Changes in Dunaliella

Changes in phosphometabolites, following osmotic shock, were analyzed by two-dimensional thin layer chromatography, in extracts of the halotolerant alga DunalielIa salina in order to clarify the regulation of glycerol synthesis from starch. The experiments were carried out in wild-type and in osmotically defective mutant cells. It is demonstrated that hyperosmotic shock induces a decrease in fructose 6-phosphate and an increase in fructose1,6-bisphosphate indicating the activation of phosphofructokinase. Two mutants, which are specifically defective in their response to hyperosmotic shock, accumulate glucose 6-phosphate or phosphogluconate following shock, and have remarkably reduced activities of glucose-6-phosphate dehydrogenase and of phosphogluconate dehydrogenase, respectively. These results indicate that the pentose-phosphate oxidative pathway has a major role in glycerol synthesis. Hyperosmotic shock leads to a transient accumulation of phosphoryicholine and to a decrease of inositolbisphosphate in D. salina extracts. Accumulation of phosphorylcholine is not detected in osmotically defective mutants. Hypoosmotic shock induces an increase in inositolbisphosphate but not in phosphorylcholine. These results are consistent with previous indications for differential activations of phospholipases by hyper or hypoosmotic shock in DunalielIa. Based on these results we suggest that (a) phosphofructokinase is an important checkpoint enzyme in the regulation of glycerol production, and (b) that the pentose-phosphate pathway has a major role in keeping oxidation-reduction balance during glycerol synthesis. The possible role of lipid breakdown products as second messengers in regulating glycerol production in DunalielIa is discussed. Dunaliella is a unicellular halotolerant green alga which adapts to a large range of NaCl concentrations, from 0.1 to 5 M, by the synthesis of intracellular glycerol. Previous studies have shown that glycerol is made primarily from starch reserves in the chloroplast (3, 4, 6, 9), but the exact metabolic pathway leading to glycerol synthesis is not known. Four enzymes which apparently are involved in glycerol metabolism have been described, three ofthem unique to Dunaliella. It has been proposed that glycerol is made from DHAP (Table I contains complete list of abbreviations) via a GP-DH and a GP-ase, or reconverted to DHAP via a G-DH and a DHA-K (reviewed in ref. 4). The exact enzymatic pathway leading to DHAP formation from starch is still unclear and the energetic requirements in ATP and reduced pyridine nucleotides are not known. The observation that glycerol production proceeds either in the light or in the dark suggests that there is no obligatory requirement for photosynthesis neither as an energy source nor as a carbon source. It is also unclear how is glycerol synthesis and its reconversion to starch being regulated. It has been clearly established that protein synthesis is not involved in the induction of glycerol production since inhibitors of protein synthesis do not affect glycerol production in response to hyperosmotic shocks (24). It is also unknown which enzymes in the metabolic pathway for glycerol synthesis are involved in regulating the process. Previous observations of a transient increase in the level ofGP following hyperosmotic shocks in Dunaliella tertiolecta (1) points to GP-DH as a possible candidate. However, in vitro comparisons of the activity of GP-DH isolated from the alga before or after osmotic shocks gave no indications for changes in its activity and the same was observed for other suspected key enzymes in the process, suggesting that covalent modification of enzymes is not likely to be involved in the activation of glycerol synthesis (1). A further complication in the regulation of glycerol synthesis is the intracellular compartmentation of the process, between the cytoplasm and the chloroplast which calls for an integrated multicompartmental regulation (7). Several factors which have been suggested to be involved in regulating glycerol synthesis are the intracellular pH, ATP and phosphate. Intracellular pH changes following hyperosmotic shocks in Dunaliella have been reported (15, 20) and suggested to be involved in the induction of starch breakdown in view of the sharp pH dependence of starch-mobilizing enzymes in this alga (15). The rapid and transient drop in ATP following hyperosmotic shock (1, 10, 22) may contribute to the activation of GP-DH and of PFK which are inhibited by ATP concentration exceeding 1 mM (19, 21). Observations of an increase in inorganic phosphate in Dunaliella following hyperosmotic shocks (14, 15, 20) and our observations of a correlation between the rate of glycerol synthesis or elimination with the cellular phosphate level, have led us to propose that the influx of phosphate from the cytoplasm into the chloroplast, in exchange for triose phosphates via the phosphate translocator, is an important link in the regulation of glycerol synthesis (5). Another interesting set ofobservations in Dunaliella, whose connection to the regulation of glycerol synthesis is not clear, is the enhanced turnover of specific phospholipids following osmotic changes in this alga. It has been observed by the group of Thompson that hypoosmotic shocks induced increased turnover of phosphoinositol phospholipids in Dunaliella salina, indicating the activation of a specific PL-C in the plasma membrane of this alga (1 1). Conversely, hyperosmotic shocks inhibit the turnover of phosphoinositol phos-