis 1  1  A version of this chapter has been published.  Guo J, Wang S, Wang J, Huang WD, Liang J, and Chen JG (2009b) Dissection of the Relationship between RACK1 and Heterotrimeric G-proteins in Arabidopsis. Plant & Cell Physiology 50:1681-1694. 58  3.1 Introduction RACK1 was originally identified as a receptor for activated Protein Kinase C (PKC) in mammals (Mochly-Rosen et al., 1991; Ron et al., 1994). However, subsequent studies demonstrated that RACK1 can physically interact with many other proteins and facilitate their interactions (reviewed by McCahill et al. 2002; Sklan et al. 2006). Therefore, RACK1 is now regarded as a versatile scaffold protein that plays regulatory roles in multiple signal transduction pathways. RACK1 is structurally similar to the heterotrimeric G-protein β subunit (Gβ), both of which contain seven tryptophan-aspartic acid-domain (WD40) repeats (Ullah et al., 2008). More importantly, RACK1 interacts with Gβ (Dell et al., 2002; Chen S et al., 2004a, 2004b, 2005). The protein sequences of both RACK1 and Gβ are highly conserved in plants (Weiss et al., 1994; Chen et al., 2006b; Guo et al., 2007). However, little is known about the relationship between RACK1 and heterotrimeric G-proteins (G-proteins) in plants. The first plant RACK1 gene was cloned from tobacco BY-2 cells as an auxin-induced gene, arcA (Ishida et al., 1993). Gene expression and induction studies implied that plant RACK1 may be involved in hormone-mediated cell division (Ishida et al., 1993; McKhann et al., 1997), and Ultra Violet and salicylic acid responses (Perennes et al., 1999). Rice RACK1, originally named RWD (Iwasaki et al., 1995), has recently been demonstrated as a key regulator of innate immunity through interaction with multiple proteins in the Rac1 immune complex (Nakashima et al., 2008). G-proteins consist of Gα, Gβ and Gγ subunits. In Arabidopsis, the Gα subunit is encoded by a single gene, GPA1 (Ma et al., 1990); the Gβ subunit is also encoded by a single gene, AGB1 (Weiss et al., 1994); and the Gγ subunits are encoded by two genes, AGG1 and AGG2 (Mason and Botella 2000, 2001). Substantial evidence indicated that G-proteins mediate multiple developmental processes and hormone signaling (reviewed by Ma, 2001; Fujisawa et al., 2001; Assmann, 2002; Jones, 2002; Perfus-Barbeoch et al., 2004; Jones and Assmann, 2004; Temple and Jones 2007; Chen 2008; Ding et al., 2008). GPA1 and AGB1 have also been shown as negative regulators of ABA responses during seed germination and early seedling development (Ullah et al., 2002; Assmann, 2005; Pandey et al., 2006). Three lines of evidence have prompted us to examine the relationship between 59  RACK1 and G-proteins in Arabidopsis. First, in mammalian cells, RACK1 physically interacts with Gβ (Dell et al., 2002; Chen S et al., 2004a, 2004b, 2005). Second, in yeast, RACK1/Asc1 functions as a Gβ and interacts with one of the two Gα subunits (Zeller et al., 2007). Third, in rice, RACK1 was one of the seven proteins whose expression was downregulated in d1 mutant, a loss-of-function allele of the sole rice Gα (Komatsu et al., 2005). We took several approaches to examine the relationship between RACK1 and G-proteins in Arabidopsis. First, we took a genetic approach to generate and analyze double mutants between rack1 and Gα and Gβ mutants. Second, we tested the physical interaction between RACK1 and Gβ. Finally, we examined if the expression of RACK1 is mis-regulated in Gprotein subunit mutants. Our work revealed a different, G-protein-independent role of RACK1 in Arabidopsis vs mammals.  3.2 Materials and methods  3.2.1 Plant materials and growth conditions All mutants are in the Arabidopsis Columbia (Col-0) ecotype background. The rack1a1 and rack1a-2 mutants were reported by Chen et al. (2006a). The gpa1-3 and gpa1-4 mutants were reported by Jones et al. (2003). The agb1-2 mutants were reported by Ullah et al. (2003). The rgs1-1 and rgs1-2 mutants were reported by Chen et al. (2003). The gpa1-4 agb1-2 double mutants were reported by Chen et al. (2004). Plants were grown in 5 × 5 cm pots containing moistened 1: 3 mixture of Sunshine Mix #1 (Sun Gro Horticulture Canada Ltd., Seba beach, Alberta, Canada) and Metro-Mix 220 (W.R. Grace & Co. of Canada, Ontario, Canada) with 14/10 hr or 8/16 hr photoperiod at approximately 120 µmol m-2 s-1 at 23°C. 3.2.2 Generation of rack1a-1, gpa1-4, and agb1-2 double mutants Double mutants between rack1a-1 and gpa1-4 or agb1-2 were generated by crossing gpa1-4 or agb1-2 into rack1a-1 single mutant, and isolated in the F2 progeny by PCR genotyping. For simplicity, the gpa1 rack1a, agb1 rack1a, and gpa1 agb1 double mutant nomenclatures in this report refer specifically to the gpa1-4 rack1a-1, agb1-2 60  rack1a-1 and gpa1-4 agb1-2 mutants, respectively. 3.2.3 ABA inhibition of seed germination and early seedling development assays Seeds from matched lots were surface-sterilized, sown on MS/G plates (Chen et al., 2006) that are supplemented with different concentrations of ABA, and stratified at 4°C for 2 days in dark before they were transferred to germination conditions (23°C, with 14/10 hr photoperiod, 120 µmol m-2 s-1). Germination was defined as an obvious protrusion of the radicle through the seed coat. The examination of the percentage of green seedlings was based on the presence of obvious green cotyledons. For the root growth assay, seeds were germinated on MS/G plates covered with one layer of sterilized filter paper. Twenty-four to sixty hours later, the germinated seeds were transferred to MS/G plates supplemented with ABA, and the plates were placed vertically to monitor root growth. Five to fourteen days later, the length of primary root was measured from each genotype. 3.2.4 RT-PCR and quantitative RT-PCR Total RNA was isolated from the mature seeds, young seedlings or rosette leaf mesophyll protoplasts using the TRIzol reagent (Invitrogen Canada Inc., Burlington, Ontario, Canada). cDNA was synthesized from 1 µg total RNA by oligo(dT)20-primed reverse transcription, using THERMOSCRIPT RT (Invitrogen). RACK1A-specific primers (5’GGCATCTCCAGACACCGAAA-3’ and 5’-GCAGAGAGCAACGACAGC-3’), GPA1specific primers (5’-ATGGGCTTACTCTGCAGTA-3’ and 5’TCATAAAAGGCCAGCCTCCAGT-3’), and AGB1-specific primers (5’CTGCTGATGTACTAAGCGTCTCA-3’ and 5’CTGCATGTTCCATCGTCTGA-3’) were used to amplify the transcripts of these genes. The expression of ACTIN2 (amplified by primers 5’CCAGAAGGATGCATATGTTGGTGA-3’ and 5’-GAGGAGCCTCGGTAAGAAGA-3’) was used as a control in PCR reactions. Quantitative RT-PCR analysis was performed as described by Guo and Chen (2008). 3.4.5 Western blot The RACK1A protein level was examined by using immunoblot analysis. Briefly, 10day old, light-grown seedlings or mature seeds of wild-type (Col) and mutant were ground into 61  powder with liquid nitrogen. Total protein was isolated by incubating the samples with 100 µl of freshly made lysis buffer [50mM Tris, 50mM NaCl, 5mM EGTA, 2mM DTT, 1% Triton X-100, and 1 × protease inhibitor cocktail (Sigma), pH 7.5] at 4°C for 30 min, followed by rocking at 4°C for another 30 min. Total proteins in the supernatant were collected by centrifuging at 14,000 rpm for 15 min at 4°C. Protein samples were separated by SDS-PAGE, electroblotted onto PVDF membrane, and immunoblotted with 1:1,000 anti-RACK1A peptide antibodies (Chang et al., 2005). Goat-anti-rabbit immunoglobulin conjugated to horse radish peroxidase (Sigma) was used as secondary antibody. The blot was developed using SuperSignal West Pico Chemiluminescent Substrate (PIERCE Biotechnology Inc., Rockford, Illinois). 3.2.6 Yeast two-hybrid assay The interaction between AGB1 and RACK1 was tested by using the ProQuest yeast twohybrid system (Invitrogen). AGB1 was cloned into the bait vector (pDEST32) and RACK1 (RACK1A, RACK1B, or RACK1C) was cloned into the prey vector (pDEST22). The known interaction between AGB1 and AGG1 or AGG2 (Mason and Botella, 2000, 2001) and the known interaction between TT8 and MYB75 (Zimmermann et al., 2004) were used as positive controls. The interaction between AGB1 and the empty prey vector, and the interaction between the empty bait vector and RACK1 were used as negative controls. The ability of yeast transformats to grow on minimal SD medium lacking both leucine and tryptophan (SD-LT) is indicative of the presence of both prey and bait constructs. Positive interactions are indicated by the growth of yeast cells on the triple-selective minimal SD medium lacking leucine, tryptophan and histidine but supplemented with 25 mM 3-AT (3-amino-1,2,4-triazole) (SD-LTH+3-AT). In the second assay, the split-ubiquitin yeast two-hybrid system developed by Dr. Imre Somssich (Max Planck Institute for Plant Breeding Research, Cologne, Germany) was used. AGB1 and RACK1 genes were cloned into the Gateway entry vector using the pCR®8/GW/TOPO cloning kit (Invitrogen) and then subcloned into yeast expression vector NuI (with an N-terminal fusion of N-half of the ubiquitin gene) and pMKZ (with an N-terminal fusion of C-half of the ubiquitin gene), respectively. The known interaction between AGB1 and AGG1 was used as a positive control. The interaction between AGB1 and the empty prey vector, and the interaction between the empty bait vector and RACK1 were used as negative controls. The growth of yeast transformants on the plates lacking both histidine and tryptophan (SD-HT) indicates the presence of both bait vector and prey vector. Positive interactions are 62  indicated by the growth of yeast cells on the minimum yeast medium lacking HT but supplemented with 0.1% (w/v) FOA (Fluoroorotic acid) (M-HT+FOA). 3.2.7 Yeast three-hybrid assay RACK1A and AGG1 were cloned into the pBridge vector (Clontech, Mountain View, CA) using restriction enzyme digestion and ligation methods. RACK1A was cloned to the N-terminus of GAL4 BD domain and the expression of AGG1 was driven by a methionine-repressive promoter, Pmet25. AGB1 was first cloned into pCR®8/GW/TOPO cloning kit (Invitrogen) and then subcloned into pACTGW-attR vector (Nakayama et al., 2002) fused with AD domain. The growth of yeast transformants on double-selective medium SD-LT (lacking leucine and tryptophan) indicates the presence of both vectors. Positive interactions are indicated by the growth of yeast cells on the triple selective yeast medium supplemented with methionine (SDLTH+M, lacking leucine, tryptophan and histidine) or without methionine (SD-LTH). On SDLTH+M medium, methionine was used at 1 mM to repress the expression of AGG1 gene. On SD-LTH medium, the expression of AGG1 gene is activated by methionine starvation. Vectors were co-transformed into HF7c host strain (a courtesy of Dr. Crisanto Gutierrez, Centro de Biologia Molecular "Severo Ochoa", Universidad Autonoma de Madrid, Madrid, Spain). 3.2.8 Plant two-hybrid protein-protein interaction assay The test of interaction between AGB1 and RACK1A in plant cells was conducted by using the two-hybrid protein-protein interaction assay (Ehlert et al., 2006). The procedures of Arabidopsis protoplast isolation, transfection and GUS activity assays were described previously (Wang et al., 2007). Plasmid DNAs for reporter and effector genes were isolated using Endofree Plasmid Maxi Kits (Qiagen, Mississauga, Ontario, Canada). Ten micrograms of each effector plasmid or reporter plasmid were used in co-transfection assays. Arabidopsis mesophyll protoplasts were co-transfected with AGB1 fused with Gal4 DNA binding domain (GD-AGB1) and RACK1A fused with VP16 transactivator (VP16-RACK1A), together with Gal4:GUS reporter in the absence or presence of 5 µM exogenous ABA. As a positive control, GD-AGB1 was co-transformed with VP16-AGG1 or VP16-AGG2. Each transfection assay was performed in triplicate and each experiment was repeated at least twice. GUS activities were measured by using a Fluoroskan Finstruments Microplate Reader (MTX Lab Systems Inc., Vienna, Virginia). 63  3.2.9 Co-immunoprecipitation (Co-IP) The full-length open reading frames of RACK1A and AGB1 were cloned in frame with an N-terminal Myc or C-terminal HA tag, respectively, into the pUC19 vector under the control of the double 35S enhancer promoter of CaMV followed by the translational enhancer from the 5’-leader of tobacco mosaic virus, and terminated by a 3’-untranslated region derived from the nopaline synthetase gene (Tiwari et al., 2003). Protoplasts were co-transfected with plasmid DNAs containing Myc-RACK1A and AGB1-HA. The expression of tag-fused proteins in transfected protoplasts were examined by immunoblotting using anti-Myc monoclonal antibody (Sigma) or anti-HA polyclonal antibodies (Abgent, San Diego, CA). In the Co-IP assay, protein extracts prepared from the Myc-RACK1A and AGB1-HA co-transfected protoplasts were immunoprecipitated with anti-HA polyclonal antibodies, separated by SDS-PAGE, electroblotted onto PVDF membrane and immunoblotted with anti-Myc monoclonal antibody. 3.3 Results 3.3.1 Double mutants between rack1a and gpa1 or agb1 There are three RACK1 homologous genes in the Arabidopsis genome, designated as RACK1A, RACK1B and RACK1C, respectively (Chen et al., 2006b). Among these three RACK1 genes, RACK1A is the most abundantly-expressed member (Guo and Chen, 2008). Furthermore, loss-of-function mutations in RACK1A, but not in RACK1B or RACK1C, conferred defects in plant development (Guo and Chen, 2008). Therefore, we mainly focus on RACK1A in this study. There are single Gα (GPA1) and single Gβ (AGB1) in Arabidopsis (Ma et al., 1990; Weiss et al., 1994), and it has been demonstrated that GPA1 interact with AGB1 physically, genetically and biochemically (Chen et al., 2004; Chen et al., 2006a; Adjobo-Hermans et al., 2006; Wang et al., 2008, Fan et al., 2008).  64  In order to get an insight into the relationship between RACK1 and the G-proteins, we first compared the phenotypes between loss-of-function alleles of RACK1A, rack1a-1 (Chen et al., 2006b), and loss-of-function alleles of Gα and Gβ subunits, gpa1-4 (Jones et al., 2003) and agb1-2 (Ullah et al., 2003). All of these mutants are in Columbia (Col) ecotypic background. We reasoned that if RACK1A and G-proteins function genetically in the same pathway, we might observe shared phenotypic traits between these mutants. As have been reported previously (Ullah et al., 2001, 2003; Chen et al., 2006a), both gpa1 and agb1 mutants had characteristic, round shaped rosette leaves, whereas the shape of rosette leaves of rack1a mutants was near wild-type (Figure 3.1). On the other hand, rack1a mutants produced fewer rosette leaves due to a reduced rate of rosette leaf production (Chen et al., 2006b) whereas gpa1 and agb1 mutants have near wild-type traits of these phenotypes (Chen et al., 2004). Therefore, morphologically, rack1a mutants are different from gpa1 and agb1 mutants. Then, we generated double mutants between rack1a-1 and gpa1-4 or agb1-2 alleles. As shown in Figure 3.1, gpa1 rack1a and agb1 rack1a double mutants showed combined morphological traits of their parental single mutants. For example, the rosette leaves of gpa1-4 rack1a-1 and agb1-2 rack1a-1 double mutants were round-shaped, similar to gpa1-4 and agb1-2 single mutants (Figure 3.1A-C). On the other hand, compared with Col or gpa1-4 or agb1-2 single mutants, the number of rosette leaves of gpa1-4 rack1a-1 and agb1-2 rack1a-1 double mutants was reduced, similar to rack1a1 single mutant (Figure 3.1A, D). We also noticed that although the overall shape of the rosette leaves of gpa1-4 rack1a-1 and agb1-2 rack1a-1 double mutants was round (evident by the increased leaf width/length ratio), these leaves appeared to be more wrinkled than those in gpa14 and agb1-2 single mutants (Figure 3.1B). Taken together, these results suggested that the effect of RACK1A on these morphological and developmental traits likely occur independently of the presence or absence of the G protein subunits.  65  Figure 3.1 agb1 rack1a and gpa1 rack1a double mutants. A. Phenotypes of 43 day-old plants grown under 8/16 hr photoperiod. B. Phenotypes of rosette leaves. Shown are the 10th rosette leaves taken from 58 day-old plants grown under 8/16 hr photoperiod. C. The width/length ratio of fully expanded rosette leaves. Shown are means ± S.E. for 10 plants each genotype. *, significantly different from Col, P<0.05. D. Number of rosette leaves. The total number of rosette leaves was counted from 43 day-old plants grown under 8/16 hr photoperiod. Shown are means ± S.E. for 10 plants each genotype. *, significantly different from Col, P<0.05.  66  3.3.2 An enhanced effect on ABA hypersensitivity was observed between rack1a and gpa1 or agb1 mutants in the ABA inhibition of cotyledon greening and root growth Although the overall morphology of the rack1a and G-protein subunit mutants was different and independent effects on morphological and developmental traits were observed in the double mutants, we could not exclude the possibility that RACK1A and G-proteins may act together genetically in a specific process or under specific conditions. Therefore, we sought conditional phenotypes shared between rack1a and G-protein subunit mutants. Because one of the best known roles of G-proteins in Arabidopsis is that GPA1 and AGB1 function as negative regulators of ABA responses during seed germination and early seedling development (Ullah et al., 2002; Pandey et al., 2006) and we have previously shown that rack1 mutants are hypersensitive to ABA (Chen et al., 2006b; Guo et al., 2009), we wanted to examine if RACK1A and GPA1 and AGB1 may interact genetically in these ABA-mediated processes. Although it has been shown that gpa1, agb1 and rack1a mutants are hypersensitive to ABA in the ABA-inhibition of seed germination and early seedling development (Ullah et al., 2002; Pandey et al., 2006; Chen et al., 2006b; Guo et al., 2009), a direct comparison of their ABA hypersensitivity had not been performed. Therefore, the seeds of gpa1, agb1 and rack1a mutants were sown side-by-side in MS/G medium containing different concentrations of ABA. We used three different assays to examine and compare the ABA sensitivity of these mutants, and these results are presented in Figure 3.2. We found that compared with gpa1 and agb1 mutants, rack1a mutants displayed ABA hypersensitivity to a less extent (Figure 3.2A, B). For example, the hypersensitivity of gpa1 and agb1 mutants could be observed at as low as 0.2 µM ABA whereas at this concentration, rack1a mutants had wild-type sensitivity to ABA (Figure 3.2A). At 1.0 µM ABA, a significant hypersensitivity was observed in rack1a mutants (Figure 3.2A). Further, the ABA hypersensitivity of rack1a mutants was more pronounced in the first three days after imbibed seeds had been transferred to germination conditions whereas gpa1 and agb1 mutants constantly displayed ABA hypersensitivity (Figure 3.2B). Subsequently, we compared the ABA hypersensitivity of rack1a with gpa1 and agb1 mutants in the ABA inhibition of early seedling development. First, we scored the percentage of green seedlings in the presence of ABA, based on the obvious greening of cotyledons. We found that the ABA hypersensitivity of rack1a mutants was comparable with gpa1 and agb1 mutants. Consistent with the report by 67  Figure 3.2 ABA hypersensitivity of loss-of-function alleles of RACK1A, GPA1 and AGB1. A. ABA dose-response of mutants in the seed germination assay. B. ABA time-course response of mutants in the seed germination assay. C. ABA dose-response of mutants in the cotyledon greening assay. D. ABA time-course response of mutants in the cotyledon greening assay. E. ABA doseresponse of mutants in the root growth assay. F. ABA time-course response of mutants in the root growth assay. Shown in (A) to (D) are the averages of three replicates ± S.E. Shown in (E) and (F) are the averages ± S.E of at least 10 seedlings. The percentage of seeds with radicle emergence in (A) was scored 2 days after the imbibed seeds were transferred to germination conditions (23C, with 14/10 hr photoperiod at 120 mol m-2 s-1). The percentage of seedlings with green cotyledons in (C) was scored 10 days after the imbibed seeds were transferred to germination conditions. The length of primary root in (E) was measured 5 days after the seedlings were transferred to medium containing ABA at indicated concentrations. ABA was used at 1 M in (B), (D) and (F). *, P<0.05, significantly different from wild-type (Col). 68  Pandey et al. (2006), agb1 mutants displayed a stronger ABA hypersensitivity than gpa1 mutants. Compared with G-protein subunit mutants, rack1a mutants appeared to be more hypersensitive to ABA than gpa1-4 mutant but less hypersensitive than agb1-2 mutant (Figure 3.2C, D). Second, we measured the length of primary root with and without ABA application. Similar to that of green seedling assay, the ABA hypersensitivity of rack1a mutants was comparable with gpa1 and agb1 mutants (Figure 3.2E, F). Because this ABA hypersensitivity (ABA inhibition of cotyledon greening and root growth) of rack1a mutant is more comparable with the G-protein subunit mutants, in subsequent studies, we used ABA these assays to examine the ABA sensitivity of agb1 rack1a and gpa1 rack1a double mutants. In the green seedling assay, without exogenously applied ABA, all single and double mutant seeds, like wild-type, could develop near 100% green seedlings (Figure 3.3A). When exogenous ABA was applied, gpa1 rack1a double mutants were more hypersensitive to ABA than gpa1-4 and rack1a-1 single mutants (Figure 3.3B-D). Similarly, agb1 rack1a double mutants were more hypersensitive to ABA than agb1-2 and rack1a-1 single mutants, and displayed the strongest ABA hypersensitivity among all genotypes tested (Figure 3.3BD). In the root growth assay, without ABA application, rack1a mutant had slightly shorter primary root, gpa1 mutants had near wild-type length of primary root, whereas agb1 mutants had longer primary root (Figure 3.4A). With ABA application, the length of primary root was reduced in all genotypes (Figure 3.4B). However, compared with their corresponding single mutants, gpa1 rack1a and agb1 rack1a double mutants were more hypersensitive to ABA (Figure 3.4B). The strongest ABA hypersensitivity was observed in agb1 rack1a double mutants (Figure 3.4B-D). Taken together, these results suggested that rack1a and G-protein subunit mutants may have additive effect on ABA hypersensitivity in the ABA-inhibition of early seedling development.  69  Figure 3.3 ABA hypersensitivity of agb1 rack1a and gpa1 rack1a double mutants in the ABA inhibition of cotyledon greening assay. Sterilized wild type (Col) and mutant seeds from matched seed lots were sown on MS/G medium containing 0 µM (A), 1.0 µM (B), or 1.5 µM (C) ABA, and cultured at 23 °C, with 14/10 hr photoperiod (120 µmol m-1 s-2). After 14 days, the percentage of green seedlings at 1.0 µM ABA was scored (D). Shown are mean values ± S.E. of three replicates. *, P<0.05, significantly different from Col; #, P<0.05, significantly different from corresponding single mutants.  70  Figure 3.4 ABA hypersensitivity of agb1 rack1a and gpa1 rack1a double mutants in the ABA inhibition of root growth assay. A. The length of primary root of seedlings grown on MS/G plates without ABA for 14 days. B. The length of primary root of seedlings grown on MS/G plates with 1.0 µM ABA for 14 days. C. Seedlings grown for 10 days on MS/G plates without ABA. D. Seedlings grown for 14 days on MS/G plates with 1.0 µM ABA. Shown in (A) and (B) are mean values ± S.E. from at least 10 seedlings. *, P<0.05, significantly different from Col; #, P<0.05, significantly different from the corresponding single mutants.  71  3.3.3 RACK1 and AGB1 may not physically interact with each other Genetic studies implied that RACK1A may or may not function in the same pathway with G-proteins to regulate ABA responses. Therefore, we sought additional evidence that may shed light on the relationship between RACK1 and G-proteins. Because RACK1 physically interacts with G-proteins in mammalian cells (Dell et al., 2002; Chen S et al., 2004a, 2004b; Chen et al., 2005), it was necessary to examine if such interaction is conserved in plants. Because in mammalian cells, the Gβ of the heterotrimeric G-protein complex is responsible for binding RACK1 (Chen S et al., 2004b), we used a conventional ProQuest yeast two-hybrid system (Invitrogen) to directly test the interaction between RACK1 and AGB1. The known interactions between AGB1 and AGG1 or AGG2 (Mason and Botella 2000, 2001) and the known interaction between TT8 and MYB75 (Zimmermann et al., 2004) were used as positive controls in our assays. As shown in Figure 3.5, we detected no interaction between RACK1 (RACK1A, RACK1B or RACK1C) and AGB1. Because in the conventional yeast two-hybrid system, the interactions inevitably take place in the yeast nucleus and it has been known that the membrane localization is important for the proper function of G-proteins, it was necessary to further test the interaction between RACK1 and AGB1 using the split-ubiquitin yeast two-hybrid system in which the interactions take place at the plasma membrane (Johnsson and Varshavsky, 1994; Stagljar et al., 1998). The known interaction between AGB1 and AGG1 (Mason and Botella 2000) was used as a positive control in our assays. Again, no interaction was detected between RACK1 (RACK1A, RACK1B or RACK1C) and AGB1 (Figure 3.5B).  72  Figure 3.5 Test of the physical interaction between RACK1 and AGB1 using yeast twohybrid assay. A. Conventional yeast two-hybrid assay. AGB1 was cloned into the bait vector, and individual RACK1 gene (RACK1A, RACK1B, or RACK1C) was cloned into the prey vector. The interactions between AGB1 and AGG1 or AGG2 and the interaction between TT8 and MYB75 were used as positive controls. The interaction between AGB1 and empty prey vector, and the interaction between empty bait vector and RACK1 were used as negative controls. The ability of yeast cells to grow on double-selective plates (SD-LT, lacking leucine and tryptophan) is indicative of the presence of both prey and bait constructs. Positive interactions are indicated by the growth of yeast cells on the triple-selective plates (lacking leucine, tryptophan and histidine) supplemented with 25 mM 3-AT (SD-LTH+3-AT). B. Split-ubiquitin yeast two-hybrid assay. AGB1 and RACK1 genes were fused with N-half (Nub) and C-half (Cub) of ubiquitin gene, respectively. The known interaction between AGB1 and AGG1 was used as a positive control and the interaction between AGB1 and empty prey vector, and the interaction between empty bait vector and RACK1 were used as negative controls. The ability of yeast cells to grow on double-selective plates (SD-HT, lacking histidine and tryptophan) is indicative of the presence of both prey and bait constructs. Positive interactions are indicated by the growth of yeast cells on the minimum yeast medium lacking histidine and tryptophan but supplemented with 0.1% (w/v) FOA (M-HT+FOA). The number on the top indicates the number of yeast cells used.  73  Because mammalian RACK1 prefers to bind to Gβγ dimer and Gαβγ trimer (Dell et al., 2002; Chen et al., 2005) and AGB1 may not be able to use yeast Gγ to form a stable dimer to bind RACK1, we decided to further test the interaction between RACK1A and G-proteins using yeast three-hybrid system (Clontech). Specifically, we wanted to test the interaction between RACK1A and AGB1-AGG1. In this assay, the expression of AGG1 was driven by a methionine-repressive promoter: in the absence of methionine, the expression of AGG1 was activated whereas in the presence of methionine, the expression of AGG1 was repressed. As shown in Figure 3.6 (panel A), we detected no interaction between RACK1A and AGB1-AGG1. Because interactions detected or not detected in yeasts may not always reflect the true situation in living plant cells (Ehlert et al., 2006) and AGB1 may not be able to use yeast Gγ to form a stable dimer, we decided to further test the interaction between RACK1A and AGB1 in plant cells using the Arabidopsis mesophyll protoplast transfection system. Our RT-PCR analysis indicated that endogenous AGG1 and AGG2 were present in Arabidopsis mesophyll protoplasts (Figure 3.6B). In our assays, effector plasmids containing AGB1 fused in-frame with the Gal4 DNA-binding domain (GD) together with the RACK1A fused in-frame with VP16 transactivator were co-transfected with the Gal4::GUS reporter into Arabidopsis rosette leaf mesophyll protoplasts. We found that while AGG1 or AGG2 interact with AGB1 in these assays, RACK1A did not interact with AGB1 in the absence or the presence of exogenous ABA (Figure 3.6B). Finally, we used an in vivo co-immunoprecipitation (Co-IP) assay to test the interaction between RACK1A and AGB1 by transiently co-expressing Myc-RACK1A and AGB1-HA tag fusion proteins in Arabidopsis mesophyll protoplasts. Although both tag-fused proteins were expressed well in Arabidopsis protoplasts, again, we failed to detect an interaction between AGB1 and RACK1A (Figure 3.6C).  74  Figure 3.6 Test of the interaction between RACK1A and AGB1 using yeast three-hybrid assay, plant two-hybrid assay, and co-immunoprecipitation assay. A. Yeast three-hybrid assay. RACK1A and AGG1 were cloned into pBridge vector in which RACK1A was placed to the N-terminus of GAL4 BD domain and the expression of AGG1 was driven by a methionine-repressive promoter. AGB1 was cloned into the pACTGW-attR vector and fused with AD domain. The ability of yeast cells to grow on double-selective plates (SD-LT, lacking leucine and tryptophan) is indicative of the presence of both prey and bait constructs. Positive interactions are indicated by the growth of yeast cells on the triple selective yeast medium supplemented with methionine (SD-LTH+M, lacking leucine, tryptophan and histidine) or without methionine (SD-LTH). On SD-LTH+M plates, methionine was used at 1 mM to repress the expression of AGG1 gene. On SD-LTH plates, the expression of AGG1 gene was activated by methionine starvation. The number on the top indicates the number of yeast cells used. B. Plant protein-protein two-hybrid assay. Shown in the left are effectors and reporter constructs used in the transfection assays. Effector genes and reporter genes were co-transfected into protoplasts derived from Arabidopsis rosette leaves. Shown in the right are averages of the relative GUS activities of three replicates ± S.E. in the absence of exogenous ABA. Similar results were obtained in the presence of 5 µM exogenous ABA. Inset, RT-PCR analysis of AGG1 and AGG2 transcripts in Arabidopsis mesophyll protoplasts. The expression of ACTIN2 was used as a control. RT-PCR was performed at 35 cycles. C. Co-immunoprecipitation (Co-IP) assay. Constructs of 35S:AGB1-HA and 35S:Myc-RACK1A were co-transfected into Arabidopsis protoplasts. The expression of tag-fused proteins were examined by anti-HA polyclonal antibodies and anti-Myc monoclonal antibodies, respectively. In the Co-IP assay, anti-HA antibody was used for immunoprecipitation (IP), and anti-Myc antibody was used for immunoblotting (IB). 75  3.3.4 The regulation of RACK1 expression by G-proteins Because rice RACK1 protein was found to be one of the seven proteins whose expression was down-regulated in d1 mutant (Komatsu et al., 2005), a loss-of-function allele of the sole Gα subunit in rice (Fujisawa et al., 1999; Ueguchi-Tanaka et al., 2000), it was necessary to test if such regulation is conserved in Arabidopsis. First, we examined the transcript of RACK1A in the young seedlings of G-protein subunit mutants. As shown in Figure 3.7, no apparent difference in the RACK1A transcript level was observed between wild-type and gpa1-4 and agb1-2 single and double mutants. Subsequently, we examined the RACK1A protein level in gpa1 mutants. Again, no apparent difference in RACK1A protein level was detected between wild-type and two independent loss-of-function alleles of GPA1 (Figure 3.7B). We extended our examination to other G-protein subunit and signaling mutants including agb1-2 single mutant, gpa1-4 agb1-2 double mutant, and two independent loss-of-function alleles of Regulator of Gprotein Signaling (RGS), rgs1-1 and rgs1-2 (Chen et al., 2003). Similarly, no apparent reduction of RACK1A protein was detected in any of these mutants (Figure 3.7C). To test if RACK1A may regulate the transcription of GPA1 and AGB1, we examined the transcripts of GPA1 and AGB1 in rack1a mutants by RT-PCR. We found no apparent alternation in the level of GPA1 and AGB1 transcripts in two independent alleles of rack1a mutants, compared with that in wildtype (Figure 3.7D). Next, we wanted to specifically examine the transcript and protein levels of RACK1A in the mature seeds (e.g. with mature embryos) of gpa1 mutants because Komatsu et al. (2005) reported a significant reduction of RACK1 protein level in the rice embryos of Gα mutant. Only the transcripts of RACK1A and RACK1B, but not RACK1C, were detected in the mature seeds under our experimental conditions, and we found that the transcript levels of RACK1A and RACK1B were significantly reduced in gpa1-4 mutants, compared with wild-type (Figure 3.8A). While RACK1A protein could be readily detected in the wild-type seeds, we found that RACK1A protein was at an undetectable level in the seeds of gpa1-4 mutants (Figure 3.8B). Taken together, these results suggested that the regulation of RACK1A expression is likely regulated by G-proteins in a tissue- or organ-specific manner and may be developmental stagedependent.  