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

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

This thesis titled

INVESTIGATING THE ROLE OF SYN3 IN CHLOROPLASTS

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

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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

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

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 ...... 43 Figure 2.10: Immunoblot analysis of SYN3 protein in transgenic plants...... 45 Figure 2.11: qRT-PCR analysis of relative SYN3 expression in SYN3-YFP O/E plants. 46 Figure 2.12: Observation of chloroplast division phenotypes of mesophyll cells...... 47 Figure 2.13: Graph of chloroplast number relative to mesophyll cell area...... 48 Figure 2.14: Total chloroplast area per mesophyll cell and mesophyll cell area...... 49 Figure 2.15: Effective PSII quantum yield of SYN3-YFP O/E plants...... 52 Figure 2.16 : Non-photochemical quenching of SYN3-YFP O/E plants...... 54

Dedication

I would like to dedicate this thesis to my late sister, Niraja Sritharan.

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Acknowledgements

Foremost, I would like to acknowledge my advisors Dr. Christopher A. Makaroff and Dr. Carole Dabney-Smith for their continuous support, patience, immense knowledge, and guidance throughout my research project. I would like to thank the rest of my committee: Dr. Richard C. Page, Dr. Michael W. Crowder, and Dr. Rachael Morgan-Kiss for their encouragement and insightful comments.

I would like to thank Martin Smith for teaching me the lab techniques and making our lab well organized and, the best environment in which to work. I would also like to thank Carole and Martin for being there for me whenever I needed help and supporting me as my academic parents during the past years.

I would like to acknowledge Dr. Li Yuan who completed the initial work of this study. My sincere thanks also go to Matt Duley for teaching me microscopic techniques and Gregory Cook for helping me with photosynthetic measurements.

I would also like to thank my fellow labmates Paul New, Aman Habtemichael, Jorge Escobar, Katie Eudy and Shiny Weerakoon for being great friends, and I cannot imagine having better labmates. Paul New, thank you so much for all your help and advice in writing and being great fellow graduate student. I would also like to thank Dr. Xiaohui Yang, Dr. Kuntal De, Sayanthan Mitra, Nefertiti Muhammad, Dr. QianQian Ma, Dr. Debjani Pal, Dr. Lei Zhang, and Dr. Amanda Storm. I would also like to thank my undergraduates Maren Osterholt, Elias Zayer, and Kendyl Kennon for helping me with my experiments and other undergraduates Gwendolyn Thomas, Katherine Sieman, Kirsten Gonzales, and Krystina Hird for being lovely labmates.

I would like to thank all friends in Miami for making my life at Miami fun and wonderful. Especially my best friend Katie Eudy, thank you so much for being there for me whenever I needed and for sharing your thoughts with me, going on canoeing trips, playing tennis and badminton, and teaching me to cook and drive. Life in Oxford would not have been as wonderful without you.

Last but not least, I would like to acknowledge my family: my parents Sritharan and Vimalambikai, and my sisters Poorvaja and Niraja for supporting me throughout my life.

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Chapter-1

1. Introduction

This thesis focuses on exploring the function of SYN3 in chloroplasts. The first chapter serves as an introduction to the important concepts, including cell division, an overview of nuclear cohesin proteins function, structure and function of chloroplasts, chromosome maintenance and division in bacteria and chloroplasts, and finally, how nuclear cohesion proteins may function in chromosome maintenance and division processes.

1.1 Cell cycle

The cell cycle is a complex series of events that a proliferative cell must go through before it can divide (Schafer, 1998). The eukaryotic cell cycle has four sequential phases: interphase (G1), DNA synthetic phase (S), post-synthetic phase (G2), and mitosis (M), nuclear cell division (Francis, 2009; Schafer, 1998; Tyson, 2002). The specific phases of the cell cycle and the cell cycle check-points that govern the transitions between them prevent abnormal cells from replicating (Amanda, 2014).

In the G1 phase, the cell prepares for DNA synthesis based on the signals from growth signaling networks. Following completion of G1 phase, the cells enter S-phase. During S-phase, the chromosomes undergo DNA replication to produce identical sister chromatids held together by proteins called cohesins (Francis, 2009; Nasmyth, 1994; Nasmyth, 2000). Following the completion of chromosomal replication, the cell enters the G2 phase during which the cell prepares for entry into mitosis. Finally, the cell undergoes mitosis and cell division (Nasmyth, 1994; Schafer, 1998; Vermeulen, 2003)

As the primary focus of mitosis, the proper organization of chromosomes is key to successful replication. Chromosomes are the universal carriers of genetic information in cellular systems. They are formed as huge macromolecular assemblies of genomic DNA and a collection of histone proteins. During cell division, these chromosomes are divided such that a copy of each chromosome is transferred to the two new daughter cells that are produced (Uhlmann, 2004).

Cell division is accompanied by the segregation of the two replicated sister genomes in S-phase. Cell division is essential for proliferation and growth of all living organisms. Errors in this process leads to aneuploidy (cells with multiple numbers of chromomosomes) or a lack of chromosomes. Aneuploidy later in human life is often associated with the development of cancer (Uhlmann, 2004).

1.2 Cell Division: Mitosis and meiosis

Meiosis and mitosis are two different types of cell division processes that occur during development in eukaryotic cells (Amanda, 2014). Mitosis is a fundamental process for vegetative reproduction and tissue formation, while meiosis is the basis for sexual reproduction through the production of gametes. Mitosis takes place to produce daughter cells that are equal to their mother cells genetic content. Meiosis generates cells that contain half their mother cells genetic content, which ultimately leads to the production of gametes. Upon fertilization, gametes can fuse to combine their genetic contents in a zygote, thereby closing the sexual reproduction cycle (Gunjan, 2012).

Figure 1.1: Schematic diagram of the mitotic cell cycle. The chromosomes (shown in red and light green) are replicated during S phase and segregate during M phase to produce two new diploid daughter cells (Adapted from Marston, 2004)

Mitosis has four sub phases: prophase, metaphase, anaphase, and telophase. During prophase, replicated chromosomes condense into compact structures, while the spindle apparatus migrates

2 to opposite poles of the cell. In early metaphase, condensed chromosomes are aligned on the midplane of the mitotic spindle. In late metaphase, condensed chromosomes start to separate as the cell transitions into anaphase. During anaphase, the sister chromatids begin to separate to opposite poles of the spindle microtubules. In telophase, daughter nuclei form and the cell begins to divide through cytokinesis yielding two daughter cells which are identical to the parent cell (Figure 1.1) (Tyson, 2002).

Meiosis is a complicated cell division process where cells undergo two rounds of cell divisions named meiosis I and meiosis II. During meiosis I, the homologous chromosomes segregate and the sister chromatids are held together by cohesin complexes (Lee, 2001a; Lee, 2001b; Miyazaki, 1994). The sister chromatids of each chromosome segregate at meiosis II (Armstrong, 2003; Dawe, 1998; Miyazaki, 1994). Meiosis produces four haploids that are not identical to each other or to the parent cell.

Figure 1.2: Schematic diagram of the meiotic cell cycle. In meiotic cell cycle, chromosomes replicate during S phase followed by two chromosome-segregation phases, meiosis I and meiosis II. Chromosomes (shown in red and light blue) are segregated in meiosis I and sister chromatids are segregated during meiosis II producing non-identical haploid cells (Adapted from Marston, 2004)

In all organisms, premeiotic S-phase is longer than pre-mitotic S-phase to ensure proper segregation of homologous chromosomes. During S-phase, chromosomes are replicated, and sister chromatid cohesion is established. Meiotic prophase I is subdivided into five stages: leptotene, zygotene, pachytene, diplotene, and diakinesis. During leptotone, homologous

3 chromosomes begin aligning together. During zygotene and pachytene DNA crossovers occur to facilitate recombination between homologous chromosomes. crossovers involve chromosomal synapsis and the formation of the synaptonemal complex (Amanda, 2014; McKee, 2004). In diplotene, the chromosomes begin to desynapse and condense to form bivalents. These bivalents align at the metaphase plate during metaphase I followed by segregation of homologous chromosomes that starts at anaphase I. Finally, the homologous chromosomes move to opposite poles of the cell during telophase I (Figure 1.2) (Amanda, 2014).

The second meiotic division is quite similar to mitosis and the separation of sister chromatids. However, during meiosis I and interkinesis (which is the period between meiosis I and meiosis II), centromeric cohesion must be protected to ensure cohesion between sister chromatids at metaphase II (Laurence, 2013). The sister chromatids remain attached at their centromeres by cohesin (Amanda, 2014). During prophase II, chromosomes start to decondense and then recondese. At metaphase II, sister chromatids attach to the spindles and during anaphase II spindles pull the sister chromatids apart. Cytokinesis finishes meiosis II with the production four haploid cells (Laurence, 2013).

1.3 Sister chromatid cohesion

Sister chromatid cohesion is established and maintained by a protein complex called cohesin. Sister chromatid cohesion ensures that replicated chromosomes are distributed properly into each daughter cell during cell division (Díaz-Martínez, 2008; Miyazaki, 1994; Uhlmann, 2004). During meiosis and mitosis, cohesion is required to keep the sister chromatids together until their separation at anaphase (Gerton, 2005; Hirano, 2000; Miyazaki, 1994). Sister chromatids remain physically linked as pairs from the time of their synthesis by sister chromatid cohesion (Guacci, 1994; Hirano, 2000; Miyazaki, 1994).

There are at least two types of cohesin sites. Cohesin associated with the arms of the chromosome and cohesin associated with the centromere and pericentric domain (Figure 1. 3).

Figure 1. 3: Cohesion sites for sister chromatids in a chromosome. Cohesion sites (shown as red ovals) are highly concentrated at centromere region where the two chromatids are linked together. In addition to centromere cohesion, cohesin is also present along the arms of the chromatids (Adapted from Gerton, 2005).

1.4 Cohesin complex

Cohesin complexes are important for the pairing of sister chromatids and the establishment of sister chromatid cohesion. Cohesin complexes have several additional functions, including regulation of gene expression, DNA double-strand break (DSB) repair, chromosome condensation and bipolar attachment to the spindle apparatus (Mehta, 2013; Peters, 2008). Defects in cohesin or cohesion regulators can lead to human diseases termed “cohesinopathies”, where defects in sister-chromatid cohesion can lead to genome instability in the form of aneuploidy, abnormal chromosomal translocations, and defects in DNA repair (Revenkova., 2005; Liu, 2008; Peters, 2008).

Cohesin is a multi-protein subunit complex, which is highly conserved in eukaryotes (Peters, 2008). The core cohesin complex consists of four subunits (Figure 1.4): the structural maintenance of chromosome (SMC) proteins, SMC1 and SMC3, and two non-SMC proteins, sister chromatid cohesion 3 (SCC3) and an α-kleisin family protein SCC1 or its meiotic counterpart REC8 (Guacci, 1997; Hirano, 2000; Lee, 2001b; Michaelis, 1997; Uhlmann, 2001).

Figure 1.4: Schematic diagram of the core cohesin complex. SMC complex fold back at the hige domain and form a trimer when it bridges with α-kleisin (SCC1). SCC3 associates to SMC1 and SCC1 (Adapted from Campbell, 2002)

SMC1 and SMC3 are part of large family proteins called the structural maintenance of chromosomes super family proteins (Hirano, 2002). SMC proteins have been found in almost all organisms from bacteria to humans (Revenkova, 2005; Hirano, 2002; Nasmyth, 2001; Thomas, 2016). SMC proteins have five distinct domains: an amino-terminal and carboxy-terminal globular domains, two long coiled-coil domains, and a hinge domain. SMC polypeptides form a long intramolecular antiparallel coiled-coil structure by folding back at the hinge domain. Two self-folded SMC monomers associate via hinge-hinge interactions to form V-shaped dimers, which represent the functional unit of SMC (Haering, 2002; Hirano, 2002). These hetero dimers associate with different proteins to form a specific cohesin complex that maintains sister chromatid cohesion during, chromosome condensation, DNA repair, and recombination (Hirano, 2002; Nasmyth, 2002). SCC3 binds and associates with C-terminus of SCC1 (Haering, 2002). SCC3 contain a heat repeat domain which is responsible for protein interactions (Neuwald, 2000). The structural and functional roles of SCC3 are poorly understood. However, multiple different forms of the SCC3 have been identified in human cells (Losada, 2000).

α-kleisins are important components of mitotic and meiotic cohesin complexes. The α-kleisins include SCC1/REC8, which are important for chromosome segregation. They help create a ring- like structure by their interactions with SMC proteins. The N- and C-termini of SCC1 bind the head domains of SMC3 and SMC1, respectively (Bose, 2010; Hirano, 2000; Nasmyth, 2002). The meiotic cohesin complex is different from the mitotic cohesin complex in that the mitotic α- kleisin subunit SCC1 is replaced by a meiosis specific paralog called REC8 ( Klein, 1999).

In Arabidopsis, four SCC1 orthologs have been identified and termed synaptic mutant (SYN) proteins 1 to 4: SYN1, SYN2, SYN3, and SYN4 (Bai, 1999; Dong, 2001; Hirano, 2000; Jiang , 2007). SYN1 is the Arabidopsis REC8 ortholog. A mutation in SYN1 leads to defects in male and female fertility with meiocytes exhibiting defects in sister chromatid cohesion, chromosome condensation, and homologous pairing (Bai, 1999; Cai, 2003; Peirson , 1997) SYN2 and SYN4 are SCC1 orthologs and function in mitosis and in DNA repair after exposure to ionizing radiation (Costa-Nunes, 2006; Dong, 2001).

A BLAST alignment analysis confirmed that SYN3 belongs to the RAD21/REC8 family (Dong, 2001). Localization and genetics studies showed that SYN3 is found in the nucleolus of both mitotic and meiotic cells and is important for megagametogenesis (Ling, 2007). Moreover, SYN3 RNA interference (RNAi) plants showed defects in both male and female meiosis (Yuan, 2012). Furthermore, knockdown of SYN3 affected the transcription of genes that are involved in recombination and synapsis during female and male meiosis (Yuan, 2012). Recent studies have suggested that plant growth and development are sensitive to high level SYN3 expression, which can affect vegetative growth (Yuan, 2014). Further, expression of SYN3 from the 35S promoter leads to a reduction in SYN1 mRNA levels, which in turn appears to lead to alterations in sister chromatid cohesion, homologous chromosome synapsis and synaptonemal complex formation during both male and female meiosis (Yuan, 2014).

1.5 Cohesion models on how cohesin complex holds sister chromatids together

Various models have been proposed on how cohesin complexes hold sister chromatids together. These models include the multimeric rod shape model (Surcel, 2008), the one ring model

(Gruber, 2003; Haering, 2002), the two ring model (Campbell, 2002; Nasmyth, 2005), and the multimeric bracelet model (Huang, 2005; Nasmyth, 2005). Among these four models, the one ring model is the most widely accepted model (Campbell, 2002; Gunjan, 2012; Haering, 2002; Nasmyth, 2005).

