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Abstract Investigating the Role of Syn3 In ABSTRACT INVESTIGATING THE ROLE OF SYN3 IN CHLOROPLASTS by Ramja Sritharan Cohesins play a significant role in chromosome segregation during mitosis and meiosis. However, cohesion-involved proteins have never been characterized in organelles like chloroplasts. In this study, we investigated the subcellular localization and function of an Arabidopsis (Arabidopsis thaliana) SYN3 protein. SYN3 is an Arabidopsis α-kleisin that is essential for megagametogenesis and is enriched in the nucleolus of mitotic and meiotic cells. Previous work has established the role of α-kleisins, including SYN3, in the nucleus; however, α-kleisins have not been shown to be localized to the organelles. SYN3 protein was found to interact with the FtsZ through yeast two-hybrid screening. FtsZ is essential for chloroplast division. To that end, we generated transgenic Arabidopsis plants that over-expressed SYN3 proteins fused to yellow fluorescent protein (YFP). SYN3 was found to be localized to the thylakoids in chloroplasts isolated from Arabidopsis. Over-expression of SYN3-YFP resulted in stunted growth, abnormal phenotype with significantly reduced chlorophyll b and total chlorophyll content of plants. Further, our results show that over-expression of SYN3-YFP may have effects on chloroplast division. INVESTIGATING THE ROLE OF SYN3 IN CHLOROPLASTS A Thesis Submitted to the Faculty of Miami University in partial fulfillment, of the requirements for the degree of Master of Science Department of Chemistry and Biochemistry by Ramja Sritharan Miami University Oxford, Ohio 2017 Advisor: Dr. Carole Dabney-Smith Advisor: Dr. Christopher A. Makaroff Reader: Dr. Michael W. Crowder Reader: Dr. Richard C. Page ©2017 Ramja Sritharan This thesis titled INVESTIGATING THE ROLE OF SYN3 IN CHLOROPLASTS by Ramja Sritharan has been approved for publication by Biochemistry and Department of Chemistry and Biochemistry ____________________________________________________ Advisor: Dr. Carole Dabney-Smith ______________________________________________________ Advisor: Dr. Christopher A. Makaroff _______________________________________________________ Committee chair: Dr. Richard C. Page ____________________________________________________ Reader: Dr. Michael W. Crowder Table of Contents Chapter-1 1 1. Introduction .............................................................................................................. 1 1.1 Cell cycle .............................................................................................................. 1 1.2 Cell Division: Mitosis and meiosis ....................................................................... 2 1.3 Sister chromatid cohesion ..................................................................................... 4 1.4 Cohesin complex ................................................................................................... 5 1.5 Cohesion models on how cohesin complex holds sister chromatids together ...... 7 1.6 Cohesin loading, maintenance and its removal..................................................... 8 1.7 Structure of chloroplast and its function ............................................................... 9 1.8 Protein import into chloroplast ........................................................................... 10 1.9 Prokaryotic chromosome maintenance and segregation ..................................... 11 1.10 Chloroplast chromosomes maintenance ........................................................... 13 1.11 FtsZ protein and its function in chloroplast cell division ................................. 15 1.12 References ......................................................................................................... 16 Chapter 2: Investigating the role of SYN3 in chloroplasts 23 2.1 Introduction ............................................................................................................. 23 2.2 Materials and methods ............................................................................................ 24 2.2.1 Preparation of chloroplast ................................................................................ 24 2.2.2 Preparation of radiolabeled precursors ............................................................ 25 2.2.3 Protein import assays into chloroplasts............................................................ 25 2.2.4 Subcellular fractionation of chloroplasts ......................................................... 26 2.2.5 Immunoblotting................................................................................................ 26 2.2.6 Immunoprecipitation of chloroplast lysates proteins under denaturing conditions .................................................................................................................. 27 2.2.7 Generating transgenic plants using Agrobacterium mediated floral dip method ................................................................................................................................... 28 2.2.8 BASTA selection and genotyping PCR for positive transformants ................ 28 2.2.9 Sample preparation for difference interference contrast microscopy .............. 29 2.2.10 RNA Extraction and qRT-PCR analysis ........................................................ 29 iii 2.2.11 Total protein extraction and immunoblotting ................................................ 30 2.2.12 Measurement of chlorophyll content and chlorophyll fluorescence .............. 30 2.3 Results and Discussion ........................................................................................... 31 2.3.1 Identification of FtsZ 1-1 and FtsZ 2-1 proteins as interacting partners of SYN3......................................................................................................................... 31 2.3.2 Endogenous SYN3 localizes to thylakoid within chloroplasts ........................ 34 2.3.3 SYN3 is not imported into isolated chloroplasts in vitro ................................. 35 2.3.4 Immunoprecipitation of SYN3 in vivo and in vitro ......................................... 37 2.3.5 Screening of transgenic plants by BASTA selection and genotyping PCR..... 40 2.3.6 Over-expression of SYN3 resulted in stunted growth and abnormal phenotype plants ......................................................................................................................... 41 2.3.7 SYN3 expression level in WT and SYN3-YFP O/E lines ............................... 44 2.3.8 Over-expression of SYN3 has no effect on chloroplast division ..................... 46 2.3.9 Reduced total chlorophyll and chl b content of plants due to over expression of SYN3......................................................................................................................... 51 2.4 References ............................................................................................................... 56 Chapter 3: Conclusions 60 3.1 Conclusions ................................................................................ ………………… 60 3.2 References ............................................................................................................... 62 iv List of Tables Chapter-2 Page Table 1 Primers used in this study .................................................................................... 30 Table 2: Interacting partners of SYN3 through yeast-two-hybrid analysis. ..................... 31 Table 3 : Phenotypes of SYN3-YFP O/E plants. .............................................................. 42 Table 4 : Chloroplast and cell characteristics of SYN3-YFP O/E plants ......................... 50 Table 5: The content of total chlorophyll (Chl), Chl a, Chl b, and Chl a/b ratios. ........... 55 v List of Figures Chapter-1 Page Figure 1.1: Schematic diagram of the mitotic cell cycle ................................................... 2 Figure 1.2: Schematic diagram of the meiotic cell cycle. ................................................... 3 Figure 1.3: Cohesion sites for sister chromatids in a chromosome.. .................................. 5 Figure 1.4: Schematic diagram of the core cohesin complex. ............................................ 6 Figure 1.5: A model for cohesin loading and regulators…. ............................................... 9 Figure 1.6 : The interior structure of a chloroplast.. ......................................................... 10 Figure 1.7 : The E. coli SMC complex, MukBEF complex. .......................................... 13 Figure 1.8 : A model for chloroplast division in plants.. .................................................. 15 Chapter-2 Figure 2.1: SYN3 immunoprecipitates FtsZ 1-1 and 2-1 in vitro.. ................................... 32 Figure 2.2: SYN3 localizes in chloroplasts. ...................................................................... 33 Figure 2.3: Sub-organellar localization of SYN3 in P. sativum chloroplasts. .................. 34 Figure 2.4: Sub-organellar localization of SYN3 in A. thaliana chloroplasts. ................. 35 Figure 2.5: In vitro expressed SYN3 failed to import into isolated chloroplasts ............. 37 Figure 2.6: An immunoprecipitation of SYN3 in vitro. ................................................... 38 Figure 2.7: In vivo immunoprecipitation of chloroplast lysate samples ........................... 39 Figure 2.8: Genotyping PCR screening of BASTA resistant SYN3-YFP lines. .............. 41 Figure 2.9: Phenotypic analysis of 35S-SYN3-YFP transgenic Arabidopsis plants .......
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