76  Figure 3.7 Expression analysis of RACK1A in the young seedlings of G-protein mutants. A. RT-PCR analysis of RACK1A transcript in gpa1 and agb1 mutants. B. Western blot analysis of RACK1A protein in gpa1 mutants. C. Western blot analysis of RACK1A protein in agb1 and rgs1 mutants. D. RT-PCR analysis of GPA1 and AGB1 transcripts in rack1a mutants. In (A) and (D), the expression of ACTIN2 was used as a control. RT-PCR was performed at 35 cycles.  77  Figure 3.8 Expression analysis of RACK1A in the mature seeds of gpa1 mutants. A. The transcript levels of RACK1A and RACK1B in wild-type (Col) and gpa1-4 mutants analyzed by quantitative RT-PCR. The expression of ACTIN2 was used as a control. All transcript levels are normalized against Col with the value of the first biological replicate set as 1. Shown are the mean values of three biological replicates ± S.E. B. The protein level of RACK1A in Col and gpa1-4 mutants analyzed by western blot. The experiment was repeated twice and similar results were obtained. Shown on top: the amount of total proteins loaded in each well.  78  3.4 Discussion Our genetic and biochemical analyses suggested that, different from their mammalian and yeast counterparts, Arabidopsis RACK1 and G-proteins work independently in regulating plant morphological traits and ABA responses. This view is directly or indirectly supported by the following four lines of evidence. (i) Loss-offunction alleles of RACK1A were morphologically different from gpa1 and agb1 mutants (Figure 3.1). (ii) Combined effects on morphological and developmental traits were observed in gpa1 rack1a and agb1 rack1a double mutants, compared with parental single mutants (Figure 3.1). (iii) Additive effect on ABA response was observed in gpa1 rack1a and agb1 rack1a double mutants (Figure 3.3, Figure 3.4). (iv) A direct physical interaction between RACK1 and AGB1 was not observed in yeast twohybrid (conventional system or split-ubiquitin system) or yeast three-hybrid assays (between RACK1A and AGB1-AGG1) (Figure 3.5, Figure 3.6). (v) A direct physical interaction between RACK1A and AGB1 was not observed in plant two-hybrid assay or in vivo Co-IP assay (Figure 3.6). Although we have attempted several different assays to test the physical interaction between RACK1A and AGB1, we are cautious that our investigation is not exhaustive. The lack of direct physical interaction between RACK1A and AGB1 in our assays does not exclude the possibility that these proteins may interact genetically or physiologically in a specific manner, such as under biotic or abiotic stress conditions. We observed an enhanced ABA hypersensitivity in agb1 rack1a and gpa1 rack1a double mutants, compared with single mutants (Figure 3.3, Figure 3.4). However, this genetic analysis does not allow us to draw a solid conclusion regarding the precise relationship between RACK1A and G-proteins in ABA responses. In rice, RACK1A interacts with Rac1 (Nakashima et al., 2008) which acts downstream of Gα in innate immunity (Suharsono et al., 2002). Therefore, in rice innate immunity, RACK1A may interact genetically with G-proteins via Rac1, although a direct, physical interaction between G-proteins and RACK1A or Rac1 has not been established. It remains unknown whether Arabidopsis RACK1 proteins have a role in innate immunity. It is worth noting that the regulation of RACK1 expression by Gα may occur in a tissueor organ-specific manner or may be developmental stage-dependent in Arabidopsis. Komatsu et 79  al. (2005) found that the protein level of RACK1 was down-regulated in the rice embryos of Gα mutant. Initially, we examined the transcript and protein level of RACK1A in the young seedlings of Arabidopsis Gα and other G-protein signaling mutants. However, we failed to detect any apparent differences (Figure 3.7). Interestingly, when we specifically examined the transcript and protein levels of RACK1A in the mature seeds of Gα mutants (gpa1-4), we found that the transcript and protein levels of RACK1A were dramatically reduced in gpa1-4 (Figure 3.8). Researches on plant G-proteins have already revealed that G-proteins function in a celltype or developmental stage-specific manner (reviewed by Perfus-Barbeoch et al., 2004; Ding et al., 2008). Here we showed that the regulation of RACK1 expression by G-proteins is also likely to be tissue- or organ-specific or developmentally stage-dependent. In yeast, RACK1/Asc1 functions as a Gβ and interacts with one of the two Gα subunits, acting as a negative regulator of G-protein signaling in glucose response (Zeller et al., 2007). However, Arabidopsis RACK1A did not physically interact with the sole Gα, GPA1 (Chen et al., 2006). Here we provide genetic evidence that rack1a and gpa1 mutants are morphologically different, and a combined effect on morphological and developmental traits was observed in gpa1 rack1a double mutants (Figure 3.1). Further, rack1a-1 and gpa1-4 did not appear to be epistatic to each other in the ABA-mediated inhibition of early seedling development (Figure 3.3, Figure 3.4). Therefore, it appears that there may also be some fundamental differences in the relationship of RACK1 and G-proteins between Arabidopsis and yeast. In summary, loss-of-function alleles of RACK1A and G-protein α and β subunits have distinct morphological and developmental traits, and a combined effect on these traits was observed in gpa1 rack1a and agb1 rack1a double mutants. Both RACK1A and G-proteins negatively regulate ABA responses in the ABA inhibition of early seedling development, but an additive effect on ABA hypersensitivity was observed in gpa1 rack1a and agb1 rack1a double mutants. Biochemical analysis indicated that RACK1 may not physically interact with Gproteins. Taken together, these finding revealed some fundamental differences in the relationship of RACK1 and G-proteins between Arabidopsis and mammals, and between Arabidopsis and yeast.  80  3.5 References Adjobo-Hermans, M.J., Goedhart, J. and Gadella, T.W. Jr. (2006) Plant G protein heterotrimers require dual lipidation motifs of Gα and Gγ and do not dissociate upon activation. J. Cell. Sci., 119, 5087-5097. Assmann, S.M. 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Ullah, H., Chen, J.G., Temple, B., Boyes, D.C., Alonso, J.M., Davis, K.R., Ecker, J.R. and Jones, A.M. (2003) The β-subunit of the Arabidopsis G protein negatively regulates auxin-induced cell division and affects multiple developmental processes. Plant Cell, 15, 393-409. Ullah, H., Chen, J.G., Wang, S. and Jones, A.M. (2002) Role of a heterotrimeric G protein in regulation of Arabidopsis seed germination. Plant Physiol., 129, 897-907. Ullah, H., Chen, J.G., Young, J.C., Im, K.H., Sussman, M.R. and Jones, A.M. (2001) Modulation of cell proliferation by heterotrimeric G protein in Arabidopsis. Science, 292, 2066-2069. Ullah, H., Scappini, E.L., Moon, A.F., Williams, L.V., Armstrong, D.L. and Pedersen L,C. (2008) Structure of a signal transduction regulator, RACK1, from Arabidopsis thaliana. Protein Sci., 17, 1771-1780. Wang, S., Assmann, S.M. and Fedoroff, N.V. (2008) Characterization of the Arabidopsis heterotrimeric G protein. J. Biol. Chem., 283, 13913-13922. 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Guo J, Wang J, Xi L, Huang WD, Liang J, and Chen JG (2009a) RACK1 is a negative regulator of ABA responses in Arabidopsis. Journal of Experimental Botany 60:3819-3833. 85  4.1 Introduction Receptor for Activated C Kinase 1 (RACK1) was originally identified as a receptor for activated protein kinase C (PKC) in mammalian cells (Mochly-Rosen et al., 1991; Ron et al., 1994), but now it is viewed as a multi-functional protein that plays regulatory roles in diverse signal transduction pathways (reviewed by McCahill et al. 2002; Sklan et al. 2006). The protein sequences and the structure of RACK1 are highly conserved in plants (Chen et al., 2006; Guo et al., 2007; Ullah et al., 2008). However, the research on plant RACK1 lags behind its counterparts in mammals and yeasts, and the function of plant RACK1 remains poorly understood. The first plant RACK1 was discovered as a G-protein β subunit-like protein in tobacco BY-2 cells (Ishida et al., 1993). Subsequently, RACK1 gene was cloned from rice (Iwasaki et al., 1995), alfalfa (McKhann et al., 1997), rape (Kwak et al., 1997) and Arabidopsis (Vahlkamp and Palme, 1997). The amino acid sequence homologs of RACK1 can be found in all plant species examined (Chen et al., 2006; Guo et al., 2007). In earlier studies, the characterization of plant RACK1 was mainly concentrated on gene expression and induction studies (Ishida et al., 1993; McKhann et al., 1997; Perennes et al., 1999). Recent genetic studies using model plant Arabidopsis revealed that RACK1 may have multiple functions in plants (Chen et al., 2006; Guo and Chen, 2008). Like their counterparts in mammals and yeasts, plant RACK1 proteins are also associated with ribosomes (Chang et al., 2005; Giavalisco et al., 2005). Nakashima et al. (2008) demonstrated that RACK1 proteins are key regulators of innate immunity in rice. Furthermore, rice RACK1 physically interacts with multiple proteins in the Rac1 immune complex (Nakashima et al., 2008), providing evidence that the scaffolding feature of RACK1 protein may be conserved in plants. Accumulating evidence suggested that plant RACK1 may have a role in hormone responses. For example, the first plant RACK1 gene was discovered as an auxin-induced gene in tobacco BY-2 cells (Ishida et al., 1993). In these tobacco cells, salicylic acid (SA) can block the UV irradiation-induced RACK1 expression (Perennes et al., 1999). Interestingly, in alfalfa, RACK1 was induced by cytokinin, but not by auxin (McKhann et al., 1997). In rice, ABA can induce the protein expression of RACK1 in imbibed seeds (Komatsu et al., 2005), whereas in rice cell culture, the expression of RACK1 can also be induced by methyl jasmonate, auxin and ABA (Nakashima et al., 2008). In Arabidopsis, loss-of-function mutations in RACK1A conferred altered sensitivities to auxin, ABA, gibberellin and brassinolide (Chen et al., 2006). 86  Despite of these findings, the exact role of RACK1 in any hormone responses has not been well characterized. Here we define Arabidopsis RACK1 as a negative regulator of ABA responses.  4.2 Materials and methods  4.2.1 Plant materials and growth conditions All mutants are in the Arabidopsis Columbia (Col-0) ecotype background. The rack1a-1 and rack1a-2 single mutants have been reported previously (Chen et al., 2006). The rack1b-1, rack1b-2, rack1c-1 and rack1c-2 single mutants, as well as rack1a-1 rack1b-2, rack1a-1 rack1c-1 and rack1b-2 rackc-1 double mutants have been reported by Guo and Chen (2008). For simplicity, the rack1a rack1b, rack1a rack1c and rack1b rack1c double mutant nomenclatures in this report refer specifically to the rack1a-1 rack1b-2, rack1a-1 rack1c-1 and rack1b-2 rack1c-1 mutants, respectively. Unless specified elsewhere, wild-type and mutant plants were grown under identical conditions with 14/10 hr photoperiod at approximately 120 μmol m-2 s-1 at 23°C. 4.2.2 Generation of RACK1A overexpression lines The whole open reading frame of RACK1A (At1g18080) was amplified by PCR from a cDNA library made from seedlings grown in light for 10 days and cloned into the pENTR/D-TOPO vector (Invitrogen, Carlsbad, CA) and then subcloned into Gateway plant transformation destination vectors pB2GW7 (Karimi et al., 2002) by LR recombination reaction. In this construct, the expression of RACK1A was driven by the 35S promoter of cauliflower mosaic virus. The 35S::RACK1A binary vector was transformed into Col-0 by Agrobacterium-mediated transformation (Clough and Bent, 1998). The RACK1A protein level in 35S::RACK1A transgenic lines was examined by western blot analysis using anti-RACK1A peptide antibodies (Chang et al., 2005). Goat-antirabbit immunoglobulin conjugated to horse radish peroxidase (Sigma-Aldrich Canada Ltd., Oakville, Ontario, Canada) was used as secondary antibody. The blot was developed using SuperSignal West Pico Chemiluminescent Substrate (PIERCE Biotechnology Inc., Rockford, Illinois).  87  4.2.3 Generation of PRACK1::GUS reporter lines Previously, we selected the genomic DNA 2740 bp upstream of RACK1A start codon, 2215 bp upstream of the RACK1B start codon, and 1091 bp upstream of the RACK1C start codon as putative promoter sequences for RACK1A, RACK1B and RACK1C, respectively (Chen et al., 2006). In this study, we selected shorter genomic DNA sequences, the regions between each RACK1 gene and it’s nearest upstream gene. Specifically, the genomic DNA 1491 bp upstream of the RACK1A start codon, 682 bp upstream of the RACK1B start codon, and 371 bp upstream of the RACK1C start codon was amplified by PCR, respectively, and cloned into the PZP211 binary vector (Hajdukiewicz et al., 1994) upstream of the GUS gene that was ligated into the vector earlier. The binary vectors containing PRACK1::GUS constructs were transformed into Col-0 by Agrobacterium-mediated transformation (Clough and Bent, 1998). GUS staining revealed no significant difference in expression patterns between this new set of promoter::GUS reporter lines and the lines generated earlier. These short genomic DNA sequences were also used for generating GFP/CFP/YFP reporter lines described below. Furthermore, expression of RACK1-GFP/CFP/YFP fusion proteins under the control of these short genomic DNA sequences complemented rack1 mutants. Taken together, these results suggested that these short genomic DNA sequences probably contain most cis-acting regulatory elements that are required for the proper expression of RACK1 genes.  4.2.4 Generation of PRACK1::RACK1-GFP/CFP/YFP reporter lines The genomic DNA starting from the beginning of the promoter region (same regions as used for the PRACK1::GUS constructs) prior to the stop codon of each RACK1 gene was amplified by PCR and cloned into the Gateway entry vector using the pCR®8/GW/TOPO cloning kit (Invitrogen Inc.). The cloned fragments were then transferred into the Gateway compatible binary vectors pGWB4, pGWB43 and pGWB40 (Nakagawa et al., 2007) by LR recombination reactions for constructing PRACK1A::RACK1A-GFP, PRACK1B::RACK1B-CFP and PRACK1C::RACK1C-YFP, respectively. These vectors allow the fusion of the fluorescent proteins to the C-terminal of RACK1 proteins. The binary vectors containing PRACK1::RACK1-GFP/CFP/YFP constructs were transformed into Col-0, rack1a single mutant, rack1a rack1b or rack1a rack1c double mutants by Agrobacterium-mediated transformation (Clough and Bent, 1998).  88  4.2.5 ABA inhibition of seed germination and cotyledon greening assays Wild-type and mutant seeds from matched lots were surface-sterilized and sown on MS/G plates consisting of ½ Murashige & Skoog (MS) basal medium supplemented with vitamins (Plantmedia, Dublin, Ohio), 1 % (w/v) sucrose, 0.6 % (w/v) phytoagar (Plantmedia), pH adjusted to 5.7 with 1N KOH, and supplemented with different concentrations of ABA. Imbibed seeds were cold-treated at 4°C in dark for 2 days, and then moved to 23°C, with 14/10 h photoperiod (120 μmol m-2 s-1). Germination is defined as an obvious protrusion of the radicle through the seed coat. Green seedling is defined as the presence of obvious two green cotyledons.  4.2.6 ABA inhibition of root growth assay For root growth assay, sterilized seeds were sown on MS/G plates and cold-treated at 4°C in dark for 2 days. The plates were then moved to germination conditions (23°C, 14/10h photoperiod at 120 μmol m-2 s-1) and placed vertically to allow the root to grow along the surface of agar. Sixty hours later, the evenly-grown seedlings were transferred to MS/G plates supplemented with or without 5 μM ABA. The plates were placed under the same conditions with a vertical orientation for monitoring root growth. Seven days later, the length of primary root was measured from each genotype. 4.2.7 Water loss assay Water-loss from the detached whole rosettes (with roots removed) of Col and rack1a single and double mutants was measured according to the method described by Tian et al. (2004) with minor modifications. Briefly, 20d-old Col and rack1a mutants grown under short-day conditions (8/16h photoperiod) were transferred from the growth chamber to the laboratory. The rosette from each plant was cut from its roots and weighed at different time points. The assay was performed at room temperature (~23°C) under dim light conditions (6 µmol m-2s-1) with 35% relative humidity. Three plants of each genotype were used and the water loss was calculated as the percentage of initial fresh weight at each time point.  4.2.8 Salt stress germination assay Sterilized wild-type and rack1 mutant seeds were sown on MS/G plates supplemented with different concentrations of NaCl. Imbibed seeds were cold-treated at 4°C in dark for 2 days, 89  and then moved to 23°C, with 14/10 h photoperiod (120 μmol m-2 s-1). Germination is defined as an obvious protrusion of the radicle through the seed coat. 4.2.9 RT-PCR and quantitative real-time RT-PCR For the analysis of transcripts of RACK1 genes in imbibed, germinating and germinated seeds, the procedure of sampling has been described by Gao et al. (2007). For extracting total RNA from imbibed, germinating and germinated seeds, hot borate RNA extraction method (Wilkins and Smart, 1996) was used. cDNA was synthesized using 1 μg total RNA by Oligo(dT)primed reverse transcription using OMNISCRIPT RT Kit (QIAGEN, Mississauga, Ontario, Canada). Primers used for RT-PCR and quantitative RT-PCR are listed in Table 4.1. The expression of ACTIN2 was used as a control. For ABA induction assay, four-and-half-day-old light-grown seedlings of wild-type and rack1a single mutant and rack1a rack1b and rack1a rack1c double mutants were used. Seedlings grown vertically on MS/G plates were moved to MS/G liquid medium without phytoagar and grown for another 2h prior to ABA induction in an orbital shaking incubator. ABA was added at 10 μM (for RD29B and RAB18 induction) or 50 μM (for putative RACK1 interactors induction) for 2h. Total RNA was isolated from ABA-treated and -untreated whole seedlings using the RNeasy Plant Mini Kit (QIAGEN). cDNA was synthesized as described above. Gene-specific primers (Table 4.1) for ABA-responsive marker genes, RD29B and RAB18, and putative RACK1 interacting, ABA- or abiotic stresses-regulated genes, At1g54510 (ATNEK1), At2g35940 (EDA29), At3g24080, At4g23570 (SGT1A), At4g27560, At5g03730 (CTR1), At5g08415 and At5g08420 were used for quantitative RT-PCR analyses to compare the expression of these genes in wild-type and rack1a mutants, with and without ABA treatment. The expression of ACTIN2 was used to normalize the expression of each gene. The quantitative real-time PCR was performed using the MJ MiniOpticon real-time PCR system (Bio-Rad, http://www.biorad.com) and IQ SYBR Green Supermix (Bio-Rad).  90  Table 4. 1 Primers used in this study  Primers  Sequence  RACK1A FW  5’-CTGAGGCTGAAAAGGCTGACAACAG-3’  RACK1A RV  5’-CTAGTAACGACCAATACCCCAAACTC-3’  RACK1B FW  5’-GGTTCTACTGGAATCGGAAACAAGACC-3’  RACK1B RV  5’-CTAGTAACGACCAATACCCCAGACCC-3’  RACK1C FW  5’-GCAGAGAAGAATGAAGGTGGTGT-3’  RACK1C RV  5’-CTAGTAACGACCAATACCCCAGACCC-3’  RD29B FW  5’-CACCAGAACTATCTCGTCCCAA -3’  RD29B RV  5’-GCTTTGAGGCAACGACGTTCT-3’  RAB18 FW  5’-TCCAGCAGCAGTATGACGAGTA-3’  RAB18 RV  5’-CCAGTTCCAAAGCCTTCAGTC-3’  ACTIN2 FW  5’-CCAGAAGGATGCATATGTTGGTGA-3’  ACTIN2 RV  5’-GAGGAGCCTCGGTAAGAAGA-3’  At1g54510 FW  5’-AACATAAGCGACGGGTCATC-3’  At1g54510 RV  5’-GACACCTTTCCTGGACCAAA-3’  At2g35940 FW  5’- TTGGGGAGGAAGATTCAGTG-3’  At2g35940 RV  5’- CTAGCATGTGCTTGTCCGAA-3’  At3g24080 FW  5’-GATTGGGAGGTGAATAGGCA -3’  At3g24080 RV  5’- GCCTTCTTCTCTTGGCCTTT-3’  At4g23570 FW  5’- TGAGTTCTTCGCTGATCGTG-3’  At4g23570 RV  5’- CTTGAAGGCAAAGTCGAAGG-3’  At4g27560 FW  5’- GTCTCATCCATCAGTCGGGT-3’  At4g27560 RV  5’- TTCACCAGATTCCCGATCTC-3’  At5g03730 FW  5’- CAGTTCCAAACAGGGCAAAT-3’  At5g03730 RV  5’- TAACACGCTCAGCATGGAAG-3’  At5g08415 FW  5’- CAGCGTAGACCGTGACGATA-3’  At5g08415 RV  5’- GCTGGAGCCTTTTCACAGTC-3’  At5g08420 FW  5’- CAGAAGTGTCCCTGCTCCTC-3’  At5g08420 RV  5’- TGGTTAATATTTCCAGCGCC-3’  91  4.3 Results 4.3.1 RACK1 genes act redundantly to negatively regulate ABA responses during seed germination and early seedling development The Arabidopsis genome contains three RACK1 genes, designated as RACK1A, RACK1B and RACK1C, respectively, which encode three proteins with near 90% identity at the amino acid level (Chen et al., 2006). Previously, we showed that three Arabidopsis RACK1 genes function in an unequally redundant manner to regulate rosette leaf production and root development (Guo and Chen, 2008). Because preliminary analysis using rack1a single mutants suggested that RACK1A also mediates hormone response (Chen et al., 2006), it is likely that similar to the situation in plant development, three Arabidopsis RACK1 genes may also function redundantly to regulate hormone responses although this has not been tested experimentally. Therefore, in this study, we specifically focus on the characterization of the role of RACK1 genes in ABA responses. Loss-of-function alleles of RACK1A, rack1a-1 and rack1c-2, are hypersensitive to ABA in seed germination and early seedling development (Chen et al., 2006). Although loss-of-function alleles of RACK1B (rack1b-1 and rack1b-2) and RACK1C (rack1c-1 and rack1c-2) displayed wild-type morphology (Guo and Chen, 2008), it had remained unknown if rack1b and rack1c mutants have altered sensitivity to ABA. In order to address this question, seeds of two independent loss-of-function alleles of each RACK1 gene were sown side-by-side on MS/G plates supplemented with different concentrations of ABA. Consistent with our previous findings (Chen et al., 2006), rack1a mutants displayed ABA hypersensitivity in seed germination assay (Figure 4.1A, B). However, rack1b and rack1c mutants exhibited wild-type sensitivity to ABA (Figure 4.1A, B). These results suggested that among three RACK1 genes, RACK1A is the prominent one that regulates ABA response in seed germination.  92  Figure 4.1 ABA sensitivity of rack1 single and double mutants in seed germination assay. Sterilized wild-type (Col) and rack1 single (A, B) and double mutant (C, D) seeds were sown on MS/G plates supplemented with different concentrations of ABA. The percentage of seeds with radicle emergence was scored 48h (A, C) and 96h (B, D) after the imbibed seeds were transferred from stratification conditions (4°C, dark for 2d) to germination conditions (23°C, with 14/10 hr photoperiod at 120 μmol m-2 s-1). Shown are the averages of three replicates ± S.E.  Because previously we have shown that loss-of-function mutations in RACK1B or RACK1C can enhance the developmental defects observed in rack1a mutants, we wanted to examine if the ABA hypersensitivity of rack1a mutants in seed germination can also be enhanced by rack1b or rack1c mutations. Therefore, seeds of rack1a-1 rack1b-2, rack1a-1 rack1c-1 and rack1b-2 rack1c-1 double mutants (Guo and Chen, 2008) were sown side-byside with rack1a single mutant seeds on ABA plates. We found that similar to the scenario of developmental traits (Guo and Chen, 2008), rack1a-1 rack1b-2 and rack1a-1 rack1c-1 double mutants displayed much stronger ABA hypersensitivity than rack1a-1 single mutants whereas 93  rack1b-2 rack1c-1double mutants exhibited wild-type sensitivity to ABA in seed germination assay (Figure 4.1C, D). Next, we scored the percentage of seedlings with green cotyledons in the presence of ABA to measure and compare the impact of these mutations on the ABA responsiveness of seedlings during early development. Without ABA treatment, almost all seeds of rack1 single and double mutants, similar to wild-type, could germinate and develop into green seedlings (Figure 4.2). We found that similar to the situation of seed germination assay, rack1a-1 rack1b-2 and rack1a-1 rack1c-1 double mutants displayed much stronger ABA hypersensitivity than rack1a-1 single mutants whereas rack1b-2 rack1c-1 double mutants exhibited wild-type sensitivity to ABA in the ABA inhibition of cotyledon greening assay (Figure 4.2). These results suggested that RACK1 genes function redundantly to regulate ABA responses in seed germination and early seedling development, and supported the view that RACK1A gene is the prominent member of RACK1 gene family that regulates ABA responses in Arabidopsis. We extended our analysis of ABA sensitivity of rack1 single and double mutants to postgermination root growth. Wild-type and mutant seeds were imbibed on MS/G medium without ABA for 60h under normal germination conditions (23°C, 14/10h photoperiod at 120 μmol m-2 s 1  ). Then, germinated seeds with emerged radicles were transferred to MS/G medium  supplemented with ABA. This assay allows us to specifically examine post-germination ABA sensitivity of these mutants. Because without ABA treatment, rack1a single mutants and rack1a rack1b and rack1a rack1c double mutants had shorter primary roots, compared with the wildtype (Guo and Chen, 2008), the percentage of root elongation inhibition (ABA treatment versus non-ABA treatment) was used to reflect the difference in ABA sensitivity in different genotypes more precisely.  94  Figure 4.2 ABA sensitivity of rack1 double mutants in cotyledon greening assay. Sterilized wild type (Col) and mutant seeds from matched seed lots were sown on MS/G medium containing 0 μM (A), 1.0 μM (B), 1.5 μM (C) and 2.0 μM ABA (D), and cultured at 23 °C, with 14/10 h photoperiod (120 μmol m-2 s-1 ). After 10 days, the percentage of seedlings with green cotyledons was scored. Shown in (E) are the mean values of the percentage of seedlings with green cotyledons ± S.E. of three replicates at 1.0 μM ABA. *, P<0.05, significantly different from Col; #, P<0.05, significantly different from rack1a-1 single mutant.  We found that like that in seed germination and cotyledon greening assays, rack1a-1 single mutants were hypersensitive to ABA, rack1a-1 rack1b-2 and rack1a-1 rack1c-1 double mutants displayed enhanced ABA hypersensitivity than rack1a single mutants, and rack1b-2 rack1c-1 double mutants exhibited wild-type sensitivity to ABA in this ABA inhibition of root growth assay (Figure 4.3). Collectively, these results suggested that three RACK1 genes act redundantly to negatively regulate ABA responses in the ABA inhibition of seed germination, cotyledon greening and root growth.  95  Figure 4.3 ABA sensitivity of rack1 double mutants in root growth assay. Sterilized wild type (Col) and mutant seeds from matched seed lots were sown and germinated on MS/G medium without ABA for 60h. Then, seedlings were transferred onto MS/G plates with or without 5 μM ABA and grown for another 7 days before the root length was scored. Shown are mean values of the percentage of root length reduction ± S.E. (n=16). *, P<0.05, significantly different from Col; #, P<0.05, significantly different from rack1a-1 single mutant.  4.3.2 Overexpression of RACK1A conferred ABA hyposensitivity To further study the role of RACK1 genes in ABA responses, we generated transgenic lines overexpressing RACK1A (35S::RACK1A), the prominent member of RACK1 gene family. Western blot analysis indicated that the RACK1A protein level was elevated in three independent transgenic lines, designated as RACK1A Overexpressor lines 2-4, 5-6 and 9-6 (AOX2-4, AOX5-6 and AOX9-6), respectively (Figure 4.4A). We found that consistent with the view that RACK1 functions as a negative regulator of ABA response, these independent lines displayed significant reduced sensitivity to ABA in root growth assay (Figure 4.4B) and in seed germination assay (Figure 4.5).  96  Figure 4.4 Overexpression of RACK1A conferred ABA hyposensitivity. (A) Western blot analysis of RACK1A protein level in 35S::RACK1A plants. Total proteins were isolated from 7d-old, light-grown seedlings. Lines 1-2, 2-4, 5-6, 6-1, 9-6, 17-6 and 20-6 are independent 35S::RACK1A transgenic lines. *, lines in which RACK1A is overexpressed and are used in subsequent studies. These lines are designated as AOX2-4, AOX5-6 and AOX9-6. (B) ABA sensitivity of RACK1A overexpressors in root growth assay. Sterilized wild type (Col), rack1a1 mutant and 35S::RACK1A seeds from matched seed lots were sown and germinated on MS/G medium without ABA for 60h. Then, seedlings were transferred onto MS/G plates with or without 5 μM ABA and grown for another 7 days before the root length was scored. Shown are means values of the percentage of root length reduction ± S.E. (n=16). *, P<0.05, significantly different from Col.  4.3.3 Expression of RACK1 in imbibed, germinating and germinated seeds Because our genetic analyses demonstrated that RACK1 genes regulate seed germination and early seedling development, we wanted to examine whether the expression of RACK1 genes is 97  correlated with seed germination and early seedling development. We used three different assays to examine the expression of RACK1 genes. First, we used RT-PCR to directly examine the presence of RACK1 transcript in imbibed, germinating and germinated seeds. We found the transcripts of RACK1A gene could be clearly detected during the whole process of seed  Figure 4.5 ABA sensitivity of RACK1A overexpression lines in seed germination assay. Sterilized seeds of wild-type (Col) and RACK1A overexpression lines (AOX2-4 and AOX5-6) were sown on MS/G plates supplemented with different concentrations of ABA. The percentage of seeds with radicle emergence was scored 48h and 96h after the imbibed seeds were transferred from stratification conditions (4ºC, dark for 2d) to germination conditions (23ºC, with 14/10 hr photoperiod at 120 µmol m-2 s-1). Shown are the averages of three replicates ± S.E. germination (Figure 4.6A). The transcripts of RACK1B and RACK1C appeared to be only weakly expressed in imbibed seeds under stratification conditions, but were readily detectable in germinating and germinated seeds (Figure 4.6A).  98  Figure 4.6. Analysis of RACK1 expression in imbibed, germinating and germinated seeds by using RT-PCR and PRACK1::GUS reporter lines. (A) RT-PCR analysis of the expression of RACK1 genes. Sterilized seeds were placed under stratification conditions, and sampled 0, 6, 12, 24, or 48 h later. After being stratified for 48h, seeds were then transferred to germination conditions (23°C, 14/10 hr photoperiod at 120 μmol m-2 s-1) for 24, 48, or 72h. The expression of ACTIN2 was used as control. PCR was performed at 30 cycles. (B) Analysis of RACK1 promoter activity. GUS staining was performed in seeds placed under stratification conditions for 24h and 48h, and in seeds that had been placed in stratification conditions for 48h and subsequently transferred to germination conditions for 24h and 48h, respectively. In the second assay, we examined the promoter activity of each of three RACK1 genes using the RACK1 promoter::GUS (PRACK1::GUS) reporter lines. Consistent with the RT-PCR results, the promoters of RACK1 genes were active in imbibed, germinating and germinated seeds (Figure 4.6B). The GUS staining was readily detected in the cotyledons of seeds 24h after 99  imbibition, and in the cotyledons and radicles of seeds 48h after imbibition (under stratification conditions, 4°C, dark). One day after the imbibed seeds had been transferred from stratification conditions to germination conditions (23°C, 14/10h photoperiod, 120 μmol m-2 s-1), RACK1 promoters were active in the protruding radicles of germinating seeds. Another day later, when radicle protrusion through seed coats was apparent in most seeds, the GUS staining appeared to be stronger in roots (particularly, in the root apical meristem) than in shoots (Figure 4.6B), consistent with a role of RACK1 in root development (Guo and Chen, 2008). In the third assay, we examined the RACK1 protein expression during seed germination and early seedling development. We generated fusion proteins between RACK1 and green, cyan or yellow fluorescent protein (GFP, CFP or YFP). Specifically, we generated RACK1A promoter::RACK1A-GFP (PRACK1A::RACK1A-GFP), RACK1B promoter::RACK1B-CFP (PRACK1B::RACK1B-CFP) and RACK1C promoter::RACK1C-YFP (PRACK1C::RACK1C-YFP) lines, in which the expression of the fusion proteins was driven by the native promoters of RACK1A, RACK1B and RACK1C, respectively. Because among rack1 mutants, only rack1a single mutants (but not rack1b or rack1c single mutants) and rack1a rack1b and rack1a rack1c double mutants displayed morphological and ABA phenotypes, the functionalities of these fusion proteins were tested by transforming the related constructs into rack1a single mutants (for PRACK1A::RACK1A-GFP), rack1a rack1b (for PRACK1B::RACK1B-CFP) or rack1a rack1c (for PRACK1C::RACK1C-YFP) double mutants. In each case, we found that the fusion proteins could function equivalently as the corresponding wild-type form of RACK1 protein (data not shown). By using these reporter lines, we found that the GFP/CFP/YFP fluorescence could be detected in the imbibed, germinating and germinated seeds of PRACK1A::RACK1A-GFP, PRACK1B::RACK1BCFP and PRACK1C::RACK1C-YFP lines (Figure 4.7). Similar to the situation of PRACK1::GUS reporter lines, RACK1-GFP/CFP/YFP proteins were expressed strongly in the protruding radicles of germinating seeds and the root apical meristem of germinated seeds. Taken together, the expression of RACK1 genes, the activity of RACK1 promoter and the expression of RACK1 proteins in imbibed, germinating and germinated seeds are consistent with their proposed roles in seed germination and early seedling development.  