Regulation of Glycerol Synthesis in Response to Osmotic Changes in Dunaliella

Abstract

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 fructose-1,6-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 of them 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 ...

Metabolic Response of Maize Roots to Hyperosmotic Shock

“…The appearance of phosphocholine in D. salina is likely to have physiological significance because osmotic shock does not decrease the surface area of the plasma membrane (6) and phosphocholine accumulation is not observed in osmotically defective mutants that have very slow glycerol synthesis (4).…”

“…In mammalian cells, PtdCho breakdown products (DAG and phosphatidic acid) have been implicated in agonist-induced signal transduction (7,17). Chitlaru and Pick (4) suggested that signal transduction involving PtdCho hydrolysis may be important in osmoregulation in Dunaliella, and it is possible that this pathway could exist in the system studied here. However, these processes are usually thought to involve low concentrations of messengers and to occur on a time scale of minutes, although Cabot et al (2) observed phorbol-induced release of choline metabolites during a period of 2 h. Hence, it is not possible to obtain information about these processes from the experiments described here.…”

Stimulation of fructose 1,6‐bisphosphate production in melanoma cells by α‐melanocyte‐stimulating hormone

“…: 0.5/g) and the second dimension with n-butanol/n-propanol/npropionic acid/H 2 O (400:175:285 :373, by vol.) [24,25].…”

“…Unlabeled spots were detected by exposing the TLC plates to ninhydrin reagent, (amine-containing metabolites), ultraviolet light (nucleotides), or iodine vapor (lipids). Phosphates on poly(ethylimine) cellulose plates (which had been washed with absolute methanol and dried) and cellulose TLC plates were detected by spraying with phosphomolybdate reagent [25] and then heated (85°C for Ϸ10 min). For quantitative autoradiographic analysis of radioactive spots, the TLC plates were imaged in a FUJI-X Bio-imaging analyzer (BAS1000 MacBas, Fuji Photo Film Co., Ltd), and the results were quantitated after subtracting background values.…”

Paving the Road to Algal Biofuels with the Development of a Genetic Infrastructure

“…Volvox carteri, a multicellular microalga, is another well-established model species for the elucidation of the genetic basis of cellular differentiation (Miller, 2002). In recent years, the green alga Dunaliella salina has been utilized to complement the study of photosynthesis, osmoregulation, carotenogenesis, and glycerol production (Jin et al, 2001;Liska et al, 2004;Thompson, 2005;Shaish et al, 1992;Chitlaru et al, 1991). Dunaliella salina is an attractive platform for both commercial and academic pursuits owing to its intriguing and advantageous abilities to survive in conditions of extreme salinity and produce significant amounts of -carotene.…”