Abstract Detail

Developmental and Structural Section

Stempinski, Erin S. [1], Edelmann, Richard E. [2], Makaroff, Christopher A. [3].

SYN3, a Cohesin Subunit, Localizes to Chloroplasts and Alters Senescence in Arabidopsis thaliana.

Cohesin is a complex of proteins that functions in sister chromatid cohesion during mitosis and meiosis. Cohesin is made up of four proteins: two Structural of Maintenance of Chromosome (SMC) proteins, a Sister Chromatid Cohesion (SCC) protein, and an α-kleisin. Arabidopsis thaliana contains four α-kleisin proteins termed SYN1-4. One of these α-kleisins, SYN3, may have a unique role in plant development. SYN3 has previously been shown to function in gametogenesis and localize to the nucleolus, whereas other SYN proteins are found throughout the nucleus (Jiang et al., 2007). Further localization efforts in isolated protoplasts found SYN3 in chloroplasts, a novel location for cohesin subunits. Knockdown of SYN3 by RNAi resulted in plants with leaves that yellowed earlier compared to wildtype. We therefore hypothesized that SYN3 functions in chloroplast development. In an effort to further understand the role of SYN3 in chloroplasts, whole leaves of wildtype and RNAi plants at varying stages of development were fixed in 2.5% glutaraldehyde, 2.0% formaldehyde, and 5 mM sucrose in sodium cacodylate buffer overnight and in 1% osmium tetroxide in half-strength buffer for six hours. The leaves were then exposed to 0.05% uranyl acetate overnight, dehydrated in an acetone series, embedded in Dr. Spurr's resin, sectioned, and post-section stained with uranyl acetate and lead citrate. The location of SYN3 within chloroplasts was further characterized using gold immunolabeling at the electron microscopy level. Whole leaves were fixed in an aldehyde solution, dehydrated in an ethanol series, and embedded in LR White resin. Immunolabeling was performed by floating grids on blocking solution, exposing the grids to the primary antibody overnight, rinsing in buffer, exposing the grids to the secondary antibody for an hour, then finally rinsing in buffer and water. Immunolocalization of SYN3 found SYN3 in both the nucleolus and the chloroplast stroma, verifying previous results. RNAi plants late in development exhibited far fewer autolysosomes within degrading cells compared to wildtype plants. The cytoplasmic material within RNAi plant cells at late developmental stages was less dense than wildtype plants. These results suggest an alteration in autolytic activity during leaf senescence. Throughout each developmental stage,chloroplasts in RNAi plants contained larger and more darkly-staining plastoglobuli than wildtype plants, indicating a change in chloroplast development. Taken together, these results show that the cohesin subunit SYN3 has a novel location outside of the nucleus as well as a novel role in organelle function and cell degradation.

Broader Impacts:

1 - Miami University, Department of Botany, 316 Pearson Hall, Oxford, OH, 45056, USA
2 - Miami University, Center for Advanced Microscopy and Imaging, 9C Upham Hall, Oxford, OH, 45056, USA
3 - Miami University, Department of Chemistry and Biochemistry, 160 Hughes Laboratories, Oxford, OH, 45056, USA

Keywords:
SYN3
Cohesin
Arabidopsis thaliana
Transmission electron microscopy
TEM
Immunolocalization.

Presentation Type: Poster:Posters for Sections
Session: P
Location: Grand Salon A - D/Riverside Hilton
Date: Monday, July 29th, 2013
Time: 5:30 PM
Number: PDS001
Abstract ID:100
Candidate for Awards:None