100  Figure 4.7 Analysis of RACK1 protein expression in imbibed, germinating and germinated seeds using PRACK1::RACK1-GFP/CFP/YFP reporter lines. GFP/CFP/YFP fluorescence was examined in seeds placed under stratification conditions for 24h and 48h, and in seeds that had been placed in stratification conditions for 48h and subsequently transferred to germination conditions for 24h and 48h, respectively. 4.3.4 ABA marker genes, RD29B and RAB18, were up-regulated in rack1a mutants To get insights into the role of RACK1 in ABA responses, we wanted to investigate if RACK1 is involved in the regulation of ABA-induced gene expression in young seedlings. We chose to examine the expression of two well-known ABA marker genes, RESPONSIVE TO DESSICATION29B (RD29B) and RESPONSIVE TO ABA18 (RAB18) whose expression are under direct regulation through ABA-responsive elements (ABRE) (Yamaguchi-Shinozaki and Shinozaki, 1994; Mantyla et al., 1995; Uno et al., 2000; Umezawa et al., 2006). Quantitative RT-PCR analysis revealed that without ABA induction, the transcript levels of RD29B and RAB18 were up-regulated 2- to 5-fold in rack1a single mutant and rack1a rack1b and rack1a rack1c double mutants (Figure 4.8). Upon ABA treatment, the transcripts of RD29B and RAB18 101  were dramatically increased in both wild-type and in rack1a single and double mutants (Figure 4.8). Although the differences in the transcript levels of RD29B and RAB18 between wild-type and rack1a mutants with ABA treatment was not as large as those without ABA treatment, the rack1a rack1b and rack1a rack1c double mutants accumulated more RD29B and RAB18 transcripts than wild-type in response to ABA (approximately 50% increase) (Figure 4.8). These results support the view that RACK1 negatively regulates ABA responses.  Figure 4.8 Expression of ABA marker genes, RD29B and RAB18, in rack1 mutants. The transcript levels of RD29B and RAB18 in wild-type and rack1a mutant without or with ABA treatment (10 μM for 2h) were analyzed by quantitative RT-PCR. The expression of ACTIN2 was used as control. All transcript levels are normalized against Col without ABA treatment, with the value of the first biological replicate set as 1. Shown are the mean values of three biological replicates ± S.E. 102  4.3.5 The transcription of three RACK1 genes was down-regulated by ABA Our genetic and molecular analyses demonstrated that RACK1 negatively regulates ABA responses in the ABA-mediated inhibition of seed germination, cotyledon greening and root growth, and ABA-induced gene expression. Because the expression of some negative regulators of ABA signaling, such as Rop 10 (Zheng et al., 2002), is also negatively regulated by ABA. We wanted to examine the possibility whether the expression of RACK1 genes themselves may be regulated by ABA. Interestingly, we found that the transcription of all three RACK1 genes was significantly down-regulated by ABA treatment in young seedlings (Figure 4.9), in contrast to that in rice cell culture or in imbibed rice seeds where RACK1A transcript or RACK1A protein were shown to be up-regulated by ABA (Komatsu et al., 2005; Nakashima et al., 2008).  Figure 4.9 Regulation of the transcription of RACK1 by ABA. The transcript levels of RACK1 genes in wild-type (Col) with ABA treatment (10 μM for 2h), compared with no ABA treatment, were analyzed by quantitative RT-PCR. The expression of ACTIN2 was used as control. Each RACK1gene was normalized against Col without ABA treatment, with the value of the first biological replicate set as 1. Shown are the mean values of three biological replicates ± S.E.  103  4.3.6 rack1 mutants display reduced water loss Our genetic and molecular analyses suggested that RACK1 genes are negative regulators of ABA responses. We sought additional evidence to support this conclusion. Because ABA is a critical regulator of stomatal movements (opening and closure), we wanted to examine if rack1 mutants may display alternations in water loss from rosettes. We measured the water loss from the detached whole rosette (with root removed) of rack1a single mutant and rack1a rack1b and rack1a rack1c double mutants, compared with wild-type. We found that rack1a single mutant and rack1a rack1b and rack1a rack1c double mutants lost water significantly slower than wild-type (Figure 4.10), whereas the RACK1A overexpressors lost water significantly faster than wild-type (Figure 4.11). These results implied that rack1 mutants may have extensive stomatal closure, probably because of their hypersensitivity to ABA, although this has not been experimentally tested.  Figure 4.10 Water loss assay of rack1 mutants. Whole rosettes of 20d-old plants grown under short day conditions (8/16h photoperiod) were cut off from the base and used for water loss assay. Shown are the mean values of three replicates ± S.E.  104  Figure 4.11 Water loss assay of RACK1A overexpression lines. Water loss assay of RACK1A overexpression lines. Whole rosettes of 20d-old plants grown under short day conditions (8/16h photoperiod) were cut off from the base and used for water loss assay. Shown are the mean values of three replicates ± S.E.  4.3.7 rack1 mutants display hypersensitivity to salt during seed germination Both drought and salt stress signal transduction pathways involve osmotic homeostasis and ABA plays an important role in some of these processes (reviewed by Zhu, 2002; Xiong et al., 2002). For example, many studies have observed that mutants with altered ABA sensitivity are affected in germination on salt containing media. Therefore, we wanted to extend our analysis of rack1a mutant to salt stress response. We examined the sensitivity of rack1a single mutant and rack1a rack1b and rack1a rack1c double mutants to different concentrations of NaCl during seed germination. We found that rack1a single mutant and rack1a rack1b and rack1a rack1c double mutants displayed hypersensitivity to NaCl (Figure 4.12), consistent with the view that these mutants are hypersensitive to ABA. As expected, RACK1A overexpressors displayed hyposensitivity to NaCl (Figure 4.13).  105  Figure 4.12 Salt stress sensitivity of rack1 mutants during seed germination. Sterilized wild-type (Col) and mutant seeds were sown on MS/G plates supplemented with different concentrations of NaCl. The percentage of seeds with radicle emergence was scored 48h (A) and 72h (B) after the imbibed seeds had been transferred to germination conditions (23°C, with 14/10 h photoperiod at 120 μmol m-2 s-1). Shown are the averages of three replicates ± S.E.  106  Figure 4.13 Salt stress sensitivity of RACK1A overexpression lines during seed germination. Sterilized seeds of wild-type (Col) and RACK1A overexpression lines (AOX2-4 and AOX5-6) were sown on MS/G plates supplemented with different concentrations of NaCl. The percentage of seeds with radicle emergence was scored 48h and 72h after the imbibed seeds had been transferred to germination conditions (23ºC, with 14/10 h photoperiod at 120 µmol m-2 s-1). Shown are the averages of three replicates ± S.E.  4.3.8 RACK1 interaction network Our genetic and molecular characterization provided strong evidence that RACK1 regulates ABA responses. In an attempt to understand the molecular mechanism by which RACK1 regulates ABA responses, we sought proteins that may interact with RACK1. We generated 107  RACK1 interaction network using BAR Arabidopsis Interactions Viewer (Geisler-Lee et al., 2007). This tool predicts interactome of protein of interest in Arabidopsis. Only RACK1A and RACK1C are present in the BAR Arabidopsis Interactions Viewer database (http://bar.utoronto.ca/interactions/cgi-bin/arabidopsis_interactions_viewer.cgi). The database predicts 53 potential interactors for RACK1A (Table 4.2) and 68 potential interactors for RACK1C (Table 4.3). Among the indentified 121 interactors, 35 proteins interact with both RACK1A and RACK1C, whereas RACK1A has 18 unique interactors and RACK1C has 33 (Table 4.2, Table 4.3). To examine the possibility that RACK1 may work together with these potential RACK1 interactors regulating ABA responses, these 86 interactors identified through the BAR Arabidopsis Interactions Viewer were searched against the Genevestigator (Zimmermann et al., 2004; https://www.genevestigator.com/gv/index.jsp) and the available literature for their potential roles in ABA or abiotic stress responses. We identified a total of eight genes from these 86 candidates. These genes are briefly summarized here. At1g54510 (ATNEK1) encodes a member of the NIMA-related serine/threonine kinases that have been linked to cell-cycle regulation in fungi and mammals, and was shown to be up-regulated by ABA (Nishimura et al., 2007). At2g35940 (EDA29, embryo sac development arrest 29) encodes a putative homeodomain transcription factor and was identified as an ABA-induced gene (Hoth et al., 2002). At3g24080 was identified as a salt-induced gene in a differential subtraction screening (Gong et al., 2001) and was shown to be up-regulated by ABA according to Genevestigator. At4g23570 (SGT1A, suppressor of G2 allele of skp1) is involved in plant disease resistance (Austin et al., 2002; Nakashima et al., 2008) and its rice ortholog physically interacts with RACK1 protein (Nakashima et al., 2008). SGT1 is slightly down-regulated by ABA according to Genevestigator. At4g27560 encodes a putative glycosyltransferase and was shown to be a  108  Table 4.2 RACK1A-interacting proteins identified by using the BAR Arabidopsis Interactions Viewer. Gene Description Locus number At1g03190 ATXPD_UVH6_ATXPD__ATXPD/UVH6 (ULTRAVIOLET HYPERSENSITIVE 6); ATP binding / ATP-dependent DNA helicase/ ATP-dependent helicase/ DNA binding / hydrolase; acting on acid anhydrides; in phosphorus-containing anhydrides / nucleic acid binding At1g03930 ADK1_CKL9ALPHA_CKL9BETA__ADK1 (DUAL SPECIFICITY KINASE 1); kinase At1g04290 thioesterase family protein At1g04510 transducin family protein / WD-40 repeat family protein At1g04730 AAA-type ATPase family protein At1g10930 ATSGS1_RECQL4A__ATRECQ4A/ATSGS1/RECQL4A; ATP-dependent helicase At1g11660 heat shock protein; putative At1g16470 PAB1__PAB1 (PROTEASOME SUBUNIT PAB1); peptidase At1g24706 similar to putative THO complex 2 [Oryza sativa (japonica cultivar-group)] (GB:BAD87730.1); similar to hypothetical protein OsI_004810 [Oryza sativa (indica cultivar-group)] (GB:EAY76963.1); similar to predicted protein [Physcomitrella patens subsp. patens] (GB:EDQ69221.1); contains domain THO2 PROTEIN (PTHR21597) At1g25490 RCN1, A subunit of protein phosphatase 2A At1g54510 ATNEK1; kinase At1g55255 HUB2 (HISTONE MONO-UBIQUITINATION 2) At1g56110 NOP56__NOP56 (ARABIDOPSIS HOMOLOG OF NUCLEOLAR PROTEIN NOP56) At1g63810 similar to unnamed protein product [Vitis vinifera] (GB:CAO22840.1); similar to Nrap protein; expressed [Oryza sativa (japonica cultivar-group)] (GB:ABA95898.2); similar to hypothetical protein [Vitis vinifera] (GB:CAN72980.1); contains InterPro domain Nrap protein (InterPro:IPR005554) At1g69220 SIK1__SIK1 (ERINE/THREONINE KINASE 1); kinase At1g77670 aminotransferase class I and II family protein At1g79210 20S proteasome alpha subunit B; putative At1g79930 HSP91__HSP91 (Heat shock protein 91) At1g80410 EMB2753__EMB2753 (EMBRYO DEFECTIVE 2753); binding At2g06510 replication protein; putative At2g35940 EDA29_BLH1__BLH1 (embryo sac development arrest 29) At2g36300 integral membrane Yip1 family protein At2g39460 ATRPL23A_RPL23A__ATRPL23A (RIBOSOMAL PROTEIN L23A); RNA binding / structural constituent of ribosome At2g44580 protein binding / zinc ion binding At2g47250 RNA helicase; putative At3g07790 DGCR14-related At3g11450 DNAJ heat shock N-terminal domain-containing protein / cell division protein-related At3g12860 nucleolar protein Nop56; putative At3g20860 ATNEK5; kinase  109  Table 4.2 (continued) RACK1A-interacting proteins identified by using the BAR Arabidopsis Interactions Viewer. Gene Locus number At3g22630 At3g24080 AT3G48040 At3g49830 At3g55620 At3g52140 At3g56990 At3g59410 At3g60240 At3g62310 AT4G23570 At4g27560 At4g32510 At5g08415 At5g08420 At5g11900 At5g13780 At5g25780 At5g41190  AT5G51060 AT5G51700 At5g58575 At5g60040  Description PBD1_PRCGB__PBD1 (PROTEASOME SUBUNIT PRGB); peptidase KRR1 family protein Rho GTPases in Arabidopsis that contains a putative farnesylation motif DNA helicase-related EMB1624, homolog of eukaryotic initiation factor 6 tetratricopeptide repeat (TPR)-containing protein EDA7__EDA7 (embryo sac development arrest 7) protein kinase family protein EIF4G_CUM2__EIF4G (EUKARYOTIC TRANSLATION INITIATION FACTOR 4G) RNA helicase; putative Closely related to SGT1B, may function in SCF(TIR1) mediated protein degradation. glycosyltransferase family protein anion exchanger lipoic acid synthase family protein RNA binding eukaryotic translation initiation factor SUI1 family protein GCN5-related N-acetyltransferase; putative EIF3B-2__EIF3B-2 (eukaryotic translation initiation factor 3B-2); nucleic acid binding / translation initiation factor similar to hypothetical protein [Vitis vinifera] (GB:CAN66411.1); similar to unnamed protein product [Vitis vinifera] (GB:CAO62795.1); contains InterPro domain D-site 20S pre-rRNA nuclease (InterPro:IPR017117); contains InterPro domain Nin one binding (NOB1) Zn-ribbon like (InterPro:IPR014881) ATRBOHB (RESPIRATORY BURST OXIDASE HOMOLOG B); FAD binding / calcium ion binding / iron ion binding / oxidoreductase; Identical to Respiratory burst oxidase homolog protein B (RBOHB) ATRAR1, encodes a resistance signaling protein with two zinc binding (CHORD) domains that are highly conserved across eukaryotic phyla similar to hypothetical protein [Platanus x acerifolia] (GB:CAL07976.1); contains InterPro domain Sgf11; transcriptional regulation (InterPro:IPR013246) NRPC1__NRPC1 (nuclear RNA polymerase C 1); DNA binding / DNA-directed RNA polymerase  110  Table 4.3 RACK1C-interacting proteins identified by using the BAR Arabidopsis Interactions Viewer. Gene Locus number At1g03190  At1g04730 At1g07770 At1g10930 At1g16470 At1g24706  At1g31660 At1g49760 At1g63810  At1g73570 At1g77670 At1g79930 At1g80410 At2g06510 At2g17360 At2g20040 At2g29100 At2g33340 At2g37270 At2g44580 At2g44950 At3g01490 At3g07790 At3g11450 At3g12280 At3g12860 At3g18370 At3g24080 At3g26500 At3g52140  Description ATXPD_UVH6_ATXPD__ATXPD/UVH6 (ULTRAVIOLET HYPERSENSITIVE 6); ATP binding / ATP-dependent DNA helicase/ ATP-dependent helicase/ DNA binding / hydrolase; acting on acid anhydrides; in phosphorus-containing anhydrides / nucleic acid binding AAA-type ATPase family protein RPS15A__RPS15A (RIBOSOMAL PROTEIN S15A); structural constituent of ribosome ATSGS1_RECQL4A__ATRECQ4A/ATSGS1/RECQL4A; ATP-dependent helicase PAB1__PAB1 (PROTEASOME SUBUNIT PAB1); peptidase similar to putative THO complex 2 [Oryza sativa (japonica cultivar-group)] (GB:BAD87730.1); similar to hypothetical protein OsI_004810 [Oryza sativa (indica cultivar-group)] (GB:EAY76963.1); similar to predicted protein [Physcomitrella patens subsp. patens] (GB:EDQ69221.1); contains domain THO2 PROTEIN (PTHR21597) similar to unknown [Populus trichocarpa] (GB:ABK93690.1); contains InterPro domain Bystin (InterPro:IPR007955) PAB8__PAB8 (POLY(A) BINDING PROTEIN 8); RNA binding / translation initiation factor similar to unnamed protein product [Vitis vinifera] (GB:CAO22840.1); similar to Nrap protein; expressed [Oryza sativa (japonica cultivar-group)] (GB:ABA95898.2); similar to hypothetical protein [Vitis vinifera] (GB:CAN72980.1); contains InterPro domain Nrap protein (InterPro:IPR005554) suppressor of lin-12-like protein-related / sel-1 protein-related aminotransferase class I and II family protein HSP91__HSP91 (Heat shock protein 91) EMB2753__EMB2753 (EMBRYO DEFECTIVE 2753); binding replication protein; putative 40S ribosomal protein S4 (RPS4A) kinase ATGLR2.9_GLR2.9__ATGLR2.9 (Arabidopsis thaliana glutamate receptor 2.9) transducin family protein / WD-40 repeat family protein ATRPS5B__ATRPS5B (RIBOSOMAL PROTEIN 5B); structural constituent of ribosome protein binding / zinc ion binding HUB1__HUB1 (HISTONE MONO-UBIQUITINATION 1); protein binding / ubiquitin-protein ligase/ zinc ion binding protein kinase; putative DGCR14-related DNAJ heat shock N-terminal domain-containing protein / cell division protein-related RBR_RB__RBR1 (RETINOBLASTOMA-RELATED 1) nucleolar protein Nop56; putative ATSYTF_NTMCTYPE3_SYTF__ATSYTF/NTMC2T3/NTMC2TYPE3/SYTF KRR1 family protein leucine-rich repeat family protein tetratricopeptide repeat (TPR)-containing protein  111  Table 4.3 (continued) RACK1C-interacting proteins identified by using the BAR Arabidopsis Interactions Viewer. Gene Locus number At3g52760 At3g55280 At3g56990 At3g59410 At3g60240 At3g61200 At3g62310 At4g00100 At4g14800 At4g34110 At4g34460 At5g03730 At5g03760 At5g08415 At5g08420 At5g11300 At5g11900 At5g13780 At5g15200 At5g20290 At5g25780 At5g27850 At5g41190  At5g42080 At5g43080 At5g52640 At5g57015 At5g58575 At5g58690 At5g60040 At5g62690 At5g67630  Description integral membrane Yip1 family protein 60S ribosomal protein L23A (RPL23aB) EDA7__EDA7 (embryo sac development arrest 7) protein kinase family protein EIF4G_CUM2__EIF4G (EUKARYOTIC TRANSLATION INITIATION FACTOR 4G) thioesterase family protein RNA helicase; putative ATRPS13A_PFL2_RPS13__ATRPS13A (RIBOSOMAL PROTEIN S13A); structural constituent of ribosome PBD2__PBD2 (20S PROTEASOME BETA SUBUNIT 2); peptidase PAB2_PABP2__PAB2 (POLY(A) BINDING PROTEIN 2); RNA binding AGB1_ELK4__AGB1 (GTP BINDING PROTEIN BETA 1) CTR1_SIS1__CTR1 (CONSTITUTIVE TRIPLE RESPONSE 1); kinase/ protein serine/threonine/tyrosine kinase ATCSLA09_ATCSLA9_CSLA09_CSLA9_RAT4__ATCSLA09 (RESISTANT TO AGROBACTERIUM TRANSFORMATION 4); transferase; transferring glycosyl groups lipoic acid synthase family protein RNA binding CYC3B_CYC2BAT_CYCA2;2__CYC3B (MITOTIC-LIKE CYCLIN 3B FROM ARABIDOPSIS); cyclin-dependent protein kinase regulator eukaryotic translation initiation factor SUI1 family protein GCN5-related N-acetyltransferase; putative 40S ribosomal protein S9 (RPS9B) 40S ribosomal protein S8 (RPS8A) EIF3B-2__EIF3B-2 (eukaryotic translation initiation factor 3B-2); nucleic acid binding / translation initiation factor 60S ribosomal protein L18 (RPL18C) similar to hypothetical protein [Vitis vinifera] (GB:CAN66411.1); similar to unnamed protein product [Vitis vinifera] (GB:CAO62795.1); contains InterPro domain D-site 20S pre-rRNA nuclease (InterPro:IPR017117); contains InterPro domain Nin one binding (NOB1) Zn-ribbon like (InterPro:IPR014881) ADL1_AG68_DRP1A__ADL1 (ARABIDOPSIS DYNAMIN-LIKE PROTEIN); GTP binding CYCA3;1__CYCA3;1 (CYCLIN A3;1); cyclin-dependent protein kinase regulator ATHSP90.1_HSP81-1_ATHS83_HSP81.1_HSP83__HSP81-1 (HEAT SHOCK PROTEIN 81-1); ATP binding / unfolded protein binding CKL12__CKL12 (Casein Kinase I-like 12); casein kinase I/ kinase similar to hypothetical protein [Platanus x acerifolia] (GB:CAL07976.1); contains InterPro domain Sgf11; transcriptional regulation (InterPro:IPR013246) phosphoinositide-specific phospholipase C family protein NRPC1__NRPC1 (nuclear RNA polymerase C 1); DNA binding / DNA-directed RNA polymerase TUB2__TUB2 (Tubulin beta-2); structural molecule DNA helicase; putative  112  Figure 4.14 Quantitative RT-PCR analysis of the expression of selected putative RACK1 interactors in response to ABA. The transcript levels of At1g54510 (ATNEK1), At2g35940 (EDA29), At3g24080, At4g23570 (SGT1A), At4g27560, At5g03730 (CTR1), At5g08415 and At5g08420 in wild-type (Col) and rack1a rack1b double mutants with ABA treatment (50 μM for 2h), compared with no ABA treatment, were analyzed by quantitative RT-PCR. The expression of ACTIN2 was used as control. The transcript level of each gene was normalized against that in Col without ABA treatment, with the value of the first biological replicate set as 1. Shown are the mean values of three biological replicates ± S.E.  113  salt-induced gene (Gong et al., 2001). At5g03730 (CTR1, CONSTITUTIVE TRIPLE RESPONSE 1) encodes a serine/threonine protein kinase and is a negative regulator of ethylene signaling (Kieber et al., 1993). At5g08415 belongs to the lipoic acid synthase family and is shown to be down-regulated by ABA according to Genevestigator. At5g08420 encodes an RNA-binding protein and is shown to be up-regulated by ABA according to Genevestigator. Then, we examined the ABA induction of these eight genes in rack1a rack1b double mutant seedlings, compared with that in wild-type. Among these eight genes, the transcript levels of two genes, At1g54510 (ATNEK1) and At3g24080, were reduced in response to ABA in rack1a rack1b mutant background, compared with wild-type (Figure 4.14). The transcript levels of five genes, At2g35940 (EDA29), At4g23570 (SGT1A), At5g03730 (CTR1), At5g08415 and At5g08420 were increased in response to ABA in rack1 rack1b mutant background (Figure 4.14). One gene, At4g27560, responded to ABA similarly in wild-type and in rack1a-1 rack1b2 mutant background (Figure 4.14). The alternation of ABA responses of these genes in rack1a rack1b mutant background implied that RACK1 may be involved in the ABA signaling route towards induction of these genes.  4.4 Discussion Accumulating evidence suggested that RACK1 regulates plant development and that RACK1 may be involved in hormonal responses in plants. However, the role of RACK1 in any hormone signaling pathways has not been defined prior to this study. Six lines of evidence directly or indirectly support the conclusion that Arabidopsis RACK1s are negative regulators of ABA responses. (i) rack1a single mutants and rack1a rack1b and rack1a rack1c double mutants were hypersensitive to ABA in the ABA inhibition of seed germination, cotyledon greening and root growth. (ii) Overexpression of RACK1A conferred ABA hyposensitivity. (iii) The expression of ABA marker genes, RD29B and RAB18, was up-regulated in the young seedlings of rack1a single mutants and rack1a rack1b and rack1a rack1c double mutants. (iv) rack1a single mutants and rack1a rack1b and rack1a rack1c double mutants lost water from detached rosettes significantly slower than wild-type plants. (v) rack1a single mutants and rack1a rack1b and rack1a rack1c double mutants were hypersensitive to NaCl during seed germination. Our discovery of RACK1 as a negative regulator of ABA responses in seed germination and early seedling development expanded the long list of negative regulators of 114  ABA responses. ABA INSENSITIVE 1 (ABI1) and ABI2, both protein phosphatase 2Cs (PP2Cs), are among the first negative regulators of ABA signaling identified through genetic screens (reviewed by Finkelstein et al., 2002; Himmelbach et al., 2003; Hirayama and Shinozaki, 2007; Wasilewska et al., 2008; McCourt and Creelman, 2008). Subsequently, other PP2Cs, including ABA HYPERSENSITIVE GERMINATION1 (AHG1) (Nishimura et al., 2004; Nishimura et al., 2007) and AHG3/AtPP2CA (Nishimura et al., 2004; Yoshida et al., 2006), HYPERSENSITIVE TO ABA 1 (HAB1) (Saez et al., 2004; Saez et al., 2006) and HAB2 (Saez et al., 2004; Yoshida et al., 2006) have also been demonstrated as negative regulators of ABA signaling in seed germination and early seedling development (reviewed by Hirayama and Shinozaki, 2007). Many other negative regulators of ABA responses in seed germination and early seedling development have been discovered through reverse genetics or gene expression studies (Finkelstein et al., 2002). For example, through reverse genetics, the heterotrimeric G-proteins are proposed to be negative regulators of ABA responses in seed germination and early seedling development (reviewed by Perfus-Barbeoch et al., 2004; Assmann, 2005; Chen, 2008), because the loss-of-function alleles of Arabidopsis heterotrimeric G-protein α (Gα) and β (Gβ) subunits are hypersensitive to ABA in these processes (Ullah et al., 2002; Pandey et al., 2006). Similarly, a putative G-protein-coupled receptor (GPCR) in Arabidopsis, GCR1, is a negative regulator of ABA responses in seed germination and early seedling development (Pandey and Assmann, 2004; Pandey et al., 2006). A small GTPase, Rop10, was also characterized as a negative regulator of ABA responses in Arabidopsis (Zheng et al., 2002). Genetic screens have also yielded many other critical components of ABA signaling, including ABI3, ABI4 and ABI5 (reviewed by Finkelstein et al., 2002; Himmelbach et al., 2003; McCourt and Creelman, 2008). ABI3 is a B3 domain transcription factor. ABI4 is an APETALA2 domain transcription factor. ABI5 is a basic leucine zipper transcription factor. It is believed that these transcription factors mediate ABA responses by controlling ABAinduced gene expression. Although alt least three different types of proteins had been proposed as ABA receptors in the last three years, including FLOWERING TIME CONTROL PROTEIN A (FCA) (Razem et al. 2006), a nuclear RNA-binding protein, the H subunit of Mgchelatase (CHLH) (Shen et al., 2006), a chloroplast protein, and G-PROTEIN-COUPLED RECEPTOR 2 (GCR2), a proposed seven-transmembrane GPCR (Liu et al., 2007), as reviewed by McCourt and Creelman (2008), none of these proposed ABA receptors appeared to function as the major receptor mediating ABA signaling in seed germination and early 115  seedling development. Furthermore, the structure and functionality of GCR2 have been challenged by other studies (Gao et al., 2007; Johnston et al., 2007; Illingworth et al., 2008; Guo et al., 2008; Risk et al., 2009). Subsequent studies also do not support the claim that FCA is an ABA receptor (Jang et al., 2008; Risk et al., 2008; Razem et al., 2008). At the cell surface, there are a few candidate ABA receptors perceiving the ABA signal. For example, a leucinerich repeat (LRR) receptor-like kinase 1, RPK1, has been shown to function as a positive regulator of ABA signaling in seed germination, early seedling growth, stomatal closure and ABA-induced gene expression in Arabidopsis (Osakabe et al., 2005). Recently, the Arabidopsis A4 subfamily of lectin receptor kinases, LecRKs, have been shown to function as negative regulators of ABA response in seed germination (Xin et al., 2008). However, the ability of these candidate receptors to bind ABA has not been established. In 2009, two new types of ABA receptors have been proposed, including two novel GPCR-type G-proteins, GTG1 and GTG2 (Pandey et al., 2009), and the PYR/PYL (RCAR) family of START proteins (Ma et al., 2009; Park et al., 2009), which has led to new discussions on ABA receptors (Pennisi, 2009). As discussed above, molecular and genetic studies have already identified a rich collection of signaling components, positive or negative regulators, involved or required in ABA signaling (reviewed by Finkelstein et al., 2002; Himmelbach et al., 2003; Hirayama and Shinozaki, 2007; Wasilewska et al., 2008; McCourt and Creelman, 2008). However, it remains elusive how these signaling components are coordinated to regulate ABA responses. The identification of RACK1 proteins, whose mammalian counterparts function as multi-functional scaffold proteins, as redundant, negative regulators of ABA responses may help provide new insights into the complex ABA signaling networks. In a preliminary analysis, we generated RACK1 interaction network through bioinformatics approach (Table 4.2, Table 4.3). The transcription of a total of eight genes among those 86 candidate interactors has been shown to be regulated by ABA or abiotic stresses. We found that ABA induction of seven of these eight ABA-regulated genes was altered in rack1a rack1b double mutant background, implying that RACK1 may be involved in the ABA signaling route towards induction of these genes. One of the well characterized proteins in our list is CTR1 which has been shown to be a negative regulator of ethylene signaling (Kieber et al., 1993). CTR1 was down-regulated by ABA in wild-type whereas was up-regulated in rack1a rack1b double mutant background (Figure 4.14). Because ethylene is considered to be a negative regulator of ABA signaling in seed germination and a positive regulator of ABA signaling in root growth (Gazzarrini and McCourt, 116  2001; Wang et al., 2007), it raises the possibility that RACK1 may serve as a nexus for these two hormone signaling pathways. This will be investigated further in future studies. On the other hand, the relationship between RACK1 and other known components in the ABA signaling pathway is unknown. The next major challenge is to precisely position RACK1 in the intricate ABA signal transduction network. In summary, we demonstrated that rack1a single and double mutants are hypersensitive to ABA in the ABA inhibition of seed germination, cotyledon greening and root growth whereas overexpression of RACK1A confers ABA hyposensitivity. We showed that the expression of ABA-responsive marker genes, RD29B and RAB18, are up-regulated in rack1a mutants and the RACK1 genes are down-regulated by ABA. Consistent with the ABA hypersensitivity, rack1a mutants lost water significantly slower from the rosettes and are hypersensitive to high concentrations of NaCl during seed germination. Furthermore, the expression of some known ABA- or abiotic stresses-regulated genes which encode putative RACK1 interactors was altered in rack1a rack1b mutant background, in response to ABA. Collectively, these results have defined RACK1s as critical regulators of ABA responses. Because RACK1 functions as a scaffold protein in mammalian cells, our work may help provide new insights into the complex ABA signaling network.  117  4.5 References Assmann SM. 2005. G protein signaling in the regulation of Arabidopsis seed germination. Science STKE 2005, cm11. Austin MJ, Muskett P, Kahn K, Feys BJ, Jones JDG, Parker JE. 2002. Regulatory role of SGT1 in early R gene-mediated plant defenses. Science 295, 2077-2080. Chang IF, Szick-Miranda K, Pan S, Bailey-Serres J. 2005. 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Zimmermann P, Hirsch-Hoffmann M, Hennig L, Gruissem W. 2004. GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox. Plant Physiology 136, 2621-2632.  122  CHAPTER 5  Involvement of Arabidopsis RACK1 in Protein Translation and Its Regulation by Abscisic Acid 1  1  A version of this chapter has been submitted for publication.  Guo J, Wang S, Valerius O, Hall H, Zeng Q, Ellis B and Chen JG. Involvement of Arabidopsis RACK1 in protein translation and its regulation by abscisic acid. Plant Physiology. In revision. 123  5.1 Introduction Living organisms need to maintain their cellular homeostasis in facing various environmental stresses. This process involves multiple cellular regulating mechanisms including the regulation of protein translation. Protein translation is regulated at three steps: initiation, elongation and termination (Scheper et al., 2007). Translation initiation is a complex multi-reaction process. Briefly, in mamalian cells, a pre-initiation complex (containing the 40S ribosome subunit) first binds to the 5’-cap of target mRNA and scans for the AUG start condon. Subsequently, the 60S subunit joins to assemble a functional 80S ribosome complex, which is ready to accept the appropriate aminoacyl-tRNA and form the first peptyl bond to initiate the translation elongation (Sonenberg and Hinnebusch, 2009). Early studies in plants identified a variety of abiotic stresses, including drought, cold and salt stresses, could lead to inhibition of global protein translation (Ben-Zioni et al., 1967; Aspinall, 1986; Kawaguchi and Bailey-Serres, 2002; Kawaguchi et al., 2003). Although the regulation of gene expression at the translation initiation stage plays an important role in adaptation of organisms to various environmental stresses (Brostrom and Brostrom, 1998; Yamasaki and Anderson, 2008), there has been report on the effect of stress conditions on regulating protein translation at elongation and termination stages (Shenton et al., 2006). In addition, ribosome biogenesis, one of the major energy consuming cellular processes, is also under tight regulation in response to environmental signals. The evolutionarily-conserved TOR kinase (Martin et al., 2004; Deprost et al., 2007) plays a central role in regulating ribosome protein biogenesis. However, the identity of the specific molecular players that link stress responses, the stress-signaling hormone abscisic acid (ABA), and the regulation of global translation has remained elusive. Mammalian RACK1 was initially identified as a Receptor for Activated C protein Kinase 1 (Ron et al., 1994) and later found to interact with numerous proteins involved in multiple signal transduction pathways (reviewed by McCahill et al., 2002; Sklan et al., 2006; Guo et al., 2007). In plants, RACK1 homologs appear to play multiple roles. The first RACK1 homolog was initially identified as an auxin-responsive gene in tobacco BY-2 cells (Ishida et al., 1993) and a related gene was subsequently isolated from alfalfa (McKhann et al., 1997). The tobacco RACK1 homolog was found to mediate cell cycle arrest triggered by salicylic acid and UV irradiation (Perennes et al., 1999). More recently, RACK1 was identified as a component of the plant 40S ribosome subunit (Chang et al., 2005; Giavalisco et al., 2005), and as an interacting 124  partner within a rice Rac1 immune complex that mediates the innate immunity response (Nakashima et al., 2008). The crystal structure of the Arabidopsis RACK1A protein was also recently resolved (Ullah et al., 2008). In earlier studies, we found that a loss-of-function mutation in one of the three RACK1 genes in Arabidopsis, RACK1A, conferred altered responses to multiple plant hormones (Chen et al., 2006). Later, we provided evidence to support the view that three RACK1 genes regulate plant development in a manner of unequal genetic redundancy (Guo and Chen, 2008). More recently, we found that RACK1 genes work redundantly as negative regulators of ABA responses and mediate stress responses (Guo et al., 2009a). Interestingly, although Arabidopsis possesses homologs of both mammalian RACK1 and heterotrimeric G-proteins, the plant homologs appear to act through a mechanism that is distinct from their counterparts in mammals (Guo et al., 2009b). One of the best characterized roles for RACK1 in Arabidopsis is acting as a regulator of ABA and abiotic stress responses (Guo et al., 2009a), and in this study, we investigate its molecular mechanism of action. By taking a combination of molecular, genetic, biochemical and pharmacological approaches, we show that RACK1 is involved in protein translation and 60S ribosome subunit biogenesis, and that its action in these processes may be regulated by ABA. These findings provide new insights into the molecular mechanism of action of RACK1 in modulating ABA responses, and into the regulation of protein translation, a fundamental cellular process in plants. 