In the one ring model, cohesin forms a ring like structure that encircles DNA to facilitate sister chromatid cohesion (Gruber, 2003; Haering, 2008; Haering, 2002; Hirano, 2000; Nasmyth, 2011; Nasmyth, 2009). SMC dimeric complexes form these ring complexes entrapping DNA by combining with a subunit of an α-kleisin at the nucleotide binding domain (Campbell, 2002; Thomas, 2016). The SMC1/SCC1/SMC3 complex forms a ring like tripartite structure with a central hole large enough for two nucleosomal DNA fibers (Figure 1.4) (Suzuki, 2013; Thomas, 2016). Once the DNA strands are trapped inside the cohesin complex, a mechanism exists to ensure that sister chromatids cannot exit due to the tension produced by the metaphase spindle (Gruber, 2003; Haering, 2002; Thomas, 2016)

1.6 Cohesin loading, maintenance and its removal

Cohesin loading appears to occur throughout the cell cycle, whereas, cohesion establishment is coupled with DNA replication. Several peripheral proteins are responsible for establishing and maintaining sister chromatid cohesion. The function of these proteins varies depending on the stage of cell cycle, including whether establishing cohesion during S-phase or maintaining cohesion during mitosis/meiosis (Lee, 2001(b)).

The SCC2/SCC4 complex directs cohesin onto the chromatin throughout the cell cycle (Ciosk, 2000). Cohesion establishment is coupled to DNA replication and requires Eco1/Ctf7 (Lee, 2001(b)). The Eco1/Ctf7 acetyltransferase acetylates conserved lysine residues in SMC3, which in turn inhibits the anti-establishment function of the Wapl-Pds5-SCC3 complex and therefore helps in establishing cohesion during S phase (Lee, 2001(b)).

In the beginning of mitosis, cohesion is removed from the chromosomes arms in a process mediated by Wapl. At the end of mitosis, proteolytic cleavage of SCC1 leads to the removal of cohesin from chromosomes (Nasmyth, 2011). Removal of cohesin allows the separation of sister chromatids by pulling them to opposite poles of the cell (Hirano, 2000). However, in meiosis,

8 cohesion removal from chromosomes occur in a stepwise manner. During the metaphase I to anaphase I transition, REC8 subunits of arm-associated cohesin complexes is cleaved. REC8 in centromeric and pericentromeric cohesin complexes remains protected until the metaphase II to anaphase transition (Pasierbek, 2001).

Figure 1.5: A model for cohesin loading and regulators. The SCC2/SCC4 complex loads cohesin on-to chromosomes and Pds5-Wapl-Scc3 releases cohesin from chromosomes. During S phase, cohesion is established by the acetylation of lysine residues on SMC3 by Eco1 (Adapted from Thomas, 2016).

1.7 Structure of chloroplast and its function

Chloroplasts evolved from an event called endosymbiosis in which a photosynthetic prokaryotic was taken up by an eukaryotic host cell (Bogorad, 1975; McFadden, 2001). Chloroplasts are double membrane bound organelles that have a system of three membranes: the outer membrane, the inner membrane, and thylakoid membranes. The outer and inner membrane system encapsulates a structure called the stroma, which is the dense fluid matrix of the chloroplast that makes up most of the internal volume. The thylakoid membrane is the network of internal membranes where photosynthetic pigments and protein complexes are embedded (Cooper, 2000)

The most important function of the chloroplast in a plant cell is photosynthesis. In addition to chloroplast function in photosynthesis, chloroplasts are the central hubs of plant cell metabolism. The metabolic functions of the chloroplast include nitrogen and sulfur metabolism and synthesis of amino acids, fatty acids, lipids, aromatic and nonaromatic amino acids, isoprenoids (sterols and carotenoids), purine and pyrimidine bases, and tetrapyrroles, including chlorophyll and heme (Maple., 2004; Lopez-Juez, 2007; Mullet, 1988; Neuhaus, 2000).

Figure 1.6 : The interior structure of a chloroplast. In addition to chloroplast envelope double membrane, chloroplasts have thylakoid membrane. These three membrane systems jointly make three compartments in chloroplasts (Adapted from Cooper, 2000).

1.8 Protein import into chloroplast

Chloroplasts are ubiquitous organelles that carry out essential functions. Following the endosymbiotic event, most of the internalized photosynthetic organisms genes were either lost or transferred into the host nuclear genome (Martin, 1998; Miyagishima, 2011; Sugiura, 1989). Therefore, the size of the plastid genome has been reduced to less than one tenth of the free living cyanobacterial genome (Miyagishima, 2011).

The plastid genome encodes only about 100 proteins. Most plastid proteins are encoded by nuclear genes and synthesized as precursor proteins with a transit peptide in the cytoplasm (Chua, 1978; Highfield, 1978; Jarvis, 2008; Keegstra, 1989). Cytosolic precursor proteins have six possible destinations: outer envelope membrane (OEM), inner envelope membrane (IEM), 10 stroma, thylakoid membrane, thylakoid lumen, and intermembrane space (IMS). Because of this structural complexity, the routing of chloroplast proteins is a complex process (Jarvis, 2008; Keegstra, 1999).

Most of the proteins required for fully functional chloroplasts are synthesized in the cytosol. Therefore, protein import mechanisms are required to transport these precursor proteins from the cytoplasm to the internal compartments of chloroplasts. The overall process of protein import into the chloroplast can be separated into several steps. They include the association of precursor proteins with the outer envelope membrane, transport of the polypeptide across the outer and inner envelope membrane, and the removal of transit peptide by a stromal processing peptidase (Keegstra, 1989).

The transport of polypeptides across these envelopes is carried out by the Toc (translocase of the outer envelope of chloroplasts) and Tic (translocase of the inner envelope of chloroplasts) complexes (Jarvis, 2004; Keegstra, 1999; Ma, 1996). Once the precursor proteins are at the chloroplast surface, transport through the Toc/Tic begins. Transport through the Toc/Tic complex is an active process that consumes energy at distinct stages where the transport process itself can be divided into three distinct steps. The first step is the energy independent reversible binding of a transit peptide with the receptor components of Toc complex. The second is an ATP and GTP energy requiring irreversible step, in which proteins get inserted into the Toc complex and interact with Tic complex. In the last step, precursor proteins are transported followed by the transit peptide being cleaved by a stromal processing peptidase in the stroma (Jarvis, 2008).

Transit peptides are an N-terminal sequence of between 20 and 100 amino acids; they have a high content of hydroxylated (serine and threonine) and small amino acids and a low content of large and acidic amino acids. Therefore, transit peptides generally have a positive charge overall (Keegstra, 1989; Lopez-Juez, 2007)

1.9 Prokaryotic chromosome maintenance and segregation

In unicellular bacteria, chromosome segregation is strictly coordinated with the cell cycle. Most bacteria contain a single circular chromosome that replicates uni or bi-directionally from a unique origin called the origin of replication (oriC). In bacteria, the spatial chromosome

11 organization is determined by the position of oriC (Adams, 2014; Badrinarayanan, 2015; Wang , 2013). Chromosomal DNA is replicated and daughter chromosomes are segregated into daughter cells prior to cell division (Hiraga, 1992).

In most bacterial species except Escherichia coli and some γ-proteobacteria, chromosome segregation depends on two proteins, ParA and ParB (Easter, 2002; Gerdes, 2010; Mierzejewska, 2012). ParB assembles the nucleoprotein complex by binding to parS sites which are clustered around oriC. After the initiation of replication, ParB complexes interact with the ATPase ParA which moves the ParB nucleoprotein complexes bi- or unidirectionally toward the cell pole (Easter, 2002; Gerdes, 2010; Leonard, 2005; Lutkenhaus, 2012).

Another key player in chromosome segregation is the SMC/ScpA/ScpB complex, or the related MukB/MukE/MukF complex found in E. coli and other γ-proteobacteria. The SMC/ScpA/ScpB complex consists of a SMC protein, a kleisin subunit called ScpA, and third protein called ScpB. In the MukB/MukE/MukF condensin complex, MukB, is the structural and functional analog of SMC; it is composed of a hinge domain, long coiled-coil arms, and head domains that form ATP binding pockets. MukE binds to the head domain of MukB via binding to MukF to form the complete complex (Figure 1.7) which contributes to chromosome segregation. MukF, which is a member of the kleisin protein family, forms a stable complex with MukE in vivo (Graumann, 2009; Hiraga, 1992; Hiraga, 2000; Weitao, 2000; Yamanaka, 1996). The E. coli MukBEF complex has been shown to play an important role in organizing chromosome arms into separate cell halves in E.coli and contributes to the symmetry of segregation of chromosome arms (Danilova, 2007).

Figure 1.7 : The E. coli SMC complex, MukBEF complex. MukB consists of three domains, a head ATPase, a hinge dimerization and a long intramolecular coiled-coil domain. MukBEF complex is formed by a MukB dimer bridged by a kleisin MukF which associates with MukE (Adapted from Nolivos, 2014).

The MukBEF complex clusters around the oriC region; however, its mechanism of recruitment is unknown (Danilova, 2007). It is also not clear how MukBEF contributes to chromosome segregation. Recent studies have shown that MukBEF directly stimulates Topoisomerase IV, which implies that MukBEF may play a role in disentangling sister chromosomes (Hayama., 2010; Li, 2010; Nicolas, 2014) Once chromosome replication and segregation are completed, the cell undergoes division. In bacteria, division occurs by binary fission and in most species, division is initiated by the assembly of the FtsZ into a ring like structure (Z-ring) at the nascent division site (Bi, 1991). The Z-ring then acts as a dynamic platform for the assembly of the division machinery (Egan, 2013; Lutkenhaus, 2012).

1.10 Chloroplast chromosomes maintenance

The chloroplast requires a substantial number of nuclear encoded proteins to be imported from cytoplasm to carry out photosynthesis. The chloroplast genome (cpDNA) encodes a considerable number of genes required for photosynthesis as well as transcription and translation. Therefore, maintenance of the chloroplast genome is necessary for chloroplast function (Sugiura, 1992).

The chloroplast genome exists as circular as well as linear molecules. cpDNA is associated with proteins and RNA as a nucleoid and is inherited when nucleoids are segregated into daughter cells during plastid division (Hansmann, 1985; Nemoto, 1988). The chromosome in plastids is not constant during development, as it is in the nucleus of the cell. cpDNA copy number and integrity decreases as cells age (Oldenburg. 2004; Shaver, 2006). The controls that regulate cpDNA replication initiation, replication, and copy number are not well understood (Nielsen, 2010).

Studies have shown that cpDNA in higher plants replicates by a double displacement loop (D- loop) mechanism from two specific replication origins (Kunnimalaiyaan, 1997). It may also replicate by a recombination dependent process of cpDNA replication (Nielsen, 2010; Oldenburg, 2004; Rowan, 2010) and a rolling circle replication (Kolodner, 1975). The use of two or more mechanisms for cpDNA replication in plants has been discussed as a possibility (Nielsen, 2010). Replication using the D-loop mechanism may be involved in maintaining a low level of chloroplast genomes in mature cells, whereas recombination dependent replication may help in rapid replication to produce high copy numbers of the genome during early stages of development (Morley, 2016).

In order to maintain cpDNA integrity, replication, recombination, and repair take place only on DNA firmly associated with membrane-attached nucleoids (Oldenburg, 2015). During development, changes in nucleoid protein composition can result in the release of damaged and unrepaired DNA from the nucleoid. This unbound DNA is susceptible to further degradation by nucleases and leads to reduced copy number (Oldenburg, 2015). Several studies have suggested that the existence of homologous recombination (HR) in chloroplasts is for the repair of DNA, possibly to cope with damage from photo-oxidation and other environmental stresses (Cerutti, 1995). Plant nuclear genomes encode bacterial type RecA homologs, including cpRecA that is involved in the maintenance of chloroplast genome stability by repairing damaged DNA and by suppressing aberrant recombination (Rowan, 2010). However, a mechanism and role of homologous recombination (HR) in chloroplasts is still unclear (Cerutti, 1995; Cerutti, 1992; Cerutti, 1993; Nielsen, 2010; Odahara, 2015; Rowan, 2010).

1.11 FtsZ protein and its function in chloroplast cell division

Figure 1.8 : A model for chloroplast division in plants. First, FtsZ proteins assemble to form a Z- ring after which ARC5/DRP5B ring is formed. Finally, the PD ring forms and constricts the IEM and OEM at the division site to form two new daughter chloroplasts (Adapted from TerBush, 2013).

The chloroplast cell division machinery has three contractile components. FtsZ1 and FtsZ2 assemble and form the FtsZ ring (Z ring) which is localized on the stromal side of the inner envelope membrane (IEM). Following Z-ring formation, the ARC5/DRP5B dynamin related protein complex forms an ARC5/DRP5B ring at the outer membrane. These ring structures constrict the outer and inner membrane at the plastid midline until final scission occurs forming two new chloroplasts (Maple, 2005; Min, 2016; Osteryoung, 2003; Stanislav Vitha., 2001; TerBush, 2013).

Accumulation and Replication of Chloroplast 6 (ARC6) is an IEM protein derived from cyanobacterial endosymbiosis (Kumiko, 2015). FtsZ2 interacts with the N- terminus of ARC6 and this interaction serves to recruit the Z-ring to the IEM and help FtsZ polymerization at the division site. Plastid Division 2 (PDV2) protein interacts with the extended C- termini of ARC6 in the intermembrane space (IMS). This interaction is essential for PDV2 to localize to the midline of the plastid.

Plastid Division 1 (PDV1) is a paralog of PDV2. Both proteins recruit ARC5/DRP5B from the cytosol to the OEM. Interaction between ARC6-PDV2 in the IMS, FtsZ-ARC6 in stroma, and PDV2 (PDV1) -DRP5B in the cytosol is important to coordinate the Z ring and ARC5/DRPB5B ring across the IEM and OEM, leading to the formation of two daughter plastids during plastid division (Min, 2016; Osteryoung, 2014; TerBush, 2013).

The main purpose of this study was to investigate the function of SYN3 in chloroplasts. A better understanding of how SYN3 functions in chloroplasts will give more information on chloroplast biogenesis and photosynthesis. This study mainly focused on localizing SYN3 in chloroplasts and investigating a potential role for SYN3 in chloroplast division. Chapter 2 includes the experimental results for SYN3 chloroplast localization studies, as well as results on SYN3’s possible function in chloroplast division and photosynthesis. Chapter 3 summarizes the results of this study and provides conclusions about the role of SYN3 in chloroplasts and some potential future directions to explore the function of SYN3 in chloroplasts.

1.12 References

1. Adams, D.W., Wu, L.J., Errington, J. 2014. Cell cycle regulation by the bacterial nucleoid. Current Opinion in Microbiology.Elsevier Ltd;. 22:94±101. 2. Marston. A.L., Angelika Amon. 2004. Meiosis: cell-cycle controls shuffle and deal. Nature Reviews. 5: 983-997. 3. Amanda, S.B., Berkowitz, K.M. 2014. The roles of cohesins in mitosis, meiosis, and human health and disease. Methods in molecular biology. 1170:229–266. 4. Armstrong, S.J., G.H.J. 2003. Meiotic cytology and chromosome behaviour in wild-type Arabidopsis thaliana. Journal of Experimental Botany. 54: 1±10. 5. Badrinarayanan A., L.T., Laub MT. 2015. Bacterial Chromosome Organization and Segregation. Annual Review of Cell and Developmental Biology. 31:171±199. 6. Bai, X., Peirsion, B.N., Dong, F., Cai, X., Makaroff, C.A.,. 1999. Isolation and characterization of SYN1, a RAD21-like gene essential for meiosis in Arabidopsis. Plant Cell 11:417±430. 7. Bi EF, L.J. 1991. FtsZ ring structure associated with division in Escherichia coli. Nature. 354:161-164. 8. Bogorad, L. 1975. Evolution of organelles and eukaryotic genomes. Science. 188:891- 898. 9. Bose, T., Jennifer L. Gerton. 2010. Cohesinopathies, gene expression, and chromatin organization. Journal of Cell Biology. 189 201–210.

