RESUMO
BACKGROUND: Some genera of the family Symbiodiniaceae establish mutualistic endosymbioses with various marine invertebrates, with coral being the most important ecologically. Little is known about the biochemical communication of this association and the perception and translation of signals from the environment in the symbiont. However, specific phosphorylation/dephosphorylation processes are fundamental for the transmission of external signals to activate physiological responses. In this work, we searched phosphorylatable proteins in amino acids of Ser, Thr and Tyr from three species of the family Symbiodiniaceae, Symbiodinium kawagutii, Symbiodinium sp. Mf11 and Symbiodinium microadriaticum. METHODS: We used specific antibodies to the phosphorylated aminoacids pSer, pThr and pTyr to identify proteins harboring them in total extracts from three species of Symbiodinium in culture. Extractions were carried out on logarithmic phase growing cultures under a 12 h light/dark photoperiod. Various light/dark, nutritional and other stimuli were applied to the cultures prior to the extractions, and proteins were subjected to SDS-PAGE and western immunoblotting. Partial peptide sequencing was carried out by MALDI-TOF on specific protein spots separated by 2D electrophoresis. RESULTS: At 4 h of the light cycle, several Thr-phosphorylated proteins were consistently detected in the three species suggesting a genus-dependent expression; however, most Ser- and Tyr-phosphorylated proteins were species-specific. Analysis of protein extracts of S. microadriaticum cultures demonstrated that the level of phosphorylation of two Thr-phosphorylated proteins with molecular weights of 43 and 75 kDa, responded inversely to a light stimulus. The 43 kDa protein, originally weakly Thr-phosphorylated when the cells were previously adapted to their 12 h dark cycle, underwent an increase in Thr phosphorylation when stimulated for 30 min with light. On the other hand, the 75 kDa protein, which was significantly Thr-phosphorylated in the dark, underwent dephosphorylation in Thr after 30 min of the light stimulus. The phosphorylation response of the 43 kDa protein only occurred in S. microadriaticum, whereas the dephosphorylation of the 75 kDa protein occurred in the three species studied suggesting a general response. The 75 kDa protein was separated on 2D gels as two isoforms and the sequenced spots corresponded to a BiP-like protein of the HSP70 protein family. The presence of differential phosphorylations on these proteins after a light stimulus imply important light-regulated physiological processes in these organisms.
RESUMO
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.