5.2 Materials and methods 5.2.1 Plant materials and growth conditions  All mutants are in the Arabidopsis Columbia (Col-0) ecotype background. Plants were grown in 5 x 5 cm pots containing Sunshine Mix #4 (Sun Gro Horticulture Canada Ltd., http://www.sungro.com) with 14/10 hr photoperiod at approximately 120 μmol m-2s-1 at 23°C.  5.2.2 DNA microarray assay Seeds of Columbia (Col-0) and rack1a rack1b mutant were germinated on ½ Murashige & Skoog (MS) basal medium with vitamins (Plantmedia, Dublin, Ohio, 125  http://www.plantmedia.com), 1 % (w/v) sucrose, 0.6 % (w/v) phytoagar (Plantmedia), pH adjusted to 5.7 with 1N KOH. The plates were vertically placed to allow root growth along the surface of the agar. Four and half day-old seedlings were harvested and then incubated in either liquid ½ MS medium containing 50 µM ABA or solvent only for 4 h before they were snapfrozen in liquid nitrogen. A custom-made full-genome (30K) Arabidopsis 70-mer oligo arrays (Douglas and Ehlting, 2005; Ehlting et al., 2005) was used in the assay. Total RNA was extracted using QIAGEN Plant Mini RNA extraction kit (Qiagen Inc. http://www1.qiagen.com). The quantity and quality of total RNA were determined by the Agilent 2100 Bioanalyzer (Agilent technologies. http://www.home.agilent.com) with the Agilent RNA 6000 Nano kit and reagents. Twenty µg total RNA was used for reverse transcription with SuperScript II RT kit (Invitrogen Inc. http://www.invitrogen.com). The 3DNA Array 350 kit (Genisphere. http://www.genisphere.com) was used according to manufacturer specifications for cDNA labeling, cDNA hybridizations, and subsequent 3DNA (dendromer) fluorescent probe hybridizations. Hybridizations were carried out using a Slidebooster SB401 (Advalytix. http://www.advalytix.com), while scanning was conducted on a Scanarray Express (Perkin Elmer. http://www.perkinelmer.com/). Scanned images were quantified using Imagene (BioDiscovery. http://www.biodiscovery.com/), and data were analyzed in the R package using Bioconductor tools and custom scripts. For background correction, the mean of the dimmest five percent of spots in a particular sub-grid (grouping of 26 x 27 spots) was used as the background value for the spots in that sub-grid. Backgroundcorrected spot intensities were then normalized on each array using the robust local-linear regression algorithm LOWESS (or ‘LOESS’) included in the R package, with a span of 0.7 (Yang and Speed, 2002). A common reference design was employed with the sample of 50 µM ABA treated Col used as the common reference which was hybridized onto each slide with another sample of various treatments/genotypes combination. The relative expression ratio for each gene represents the average of three biological replicates, where p-value significance estimates were computed using either two-tailed Student’s (direct hyb comparisons) or paired (indirect channel comparisons across slides) t-tests (alpha = 0.05) and adjusted for false discovery rate using the q-value correction (Storey, 2002).  126  5.2.3 Yeast complementation assay The S. cerevisiae strains of 1278b background used were RH2656 (wild type diploid, MAT a/  ura3-52/ura3-5 trp1::hisG/TRP1, Braus et al., 2003) and RH3264 (homozygous diploid cpc2/rack1 mutant, MATa/ GCRE6-lacZ::URA3/ura3-52 trp1::hisG/ trp1::hisG leu2::hisG/leu2::hisG cpc2::LEU2/cpc2::LEU, Valerius et al., 2007). The plasmids used were p424MET25, a TRP1-marked centromere vector (Mumberg et al., 1994). The protein coding sequences of CPC2, RACK1A, RACK1B and RACK1C were cloned into p424MET25 using restriction enzyme digestion and ligation method. A lithium acetate mediated transformation method was used to transfer the plasmid into host yeast strain and the successful transformants were selected on appropriate nutrient-selective media. For the pseudohyphal growth assay, the transformed yeast strains were grown on nitrogen starvation plates (0.15% (w/v) yeast nitrogen base (w/o amino acids and ammonium sulfate, BD Difco. http://www.bd.com/ds/), 50 M ammonium sulfate, 2% (w/v) glucose, 2.5% (w/v) agar (Sigma. http://www.sigmaaldrich.com) and (350 mg/L) Uracil) for 5 days at 30ºC before the morphology of individual yeast colonies was examined and photographed under compound light microscope.  5.2.4 Isolation of eif6a and eif6b T-DNA insertional mutants  All the T-DNA insertional mutants of RACK1 genes have been described previously (Chen et al., 2006; Guo and Chen, 2008). The T-DNA insertion mutant of eIF6A (At3g55620), eif6a-1 (GABI_817H01), and the T-DNA insertion mutants of eIF6B (At2g39820), eif6b1(SALK_017008), eif6b-2 (SALK_057424), were identified from the SALK T-DNA Express database (http://signal.salk.edu/cgi-bin/tdnaexpresses). The second mutant allele of eIF6A, emb1624 (Tzafrir et al., 2004) was originally identified within a collection of mutants defective in embryo development, and was here renamed eif6a-2. For each SALK T-DNA insertional mutant (Alonso et al., 2003), the insertion locus was confirmed by PCR and sequencing using eIF6B-specific primers (5’-ATGGCGACTCGTCTTCAGTTTGTGAACAAC-3’ and 5’TATCGATCGAAGACTTCCTCATTTCACTAC-3’) and a T-DNA left border-specific primer JMLB1 (5’-GGCAATCAGCTGTTGCCCGTCTCACTGGTG-3’). For the GABI-Kat T-DNA insertional mutant eif6a-1 (Rosso et al., 2003), the eIF6A-specific primers (5’ATGGCGACTCGTCTTCAATATGATAACAAATA-3’ and 5’AGATATTCACCAAAACTCTACAATC-3’) and another T-DNA left border-specific primer 127  Garbi-LB-o2588 (5’- CGCCAGGGTTTTCCCAGTCACGACG-3’) were used to confirm the insertion position by PCR and sequencing. For eif6a-2 (emb 1624), the eIF6A-specific primers (5’-CTCTACAATACCTCATTTTACATGCTCC-3’ and 5’AGGCTAACGTACACCTGCGTAG-3’) and T-DNA left border-specific primer LB3 (5’- TAG CAT CTG AAT TTC ATA ACC AAT CTC GAT ACA C-3’) (McElver et al., 2001) were used to confirm the insertion position by PCR and sequencing.  5.2.5 Yeast two-hybrid assay  The interactions between eIF6s and RACK1s were tested by using the ProQuest yeast twohybrid system (Invitrogen Canada Inc. http://www.invitrogen.com). eIF6s were cloned into bait vector pDEST32 and RACK1s were cloned into prey vector pDEST22. The yeast transformants that contain both prey and bait were able to grow on minimum SD (Synthetic Dextrose) dropout medium lacking both leucine and tryptophan (SD-LT). A positive interaction between two proteins is indicated by the growth of yeast colony on the minimum SD medium lacking leucine, tryptophan and histidine and containing 10 mM 3-amino-1,2,4-triazolium.  5.2.6 Bi-molecular Fluorescence Complementation (BiFC) assay in Arabidopsis mesophyll protoplasts  The coding sequences of RACK1s were cloned into pSAT1A-nEYFP-N1 and fused to the Nterminal half of the YFP (yellow fluorescent protein) molecule. eIF6s were cloned into pSAT4AcEYFP-N1 and fused to the C-terminal half of the YFP molecule. The coding sequences of RACK1s and eIF6s were also cloned into the pSAT6-EYFP-N1 vector in which the full-length (YFP) is fused to the C-terminus of the proteins for studying subcellular localization of each protein (Citovsky et al., 2006). The isolation and transfection of Arabidopsis leaf mesophyll protoplast cells were conducted the same way as previously described (Wang et al., 2005; Yoo et al., 2007). In brief, protoplasts were isolated from rosette leaves of 3-week-old plants. Constructs prepared as described above were transfected (for subcellular localization) or co-transfected (for BiFC) into protoplasts and incubated in the dark for 20 h to allow expression of the introduced genes. The 128  YFP fluorescence was examined and photographed using a Leica DM-6000B upright fluorescent microscope with phase and differential interference contrast (DIC) equipped with a Leica FW4000 digital image acquisition and processing system (Leica Microsystems, www.leica-microsystems.com).  5.2.7 Root growth assay with anisomycin  Seeds of Col and rack1 mutants were germinated on ½ MS media plates for 60h at 14/10h photoperiod. The seedlings were then transferred to ½ MS media containing various concentrations of anisomycin and grown vertically for another five days before data were collected. The Image J software was used to measure the primary root length from pictures of each plate.  5.2.8 Analysis of embryo development  Siliques of different developmental stages from heterozygous eif6a-1 and eif6a-2 were dissected under a dissecting microscope with a fine-tip pin. Since all the seeds from the same silique are at the same developmental stage, the numbers of white seeds and green seeds in each silique were scored and the seeds were then individually immersed in fixation/clearing solution (chloral hydrate:H2O:glycerol=8:2:1). The cleared green seeds were then examined under a compound microscope to assess their developmental stage. For each representative developmental stage of the green seeds, the white seeds from the same silique were observed microscopically and photographed.  5.2.9 Ribosome profiling assay  The procedure used for the ribosome profiling assay was essentially the same as previously described (Kawaguchi et al., 2003). In summary, a 2 g sample of 4 1/2 - day-old seedlings were ground to a fine powder under liquid nitrogen. For each sample, 750 µl frozen ground tissue was 129  quickly homogenized in 750 µl ribosome extraction buffer (Kawaguchi et al., 2003) and incubated on ice for 10 min. The supernatant (500 µl) was layered on top of a 5 ml (20%-60%) sucrose gradient (Fennoy and Bailey-serres, 1995) and centrifuged for 90 min at 45,000 rpm at 4 ºC. Gradient fractions (200 µl) were collected manually, starting from top of the gradient and the OD260 for each fraction was measured using a Synergy HT multi-mode microplate reader (BioTek Instruments, Inc. http://www.biotek.com). The baseline absorbance of a gradient loaded only with extraction buffer was subtracted and the profiles were normalized to equal total OD absorption units to allow for comparison between samples. For ABA treatment, 4 ½-day-old Col seedlings were incubated in ½ MS liquid medium containing 50 µM ABA for 4 h or 8 h before they were snap-frozen in liquid nitrogen and assayed later.  5.2.10 Gene expression analysis  For the quantitative RT-PCR assay, Col seeds were germinated on ½ MS medium and plates were placed vertically to allow the roots grow along the surface of the agar. Col seedlings (4 ½ days-old) were gently removed from the agar surface and incubated in liquid ½ MS medium with or without 20 µM ABA for different periods of time. They were then harvested and snapfrozen in liquid nitrogen. Total RNA was isolated using the Qiagen Plant Mini RNA isolation kit (Qiagen Inc.) and cDNA was synthesized with Omniscript RT kit (Qiagen Inc.). Quantitative real-time PCR was performed using the MJ MiniOpticon real-time PCR system (Bio-Rad Laboratories (Canada) Ltd., Mississauga, Ontario, Canada, http://www.bio-rad.com) and IQ SYBR Green Supermix (Bio-Rad Laboratories). The real-time PCR primers used for analyzing the transcript levels of RACK1A, RACK1B, RACK1C and ACTIN2 (used for normalization) were the same as described (Guo and Chen, 2008). The experiments were repeated three times and data with similar trends were obtained. For promoter::GUS assay, the PRACK1::GUS lines described previously (Guo et al., 2009a) were used. Seeds were germinated on ½ MS medium and plates were placed vertically. Seedlings (4 ½ days-old) were incubated in liquid ½ MS medium with or without ABA for 6 h and then subjected to GUS staining as described previously (Guo et al., 2009a). Pictures of seedlings were taken under a dissecting microscope. 130  5.3 Results  5.3.1 A group of genes co-regulated by ABA and rack1 mutation  To characterize the role of RACK1 in ABA responses in more detail, a global gene expression profiling assay was conducted, using rack1a rack1b double mutants. We specifically looked for genes that are up- or down-regulated 2.0 fold in the rack1a rack1b mutant background, and compared these responses with the list of genes that are up- or down-regulated by ABA treatment in wild-type (Col-0) background. Three biological replicates were used for each sample. This analysis identified a total of 1,254 genes that were up-regulated 2-fold in the rack1a rack1b mutant plants and a total of 1,312 genes that were down-regulated (Appendix 1, Figure 5.1). Under our experimental conditions, a total of 968 genes were up-regulated, and 1,253 genes were down-regulated, by ABA treatment in the wild-type plants (Appendix 1, Figure 5.1). Functional categorization of the genes that were differentially expressed in rack1a rack1b mutant background revealed a relatively high percentage of genes whose predicted biological function is involved in stress responses (4.7% of up-regulated genes and 4.6% of down-regulated genes), in response to abiotic and biotic stimulus (4.1% of up-regulated genes and 4.8% down-regulated genes), in protein metabolism (6.7% of up-regulated genes and 5.8% of down-regulated genes), and in developmental processes (4.3% of up-regulated genes and 4.9% down-regulated genes) (Figure 5.1), suggesting an important role for RACK1 genes in mediating these biological processes. Interestingly, we found that approximately 41% (400 out of 968) of the ABA-up-regulated genes in wild-type plants were also up-regulated in rack1a rack1b double mutant background even in the absence of ABA treatment. Similarly, we found that approximately 41% (519 out of 1253) of the ABA-down-regulated genes in wild-type plants were also down-regulated in rack1a rack1b mutant plants without ABA treatment (Figure 5.2, Appendix 1). In contrast, only 7 ABA-down-regulated genes were up-regulated in rack1a rack1b mutant and 26 ABA-up-regulated genes were down-regulated in rack1a rack1b mutant. These results reinforced the view that RACK1 is an important regulator of ABA responses.  131  Figure 5.1 Gene ontology distribution of the genes that are differentially regulated in rack1a rack1b mutants. A. Functional categorization of genes that were up-regulated two-fold or more in the rack1a rack1b mutant background. B. Functional categorization of genes that were down-regulated two-fold or more in rack1a rack1b mutant background. Functional categorization of genes was obtained through the TAIR Gene Ontology (GO) Annotations tool (http://www.arabidopsis.org/tools/bulk/go/index.jsp).  132  Figure 5.2 A Venn diagram shows the number of genes that are co-regulated by 50 µM ABA treatment and by rack1 mutation. The number of genes that were co-regulated by ABA treatment and rack1 mutation appears in the overlapped portion of the circles and the number of genes that were not co-regulated appears in the non-overlapping portions. A. Diagram for the 2fold upregulated genes. B. Diagram for the 2-fold downregulated genes.  5.3.2 Co-expression analysis of RACK1 genes  To gain further insight into the biochemical/molecular function of RACK1 in Arabidopsis, we performed global co-expression data analysis (PRIME, http://prime.psc.riken.jp/) to identify genes that are co-expressed with all three RACK1 genes. Surprisingly, we found that more than 80% (128 out of 154) of the genes that are co-expressed with RACK1 encode ribosomal proteins (Figure 5.3 and Appendix 2), implying a potential relationship between RACK1 function and the ribosome complex. RACK1 proteins were previously reported to be associated with ribosomes in Arabidopsis (Chang et al., 2005; Giavalisco et al., 2005) and one of the major functions of RACK1 in mammalian cells and yeast is to regulate translation initiation at the ribosome assembly reaction (Ceci et al., 2003; Shor et al., 2003). These findings prompted us to examine the function of Arabidopsis RACK1 in ribosome assembly and translation initiation, and the relationship, if any, between such a role and cellular responses to ABA.  133  Figure 5.3 RACK1 co-expression analysis. RACK1A, RACK1B and RACK1C were submitted to the Correlated Gene Search of the Platform for RIKEN Metabolomics, PRIME (http://prime.psc.riken.jp/). Threshold value was set at 0.4, and display limit was set at 300. Group A includes genes that are co-expressed with all three RACK1 genes; group B comprises the genes that co-expressed with RACK1A and RACK1B; group C contains genes that are co-expressed with RACK1B and RACK1C; groups D,E and F contain genes that are co-expressed with RACK1A, RACK1B or RACK1C, respectively.  5.3.3 Arabidopsis RACK1 complements the S. cerevisiae cpc2/rack1 mutant  Because a large amount of information about the molecular function of RACK1 has been accumulated for mammals and yeast, and many of the signaling pathways and cellular processes that RACK1 is involved in appear to be conserved across eukaryotic kingdoms (McCahill et al., 2002; Sklan et al., 2006; Guo et al., 2007), we asked whether the Arabidopsis RACK1 genes can rescue the yeast cpc2/rack1 mutant phenotypes. Diploid S. cerevisiae strains of the genetic 1278b background are dimorph and develop from single yeast cells to filament-like pseudohyphal cells under nitrogen starvation condition (Gimeno et al., 1992). A homozygous deletion of CPC2 results in the loss of pseudohyphae development under nitrogen starvation condition and remained in a smooth-border round colony (Figure 5.4A, B, Valerius et al., 2007). We first expressed the full length of yeast CPC2 gene in 134  the cpc2 mutant with the yeast expression vector p424MET25 (Mumberg et al., 1994) and observed the recovered growth of pseudohyphae (Figure 5.4C). With this validated system, we found that when any of the three Arabidopsis RACK1 genes was expressed in yeast cpc2 diploid  Figure 5.4 Complementation assay for failed pseudohyphal growth in the diploid S. cerevisiae cpc2 mutant, using three Arabidopsis RACK1 genes. (a) RH2656 (wild type) + p424MET25 (empty vector), (b) RH3246 (cpc2) + p424MET25 (empty vector), (c) RH3246 (cpc2) + p424MET25-CPC2, (d) RH3246 (cpc2) + p424MET25-RACK1A, (e) RH3246 (cpc2) + p424MET25-RACK1B, (f) RH3246 (cpc2) + p424MET25-RACK1C. Transformants were patched on nitrogen starvation plates and grown for 5 days before pictures were taken.  mutant background, the transformant regained the ability to produce the filament-like structures (pseudohyphae) (Figure 5.4D-F). These results demonstrated that Arabidopsis RACK1 genes are functional equivalent to yeast CPC2/RACK1. In another study, Gerbasi et al. (2004) provided evidence to support that the mammalian RACK1 is a functional orthologue of yeast CPC2 gene. In agreeing with the genetic data, both the amino acid sequences (Chen et al., 2006) and crystal structure (Ullah et al., 2008) of RACK1 are also highly conserved in different eukaryotic organisms. Taken together, these results supported the view that some functions of RACK1 gene may be conserved in mammals, yeast and Arabidopsis.  135  5.3.4 RACK1 physically interacts with eukaryotic initiation factor 6 (eIF6)  In mammalian ribosomes, it has been proposed that RACK1 acts as a scaffold protein to bring together activated protein kinase C (PKC) and eukaryotic initiation factor 6 (eIF6). eIF6 is then phosphorylated by PKC and subsequently dissociates from the 60S ribosome subunit, which allows the 40S and 60S ribosome subunits to form the functional 80S ribosome (Ceci et al., 2003). Despite the lack of obvious PKC homologs in the Arabidopsis genome, two homologs of eIF6, encoded by loci At3g55620 (hereafter named as eIF6A) and At2g39820 (hereafter named as eIF6B), are present. We therefore decided to test whether physical interaction can be detected between the Arabidopsis RACK1 and eIF6 proteins. When the interaction was tested in a yeast two-hybrid system, each of the three RACK1 proteins was found to physically interact with each of the two eIF6 proteins (Figure 5.5). To establish whether the physical interaction also occurs in plant cells, a bimolecular fluorescence complementation system (BiFC) (Citovsky et al., 2006) was used in combination with Arabidopsis leaf mesophyll protoplast transient expression system (Yoo et al., 2007). Again, positive interactions were detected for each pair of RACK1 and eIF6 proteins (Figure 5.5B). The interaction was primarily detected in the cytoplasm and nucleus, which is consistent with the respective subcellular localization of each protein (Figure 5.6) and resembles the subcellular localizations of their mammalian counterparts (Ceci et al., 2003).  136  Figure 5.5 Physical interaction between RACK1 and eIF6 detected in yeast two-hybrid assays and in the BiFC system. A Interactions between RACK1s and eIF6s in the yeast two-hybrid assay. eIF6 genes were cloned into pDEST32 and RACK1s were cloned into pDEST22. The interaction between eIF6s and the empty prey vector was used as a negative control. The ability of yeast cells to grow on synthetic medium lacking leucine, tryptophan and histidine, and containing 10 mM 3-AT, is scored as a positive interaction. B Interactions between RACK1s and eIF6s in BiFC. RACK1 proteins were fused with the Nterminal half of YFP and eIF6 proteins were fused with C-terminal half of YFP. The interaction between OFP1 (Wang et al., 2007) and eIF6 proteins was used as a negative control. Image shown are the same transformants pictured under YFP fluorescent and DIC microscopic setups.  137  Figure 5.6 Subcellular localization of RACK1 and eIF6. RACK1 and eIF6 were fused with full length YFP at the C-terminus. The construct was introduced into protoplasts and the transformants were incubated in the dark for 24 h to allow the propagation of proteins. The transformed protoplast population was examined by fluorescent microscopy and images of positive cells were recorded with a digital camera attached to the microscope. For each transformation, the two pictures shown were taken with YFP fluorescent exiting light and DIC, respectively.  5.3.5 eIF6 homologs in Arabidopsis  The proteins predicted to be encoded by the two Arabidopsis eIF6 genes share 86% amino acid sequence similarity and 72% identity to each other at the amin acid level (Figure 5.7A, B). The protein sequence of eIF6A appears to be highly conserved within the plant kingdom. Moreover, Arabidopsis eIF6A shares about 73% identity and 85% similarity with its homologs in humans (Homo sapiens) and yeast (Saccharomyces cerevisiae) (Figure 5.7A, B). eIF6B is somewhat more divergent, and shares about 60% identity and 78% similarity to its homologs in human and 138  yeast. RT-PCR analysis revealed that the expression of eIF6A is ubiquitous across various tissues and organs in Arabidopsis, whereas eIF6B is only expressed in flower buds (Figure 5.8A). These results are largely consistent with the in silico data from the Genevestigator Arabidopsis thaliana microarray database (Zimmermann et al., 2004) (Figure 5.8B). The higher amino acid sequence homology of eIF6A to its counterparts in other organisms, as well as its ubiquitous expression pattern, implies that eIF6A may be the predominant functional copy of the two eIF6 genes. To further study the function of eIF6 genes in Arabidopsis, we obtained two independent T-DNA insertional alleles for each eIF6 gene, all in the Columbia (Col-0) ecotypic background. The two mutant alleles of eIF6A were designated as eif6a-1 (GABI_817H01) and eif6a-2 (emb 1624, Syngenta) and the two mutant alleles of eIF6B as eif6b-1 (SALK_017008) and eif6b-2 (SALK_057424). RT-PCR analysis indicated that eif6b-1 allele is a full-transcript null allele whereas eif6b-2 is a knock-down allele (Figure 5.9B). All insertion positions were validated by sequencing. When we examined the phenotypes of these mutant alleles, we were unable to recover plants homozygous for the eif6a-1 or eif6a-2 loci. We found that within the siliques of the eif6a+/- parent plants, the ratio of white seeds (containing developmentally-halted embryo) to green seeds (containing normally developing embryo) was approximately 1:3 (n=500), indicative of a homozygous embryo lethal outcome (Figure 5.9C). By examining the white seeds microscopically, we found that the development of the embryo was arrested at the globular stage (Figure 5.9C). These results are consistent with the fact that eif6a-2/emb1624 allele was originally identified in the collection of mutants defective in embryo development (Tzafrir et al., 2004). We have observed such defects in both T-DNA insertional alleles of eIF6A gene. The eif6b-1 and eif6-2 alleles, on the other hand, did not display any apparent developmental defects (Figure 5.9D), which supports the view that eIF6A, but not eIF6B, may be the predominant member of the small eIF6 gene family in Arabidopsis.  139  Figure 5.7 Arabidopsis eIF6 homologs. (A) Protein sequence alignment of eIF6 homologs in plants, yeast and human. The Arabidopsis (Arabidopsis thaliana) genome contains two eIF6 homologs, At3g55620 and At2g39820, designated here as AteIF6A and AteIF6B, respectively. The rice (Oryza sativa) genome also contains two eIF6 homologs, Os07g0639800 and Os01g0280500, designated here as OseIF6A and OseIF6B, respectively. The poplar (Populus trichocarpa) and grape vine (Vitis vinifera) genomes each contains at least one eIF6 homolog, designated as PteIF6 and VteIF6, respectively. The accession numbers for yeast (Saccharomyces cerevisiae, SceIF6) and human (Homo sapiens, HseIF6) eIF6 are NP_015341 and NP_852133, respectively. Amino acids that are identical or similar are shaded with black or grey, respectively. The sequence alignment was generated by the ClustalW multiple alignment of BioEdit Sequence Alignment Editor (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). (B) Pair-wise analysis of the identity and similarity at the amino acid level of eIF6 homologs in plants, yeast and humans.  140  Figure 5.8 Expression of Arabidopsis eIF6 homologs. A RT-PCR assay for expression of eIF6 genes in different Arabidopsis tissues. PCR was performed with 30 cycles. B In silico analysis of the relative transcript levels of eIF6A (At3g55620) and eIF6B (At2g39820) in various tissue and organs in Arabidopsis. Data were imported from the Genevestigator Arabidopsis thaliana microarray database (https://www.genevestigator.ethz.ch/; Zimmermann et al., 2004).  141  Figure 5.9 eif6 mutant alleles. A. T-DNA insertion mutant alleles of eIF6A and eIF6B in Arabidopsis. The exons are depicted by boxes and the intron and intergenic regions are depicted by lines. The T-DNA insertion sites are drawn as triangular boxes (not to scale). LB, T-DNA left border. B. RT-PCR analysis of eif6b-1 and eif6b-2 alleles. C. The eif6a mutants are embryo lethal. Each pair of pictures is representative of the green seeds (top) and white seed (bottom) from the same silique. D. Three-week-old eif6b-1 and eif6b-2 mutant plants grown under 14 /10 h photoperiod.  142  5.3.6 rack1 mutants are hypersensitive to anisomycin, an inhibitor of protein translation Our co-expression analysis indicated that the majority of genes co-expressed with RACK1 encode ribosomal proteins (Figure 5.3 and Appendix 2), and we have shown that RACK1 physically interacts with eIF6 (Figure 5.5), a key protein regulating functional 80S ribosome assembly in mammals. We therefore sought additional evidence that might support a role of RACK1 in protein translation. Anisomycin is a drug that inhibits peptide bond formation, presumably by competing with amino acids for access to the peptidyltransferase centre (A-site, the entry point of amino acid-charged tRNA) (Meskauskas et al., 2005). This drug has been used in other eukaryotic cells to functionally implicate specific proteins in translation process (Nelson et al., 1992; Spence et al., 2000; Regmi et al., 2008), although no study of the effect of anisomycin in plants has been reported. When we used Arabidopsis primary root elongation as the metric to assay the effect of anisomycin on plant growth, we established that the halfmaximal inhibitory concentration (IC50) of anisomycin for root elongation is ~ 5 µM (Figure 5.10A), and that the growth of the primary root was completely halted by 15 µM anisomycin (Figure 5.10A). We then compared the sensitivity of wild-type and rack1 mutant plants to anisomycin. Because rack1a single mutants and rack1a rack1b and rack1a rack1c double mutants already displayed shorter primary root without any treatment (Guo and Chen, 2008), we used percentage of root reduction as a way to more precisely compare the relative sensitivity between each genotype to anisomycin. We found that rack1a single and rack1a rack1b and rack1a rack1c double mutants all displayed hypersensitivity to anisomycin (Figure 5.10A, B). rack1b rack1c double mutants also displayed hypersensitivity to anisomycin, but to a lesser extent (Figure 5.10B). Among all genotypes examined, the rack1a rack1b double mutants displayed the greatest hypersensitivity to anisomycin (Figure 5.10). While not definitive, these results are consistent with a role for RACK1 in protein translation in Arabidopsis.  143  Figure 5.10 The synergistic effect of anisomycin treatment and rack1 mutation on Arabidopsis seedling root growth. A. Root growth of rack1 single mutants in the present of 10 M anisomycin. B. Root growth of rack1 double mutants in the present of 5 M anisomycin. The experiments were repeated three times and the same data trends were obtained. Data from one experiment are presented here with the standard error (n=20) indicated on the top of each column. * indicates significant difference from that of Col using Student’s t-test (p<0.05).  144  5.3.7 RACK1 in functional 80S ribosomal subunit assembly and 60S ribosome biogenesis To assess the role of RACK1 in protein translation in vivo, we compared the polyribosome profile of extracts prepared from wild type (Col-0) and rack1a-1 rack1b-2 double mutant plants. This assay provides a relative measurement of efficiency in mRNA translation, as controlled by ribosome biogenesis and assembly (Lorsch, 2007). The profiling assay revealed a decrease in the abundance of both 60S ribosomal subunits and 80S monosomes (Figure 5.11A) in the rack1a rakc1b double mutant plants, compared with Col, but no significant difference was observed at the level of polysomes, indicative of a role for RACK1 in the reaction in which ribosome subunits join to initiate translation, as well as in ribosome subunit biogenesis.  5.3.8 ABA inhibits global protein translation  In view of the fact that RACK1 genes are negative regulators of ABA responses (Guo et al., 2009a), that our global gene expression profiling had revealed a convergent group of genes coregulated by both ABA and rack1 mutation (Figure 5.2), and that RACK1 appeared to be involved in ribosomal subunit assembly and 60S ribosome biogenesis (Figure 5.11A), we next asked whether ABA might also affect translation initiation in Arabidopsis. By using the ribosome profiling assay, we found that 50 µM ABA caused a dramatic reduction in the relative abundance of polysomes (Figure 5.11B). An increase in 80S monosome abundance was also observed, probably as a consequence of reduced progression into the elongation step (Naranda et al., 1997). These data agree with what was reported much earlier in soybean hypocotyls (Bensen et al., 1988), and support a model in which ABA plays a direct role in regulating protein translation.  145  Figure 5.11 Ribosome profiling of rack1a rack1b mutant and ABA-treated Arabidopsis seedlings. A. the overlay of the ribosome profiles of Col and rack1a rack1b mutant without ABA treatment. B. the overlay of the ribosome profiles of Col with or without ABA treatment. The position of 40S ribosomal subunits, 60S ribosomal subunits and 80S ribosomes was located based on the absorbance peaks at 260nm and is indicated with arrows. Profiles are average of four independent experiments with standard error bar. Shown is sucrose density gradient analysis of polysomes extracted from four and half-day old Col seedlings, with or without 50 µM ABA treatment for 8 h.  146  5.3.9 ABA regulates the expression of both RACK1 and eIF6 genes  Since both ABA and RACK1 appear to be involved in the regulation of protein translation (Figure 5.11A, B), we further investigated the functional relationship between ABA and RACK1 in these processes. A preliminary experiment had shown that ABA negatively regulates the expression of RACK1 genes (Guo et al., 2009a), leading us to hypothesize that ABA might regulate ribosome assembly and translation initiation through down-regulation of RACK1 genes. Using quantitative RT-PCR, we conducted a detailed analysis of the expression of RACK1 gene family members in response to ABA treatment. The level of transcripts for all three RACK1 genes was down-regulated as early as 1 h after ABA treatment, and remained suppressed thereafter (Figure 5.12A). Consistent with these findings, the activities of all three RACK1 gene promoters in the root tip were inhibited by ABA treatment (Figure 5.12B). We then extended our analysis to examine the possible regulation of eIF6 expression by ABA. We found that a reduction of eIF6A expression could be detected as early as 15 min after ABA treatment, and expression of eIF6A continued to decline for up to six hours (Figure 5.12A). Consistently, the three RACK1s and eIF6A were shown to be down-regulated by ABA treatment in DNA microarray data (data not shown). The expression of eIF6B gene was too low to be detected in seedlings used for qRT-PCR and DNA microarray assay. These results suggested that ABA might regulate translation initiation at least in part through regulation of expression of RACK1 and eIF6.  