The role of serine hydroxymethyltransferase 1 (CHSHMT1) in the regulation of the key osmolyte glycine in the estuarine Hong Kong oyster Crassostrea hongkongensis under osmotic stress

The estuarine Crassostrea hongkongensis is the major cultured oyster species in the South China, and mass mortality induced by hyper-salinity has heavily damaged the oyster farming, especially during dry season. To benefit the genetic improvement of hyper-salinity tolerance, three genes related to the intracellular concentration of glycine (Gly) that functions as important osmolyte were identified and investigated in C. hongkongensis. Serine hydroxymethyltransferase 1 in C. hongkongensis (CHSHMT1) was found to be upregulated in gill since 48 h transferred from 18%o to 30%o filtered seawater (FSW). While glycine dehydrogenase (CHGLDC) and ALA synthase gene (CHALAS) were significantly upregulated in oyster from 8 to 48 h exposed to 6%o FSW when compared with that in 18%o and 30%o FSW. And the expression of the three genes agreed with that Gly content decreased under hypo-osmotic stress and increased under hyper-osmotic stress. RNA in situ hybridization (ISH) revealed that CHSHMT1 and CHGLDC were mainly expressed in the ciliated columnar cell of gill filament and epithelial cell of water tube wall, where oyster gill directly contacts with seawater when the animal is filtering. But positive signal of CHALAS mRNA showed no significant difference in intensity between various gill cells. Based on these findings, CHSHMT1, CHGLDC and CHALAS were supposed to contribute to the variation of Gly content under osmotic stress. Subsequent RNA interference (RNAi) confirmed the function of CHSHMT1 in the transformation from serine (Ser) to Gly under hyper-osmotic stress. Moreover, CHGLDC and CHALAS were found to be significantly upregulated when CHSHMT1 was silenced under hyper-osmotic stress. This was hypothesized that the expression of CHGLDC and CHALAS might be suppressed by CHSHMT1, but more evidence should be provided. This work enriched the understanding of oyster osmolyte system that prevent cell shrinking or swelling under osmotic stress and provided potential genes for the breeding of Hong Kong oyster strain with hyper-salinity tolerance.