REGULATION OF INTRACELLULAR OSMOTIC PRESSURE AND SOME FACTORS THAT INFLUENCE THE PROMOTION OF GLYCEROL SYNTHESIS IN A RESPIRATION-DEFICIENT MUTANT OF THE SALT-TOLERANT YEAST ZYGOSACCHAROMYCES ROUXH DURING SALT STRESS

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TThe accumulation of glycerol and inorganic ions were examined in a respiration-deficient (RD) mutant isolated from the salt-tolerant yeast Zygosaccharomyces rouxii for 3h after salt stress due to 1M NaCl. After the start of salt stress, intracellular levels of glycerol continued to increase for up to 3h, while the levels of Na⁺ and Cl^- ions in cells reached maximum values within 1h and then decreased gradually. Increases in intracellular concentrations of solutes resulted in an osmotic pressure that was almost equivalent to the external osmotic pressure within 2h after salt stress. The RD strain had the same ability to tolerate salt as the wild-type strain. Therefore, we used the RD strain to examine the mechanism in the glycolytic pathway that is responsible for the promotion of glycerol synthesis that is induced by NaCl. When exposed to medium with 1M NaCl, RD cells diverted about one-sixteenth of the amount of ethanol that was produced in the medium without NaCl to the production of glycerol. This result suggests the presence of factors that mediate a change from the normal metabolism of glucose to the promotion of glycerol synthesis in response to external NaCl. The specific activities of glycerol-3-phosphate dehydrogenase (GPDH) in extracts of cells grown with and without 1M NaCl were very low in reaction mixtures with NADH or NADPH, although the cellular activity of alcohol dehydrogenase (ADH) was high and was repressed by external NaCl. This result indicates that the pathway involving GPDH makes only a small contribution to the synthesis of glycerol and that an alternative pathway functions for the synthesis in Z. rouxii. The addition of sodium sulfite, which binds to acetaldehyde, and of glycidol, an inhibitor of triosephosphate isomerase (TPI), to the medium promoted the synthesis of glycerol in RD strain. These results suggest the possibility that the extra NADH resulted from the binding of
sulfite to acetaldehyde, or the inhibition of ADH and/or TPI under the NaCl-stressed condition lead to the promotion of glycerol synthesis by Z. rouxii.