147  Figure 5.12 The regulation of RACK1 and eIF6 expression by ABA. A. Quantitative RT-PCR analysis of RACK1 and eIF6 gene expression. The transcript levels of RACK1 and eIF6A genes were normalized against the transcript level of ACTIN2 for each sample. Total RNA was extracted from four and half day old Arabidopsis seedlings and used for qRT-PCR analysis. The presented are the average of three biological replicates ±standard error. B. Promoter::GUS assay. Four and half-day old seedlings were incubated in ½ MS liquid medium with or without 50 µM ABA for 6 h and then subjected to GUS staining.  5.4 Discussion  To answer the question of how RACK1 genes are involved in ABA responses, we employed a combination of experimental approaches. First, by using global gene expression profiling, we detected a strong correlation between gene expression patterns invoked by ABA treatment and those associated with loss of function at the RACK1 loci. Second, yeast genetic complementation assays demonstrated that the function of RACK1 genes may be conserved across different kingdoms. Third, gene co-expression analysis provided evidence that RACK1’s function might be associated with the ribosome complex. Therefore, we specifically focused on  148  investigation of the role of RACK1 in protein translation as a candidate mechanism through which RACK1 might negatively regulate ABA responses. Four lines of evidence directly or indirectly support the idea that RACK1 regulates protein translation, and that these regulatory processes involve ABA. First, RACK1 physically interacts with eIF6 (Figure 5.5), a key regulator of ribosome assembly reaction of translation initiation in mammals. Second, rack1 mutants are hypersensitive to anisomycin (Figure 5.10), a protein translation inhibitor. Third, a decrease in the relative abundance of 60S ribosome subunits and 80S ribosome was observed in rack1a rakc1b plants (Figure 5.11A). And finally, ABA inhibits the expression of both RACK1 and eIF6 (Figure 5.12).  5.4.1 Arabidopsis RACK1 genes are functional equivalent to S. cerevisiae CPC2  RACK1 is a versatile scaffold protein that is involved in numerous signaling pathways and cellular processes in mammals and yeast (McCahill et al., 2002; Sklan et al., 2006; Guo et al., 2007). A few earlier studies have implied that Arabidopsis RACK1 may be a functional orthologue of mammalian RACK1 and yeast CPC2 gene. For example, the amino acid sequence of RACK1 is highly conserved between Arabidopsis and other taxa (Chen et al., 2006), as is the protein structure (Ullah et al., 2008). That proposed close relationship has been confirmed in the present study where Arabidopsis RACK1 was found to complement a genetic lesion at the yeast CPC2 locus (Figure 5.4). These results help provide a logical ground for utilizing the vast information available in the mammalian and yeast systems to probe the function of RACK1 in Arabidopsis. However, we are cautious that our findings do not exclude the possibility that some aspects of RACK1’s function are not conserved across different kingdoms. Indeed, the majority of the identified RACK1 interacting partners in mammals and yeast do not have obvious homologs in Arabidopsis (Guo et al., 2007). Even for those with obvious plant homologs, there is evidence that their interaction with RACK1 is not necessarily conserved in Arabidopsis. For example, RACK1 interacts with the β subunit of the heterotrimeric G-proteins and mediates a subset of the downstream signaling events in mammals (Dell et al., 2002; Chen et al., 2004b; Chen et al., 2004a; Chen et al., 2005). However, genetic and biochemical analyses indicate that RACK1 may not directly interact with G proteins in Arabidopsis (Guo et al., 2009b).  149  5.4.2 RACK1 is involved in the 60 ribosome subunit biogenesis and 80S functional ribosome assembly in Arabidopsis  RACK1’s multifaceted molecular function is mainly manifested via its physical interaction with many different signaling molecules (Guo et al., 2007). Significantly, RACK1 was repeatedly identified being associated with the ribosome in different species, using different approaches (Ceci et al., 2003; Shor et al., 2003; Gerbasi et al., 2004; Nilsson et al., 2004; Sengupta et al., 2004; Chang et al., 2005; Giavalisco et al., 2005; Manuell et al., 2005; Yu et al., 2005; Regmi et al., 2008; Coyle et al., 2009). Our co-expression data also indicated that RACK1 genes are coordinately regulated with many ribosome proteins encoding genes (Figure 5.3 and Appendix 2). These observations point to a conserved function of the RACK1 protein in its association with the ribosome complex. It has been proposed in other taxa that the function of RACK1 mostly directly related to its association with ribosome is its regulatory effect on translation initiation at the functional 80S ribosome assembly reaction (Ceci et al., 2003). In mammalian cells, this regulatory role involves RACK1’s interaction with activated PKC and eIF6 (Ceci et al., 2003). By using yeast two-hybrid assays and the BiFC assay, we showed that Arabidopsis RACK1 physically interacts with eIF6 (Figure 5.5). This conserved interaction between RACK1 and eIF6 likely mediates ribosome assembly, as do their counterparts in mammalian cells. In addition, we found that a translation inhibitor, anisomycin, displayed a synergistic effect with rack1 mutation in inhibiting root elongation (Figure 5.10). The significant role of RACK1 in protein translation regulation is also supported by the polysome profiling data, where the rack1 mutation caused reduced 60S ribosome subunit biogenesis and impaired 80S ribosome assembly (Figure 5.11A). Interestingly, RACK1 homolog in S. cerevisiae is also known to play a role in ribosome biogenesis (Shor et al., 2003). Consistent with such an essential contribution of the RACK1 genes to the translation process, and the potentially same essential contribution of the eIF6 genes, the rack1 triple mutant is seedling lethal (Guo and Chen, 2008) whereas the knock out mutant of eIF6A is embryo lethal (Figure 5.9C). Intriguingly, it has been demonstrated that the eIF6 gene is also involved in 60S ribosome biogenesis in yeast (Basu, 2001). It would be interesting to know whether such impaired 80S ribosome assembly and reduced ribosome subunits biogenesis can also be observed in eif6a knock-down mutants using RNAi technique. 150  In mammals, PKC plays an important role in the PKC-RACK1-eIF6 complex in regulating ribosome assembly. Although no apparent PKC orthologue has been found in plants, searching for other plant protein kinases (e.g. those possessing a C2 domain) that can phosphorylate eIF6 and interact with RACK1 might help identify a functional equivalent protein complex that regulates the same essential process in Arabidopsis.  5.4.3 ABA inhibits translation initiation  Plants are sessile and subject to constant biotic and abiotic stresses from the environment. ABA is one of the major phytohormones that regulate plant abiotic stress responses and it also plays a role in plant growth (Zhu, 2002). A global inhibition of translation under stress conditions has been recognized for some time (Kawaguchi et al., 2004). However, little is known about the signaling mechanism responsible for linking abiotic stress signaling, ABA signaling and the inhibition of translation. In the present study, we found that RACK1 genes, earlier identified as negative regulators of ABA responses (Guo et al., 2009a), are also important regulators of the protein translation (Figure 5.10, Figure 5.11A). In addition, the ribosome profiling assay provided direct evidence that ABA has an inhibitory effect on translation initiation in Arabidopsis seedlings. The reduced polysome levels and concomitant accumulation of 80S ribosomes in ABA-treated seedlings are indicative of inefficiency in entering the translation elongation stage (Figure 5.11B). Despite that the inhibitory effect of ABA on this reaction might not be mediated by RACK1 as can be seen from the difference in the ribosome profiles in rack1 mutant (Figure 5.11A) and in ABA-treated seedlings (Figure 5.11B), ABA might exert an inhibitory effect on the 60S ribosome biogenesis and the functional 80S ribosome assembly reaction of translation initiation via RACK1. This is supported by the finding that ABA inhibits the expression of RACK1 and eIF6 over an extended period (Figure 5.12), and that the “knockdown” mutant (rack1a rack1b) of the major RACK1 genes displayed characteristics of impaired 60S ribosome subunit biogenesis and 80S ribosome assembly (Figure 5.11A), and by the report of the function of RACK1 and eIF6 in these two process in other organisms (Basu et al., 2001; Ceci et al., 2003; Shor et al., 2003). These data together support the assumption that RACK1 might be a candidate molecular link between ABA signaling and its effect on ribosome biogenesis and global translation. An inhibitory effect of ABA on 80S ribosome assembly and 151  ribosome subunit biogenesis cannot be discerned in ribosome profiling assay (Figure 5.11B), This is probably because the relative mild reduction of 60S and 80S ribosome peaks were masked by the vast accumulation of 80S ribosomes and ribosome subunits resulting from ABA blocking the entry into the translation elongation phase. Consistent with our findings, protein translation initiation efficiency was found to be reduced in tobacco leaves subjected to drought stress (Kawaguchi et al., 2003), while in soybean, ABA treatment increased the level of polysomes in hypocotyl tissue (Bensen et al., 1988). In addition, an evolutionarily-conserved protein kinase TOR, which is known to regulate ribosome biogenesis in mammalian cells, is reported to have altered response to abiotic stress (Martin et al., 2004; Deprost et al., 2007). Nevertheless, in light of its multifaceted roles in mammal and yeast biology, we cannot rule out the possibility that RACK1 may mediate ABA responses indirectly through its involvement in other signaling pathways and cellular processes.  Taken together, our study supports a role of RACK1 in regulating 60S ribosome biogenesis and protein translation in Arabidopsis. This is the first cellular process in which RACK1 gene is reported to function in plants. 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My PhD work defined the relationship between the three RACK1 genes in the Arabidopsis genome, established a role for RACK1 in plant growth and development and ABA responses, defined a distinctive relationship between Arabidopsis RACK1 and the heterotrimeric G-proteins in contrast to their counterparts in mammalian cells and in yeast, and revealed an important role for RACK1 in 60S ribosome biogenesis. My results suggest that RACK1 acts as a molecular link between ABA and its regulation on protein translation.  6.1 Conclusions 6.1.1 Three Arabidopsis RACK1s influence plant growth and development and ABA responses in a manner of unequal genetic redundancy (Chapter 2 and Chapter 4)  Prior to this thesis study, among three Arabidopsis RACK1 genes, RACK1A, RACK1B and RACK1C, loss-of-function mutants had been reported for only RACK1A (Chen et al., 2006). In order to study the relationship between the three RACK1 genes, I isolated full-transcript-null alleles of RACK1B and RACK1C and generated double and triple mutants (Figure 2.2). Mutants of RACK1B and RACK1C, along with the rack1b rack1c double mutant, did not show any apparent defects in plant growth and development (Figure 2.3 and 2.4) or ABA responses (Figure 4.1A, 4.1B, 4.2 and 4.3). However, knocking out either of these two genes enhanced the growth and developmental defects (Figure 2.3 and 2.4), as well as the response to ABA, of rack1a mutants (Figure 4.1C, 4.1D, 4.2 and 4.3). This resembles the scenario of unequal genetic redundancy of homologous genes (Briggs et al., 2006). The unequal genetic redundancy could be caused by differences in the pattern of gene expression, their level of expression or the ability of genes to cross-regulate each other’s expression (Briggs et al., 2006). To test the mechanism underlying RACK1 gene unequal genetic redundancy, I first used RT-PCR analysis to check the expression of RACK1 genes. I found that all three RACK1s are expressed ubiquitously across different organs and different developmental stages (Figure 2.6), which ruled out the differential expression pattern as a candidate mechanism. In contrast, the transcript level of any given RACK1 gene was reduced in the single or double mutants for the other two RACK1 genes 159  (Figure 2.7), suggesting that cross-regulation of expression among the three RACK1 genes may be part of the mechanism for the observed unequal genetic redundancy. Significantly, RT-PCR and quantitative RT-PCR analysis revealed that both RACK1B and RACK1C were expressed at a lower level compared with that of RACK1A across different organs and developmental stages, with a trend of RACK1A>RACK1B>RACK1C (Figure 2.6 and 2.7). This supports the notion that RACK1 genes might be functionally equivalent to each other, and that the difference in the mutant phenotypes might be due to the different contribution of each RACK1 gene to the total cellular quantity of RACK1 protein. To further investigate this hypothesis, I did a genetic complementation experiment in which I over-expressed each of the three genes under control of the constitutive 35S promoter in the rack1a mutant background. I observed complementation of all the growth and developmental defects of rack1a mutant by over-expression of each of three RACK1 genes, indicating that RACK1B and RACK1C are in principle functionally equivalent to RACK1A (Figure 2.5). This functional equivalence was further supported by the later observation that all three Arabidopsis RACK1 genes were able to complement the yeast cpc2/rack1 mutant phenotype (Figure 5.4), that all three RACK1 proteins were able to physically interact with eukaryotic initiation factor 6 (eIF6) (Figure 5.5), and that the majority of the RACK1 co-expressed genes are those that co-expressed with all three RACK1 genes simultaneously (Figure 5.3). Supported by my genetic and molecular biological data, I categorized the three RACK1 genes as functional equivalents in my study, and treated the rack1a, rack1a rack1c and rack1a rack1b mutants as effectively the same as rack1 knock down mutants, in which total pool of RACK1 gene transcripts are depleted to different levels.  6.1.2 RACK1s function as negative regulators of ABA responses (Chapter 4)  ABA is one of the plant hormones to which the rack1a mutant displayed altered responses (Chen et al., 2006). In my study, I established that RACK1 is required for negative regulation of ABA responses through the following discoveries. First, I found that rack1b and rack1c mutations can enhance the effect of rack1a mutation on the plant’s response to ABA in seed germination, early seedling development as well as root development (Figure 4.1, 42. and 4.3). Second, I generated RACK1A over-expression lines and observed that overexpression of RACK1A confers plants’ resistance to ABA treatment (Figure 4.4). Third, I discovered that, in 160  addition to ABA, rack1 mutants also displayed altered sensitivity to salt stress and drought stress, indicating the involvement of RACK1 genes in mediating abiotic stresses responses (Figure 4.5, 4.10, 4.11, and 4.12). Intriguingly, a recent study in rice indicated a role for RACK1 in biotic stress responses (Nakashima et al., 2008). Both abiotic and biotic stresses are now known to incorporate ABA signaling pathways (Fujita et al., 2006). Fourth, in a DNA microarray analysis I conducted, I discovered that 41% of those genes that respond to ABA also respond to the rack1 mutation in a similar manner (Figure 5.2). This large percentage of shared molecular responses between the rack1 mutation and ABA treatment further suggests a role for RACK1 in ABA responses. After establishment of the negative influence of RACK1 on ABA responses, I looked into the potential molecular mechanisms that could explain the action of RACK1 in ABA responses.  6.1.3 Arabidopsis RACK1 and heterotrimeric G-protein complex interact in a mechanism that is distinct from their counterparts in mammalian cells and in yeast (Chapter 3)  Considering the scaffold protein property of RACK1 in mammalian cells, one of the plausible mechanisms by which RACK1 could influence ABA responses is through its physical interaction with other known regulators of ABA signaling pathway. Among the 60 physical interactors of RACK1 in mammalian cells and in yeast (Table 1.1), only four of them were identified to have highly-conserved homologs in Arabidopsis: eukaryotic initiation factor 6, protein phosphatase 2A, 14-3-3 protein and the heterotrimeric G-protein complex (Figure 1.7, 1.8, 1.9, and 1.10). I chose to start by investigating the relationship between RACK1 and heterotrimeric G-protein complex, for the reasons discussed below. Heterotrimeric G-protein complex is comprised of three subunits (α, β, and γ) and is involved in numerous signaling pathways both in mammals and in plants (Jones and Assmann, 2004). A close relationship between RACK1 and heterotrimeric G proteins in various signaling pathways has been reported. In mammalian cells, RACK1 has a physical interaction with the βγ dimer of the heterotrimeric G-protein, and this interaction regulates a specific set of downstream signaling partners of G-proteins (Chen et al., 2005). In yeast, RACK1 functions as a Gβ in glucose signaling (Zeller et al., 2007). In plants, RACK1 proteins are down-regulated in rice Gα 161  mutant embryos (Komatsu et al., 2005). Both RACK1 and AGB1’s amino acid sequences are highly conserved between Arabidopsis and mammals (Guo, 2007) and the heterotrimeric proteins in Arabidopsis have also been reported to act as negative regulators of ABA responses in seed germination and early seedling development (Assmann, 2002). To study the relationship between RACK1 and heterotrimeric G-protein, double mutants between RACK1A and AGB1 (β subunit), GPA1 (α subunit) were generated. Double mutant analysis indicated that the hypersensitivity of both gpa1-4 and agb1-2 mutants to ABA was enhanced by the rack1a-1 mutation. This additive effect was observed in an array of ABA sensitivity tests including seed germination, cotyledon greening and root elongation (Figure 3.1, 3.2 and 3.3). However, despite many attempts in different systems, a direct physical interaction between RACK1A and AGB1 could not be detected (Figure 3.5 and Figure 3.6). In addition, it was found earlier that GPA1 also has no physical interaction with RACK1 in Arabidopsis (Chen et al., 2006). These data suggest that the additive effect of rack1a, gpa1 and agb1 mutations on the plant’s response to ABA might not be manifested through the direct physical interaction between RACK1 and the two subunits of G-protein complex. Furthermore, rack1a mutants shared little morphological similarity with gpa1 and agb1 mutants (Figure 3.1), indicating that RACK1 and heterotrimeric G-protein complex might not work together in regulating plant morphological and developmental traits. RACK1 transcript and protein levels were downregulated in mature seeds of the gpa1-4 mutant, indicating that G-proteins might regulate the expression of RACK1. Consistent with these findings, Nakashima et al. (2008) found that RACK1 gene expression is regulated by Rac1 in rice, and because the heterotrimeric G proteins regulate innate immunity through Rac1 in rice, they proposed that G proteins might regulate RACK1 expression through Rac1. Interestingly, no cross-regulation was observed between RACK1 and G-proteins in four-and-half-day-old seedlings, suggesting a temporal pattern of interaction between these two genes. My studies of the relationship between Arabidopsis RACK1 and heterotrimeric Gprotein indicate that these two protein(s) (complexes) work independently in influencing ABA responses. My data do not support a direct physical interaction between these two protein(s) (complex), in contrast to their mammalian and yeast counterpart. In addition, they are probably not working together in regulating some of the morphological traits that we studied. The only interaction that was detected in this study is that heterotrimeric G-proteins regulate the expression of RACK1 in certain developmental stages. 162  While my studies did not find any physical association between the beta subunit (AGB1) of G-proteins and RACK1, my studies do not exclude the possibility that RACK1 and Gproteins may work together in a protein complex and interact indirectly vai other proteins. This deserves further investigation. 6.1.4 Arabidopsis RACK1s are involved in protein translation regulation and might serve as a molecular link between ABA signaling and its inhibitory effect on global protein translation (Chapter 5)  Abiotic stress and ABA have long been known to inhibit global protein translation (BenZioni et al., 1967; Aspinall, 1986; Kawaguchi and Bailey-Serres, 2002; Kawaguchi et al., 2003). However, the exact mechanism linking these processes is largely unknown. One of the important cellular processes RACK1 regulates in mammalian cells is the assembly of a functional 80S ribosome at the initiation stage of protein translation (Ceci et al., 2003). These data led me to look into whether the Arabidopsis RACK1 also plays roles in regulating protein translation and whether this function can account for its negative impact on ABA responses. Despite the observation that RACK1 protein is highly conserved across different kingdoms (Figure 1.4 and 1.5), whether Arabidopsis RACK1 is the functional orthologue of mammalian or yeast RACK1 had remained unknown prior to my study. I therefore conducted a genetic complementation assay using the S. cerevisiae cpc2/rack1 mutant and found that Arabidopsis RACK1 is indeed the genetic orthologue of S. cerevisiae CPC2/RACK1 gene (Figure 5.4). On the other hand, our co-expression analysis indicates a coordinated response of RACK1 gene and ribosome protein encoding genes to a variety of stimuli (Figure 5.3), providing evidence that Arabidopsis RACK1’s function might be closely related to its association with the ribosome complex. RACK1 has been repeatedly identified to be associated with ribosomes in different species using different approaches (Ceci et al., 2003; Shor et al., 2003; Gerbasi et al., 2004; Nilsson et al., 2004; Sengupta et al., 2004; Chang et al., 2005; Giavalisco et al., 2005; Manuell et al., 2005; Yu et al., 2005; Regmi et al., 2008; Coyle et al., 2009). These data together support a potentially evolutionarily-conserved function of RACK1 in its association with the ribosome. In an attempt to dissect the role of RACK1 in regulating protein translation in Arabidopsis, I took the following approaches. First, in mammalian cells, RACK1 regulates 80S 163  functional ribosome assembly via its physical interaction with activated protein kinase C (PKC) and eukaryotic initiation factor 6 (eIF6) (Ceci et al., 2003). While there are no obvious PKC orthologues in the Arabidopsis genome, two copies of eIF6 are present. Using a yeast twohybrid assay and bi-molecular complementation assays in combination with the leaf mesophyll protoplast transient expression system, I found that all three RACK1s have physical interactions with both eIF6 proteins (Figure 5.5). The conserved interaction between RACK1 and eIF6 might indicate a conserved role in translation, as found in mammalian cells. Unfortunately, but not surprisingly considering their potentially essential role in regulating protein translation machinery, a knock-out allele of the predominant copy of eIF6 gene, eIF6A, is embryo lethal. Obtaining a knock-down allele of eif6a could provide insight on the significance of the interaction to the regulation of protein translation. Second, using a pharmacological approach, I found a synergistic effect between the rack1 mutation and a protein translation inhibitor, anisomycin (Figure 5.10) on inhibition of primary root elongation. This also supports an involvement of RACK1 in protein translation. Third, I took a direct approach to test the effect of rack1 mutation on seedling ribosome profiles. A decrease in the relative abundance of 60S ribosome subunits and 80S ribosome was observed in rack1a rack1b plants, providing evidence of impaired ribosome assembly and subunit biogenesis in the rack1 mutant background (Figure 5.11A). Fourth, I discovered that ABA inhibits the expression of both RACK1 and eIF6 (Figure 5.12). These data support a scenario in which ABA inhibits expression of RACK1 and eIF6, and the reduced RACK1 (and potentially eIF6) level causes a reduction in 60S ribosome biogenesis and in assembled 80S functional ribosomes (Figure 5.11A). Therefore, ABA might manifest its effect on protein translation at the ribosome biogenesis and 80S ribosome assembly steps via inhibiting the expression of RACK1 genes. Another interesting discovery is that ABA also inhibits translation initiation at the entry point of translation elongation, based on its effect on ribosome profiles (Figure 5.11B). This process might not be mediated by RACK1, because rack1 mutation did not have an effect on translation initiation based on the ribosome profiling assays (Figure 6.1), but this interpretation needs to be further tested. This research project thus defined the first cellular process in which Arabidopsis RACK1 can be demonstrated to play a specific role. In addition, my data support an inhibitory effect of ABA on 60S ribosome biogenesis and functional 80S ribosome assembly, a process that might be mediated by RACK1.  164  Figure 6.1 A schematic presentation of the role of RACK1 in stress/ABA responses and protein translation regulation.  6.2 General discussion and future directions 6.2.1 What is the role of RACK1 in ABA responses?  Through extensive genetic, physiological and molecular analysis, I established that the RACK1 gene family acts to negatively mediate ABA responses. Considering the scaffold protein 165  property of RACK1, I first looked into the possibility that RACK1 mediates ABA responses via its interaction with other known modulators of ABA signaling. The first candidate interacting partner I chose was the heterotrimeric G-protein. The heterotrimeric G-protein complex mediates many aspects of ABA responses including seed germination, post-germination growth and stomatal movement (Wang et al., 2001; Pandey et al., 2006). The interaction between RACK1 and the heterotrimeric G-protein complex mediates specific aspects of G-protein signaling in both mammalian cells and in yeast (Chen et al., 2004; Zeller et al., 2007). However, despite the fact that an enhanced response to ABA which was observed in double mutants between rack1 and α or β subunit of G-proteins, a direct interaction of RACK1 with the subunits of heterotrimeric G-protein was not observed in my study. This observation might indicate that a direct interaction between RACK1 and heterotrimeric G-protein complex does not exist in plants, or that the interaction was too transient or weak to be detected in our assay. It would be interesting and informative to systematically test the interaction between RACK1 and other well established ABA signaling proteins such as ABI1, ABI2, ABI3, ABI4, and ABI5 to investigate whether RACK1 negatively regulates ABA responses via modulating the action of these ABA signaling proteins. In the meantime, considering the scaffold property of mammalian RACK1 protein and the pleiotropic phenotype of Arabidopsis rack1 mutants, we’re aware of the possibility that RACK1 might not be directly involved in ABA signaling and the ABA-related phenotypes might be the reflection of some indirect effect of ABA in some cellular processes or even other signaling pathways. Besides the inhibitory effect on seed germination and post-germination growth, another important physiological function of ABA is to regulate leaf stomatal opening and closing. Some ABA signaling modulating genes have been found to play distinct roles in regulating these two physiological processes. For example, gpa1, the mutant of the α-subunit of the heterotrimeric Gprotein, displayed hyposensitivity to ABA in the inhibition of stomata opening assay, whereas it was hypersensitive to ABA in the seed germination and early seedling development assays (Wang et al., 2001; Pandy et al., 2006). Therefore, it would be interesting to test whether and how RACK1 genes are involved in ABA-regulated stomatal opening and closing.  166  6.2.2 What is the role of RACK1 in protein translation?  Protein translation is subject to tight regulation in response to diverse stimuli and developmental cues (Brostrom and Brostrom, 1998; Yamasaki and Anderson, 2008). One of the well established roles of RACK1 in mammalian cells is its regulation of ribosome assembly (Ceci et al., 2003). The data obtained in my PhD study supports a role for RACK1 in protein translation in Arabidopsis. However, a direct assay of protein translation rate (such as S35 amino acid incorporation) in rack1 mutant and in ABA-treated Arabidopsis seedlings could provide more convincing evidence for the involvement of RACK1 in protein translation. In mammalian cells, the regulation of ribosome assembly involves the interaction of three proteins: RACK1, eukaryotic initiation factor 6 (eIF6) and protein kinase C (PKC). Because the full-transcript null allele of eIF6A is embryo lethal, an RNA interference approach could be used to knock down eIF6A gene at various levels to observe its role in protein translation. In addition, although there is no overt PKC homolog present in the Arabidopsis genome, it has been recognized that the plant AGC protein kinase family members (S6 kinase, IRE, NDR) have a PKC-like kinase domain2 (Bogre et al., 2003) and thus are prominent candidates for the functional orthologues of PKC. The interaction with RACK1, the ability to phosphorylate eIF6, and evidence for involvement in protein translation regulation could all be used as criteria for identifying which one(s) of the AGC kinases is (are) executing the role of PKC in regulating the protein translation process.  6.2.3 Is RACK1 one of the molecular links between stress signaling, ABA signaling and their effect on protein translation?  The plant is constantly subjected to various abiotic stresses from the environment. ABA is one of the major phytohormones mediating cellular responses to abiotic stress. Exogenous application of ABA affects the growth and development of plants in a way similar to what is observed under stress conditions, such as increased seed dormancy and reduced growth rate. Limiting cellular energy consumption is one of the plant’s adaptive strategies in order to survive through the nutrition-deficient period usually associated with stress conditions. Protein translation and related ribosome biogenesis are major cellular energy-consuming processes. It has been reported that both stress conditions, and ABA treatment lead to reduced protein 167  translation rate (Ben-Zioni et al., 1967; Aspinall, 1986; Kawaguchi and Bailey-Serres, 2002; Kawaguchi et al., 2003), but the exact molecular link between stress/ABA signaling and its effect on protein translation machinery is not clear. My study provided evidence consistent with the hypothesis that ABA inhibits global protein translation via its inhibitory effect on the expression of RACK1 (and probably eIF6) genes. In addition, RACK1 mediates both ABA responses and salt and drought stress responses. Thus, RACK1 might serve as one of the molecular links between stress signaling, ABA signaling and their effects on global protein translation (Figure 6.1, 6.2). However, I cannot present a definite conclusion yet. First, stress signaling is mediated by multiple phytohormones including ABA and ethylene (Page and Malcolm, 1997). Without testing the role of RACK1 in ethylene signaling, for example, I cannot conclude that ABA is the only hormone that mediates stress signaling by modulating the expression of RACK1 genes. Moreover, whether the degree of repression of RACK1 expression by ABA is sufficient to cause the reduction of ribosome biogenesis that was observed in the rack1a rack1b mutant has not been tested. One intriguing observation is that ectopic expression of RACK1A caused reduced sensitivity to ABA. It would certainly be informative to test whether the alleviated growth inhibition by ABA in RACK1A overexpression lines is directly related to the reduced inhibition of protein translation by ABA.  6.2.4 Other studies that could be done to better understand the function of RACK1  With the discoveries I made in my PhD study about the physiological, molecular and cellular function of Arabidopsis RACK1, along with studies done in other labs (Chang et al., 2005; Nakashima et al., 2008), the versatility of plant RACK1 is starting to be revealed. There are a few interesting and important aspects of RACK1’s function in Arabidopsis that could have been further addressed if time had allowed. One of the major mechanisms for the multifaceted function of RACK1 is via its physical interaction with other signaling proteins. Therefore, a genome-scale screening for the physical interacting partners of Arabidopsis RACK1 could potentially contribute significantly to our understanding of how RACK1’s function is manifested in various physiological responses at different developmental stages (Chen et al., 2006).  