10. Cai, X., F.G. Dong, R.E. Edelmann., Makaroff. C.A. 2003. The Arabidopsis SYN1 cohesin protein is required for sister chromatid arm cohesion and homologous chromosome pairing. Journal of Cell Science. 116, 2999-3007. 11. Campbell, J.L.a.C.-F., O. 2002. Chromosome cohesion: ring around the sisters? Trends in Biochemical Sciences. 27:492–495. 12. Cerutti, H., Johnson,A M.,Boynton J E., Gillham, N.W. 1995. Inhibition of chloroplast DNA recombination and repair by dominant negative mutants of Escherichia coli RecA. Molecular and Cellular Biology. 15:3003–3011. 13. Cerutti, H., Osman, M., Grandoni, P. Jagendorf, A.T. 1992. A homolog of Escherichia coli RecA protein in plastids of higher plants. PNAS. 89:8068–8072. 14. Chua, N.-H., Schmidt, G. W. 1978. Post-translational transport into intact chloroplasts of a precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase. PNAS. 75:6110–6114. 15. Ciosk, R., Shirayama, M., Anna,S., Tomoyuki .T., Attila. T., Shevchenko.A., Nasmyth.K. 2000. Cohesin's Binding to Chromosomes Depends on a Separate Complex Consisting of Scc2 and Scc4 Proteins. Molecular Cell. 5:243–254. 16. Cooper, G. 2000. The Cell: A Molecular Approach. 2nd edition. Sunderland (MA): Sinauer Associates. 17. Costa-Nunes, J.A.d., A. M. Bhatt., S. O’Shea.,, C. E. West., C. M. Bray., U. Grossniklaus.,H. G. Dickinson. 2006. Characterization of the three Arabidopsis thaliana RAD21 cohesins reveals differential responses to ionizing radiation. Journal of Experimental Botany. 57:971–983. 18. Danilova.O., R.-L.R., Pinskaya M, Sherratt D, Possoz C. 2007. MukB colocalizes with the oriC region and is required for organization of the two Escherichia coli chromosome arms into separate cell halves. Molecular Microbiology. 65:1485–1492. 19. Dawe, R.K. 1998. Meiotic chromosomeorganization and segregation in plants. Annual Review of Plant Physiology and Plant Molecular Biology 49:371–395. 20. Dong, F., X. Cai., Makaroff. C.A. 2001. Cloning and characterization of two Arabidopsis genes that belong to the RAD21/REC8 family of chromosome cohesin proteins. Gene. 271:99±108. 21. Díaz-Martínez, L.A., Juan,F. Giménez-Abián., Clarke, D. J. 2008. Chromosome cohesion – rings, knots, orcs and fellowship. Journal of Cell Science. 121:2107-2114. 22. E Revenkova., R.J. 2005. Keeping sister chromatids together: cohesins in meiosis. Reproduction. 130:783–790. 23. Easter. J., G.J. 2002. ParB-stimulated nucleotide exchange regulates a switch in functionally distinct ParA activities. . Molecular Cell. 10:427–434. 24. Egan. AJ., V.W. 2013. The physiology of bacterial cell division. Annals of the New York Academy of Sciences. 1277:8-28. 25. F. Klein, P.M., M. Galova, S.B. Buonomo, C. Michaelis, K. Nairz, K. Nasmyth. 1999. A central role for cohesins in sister chromatid cohesion, formation of axial elements, and recombination during yeast meiosis. Cell. 98:91–103. 26. Francis, D. 2009. What’s New in the Plant Cell Cycle? Progress in Botany. 70:33-49. 27. Gerdes. K., H.M., Szardenings, F. 2010. Pushing and pulling in prokaryotic DNA segregation. Cell. 141:927±942. 28. Gerton, J. 2005. Chromosome Cohesion: A Cycle of Holding Together and Falling Apart. PLoS Biol. 3:e94.

29. Graumann.Peter. L., T.K. 2009. Dynamics of the bacterial SMC complex and SMC-like proteins involved in DNA repair. Chromosome Research. 17:265–275. 30. Gruber, S., . Christian, H. Haering.,Nasmyth.K. 2003. Chromosomal Cohesin Forms a Ring. Cell.112:765–777. 31. Guacci, V., Douglas K., A.Strunnikov. 1997. A Direct Link between Sister Chromatid Cohesion and Chromosome Condensation Revealed through the Analysis of MCD1 in S. cerevisiae. Cell. 91:47–57. 32. Guacci, V., Eileen H., Douglas K. 1994. Chromosome Condensation and Sister Chromatid Pairing in Budding Yeast. The Journal of Cell Biology. 125:517-530. 33. Gunjan, M., . Syed, Rizvi,. Santanu K.G. 2012. Cohesin: A guardian of genome integrity. Biochimica et Biophysica Acta. 1823:1324–1342. 34. H. Cerutti, H.Z.I., Jagendorf. A. T. 1993. Treatment of Pea (Pisum sativum L.) Protoplasts with DNA-Damaging Agents Induces a 39-Kilodalton Chloroplast Protein Immunologically Related to Escherichia coli RecA. Plant Physiology. 102:155-163. 35. Haering, C.H., . Ana-Maria Farcas.,, Prakash A., Jean M.,Nasmyth.K. 2008. The cohesin ring concatenates sister DNA molecules. Nature. 454. 36. Haering, C.H., Jan Löwe., Andreas H., Nasmyth.K. 2002. Molecular Architecture of SMC Proteins and the Yeast Cohesin Complex. Molecular Cell 9:773-788. 37. Hansmann P, F.H., Ronai K, Sitte P. 1985. Structure, composition, and distribution of plastid nucleoids in Narcissus pseudonarcissus. Planta 164:459-472. 38. Hayama. R., M.K. 2010. Physical and functional interaction between the condensin MukB and the decatenase topoisomerase IV in Escherichia coli. PNAS. 107:18826– 18831. 39. Highfield, P.E., Ellis, R. John. 1978. Synthesis and transport of the small subunitof chloroplast ribulose bisphosphate carboxylase. Nature. 271:420-424. 40. Hiraga.S. 1992. Chromosome and plasmid partition in escherichia coli. Annual Review of Biochemistry. 61:283-306. 41. Hiraga.S. 2000. Dynamic localization of bacterial and plasmid chromosomes. Annual Review of Genetics. . 34:21–59. 42. Hirano, T. 2000. Chromosome cohesion, condensation and separation. Annual Review of Biochemistry. 2000. 69:115–144. 43. Hirano, T. 2002. The ABCs of SMC proteins: two-armed ATPases for chromosome condensation, cohesion, and repair. Genes & Development. 16:399–414. 44. Huang, C.E., Mark M., Douglas K. 2005. Rings, bracelet or snaps: fashionable alternatives for Smc complexes. Philosophical Transactions of the Royal Society B. 360:537–542. 45. Jarvis, P. 2008. Targeting of nucleus-encoded proteins to chloroplasts in plants. New Phytologist. 179:257–285. 46. Jarvis, P., Robinson.C. 2004. Mechanisms of protein import and routing in chloroplasts. Current Biology. 14:R1064-1077. 47. Jiang, L., M. Xia, L.I. Strittmatter, Makaroff. C. A. 2007. The Arabidopsis cohesin protein SYN3 localizes to the nucleolus and is essential for gametogenesis. Plant Journal. 50:1020-1034. 48. Jodi Maple., M.T.F., Nobutaka K, Tracy L.,Neil R. B, Shigeo Y.,Simon G.Møller. 2004. Giant chloroplast 1 is essential for correct plastid division in arabidopsis. Current Biology. 14:776–781.

49. Keegstra, K., K. Cline. 1999. Protein import and routing systems of chloroplasts. Plant Cell. 11:557-570. 50. Keegstra, K., Olsen, L.J., Theg, S.M. 1989. Chloroplastic precursors and their transport across the envelope membranes. Annual Review of Plant Physiology and Plant Molecular Biology. 40:471–501. 51. Kolodner, R., Tewari. KK. 1975. Chloroplast DNA from higher plants replicates by both the cairns and rolling circle mechanism. Nature. 256:708–711. 52. Kumiko, O., .Shin-ya, Miyagishima,. Wadaa.H. 2015. Phosphatidylinositol 4-phosphate negatively regulates chloroplast division in Arabidopsis. The Plant Cell. 27:663–674. 53. Kunnimalaiyaan, M., F. Shi, and B.L. Nielsen. 1997. Analysis of the tobacco chloroplast DNA replication origin (oriB) downstream of the 23 S rRNA gene. Journal of Molecular Biology. 268:273-283 issn: 0022-2836. 54. Laurence, C., Jolivet.S., Horlow.C., Chelysheva.L., Heyman.J.,,G. D. Jaeger, C. Koncz, L. D. Veylder,. Raphael,M. 2013. Centromeric cohesion is protected twice at meiosis, by shugoshins at anaphase i and by patronus at interkinesis. Current Biology. 23:2090–2099. 55. Lee, B., Angelika, A. 2001a. Meiosis: how to create a specialized cell cycle. Current Opinion in Cell Biology. 13:770–777. 56. Lee, J.Y., Terry L. Orr-Weaver. 2001b. The molecular basis of sister-chromatid cohesion. Annual Review of Cell and Developmental Biology . 17:753–777. 57. Leonard. TA., M.-J.J., . LoÈwe. J. 2005. Towards understanding the molecular basis of bacterial DNA segregation. Philosophical Transactions of the Royal Society of London series B, Biological Sciences. 360: 523±535. 58. Li Y, S.N., Berger AJ, Vos S, Schoeffler AJ, et al. 2010. Escherichia coli condensin MukB stimulates topoisomerase IV activity by a direct physical interaction. PNAS. 107:18832–18837. 59. Ling, J., . Ming, Xia., Lara, I. Strittmatter., Makaroff. C. A.. 2007. The Arabidopsis cohesin protein SYN3 localizes to the nucleolus and is essential for gametogenesis. The Plant Journal. 60. Liu, J., Ian D. Krantz. 2008. Cohesin and human disease. Annual Review of Genomics and Human Genetics. 9:303–320. 61. Lopez-Juez, E. 2007. Plastid biogenesis, between light and shadows. Journal of Experimental Botany. 58:11-26. 62. Losada, A., T. Yokochi, R. Kobayashi, and T. Hirano. 2000. Identification and characterization of SA/Scc3p subunits in the Xenopus and human cohesin complexes. Journal Cell Biology . 150:405-416. 63. Lutkenhaus.J. 2012. The ParA/MinD family puts things in their place. Trends in Microbiology. Elsevier ltd. 20:411±418. 64. Ma, Y., A. Kouranov, S.E. LaSala, Schnell. D.J. 1996. Two components of the chloroplast protein import apparatus, IAP86 and IAP75, interact with the transit sequence during the recognition and translocation of precursor proteins at the outer envelope. Journal Cell Biology.. 134:315-327. 65. Maple.J., A.C., , Møller, SG. 2005. Plastid division is mediated by combinatorial assembly of plastid division proteins. Plant Journal. 43:811–823. 66. Martin, W., and R.G. Herrmann. 1998. Gene transfer from organelles to the nucleus: how much, what happens, and Why? Plant Physiology. 118:9-17. 67. McFadden, G.I. 2001. Chloroplast origin and integration. Plant Physiology. 125:50–53.

68. McKee, B.D. 2004. Homologous pairing and chromosome dynamics in meiosis and mitosis. Biochimica et Biophysica Acta. 1677 165– 180. 69. Mehta, G.D., Ravinder K., Sanjeeva S., Santanu K. G. 2013. Cohesin: functions beyond sister chromatid cohesion. FEBS Letters. 587:2299–2312. 70. Michaelis C, C.R., Nasmyth K. 1997. Cohesins: Chromosomal proteins that prevent premature separation of sister chromatids. Cell. 91:35–45. 71. Mierzejewska. J., J.-B.G. 2012. Prokaryotic ParA-ParB-parS system links bacterial chromosome segregation with the cell cycle. Plasmid. :1-14. 72. Min, Z., Cheng, C., John,E. Froehlich., Allan, D. TerBush., K, W. Osteryoung. 2016. Roles of arabidopsis parc6 in coordination of the chloroplast division complex and negative regulation of ftsz assembly. Plant Physiology. 170:250–262. 73. Miyagishima, S.-y. 2011. Mechanism of plastid division: from a bacterium to an organelle. Plant Physiology. 155:1533–1544. 74. Miyazaki, W.Y., Orr-Weaver,T .L. 1994. Sister-Chromatid cohesion in mitosis and meiosis. Annual Review of Genetics. 28:167-187. 75. Morley.Stewart A., B.L.N. 2016. Chloroplast DNA copy number changes during plant development in organelle dna polymerase mutants. Frontiers in Plant Science. 7:1-10. 76. Mullet, J.E. 1988. Chloroplast development and gene expression. Annual Review Plant Physiology. Plant Molecular Biology. 39:475–502. 77. Nasmyth, K. 2001. Disseminating the genome: joining, resolving, and separating sister chromatids during mitosis and meiosis. Annual Review Genetics. 35:673–745. 78. Nasmyth, K. 2005. How might cohesin hold sister chromatids together? Philosophical Transactions of the Royal Society B Biological Sciences.. 360:483–496. 79. Nasmyth, K. 2011. Cohesin: a catenase with separate entry and exit gates? Nature Cell Biology. 13:1170-1177. 80. Nasmyth, K., Christian H. Haering. 2009. Cohesin: its roles and mechanisms. Annual Review of Genetics. 43:525–558. 81. Nasmyth, K.A. 1994. Book review An egg-ocentric view of the cell cycle. By Andrew M. Murray and Timothy Hunt. Cell. 78:11-12. 82. Nasmyth., K.a.P., Jan-Michael., and Frank,Uhlmann. 2000. Splitting the chromosome: cutting the ties that bind sister chromatids. Science. 288:1379-1384. 83. Nasmyth.K. 2002. Segregating sister genomes: the molecular biology of chromosome separation. Science. 297:559–565. 84. Nemoto Y, K.S., Nakamura S, Mita T, Nagata T, Kuroiwa T. 1988. Studies on plastid nuclei (nucleoids) in Nicotiana tabacure L. I. Isolation of proplastid-nuclei from cultured cells and identification of proplastid-nuclear proteins. Plant Cell Physiology. 29:167-177. 85. Neuhaus.H. E., M.J.E. 2000. Nonphotosynthetic metabolism in plastids. Annual Review of Plant Physiology. Plant Molecular Biology. 51:111–140. 86. Neuwald, A.F., Hirano.T. 2000. HEAT repeats associated with condensins, cohesins and other complexes involved in chromosome-related functions. Genome Research. 10:1445– 1452. 87. Nicolas.E., U.A., Uphoff S, Henry O, Badrinarayanan A, Sherratt D. 2014. The SMC complexMukBEF recruits topoisomerase IV to the origin of replication region in live Escherichia coli. Mbio. 5:e01001–01013.

