Role of AtCIPK16 in Arabidopsis abiotic tolerance Wenmian Huang A thesis submitted for the degree of Doctor of Philosophy School of Agriculture, Food and Wine Faculty of Sciences The University of Adelaide May 2015 Table of Contents Table of Contents………………………………………………………..……………………i List of Figures………………………………………………………..……………………….vii List of Tables…………………………………………………………….…………………..xi List of Abbreviations……………………………………………...……………………….xiii Abstract……………………………………………………………………………………..xvii Declaration………………………………………………………………………………….xix Acknowledgments…………………………………………………………………………..xxi Chapter 1: Literature Review and Research Aims…………………………………………..1 1.1 Salinity……………………………………………………………………………. ….1 1.1.1 Impacts of salinity………………………………………………………………… ….1 1.1.2 Effects of salinity stress on plants………………………………………………… ….1 1.1.3 The plants’ tolerance mechanism to salt stress………………………………………..2 1.2 Calcium signalling pathways….……………………………………………………..7 1.2.1 Structural characterisation of CBL……………………………………………….....8 1.2.2 Structural characterisation of CIPK…………………………………………………..9 1.2.3 Specificity of the CBL-CIPK signalling pathway…………………………………..12 1.2.4 Function of the CBL-CIPK signalling pathway……..………………………………13 1.3 AtCIPK16……………………………………..……………………………………..18 1.3.1 Potential role of AtCIPK16 in salinity tolerance………………………………….19 1.4 Research aims………………………………………………………………... ……...20 Chapter 2: General materials and methods……………………………………………..21 2.1 Plant materials……………………………………………………………………..21 2.2 Plant growth facilities……………………………………………………………….21 2.3 Plant growth in soil…………………………………………………..….………….21 2.4 Plant growth in hydroponics……………………………………………………….23 2.5 Plant growth on plates containing Murashige and Skoog media………….……...…25 2.6 DNA extractions………………………………………………………………..……25 2.6.1 Phenol/chloroform/iso-amyl alcohol method………………………………….…..25 2.6.2 Edwards DNA extraction method…………………………………………… ……...26 2.7 Agarose gel electrophoresis - DNA……………………………………………….27 2.8 DNA extraction from agarose gels…………………………………………………..27 2.9 DNA sequencing…………………………………………………………….………27 2.10 RNA extractions and agarose gel electrophoresis………………………….………..28 2.11 cDNA synthesis…………………………………………………………..…………29 2.12 Polymerase chain reaction (PCR)…………………………………………...………30 2.12.1 Routine gDNA/cDNA PCR…………………………………………………...……..30 i 2.12.2 High-fidelity PCR………………………………………………………………...…31 2.12.3 Colony PCR…………………………………………………………………..……32 2.13 Cloning PCR products into entry vectors…………………………………………33 2.14 Preparation of competent cells (Escherichia coli)…………………..………………36 2.15 Transformation of plasmid DNA into E.coli cells…………………………………36 2.16 Isolation of plasmid DNA from E.coli cells…………………………………………37 2.17 Restriction enzyme digestion of plasmid DNA……………………..………………37 2.18 LR reactions…………………………………………………………………………38 2.19 Agrobacterium-mediated stable transformation of Arabidopsis………….…………40 2.19.1 Preparation of competent A. tumefaciens AGL1 cells………………………………40 2.19.2 Transformation of plasmid DNA into A.tumefaciens AGL1 cells………..…………40 2.19.3 Transformation by floral dipping……………………………………………………41 2.20 Selection of transformants………………………………………..…………………41 2.20.1 Selection in soil…………………………………………………..…………………41 2.20.2 Selection on MS plate…………………………………………….…………………41 2.21 Statistical analysis…………………………………………………..………………42 Chapter 3: Identification of upstream regulators of AtCIPK16…………………………43 3.1 Introduction……………………………………………………………………..…..43 3.2 Chapter aims…………………………………………………………………………46 3.3 Materials and methods……………………………………………………………..46 3.3.1 Yeast two hybrid assays………………………………………………………..……46 3.3.1.1 Cloning for yeast two hybrid assays…………………………………………….…..46 3.3.1.2 Preparation of yeast strain AH109 from stock………………………………..……..51 3.3.1.3 Transformation of constructs into S. cerevisiae………………………………………….51 3.3.1.4 Yeast two-hybrid assay………………………………………………………………52 3.3.1.5 Isolation of plasmid DNA from S. cerevisiae…………………………………………….53 3.3.2 Bimolecular fluorescence complementation (BiFC) assay using both transient expression and stable expression………………………………………………...….53 3.3.2.1 Cloning of AtCBLs and AtCIPK16 into BiFC assay vector for transient expression in mesophyll protoplast……………………………………………………………...