168  Figure 6.2 ABA signaling regulation of seed germination and early seedling development and the putative role of RACK1 in facilitating ABA responses. The pointed-end arrow indicates a directional signal transduction route that involves direct biochemical interaction (such as physical binding, phosphorylation etc.) or extrapolated through epistatic study. The blunt-end arrow indicates an inhibitory effect. ABI, ABA insensitive ; PP2Cs, protein phosphatase 2Cs; PYR, pyrabatin resistance; SnRK, sucrose non-fermenting kinase; ABF, ABA-responsive elements (ABRE) binding factors; ICK1, inhibitor of cyclin-dependent kinase 1; GTG, GPCR-type G protein; CHLH, H subunit of the magnesium-protoporphyrin-IX chelatase; Rop, plant-specific subfamily of RHO GTPase; CPK, calcium-dependent protein kinase; GCR, putative G-protein coupled receptor; GPA1, G-protein alpha subunit 1; AGB1, Gprotein beta subunit 1. RACK1, receptor for activated C-protein kinase 1. I discovered a role for the RACK1 gene in influencing plant growth and development, specifically in leaf production and root elongation (Chapter 2). An interesting preliminary observation is that the rack1 triple mutant does not possess visible root quiescent center (QC) cells. Since the QC cells act to maintain the stem cell identity of its surrounding stem cells (van den Berg et al., 1997), it would be interesting to examine the role of the RACK1 gene in root stem cell population maintenance. 169  6.4 References Aspinall D (1986). Metabolic effects of water and salinity stress in relation to expansion of the leaf surface. Funct Plant Biol 13: 59-73 Assmann SM (2002) Heterotrimeric and unconventional GTP binding proteins in plant cell signaling. Plant Cell 14 Suppl: S355-373 Ben-Zioni A, Itai C, and Vaadia Y (1967) Water and salt stresses, kinetin and protein synthesis in tobacco leaves. Plant Physiol 42: 361-365 Bogre L, Okresz L, Henriques R, Anthony RG (2003) Growth signaling pathways in Arabidopsis and the AGC protein kinases. Trends in Plant Science 8: 424-431 Brostrom CO, Brostrom MA (1998) Regulation of translational initiation during cellular responses to stress. Prog Nucleic Acid Res Mol Biol 58: 79-125 Briggs GC, Osmont KS, Shindo C, Sibout R, Hardtke CS (2006) Unequal genetic redundancies in Arabidopsis - a neglected phenomenon? Trends Plant Sci 11: 492-498 Ceci M, Gaviraghi C, Gorrini C, Sala LA, Offenhauser N, Marchisio PC, Biffo S (2003) Release of eIF6 (p27BBP) from the 60S subunit allows 80S ribosome assembly. Nature 426: 579-584 Chang IF, Szick-Miranda K, Pan S, Bailey-Serres J (2005) Proteomic characterization of evolutionarily conserved and variable proteins of Arabidopsis cytosolic ribosomes. Plant Physiol 137: 848-862 Chen JG, Ullah H, Temple B, Liang J, Guo J, Alonso JM, Ecker JR, Jones AM (2006) RACK1 mediates multiple hormone responsiveness and developmental processes in Arabidopsis. J Exp Bot 57: 2697-2708 Chen S, Spiegelberg BD, Lin F, Dell EJ, Hamm HE (2004) Interaction of Gbetagamma with RACK1 and other WD40 repeat proteins. J Mol Cell Cardiol 37: 399-406 Chen S, Lin F, Hamm HE (2005) RACK1 binds to a signal transfer region of G betagamma and inhibits phospholipase C beta2 activation. J Biol Chem 280: 33445-33452 Coyle SM, Gilbert WV, Doudna JA (2009) Direct link between RACK1 function and localization at the ribosome in vivo. Mol Cell Biol 29: 1626-1634 Fujita M, Fujita Y, Noutoshi Y, Takahashi F, Narusaka Y, Yamaguchi-Shinozaki K, Shinozaki K (2006) Crosstalk between abiotic and biotic stress responses: a current view from the points of convergence in the stress signaling networks. Curr Opin Plant Biol 9: 436-442 Gerbasi VR, Weaver CM, Hill S, Friedman DB, Link AJ (2004) Yeast Asc1p and mammalian RACK1 are functionally orthologous core 40S ribosomal proteins that repress gene expression. Mol Cell Biol 24: 8276-8287 Giavalisco P, Wilson D, Kreitler T, Lehrach H, Klose J, Gobom J, Fucini P (2005) High heterogeneity within the ribosomal proteins of the Arabidopsis thaliana 80S ribosome. Plant Mol Biol 57: 577-591 Guo J, Liang, J., Chen, J.G. (2007) RACK1: a versatile scaffold protein in plants? Int J Plant Dev Biol 1: 95-105  170  Jones AM, Assmann SM (2004) Plants: the latest model system for G-protein research. EMBO Rep 5: 572-578 Kawaguchi R, Bailey-Serres J (2002) Regulation of translational initiation in plants. Curr Opin Plant Biol 5: 460-465 Kawaguchi R, Williams AJ, Bray EA, Bailey-Serres J (2003) Water-deficit-induced translational control in Nicotiana tabacum. Plan Cell Environ 26: 221-229 Manuell AL, Yamaguchi K, Haynes PA, Milligan RA, Mayfield SP (2005) Composition and structure of the 80S ribosome from the green alga Chlamydomonas reinhardtii: 80S ribosomes are conserved in plants and animals. J Mol Biol 351: 266-279 McCahill A, Warwicker J, Bolger GB, Houslay MD, Yarwood SJ (2002) The RACK1 scaffold protein: a dynamic cog in cell response mechanisms. Mol Pharmacol 62: 12611273 Nakashima A, Chen L, Thao NP, Fujiwara M, Wong HL, Kuwano M, Umemura K, Shirasu K, Kawasaki T, Shimamoto K (2008) RACK1 functions in rice innate immunity by interacting with the Rac1 immune complex. Plant Cell 20: 2265-2279 Nilsson J, Sengupta J, Frank J, Nissen P (2004) Regulation of eukaryotic translation by the RACK1 protein: a platform for signaling molecules on the ribosome. EMBO Rep 5: 1137-1141 Regmi S, Rothberg KG, Hubbard JG, Ruben L (2008) The RACK1 signal anchor protein from Trypanosoma brucei associates with eukaryotic elongation factor 1A: a role for translational control in cytokinesis. Mol Microbiol 70: 724-745 Sengupta J, Nilsson J, Gursky R, Spahn CM, Nissen P, Frank J (2004) Identification of the versatile scaffold protein RACK1 on the eukaryotic ribosome by cryo-EM. Nat Struct Mol Biol 11: 957-962 Shor B, Calaycay J, Rushbrook J, McLeod M (2003) Cpc2/RACK1 is a ribosome-associated protein that promotes efficient translation in Schizosaccharomyces pombe. J Biol Chem 278: 49119-49128 Sklan EH, Podoly E, Soreq H (2006) RACK1 has the nerve to act: structure meets function in the nervous system. Prog Neurobiol 78: 117-134 Page WM, Malcolm CD (1997) Ethylene and plant responses to stress. Physiol Plantarum 100: 620-630 Pandey S, Chen J-G, Jones AM, Assmann SM (2006) G-Protein complex mutants are hypersensitive to abscisic acid regulation of germination and postgermination development. Plant Physiol. 141: 243-256 van den Berg C, Willemsen V, Hendriks G, Weisbeek P, Scheres B (1997) Short-range control of cell differentiation in the Arabidopsis root meristem. Nature 390: 287-289 Wang X-Q, Ullah H, Jones AM, Assmann SM (2001) G protein regulation of ion channels and abscisic acid signaling in Arabidopsis guard cells. Science 292: 2070-2072 Yamasaki S, Anderson P (2008) Reprogramming mRNA translation during stress. Curr Opin Cell Biol 20: 222-226 Yu Y, Ji H, Doudna JA, Leary JA (2005) Mass spectrometric analysis of the human 40S ribosomal subunit: native and HCV IRES-bound complexes. Protein Sci 14: 1438-1446 171  Zeller CE, Parnell SC, Dohlman HG (2007) The RACK1 ortholog Asc1 functions as a Gprotein beta subunit coupled to glucose responsiveness in yeast. J Biol Chem 282: 25168-25176 Zhu JK (2002) Salt and drought stress signal transduction in plants. Annu Rev Plant Biol 53: 247-273  172  APPENDIX 1A. Genes that are co-up-regulated by rack1 mutation and ABA treatment GeneID  Locus  TAIR6 Annotation  A203810_01  At3g14510  A202158_01 A201049_01 A201002_01 A000019_01 A203195_01 A203014_01 A200487_01 A203216_01 A009424_01 A200726_01 A001627_01 A202194_01 A200490_01 A001700_01 A013577_01 A202102_01 A004843_01 A008351_01 A202196_01  At4g07706 At1g76825 At1g69550 At1g12150 At5g36840 At5g28410 At1g21300 At5g38240 At3g15240 At1g45145 At1g68460 At4g08874 At1g21330 At1g64270 At4g16710 At4g04525 At1g05805 At2g37310 At4g08895  A200847_01 A202479_01 A203516_01 A204068_01 A202742_01 A200177_01  At1g55365 At4g33980 At5g27930 At5g36020 At5g20260 At5g43470  A203042_01 A005637_01 A013362_01 A200511_01 A011097_01 A203908_01 A006227_01 A201167_01 A202071_01 A201145_01 A200194_01 A201931_01 A001338_01 A011998_01 A203167_01 A000436_01 A200436_01 A200865_01 A201054_01  At5g28700 NA At4g37690 At1g23150 At3g30770 At4g03910 At2g30230 At2g07500 At4g01935 At2g06255 At1g01310 At3g43302 At1g12940 At3g24510 At5g35145 At1g03750 At1g18220 At1g56145 At1g76965  geranylgeranyl pyrophosphate synthase (GGPS3)(farnesyltranstransferase), putative En/Spm-related transposon protein translation initiation factor IF-2 -related disease resistance protein (TIR-NBS class), putative hypothetical protein hypothetical protein common family hypothetical protein hypothetical protein serine/threonine protein kinase, putative expressed protein thioredoxin H-type 5 (TRX-H-5) tR isopentenyl transferase -related hypothetical protein hypothetical protein expressed protein glycosyltransferase family 28 hypothetical protein bHLH protein pentatricopeptide (PPR) repeat-containing protein inorganic phosphate transporter (dbj|BAA34390.1) related hypothetical protein expressed protein protein phosphatase 2C (PP2C), putative hypothetical protein common family Exostosin family disease resistance protein (CC-NBS-LRR class), putative hypothetical protein EST transferase - related expressed protein hypothetical protein plant transposase (Ptta/En/Spm) family hypothetical protein Mutator-related transposase hypothetical protein expressed protein pathogenesis-related protein family retroelement pol polyprotein -related nitrate transporter -related hypothetical protein see GB:AF077408 -related helicase, putative hypothetical protein receptor protein kinase -related glycine-rich protein  mean_ratio_abABA0 _vs_Col-ABA0 49.06124596 27.43575244 25.40243741 24.90312486 24.27646736 24.25524986 22.10523983 21.78614175 21.43168687 20.08010639 16.99446054 16.40881648 13.98866541 13.05611132 13.02990346 12.90687442 12.82483313 12.66867052 12.65483121 12.45312238 12.20237308 12.1216635 12.03612768 12.0140197 11.92308017 11.83069553 11.65148847 11.43322106 11.23601411 10.67391764 10.55145531 10.33752082 9.971397661 9.746749525 9.673442649 9.454994264 9.357121204 9.197058251 9.055114877 9.042308691 8.935157284 8.754220707 8.691086812 8.673102743 8.577420487  173  GeneID  Locus  TAIR6 Annotation  A017412_01  At5g63930  A000373_01 A201323_01 A019520_01 A002122_01 A200457_01 A201743_01 A201070_01 A004775_01 A002053_01 A025246_01 A010098_01 A201149_01 A007007_01 A016532_01 A202978_01 A022570_01 A011675_01 A200058_01 A203383_01 A017492_01 A200426_01 A201887_01 A201932_01 A201640_01 A019564_01 A023918_01 A010146_01 A001806_01 A201830_01 A200945_01 A002861_01 A201913_01  At1g20770 At2g20625 At4g21900 At1g21880 At1g19460 At3g23295 At1g78360 At1g67790 At1g68610 At3g53640 At3g52260 At2g06555 At2g35350 At5g49470 At5g27845 At4g05240 At3g02350 At2g42200 At5g60400 At5g66940 At1g17545 At3g32010 At3g43355 At3g13062 At3g48330 At1g67760 At3g28470 At1g51540 At3g29570 At1g64560 At1g56010 At3g42535  leucine-rich repeat transmembrane protein kinase, putative expressed protein hypothetical protein MATE efflux protein family expressed protein Kelch repeat containing F-box protein family hypothetical protein glutathione transferase, putative F12A21.8 hypothetical protein protein kinase family pseudouridine synthase -related hypothetical protein expressed protein protein kinase non-LTR reverse transcriptase -related ubiquitin family glycosyltransferase family 8 squamosa-promoter binding protein -related expressed protein Dof zinc finger protein protein phosphatase 2C -related hypothetical protein see GB:AF077408 -related expressed protein protein-L-isoaspartate(D-aspartate) -related F12A21.11 myb family transcription factor Kelch repeat-containing protein hypothetical protein hypothetical protein C1 / No apical meristem (NAM) protein family Mutator-related transposase  A010142_01  NA  A018238_01 A000598_01 A202956_01 A203000_01 A008352_01 A201263_01 A202061_01 A202167_01 A203566_01 A200061_01 A203956_01 A019238_01 A021901_01  At5g11930 At1g66250 At5g27500 At5g28160 At2g37340 At2g14120 At4g01390 At4g07920 At3g18700 At2g45560 At4g09590 At5g59460 At5g15960  Arabidopsis thaliana D chromosome 3, BAC clone F26K9 glutaredoxin protein family glycosyl hydrolase family 17 hypothetical protein Kelch repeat containing F-box protein family splicing factor RSZ33, putative dynamin-related protein hypothetical protein hypothetical protein hypothetical protein cytochrome P450 family harpin-induced protein 1 family (HIN1) expressed protein stress-induced protein KIN1  mean_ratio_abABA0 _vs_Col-ABA0 8.559014466 8.414215786 8.399431412 8.390560304 8.375879268 8.333212368 8.321919466 8.305070304 8.267393914 8.20138873 8.168695684 8.04133405 7.771065657 7.653501398 7.581482753 7.544263112 7.51489931 7.485282917 7.379970237 7.360215568 7.274729719 7.271343085 7.154532437 7.096818052 6.818281288 6.814897697 6.770964663 6.575535474 6.518910395 6.488704908 6.465356184 6.464198014 6.399286149 6.36425436 6.229760618 6.221326254 6.220072193 6.19208625 6.165153986 6.019588485 5.919150582 5.904136934 5.88984698 5.865432812 5.843003202 5.828825299 5.802225222  174  GeneID  Locus  TAIR6 Annotation  A000626_01  At1g50610  A013387_01 A004879_01  At4g22420 NA  A025413_01 A201720_01 A203235_01 A013149_01 A000229_01  At2g28930 At3g21020 At5g39920 At4g28570 NA  A201486_01 A000672_01  At2g38465 NA  A203306_01 A203652_01 A020440_01  At5g50115 At1g37537 NA  A002653_01 A011621_01 A203746_01 A201239_01 A024572_01 A000698_01 A023907_01  At1g70390 At3g20550 At2g07776 At2g11270 At1g72320 At1g34260 NA  A202688_01 A010112_01 A203938_01 A006524_01  At5g19420 At3g29000 At4g07458 At2g37870  A203693_01 A200694_01 A200977_01 A201572_01 A203639_01 A001742_01 A022854_01  At1g54923 At1g42380 At1g67690 At3g03305 At1g34730 At1g03320 NA  A202725_01 A203734_01 A201198_01 A019574_01 A010115_01 A004926_01 A201880_01 A200528_01 A202883_01 A200670_01 A200690_01 A000572_01  At5g19970 At2g05890 At2g07706 At4g23980 At3g18524 At1g02010 At3g31403 At1g24010 At5g25560 At1g37037 At1g41860 At1g19430  leucine-rich repeat transmembrane protein kinase, putative hypothetical protein Genomic sequence for Arabidopsis thaliana BAC F22O13 from chromosome I protein kinase (APK1b) expressed protein hypothetical protein alcohol oxidase-related Genomic sequence for Arabidopsis thaliana BAC T32E20 from chromosome I expressed protein Arabidopsis thaliana chromosome I BAC F7A19 genomic sequence DnaJ protein-related conserved hypothetical protein Housekeeping 06, Arabidopsis thaliana ribosomal protein L32 (K11I1.2) mR F-box protein family expressed protein hypothetical protein citrate synthetase -related pumilio-family R-binding protein, putative expressed protein Genomic sequence for Arabidopsis thaliana BAC F18O14 from chromosome I expressed protein calcium-binding EF-hand family protein Athila retroelement ORF1 protein -related protease inhibitor/seed storage/lipid transfer protein (LTP) family hypothetical protein hypothetical protein F12A21.16 calcineurin-related phosphoesterase family hypothetical protein common family hypothetical protein Arabidopsis thaliana genomic D, chromosome 5, P1 clone:MIJ24 hypothetical protein hypothetical protein common family hypothetical protein auxin response transcription factor (ARF9) D mismatch repair protein MSH2 hypothetical protein mutator-related transposase hypothetical protein hypothetical protein hypothetical protein hypothetical protein dehydration-induced protein-related  mean_ratio_abABA0 _vs_Col-ABA0 5.771873827 5.744455076 5.658596017 5.525115746 5.520187011 5.514128859 5.486438891 5.433909718 5.38770308 5.347082551 5.279462435 5.27939913 5.253795151 5.240149188 5.217771995 5.194914461 5.028620189 5.010325238 5.004995729 4.997933691 4.988229474 4.975730786 4.928211926 4.915191826 4.908831273 4.886614827 4.878829986 4.862869372 4.857648136 4.829047897 4.827972401 4.823720855 4.794275118 4.729877069 4.724784665 4.716056182 4.678029139 4.664928171 4.62913321 4.62530253 4.592773451 4.573364705 4.552925189  175  GeneID  Locus  TAIR6 Annotation  A008600_01 A019514_01 A203049_01 A004917_01  At2g05420 At4g32240 At5g28790 At1g69190  A010106_01 A004073_01 A201087_01  At3g26780 At1g17690 At1g79780  A005838_01 A005759_01  At1g75770 NA  A203852_01 A000638_01 A014480_01 A008468_01 A203935_01 A200876_01 A202124_01 A023106_01  At3g31900 At1g29330 At4g03500 At2g15670 At4g07360 At1g59620 At4g06534 NA  A202623_01 A201947_01 A018332_01 A022137_01 A001861_01  At5g18530 At3g44716 At5g42900 At3g48720 At1g78510  A201278_01 A014555_01 A202954_01 A004004_01  At2g15700 At4g23310 At5g27460 NA  A023890_01  NA  A202284_01  At4g16095  A023642_01 A203518_01 A201885_01 A202610_01  At2g20550 At5g28340 At3g31910 At5g18350  A203724_01 A001555_01 A019454_01 A203132_01 A200702_01 A006394_01  At2g04770 At1g30740 At4g10250 At5g33395 At1g43245 NA  A200251_01 A201280_01  At1g05400 At2g16015  hypothetical protein expressed protein hypothetical protein dihydropterin pyrophosphokinase dihydropteroate synthase -related expressed protein expressed protein hypothetical integral membrane protein common family expressed protein ESTs, Highly similar to T48118 hypothetical protein F16M2.140 - Arabidopsis thaliana [A.thaliana] hypothetical protein ER lumen protein retaining receptor expressed protein hypothetical protein may be a pseudogene disease resistance protein (CC-NBS class), putative predicted protein Arabidopsis thaliana genomic D, chromosome 5, P1 clone:MJG14 WD-40 repeat protein family hypothetical protein expressed protein transferase family geranyl diphosphate synthase (GPPS)(dimethylallyltransferase), putative copia-related retroelement pol polyprotein receptor-like protein kinase, putative pentatricopeptide (PPR) repeat-containing protein Arabidopsis thaliana chromosome 1 BAC F6D8 sequence Genomic sequence for Arabidopsis thaliana BAC F4N2 from chromosome I disease resistance protein (CC-NBS-LRR class) related DnaJ protein family pentatricopeptide (PPR) repeat-containing protein hypothetical protein disease resistance protein (TIR-NBS-LRR class), putative plant transposase (Ptta/En/Spm) family FAD-linked oxidoreductase family endomembrane-localized small heat shock protein plant transposase (Ptta/En/Spm) family hypothetical protein Arabidopsis thaliana chromosome II section 253 of 255 of the complete sequence. Sequence from clones T8I13, T30B22 hypothetical protein hypothetical protein  *mean_ratio_abABA0 _vs_Col-ABA0 4.543085995 4.529830363 4.528599233 4.527800937 4.50331237 4.476236694 4.460059288 4.448213795 4.436961688 4.433859875 4.430473015 4.425004931 4.389606493 4.371632641 4.331708446 4.247245747 4.239674931 4.229130565 4.225601214 4.205645896 4.20402726 4.19895408 4.172168628 4.17140959 4.167186146 4.118453406 4.116300824 4.093465698 4.064400046 4.050400165 4.03912975 4.0215463 4.00649255 3.996046322 3.983964887 3.973411259 3.967615078 3.962749796 3.927688342 3.916632957  176  GeneID  Locus  TAIR6 Annotation  A019530_01  NA  A200518_01 A023167_01 A020057_01  At1g23450 NA NA  A001247_01 A200777_01 A200449_01 A202119_01 A201832_01 A200781_01 A012793_01 A025532_01 A203328_01 A009555_01  At1g50420 At1g50000 At1g18910 At4g06478 At3g29620 At1g50230 At3g27660 At1g20570 At5g54585 NA  A001385_01 A202010_01 A000703_01  At1g63450 At3g57790 NA  A011996_01 A202100_01 A201115_01 A203620_01 A203588_01 A203950_01 A013376_01 A004897_01  At3g09650 At4g04423 At2g03560 At1g21020 At5g25010 At4g08260 At4g30240 At1g53420  A025558_01 A000499_01  At1g26390 NA  A023311_01  NA  A202783_01  At5g20810  A202748_01  At5g20360  A021985_01 A201359_01 A003539_01 A013450_01  NA At2g24255 At1g35440 NA  A202420_01  At4g26900  A013460_01 A201114_01 A019522_01 A203854_01 A022855_01  At4g03130 At2g03505 At4g34040 At3g31960 At5g39560  Arabidopsis thaliana D chromosome 4, BAC clone T4L20 (ESSA project) pentatricopeptide (PPR) repeat-containing protein Arabidopsis thaliana BAC IG002N01 ESTs, Highly similar to T46189 calcium-dependent protein kinase - Arabidopsis thaliana [A.thaliana] scarecrow-like transcription factor 3 (SCL3) hypothetical protein hypothetical protein predicted protein hypothetical protein protein kinase family oleosin tubulin family expressed protein Arabidopsis thaliana genomic D, chromosome 3, P1 clone: MDC16 hypothetical protein polygalacturonase, putative Arabidopsis thaliana chromosome I BAC F28G4 genomic sequence pentatricopeptide (PPR) repeat-containing protein hypothetical protein F-box protein family FBX7 hypothetical protein common family hypothetical protein protein phosphatase 2C (PP2C), putative expressed protein leucine-rich repeat transmembrane protein kinase, putative FAD-linked oxidoreductase family Arabidopsis thaliana chromosome 1 BAC F25P22 genomic sequence Arabidopsis thaliana chromosome I BAC T6H22 genomic sequence auxin-induced (indole-3-acetic acid induced) protein, putative (SAUR_b) octicosapeptide/Phox/Bem1p (PB1) domain/tetratricopeptide repeat (TPR)-containing protein EST hypothetical protein cyclin family Arabidopsis thaliana D chromosome 4, contig fragment No. 7 imidazole glycerol phosphate synthase hisHF, chloroplast (bifunctional glutamine amidotransferase/cyclase) hypothetical protein glycosyl hydrolase family 17 expressed protein hypothetical protein expressed protein  *mean_ratio_abABA0 _vs_Col-ABA0 3.883820835 3.865043494 3.862245348 3.84652713 3.835229492 3.824705228 3.809657292 3.800952963 3.785219656 3.779625985 3.778315102 3.773632183 3.763508727 3.762201434 3.754508797 3.744584207 3.743618118 3.70383666 3.690429285 3.678077936 3.639494936 3.629141597 3.625367731 3.620580568 3.609535815 3.603375153 3.598564946 3.597091841 3.583484 3.57425476 3.572428453 3.534167516 3.517281375 3.493950284 3.483720201 3.461962712 3.461398404 3.439334392 3.430503466 3.416928061  177  GeneID  Locus  TAIR6 Annotation  A001083_01  NA  A202668_01 A204062_01 A202110_01 A025508_01  At5g19130 At5g35250 At4g05581 NA  A200862_01 A203473_01 A013326_01 A201507_01 A200200_01 A203186_01 A022498_01  At1g56085 At3g05675 At4g11550 At2g40720 At1g01670 At5g35775 NA  A201484_01 A203171_01 A004056_01 A203491_01 A200599_01 A203716_01  At2g38365 At5g35300 At1g51890 At3g47770 At1g31390 At2g01028  A025420_01 A022875_01  At2g38590 NA  A201814_01 A201875_01 A203866_01 A010407_01  At3g28958 At3g31320 At3g33448 NA  A013160_01 A200764_01 A203666_01 A201128_01 A200494_01 A200975_01 A200812_01 A005919_01  NA At1g48690 At1g43080 At2g04495 At1g21390 At1g67270 At1g53265 NA  A006393_01  NA  A202904_01 A203099_01 A203682_01 A202901_01 A203905_01 A201736_01 A201860_01 A203152_01 A203437_01  At5g26050 At5g31685 At1g47700 At5g25850 At4g03305 At3g22710 At3g30710 At5g34850 At1g14070  Arabidopsis thaliana D chromosome 3, BAC clone T20E23 GPAA1 - like protein hypothetical protein common family hypothetical protein Sequence of BAC F16P17 from Arabidopsis thaliana chromosome 1 hypothetical protein expressed protein CHP-rich zinc finger protein, putative pentatricopeptide (PPR) repeat-containing protein expressed protein retroelement pol polyprotein -related Arabidopsis thaliana D chromosome 4, BAC clone F25E4 (ESSA project) hypothetical protein hypothetical protein leucine rich repeat protein kinase, putative ABC transporter family protein hypothetical protein reverse transcriptase (R-dependent DNA polymerase) family protein expressed protein Arabidopsis thaliana genomic D, chromosome 5, P1 clone:MZA15 plastocyanin-like domain containing protein hypothetical protein hypothetical protein Arabidopsis thaliana genomic D, chromosome 3, BAC clone:F21A17 Arabidopsis thaliana BAC T4B21 auxin-regulated protein -related polygalacturonase, putative expressed protein expressed protein hypothetical protein hypothetical protein EST, Moderately similar to T51543 TOM (target of myb1)-like protein - Arabidopsis thaliana [A.thaliana] Arabidopsis thaliana chromosome II section 90 of 255 of the complete sequence. Sequence from clones F26H6 hypothetical protein predicted protein hypothetical protein F-box protein family transposase -related F-box protein family hypothetical protein calcineurin-like phosphoesterase family xyloglucan fucosyltransferase, putative  *mean_ratio_abABA0 _vs_Col-ABA0 3.406650417 3.397995901 3.397236226 3.385297342 3.372459381 3.353107606 3.329644402 3.327890082 3.32191303 3.320069479 3.316004342 3.315176248 3.302764249 3.289556536 3.281211277 3.274969453 3.268267859 3.26780139 3.262923438 3.261382922 3.256476248 3.248978341 3.248589183 3.241303363 3.232267466 3.219767391 3.210160853 3.206332475 3.196055229 3.183661406 3.178052649 3.167053668 3.16636179 3.165165927 3.143990778 3.138243591 3.133026972 3.132188164 3.09997158 3.084662193 3.083758162 3.079441433  178  GeneID  Locus  TAIR6 Annotation  A202545_01 A023711_01  At5g04290 NA  A016918_01 A201025_01 A200160_01 A201223_01 A200290_01 A019552_01 A022173_01 A201858_01 A203169_01 A018601_01 A203050_01 A201222_01 A204048_01 A202107_01 A203164_01 A200700_01 A200045_01 A202621_01 A003400_01  At5g05750 At1g73750 At5g42080 At2g08785 At1g07860 At4g27080 At3g47150 At3g30650 At5g35240 At5g59200 At5g28800 At2g08584 At5g34885 At4g05095 At5g35070 At1g43140 At2g24600 At5g18510 NA  A010832_01 A203819_01 A019411_01 A012638_01 A010230_01  At3g27070 At3g24982 At5g18250 At3g51280 NA  A000635_01 A203961_01 A203839_01 A201360_01 A202029_01 A203708_01 A006497_01 A001397_01  At1g16250 At4g10660 At3g30430 At2g24285 At3g62570 At1g64410 At2g41190 At1g21960  A201204_01 A004834_01  At2g07718 NA  A203407_01 A006841_01  At5g64905 NA  A020780_01  NA  A001730_01  At1g75050  A025580_01  NA  A203787_01  At2g15940  glycine-rich protein Genomic sequence for Arabidopsis thaliana BAC T10O22 from chromosome I DnaJ protein family expressed protein dynamin-related protein (pir||S59558) predicted protein expressed protein expressed protein F-box protein family hypothetical protein hypothetical protein pentatricopeptide (PPR) repeat-containing protein hypothetical protein predicted protein expressed protein reverse transcriptase -related hypothetical protein hypothetical protein expressed protein hypothetical protein Arabidopsis thaliana chromosome I BAC T14P4 genomic sequence TOM20 -related leucine rich repeat protein family, 5' fragment expressed protein male sterility MS5, putative Arabidopsis thaliana D chromosome 3, BAC clone F18P9 Kelch repeat containing F-box protein family hypothetical protein F-box protein family hypothetical protein DnaJ domain-containing protein hypothetical protein amino acid transporter family zinc finger (C3HC4-type RING finger) protein family expressed protein Sequence of BAC T20M3 from Arabidopsis thaliana chromosome 1 expressed protein Arabidopsis thaliana chromosome II section 65 of 255 of the complete sequence. Sequence from clones F23M2, T10J7, F24C20 Housekeeping 04, Arabidopsis thaliana putative ubiquitin protein (T23A1.5/At2g17190) mR thaumatin-like protein (pathogenesis-related protein), putative Arabidopsis thaliana BAC T1F9 chromosome 1, complete sequence Ac-related transposase  *mean_ratio_abABA0 _vs_Col-ABA0 3.073155714 3.056040668 3.051151998 3.046368897 3.024270202 3.016296189 3.014011666 2.999419489 2.991879129 2.988741521 2.986659308 2.983993339 2.972776745 2.970826975 2.96101742 2.947144222 2.945457822 2.944205514 2.935621473 2.924635823 2.924248448 2.910870325 2.886347125 2.879974495 2.865091244 2.858753892 2.844991722 2.844332256 2.836320032 2.832544095 2.825987466 2.822442676 2.808443667 2.80832626 2.797638415 2.79151142 2.785071269 2.765540847 2.756301869 2.736332726 2.7348632 2.722531977  179  GeneID  Locus  TAIR6 Annotation  A000654_01  NA  A008503_01 A002114_01 A204045_01 A202687_01 A200951_01 A006878_01 A023737_01  At2g31460 At1g10460 At5g34838 At5g19410 At1g65342 At2g26460 NA  A202955_01 A201295_01 A203157_01 A016544_01  At5g27490 At2g17490 At5g34875 NA  A019959_01 A203254_01 A018782_01 A015892_01 A014906_01 A009671_01  At1g30070 At5g42785 At5g23240 At5g58070 At4g25200 NA  A203057_01 A009017_01  At5g28885 NA  A002748_01 A003754_01 A019670_01 A200682_01  At1g03780 At1g75980 At3g20470 At1g40111  A200442_01 A022552_01  At1g18390 NA  A021821_01 A017388_01 A023462_01  NA At5g44390 NA  A021963_01  NA  A009681_01 A000492_01 A001088_01  At3g21150 At1g03530 NA  A005327_01 A201763_01 A202468_01  NA At3g24750 At4g33355  A200949_01 A002029_01 A200685_01  At1g65200 At1g47610 At1g40129  Arabidopsis thaliana chromosome I BAC F28N24 genomic sequence hypothetical protein germin-like protein (GLP7) hypothetical protein ABC transporter family protein hypothetical protein expressed protein Genomic sequence for Arabidopsis thaliana BAC F16A14 from chromosome I expressed protein retroelement pol polyprotein -related mutator-related transposase -related Arabidopsis thaliana genomic D, chromosome 5, P1 clone:MSK10 expressed protein hypothetical protein DnaJ protein family outer membrane lipo protein - like mitochondrion-localized small heat shock protein Arabidopsis thaliana genomic D, chromosome 3, TAC clone:K13E13 hypothetical protein Arabidopsis thaliana genomic D, chromosome 3, P1 clone:MKA23 expressed protein expressed protein glycine-rich protein polyprotein (gypsy_Ty3-element) [Sorghum bicolor] -related (GB:AAD19359) protein kinase family Arabidopsis thaliana D chromosome 4, contig fragment No. 6 EST FAD-linked oxidoreductase family Arabidopsis thaliana chromosome II section 114 of 255 of the complete sequence. Sequence from clones F3P11, F6F22 ESTs, Weakly similar to T05133 hypothetical protein F7H19.200 - Arabidopsis thaliana [A.thaliana] CONSTANS B-box zinc finger family protein expressed protein Arabidopsis thaliana chromosome 1 BAC F14O23 sequence ESTs hypothetical protein protease inhibitor/seed storage/lipid transfer protein (LTP) family hypothetical protein transducin / WD-40 repeat protein family predicted protein  *mean_ratio_abABA0 _vs_Col-ABA0 2.713929838 2.705169144 2.70079087 2.699854163 2.69359893 2.689822764 2.684437275 2.681809544 2.679052693 2.660873175 2.659099305 2.653188666 2.649869832 2.640898401 2.625875814 2.615708373 2.612863185 2.600317571 2.596723756 2.58118183 2.576730143 2.567036983 2.566631909 2.553416552 2.538387873 2.536789458 2.536291853 2.530607358 2.528521101 2.528421958 2.523527526 2.520990481 2.518996707 2.517925138 2.515002578 2.509925182 2.502692015 2.501344556 2.495122821  180  GeneID  Locus  TAIR6 Annotation  A201072_01 A200393_01 A004055_01  At1g78540 At1g14820 NA  A015353_01  At4g12150  A001809_01 A203738_01 A202258_01  At1g75240 At2g06620 At4g14905  expressed protein SEC14 cytosolic factor, putative Genomic sequence for Arabidopsis thaliana BAC F15H18 from chromosome I zinc finger (C3HC4-type RING finger) protein family expressed protein hypothetical protein F-box domain / Kelch motif protein  A006438_01  NA  A202832_01 A013947_01 A017556_01 A203632_01 A025146_01 A201940_01 A002799_01  At5g24850 At4g34310 At5g16860 At1g33000 At5g38010 At3g43686 NA  A200411_01 A018317_01 A024894_01  At1g16225 At5g63920 NA  A021509_01 A201935_01  At5g54700 At3g43546  A203206_01 A202616_01 A203667_01 A005555_01  At5g37210 At5g18450 At1g43205 NA  A024829_01 A005861_01 A017745_01 A005630_01 A011482_01 A001114_01  At3g53130 NA At5g40410 NA At3g20660 NA  A203096_01 A016556_01 A202481_01 A201943_01  At5g30470 At5g15540 At4g34131 At3g44035  A021967_01 A006552_01  NA NA  A006912_01 A201109_01  At2g44660 At2g02690  Arabidopsis thaliana chromosome II section 133 of 255 of the complete sequence. Sequence from clones F26B6 D photolyase - like protein hypothetical protein pentatricopeptide (PPR) repeat-containing protein hypothetical protein glucosyltransferase-related protein transposase-related Genomic sequence for Arabidopsis thaliana BAC F16A14 from chromosome I hypothetical protein D topoisomerase III Arabidopsis thaliana D chromosome 4, BAC clone F18E5 (ESSAII project) ankyrin repeat protein family reverse transcriptase (R-dependent DNA polymerase) family protein CHP-rich zinc finger protein, putative AP2 domain transcription factor, putative hypothetical protein Arabidopsis thaliana YABBY2 (YABBY2) mR, complete cds cytochrome p450 family ESTs pentatricopeptide (PPR) repeat-containing protein EST organic cation transporter family Sequence of BAC F14L17 from Arabidopsis thaliana chromosome 1 transposase-related expressed protein glycosyltransferase family reverse transcriptase (R-dependent DNA polymerase) family protein EST Arabidopsis thaliana chromosome II section 241 of 255 of the complete sequence. Sequence from clones T13E15, T14P1 ALG6, ALG8 glycosyltransferase family CHP-rich zinc finger protein, putative  *mean_ratio_abABA0 _vs_Col-ABA0 2.49171532 2.475312561 2.469965697 2.462957414 2.461570822 2.460309984 2.456290662 2.452614549 2.439372346 2.432865182 2.428813923 2.424985936 2.414189711 2.413465425 2.41303887 2.412140531 