88. Nielsen, B.L., John D. Cupp., Jeffrey B. 2010. Mechanisms for maintenance, replication, and repair of the chloroplast genome in plants. Journal of Experimental Botany. 61:2535– 2537. 89. Nolivos.S., Sherratt D. 2014. The bacterial chromosome: architecture and action of bacterial SMC and SMC-like complexes. FEMS Microbiology Review. 38:380-392. 90. Odahara, M., Takayuki, Inouye., Yoshiki, Nishimura,.Yasuhiko ,Sekine. 2015. RECA plays a dual role in the maintenance of chloroplast genome stability in Physcomitrella patens. The Plant Journal. 84:516–526. 91. Oldenburg, D.J., Arnold, J. Bendich. 2015. DNA maintenance in plastids and mitochondria of plants. Frontiers in Plant Science. 6:1-15. 92. Oldenburg.Delene J. ., A.J.B. 2004. Changes in the structure of DNA molecules and the amount of dna per plastid during chloroplast development in maize. Journal of Molecular Biology. 344:1311–1330. 93. Oldenburg.Delene J. ., A.J.B. 2015. DNA maintenance in plastids and mitochondria of plants. Fronteirs in Plant Science. 94. Osteryoung, K., W., Kevin A. Pyke. 2014. Division and dynamic morphology of plastids. Annual Review of Plant Biology. 65. 95. Osteryoung, K.W., Jodi Nunnari. 2003. The division of endosymbiotic organelles. Science. 302. 96. P. Pasierbek, M.J., M. Melcher, A. Schleiffer, D. Schweizer, J.A. Loidl. 2001. Caenorhabditis elegans cohesion protein with functions in meiotic chromosome pairing and disjunction. Genes & Development. 15:1349–1360. 97. Peirson, B.N., S.E. Bowling, Makaroff. C. A. 1997. A defect in synapsis causes male sterility in a T-DNA-tagged Arabidopsis thaliana mutant. Plant journal. 11:659-669. 98. Peters, J.-M., Antonio Tedeschi, Julia, Schmitz. 2008. The cohesin complex and its roles in chromosome biology. Genes & Development. 22:3089–3114. 99. Rowan, B.A., Oldenburg, D.J. and Bendich, A.J. 2010. RecA maintains the integrity of chloroplast DNA molecules in Arabidopsis. Journal of Experimental Botany. 61:2575– 2588. 100. Schafer, K.A. 1998. The cell cycle: a review. Veterinary Pathology. 35:461-478. 101. Shaver. JM., O.D., Bendich AJ. 2006. Changes in chloroplast DNA during development in tobacco, Medicago truncatula, pea, and maize. Planta. 2006. 224:72-82. 102. Stanislav Vitha., R.S.M., Osteryoung.K.W. 2001. FtsZ ring formation at the chloroplast division site in plants. The Journal of Cell Biology. 153:111–119. 103. Sugiura, M. 1989. The chloroplast chromosomes in land plants. Annual Review. Cell Biology. 5:51–70. 104. Sugiura, M. 1992. The Chloroplast Genome. Plant Molecular Biology. 19:149- 168. 105. Surcel, A., . D. Koshland, H. Ma, R.T. Simpson. 2008. A. Surcel, D. Koshland, H. Ma, R.T. Simpson, Cohesin interaction with centromeric minichromosomes shows a multi-complex rod-shaped structure, . PLoS One: e2453. 106. Suzuki, G., Chikage, Nishiuchi., Asami ,Tsuru.,, Eri Kako., Jian Li .,Maki Yamamoto., Yasuhiko Mukai. 2013. Cellular localization of mitotic RAD21 with repetitive amino acid motifs in Allium cepa. Gene.514(2):75-81. 107. TerBush AD, Y.Y., Osteryoung KW. 2013. FtsZ in chloroplast division: structure, function and evolution. Current Opinion Cell Biology. 25:461-470.

108. Thomas, G., Jan Löwe. 2016. Structural insights into ring formation of cohesin and related smc complexes. Trends in Cell Biology. 26:680-693. 109. Tyson, J.J., Csikasz-Nagy.A., Novak.B. 2002. The dynamics of cell cycle regulation. BioEssays. 24:1095–1109. 110. Uhlmann, F. 2001. Chromosome cohesion and segregation in mitosis and meiosis. Current Opinion in Cell Biology. 13:754–761. 111. Uhlmann, F. 2004. The mechanism of sister chromatid cohesion. Experimental Cell Research. 296:80– 85. 112. Vermeulen, K., Dirk,R. Van B., Zwi N. Berneman. 2003. The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer. Cell Proliferation. 2003. 36:131–149. 113. Wang X, M.L.P., Rudner DZ. 2013. Organization and segregation of bacterial chromosomes. Nature Reviews Genetics. 14:191±203. 114. Weitao, T., Dasgupta.S., NordstroÈm,K. 2000. Role of the mukB gene in chromosome and plasmid partition in Escherichia coli. Molecular Microbiology. 38:392- 400. 115. Yamanaka K, O.T., Niki H, Hiraga S. 1996. Identification of two new genes, mukE and mukF, involved in chromosome partitioning in Escherichia coli. Molecular and General Genetics. 250:241–251. 116. Yuan, L., Yang,X., Ellis, J. L., Fisher,N.M., Makaroff. C. A. 2012. The Arabidopsis SYN3 cohesin protein is important for early meiotic events. The Plant Journal. 71:147–160. 117. Yuan, L., Yang,X., Auman,D., Makaroff. C. A. 2014. Expression of epitope- tagged SYN3 cohesin proteins can disrupt meiosis in arabidopsis. Journal of Genetics and Genomics. 41:153-164.

Chapter 2: Investigating the role of SYN3 in chloroplasts

2.1 Introduction

Cohesin complexes are chromosome-associated complexes that facilitate the cohesion between chromosomes that is necessary for proper chromosome separation and segregation during mitosis and meiosis (Gerton, 2005; Miyazaki, 1994; Hirano,2000). These complexes are also important for proper spindle attachment to chromosomes, DNA double-strand break (DSB) repair, and transcriptional regulation (Mehta, 2013; Peters, 2008).

Cohesin complexes consist of four proteins: two long-coiled proteins, SMC1 & SMC3; SCC3; and an α-kleisin family protein, SCC1 (Hirano, 2000; Lee, 2001; Michaelis, 1997; Uhlmann, 2001). α-kleisins are essential components of the complex that play a key role in cohesion by creating a ring-like structure through their interactions with the SMC proteins. Arabidopsis has four α-kleisin proteins: SYN1, SYN2, SYN3, and SYN4 (Bai, 1999; Dong, 2001; Hirano, 2000; Ling, 2007).

SYN1 is the Arabidopsis REC8 ortholog that functions in meiosis (Bai, 1999; Peirson, 1997; Cai, 2003), whereas SYN2 and SYN4 are SCC1 orthologs that are essential for mitosis with SYN2 also playing a role in DNA repair (Dong, 2001; Costa-Nunes, 2006). SYN3 localizes to the nucleolus in both mitotic and meiotic cells and is important for megagametogenesis (Ling, 2007). Moreover, knock down of SYN3 mRNA levels by RNA interference (RNAi) resulted in defects in both male and female meiosis. Further, knockdown of SYN3 affected transcription of genes involved in recombination and synapsis which, in turn, could inhibit recombination (Yuan, 2012). Recent studies suggested that plant growth and development are sensitive to elevated levels of SYN3 expression, specifically affecting the vegetative growth of plants (Yuan, 2014).

Previous work established the presence of α-kleisins in the nucleolus and showed a requirement for SYN3 in meiosis and mitosis. In an attempt to characterize the role of SYN3 in the nucleolus, a yeast two-hybrid screen was used to identify potential interacting partners (Yuan and Makaroff, unpublished). Interestingly, Ftz1-1 and 2-1 were identified as SYN3 interacting proteins (Yuan

23 and Makaroff, unpublished). FtsZ is a vital component of the chloroplast division machinery that is a homolog of the bacterial protein FtsZ, an essential element in prokaryotic cell division machinery. Bacteria have a single FtsZ gene, while there are three FtsZ genes present in Arabidopsis: FtsZ1-1, FtsZ2-1, and FtsZ2-2 (Osteryoung, 1998 (a); Osteryoung, 1995; Osteryoung, 1998 (b). In Arabidopsis, both depletion and over-expression of FtsZ-1 or FtsZ-2 exhibited dose dependent effects as indicated by a reduction in chloroplast number. Both FtsZ-1 and FtsZ-2 play distinct roles in chloroplast division (Karamoko, 2011; Kiessling, 2000; Osteryoung, 1998 (b); Schmitz, 2009; Stokes, 2000).

In addition, the bacterial kleisin MukF (Fennell-Fezzie, 2005) is important for chromosome segregation, transcriptional regulation, and DNA repair (Dervyn, 2004). Therefore, we hypothesize that the eukaryotic SYN3 may function as prokaryotic kleisin homolog of MukF in chloroplast and may be involved in cellular processes including in chromosome segregation during chloroplast division.

The primary focus of this project was to unravel the potential roles of SYN3 in chloroplasts by studying the localization of SYN3 in chloroplasts and understanding the function of SYN3 in chloroplast division. Here, we show that SYN3 localizes to the thylakoid within chloroplasts. Furthermore, over-expression of SYN3 caused abnormal, stunted growth and plants with significantly reduced total chloroplast and chlorophyll b content. Differential interference contrast microscopy (DIC) results showed that SYN3 over-expression may have effects on chloroplast division.

2.2 Materials and methods

2.2.1 Preparation of chloroplast

Intact chloroplasts were isolated from 12-14 day old garden pea plants (Pisum sativum var. Little Marvel) as previously described (Cline, 1986). Briefly, intact chloroplasts were separated from broken chloroplasts in leaf homogenates using density gradient centrifugation. Isolated

24 chloroplasts were suspended to 1 mg/ml chlorophyll (chl) in import buffer (IB; 50 mM Hepes/KOH, pH 8, containing 0.33 M sorbitol) and kept on ice.

2.2.2 Preparation of radiolabeled precursors

Plasmid, pGBKT7 containing the SYN3 cDNA inserted downstream of the T7 RNA polymerase promoter was digested with SmaI restriction enzyme to linearize the plasmid at the 3’end of the SYN3 cDNA in preparation of in vitro transcription. PCR cleanup of restriction digested samples was done using the Wizard SV Gel and PCR Clean-Up kit (Promega) according to manufacturer’s instructions. The plasmid containing SYN3 cDNA was kindly provided by Dr. Makaroff’s lab.

The linearized plasmid was used in a coupled in vitro transcription/translation reaction using the TNT® Wheat Germ Extract System (Promega). Reactions contained [3H] leucine amino acid. Following one hour incubation at 25 оC, reactions were terminated by the addition of an equal volume of 2 x IB which also contained 60 mM unlabeled leucine.

The gene for the precursor to the 17 kDa subunit of the extrinsic oxygen evolving complex of photosystem II, pre OE17, was transcribed by SP6 polymerase and mRNA translated separately in the presence of [3H] leucine using the Wheat Germ Extract System (Promega). Translations were diluted and adjusted to 1x IB containing 30 mM unlabeled leucine before use.

2.2.3 Protein import assays into chloroplasts

Assays for import of precursor proteins were carried out in vitro. Reactions contained 200 µg of isolated chloroplast, 100 µl of translated product, 5 mM DTT, 5 mM Mg2+-ATP in a 400 µl reaction mixture. Import reactions were initiated by addition of precursors and incubated at 25 оC in the presence of 150 µmol/m2/s of white light. Assays were incubated for 15 minutes with gentle mixing at five minute intervals. Reactions were terminated by adding two volumes of cold 1x IB and placement on ice. Chloroplasts were recovered from import reaction mixtures by centrifugation at 1000xg for five minutes and re-suspended in 1x IB with 1 mM thermolysin for 40 minutes on ice. Control reactions were treated identically but received no protease. Reactions were terminated by the addition 50 mM EDTA. Intact chloroplasts were re-purified by centrifugation through 35% Percoll (GE Healthcare) in IB with 5 mM EDTA. 2x sample

25 solubilizing buffer (2x SSB: 0.1M Tris-HCl, pH 6.8, 5%SDS, 30% glycerol, 0.1% bromophenol blue) was added to recovered chloroplasts, and the mixture heated for 5 minutes at 95 oC before being subjected to SDS- PAGE under denaturing conditions. Gels were analyzed by fluorography and exposure to X-ray film.

2.2.4 Subcellular fractionation of chloroplasts

Cell lysates from both Arabidopsis and peas plants were prepared by adding 100 µl of 1x grinding buffer (0.1 M Hepes-KOH, 0.66 M sorbitol, 2 mM MgCl2, 2 mM MnCl2, 4 mM EDTA,10 mM Na-ascorbate, 0.2% BSA) to 1 gram of leaf tissue and ground with beads in a bullet blender homogenizer for two minutes followed by centrifugation for 5 minutes at 14000 rpm and the supernatant was collected.

Chloroplast lysates were obtained by hypotonic lysis of intact chloroplasts by re-suspension of pelleted chloroplast (1 mg chl/ml) in 20 mM Hepes/KOH (pH 8), 10 mM MgCl2 and incubation for 5 minutes on ice.

Thylakoids were prepared from chloroplast lysates by centrifugation at 3200xg for 8 min at 4 оC. Stromal extracts were prepared from the resulting supernatant by further centrifugation at 50000xg for 20 minutes at 4 оC. Lysates prepared at a stoichiometry of 1 mg chl/mL were arbitrarily referred to as lx lysate and the stromal extract resulting from such lysates as 1x stroma.

2.2.5 Immunoblotting

Cell lysates, isolated organelles, and fractionated organelle samples were subjected to SDS- PAGE and electro-transfer to nitrocellulose membrane for immunodetection. Membranes were treated with antibodies derived against SYN3, HCF106, and histone H3 at titers of 1:3000, 1:3000 and 1:5000, respectively. Following the primary antibody wash was a horse radish peroxidase conjugated goat anti-rabbit IgG secondary antibody incubation at a titer of 1:3000. Membranes were subjected to Clarity Max™ Western ECL Blotting Substrates (BIO-RAD) according to the manufacturer’s protocol. Fluorescence was detected by exposure of membranes to X-ray film. Antibodies raised against SYN3 by injecting rabbits with overexpressed, purified peptide corresponding to amino acids region 239-510 of SYN3 expressed as a 6x His-tagged

26 protein have been described (Ling, 2007). Antibodies raised against P. sativum OE23 and HCF106 were available in Dr. Dabney-Smith’s lab. OE23 produced in E. coli was used for antibody preparation in rabbits (Cline, 1993). E. coli expressed HCF106 was used for antibody preparation in rabbits (Mori, 1999). Antibodies against histone H3 were purchased from Agrisera.

2.2.6 Immunoprecipitation of chloroplast lysates proteins under denaturing conditions

Immunoprecipitation of chloroplast lysate proteins under denaturing conditions was carried out as previously described (Cline, 2001). Chloroplasts lysates were re-suspended in TBS to 1 mg chl/mL and added 2% SDS, 1mM EDTA, TBS and incubated at 37оC for 5 minutes. After five minutes of centrifugation at 14000 rpm, supernatants were transferred to tubes containing IP buffer (1% Triton X-100, 0.5% deoxycholate, 1mM EDTA, TBS) and a 25% slurry IgG-beads. The mixture was incubated overnight at 4 оC with end-over-end mixing. The supernatant was removed by centrifugation at 500xg for 5 minutes. The beads then were washed with IP buffer three times followed by a washing with (0.05 % Triton X-100, TBS). The bound protein was eluted directly into 2x SSB and analyzed by SDS-PAGE and immunoblotting with antibodies derived against SYN3 and pOE23 (ie, α-SYN3, α-pOE23, respectively).