…54 3.3.2.2 Cloning of AtCBLs and AtCIPK16 into BiFC assay vector for Agrobacterium-infiltration in Arabidopsis leaves, tobacco leaves and stable constitutive over-expression in Arabidopsis plants……………………………….....58 3.3.2.3 Transient expression of AtCBLs-AtCIPK16 in Arabidopsis mesophyll protoplasts...60 3.3.2.4 Transient expression of AtCBLs-AtCIPK16 in Arabidopsis leaves using Agro-infiltration……………………………………………………………………..61 3.3.2.5 Transient expression of AtCBLs-AtCIPK16 in tobacco leaves (Nicotiana benthamiana) using Agro-infiltration……………………………………………….62 3.3.2.6 Stable constitutive over-expression of AtCBLs-AtCIPK16 in Arabidopsis ecotype ii Col-0…………………………………………………………………………………63 3.3.2.7 Fluorescence imaging by confocal microscopy……………………………………64 3.4 Results………………………………………………………………………………65 3.4.1 Vector construction for a yeast two hybrid assay…………………………………..65 3.4.2 Yeast two hybrid assay shows AtCIPK16 interacts with 6 AtCBL proteins………..67 3.4.3 Vector construction for Bimolecular Fluorescence Complementation (BiFC) assay in Arabidopsis mesophyll protoplast…………………………………………………..68 3.4.4 Bimolecular fluorescent complementation (BiFC) assay in Arabidopsis mesophyll protoplast……………………………………………………………………………71 3.4.5 Vector construction for a Bimolecular Fluorescence Complementation (BiFC) assay using either Agro-infiltration of Arabidopsis and tobacco leaves, or stable expression in Col-0………………………………………………………………………………75 3.4.6 Subcellular localization using Agro-infiltration in Arabidopsis leaves…………….78 3.4.7 Subcellular localization using Agro-infiltration in tobacco leaves……………….88 3.4.8 Localization of AtCBLs-AtCIPK16 complexes using stable expression in Arabidopsis ecotype Col-0…………………………………………………………..91 3.5 Discussion……………………………………………………………………...……93 3.5.1 Interacting partners of AtCIPK16 in yeast two hybrid assays……………………93 3.5.2 Interactions and localizations of AtCBL-AtCIPK16 in BiFC assays………………..95 3.6 Summary……………………………………………………………………..……102 Chapter 4: Identification of downstream targets of AtCIPK16…………………………103 4.1 Introduction……………………………………………………………...…………103 4.2 Materials and methods……………………………………………………………105 4.2.1 Pull-down assay……………………………………………………………………105 4.2.1.1 Peptide antigen design……………………………………………………………105 4.2.1.2 Generation of a specific rabbit IgG antibody…………………………………….106 4.2.1.3 Production of recombinant protein………………………………………………107 4.2.1.4 SDS Polyacrylamide Gel Electrophoresis…………………………………………110 4.2.1.5 Western blot for identification of the expected band on the gel……………………111 4.2.1.6 Optimization of recombinant protein synthesis.…………………………………112 4.2.1.7 Purification of denatured protein…………………………………………………113 4.2.1.8 Refolding of purified denatured protein……………………………………………114 4.2.2 Yeast two hybrid assay……………………………………………………………115 4.2.2.1 Cloning for yeast two hybrid assay ………………………………………..………115 4.2.2.2 Analysis of the protein sequences of AtHKT1;1, AtSOS1 and AtAKT1 …………115 4.4 Results…………………………………………………………………………..…117 4.4.1 Alignment of the protein sequences of AtCIPK16 with 26 AtCIPKs in Arabidopsis and peptide antigen design…………………………………………………………117 4.4.2 Construction of plasmid for protein synthesis……………………………………121 iii 4.4.3 Recombinant His-AtCIPK16 was obtain from E.coli and recognized by anti-AtCIPK16 antibody in Western blot…………………………………………121 4.4.4 Optimization of recombinant protein synthesis…………………………………123 4.4.4.1 Expression of recombinant His-AtCIPK16 in two codon bias-adjusted E. coli strains showed no improvement in protein yield………………………………………..124 4.4.4.2 Low temperature induction shows no improvement on soluble protein yield……126 4.4.4.3 Induction of His-AtCIPK16 using 0.2 % L-arabinose resulted in the maximum yield of insoluble recombinant protein…………………………………………………..126 4.4.5 Recombinant His-AtCIPK16 was successfully denatured by Guanidine-HCl and purified by using cobalt chelating resin………………………………………….128 4.4.6 Purified denatured His-AtCIPK16 was refolded using gradual dialysis…………128 4.4.7 Construction of vector for yeast two hybrid assay…………………………………129 4.5 Discussion……………………………………………………………………….…133 4.5.1 Expression of recombinant protein His-AtCIPK16………………………………133 4.5.2 Potential downstream targets of AtCIPK16………………………………………134 4.5.3 The alignment of 26 AtCIPKs shows unique regions of AtCIPK16 in functional motifs…………………………………………………………………………….…135 4.5.4 Future work…………………………………………………………………..……140 4.6 Summary……………………………………………………………………..……142 Chapter 5: Dissecting the role of AtCIPK16 in salinity tolerance……………………..143 5.1 Introduction……………………………………………………………………..…143 5.2 Chapter aims………………………………………………………………………145 5.3 Methods……………………………………………………………………………145 5.3.1 Plant materials for complementary function analysis…………………………….145 5.3.2 Cloning of AtCIPK16 into constitutive expression vector…………………………145 5.3.3 Transformation of plasmid DNA into A.tumefaciens AGL1 competent cells….…146 5.3.4 Stable constitutive over-expression of AtCIPK16 in sos2 knockout lines……….146 5.3.5 Selection of transformants of AtCIPK16-sos2……………………………………146 5.3.6 Phenotyping of T2 transgenic lines that constitutively over-expresses AtCIPK16 in sos2 knockout lines under salt stress………………………………….……………147 5.3.7 Biomass and flame photometry measurements…………………………………….148 5.3.8 Genotyping……………………………………………………………………..….148 5.3.9 RT-PCR……………………………………………………………………………149