31P and 13C-NMR Studies of the Phosphorus and Carbon Metabolites in the Halotolerant Alga, Dunaliella salina1

The intracellular phosphorus and carbon metabolites in the halotolerant alga Dunaliella salina adapted to different salinities were monitored in living cells by 31Pand "3C-nuclear magnetic resonance (NMR) spectroscopy. The 13C-NMR studies showed that the composition of the visible intracellular carbon metabolites other than glycerol is not signiflcantly affected by the salinity of the growth medium. The T1 relaxation rates of the 13C-glycerol signals in intact cells were enhanced with increasing salinity of the growth medium, in parallel to the expected increase in the intracellular viscosity due to the increase in intracellular glycerol. The 31P-NMR studies showed that cells adapted to the various salinities contained inorganic phosphate, phosphomonoesters, high energy phosphate compounds, and long chain polyphosphates. In addition, cells grown in media containing up to 1 molar NaCI contained tripolyphosphates. The tripolyphosphate content was also controlled by the availability of inorganic phosphate during cell growth. Phosphate-depleted D. salina contained no detectable tripolyphosphate signal. Excess phosphate, however, did not result in the appearance of tripolyphosphate in 31P-NMR spectra of cells adapted to high (>1.5 molar NaCI) salinities. Members of the genus Dunaliella (division Chlorophycophyta, order Volvocales, family Polyblepharidaceae) are unicellular, motile, green algae, which lack a rigid cell wall. Dunaliella has the capacity to tolerate and adapt to a wide range of salt concentrations (0.1-5.5 M NaCl). This is achieved through the ability of the algae to survive the initial osmotic stress, and then adjust their intracellular osmotic content to the new required level, by synthesis or elimination of glycerol (3, 4). Little is known about the intracellular phosphate compounds in Dunaliella, and no characterization of the intracellular phosphate compounds is available. Ginzburg and Ginzburg (9) measured an intracellular inorganic phosphate concentration of 400 mm for 12 species of Dunaliella, cultured at 0.5 or 2 M NaCl and 2 mm phosphate. Pick et al. (20) showed that the cells accumulate phosphate in proportion to its concentration in the growth medium. Gimmler and Moller (8) studied the phosphates of D. parva cells following osmotic shocks. Following a hyperosmotic shock, they observed a very rapid increase in the endogenous inorganic phosphate level, and in the level of an unidentified phosphate compound, at the expense of the acid-soluble, cellular organic phosphates. A hypoosmotic shock caused a rapid decrease in the intracellular inorganic phosphate level. The ATP to ADP ratio, the phosphorylation potential (ATP/ADP x Pi), I Supported by a grant from the United States/Israel Binational Science Foundation. and the 3-phosphoglyceric acid to Pi ratio also decreased. In general, algal intracellular phosphate levels can vary widely, depending on whether the algae are growing under phosphorusrich or phosphorus-limited conditions (11). Like other microorganisms, algal cells possess the ability to accumulate excess phosphate. This is stored in the form of condensed phosphates, which can be either cyclic (metaphosphates) or linear unbranched chains (polyphosphates). The existence of intracellular polyphosphate granules, whose size (30-500 nm) and number were affected by the growth conditions, was demonstrated in the algae Chlorella pyrenoidosa (19) and Anabaenaflos-aquae (21) by electron microscopic studies combined with x-ray energy dispersive analysis. In this paper, we report the use of 31P-NMR spectroscopy to characterize the intracellular phosphate metabolites in living D. salina cells adapted to different salinities and phosphate concentrations. In vivo 31P-NMR has been used extensively in studies of mammalian cells and organs, including organs in living animals (2, 7 and references therein), and recently also in studies of unicellular photosynthetic organisms (10, 14, 15, 18, 24, 25). We also report the use of '3C-NMR spectroscopy to study D. salina grown in media containing '3C-enriched carbonate and monitored under conditions similar to those used in the 31p studies. 13C-NMR was employed previously in numerous studies of intact cells and organs (1). The glycerol in D. salina grown in 2.1 M NaCl was previously monitored by natural abundance 13CNMR studies (17). '3C-labeled D. salina permitted additional characterization of several other intracellular metabolites (5). In the latter study, it was also shown that following a hypoosmotic shock, glycerol is converted to a(1-*4)glucan, and the reverse process occurs following a hyperosmotic shock. In this study we monitored, by in vivo '3C-NMR, the intracellular glycerol, metabolites, and membranal components in cells adapted to a wide range of salinities. MATERIALS AND METHODS Growth Conditions. Dunaliella salina was grown in batch cultures in media containing NaCl at the indicated concentration, 50 mM NaHCO3, 5 mm KNO3, 5 mM MgSO4, 0.3 mm CaCl2, 0.8 /1M ZnC12, 0.02 /.M CoCl2, and 0.2 nM CuCl2 (initial pH, 7.58.2). Algal cultures were grown under continuous illumination with white fluorescent lamps (light intensity 3800 lux) at 26°C with slow (80 rpm) continuous shaking within a New Brunswick Psycrotherm incubator. Adaptations to different NaCl or phosphate concentrations were made by growing for several days under the desired conditions. Cells for 13C-NMR studies were cultured in the same medium as above, except that it contained 25 mM [13C, 10%]NaHCO3 for the last 2 to 3 division cycles. Preparation of Cells for NMR Measurements. Algae at the logarithmic phase (1-2.5 x 106 cells/mL) were concentrated by centrifugation at 4°C (2000g for 10 min). All subsequent oper-