Antibody cross linked to Protein A sepharose (α-PAS) beads (25% slurry) were centrifuged at 500xg for 2 minutes, the supernatant was removed, the beads were suspended with PBS buffer, pelleted by centrifugation, and the supernatant was removed. Three rounds of adding PBS followed the pelleting of the beads at which point the supernatant was removed. These rounds of adding PBS, pelleting the beads, and removing the supernatant are referred to as wash steps. For the immunoprecipitation assays, 500 µL of PBS and 20 µL of translated product was added to the IgG pellet. Translation product contained radiolabeled [3H]-leucine. Assays were incubated for 2 hours at 4oC. Following centrifugation, α-PAS pellet was washed three times with PBS

(136 mM NaCl, 1.76 mM KH2PO4, 10.1 Mm Na2HPO4, 2.6 mM KCl). Bound proteins were eluted from the beads by adding 2x SSB. Samples were subjected to SDS-PAGE and fluorography. Radiolabeled bands were detected using X-ray film.

2.2.7 Generating transgenic plants using Agrobacterium mediated floral dip method

Arabidopsis thaliana ecotype Col-0 wildtype plants were grown to flowering stage in a growth chamber under 130 μmol m−2 s−1 light at 22 оC with a 16 h light/8 h dark cycle. Agrobacterium tumefaciens strain GV3101 carrying the binary plasmid pFGC5941(35S-SYN3-cDNA-YFP- pFGC) designed by Li Yuan and provided by Dr. Makaroff’s lab, was grown to stationary phase (OD 1.5-2.0) in liquid culture at 28о C in sterilized LB (10 g tryptone, 5g yeast extract, 5g NaCl -1 per liter H2O) with added kanamycin selectivity (50 µg ml ).

Colony PCR was carried out with SYN3 and YFP primers (listed in table-1) to confirm the presence of the desired construct in A. tumefaciens strain and single colony was used for inoculation in 5 mL at 28оC for 2 days. This sample was used to inoculate at 500 mL feeder culture. Cultures were harvested by centrifugation for 20 minutes at room temperature at 3700xg. The pellet was gently suspended in floral dip inoculation medium (5.0% sucrose and 0.02% silwet L-77) as described previously (Zhang, 2006) (Clough, 1998)

The A. tumefaciens cell suspension was applied using a floral dip method to all plant parts including very young flower shoots and buds using pipette tips. Treated plants were left in low light overnight and returned to the growth chamber the next day. Plants were grown for a further 4-6 weeks until siliques were brown and dry. Seeds were harvested and were stored in microfuge tubes and kept at 4о C.

2.2.8 BASTA selection and genotyping PCR for positive transformants

BASTA selection was done as previously described (Zhang, 2006). The seeds were germinated on soil and transgenic plants were selected by spraying with 0.1% BASTA herbicide in the growth chamber. Spraying was performed one week after germination and repeated four times at two-day intervals. Transgenic plants were readily identified at the end of the BASTA selection. Positive transformants from BASTA selection were tested by genotyping PCR using primers against SYN3 and YFP (primers are listed in table-1). PCR positive plants were grown and seeds were collected from plants following BASTA selection to identify homozygous lines for the SYN3-YFP insert.

2.2.9 Sample preparation for difference interference contrast microscopy

Samples were prepared as previously described (Pyke, 1991) with small modifications. To characterize the chloroplast phenotype, three whole leaves from 35 day old plants were sliced and fixed in an Eppendorf tube with 3.5% glutaraldehyde for 1 h in darkness, the glutaraldehyde was then replaced with 0.1 M Na2EDTA (pH= 9.0). The tube was incubated in a water bath of 60оC for two and half hours. Six separate plants were analyzed for each SYN3 O/E lines and wildtype. The number of chloroplasts were observed in twenty-four individual mesophyll cells using difference interference contrast microscopy. Images were taken with a digital camera coupled to an Olympus AX70 microscope and chloroplast number was counted using Image-Pro Plus software.

Significance for differences in average chloroplast number and chloroplast plan area was determined by student’s paired two sample for means t-test. Statistical analyses were conducted using Microsoft Excel and graphs were plotted using prism.

2.2.10 RNA Extraction and qRT-PCR analysis

Total RNA was extracted from 100 mg of Arabidopsis leaf tissue from 2 to 3-week-old SYN3- YFP O/E lines and wildtype plants with the RNeasy Plant Mini Kit (Qiagen), according to the manufacturers protocol. Total RNA concentration in each sample was determined with a NanoDrop Spectrophotometer. Isolated RNA (200 ng) was used to synthesize complementary DNA strands (cDNA) using qScript™ cDNA Synthesis Kit (Quanta). Reverse transcribed cDNA was used as template for real time PCR using the PerfeCta® SYBR® Green Supermix for iQ Kit (Quanta) according to manufacturer’s instructions. The primers used in these studies were found from literature (Yuan, 2014) and listed in Table-1. Expression was normalized against UBQ4. Three biological replicates were performed, with three technical replicates for each sample. Relative gene expression was calculated using the 2−δδt method (Livak, 2001).

Table 1 Primers used in this study

Primer name Sequence qPCR (F) SYN3 AAA GAA ATT TGG GGC TTT CA qPCR (R) SYN3 TGG TGT TCC TAC TGG GGA AT qPCR (F) UBQ4 CTG TTC ACG GAA CCC AATT qPCR (R) UBQ4 GGA AAA AGG TCT GAC CGA CA SYN3-7 GGA GAA TGA ATA ACC AGT CAA G YFP-1 AGC TTG CCG GTG GTG CAG ATG SYN3-4 TCT TGA CTA GCT GGT GTT GTT CC

2.2.11 Total protein extraction and immunoblotting

A. thaliana cell lysates were extracted from 2-week-old wildtype and SYN3-YFP O/E leaves. Briefly, leaves were harvested and homogenized with bullet blender (Next Advance) using solubilizing buffer (125 mM Tris-HCl, pH 6.8, 2% SDS and 10% glycerol). Homogenates were centrifuged for 30 minutes at 14000 rpm and supernatant was collected. Total protein concentration in the supernatant was measured using BCA kit (Pierce) and equal amounts of protein samples were loaded and separated by electrophoresis through a 12.5% acrylamide SDS- PAGE. Proteins were then transferred to nitrocellulose membranes and probed with anti-SYN3 or anti-pOE23 antibodies at 1:2000 and 1:10000 dilutions respectively. Membranes were subjected to Clarity MaxTM ECL blotting substrates (BIO-RAD) according to the manufacturer’s protocol. This was followed by exposure of membranes to X-ray film and film development.

2.2.12 Measurement of chlorophyll content and chlorophyll fluorescence

Chlorophyll extraction was done as described previously (Rajalakshmi, 2013). Briefly, 100 mg of fresh leaves of wild type and SYN3 O/E were taken and ground with 1.5 ml of 80% cold acetone. Homogenized samples were centrifuged at 14000 rpm for 5 minutes. The supernatant was transferred and the absorbance of the solution was taken at 645nm and 663nm against the solvent (80% acetone) blank.

Chlorophyll fluorescence parameters were measured with a microscopy pulse amplitude modulation (PAM) fluorometer according to (Bang, 2008). The leaves were dark adapted for 10 minutes before each fluorescence measurement recording with the white actinic light (216 µmol m-2 s-1)

2.3 Results and Discussion

2.3.1 Identification of FtsZ 1-1 and FtsZ 2-1 proteins as interacting partners of SYN3

The yeast-two hybrid (Y2H) system is a powerful tool for detecting protein-protein interactions in vivo. It can be used to establish interactions between two known proteins or to build cDNA libraries that target interacting proteins. For a better understanding of the function of SYN3, a yeast-two hybrid assay was performed by Yuan and Makaroff to find interacting partners of SYN3 in vivo.

Table 2: Yeast-two-hybrid analysis reveals candidate interacting partners of SYN3 in the chloroplasts (Yuan and Makaroff, unpublished data).

Arabidopsis cDNA libraries, using SYN3 as bait, identified several interacting partners of SYN3 (Table-1) including FtsZ 1-1 and FtsZ 2-1. Among all the interacting partners of SYN3 (Table- 2), FtsZ 1-1 and 2-1 are particularly interesting to us because FtsZ proteins are found to be localized to chloroplasts and are involved in chloroplast division (Osteryoung, 1998 (a); Osteryoung, 1998 (b). This finding is significant as it suggests that SYN3 may be involved in chloroplast cell division as SYN3 interacts with FtsZ 1-1 and 2-1.

Figure 2.1: SYN3 immunoprecipitates FtsZ 1-1 and 2-1 in vitro. Co-immunoprecipitation was carried out with in vitro expressed FtsZ1-1 and 2-1 proteins were [3H] labeled and pulled down using cold SYN3 protein (Yuan and Makaroff, unpublished data).

A co-immunoprecipitation (Co-IP) assay was carried out by Li Yuan to confirm in vitro interaction of SYN3 with FtsZ 1-1 and FtsZ 2-1. Co-IP is the most straightforward technique to study protein interactions, whereby an antibody against a target protein is attached to Sepharose beads and is used to directly capture that protein and indirectly additional proteins that may interact with the target protein. Co-IP was performed by immunoprecipitating in vitro expressed SYN3 that had been incubated with in vitro expressed FtsZ1-1 and 2-1 to test if SYN3 interacts with either.

Analysis of immunoprecipitation by SDS-PAGE and immunoblotting using anti-FtsZ 1-1 and anti-FtsZ 2-1 antibodies showed bands near 47 kDa and 53 kDa, respectively (Figure 2.1). Immunoprecipitation of in vitro expressed SYN3 shows an interaction with FtsZ1-1 and 2-1. Co- 32

IP results were consistent with Y2H in vivo interaction study results and it further confirmed the interaction between in vitro expressed SYN3 and FtsZ 1-1 or FtsZ 2-1.

Figure 2.2: SYN3 localizes in chloroplasts. Immunolabeled sections were viewed using DAPI (blue), FITC (green), and TEXAS (red) filters sets. The yellow color in the overlay of red, blue and green signals indicates the localization of SYN3 in chloroplasts (Yuan and Makaroff, unpublished data).

Both Co-IP and Y2H results showed the interaction between SYN3 and FtsZ proteins. To get a better understanding of the function of SYN3, immunolocalization studies were carried out by Li Yuan to determine if SYN3 is in fact a chloroplast-localized protein. To study the localization of SYN3, protoplasts isolated from stably-transformed SYN3-YFP plants were used. Immunolabelling of SYN3 fused to YFP using GFP antibodies showed the localization of SYN3 in chloroplasts (Figure 2.1) The blue DAPI signal reflects staining of organelle DNA. Green shows SYN3-YFP immunolabeled with antibodies against GFP. Red is chlorophyll auto- fluorescence, and yellow is merged blue, green, and red signals. Merged yellow signal image indicates that SYN3 localizes to chloroplasts. As an alternative explanation, SYN3 may be also localized on the surface of chloroplasts. However, our immunolocalization analysis at least suggests that SYN3 is localized to chloroplasts and emphasized more on that SYN3 may have a function in chloroplasts.

2.3.2 Endogenous SYN3 localizes to thylakoid within chloroplasts

Immunolocalization studies showed that SYN3 localizes into the chloroplast. However, it is unclear whether the protein is actually within the chloroplast and if so to which sub organellar compartment of the chloroplast SYN3 is localized. The sub organellar localization of a protein is an important characteristic for functional studies. Therefore, to further study the potential localization of SYN3 in chloroplasts, immunoblotting with total cell lysates and fractionated chloroplasts from P. sativum leaves was performed.

Figure 2.3: Sub-organellar localization of SYN3 in P. sativum chloroplasts. Equal volume (10 µl) of protein samples were separated by SDS-PAGE, transferred to nitrocellulose membrane and immunoblotted with antibodies to SYN3, HCF106, and histone H3, followed by secondary antibodies.

Immunoblotting with fractionated chloroplast samples showed that SYN3 localizes to the thylakoid within chloroplasts (Figure 2.3). The size of the chloroplast localized pea SYN3 protein was found to be about 56 kDa. Histone H3 is a nuclear protein and was used as a nuclear control. As predicted, histone H3 localizes only in whole cell lysate, which has size around 17 kDa. Hcf106 is a thylakoid membrane protein and was used as a thylakoid localization control.

Figure 2.4: Sub-organellar localization of SYN3 in A. thaliana chloroplasts. Equal volumes (10 µl) of protein samples were separated by SDS-PAGE, transferred to nitrocellulose membrane and immunoblotted with primary antibodies against SYN3, HCF106, and histone H3 followed by secondary antibodies.

Results in Figure 2.3 showed that SYN3 localizes into the thylakoid within pea chloroplasts. However, SYN3 is an Arabidopsis α-kleisin. Therefore, to further confirm localization of SYN3 in Arabidopsis chloroplasts, fractionation was performed with Arabidopsis leaves. Immunoblotting results (Figure 2.4) confirmed that SYN3 localizes in the thylakoid within chloroplast. Again, the size of the chloroplast-localized SYN3 protein was found to be about 56 kDa. Therefore, immunoblotting results from P. sativum and Arabidopsis chloroplasts (Figure 2.3 and Figure 2.4) confirmed that SYN3 localizes to the thylakoid. Further studies need to be performed to determine where more specifically SYN3 localizes in the thylakoid.

Surprisingly, the size of the chloroplast-localized SYN3 protein is about 56 kDa as detected by western blot, even though full length nuclear SYN3 has protein predicted size of about 77 kDa. This result suggests that SYN3 may contain an unusually long targeting sequence that is processed during in vivo import resulting in a mature protein size of about 56 kDa.

2.3.3 SYN3 is not imported into isolated chloroplasts in vitro

Immunolocalization (Figure 2.2) and immunoblotting (Figures 2.3 and 2.4) results showed that a fraction of the cellular SYN3 is localized to chloroplasts. To test whether SYN3 could be

35 targeted to the chloroplasts, we performed in vitro chloroplast import experiments for SYN3 into isolated chloroplasts using a robust in vitro assay. The in vitro translated precursor proteins (TP) for SYN3 and pOE17 were about 92 kDa and 23 kDa, respectively (Figure 2.5, lane 1 and 4). pOE17 was imported and processed to its mature form, as expected (Figure 2.4, lane 5 and 6). The ability of the pOE17 control protein to be successfully imported, followed by processing to a protease protected mature form, indicated that the chloroplasts were active and intact. However, a mature, imported band was not observed for SYN3 (Figure 2.4, lane 2 and 3). Therefore, SYN3 failed to import into isolated chloroplasts using the same conditions that supported the successful import of the known chloroplast protein pOE17.

One reason for the lack of import efficiency could be attributed to the fact that the TOC import apparatus for SYN3 precursor import may be highly susceptible to proteolysis during the chloroplast isolation steps (Kessler, 1994; Jarvis, 2002). If SYN3 is imported via the TOC apparatus, then this could explain the negative result. Therefore, import assay conditions were varied by isolating chloroplasts in the presence of protease inhibitor. However, the same result was obtained when chloroplasts were isolated in the presence of proteases (data not shown).

