The PsbO homolog from Symbiodinium kawagutii (Dinophyceae) characterized using biochemical and molecular methods

Abstract

A photosystem II component, the PsbO protein is essential for maximum rates of oxygen production during photosynthesis, and has been extensively characterized in plants and cyanobacteria but not in symbiotic dinoflagellates. Its close interaction with D1 protein has important environmental implications since D1 has been identified as the primary site of damage in endosymbiotic dinoflagellates after thermal stress. We identified and biochemically characterized the PsbO homolog from Symbiodinium kawagutii as a 28-kDa protein, and immunolocalized it to chloroplast membranes. Chloroplast association was further confirmed by western blot on photosynthetic membrane preparations. TX-114 phase partitioning, chromatography, and SDS-PAGE for single band separation and partial peptide sequencing yielded peptides identical or with high identity to PsbO from dinoflagellates. Analysis of a cDNA library revealed three genes differing by only one aminoacid residue in the in silico-translated ORFs despite greater differences at nucleotide level in the untranslated, putative regulatory sequences. The consensus full amino acid sequence displayed all the characteristic domains and features of PsbO from other sources, but changes in functionally critical, highly conserved motifs were detected. Our biochemical, molecular, and immunolocalization data led to the conclusion that the 28-kDa protein from S. kawagutii is the PsbO homolog, thereby named SkPsbO. We discuss the implications of critical amino acid substitutions for a putative regulatory role of this protein.

The actin cytoskeleton organization and disorganization properties of the photosynthetic dinoflagellateSymbiodinium kawagutiiin culture

“…To date, the organization of the actin cytoskeleton in marine dinoflagellate algae of the genus Symbiodinium had not been reported. This was probably due to the fact that Symbiodinium are photosynthetic cells that contain a tubular membrane chloroplast surrounding the cytoplasm (Wakefield et al 2000;Castillo-Medina et al 2013). Thus, the presence of chlorophyll and other pigments hinders the detection of fluorescence from any other applied fluorescent probes (Castillo-Medina et al 2011).…”

“…This was probably due to the fact that Symbiodinium are photosynthetic cells that contain a tubular membrane chloroplast surrounding the cytoplasm (Wakefield et al 2000;Castillo-Medina et al 2013). Thus, the presence of chlorophyll and other pigments hinders the detection of fluorescence from any other applied fluorescent probes (Castillo-Medina et al 2011). In addition, a rigid proteinaceous cell wall and a complex membrane (Wakefield et al 2000) require harsh procedures to make the required probes accessible to the cytoplasm.…”

“…The freeze-fracture procedure reported by Castillo-Medina et al (2011) was applied to exponentially growing cells. Briefly, a cell aliquot from the cultures was fixed with 4% formaldehyde (freshly prepared from paraformaldehyde powder) in regular PBS or PBS adjusted to 0.6 mol/L salt for 1 h at 25°C.…”

“…We used a previously documented freeze-fracture and solvent extraction procedure developed in our laboratory (Castillo-Medina et al 2011) to see if we were able to visualize the polymerized actin cytoskeleton in S. kawagutii cells in culture using fluorescently labeled phalloidin. In these cells under culture conditions, the coccoid stage predominates, and thus, all of our observations were made on this cell type.…”

“…Our technique for fluorescent probe application also allows the entrance of antibodies to the cell (Castillo-Medina et al 2011. This allowed us to probe the population of polymeric filamentous actin plus monomeric actin in the S. kawagutii cells using immunofluorescence with anti-actin monoclonal antibodies.…”