2.399443212 2.397143647 2.39166301 2.387394054 2.377866829 2.377846302 2.374105397 2.371386968 2.366562637 2.363781219 2.363337826 2.350593738 2.349652053 2.347573608 2.3458773 2.337184533 2.337158511 2.331489283 2.323865334 2.322246565 2.322127185 2.315178987  181  GeneID  Locus  TAIR6 Annotation  A013415_01 A200920_01 A002956_01  At4g32450 At1g62820 At1g72840  A201460_01 A203371_01 A008490_01 A201739_01 A005097_01  At2g35795 At5g59330 At2g28440 At3g23172 NA  A010212_01 A202796_01 A013478_01 A024083_01  At3g60510 At5g21050 At4g04220 NA  A020780_01  NA  A007509_01 A200974_01 A005059_01 A200334_01 A203070_01 A200823_01 A005650_01 A002073_01 A022491_01  At2g43000 At1g67240 NA At1g10155 At5g29028 At1g53790 NA At1g47128 NA  A019516_01 A010093_01 A005632_01 A005320_01 A026086_01  At4g17870 At3g62140 NA NA At1g67840  A006501_01 A012197_01  At2g07520 NA  A007203_01 A201432_01 A014198_01 A024132_01  At2g38070 At2g33670 At4g37400 NA  A200653_01 A201334_01  At1g35750 At2g21100  A203560_01 A001406_01  At2g24880 NA  A200192_01 A000699_01  At1g01190 NA  A202012_01 A006528_01  At3g58690 At2g37840  pentatricopeptide (PPR) repeat-containing protein calmodulin, putative disease resistance protein (TIR-NBS-LRR class), putative DnaJ domain-containing protein hypothetical protein proline-rich protein family predicted protein Housekeeping 11, Arabidopsis thaliana putative acyl-coA dehydrogenase G6p (AtG6) mR enoyl-CoA hydratase/isomerase family expressed protein disease resistance protein family Arabidopsis thaliana chromosome III BAC F10A16 genomic sequence Housekeeping 04, Arabidopsis thaliana putative ubiquitin protein (T23A1.5/At2g17190) mR No apical meristem (M) protein family mutator-related transposase EST hypothetical protein plant transposase (Ptta/En/Spm) family F-box protein family EST cysteine proteinase RD21A Arabidopsis thaliana D chromosome 4, BAC clone F27B13 (ESSA project) expressed protein hypothetical protein EST ESTs ATP-binding region, ATPase-like domaincontaining protein hypothetical protein Arabidopsis thaliana genomic D, chromosome 3, P1 clone:MIL15 expressed protein seven transmembrane MLO protein family (MLO5) cytochrome P450 family Arabidopsis thaliana chromosome 1 BAC F28P22 genomic sequence pumilio-family R-binding protein, putative disease resistance response protein-related/ dirigent protein-related self-incompatibility protein-related Genomic sequence for Arabidopsis thaliana BAC F10B6 from chromosome I cytochrome P450, putative Arabidopsis thaliana chromosome I BAC F14M2 genomic sequence protein kinase family protein kinase family  *mean_ratio_abABA0 _vs_Col-ABA0 2.309860039 2.309474244 2.304694637 2.29613192 2.295169087 2.290643367 2.288620376 2.278495369 2.276026173 2.27212619 2.264665892 2.258515691 2.243097887 2.242763084 2.240419215 2.233788014 2.233625215 2.225419893 2.225336463 2.224634268 2.222701356 2.211102346 2.20432789 2.190999867 2.188477024 2.179589009 2.178507678 2.178114402 2.177406982 2.176327989 2.164821094 2.164372716 2.158817803 2.156862707 2.151303936 2.150146598 2.133055063 2.12890099 2.126803456 2.126438352 2.117788412  182  GeneID  Locus  TAIR6 Annotation  A015473_01  NA  A008229_01 A202701_01 A202268_01 A005180_01  At2g46735 At5g19650 At4g15260 NA  A021358_01  NA  A021562_01  NA  A019392_01 A001506_01  At5g17480 NA  A022352_01  NA  A022084_01 A201392_01 A005443_01 A201714_01 A021852_01  At2g06640 At2g28725 NA At3g20555 NA  A202195_01  At4g08878  A002326_01 A200230_01 A000575_01 A005929_01 A005203_01  At1g48390 At1g03055 At1g09350 NA NA  A019780_01 A015122_01  At3g24310 At4g39050  Arabidopsis thaliana D chromosome 4, BAC clone F20M13 (ESSA project) expressed protein hypothetical protein UDP-glycosyltransferase family Housekeeping 02, Arabidopsis thaliana actin (ACT1) gene Arabidopsis thaliana genomic D, chromosome 3, BAC clone:F16J14 Arabidopsis thaliana chromosome I BAC F7A19 genomic sequence polcalcin (calcium-binding pollen allergen), putative Arabidopsis thaliana chromosome 1 BAC F3A14 genomic sequence Arabidopsis thaliana D chromosome 4, BAC clone F6I18 (ESSA project) hypothetical protein hypothetical protein EST hypothetical protein Arabidopsis thaliana putative transcription factor (MYB75) mR, complete cds inorganic phosphate transporter (dbj|BAA34390.1) related Arabidopsis hypothetical common family expressed protein glycosyltransferase family 8 EST Housekeeping 09, Arabidopsis thaliana heat shock protein 70 (Hsc70-7) mR myb family transcription factor kinesin-related protein  A017678_01 A014657_01 A000523_01 A010530_01 A201357_01 A019603_01 A200638_01  At5g51480 At4g11670 At1g03080 At3g26010 At2g23945 At5g01670 At1g34490  A005097_01  NA  A200879_01 A008163_01 A012264_01 A021020_01 A201315_01 A020478_01 A023303_01  At1g59680 At2g16730 At3g08990 NA At2g20430 At5g50320 NA  pectinesterase (pectin methylesterase) family expressed protein expressed protein F-box protein family hypothetical protein aldose reductase, putative long-chain-alcohol O-fatty-acyltransferase (wax synthase) family Housekeeping 11, Arabidopsis thaliana putative acyl-coA dehydrogenase G6p (AtG6) mR F-box protein family glycosyl hydrolase family 35 (beta-galactosidase) Yippee-related protein EST hypothetical protein GCN5-related N-acetyltransferase (GT) family Arabidopsis thaliana chromosome 1 BAC F28J9 sequence  *mean_ratio_abABA0 _vs_Col-ABA0 2.11625995 2.108574755 2.096890203 2.096628395 2.095226049 2.094337617 2.093409768 2.086590249 2.081803778 2.075743324 2.074204573 2.064612586 2.062776343 2.062625328 2.060981747 2.055353628 2.054933112 2.054683036 2.053923131 2.052688341 2.043181052 2.040131407 2.039968781 2.036231902 2.03619534 2.026607483 2.02625184 2.019673297 2.016815527 2.01544491 2.014388789 2.012016283 2.009471741 2.006878188 2.006523528 2.004563685 2.004318424 2.00279807  183  GeneID  Locus  TAIR6 Annotation  A023705_01  NA  A203546_01  At1g56650  Sequence of BAC T3F20 from Arabidopsis thaliana chromosome 1 myb-related protein anthocyanin2, putative  *mean_ratio_abABA0 _vs_Col-ABA0 2.00190809 2.001888078  * The genes whose expression level was co-up-regulated by both ABA and rack1 mutation in comparison to Col to two times or higher were listed in this table.  184  APPENDIX 1B. Genes that are co-down-regulated by rack1 mutation and ABA treatment GeneID  Locus  TAIR6 Annotation  *mean_ratio_abABA0_vs_ColABA0  A203273_01  At5g45573  hypothetical protein  0.03395319  A203178_01  At5g35603  Drosophila suppressor of sable protein  0.037277732  (GB:M57889) -related A203591_01  At5g28960  hypothetical protein  0.041517822  A019525_01  At4g35040  bZIP protein  0.047161595  A200970_01  At1g67000  serine/threonine protein kinase, putative  0.050997456  A004823_01  At1g36950  zinc finger protein -related  0.05251902  A019824_01  At2g19350  expressed protein  0.053453715  A202988_01  At5g27980  embryonic abundant protein - like  0.057106009  A200906_01  At1g61667  expressed protein  0.058945772  A200410_01  At1g16130  WAK-like kinase (WLK)  0.05911999  A019389_01  At5g39330  hypothetical protein  0.071068708  A001699_01  At1g64390  glycosyl hydrolase family 9 (endo-1,4-beta-  0.072395731  glucanase) A203095_01  At5g30450  plant transposase (Ptta/En/Spm) family  0.076522641  A201451_01  At2g35280  F-box protein family  0.077696054  A203755_01  At2g10740  Athila retroelement ORF1 protein -related  0.080973636  A203522_01  At5g34825  transposon protein -related  0.081037425  A007199_01  At2g37820  CHP-rich zinc finger protein, putative  0.08182031  A203329_01  At5g54620  ankyrin-repeat-containing protein-related  0.083556761  A019521_01  At4g21190  pentatricopeptide (PPR) repeat-containing protein  0.086304951  A004686_01  At1g65450  anthranilate N-  0.08879725  hydroxycinnamoyl/benzoyltransferase - related A009450_01  At3g08770  lipid transfer protein 6 (ltp6)  0.091470737  A204072_01  At5g36690  hypothetical protein  0.092079076  A010215_01  At3g28330  hypothetical protein  0.092981766  A200211_01  At1g02210  hypothetical protein  0.093602775  A203740_01  At2g06730  En/Spm-related transposon protein  0.094089912  A009419_01  At3g55960  expressed protein  0.094621233  A203058_01  At5g28890  hypothetical protein  0.095299104  A000686_01  At1g59820  haloacid dehalogenase-like hydrolase family  0.095513304  A010152_01  At3g45320  hypothetical protein  0.095618709  185  GeneID  Locus  TAIR6 Annotation  *mean_ratio_abABA0_vs_ColABA0  A006326_01  At2g37030  auxin-induced (indole-3-acetic acid induced)  0.095695461  protein family A200301_01  At1g08150  cation/hydrogen exchanger -related (CHX5)  0.099382873  A005851_01  At5g02020  expressed protein  0.09998062  A025117_01  At3g20110  cytochrome P450 family  0.100045368  A000658_01  At1g77580  expressed protein  0.10614979  A203841_01  At3g30540  beta-mannan endohydrolase -related  0.110565503  A200729_01  At1g45207  conserved hypothetical protein  0.110775251  A202303_01  At4g16740  monoterpene synthase/cyclase family  0.11112787  A202858_01  At5g25290  F-box protein family  0.11143212  A203954_01  At4g08890  hypothetical protein common family  0.111571165  A001822_01  At1g09320  expressed protein  0.112714063  A021396_01  At5g38930  germin-like protein, putative  0.113116263  A202085_01  At4g03415  protein phosphatase 2C (PP2C) -related  0.115155627  A202068_01  At4g01780  hypothetical protein  0.115420847  A203628_01  At1g29620  hypothetical protein  0.117856549  A004990_01  NA  EST  0.121201771  A003955_01  NA  Arabidopsis thaliana chromosome I BAC T10P12  0.121432423  genomic sequence A202998_01  At5g28140  hypothetical protein  0.12223825  A200414_01  At1g16830  pentatricopeptide (PPR) repeat-containing protein  0.122391045  A200269_01  At1g06660  expressed protein  0.126664902  A200696_01  At1g42515  retroelement pol polyprotein -related  0.128145876  A001611_01  At1g25540  expressed protein  0.132258919  A013347_01  At4g23470  proline-rich protein family  0.132324842  A013017_01  At4g37420  hypothetical protein  0.133604822  A010205_01  At3g54790  armadillo repeat containing protein  0.137409472  A006109_01  NA  Arabidopsis thaliana putative transcription factor  0.137546695  (MYB88) mR, complete cds A200493_01  At1g21370  expressed protein  0.138406975  A200608_01  At1g32600  F-box protein-related  0.139636145  A201434_01  At2g33775  expressed protein  0.140481813  A202397_01  At4g23496  expressed protein  0.141122603  A000664_01  At1g19290  pentatricopeptide (PPR) repeat-containing protein  0.141310808  A202497_01  At4g35620  cyclin 2b (cyc2b)  0.143892086  A014319_01  At4g29500  X-Pro dipeptidase - like protein (fragment)  0.144083718  186  GeneID  Locus  TAIR6 Annotation  *mean_ratio_abABA0_vs_ColABA0  A203640_01  At1g34910  hypothetical protein  0.144568321  A017018_01  At5g40970  expressed protein  0.145029865  A013186_01  At4g24450  expressed protein  0.146750521  A024988_01  At4g04540  receptor-related protein kinase  0.146822493  A002625_01  At1g79260  expressed protein  0.146965201  A025295_01  NA  Arabidopsis thaliana putative transcription factor  0.148197685  (MYB90) mR, complete cds A203452_01  At1g51830  leucine rich repeat protein kinase, putative  0.149486495  A013339_01  At4g35080  expressed protein  0.150496395  A203406_01  At5g64810  WRKY family transcription factor  0.152386963  A012870_01  At4g37780  myb D-binding protein (AtMYB87)  0.152972775  A202580_01  At5g10540  oligopeptidase A - like protein  0.153963848  A024238_01  NA  Arabidopsis thaliana D chromosome 3, BAC clone  0.156482988  T10K17 A201985_01  At3g50290  hydroxycinnamoyl benzoyltransferase-related  0.157390292  A022723_01  At3g23450  glycine-rich protein  0.157465777  A202788_01  At5g20880  expressed protein  0.157750807  A202751_01  At5g20390  glycosyl hydrolase family 17 (beta-1,3-glucanase)  0.159284809  A010140_01  NA  Arabidopsis thaliana genomic D, chromosome 3,  0.159393163  P1 clone: MSD21 A203757_01  At2g12100  Ulp1 protease family  0.160200301  A004078_01  NA  Genomic sequence for Arabidopsis thaliana BAC  0.160703772  T2E6 from chromosome I A008215_01  At2g07040  leucine-rich repeat transmembrane protein kinase,  0.162473517  putative A201010_01  At1g70910  zinc finger (C3HC4-type RING finger) protein  0.164794716  family A202943_01  At5g27240  DnaJ domain-containing protein  0.165153795  A203863_01  At3g33064  hypothetical protein  0.166389421  A202403_01  At4g24010  cellulose synthase - related  0.171869234  A000648_01  At1g77600  expressed protein  0.172431473  A203263_01  At5g44345  F-box protein-related  0.176626988  A001923_01  At1g13130  glycosyl hydrolase family 5/cellulase  0.179674325  A002937_01  At1g34580  monosaccharide transporter, putative  0.180658836  A202640_01  At5g18770  F-box protein family  0.182293723  187  GeneID  Locus  TAIR6 Annotation  *mean_ratio_abABA0_vs_ColABA0  A201063_01  At1g77860  membrane protein, Rhomboid family  0.184168808  A011537_01  At3g49820  hypothetical protein  0.185308816  A200151_01  At5g25475  expressed protein  0.185765671  A003204_01  At1g60050  nodulin MtN21 - related  0.187409534  A203161_01  At5g34970  hypothetical protein common family  0.187591511  A007694_01  At2g39370  expressed protein  0.189511386  A200536_01  At1g25570  leucine rich repeat protein-related  0.189669154  A202104_01  At4g04745  expressed protein  0.189689502  A005853_01  At5g28900  protein phosphatase 2A regulatory subunit B-related  0.190061601  protein A013138_01  At4g36140  disease resistance protein (TIR-NBS-LRR class),  0.190746154  putative A200222_01  At1g02590  aldehyde oxidase -related  0.190788477  A015964_01  At5g24470  pseudo-response regulator, APRR5 (APRR1/TOC1  0.191229284  family) A203553_01  At2g04037  hypothetical protein  0.191687929  A010137_01  At3g05890  low temperature and salt responsive protein  0.192110784  (LTI6B) A203335_01  At5g55135  hypothetical protein  0.192425568  A003702_01  At1g44900  D replication licensing factor, putative  0.194467442  A201995_01  At3g52670  F-box protein family  0.194608743  A001532_01  At1g32740  expressed protein  0.196639779  A020856_01  NA  EST, Moderately similar to T06010 hypothetical  0.197548726  protein T25K17.70 - Arabidopsis thaliana [A.thaliana] A202680_01  At5g19300  hypothetical protein  0.199313998  A019390_01  At5g17460  expressed protein  0.20009585  A010279_01  At3g50050  expressed protein  0.200675425  A203497_01  At4g11850  phospholipase D-gamma, putative  0.201603229  A203747_01  At2g08986  predicted protein  0.202691131  A002813_01  NA  Arabidopsis thaliana chromosome I BAC F9H16  0.204348153  genomic sequence A200497_01  At1g21580  proline-rich protein-related  0.204991816  A202326_01  At4g17695  myb family transcription factor  0.205201363  A017364_01  At5g03790  homeodomain protein  0.205901314  A003464_01  At1g56590  clathrin-associated protein -related  0.206535696  188  GeneID  Locus  TAIR6 Annotation  *mean_ratio_abABA0_vs_ColABA0  A025625_01  NA  Arabidopsis thaliana chromosome 1 BAC T8F5  0.206617745  sequence A200574_01  At1g29490  auxin-induced (indole-3-acetic acid induced)  0.207910714  protein family A201819_01  At3g29050  receptor-like protein kinase-related  0.209119627  A005516_01  At1g23000  heavy-metal-associated domain-containing protein  0.210846162  A007202_01  At2g38060  transporter - related  0.21160742  A006943_01  NA  Arabidopsis thaliana chromosome II section 238 of  0.212017035  255 of the complete sequence. Sequence from clones F4I1 A202430_01  At4g28005  hypothetical protein  0.212396897  A201775_01  At3g25720  hypothetical protein  0.212809519  A200874_01  At1g58602  disease resistance protein (CC-NBS-LRR class),  0.21333869  putative A003269_01  At1g01370  histone family  0.213783064  A200798_01  At1g51920  hypothetical protein  0.215733729  A025750_01  At2g45510  cytochrome p450, putative  0.215778432  A009613_01  At3g15700  hypothetical protein  0.216187693  A005825_01  NA  EST  0.216549183  A200420_01  At1g17350  auxin-induced (indole-3-acetic acid induced),  0.216969389  pseudogene A023669_01  At2g04300  leucine rich repeat protein kinase, putative  0.218051538  A013126_01  At4g34360  protease-related  0.21818824  A015338_01  At4g17760  expressed protein  0.218464655  A203449_01  At1g35920  hypothetical protein common family  0.219140061  A013114_01  At4g17170  GTP-binding protein (At-RAB2)  0.220029543  A005626_01  NA  EST  0.2217039  A009854_01  At3g54940  cysteine proteinase  0.222424919  A000390_01  At1g56160  myb family transcription factor  0.225910872  A200892_01  At1g60970  coatomer zeta subunit -related  0.225931101  A202480_01  At4g33990  pentatricopeptide (PPR) repeat-containing protein  0.22685252  A025562_01  NA  Arabidopsis thaliana chromosome I BAC T14L22  0.229844357  genomic sequence A204024_01  At5g30520  hypothetical protein  0.230006706  A202035_01  At3g63530  zinc finger (C3HC4-type RING finger) protein  0.230297152  family  189  GeneID  Locus  TAIR6 Annotation  *mean_ratio_abABA0_vs_ColABA0  A203326_01  At5g54064  receptor protein kinase-related protein  0.231359349  A020396_01  NA  Arabidopsis thaliana serine/threonine protein  0.231384101  phosphatase (MOJ9.27) mR, complete cds A010072_01  At3g06180  expressed protein  0.231583562  A200928_01  At1g63480  D-binding protein -related  0.232034662  A204078_01  At5g37330  hypothetical protein  0.232240341  A202249_01  At4g14670  heat shock protein 101 (HSP101),putative  0.233023698  A202657_01  At5g18990  pectinesterase family  0.237250333  A010223_01  At3g63280  protein kinase family  0.237291947  A002563_01  NA  Arabidopsis thaliana chromosome I BAC T5A14  0.237860641  A200365_01  At1g12530  hypothetical protein  0.237919245  A010306_01  At3g12090  senescence-associated protein family  0.239275867  A007413_01  At2g30340  lateral organ boundaries (LOB) domain protein 13  0.240175786  (LBD13) A019868_01  At2g36050  expressed protein  0.241072278  A203474_01  At3g09170  Ulp1 protease family  0.241897047  A006919_01  At2g46950  cytochrome P450 family  0.242850709  A002429_01  At1g19750  transducin / WD-40 repeat protein family  0.243696729  A200597_01  At1g31335  expressed protein  0.243789477  A010259_01  At3g57240  glycosyl hydrolase family 17 (beta-1,3-glucanase  0.245090157  bg3) A020736_01  NA  Arabidopsis thaliana putative ABC transporter  0.246417506  protein (MCK7.14/AT5g58270) mR, complete cds A200041_01  At2g18876  expressed protein  0.246761914  A202419_01  At4g26555  peptidylprolyl isomerase, putative  0.24698646  A010248_01  At3g59680  expressed protein  0.248048525  A200162_01  At5g44930  Exostosin family  0.248311894  A200349_01  At1g11290  pentatricopeptide (PPR) repeat-containing protein  0.248342732  A200830_01  At1g54230  hypothetical protein  0.248520698  A006371_01  At2g34380  expressed protein  0.249309075  A008275_01  At2g41020  expressed protein  0.249884296  A013311_01  At4g33180  hydrolase, alpha/beta fold family  0.251943264  A019412_01  At5g15560  hypothetical protein  0.253637829  A201993_01  At3g52525  similar to unknown protein (gb|AAB63094.1) -  0.253891458  related A000603_01  At1g61360  S-locus protein kinase family  0.254458839  190  GeneID  Locus  TAIR6 Annotation  *mean_ratio_abABA0_vs_ColABA0  A012484_01  At3g42990  hypothetical protein  0.254629243  A203556_01  At2g10110  hypothetical protein  0.255856063  A003726_01  At1g72500  inter-alpha-trypsin inhibitor heavy chain-related  0.256671131  A005999_01  NA  EST  0.257819656  A200039_01  At2g17033  pentatricopeptide (PPR) repeat-containing protein  0.259133707  A009720_01  At3g57780  expressed protein  0.259646959  A006474_01  At2g26700  second messenger-dependent protein kinase -related  0.259966281  A016563_01  At5g12430  DnaJ domain-containing protein  0.262004177  A009458_01  At3g02390  expressed protein  0.26245406  A200111_01  At4g27630  expressed protein  0.262962436  A000999_01  At1g16610  arginine/serine-rich protein, putative  0.2634014  A200643_01  At1g34790  transparent testa 1 (TT1) protein  0.263602152  A000411_01  At1g32530  zinc finger (C3HC4-type RING finger) protein  0.263861999  family A010176_01  At3g59770  expressed protein  0.263920744  A200679_01  At1g39190  hypothetical protein  0.264232213  A203641_01  At1g35280  CACTA-element transposase -related  0.265040359  A203528_01  At1g20600  hypothetical protein  0.265381585  A021841_01  At1g31540  disease resistance protein (TIR-NBS-LRR class),  0.266251572  putative A010906_01  At3g22270  expressed protein  0.267125851  A019217_01  At5g51220  expressed protein  0.26714272  A010421_01  At3g07600  heavy-metal-associated domain-containing protein  0.267860102  A200846_01  At1g55320  AMP-dependent synthetase and ligase family  0.268499317  A017532_01  At5g06060  short-chain dehydrogenase/reductase family protein  0.268785092  (tropinone reductase, putative) A200584_01  At1g30660  hypothetical protein  0.269131242  A010149_01  At3g58170  Bet1/Sft1-related SRE (AtBS14a)  0.269654896  A203059_01  At5g28892  hypothetical protein  0.270720566  A019562_01  At4g31840  plastocyanin-like domain containing protein  0.270851357  A203258_01  At5g43175  bHLH protein family  0.270938686  A201013_01  At1g70985  proline-rich protein family  0.270942714  A203122_01  At5g33240  hypothetical protein  0.272299725  A013388_01  At4g13710  polysaccharide lyase family 1 (pectate lyase)  0.272608303  A200147_01  At5g23090  TATA-binding protein-associated phosphoprotein  0.273138177  Dr1 protein homolog (sp|P49592)  191  GeneID  Locus  TAIR6 Annotation  *mean_ratio_abABA0_vs_ColABA0  A019541_01  At4g32900  expressed protein  0.273605308  A201205_01  At2g07719  hypothetical protein  0.273707032  A202327_01  At4g17700  hypothetical protein  0.273830057  A008469_01  At2g15730  expressed protein  0.273838426  A200730_01  At1g45231  hypothetical protein  0.274143064  A203702_01  At1g60830  U2 snRNP auxiliary factor, large subunit, putative  0.275200279  A011037_01  At3g21090  ABC transporter family protein  0.275369006  A009206_01  At3g42570  peroxidase-related  0.276485396  A009186_01  At3g28280  hypothetical protein  0.276817058  A018751_01  At5g64630  transducin / WD-40 repeat protein family  0.277246405  A203287_01  At5g47455  expressed protein  0.277327392  A202275_01  At4g15540  nodulin MtN21 - related  0.277453775  A001256_01  At1g07740  pentatricopeptide (PPR) repeat-containing protein  0.278418808  A019257_01  At5g12900  expressed protein  0.280806041  A204002_01  At5g26590  hypothetical protein  0.281370851  A201074_01  At1g78895  expressed protein  0.281403376  A017119_01  At5g48690  hypothetical protein  0.281815363  A201919_01  At3g42725  expressed protein  0.282535997  A009473_01  NA  Arabidopsis thaliana genomic D, chromosome 3,  0.282609833  TAC clone:K24M9 A001108_01  At1g56670  GDSL-motif lipase/hydrolase protein  0.2826669  A016534_01  At5g51310  gibberellin 20-oxidase-related  0.283629062  A202445_01  At4g29800  patatin-related  0.283737411  A011236_01  At3g13770  pentatricopeptide (PPR) repeat-containing protein  0.284601273  A000587_01  At1g49870  hypothetical protein  0.284977112  A003281_01  At1g79830  expressed protein  0.285022087  A000062_01  At1g76570  light-harvesting chlorophyll a/b binding protein  0.285716938  A201076_01  At1g78955  beta-Amyrin synthase, putative  0.286604564  A011434_01  At3g09120  hypothetical protein  0.287688213  A011225_01  At3g60360  expressed protein  0.287809624  A012756_01  At3g54130  Machado-Joseph disease MJD1a -related protein  0.288025881  A203352_01  At5g57890  anthranilate synthase, beta subunit (ASB), putative  0.288161207  A012721_01  At3g47050  glycosyl hydrolase family 3  0.288190861  A020235_01  At3g54480  SKP1 interacting partner 5 (SKIP5)  0.288339204  A000676_01  NA  Arabidopsis thaliana chromosome I BAC F7A19  0.288454042  genomic sequence  192  GeneID  Locus  TAIR6 Annotation  *mean_ratio_abABA0_vs_ColABA0  A004065_01  At1g28220  purine permease, putative  0.288877848  A201888_01  At3g32050  hypothetical protein  0.290183618  A000262_01  At1g75260  isoflavone reductase family  0.2908736  A011444_01  NA  Arabidopsis thaliana genomic D, chromosome 3,  0.292685749  BAC clone:F20C19 A201169_01  At2g07744  hypothetical protein  0.293396521  A203062_01  At5g28930  hypothetical protein  0.295227505  A203874_01  At3g42760  hypothetical protein common family  0.295876119  A201839_01  At3g29785  hypothetical protein  0.296439202  A020742_01  At1g20370  expressed protein  0.296583569  A203917_01  At4g04430  plant transposase (Ptta/En/Spm) family  0.297408469  A006392_01  NA  Arabidopsis thaliana chromosome II section 253 of  0.298047383  255 of the complete sequence. Sequence from clones T8I13, T30B22 A202438_01  At4g28830  expressed protein  0.298349023  A203894_01  At3g50490  hypothetical protein  0.298468402  A202116_01  At4g06475  hypothetical protein  0.298946039  A020279_01  At4g27840  expressed protein  0.300733489  A019543_01  At4g23600  aminotransferase family  0.301181307  A203205_01  At5g37175  non-LTR retroelement reverse transcriptase -related  0.30132595  A013516_01  At4g08750  nucleolin, putative  0.303321491  A004934_01  NA  A.thaliana mR for ARP protein  0.303738303  A010107_01  At3g13530  MAP3K epsilon protein kinase  0.303818985  A200748_01  At1g47885  leucine rich repeat protein family  0.303998035  A007200_01  At2g37840  protein kinase family  0.30402518  A200416_01  At1g17090  expressed protein  0.304196008  A010194_01  At3g15370  expansin, putative (EXP12)  0.304657575  A202235_01  At4g13985  F-box protein family  0.304805728  A202432_01  At4g28365  plastocyanin-like domain containing protein  0.305103511  A024925_01  NA  Arabidopsis thaliana D chromosome 4, BAC clone  0.305964007  F17A8 (ESSA project) A000382_01  At1g11080  serine carboxypeptidase -related  0.306181037  A002193_01  At1g32850  hypothetical protein  0.308513084  A203116_01  At5g32600  hypothetical protein  0.308775537  A010341_01  At3g44260  CCR4-associated factor 1-related protein  0.310068607  A203802_01  At2g31035  hypothetical protein  0.310729947  193  GeneID  Locus  TAIR6 Annotation  *mean_ratio_abABA0_vs_ColABA0  A018728_01  NA  Arabidopsis thaliana RAR1 (Rar1) mR, complete  0.310745423  cds A000774_01  At1g31060  expressed protein  0.310755346  A020154_01  At3g23190  expressed protein  0.311299231  A009565_01  At3g42160  hypothetical protein  0.312327471  A005873_01  NA  ESTs  0.312512326  A201435_01  At2g33835  expressed protein  0.313151445  A000678_01  At1g59720  pentatricopeptide (PPR) repeat-containing protein  0.314130906  A022493_01  At4g11390  CHP-rich zinc finger protein, putative  0.314276925  A000663_01  NA  Arabidopsis thaliana chromosome 1 BAC T7P1  0.315638402  genomic sequence A010074_01  At3g56380  response regulator 17  0.315749045  A004685_01  At1g21890  nodulin N21 family protein  0.316223239  A011352_01  At3g25730  AP2 domain transcription factor, putative  0.316404232  A021437_01  NA  Arabidopsis thaliana genomic D, chromosome 5,  0.31678339  BAC clone:F16F17 A203002_01  At5g28180  Kelch repeat containing F-box protein family  0.317280753  A201840_01  At3g29786  non-LTR retroelement reverse transcriptase -related  0.31731577  A202881_01  At5g25540  expressed protein  0.317474632  A202402_01  At4g23885  expressed protein  0.318542413  A200602_01  At1g32370  (TOM2B) tobamovirus multiplication 2B  0.319661015  A023935_01  At1g60030  permease -related  0.319854705  A007321_01  At2g15030  hypothetical protein  0.320108416  A201602_01  At3g07530  hypothetical protein  0.320177064  A011911_01  At3g56900  HIRA protein-related (TUP1)  0.321168763  A005318_01  NA  EST  0.322008874  A007180_01  At2g04910  glycosyl hydrolase family 17  0.322105656  A003994_01  At1g80010  far-red impaired response protein -related  0.322169155  A000725_01  At1g15470  transducin / WD-40 repeat protein family  0.322931521  A201581_01  At3g05155  sugar transporter, putative  0.323562262  A005136_01  NA  ESTs  0.323898494  A000415_01  At1g70720  pectinesterase - related  0.325508639  A014059_01  At4g15950  hypothetical protein  0.325995474  A020749_01  NA  EST  0.326468012  A200966_01  At1g66345  pentatricopeptide (PPR) repeat-containing protein  0.326770041  A200626_01  At1g33830  AIG1-related protein  0.328323202  194  GeneID  Locus  TAIR6 Annotation  *mean_ratio_abABA0_vs_ColABA0  A201898_01  At3g32270  hypothetical protein  0.328494017  A001368_01  At1g25300  octicosapeptide/Phox/Bem1p (PB1) domain-  0.328666595  containing protein A200437_01  At1g18320  expressed protein  0.328708487  A202212_01  At4g10930  expressed protein  0.32968864  A022055_01  At5g28570  hypothetical protein  0.329761355  A001584_01  At1g27880  ATP-dependent D helicase, putative  0.330762999  A011012_01  At3g61010  glycosyl hydrolase family 85  0.331560146  A004938_01  NA  ESTs, Weakly similar to T09573 replication factor  0.331973011  C 38K chain [H.sapiens] A001734_01  At1g75090  D-3-methyladenine glycosylase I -related  0.332483818  A005360_01  NA  EST  0.333861364  A025267_01  NA  Sequence of BAC F7G19 from Arabidopsis  0.333884812  thaliana chromosome 1 A006504_01  NA  Arabidopsis thaliana chromosome II section 131 of 255 of the complete sequence. Sequence from clones T20K9, F21P24  0.33412934  A016971_01  At5g39520  expressed protein  0.335367791  A010103_01  At3g26810  transport inhibitor response 1 (TIR1), putative  0.335507977  A006494_01  At2g25090  CBL-interacting protein kinase 16  0.337617481  A002488_01  At1g76110  expressed protein  0.337795931  A200994_01  At1g69260  expressed protein  0.338618404  A007767_01  At2g27920  serine carboxypeptidase -related  0.338795532  A011318_01  At3g15280  expressed protein  0.340143349  A203442_01  At1g26730  aldehyde oxidase -related  0.340306708  A022750_01  NA  Arabidopsis thaliana genomic D, chromosome 3,  0.34039098  TAC clone:K5K13 A202836_01  At5g24900  cytochrome P450 family  0.340727844  A201062_01  At1g77765  hypothetical protein  0.340896004  A013162_01  At4g13360  3-hydroxyisobutyryl-coenzyme A hydrolase (CoA-  0.341295551  thioester hydrolase) family A007655_01  NA  Arabidopsis thaliana chromosome II section 11 of 255 of the complete sequence. Sequence from clones T8K22, T20F6, T17M13  0.341680591  A007135_01  At2g21480  protein kinase family  0.341936578  A006693_01  At2g24800  peroxidase, putative  0.342048319  195  GeneID  Locus  TAIR6 Annotation  *mean_ratio_abABA0_vs_ColABA0  A201085_01  At1g79615  expressed protein  0.343596591  A024998_01  At4g07570  see GB:AF077408  0.344371182  A009384_01  At3g16920  glycosyl hydrolase family 19 (chitinase)  0.345139111  A004068_01  At1g31800  cytochrome P450, putative  0.345230683  A008540_01  At2g14110  hypothetical protein  0.345531705  A203870_01  At3g42556  hypothetical protein  0.348296245  A202118_01  At4g06477  transposon protein -related  0.348627313  A003993_01  At1g74690  expressed protein  0.348692791  A202400_01  At4g23840  leucine rich repeat protein-related  0.350090824  A023765_01  NA  Genomic sequence for Arabidopsis thaliana BAC  0.351572279  F9C16 from chromosome I A200718_01  At1g44060  plant transposase (Ptta/En/Spm) family  0.351880981  A000628_01  NA  Genomic sequence for Arabidopsis thaliana BAC  0.352027275  F18O14 from chromosome I A201843_01  At3g30230  myosin heavy chain-related  0.352890984  A005734_01  NA  Arabidopsis thaliana At1g09870 gene, complete  0.353379612  cds A001486_01  NA  Genomic sequence for Arabidopsis thaliana BAC  0.354639345  F15O4 from chromosome I A024832_01  NA  Arabidopsis thaliana D chromosome 3, BAC clone  0.355675986  T8H10 A201183_01  At2g07771  expressed protein  0.355934317  A200466_01  At1g19980  expressed protein  0.355974408  A004071_01  At1g28320  expressed protein  0.356986996  A015714_01  At5g48670  MADS-box protein  0.357840693  A202460_01  At4g32105  expressed protein  0.357853977  A009783_01  At3g01600  No apical meristem (M) protein family  0.358361025  A201809_01  At3g28590  hypothetical protein  0.358607585  A002231_01  NA  Arabidopsis thaliana chromosome 1 BAC F24B9  0.360504959  sequence A006279_01  At2g45200  cis-Golgi SRE protein, putative  0.360551998  A025189_01  At5g38500  hypothetical protein  0.360901258  A006106_01  At2g44740  cyclin family  0.361049903  A203266_01  At5g44710  expressed protein  0.362466485  A001538_01  NA  Arabidopsis thaliana chromosome 1 BAC T16B5  0.363557568  sequence  196  GeneID  Locus  TAIR6 Annotation  *mean_ratio_abABA0_vs_ColABA0  A005169_01  NA  ESTs, Weakly similar to T47442 disease resistance  0.364321008  protein homlog - Arabidopsis thaliana [A.thaliana] A007841_01  At2g33230  flavin-containing monooxygenase (FMO), putative  0.364738006  A024014_01  At1g68880  bZIP family transcription factor  0.364816618  A025069_01  At3g26270  cytochrome P450 71B25  0.365457067  A017983_01  At5g50130  short-chain dehydrogenase/reductase family protein  0.365480438  A008361_01  At2g19490  recA protein -related  0.365846271  A012643_01  At3g25670  leucine rich repeat protein family  0.365879984  A008771_01  At2g24840  MADS-box protein  0.365976601  A005538_01  NA  EST  0.367007822  A005264_01  NA  EST  0.367084612  A012703_01  At3g52250  myb family transcription factor  0.368407207  A000747_01  At1g24420  acyltransferase family  0.368788308  A203113_01  At5g32570  hypothetical protein  0.369189793  A013258_01  At4g23950  hypothetical protein  0.371084004  A007144_01  At2g36960  myb family transcription factor  0.371729535  A000071_01  At1g70030  expressed protein  0.371849295  A006370_01  At2g34400  pentatricopeptide (PPR) repeat-containing protein  0.372508781  A202321_01  At4g17570  GATA zinc finger protein  0.372987362  A203650_01  At1g37160  hypothetical protein  0.374010888  A006187_01  At2g42930  glycosyl hydrolase family 17  0.374700164  A005537_01  At5g25870  hypothetical protein  