These results indicate that SYN3 cannot be post-translationally targeted to the chloroplasts using in vitro experiments despite its demonstrated localization in chloroplasts (Figures 2.4 and 2.5). However, based on our western blotting analysis, the SYN3 size in chloroplast is 56 kDa, but the in vitro translated product size of SYN3 is about 92 kDa, raising the possibility that the in vitro translated protein contains plasmid-derived sequences that may interfere with its import. It is unclear how SYN3 gets imported into chloroplasts. Future studies need to be performed to show which part of SYN3 is imported into chloroplasts and subsequently becomes processed into the active form of SYN3. To that end, the subcellular localization of deletion mutants of SYN3 need to be made and analyzed by transient expression of YFP fusion proteins in A. thaliana protoplasts. This study will yield more information about the mature part of SYN3 that is active in chloroplasts.

Figure 2.5: In vitro expressed SYN3 failed to import into isolated chloroplasts from P. sativum. Radiolabeled proteins SYN3 and pOE17 were synthesized in vitro (translation product: TP) and incubated with isolated, intact chloroplasts. The position of precursor and mature proteins are indicated.

2.3.4 Immunoprecipitation of SYN3 in vivo and in vitro

Previous co-IP results (Figure 2-1) showed that in vitro expressed SYN3 interacts with in vitro expressed with FtsZ 1-1 and 2-1. To further study the interaction of SYN3 with FtsZ1-1 and 2-1 in vivo, we have performed in vitro immunoprecipitation of SYN3 with antibodies against SYN3 protein as a prelude to in vivo immunoprecipitation of SYN3.

Figure 2.6, lanes 1 and 3 show that the sizes of in vitro translated SYN3 and pOE33 are about 92 kDa and 33 kDa, respectively. In vitro expression and immunoprecipitation with SYN3 antibody bound to PAS beads resulted in a strong band at 92 kDa, which is the same size as the protein produced by in vitro transcription/translation (Figure 2.6, lane 2). This result confirms the ability

37 to immunoprecipitate SYN3 that was synthesized in vitro. pOE33 was used as a negative control to confirm the specificity of the co-immunoprecipitation. As expected, the SYN3 antibody does not pull down the 33 kDa control (Figure 2.6, lane 3), confirming the specificity of the SYN3 antibody.

Figure 2.6: An immunoprecipitation of SYN3 in vitro. Radiolabeled recombinant SYN3 and pOE33 were incubated with anti-SYN3 beads. Translated product (TP) and immunoprecipitated samples were analyzed by SDS-PAGE and radio fluorography.

Y2H assay (Table 1) and in vitro immunoprecipitation (Figures 2-1) results showed that FtsZ 1-1 and FtsZ 2-1 proteins interact with SYN3. This result prompted us to perform a co- immunoprecipitation with chloroplast lysates to study if SYN3 interacts with FtsZ 1-1 and FtsZ 2-1 in vivo.

To validate the experimental conditions, we first used pOE23 (P. sativum oxygen evolving complex subunit 23 kDa) for which beads bound to specific antibodies were available. After immunoprecipitating chloroplast lysates of wild-type plants with pOE23 antibodies and immunoblotting with pOE23 antibodies (Figure 2.7), a band closer to 23 kDa, which is near the size of pOE23 input, confirms that pOE23 was pulled down with pOE23 antibodies and that the conditions for immunoprecipitation are satisfactory. The isolated supernatant has a small quantity of target protein, as bands are seen when it was blotted with antibody against pOE23. 38

Washes gave no band when it was blotted with antibody against pOE23, confirming that there is no target protein in wash.

Figure 2.7: Immunoblot showing results after immunoprecipitation (IP) of chloroplast lysate samples with anti SYN3 or pOE23 antibody bound protein A sepharose beads. Pull down samples and input were separated by SDS-PAGE and immunoblotted with α-pOE23 (1:10000) and α-SYN3 (1: 3000). This experiment was replicated 4 times.

When the immunoprecipitation experiments were repeated with SYN3 antibody bound to PAS beads, followed by western blotting with anti-SYN3 antibody, a band was observed closer to 56 kDa which is the same size as was observed in chloroplast lysate input (Figure 2.7). However, the 56 kDa band was also observed when immunoprecipitated proteins were blotted with pOE23 antibodies (indicated in asterisk), which raises the question if the 56 kDa band observed in SYN3 immunoblot experiments is due to non-specific interactions.

The antibody heavy (H) chain is approximately 50 kDa and the light chain is about 25 kDa. The one that is indicated by an asterisk may be the heavy chain. Therefore, the heavy chain might be masking an interaction of SYN3, which has target size of about 56 KDa. Addition of a YFP tag to SYN3 protein would increase the molecular weight of the SYN3 target protein and eliminate the heavy chain issues. We have used chloroplast lysates from P. sativum leaves in this experiment. Therefore, SYN3-YFP plant lines need to be used for immunoprecipitation experiments, which should remove possible size obstructions in the western blot caused by the heavy chain.

The negative result may also be due to the endogenous protein concentrations of SYN3 in chloroplast lysates not being sufficient for immunoprecipitation. Using chloroplast lysates from SYN3 over-expressed (SYN3 O/E) lines might overcome any endogenous protein level issues and increase visualization of protein-protein interactions. Furthermore, for antigen non- specificity, pre-immune serum needs to be included as a negative control to check if the antibody detects anything other than our antigen of interest (SYN3).

Previous work has also shown that SYN3 interacts with FtsZ 1-1 and FtsZ 2-1 (Yuan and Makaroff., unpublished). Therefore, we suspect that SYN3 might have specific interactions with FtsZ1-1 and FtsZ 2-1 during its function in chloroplast division. To that end, first, whether SYN3 interacts with FtsZ proteins in vivo needs to be shown in future with techniques such as co- immunoprecipitation assays using chloroplast lysates.

2.3.5 Screening of transgenic plants by BASTA selection and genotyping PCR

BASTA is an herbicide that contains glufosinate ammonium, one of the most commonly used markers for the selection of transgenic Arabidopsis plants (Weigel, 2006). The bacterial bialophos resistance gene (BAR) encodes the enzyme called phosphinotricin acetyl transferase (PAT), which confers resistance to glufosinate ammonium (Weigel, 2006). We have used this BASTA selection method to screen for transformants as this method does not require the use of sterile techniques and can be performed on plants growing in soil. SYN3 O/E, BASTA resistant transformants grew normally and were green while, untransformed plants remained small, became yellow and died one week after treatment.

Genotyping PCR is one process for screening of transformants plants for the presence of transgene (Abdalla, 2007). Genotyping PCR was performed for plants that are BASTA resistant plants.

Figure 2.8: Genotyping PCR screening of BASTA resistant SYN3-YFP lines. The expected size of cDNA was observed ~ 1.2Kb. Lane 1- 100 bp marker, Lane 2-16: BASTA resistant lines, lane 17: wild type plant and lane 18: positive control, colony #73 (35S-SYN3-cDNA-YFP). Primers used were SYN3-7 &YFP-1.

Plants that are transformed with SYN3-YFP transgene were expected to produce about 1.2 kBp fragment in PCR experiments using SYN3 forward and YFP reverse primers (lane 18). All 15 BASTA resistant plants lines tested were PCR positive and gave a band the size of 1.2 kBp (Figure 2.8, lane 2 & 4-16). Only one plant did not give a positive band (Figure 2.8, lane 3). Wild-type A. thaliana (non-transgenic plants) was used as negative control. No bands were seen when non-transgenic plant DNA was used. This result confirmed the presence of the transgene SYN3-YFP cDNA in SYN3-YFP lines. These PCR resistant plants were used for our study.

2.3.6 Over-expression of SYN3 resulted in stunted growth and abnormal phenotype plants

In an attempt to determine SYN3 function in the chloroplast, SYN3 YFP fusions expressed from the 35S promoter were generated. Plants transformed with Agrobacterium which carries 35S- SYN3-YFP vector were named as SYN3-YFP O/E lines.

Transgenic SYN3-YFP O/E T1 plants segregated into two populations or phenotypes. Class I, which had a wild-type phenotype (Figure 2.9), plants were classified as “wild-type-like” if the plants displayed a phenotype similar to the wild-type controls. In class II, which had a strong

41 phenotype, the plants were severely stunted and showed a dramatic increase in the formation of stems and branches. Growth and flowering stages were delayed. The class II plants were however able to produce siliques like wild-type (Figure 2.9). The ratio of wild-type looking to severely stunted phenotype in the T1 plants was 43%:57% (Table-3).

Generation Total Phenotype number wildtype looking Severe stunted Intermediate Mild type of growth plants

T1 14 6 8 0 0

T2 39 25 0 14 0

T3 8 3 0 0 5

Table 3 : Phenotypes of SYN3-YFP O/E plants. Plants that have a phenotype similar to the wild- type are described as wildtype looking. Plants that were severely stunted in growth were called stunted growth plants. Plants that have phenotype is milder than the T1 stunted growth phenotype plants are described as intermediate type. Plants that have a milder phenotype compared to T1 and T2 were called mild type.

T1 wild-type looking plants by self-pollination produced T2 plants with wild-type looking and intermediate phenotypes. T1 stunted growth plants produced only intermediate phenotype. Plants that displayed intermediate phenotype displayed slow growth and delayed flowering and purple leaves, while some of these plants were not able to produce seeds, some plants did not have the ability to produce siliques. One of the wild type looking T2 progeny by self-pollination (named as 13cT2) produced T3 plants that were 100% BASTA resistant, confirming that 13cT2 line was homozygous.

while in other lines the mutation was either not detectable in subsequent generations, or T-DNA were found t 42

A B C

D E F

G H

Figure 2.9: Phenotypic analysis of 35S-SYN3-YFP transgenic Arabidopsis thaliana plants. A) Phenotype of SYN3 O/E, 7 weeks old T1 plants. B&C) Phenotype of 7-week-old SYN3 O/E T2 plants. D-F) Phenotype of wild type looking 40 days old SYN3 O/E T3 plants. G) phenotype of 5 weeks old T1 wild-type A. thaliana (Col-0). H) 40 days old wild-type A. thaliana.

These results suggest that over-expression of SYN3-YFP has an effect on plant growth. The Brassica rapa NGA gene (BrNGA1) that belongs to the B3- type transcription factor superfamily, is a homolog of the AtNGA family and involved in lateral organ growth. Over-expression of a BrNGA1 gene in A. thaliana markedly reduced the growth of leaves, root, flowers, and cotyledons due to reduction in cell numbers (Kwon, 2009) have similar phenotypes to SYN3 O/E lines.

A previous study has shown that elevated levels of native SYN3 expression affects early vegetative growth in Arabidopsis plants (Yuan, 2014). We suggest that high-level expression of SYN3-YFP may also affect plant growth and developmental processes. However, it is not clear how an increased level of SYN3-YFP expression affects plant growth or development at this point. Previous studies also have suggested that SYN3 may be indirectly involved in transcriptional regulation (Yuan, 2014).

2.3.7 SYN3 expression level in WT and SYN3-YFP O/E lines

An immunblot analysis on whole cellular extracts of plant leaves was performed to determine the expression level of SYN3-YFP in SYN3-YFP O/E lines (Figure -2.10). For this experiment, SYN3 O/E T3 generation lines which have the mildest phenotypes compared to T2 and T1 lines were chosen for analysis.

SYN3 is consistently observed as a 56 kDa in wild-type chloroplasts. In contrast, a 72 kDa band was primarily detected in SYN3-YFP O/E lines (Figure 2.10, lane 2X). This 72 kDa band in SYN3-YFP O/E lines is the size expected for the full length nuclear SYN3, which is about 77kDa. However, the 72 kDa band is only observed in SYN3-YFP O/E lines and not in wild- type. Therefore, this suggests that the band represents the transgene. However, the identity of the band as the transgene still needs to be confirmed with antibodies against YFP. Microscopy studies also can be done to visualize the localization of yellow fluorescenceassociated with SYN3-YFP protein expression. pOE23 was used as a loading control for this experiment and its expression was observed in both wild type and SYN3-YFP O/E plants with the size appearing to be 23 kDa.

Figure 2.10: Immunoblot analysis of SYN3 protein in wild-type and SYN3-YFP plants. Total protein was extracted from wild-type and SYN3-YFP plant leaves. Samples were loaded in concentration decerasing fashion 2x (20 µg), 1x (10 µg). pOE23 was used as a loading control.

Quantitative RT-PCR (qRT-PCR) was next performed to study SYN3-YFP mRNA levels in SYN3-YFP O/E lines. The results of this expression analysis (Figure 2.11) show that transcript level of SYN3-YFP in SYN3-YFP O/E lines was significantly different (p < 0.05) from wild- type plants, being upregulated by about 14-fold compared with the wild type plants. This result confirms the over-expression of SYN3-YFP in mid-type SYN3-YFP O/E lines.

Therefore, both western blot and qRT-PCR results (Figures 2.10 and 2.11) confirmed the expression of the SYN3-YFP transgene in SYN3-YFP O/E lines. We suggest that the abnormal phenotype in SYN3-YFP over expression lines may be due to elevated levels of SYN3-YFP expression (Figure 2.9).

14.99 WT SYN3 0/E 15

Relative gene expression level expression gene Relative 1 0 SYN3-YFP

Figure 2.11: qRT-PCR analysis of relative SYN3 expression in wild type and SYN3 O/E plants. SYN3 expression level was normalized to the control gene UBQ4. Data are given as the mean of three biological replicates. Error bar = standard error.

2.3.8 Over-expression of SYN3 has no effect on chloroplast division

Based on Y2H and Co-IP assay results (Table-2 and Figure 2.1), we hypothesized that SYN3 may be involved in chloroplast division as SYN3, a nuclear gene involved in chromosome segregation during cell division, interacts with chloroplast division FtsZ proteins. In addition, studies have shown that increased levels of AtFtsZ-1 inhibited chloroplast division and resulted in plants that exhibit novel chloroplast morphology (Osteryoung, 1998 (b) ; Stokes, 2000). Over expression of SYN3 yielded a stunted growth phenotype (Figure 2.9), which further prompted us to question whether SYN3 affects chloroplast division.

A B

Figure 2.12: Observation of chloroplast division phenotypes of mesophyll cells. A) SYN3-YFP cells, B) wild type cells. Isolated leaf mesophyll cells from 35 days old wild type and SYN3-YFP O/E plants viewed under differential interference microscopy (DIC). Scale bar represents 5 µm.

To test if over-expression of SYN3-YFP affects chloroplast division, differential interference microscopy (DIC) was used to count the number of chloroplasts per cell, cell area, and chloroplast area per cell in transgenic SYN3-YFP with mild type phenotype and wild-type plants (Figure 2.12). The mean values for mesophyll cell area and chloroplast number and chloroplast area as directly determined from DIC images (Figure 2.12) were compared to the values for wild-type cells (Table-3). Figure 2.12 shows sample microscopy results of mesophyll cells from a leaf section of T3 homozygous SYN3-YFP O/E line and wild-type plants.

Figure 2.13: Graph of chloroplast number relative to mesophyll cell area. SYN3-YFP O/E cells do not have significantly different numbers of chloroplasts per mesophyll cell area. Each point represents the measurement from one mesophyll cell whose chloroplasts number and area were measured using ImagePro software.

