The Osmotic Adjustment Of Dunaliella Salina For Salinity Resistance And Ca²⁺ Mediated Osmotic Signal Transduction

Dunaliella salina, one member of the genus Dunaliella (Chlorophyceae, Volvocales), could survive in media containing a wide range of NaCl concentrations ranging from about 0.05 mol/L to saturation (around 5.5 mol/L). Glycerol is an important osmolyte when D. salina survives in varying salt environment. The biosynthesis of glycerol involves two specific enzymes, (NAD⁺)-dependent glycerol-3-phosphate dehydrogenase (G3PDH) and glycerol-3-phosphate phosphatase (G3PP). In the opposite direction, the dissimilation of glycerol involves an NADPH-specific dihydroxyacetone reductase (DHAR) and a dihydroxyacetone kinase (DHAK). Ca²⁺ serves as a ubiquitous intracellular second messenger in signal transduction of environmental stimuli.In this paper, to study the osmotic response process of D. salina under salinity stress, I investigated the changes of cell growth, cell shape, intracellular glycerol concentration and four enzymes activities in glycerol metabolism under different salinity stress. The isozyme activity of (NAD⁺)-dependent G3PDH in different salinities was investigated by using isozyme electrophoresis, and the full cDNA of a (NAD⁺)-dependent G3PDH with 2100 bp was cloned from D. salina and the effects of salinity changes on this gene expression were detected by real-time quantitative PCR. In addition, the effect of Ca²⁺ channel blockers on cytosolic Ca²⁺ levels and the role of Ca²⁺ in glycerol metabolism and (NAD⁺)-dependent G3PDH activity under hypoosmotic and hyperosmotic stress were investigated using the confocal laser scanning microscope and a pharmacological approach.Results showed that 2.0 mol/L NaCl was the optimal salinity for the growth of D. salina, and the changes of shape and volume of D. salina cell cultured chronically at various salinities were minor, but when the salinity was changed rapidly, the variations of cell shape and cell volume of D. salina were significant, which were recovered basically after 2 h except treating by high salinity. Also, it was found some lipid globules in the surface of D. salina cells when the salinity increased from 2.0 to 4.0-5.0 mol/L NaCl rapidly. When D. salina was cultured chronically at various salinities, the accumulation of single cell glycerol increased with increased salinity, and D. salina also could rapidly decrease or increase single cell glycerol contents to adapt to hypoosmotic or hyperosmotic shock. In glycerol metabolism pathway, all of four enzymes were activated by chronically high salt stress, but only the activity of G3PDH was significantly correlated to the salinity under hypoosmotic and hyperosmotic stress. In the isozyme electrophoresis assay two kinds of isozymes, G3PDH and superoxide dismutase, were detected synchronously. Especially, it was first found that there are five isozymes of G3PDH in D. salina, and these isozymes respectively take effects in different salinities. The expression level of G3pdh in D. salina grown chronically at various salinities was significantly inversely correlated to the salinity, but there was no significant correlation between the gene expression level and salinity after two hours of treatment by hyperosmotic or hypoosmotic shock. In addition, intracellular Ca²⁺ concentration increased rapidly when extracellular salinity suddenly decreased or increased, but the increase could be inhibited by pretreatment of three Ca²⁺ channel blockers. Blocker verapamil (VP) may block the extrusion of Ca²⁺ from cytoplasm as well. The changes of glycerol content and G3PDH activity in D. salina to respect to hypoosmotic or hyperosmotic stress were also inhibited in different degrees by pretreatment of Ca²⁺ channel blockers.Thus, it is illuminated that the osmotic response process of D. salina, including the changes of cell shape, intracellular glycerol concentration, some enzymes involved to glycerol metabolism and the expression of some genes, play important roles in osmotic adjustment to enable D. salina survive at varying salinities, and the in?ux of Ca²⁺ from the extracellular space or intracellular calcium store is required for the transduction of osmotic signal to regulate the osmotic responses of D. salina.