0.375450524  A021166_01  NA  EST  0.376353514  A003332_01  At1g63190  hypothetical protein  0.376789196  A023291_01  NA  Arabidopsis thaliana chromosome 1 BAC F6D8  0.377844649  sequence A201135_01  At2g05025  hypothetical protein  0.379257291  A200427_01  At1g17600  disease resistance protein (TIR-NBS-LRR class),  0.380512476  putative A013147_01  At4g11340  disease resistance protein (TIR class), putative  0.381214199  A001043_01  NA  Housekeeping 12, Arabidopsis thaliana alpha-1-  0.38291214  tubulin gene A008859_01  At2g17820  histidine kinase 1  0.382964511  A010059_01  At3g15095  hypothetical protein  0.384295155  A017022_01  At5g41050  expressed protein  0.385034307  A001881_01  At1g76610  hypothetical protein  0.385356323  197  GeneID  Locus  TAIR6 Annotation  *mean_ratio_abABA0_vs_ColABA0  A020410_01  At2g28470  glycosyl hydrolase family 35 (beta-galactosidase)  0.386507037  A202569_01  At5g08141  bZIP family transcription factor  0.387858049  A005138_01  NA  Genomic sequence for Arabidopsis thaliana BAC  0.388222405  F5J5 A020986_01  NA  ESTs  0.388745389  A013229_01  At4g24390  F-box protein family, AtFBX14  0.389759944  A022380_01  At4g27250  dihydroflavonol 4-reductase (dihydrokaempferol 4-  0.390172209  reductase) family A013350_01  At4g01000  ubiquitin family  0.390245048  A002319_01  NA  Genomic sequence for Arabidopsis thaliana BAC  0.39146939  T32E20 from chromosome I A201415_01  At2g31580  hypothetical protein  0.393959174  A202713_01  At5g19830  expressed protein  0.394193269  A010120_01  At3g45180  ubiquitin family  0.394991855  A200625_01  At1g33820  hypothetical protein  0.395983229  A014586_01  At4g37990  mannitol dehydrogenase (ELI3-2), putative  0.396088995  A000562_01  At1g20870  hypothetical protein  0.396873208  A201268_01  At2g14420  Mutator-related transposase  0.397565147  A008023_01  At2g12880  CCHC-type zinc finger protein -related  0.397789348  A203720_01  At2g03080  reverse transcriptase -related  0.398414312  A203851_01  At3g31415  sesquiterpene synthase/cyclase family  0.398848879  A200801_01  At1g52325  hypothetical protein  0.39967711  A004955_01  At5g27120  SAR D-binding protein, putative  0.401386747  A012615_01  At3g59550  cohesion family protein (SYN3) (RAD21-2)  0.40299694  A203540_01  At1g47395  expressed protein  0.403179794  A012114_01  At3g18670  hypothetical protein  0.403809269  A013358_01  At4g39120  Inositol monophosphatase - like protein  0.404120589  A004574_01  At1g14580  zinc finger protein -related  0.404161461  A011874_01  At3g61570  expressed protein  0.404898509  A014877_01  At4g24970  hypothetical protein  0.405148228  A200070_01  At3g13080  ABC transporter family protein  0.405305164  A006290_01  At2g14660  expressed protein  0.406839636  A202768_01  At5g20600  expressed protein  0.407023276  A200179_01  At5g48400  glutamate receptor family (GLR1.2)  0.407329253  A010198_01  At3g16660  expressed protein  0.409912928  A004873_01  At1g22500  RING-H2 zinc finger protein ATL5 -related  0.410401141  198  GeneID  Locus  TAIR6 Annotation  *mean_ratio_abABA0_vs_ColABA0  A019865_01  At2g22360  DnaJ protein, putative  0.41055022  A203953_01  At4g08876  phosphofructokinase beta subunit -related  0.410890409  A010917_01  NA  Arabidopsis thaliana genomic D, chromosome 3,  0.411005487  P1 clone: MOB24 A202794_01  At5g21020  expressed protein  0.412996747  A010180_01  NA  Arabidopsis thaliana genomic D, chromosome 3,  0.414350992  P1 clone: MSD21 A203814_01  At3g23460  hypothetical protein  0.414511738  A202636_01  At5g18730  hypothetical protein  0.414637207  A001908_01  NA  Arabidopsis thaliana chromosome I BAC F11M15  0.415534452  genomic sequence A200831_01  At1g54260  hypothetical protein  0.416493835  A019399_01  NA  Arabidopsis thaliana genomic D, chromosome 5,  0.417292506  P1 clone:MUL8 A005157_01  NA  EST  0.41754872  A203539_01  At1g43870  myosin heavy chain-related  0.42018891  A005476_01  NA  ESTs, Highly similar to T05219 hypothetical  0.420206194  protein F17I5.100 - Arabidopsis thaliana [A.thaliana] A203957_01  At4g09740  glycosyl hydrolase family 9  0.420375675  A201876_01  At3g31340  Athila ORF 1 -related  0.420890845  A203490_01  At3g47760  ABC transporter family protein  0.421789878  A201327_01  At2g20825  hypothetical protein  0.422564608  A010397_01  At3g10600  amino acid transporter -related  0.423102236  A018634_01  NA  Arabidopsis thaliana genomic D, chromosome 5,  0.42418674  P1 clone:MXF12 A013770_01  At4g01480  inorganic phosphatase -related  0.42464902  A010331_01  At3g03220  expansin, putative (EXP13)  0.425232127  A001060_01  NA  Arabidopsis thaliana chromosome 1 BAC F10O5  0.425319769  genomic sequence A202908_01  At5g26160  hypothetical protein  0.425590215  A000612_01  At1g13245  expressed protein  0.425749615  A001563_01  NA  Sequence of BAC F10K1 from Arabidopsis  0.425778235  thaliana chromosome 1 A201249_01  At2g12405  hypothetical protein  0.426525237  199  GeneID  Locus  TAIR6 Annotation  *mean_ratio_abABA0_vs_ColABA0  A005376_01  At1g51600  zinc finger protein family  0.427353746  A201293_01  At2g17250  expressed protein  0.430778242  A003840_01  At1g04730  hypothetical protein  0.431087352  A010073_01  At3g56390  hypothetical protein  0.432419832  A009276_01  NA  Arabidopsis thaliana D chromosome 3, BAC clone  0.432831303  T14K23 A021210_01  NA  EST, Weakly similar to T05630 hypothetical  0.432942956  protein F20D10.150 - Arabidopsis thaliana [A.thaliana] A007242_01  At2g30120  expressed protein  0.432970976  A200746_01  At1g47800  F-box protein family  0.434571446  A203256_01  At5g42965  non-LTR retroelement reverse transcriptase -related  0.435662224  A022470_01  At4g31900  chromatin remodeling factor, putative  0.435924938  A024462_01  NA  Genomic sequence for Arabidopsis thaliana BAC  0.436002984  T27G7 from chromosome I A010092_01  At5g04130  D topoisomerase [ATP-hydrolyzing] (DNA  0.437173722  topoisomerase II/DNA gyrase), putative A021182_01  NA  EST  0.437299227  A006825_01  At2g37950  zinc finger (C3HC4-type RING finger) protein  0.437966118  family A021195_01  NA  EST, Weakly similar to T48411 Terminal flower1  0.438145879  (TFL1) - Arabidopsis thaliana [A.thaliana] A203913_01  At4g04165  hypothetical protein  0.440237889  A010250_01  At3g48780  serine C-palmitoyltransferase, putative  0.440250581  A014199_01  At4g37480  DnaJ protein family  0.441108924  A022058_01  NA  Arabidopsis thaliana genomic D, chromosome 3,  0.441369883  BAC clone:F11I2 A203514_01  At5g25754  expressed protein  0.441561255  A002444_01  At1g02510  outward rectifying potassium channel, putative  0.442410332  (KCO4) A202495_01  At4g35335  hypothetical protein  0.444249234  A204018_01  At5g28480  hypothetical protein  0.444657405  A016879_01  At5g27730  expressed protein  0.444913161  A017714_01  NA  Arabidopsis thaliana genomic D, chromosome 5,  0.44597782  P1 clone:MNJ7  200  GeneID  Locus  TAIR6 Annotation  *mean_ratio_abABA0_vs_ColABA0  A201304_01  At2g18115  glycine-rich protein  0.446190193  A203986_01  At4g33320  hypothetical protein  0.446906419  A012635_01  NA  Arabidopsis thaliana genomic D, chromosome 3,  0.448702985  BAC clone:T5M7 A009862_01  At3g61410  hypothetical protein  0.449564574  A000443_01  At1g71930  No apical meristem (M) protein family  0.450482644  A200055_01  At2g38040  alpha-carboxyltransferase -related  0.451156452  A201155_01  At2g06902  hypothetical protein  0.451302809  A013370_01  At4g13230  late embryogenesis abundant (LEA) domain-  0.452213384  containing protein A021708_01  NA  Arabidopsis thaliana chromosome I BAC T28K15  0.452346045  genomic sequence A200852_01  At1g55750  expressed protein  0.45354588  A004057_01  NA  Genomic sequence for Arabidopsis thaliana BAC  0.45358689  F23N19 from chromosome I A020780_01  NA  Housekeeping 04, Arabidopsis thaliana putative  0.453644156  ubiquitin protein (T23A1.5/At2g17190) mR A013570_01  At4g16400  hypothetical protein  0.453764797  A006142_01  At2g46460  hypothetical protein  0.453954492  A019481_01  At3g51140  expressed protein  0.455796943  A201000_01  At1g69520  hypothetical protein  0.455900202  A013321_01  At4g35560  expressed protein  0.45735821  A021189_01  NA  Arabidopsis thaliana chromosome 1 BAC F13N6  0.457853998  genomic sequence A020935_01  NA  EST  0.458196677  A203274_01  At5g45595  hypothetical protein  0.458717051  A001616_01  At1g30170  hypothetical protein  0.459111421  A001645_01  At1g33410  expressed protein  0.459336514  A012564_01  At3g16750  hypothetical protein  0.460290365  A005352_01  NA  EST  0.461073626  A010524_01  At3g63430  hypothetical protein  0.461402196  A204076_01  At5g36850  hypothetical protein common family  0.461405292  A009104_01  At3g27240  cytochrome c -related  0.461615545  A203914_01  At4g04276  Athila retroelement ORF1 protein -related  0.462003899  A014645_01  At4g14180  hypothetical protein  0.462508533  A203945_01  At4g08013  hypothetical protein  0.462914997  201  GeneID  Locus  TAIR6 Annotation  *mean_ratio_abABA0_vs_ColABA0  A005598_01  NA  EST, Moderately similar to T48276 hypothetical  0.462993625  protein T22P11.140 - Arabidopsis thaliana [A.thaliana] A200745_01  At1g47786  hypothetical protein  0.464527249  A000412_01  At1g70570  anthranilate phosphoribosyltransferase, putative  0.465701103  A015315_01  NA  Arabidopsis thaliana D chromosome 4, BAC clone  0.465790909  F17L22 (ESSAII project) A010202_01  NA  Arabidopsis thaliana genomic D, chromosome 3,  0.466064388  BAC clone: T22B15 A003286_01  At1g19310  zinc finger (C3HC4-type RING finger) protein  0.467136823  family A021724_01  NA  Arabidopsis thaliana chromosome 1 BAC T8F5  0.46823032  sequence A000947_01  At1g26190  expressed protein  0.468446103  A008476_01  At2g41830  cyclin-related  0.468480177  A202294_01  At4g16345  adapter protein SPIKE1 -related  0.468498109  A016935_01  At5g13810  expressed protein  0.469122599  A015683_01  At5g10120  transcription factor TEIL/ethylene-insensitive - like  0.470546213  protein A000368_01  At1g74250  heat shock protein -related  0.470910729  A023716_01  NA  Genomic sequence for Arabidopsis thaliana BAC  0.471160862  F2K11 from chromosome I A008951_01  At3g45160  expressed protein  0.471722164  A000667_01  NA  Arabidopsis thaliana chromosome I BAC F7A19  0.471826331  genomic sequence A008123_01  At2g22010  zinc finger (C3HC4-type RING finger) protein  0.471847128  family A009111_01  At3g52370  predicted GPI-anchored protein  0.471871529  A009661_01  At3g19670  expressed protein  0.472941666  A015438_01  At4g02170  hypothetical protein  0.474341446  A008362_01  At2g19500  cytokinin oxidase family  0.474426677  A023270_01  NA  Arabidopsis thaliana BAC T1N24  0.47454897  A005442_01  NA  EST  0.475469641  A009819_01  At3g10950  60S ribosomal protein L37a (RPL37aB)  0.476441657  A200652_01  At1g35740  hypothetical protein  0.47662693  A201402_01  At2g30615  myb-related transcription factor -related  0.476800637  202  GeneID  Locus  TAIR6 Annotation  *mean_ratio_abABA0_vs_ColABA0  A024155_01  At2g36100  hypothetical integral membrane protein common  0.476946178  family A004848_01  At1g23090  sulphate transporter protein -related  0.477065289  A000101_01  NA  Arabidopsis thaliana chromosome I BAC F13F21  0.47776894  genomic sequence A022620_01  NA  Arabidopsis thaliana D chromosome 4, contig  0.478422086  fragment No. 45 A025126_01  At5g54550  expressed protein  0.47849644  A013139_01  NA  Arabidopsis thaliana D chromosome 4, BAC clone  0.478886999  F23K16 (ESSA project) A200901_01  At1g61400  S-locus protein kinase, putative  0.479007786  A001232_01  At1g69370  chorismate mutase (CM), putative  0.479950212  A006307_01  NA  Arabidopsis thaliana chromosome II section 240 of  0.480205539  255 of the complete sequence. Sequence from clones F16B22, T13E15 A020898_01  NA  EST  0.480688127  A023932_01  NA  Sequence of BAC T29M8 from Arabidopsis  0.481556383  thaliana chromosome 1 A000583_01  At1g47920  syntaxin SYP81  0.48186406  A013541_01  At4g14570  acylaminoacyl-peptidase like protein  0.481938056  A002127_01  NA  Genomic sequence for Arabidopsis thaliana BAC  0.482093187  T32E20 from chromosome I A005951_01  NA  EST  0.482624307  A201713_01  At3g20540  D polymerase -related  0.48297299  A017500_01  NA  Arabidopsis thaliana genomic D, chromosome 5,  0.485548031  BAC clone:F5H8 A021300_01  At4g12280  copper amine oxidase like protein (fragment2)  0.485941568  A202693_01  At5g19520  expressed protein  0.487294688  A200495_01  At1g21475  hypothetical protein  0.48744439  A001993_01  NA  Arabidopsis thaliana chromosome 1 BAC F9L1  0.487739567  sequence A024342_01  At1g29470  dehydration-induced protein-related  0.488000407  A003084_01  At1g05340  expressed protein  0.488671675  203  GeneID  Locus  TAIR6 Annotation  *mean_ratio_abABA0_vs_ColABA0  A008704_01  NA  Arabidopsis thaliana chromosome II section 221 of 255 of the complete sequence.  A201778_01  At3g26030  Sequence from clones T20B5  0.489917987  protein phosphatase 2A regulatory subunit isoform  0.490767424  B delta A201987_01  At3g50685  expressed protein  0.491169344  A011381_01  At3g51560  disease resistance protein (TIR-NBS-LRR class),  0.491210749  putative A016856_01  NA  Arabidopsis thaliana D chromosome 5, BAC clone  0.491569911  F8M21 (ESSA project) A017552_01  At5g61430  M, no apical meristem, - like protein  0.492320548  A014674_01  At4g31730  expressed protein  0.49260629  A005701_01  At3g51880  high mobility group protein 2-related  0.492950393  A010175_01  NA  Arabidopsis thaliana genomic D, chromosome 3,  0.493286823  TAC clone:K7P8 A002318_01  NA  Genomic sequence for Arabidopsis thaliana BAC  0.493482072  T32E20 from chromosome I A203606_01  At1g02440  ADP-ribosylation factor, putative  0.494381307  A005301_01  NA  Arabidopsis thaliana BAC T7I23, complete  0.494636201  sequence A000886_01  At1g32040  hypothetical protein  0.49471337  A202064_01  At4g01575  expressed protein  0.495157834  A203735_01  At2g06095  athila ORF 1 -related  0.496776423  A002860_01  At1g14120  2-oxoglutarate-dependent dioxygenase, putative  0.497154147  A026039_01  At3g44500  hypothetical protein common family  0.497628757  A200104_01  At4g23290  serine/threonine kinase - like protein  0.498396999  A025019_01  At4g16950  disease resistance protein, RPP5-like (TIR-NBS-  0.498539187  LRR class), putative A012317_01  NA  Arabidopsis thaliana genomic D, chromosome 3,  0.498624659  BAC clone: T8O3 A004959_01  NA  ESTs  0.499591448  * The genes whose expression level was co-down-regulated by both ABA and rack1 mutation in comparison to Col to half or lower were listed in this table.  204  APPENDIX 2. RACK1-coexpressed genes I. Genes that are coexpressed with RACK1A Gene locus  Annotation of co-expressed gene  number At1g01100  60S acidic ribosomal protein P1 (RPP1A) similar to 60S ACIDIC RIBOSOMAL PROTEIN P1 GB:O23095 from [Arabidopsis thaliana]  At1g02780  60S ribosomal protein L19 (RPL19A) similar to ribosomal protein L19 GI:36127 from [Homo sapiens]  At1g08360  60S ribosomal protein L10A (RPL10aA) similar to 60S ribosomal protein L10A GB:AAC73045 GI:3860277 from [Arabidopsis thaliana]  At1g14320  60S ribosomal protein L10 (RPL10A) / Wilm's tumor suppressor protein-related similar to tumor suppressor GI:575354 from [Oryza sativa]  At1g15930  40S ribosomal protein S12 (RPS12A) similar to 40S ribosomal protein S12 GI:4263712 from [Arabidopsis thaliana]  At1g33140  60S ribosomal protein L9 (RPL90A/C) similar to RIBOSOMAL PROTEIN L9 GB:P49209 from [Arabidopsis thaliana]  At1g43170  60S ribosomal protein L3 (RPL3A) identical to ribosomal protein GI:166858 from [Arabidopsis thaliana]  At1g67430  60S ribosomal protein L17 (RPL17B) similar to ribosomal protein GI:19101 from [Hordeum vulgare]  At1g69620  60S ribosomal protein L34 (RPL34B) similar to SP:Q42351 from [Arabidopsis thaliana]  At1g72370  40S ribosomal protein SA (RPSaA) identical to laminin receptor-like protein GB:U01955 [Arabidopsis thaliana]; identical to cDNA laminin receptor homolog GI:16379  At2g09990  40S ribosomal protein S16 (RPS16A) Same as GB:Q42340  At2g18020  60S ribosomal protein L8 (RPL8A)  At2g19730  60S ribosomal protein L28 (RPL28A)  At2g21580  40S ribosomal protein S25 (RPS25B)  At2g27720  60S acidic ribosomal protein P2 (RPP2A)  At2g34480  60S ribosomal protein L18A (RPL18aB)  At2g36160  40S ribosomal protein S14 (RPS14A)  At2g37270  40S ribosomal protein S5 (RPS5A) identical to GP:3043428  At2g41840  40S ribosomal protein S2 (RPS2C)  At3g02080  40S ribosomal protein S19 (RPS19A) similar to 40S ribosomal protein S19 GB:P40978 [Oryza sativa]  205  Gene locus  Annotation of co-expressed gene  number At3g04400  60S ribosomal protein L23 (RPL23C) similar to ribosomal protein L17 GB:AAA34113.1 from [Nicotiana tabacum]  At3g09200  60S acidic ribosomal protein P0 (RPP0B) similar to putative 60S acidic ribosomal protein P0 GB:P50346 [Glycine max]  At3g09680  40S ribosomal protein S23 (RPS23A) similar to 40S ribosomal protein S23 (S12) GB:P46297 from [Fragaria x ananassa]  At3g24830  60S ribosomal protein L13A (RPL13aB) similar to 60S RIBOSOMAL PROTEIN L13A GB:P35427 from [Rattus norvegicus]  At3g44010  40S ribosomal protein S29 (RPS29B) ribosomal protein S29, rat, PIR:S30298  At3g45030  40S ribosomal protein S20 (RPS20A) 40S ribsomomal proteinS20, Arabidopsis thaliana, pir:T12992  At3g49910  60S ribosomal protein L26 (RPL26A) 60S RIBOSOMAL PROTEIN L26, Brassica rapa, EMBL:BRD495  At3g52590  ubiquitin extension protein 1 (UBQ1) / 60S ribosomal protein L40 (RPL40B) identical to GI:166929, GI:166930  At3g53020  60S ribosomal protein L24 (RPL24B) 60S ribosomal protein L24, Arabidopsis thaliana, EMBL:AC006282  At3g61110  40S ribosomal protein S27 (ARS27A) identical to cDNA ribosomal protein S27 (ARS27A) GI:4193381  At4g00100  40S ribosomal protein S13 (RPS13A) similar to ribosomal protein S13; PF00312 (View Sanger Pfam): ribosomal protein S15; identical to cDNA AtRPS13A mRNA for cytoplasmic ribosomal protein S13 GI:6521011  At4g14320  60S ribosomal protein L36a/L44 (RPL36aB)  At5g10360  40S ribosomal protein S6 (RPS6B)  At5g15200  40S ribosomal protein S9 (RPS9B) 40S ribosomal protein S9, Chlamydomonas sp., EMBL:AU066528  At5g19510  elongation factor 1B alpha-subunit 2 (eEF1Balpha2) identical to elongation factor 1B alpha-subunit [Arabidopsis thaliana] GI:6686821  At5g20290  40S ribosomal protein S8 (RPS8A) ribosomal protein S8 - Zea mays, PIR:T04088  At5g27850  60S ribosomal protein L18 (RPL18C) 60S ribosomal protein L18, Arabidopsis thaliana, SWISSPROT:RL18_ARATH  At5g35530  40S ribosomal protein S3 (RPS3C)  At5g47700  60S acidic ribosomal protein P1 (RPP1C)  At5g57290  60S acidic ribosomal protein P3 (RPP3B)  206  II. Genes that are co-expressed with RACK1B Gene locus  Annotation of co-expressed gene  number At2g39390  60S ribosomal protein L35 (RPL35B)  RACK1C  guanine nucleotide-binding family protein / activated protein kinase C receptor (RACK1) identical to guanine nucleotide-binding protein; activated protein kinase C receptor; RACK1 (GI:9294068) {Arabidopsis thaliana}; contains Pfam profile: PF00400 WD domain  At3g20050  T-complex protein 1 alpha subunit / TCP-1-alpha / chaperonin (CCT1) identical to SWISS-PROT:P28769- T-complex protein 1, alpha subunit (TCP-1-alpha) [Arabidopsis thaliana]  At5g08180  ribosomal protein L7Ae/L30e/S12e/Gadd45 family protein Similar to NHP2/L7Ae family proteins, see SWISSPROT:P32495 and PMID:2063628.  At5g15520  40S ribosomal protein S19 (RPS19B) 40S RIBOSOMAL PROTEIN S19 - Oryza sativa, SWISSPROT:RS19_ORYSA  At5g41520  40S ribosomal protein S10 (RPS10B) contains similarity to 40S ribosomal protein S10  At5g48760  60S ribosomal protein L13A (RPL13aD)  At5g56710  60S ribosomal protein L31 (RPL31C)  III. Genes that are co-expressed with RACK1C Gene locus  Annotation of co-expressed gene  number RACK1B  guanine nucleotide-binding family protein / activated protein kinase C receptor, putative / RACK, putative contains 7 WD-40 repeats (PF00400); very similar to guanine nucleotide-binding protein; activated protein kinase C receptor; RACK1 (GI:9294068) {Arabidopsis thanliana}  At2g27840  histone deacetylase-related / HD-related similar to nucleolar histone deacetylase HD2-p39 [Zea mays] GI:2257756; contains non-consensus donor splice site AT at exon2 and acceptor splice site AC at exon3.  At3g62120  tRNA synthetase class II (G, H, P and S) family protein similar to SP|P07814 Bifunctional aminoacyl-tRNA synthetase [Includes: Glutamyl-tRNA synthetase (EC 6.1.1.17) (Glutamate--tRNA ligase); Prolyl-tRNA synthetase (EC 6.1.1.15) (Proline--tRNA ligase)]  207  IV. Genes that are co-expressed with RACK1A and RACK1B Gene locus  Annotation of co-expressed gene  number At1g04270  40S ribosomal protein S15 (RPS15A) Strong similarity to Oryza 40S ribosomal protein S15. ESTs gb|R29788,gb|ATTS0365 come from this gene  At1g04270  40S ribosomal protein S15 (RPS15A) Strong similarity to Oryza 40S ribosomal protein S15. ESTs gb|R29788,gb|ATTS0365 come from this gene  At1g22780  40S ribosomal protein S18 (RPS18A) Match to ribosomal S18 gene mRNA gb|Z28701, DNA gb|Z23165 from A. thaliana. ESTs gb|T21121, gb|Z17755, gb|R64776 and gb|R30430 come from this gene  At1g22780  40S ribosomal protein S18 (RPS18A) Match to ribosomal S18 gene mRNA gb|Z28701, DNA gb|Z23165 from A. thaliana. ESTs gb|T21121, gb|Z17755, gb|R64776 and gb|R30430 come from this gene  At1g23290  60S ribosomal protein L27A (RPL27aB) similar to 60S RIBOSOMAL PROTEIN L27A GB:P49637 GI:1710530 from [Arabidopsis thaliana]  At1g23290  60S ribosomal protein L27A (RPL27aB) similar to 60S RIBOSOMAL PROTEIN L27A GB:P49637 GI:1710530 from [Arabidopsis thaliana]  At1g41880  60S ribosomal protein L35a (RPL35aB) identical to GB:CAB81600 from [Arabidopsis thaliana]  At1g41880  60S ribosomal protein L35a (RPL35aB) identical to GB:CAB81600 from [Arabidopsis thaliana]  At1g52300  60S ribosomal protein L37 (RPL37B) similar to SP:Q43292 from [Arabidopsis thaliana]  At1g52300  60S ribosomal protein L37 (RPL37B) similar to SP:Q43292 from [Arabidopsis thaliana]  At2g27710  60S acidic ribosomal protein P2 (RPP2B)  At2g27710  60S acidic ribosomal protein P2 (RPP2B)  At2g33370  60S ribosomal protein L23 (RPL23B)  At2g33370  60S ribosomal protein L23 (RPL23B)  At2g43460  60S ribosomal protein L38 (RPL38A)  At2g43460  60S ribosomal protein L38 (RPL38A)  At3g02560  40S ribosomal protein S7 (RPS7B) similar to ribosomal protein S7 GB:AAD26256 from [Secale cereale]  At3g02560  40S ribosomal protein S7 (RPS7B) similar to ribosomal protein S7 GB:AAD26256 from [Secale cereale]  At3g52580  40S ribosomal protein S14 (RPS14C) ribosomal protein S14 -Zea mays,PIR2:A30097  At3g52580  40S ribosomal protein S14 (RPS14C) ribosomal protein S14 -Zea mays,PIR2:A30097  At3g62870  60S ribosomal protein L7A (RPL7aB) 60S RIBOSOMAL PROTEIN L7A - Oryza sativa, SWISSPROT:RL7A_ORYSA  208  Gene locus  Annotation of co-expressed gene  number At3g62870  60S ribosomal protein L7A (RPL7aB) 60S RIBOSOMAL PROTEIN L7A - Oryza sativa, SWISSPROT:RL7A_ORYSA  At4g16720  60S ribosomal protein L15 (RPL15A)  At4g16720  60S ribosomal protein L15 (RPL15A)  At4g18730  60S ribosomal protein L11 (RPL11C)  At4g18730  60S ribosomal protein L11 (RPL11C)  At5g03850  40S ribosomal protein S28 (RPS28B) ribosomal protein S28, Arabidopsis thaliana, EMBL:ATRP28A  At5g03850  40S ribosomal protein S28 (RPS28B) ribosomal protein S28, Arabidopsis thaliana, EMBL:ATRP28A  At5g04800  40S ribosomal protein S17 (RPS17D) 40S ribosomal protein S17, Lycopersicon esculentum, EMBL:AF161704  At5g04800  40S ribosomal protein S17 (RPS17D) 40S ribosomal protein S17, Lycopersicon esculentum, EMBL:AF161704  At5g07090  40S ribosomal protein S4 (RPS4B)  At5g07090  40S ribosomal protein S4 (RPS4B)  At5g23740  40S ribosomal protein S11 (RPS11C)  At5g23740  40S ribosomal protein S11 (RPS11C)  At5g27770  60S ribosomal protein L22 (RPL22C) ribosomal protein L22 (cytosolic), Rattus norvegicus, PIR:S52084  At5g27770  60S ribosomal protein L22 (RPL22C) ribosomal protein L22 (cytosolic), Rattus norvegicus, PIR:S52084  At5g47930  40S ribosomal protein S27 (RPS27D)  At5g47930  40S ribosomal protein S27 (RPS27D)  V. Genes that are co-expressed with RACK1B and RACK1C Gene locus  Annotation of co-expressed gene  number At1g04480  60S ribosomal protein L23 (RPL23A) identical to GB:AAB80655  At1g07070  60S ribosomal protein L35a (RPL35aA) similar to ribosomal protein L35a GI:57118 from [Rattus norvegicus]  At1g26910  60S ribosomal protein L10 (RPL10B) Nearly identical to ribosomal protein L10.e, Wilm's tumor suppressor homolog, gi|17682 (Z15157), however differences in sequence indicate this is a different member of the L10 family in sequence indicate this is a different member of the L10 family  At1g28395  expressed protein  209  Gene locus  Annotation of co-expressed gene  number At1g29250  expressed protein contains TIGRFAM TIGR00285: conserved hypothetical protein TIGR00285  At1g34030  40S ribosomal protein S18 (RPS18B) similar to ribosomal protein S18 GI:38422 from [Homo sapiens]  At1g49410  expressed protein  At1g52930  brix domain-containing protein contains Pfam domain, PF04427: Brix domain  At1g56110  nucleolar protein Nop56, putative similar to XNop56 protein [Xenopus laevis] GI:14799394; contains Pfam profile PF01798: Putative snoRNA binding domain  At1g57660  60S ribosomal protein L21 (RPL21E) similar to 60S ribosomal protein L21 GB:Q43291 GI:2851508 from [Arabidopsis thaliana]  At1g64880  ribosomal protein S5 family protein contains similarity to 30S ribosomal protein S5 GI:6969105 from [Campylobacter jejuni]  At2g20450  60S ribosomal protein L14 (RPL14A)  At2g33210  chaperonin, putative similar to SWISS-PROT:Q05046- chaperonin CPN60-2, mitochondrial precursor (HSP60-2) [Cucurbita maxima]; contains Pfam:PF00118 domain, TCP-1/cpn60 chaperonin family  At3g03960  chaperonin, putative similar to SWISS-PROT:P42932- T-complex protein 1, theta subunit (TCP-1-theta) [Mus musculus]; contains Pfam:PF00118 domain, TCP-1/cpn60 chaperonin family  At3g06680  60S ribosomal protein L29 (RPL29B) similar to 60S ribosomal protein L29 GB:P25886 from (Rattus norvegicus)  At3g10610  40S ribosomal protein S17 (RPS17C) similar to 40S ribosomal protein S17 GB:AAD50774 [Lycopersicon esculentum]  At3g16780  60S ribosomal protein L19 (RPL19B) similar to ribosomal protein L19 GB:CAA45090 from [Homo sapiens]  At3g27740  carbamoyl-phosphate synthase [glutamine-hydrolyzing] (CARA) / glutamine-dependent carbamoyl-phosphate synthase small subunit identical to carbamoyl phosphate synthetase small subunit GI:2462781 [Arabidopsis thaliana]  At3g44590  60S acidic ribosomal protein P2 (RPP2D) acidic ribosomal protein P2, maize, PIR:S54179  At3g44750  histone deacetylase, putative (HD2A) contains Pfam domain, PF00096: Zinc finger, C2H2 type; identical to cDNA putative histone deacetylase (HD2A) GI:11066134  At3g47370  40S ribosomal protein S20 (RPS20B) 40S RIBOSOMAL PROTEIN S20 ARABIDOPSIS THALIANA,PID:g1350956  At3g49080  ribosomal protein S9 family protein contains Pfam profile PF00380: ribosomal protein S9  210  Gene locus  Annotation of co-expressed gene  number At3g53890  40S ribosomal protein S21 (RPS21B) ribosomal protein S21, cytosolic - Oryza sativa, PIR:S38357  At3g55010  phosphoribosylformylglycinamidine cyclo-ligase, chloroplast / phosphoribosylaminoimidazole synthetase / AIR synthase (PUR5) identical to phosphoribosylformylglycinamidine cyclo-ligase, chloroplast precursor SP:Q05728 from [Arabidopsis thaliana]; contains  At3g55280  60S ribosomal protein L23A (RPL23aB) various ribosomal L23a proteins  At3g56070  peptidyl-prolyl cis-trans isomerase, putative / cyclophilin, putative / rotamase, putative similar to peptidyl-prolyl cis-trans isomerase, PPIase (cyclophilin, cyclosporin A-binding protein) [Catharanthus roseus] SWISS-PROT:Q39613  At3g57490  40S ribosomal protein S2 (RPS2D) 40S ribosomal protein S2 - Arabidopsis thaliana, SWISSPROT:RS2_ARATH  At4g00810  60S acidic ribosomal protein P1 (RPP1B) similar to acidic ribosomal protein p1  At4g10480  nascent polypeptide associated complex alpha chain protein, putative / alpha-NAC, putative similar to alpha-NAC, non-muscle form [Mus musculus] GI:1666690; contains Pfam profiles PF01849: NAC domain, PF00627: UBA/TS-N domain  At4g12600  ribosomal protein L7Ae/L30e/S12e/Gadd45 family protein Similar to NHP2/L7Ae family proteins, see SWISSPROT:P32495 and PMID:2063628.  At4g13170  60S ribosomal protein L13A (RPL13aC) ribosomal protein L13a -Lupinus luteus,PID:e1237871  At4g25630  fibrillarin 2 (FIB2) identical to fibrillarin 2 GI:9965655 from [Arabidopsis thaliana]  At4g25890  60S acidic ribosomal protein P3 (RPP3A) acidic ribosomal protein P3a - maize, PIR2:T02037  At4g26230  60S ribosomal protein L31 (RPL31B) ribosomal protein L31, Nicotiana glutinosa, U23784  At4g30800  40S ribosomal protein S11 (RPS11B) ribosomal protein S11, Arabidopsis thaliana,PIR2:C35542  At5g02610  60S ribosomal protein L35 (RPL35D) ribosomal protein L35- cytosolic, Arabidopsis thaliana, PIR:T00549  At5g22440  60S ribosomal protein L10A (RPL10aC)  At5g58420  40S ribosomal protein S4 (RPS4D) ribosomal protein S4, Arabidopsis thaliana, PIR:T48480  At5g60670  60S ribosomal protein L12 (RPL12C) 60S RIBOSOMAL PROTEIN L12 (like), Arabidopsis thaliana, PIR:T45883  At5g64140  40S ribosomal protein S28 (RPS28C)  At5g67510  60S ribosomal protein L26 (RPL26B)  211  VI. Genes that are co-expressed with RACK1A, RACK1B and RACK1C Gene locus  Annotation of co-expressed gene  number At1g09690  60S ribosomal protein L21 (RPL21C) Similar to ribosomal protein L21 (gb|L38826). ESTs gb|AA395597,gb|ATTS5197 come from this gene  At1g18540  60S ribosomal protein L6 (RPL6A) similar to 60S ribosomal protein L6 GI:7208784 from [Cicer arietinum]  At1g26880  60S ribosomal protein L34 (RPL34A) identical to GB:Q42351, location of EST 105E2T7, gb|T22624  At1g27400  60S ribosomal protein L17 (RPL17A) similar to GB:P51413 from [Arabidopsis thaliana]; similar to ESTs gb|L33542 and gb|AA660016  At2g01250  60S ribosomal protein L7 (RPL7B)  At2g27530  60S ribosomal protein L10A (RPL10aB)  At2g32060  40S ribosomal protein S12 (RPS12C)  At2g36170  ubiquitin extension protein 2 (UBQ2) / 60S ribosomal protein L40 (RPL40A) identical to GI:166930, GI:166931  At2g36620  60S ribosomal protein L24 (RPL24A)  At2g37190  60S ribosomal protein L12 (RPL12A)  At2g39460  60S ribosomal protein L23A (RPL23aA) identical to GB:AF034694  At2g47610  60S ribosomal protein L7A (RPL7aA)  At3g04840  40S ribosomal protein S3A (RPS3aA) similar to 40S ribosomal protein S3A (S phase specific protein GBIS289) GB:P49396 [Brassica rapa]  At3g05560  60S ribosomal protein L22-2 (RPL22B) identical to 60S ribosomal protein L22-2 SP:Q9M9W1 from [Arabidopsis thaliana]  At3g05590  60S ribosomal protein L18 (RPL18B) similar to GB:P42791  At3g07110  60S ribosomal protein L13A (RPL13aA) similar to ribosomal protein L13A GB:O49885 [Lupinus luteus]  At3g09630  60S ribosomal protein L4/L1 (RPL4A) strong similarity to 60S ribosomal protein L1 GB:P49691  At3g11250  60S acidic ribosomal protein P0 (RPP0C) similar to 60S acidic ribosomal protein P0 GI:2088654 [Arabidopsis thaliana]  At3g11510  40S ribosomal protein S14 (RPS14B) similar to 40S ribosomal protein S14 GB:P19950 [Zea mays]  At3g16080  60S ribosomal protein L37 (RPL37C) similar to ribosomal protein L37 GB:BAA04888 from [Homo sapiens]  At3g23390  60S ribosomal protein L36a/L44 (RPL36aA) similar to ribosomal protein L41 GB:AAA34366 from [Candida maltosa]  212  Gene locus  Annotation of co-expressed gene  number At3g25520  60S ribosomal protein L5 similar to 60S ribosomal protein L5 GB:P49625 from [Oryza sativa]  At3g48930  40S ribosomal protein S11 (RPS11A)  At3g49010  60S ribosomal protein L13 (RPL13B) / breast basic conserved protein 1-related (BBC1)  At3g51800  metallopeptidase M24 family protein similar to SP|P50580 Proliferation-associated protein 2G4 {Mus musculus}; contains Pfam profile PF00557: metallopeptidase family M24  At3g53430  60S ribosomal protein L12 (RPL12B) 60S RIBOSOMAL PROTEIN L12, Prunus armeniaca, SWISSPROT:RL12_PRUAR  At3g56340  40S ribosomal protein S26 (RPS26C) several 40S ribosomal protein S26  At3g60245  60S ribosomal protein L37a (RPL37aC)  At3g60770  40S ribosomal protein S13 (RPS13A) AtRPS13A mRNA for cytoplasmic ribosomal protein S13, Arabidopsis thaliana,AB031739  At4g10450  60S ribosomal protein L9 (RPL90D) ribosomal protein L9, cytosolic - garden pea, PIR2:S19978  At4g15000  60S ribosomal protein L27 (RPL27C)  At4g17390  60S ribosomal protein L15 (RPL15B)  At4g25740  40S ribosomal protein S10 (RPS10A) 40S ribosomal protein S10 - Lumbricus rubellus, PID:e1329701  At4g29410  60S ribosomal protein L28 (RPL28C) unknown protein chromosome II BAC F6F22 Arabidopsis thaliana,PID:g3687251  At4g31700  40S ribosomal protein S6 (RPS6A) ribosomal protein S6, Arabidopsis thaliana, PID:g2662469  At5g02870  60S ribosomal protein L4/L1 (RPL4D) 60S roibosomal protein L4, Arabidopsis thaliana, EMBL:CAA79104  At5g16130  40S ribosomal protein S7 (RPS7C) 40S ribosomal protein S7 homolog - Brassica oleracea, EMBL:AF144752  At5g39740  60S ribosomal protein L5 (RPL5B) ribosomal protein L5, rice  At5g47210  nuclear RNA-binding protein, putative similar to nuclear RNA binding protein GI:6492264 from [Arabidopsis thaliana]  At5g52650  40S ribosomal protein S10 (RPS10C) contains similarity to 40S ribosomal protein S10  At5g56670  40S ribosomal protein S30 (RPS30C)  At5g59850  40S ribosomal protein S15A (RPS15aF) cytoplasmic ribosomal protein S15a, Arabidopsis thaliana, EMBL:ATAF1412  At5g61170  40S ribosomal protein S19 (RPS19C) 40S ribsomal protein S19, Oryza sativa, SWISSPROT:RS19_ORYSA  213  

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