Figure-2.13 shows the relationship between chloroplast number per mesophyll cell and mesophyll cell area. The best fit lines have slopes of 0.2569 (r2 = 0.92) for wild-type and 0.2278 (r2 = 0.94) for SYN3-YFP O/E. During wild-type mesophyll cell development, the number of chloroplasts per cell is closely corelated with the size of the cell (slope =0.2569, r2= 0.92). Therefore, values of slope and correlation for wild type and SYN3-YFP O/E suggest that chloroplast number and cell area are closely correlated in both wild type and SYN3-YFP O/E (Figure 2.13).

Figure 2.14: Total chloroplast area per mesophyll cell and mesophyll cell area in wild type and SYN3-YFP O/E leaves. For each cell, total chloroplast plan area was calculated by using equation, total chloroplast plan area = no of chloroplasts multiplied by mean chloroplast plan area. Each point represents the measurement from one mesophyll cell whose chloroplasts total area and cell area were measured using ImagePro software.

The best fit lines of wild-type (slope= 0.4194, r2 = 0.92) shows that the chloroplast area and cell area are closely correlated with each other (Figure 2.14). In SYN3-YFP O/E lines, this relationship between chloroplast area and cell area (slope= 0.5462, r2 = 0.94) is different from that shown in wild-type cells (Figure 2.14).

Table 3 summarizes the results for mean values of mesophyll cell area, chloroplast number, and chloroplast area for wild-type and SYN3-YFP O/E lines as directly determined from DIC images (Figure 2.12). The relationship between chloroplast number per mesophyll cell and mesophyll cell size in SYN3 O/E leaves (Table-3) shows that chloroplast number remains at an average of 28 per cell (p > 0.05). This is not different from wild-type cells. The relationship between chloroplast area per cell area for SYN3 O/E cells (Table-3) is different from the wild-type (P< 0.05). Further, cell area of SYNE O/E lines also was different from wild-type cell area (P< 0.05).

Mesophyll cells

Mean cell area Mean chlroplasts Mean chloroplast

(µm2) # area Genotype (µm2)

Wild-type 90 (45) 27 (11) 50 (20)

SYN3 O/E 100 (55) 28 (15) 60 (31)

Table 4 : Chloroplast and Cell Characteristics of SYN3 O/E and wild type plants. Mesophyll cell areas and chloroplast areas were measured by image analysis. Mean was caluculated for 144 cells from each plants. Six different plants were analyzed from wild type and SYN3-YFP O/E. Standard deviation are shown in parentheses.

In the future, it will be necessary to compare changes in the relationship between chloroplast number and cell size in over a range of cell sizes within the leaf and in developmentally-staged leaves. This is important to show whether the chloroplast division in SYN3-YFP O/E lines differs markedly from the wild type. Our DIC results (Table-3, Figures 2.13 and 2.14) showed that there is a trend in difference in chloroplast area and cell area between SYN3-YFP O/E lines and wild-type even though the chloroplasts number per mesophyll cell between wild type and SYN3-YFP O/E remains about the same.

Chloroplasts are generated through the binary fission of preexisting chloroplasts. In mutants with division defects, chloroplasts are generally enlarged in size and reduced in number. An increased number of chloroplasts per unit cell area is associated with a reduction in chloroplast size compared with wild-type, while decreased mean chloroplast number per cell are associated with an increase in chloroplast size (Pyke, 1987; Pyke, 1992; Pyke, 1994 ; Osteryoung, 1998 (b). With increased cell area, SYN3-YFP O/E lines should have an increased number of chloroplasts

50 compared to wild-type. The increased chloroplasts area in SYN3-YFP O/E lines maintain a constant number of chloroplasts number per mesophyll cell volume as wild-type. Whereas, total chloroplast area is increased in SYN3-YFP O/E lines compared to wild-type.

Therefore, it is possible that a failure of the chloroplasts to divide normally, followed by continued chloroplast expansion leads chloroplasts to accumulate and increase in chloroplast area in the mature mesophyll cell. Therefore, it is possible that over-expression of SYN3-YFP affects chloroplast division and that during mesophyll cell development, chloroplast area increases while chloroplasts number remains similar to wild-type. We hypothesized a role for SYN3 in chloroplast division based on its interaction with the FtsZ 1-1 and FtsZ 2-1, and the immunolocalization results demonstrating that SYN3 is localized in the chloroplast (Figure 2-2). These results support our hypothesis. However, whether SYN3 or SYN3-YFP has an effect on chloroplast division is currently unknown.

Our DIC results are consistent with those of the previous data that found that SYN3-YFP O/E cells have fewer number, larger chloroplasts (Yuan and Makaroff, unpublished). However, our results contradict those of a second study where they found that knockdown or over expression of SYN3 has no effect on chloroplast cell division (Stempkensi and Edelmann, unpublished). Therefore, further studies need to be done to determine if SYN3 influences chloroplast cell division using SYN3 knock down lines.

2.3.9 Reduced total chlorophyll and chl b content of plants due to over expression of SYN3

Chloroplast development is an important determinant of the photosynthetic capacity of leaves (Webber, 1996). Further, the thylakoid membrane is an important site for protein synthesis and most of the photosynthetic related proteins are localized in thylakoids (Friso, 2004; Jarvi, 2016). Immunoblot results (Figures 2.3 and 2.4) showed that SYN3 localizes into the thylakoid and over expression of SYN3-YFP affected chloroplast division. Therefore, we examined effects of over expression of SYN3-YFP on chlorophyll synthesis and photosynthetic efficiency in SYN3-YFP

O/E plants compared to wild type. Comparison of chlorophyll fluorescence parameters are summarized in Figures 2.15 and 2.16.

Figure 2.15: Fluorescence parameters, effective PSII quantum yield (фPSII) of wild type and SYN3 O/E plants. Mean and SE bars are shown for three independent measurements (n=3).

The ratio of variable fluorescence/maximal fluorescence from dark-adapted leaves (Fv/Fm) can be used to measure the maximum photochemical efficiency of PSII (Baker, 2008). The maximum quantum yield of PSII (Fv/Fm) values for non-stressed leaves are around 0.83 which is the normal value shown in mesophyll cells of most plant species (Björkman, 1987). The Fv/Fm of SYN3 O/E plants was about 0.79 +/- 0.01 which is closer to wild-type Fv/Fm 0.80 +/- 0.01 and was not significantly different from wild-type.

Immediately after application of illumination of actinic light, maximum quantum yield of PSII (Fv/Fm) values of wild-type and SYN3-YFP O/E plant leaves dropped from 0.80. At the end of the exposure interval, the effective quantum yield of PSII (фPSII) in wild-type reached a value about 0.42 whereas the value in SYN3-YFP O/E plants was closer to 0.30 (Figure 2.15). фPSII measures the proportion of light absorbed by PSII that is used in photochemistry. It gives a measure of the rate of linear electron transport and so indicates overall efficiency of photosystem II in photosynthesis (Murchie, 2013).

The фPSII of SYN3-YFP O/E plants was not significantly different from wild-type plants as standard errors overlap. These фPSII values indicate that the SYN3-YFP O/E plants exhibit photosynthetic activity similar to wild-type plants. Studies have shown that the photosynthetic efficiency in leaves depends on the development of chloroplasts (Pyke, 1992; Boffey, 1992).

The normal value for maximum effective PSII quantum yield (фPSII) for wild-type Arabidopsis plants has been reported to be close to 0.50 (50%) by most studies. Figure 2.15 shows a фPSII value of 0.42 for wild-type. We suggest that this is caused by an external stress for plants. This suggest that SYN3-YFP O/E plants may also have been under stress and could be more sensitive to the stress than wild-type. Therefore, it is currently unclear whether over expression of SYN3- YFP has a direct effect on photosynthetic efficiency.

Figure 2.16 : Fluorescence parameters, non-photochemical quenching (NPQ) of wild type and SYN3 O/E plants. Mean and SE bars are shown for three independent measurements (n=3).

Non-photochemical quenching (NPQ) is a photoprotective mechanism that dissipates excess energy of photosystem II (PSII) into heat. Therefore, we have measured NPQ to estimate the rate constant for heat loss from PSII (Muller, 2001). Figure 2.16 shows that the initial NPQ value for SYN3-YFP O/E and wild-type plants is zero. Studies have shown that all PSII centers are open and no NPQ is present in healthy non-stressed plants in the dark-adapted state (Murchie, 2013).

The application of a saturating pulse of light to dark-adapted leaves induced a maximum value of fluorescence about 1.2 in both wild-type and SYN3-YFP O/E plants by closing the reaction centers. Standard error bars of both SYN3 O/E and wild-type NPQ overlap considerably, and statistical analysis confirmed there is no difference (p > 0.05) between the NPQ values of SYN3 O/E and wild-type plants.

During the application of actinic light, the reaction centers were closed. So, the level of photochemical efficiency was reduced and only non-photochemical processes were remained. Therefore, the NPQ values were increased from zero. These NPQ results indicate that the SYN3- YFP O/E plants dissipate excess excitation energy via nonphotochemical pathways as healthy wild-type plants and NPQ is not affected by SYN3 over-expression in SYN3 O/E plants.

Chl (µg/ml) Line Total Chl Chl a Chl b Chl a/ Chl b

Wild type 53.30±0.03 19.13±0.03 34.17±0.30 0.56±0.01

SYN3 O/E 51.71±0.02 19.24±0.29 32.47±0.31 0.59±0.01

Table 5: The content of total Chlorophyll (Chl), Chl a, Chl b, and Chl a/b ratios. The concentrations of chlorophyll a, chlorophyll b, and total chlorophyll were calculated using the following equation: Total Chlorophyll: 20.2(A645) + 8.02(A663), Chlorophyll a: 12.7(A663) – 2.69(A645), Chlorophyll b: 22.9(A645) – 4.68(A663). Data are means +/- SD (n=3).

Based on our findings, we found that over expression of SYN3-YFP has an effect on the photosynthetic efficiency of plants. One interesting feature of the SYN3-YFP O/E plants is the presence of light green and purple color leaves, in contrast to the dark green color observed in wild-type (Figure 2.9). To understand the basis for the impaired photosynthetic capacity in the SYN3-YFP O/E mutants, we have studied chloroplast composition. For this reason, the chlorophyll content was estimated using 100 mg of fresh leaves of T3 homozygous, SYN3-YFP O/E mild-type lines.

No significant differences (p > 0.05) were observed in the chlorophyll a content and Chl a/b ratio between wild-type and SYN3 O/E lines. However, the total chlorophyll content in SYN3 O/E

55 plants was statistically lower than the wild-type plants (p < 0.05). There was also a reduction (p < 0.05) of chlorophyll b content in SYN3 O/E lines (Table 4) compared to wild-type.

These results (Table 4) indicate that the SYN3-YFP over expression influences chlorophyll b and total chlorophyll content in SYN3-YFP O/E lines. Changes in total chlorophyll content might be observed due to a reduction of chlorophyll b content. We have estimated chlorophyll content based on three separate plant lines. Additional data needs to be obtained to determine if over expression of SYN3-YFP affects the biosynthesis of chlorophyll b in any way and to draw a valid conclusion. To that end, whether these plants have reduced light harvesting complexes (LHCs) and develop abnormal thylakoid membranes because of limited chlorophyll b synthesis need to be examined.

Taken together, our study results altogether suggest that over expression of SYN3-YFP may have effects on chloroplast number and chlorophyll b content.

2.4 References

1. Abdalla, K.S. 2007. A simple and reliable PCR-based method for detection and screening of transgenic plants transformed with the same endogenous gene. Arab Journal of Biotechnology. 10 :155-160. 2. Bai, X., Peirsion, B.N., Dong, F., Cai, X., Makaroff, C.A. 1999. Isolation and characterization of SYN1, a RAD21-like gene essential for meiosis in Arabidopsis. Plant Cell. 11:417±430. 3. Baker, N.R. 2008. Chlorophyll fluorescence: A probe of photosynthesis in vivo. In Annual Review of Plant Biology. Vol. 59. Annual Reviews, Palo Alto. 89-113. 4. Bang, W.Y., Son, Y.S., Kim, S.W., Jeong, J. ., Bahk, J.D.., Shiina, T., Kim, D.W., Jeong, I.S., Im, C.H., Hwang, S.M., Ji. C. 2008. Role of arabidopsis chl27 protein for photosynthesis, chloroplast development and gene expression profiling. Plant Cell Physiology. 49:1350–1363. 5. Björkman, O., Demmig. B. 1987. Photon yield of O2 evolution and chlorophyll fluorescence characteristics at 77 K among vascular plants of diverse origins. Planta. 170:489-504. 6. Boffey,S. 1992. Chloroplast replication.In N Baker, H Thomas, eds, Crop photosynthesis: spatial and temporal determinants. Elsevier, Amsterdam.361-379. 7. Bose, T., Jennifer,L., Gerton. 2010. Cohesinopathies, gene expression, and chromatin organization. Journal of Cell Biology. 189:201–210.

8. Cai, X., Dong. F.G., Edelmann, R.E., Makaroff. C.A. 2003. The Arabidopsis SYN1 cohesin protein is required for sister chromatid arm cohesion and homologous chromosome pairing. Journal of Cell Science., 2003, 116, 2999-3007. 9. Cline, K. 1986. Import of Proteins into Chloroplasts. The Journal of Biological Chemistry. 261: 14804-14810. 10. Cline, K., Henry, R., Li, C.J. Yuan, J. (1993). Multiple pathways for protein transport into or across the thylakoid membrane. EMBO Journal. 12 : 4105-4114. 11. Cline.K., Mori.H 2001. Thylakoid ΔpH-dependent precursor proteins bind to a cpTatC– Hcf106 complex before Tha4-dependent transport. Journal of Cell Biology. 154:719–730. 12. Clough, S.J., Bent. A.F. 1998. Floral dip: a simplified method for Agrobacterium- mediated transformation of Arabidopsis thaliana. Plant Journal. 16:735-743. 13. Costa-Nunes, J.A., Bhatt. A. M., O’Shea. S., West.C.E., Bray.C. M., Grossniklaus. U., Dickinson.H.G. 2006. Characterization of the three Arabidopsis thaliana RAD21 cohesins reveals differential responses to ionizing radiation. Journal of Experimental Botany. 57:971–983. 14. Dervyn, E., Noirot-Gros, M., Mervelet, P., McGovern, S., Ehrlich, S. D., Polard, P., Noirot,P. 2004. The bacterial condensin/cohesin-like protein complex acts in DNA repair and regulation of gene expression. Molecular Microbiology. 51:1629-1640. 15. Dong, F., X. Cai, Makaroff. C.A. 2001. Cloning and characterization of two Arabidopsis genes that belong to the RAD21/REC8 family of chromosome cohesin proteins. Gene. 271:99-108. 16. Fennell-Fezzie, R., Gradia, S.D., Akey, D. Berger. J.M. 2005. The MukF subunit of Escherichia coli condensin: architecture and functional relationship to kleisins. Embo Journal. 24:1921-1930. 17. Friso, G., Giacomelli, L., Ytterberg, A.J., Peltier, J.B., Rudella, A.., Sun, Q.., van Wijk. K.J. 2004. In-depth analysis of the thylakoid membrane proteome of Arabidopsis thaliana chloroplasts: New proteins, new functions, and a plastid proteome database. Plant Cell. 16:478-499. 18. Gerton, J. 2005. Chromosome cohesion: a cycle of holding together and falling apart. PLoS Biol. 3:e94. 19. Hirano, T. 2000. Chromosome cohesion, condensation and separation. Annual Review of Biochemistry. 2000. 69:115–144. 20. Jarvi, S., Isojarvi, J., Kangasjarvi, S., Salojarvi, J., Mamedov, F., Suorsa, M. ., Aro. E.M. 2016. Photosystem II repair and plant immunity: lessons learned from arabidopsis mutant lacking the thylakoid lumen protein 18.3. Frontiers in Plant Science. 7:13. 21. Jarvis, P., Soll.J. 2002. Toc, Tic, and chloroplast protein import (vol 1541, pg 64, 2001). Biochimica et Biophysica Acta-molecular Cell Research. 1590:177-189. 22. Karamoko, M., El,K. E., Mandaron, P., Lerbs-Mache, S., Falconet, D. 2011. Multiple FtsZ2 isoforms involved in chloroplast division and biogenesis are developmentally associated with thylakoid membranes in Arabidopsis. FEBS Letters. 585:1203-1208. 23. Keegstra, K., Cline.K. 1999. Protein import and routing systems of chloroplasts. Plant Cell. 11:557-570. 24. Kessler, F., Schnell, D.J. Patel, H.A. Blobel.G. 1994. Identification of two GTP-binding proteins in the chloroplast protein import machinery. Science. 266:1035-1039.

25. Kiessling, J., Kruse, S., Rensing, S. A., Harter, K., Decker, E. L., Reski, R. 2000. Visualization of a cytoskeleton-like FtsZ network in chloroplasts. Journal of Cell Biology. 151:945-950. 26. Kwon, S.H., Lee, B.H. Kim, E.Y,. Seo, Y.S., Lee, S., Kim, W.T., Song, J.T., Kim. J.H. 2009. Overexpression of a Brassica rapa NGATHA Gene in Arabidopsis thaliana negatively affects cell proliferation during lateral organ and root growth. Plant and Cell Physiology. 50:2162-2173. 27. Lee, J.Y., Terry L. Orr-Weaver. 2001. The molecular basis of sister-chromatid cohesion. Annual Review of Cell and Developmental Biology. 17:753–777. 28. Ling, J., Xia.M., Strittmatter.L.I, Makaroff. C. A. 2007. The Arabidopsis cohesin protein SYN3 localizes to the nucleolus and is essential for gametogenesis. The Plant Journal. 29. Livak, K.J., Schmittgen.T.D. 2001. Analysis of relative gene expression data using real- time quantitative PCR and the 2(T)(-delta delta C) method. Methods. 25:402-408. 30. May, T.,Soll.J. 2000. 14-3-3 proteins form a guidance complex with chloroplast precursor proteins in plants. Plant Cell. 12:53-63. 31. Mehta, G.D., Kumar. R., Srivastava.S., Ghosh.S.K. 2013. Cohesin: Functions beyond sister chromatid cohesion. FEBS Letters. 587:2299–2312. 32. Michaelis C., Nasmyth, K. 1997. Cohesins: chromosomal proteins that prevent premature separation of sister chromatids. Cell. 91:35–45. 33. Miyazaki, W.Y., Orr-Weaver,T.L. 1994. Sister-Chromatid Cohesion in Mitosis and Meiosis. Annual Review of Genetics. 28:167-187. 34. Mori,H., Summer,E.J., Ma,X.,Cline,K. (1999). Component specificity for the thylakoidal Sec and delta pH–dependent protein transport pathways. The Journal of Cell Biology.146(1):45-56. 35. Murchie, E.H.,Lawson.T. 2013. Chlorophyll fluorescence analysis: a guide to good practice and understanding some new applications. Journal of Experimental Botany. 64:3983-3998. 36. Müller, P., Li, X.P., Niyogi. K.K. 2001. Non-photochemical quenching. A response to excess light energy. Plant Physiology. 125:1558-1566. 37. Osteryoung, K.W., Stokes, K.D., Rutherford.S.M., Percival.A.L., Lee.W.Y. 1998. Chloroplast division in higher plants requires members of two functionally divergent gene families with homology to bacterial ftsZ. The Plant Cell. 10:1991-2004. 38. Osteryoung,K.W., Vierling.E. 1995. Conserved cell and organelle division. Nature. 376:473-474. 39. Osteryoung.K.W. Pyke. K .A.1998(a). Plastid division: evidence for a prokaryotically derived mechanism. Current Opinion in Plant Biology. 1:475-479. 40. Osteryoung, K.W., Kevin, D. Stokes., Rutherford.S.M., Percival,A.L.,Lee.W.Y. 1998. (b) Chloroplast division in higher plants requires members of two functionally divergent gene families with homology to bacterial ftsZ. The Plant Cell. 10:1991-2004. 41. Peirson, B.N., Bowling, S.E. Makaroff. C.A. 1997. A defect in synapsis causes male sterility in a T-DNA-tagged Arabidopsis thaliana mutant. Plant Journal. 11:659-669. 42. Peirson, B.N., Owen, H.A., Feldmann, K.A., Makaroff.C.A. 1996. Characterization of three male-sterile mutants of Arabidopsis thaliana exhibiting alterations in meiosis. Sexual Plant Reproduction. 9:1-16. 43. Peters, J.-M., Tedeschi,A., Schmitz.J.2008. The cohesin complex and its roles in chromosome biology. Genes & Development. 22:3089–3114.

44. Pyke, K.A., Leech. R.M. 1991. Rapid image analysis screening procedure for identifying chloroplast number mutants in mesophyll cells of arabidopsis thaliana (l.) heynh. Plant Physiology. 96:1193-1195. 45. Pyke, K.A., Leech R. M. 1994. A Genetic analysis of chloroplast division and expansion in arabidopsis thaliana. vol. 104(1). Plant Physiology. 201–207. 46. Pyke, K.A., Leech, RM. 1987. The control of chloroplast number in wheat mesophyll cells. Planta. 170:416-420. 47. Pyke, K.A., Leech R.M.. 1992. Chloroplast division and expansion is radically altered by nuclear mutations in arabidopsis thaliana. Plant Physiology. 99:1005-1008. 48. Rajalakshmi.K.,Banu.N. 2013. Extraction and estimation of chlorophyll from medicinal plants. International Journal of Science and Research . 4:209-212. 49. Schmitz, A.J., Glynna, J. M., Olson, B. J. S. C., Stokes. K.D., Osteryoung, K. W. 2009. Arabidopsis FtsZ2-1 and FtsZ2-2 are functionally redundant, but FtsZ-based plastid division is not essential for chloroplast partitioning or plant growth and development. Molecular Plant. 2:1211-1222. 50. Stokes. K.D., McAndrew, R.S., Figueroa. R., Vitha. S., Osteryoung,K.W. 2000. Chloroplast division and morphology are differentially affected by overexpression of FtsZ1 and FtsZ2 genes in Arabidopsis. Plant Physiology. 124:1668-1677. 51. Uhlmann, F. 2001. Chromosome cohesion and segregation in mitosis and meiosis. Current Opinion in Cell Biology. 13:754–761. 52. Webber, A., Baker, N.R. 1996. Control of thylakoid membrane development and assembly. In: ort dr and yocum cf (eds) oxygenic photosynthesis: the light reactions. Kluwer academic publishers, dordrecht, The Netherlands. 41–58. 53. Weigel, D.,Glazebrook. J. 2006. Glufosinate ammonium selection of transformed Arabidopsis. CSH Protocol. 2006. 54. Yuan, L., Yang.X., Ellis.J.L., Fisher.N.M., Makaroff.C.A 2012. The Arabidopsis SYN3 cohesin protein is important for early meiotic events. The Plant Journal. 71:147–160. 55. Yuan. L., X.Y., Auman. D., Makaroff. C.A. 2014. Expression of epitope-tagged syn3 cohesin proteins can disrupt meiosis in arabidopsis. Journal of Genetics and Genomics. 41:153-164. 56. Zhang, X., Henriques.R., Lin.S., Niu.Q., Nam-Hai.C. 2006. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nature Protocols. 1.

Chapter 3: Conclusions

3.1 Conclusions

Arabidopsis contains genes encoding four α-kleisins that are essential components of meiotic and mitotic cohesin complexes. Several studies from many model organisms have established that cohesin proteins play a key role in sister chromatid cohesion and the maintenance of genome integrity during cell division (Gerton, 2005; Guacci, 1994; Hirano, 2000; Miyazaki, 1994). Studies also have showed that cohesins are involved in transcriptional regulation, DNA repair and proper spindle attachment of chromosomes (Mehta, 2013; Peters, 2008).

Previous studies have established the function of cohesin subunits, including a role for SYN3 in nucleus. However, no studies have investigated the role of cohesin subunits in organelles such as in the chloroplast. A better understanding of how SYN3 functions in chloroplasts would give more information on chloroplast biogenesis and photosynthesis. Recent work has shown that SYN3 interacts with FtsZ and localizes to the chloroplast. However, it is currently unknown if SYN3 has a possible role inside the chloroplast.

This study focused on finding the localization and the function of SYN3 in chloroplasts. As a first step in this study, I completed western blot analysis on fractionated chloroplast samples, and the results showed the localization of SYN3 into the thylakoid within the chloroplast. However, the results of our in vitro import assays showed that SYN3 was not able to be imported into isolated chloroplasts. Future studies need to be performed to determine which sub-organellar compartment of the thylakoid SYN3 localizes and whether SYN3 protein gets imported into the chloroplast. In addition, over-expression of SYN3-YFP in plants caused a stunted growth phenotype. Previous studies have shown that photosynthetic efficiency is dependent on proper chloroplast division and development (Pyke, 1992). Therefore, we suggest that defects in chloroplast division could be the reason for reduced total chlorophyll and chlorophyll b content.

The localization of SYN3-YFP using microscopy studies and troubleshooting in vitro SYN3 import into isolated chloroplasts will be the aim of future studies. Previous studies have shown that precursor bound with the guidance complex is much more competent for import into isolated chloroplasts than is the free precursor protein. Phosphorylated chloroplast targeting signals bind to the cytosolic guidance complex consisting of Hsp70 and 14-3-3 proteins (May, 2000). Import studies need to be optimized and performed in the presence of guidance complex to test if SYN3 gets imported into isolated chloroplasts. These studies will give new insights and firmly establish the subcellular localization of SYN3 in chloroplasts, which will help to explore the function of SYN3 in chloroplast division.

In eukaryotic cohesin complex, the N- and C-termini of Rad21 / Scc1 have been shown to bind directly to Smc3 and Smc1, respectively (Haering, 2002 and Haering, 2004). In prokaryotes, MukB is an ATPase and DNA-binding protein of the SMC superfamily (Fennell-Fezzie, 2005). E. coli MukF function as a kleisin; N-terminal of MukF region folds into a winged-helix domain and an extended coiled-coil domain that self-associate to form a stable dimer. C-terminal of MukF binds to MukB and MukE to form MukBEF complex. N-terminal domain of MukF does not interact with MukB or MukE tightly (Fennell-Fezzie, 2005) while the N-terminal domain of SCC1 has been reported to bind to the ATPase domain of SMC3.

Previous studies have shown that the bacterial kleisin MukF is important for chromosome segregation, transcriptional regulation, and DNA repair (Dervyn, 2004; Danilova, 2007; Graumann, 2009; Hiraga, 1992; Hiraga, 2000; Weitao, 2000; Yamanaka, 1996 ). Studies also have shown that the N-terminus of MukB binds to E.coli FtsZ (Lockhart, 1998 (a)) and eukaryotic microtubules in vitro in the presence of nucleotides (Lockhart, 1998 (b)). Bacterial FtsZ is an important player in chromosomal segregation at the future site of septum formation. However, the biological significance of the in vitro binding of the MukB head domain to FtsZ is still unclear (Hiraga, 2000). Therefore, we suggest that SYN3 may function as prokaryotic kleisin homolog of MukF in chloroplasts after N-terminal signal peptide cleavage and may be involved in chromosome segregation during chloroplast division.

Apart from SYN3 function on chloroplast division, studies also need to be done to test if SYN3 binds to chloroplast DNA. This will help to identify the SYN3 binding sites, if any, on chloroplast DNA. Further exploration of this binding location could show how SYN3 interacts with chloroplast DNA to regulate gene expression or how it is involved in DNA repair.

3.2 References

1. Danilova.O., Reyes-Lamothe.R., Pinskaya.M., Sherratt.D., Possoz.C. 2007. MukB colocalizes with the oriC region and is required for organization of the two Escherichia coli chromosome arms into separate cell halves. Molecular Microbiology. 65:1485– 1492. 2. Dervyn, E., Noirot-Gros, M., Mervelet, P., McGovern, S., Ehrlich, S. D., Polard, P., Noirot,P. 2004. The bacterial condensin/cohesin-like protein complex acts in DNA repair and regulation of gene expression. Molecular Microbiology. 51:1629-1640. 3. Fennell-Fezzie, R., Gradia, S.D., Akey, D., Berger. J.M. 2005. The MukF subunit of Escherichia coli condensin: architecture and functional relationship to kleisins. Embo Journal. 24:1921-1930. 4. Gerton, J. 2005. Chromosome Cohesion: A cycle of holding together and falling apart. Plos Biology. 3:e94. 5. Graumann.P. L., Knust,T. 2009. Dynamics of the bacterial SMC complex and SMC-like proteins involved in DNA repair. Chromosome Research. 17:265–275. 6. Guacci, V., Hogan,E., Koshland.D. 1994. Chromosome condensation and sister chromatid pairing in budding yeast. The Journal of Cell Biology. 125:517-530. 7. Hiraga.S. 1992. Chromosome and plasmid partition in Escherichia Coli. Annual Review of Biochemistry. 61:283-306. 8. Hiraga.S. 2000. Dynamic localization of bacterial and plasmid chromosomes. Annual Review of Genetics. 34:21–59. 9. Hirano, T. 2000. Chromosome cohesion, condensation and separation. Annual Review of Biochemistry. 2000. 69:115–144. 10. Lockhart A, Kendrick-Jones A. 1998(a). Interaction of the N-terminal domain of MukB with the bacterial tubulin homologue FtsZ. FEBS Letter. 430:278–282. 11. Lockhart A, Kendrick-Jones A. 1998(b). Nucleotide-dependent interaction of the N- terminal domain of MukB with microtubules. Journal of Structural Biology. 124:303– 310. 12. Mehta, G.D., Kumar.R., Srivastava.S., Ghosh.S.K 2013. Cohesin: Functions beyond sister chromatid cohesion. FEBS Letters. 587:2299–2312. 13. Miyazaki, W.Y., Orr-Weaver,T.L. 1994. Sister-chromatid cohesion in mitosis and meiosis. Annual Review of Genetics. 28:167-187. 14. Peters, J.-M., Tedeschi.A., Schmitz.J. 2008. The cohesin complex and its roles in chromosome biology. Genes & Development. 22:3089–3114. 15. Pyke, K.A., Leech. R.M. 1992. Chloroplast division and expansion is radically altered by nuclear mutations in Arabidopsis thaliana. Plant Physiology. 99:1005-1008.

16. Weitao, T., Dasgupta.S., NordstroÈm.K. 2000. Role of the mukB gene in chromosome and plasmid partition in Escherichia coli. Molecular Microbiology, 38,. 38:392-400. 17. Yamanaka K, O.T., Niki. H., Hiraga. S. 1996. Identification of two new genes, mukE and mukF, involved in chromosome partitioning in Escherichia coli. Molecular Genetics and Genomics. 250:241–251.