Modulation of Na/H Antiporter Activity by Extreme pH and Salt in the Halotolerant Alga Dunaliella salina.

The effect of different growth conditions on the activity of the Na(+)/H(+) antiporter in Dunaliella salina has been investigated. Adaptation of D. salina cells to ammonia at alkaline pH or to high NaCl concentrations is associated with a pronounced increase in the plasma membrane Na(+)/H(+) exchange activity. The enhanced activity is manifested both in vivo, by stimulation of Na(+) influx into intact cells in response to internal acidification, and in vitro, by a larger (22)Na accumulation in plasma membrane vesicles in response to an induced pH gradient. Kinetic analysis shows that the stimulation does not result from a change of the K(m) for Na(+) but from an increase in the V(max). In contrast, adaptation of cells to a high LiCl concentration (0.8 m) depresses the activity of the Na(+)/H(+) antiporter. Adaptation to ammonia is also associated with a large increase of three polypeptide bands in purified plasma membrane preparations, indicating that they may compose the antiporter polypeptides. These results suggest that adaptation to ammonia or to high salinity induces overproduction of the plasma membrane Na(+)/H(+) antiporter in Dunaliella.

Proton flow through the ATP synthase in chloroplasts regulates the distribution of light energy between PS I and PS II.

The involvement of ATP synthase in the imbalance between the photoactivities of PS I and PS II under light-limiting conditions, was examined in broken lettuce chloroplasts using modulated fluorimetry. The imbalance, in favor of PS II, was minimal and roughly constant between pH 6.5-7.3 (ratio of PS II/PS I activities about 1.1), and maximal at pH 8.5 (ratio of PS II/PS I activities about 1.4). This increase was strongly inhibited by a treatment of the chloroplasts with the CF0 ATP synthase inhibitor DCCD, but unaffected by the CF1 ATPase inhibitor, tentoxin. However, tentoxin plus ADP-P1 did inhibit the high pH-induced increased imbalance. These results, when considered with the previous results on the effect of high pH on proton flux through the ATP synthase, suggest that the rate of such proton flow controls the imbalance between the two photo-systems. It is possible that there is an in vivo fine-tuning regulating mechanism of the photosystems imbalance via the opening and closing of proton gradient dissipation through the ATP synthase. This mechanism may help alleviate photoinhibitory damage.

Polyphosphate metabolism in the alga Dunaliella salina studied by 31P-NMR.

Polyphosphate synthesis and the state of the intracellular polyphosphates in the unicellular green alga Dunaliella salina were studied using in vivo 31P-NMR spectroscopy. By perfusing phosphate-depleted algal cells trapped inside agarose beads with orthophosphate (Pi) containing medium, we were able to follow the process of polyphosphate synthesis in whole, living cells. The results suggest that, in Dunaliella, low molecular weight, probably cyclic, polyphosphate intermediates are synthesized from Pi, and are then condensed to high molecular weight polymers. Studies of the intracellular organization of the polyphosphates by electron microscopy and solid-state NMR techniques indicate that most of these polymers are stored in the cell in a soluble form, and not in solid-like structures.

Metabolic studies with NMR spectroscopy of the alga Dunaliella salina trapped within agarose beads.

A technique for the entrapment of the unicellular algae Dunaliella salina in agarose beads and their perfusion during NMR measurements is presented. The trapped cells maintained their ability to proliferate under normal growth conditions, and remained viable and stable under steady-state conditions for long periods during NMR measurements. Following osmotic shock in the dark, prominent changes were observed in the intracellular level of ATP and polyphosphates, but little to no changes in the intracellular pH or orthoposphate content. When cells were subjected to hyperosmotic shock, the ATP level decreased. The content of NMR-visible polyphosphates decreased as well, presumably due to the production of longer, NMR-invisible structures. Following hypoosmotic shock, the ATP content increased and longer polyphosphates were broken down to shorter, more mobile polymers.

Bioavailability of a natural isomer mixture as compared with synthetic all-trans-beta-carotene in rats and chicks.

The unicellular halotolerant alga Dunaliella bardawil was previously shown to contain high concentrations of beta-carotene composed of about equal amounts of the all-trans and 9-cis isomers. One-d-old chicks and 7-wk-old male rats were fed diets supplemented with synthetic all-trans-beta-carotene or dry D. bardawil at equivalent levels of beta-carotene. The chicks were fed diets containing up to 0.025% beta-carotene for 2 mo, and the rats up to 0.1% beta-carotene for 2 wk. Liver analyses at the end of these periods indicated that both species showed at least a tenfold higher accumulation of the algal beta-carotene isomer mixture than of the synthetic all-trans-beta-carotene. The ratio of 9-cis-beta-carotene to the all-trans isomer in the livers of the algae-fed rats and chicks was similar to or higher, respectively, than that present in the algae. Retinol plus retinyl ester accumulated to a similar extent in the rats and chicks fed diets supplemented with synthetic all-trans or the natural isomer mixture of beta-carotene. The preferable accumulation of the natural isomer mixture of beta-carotene suggests that attention should be paid to the different sources of beta-carotene when testing their efficacy in effects other than providing retinol, such as in their possible role in the prevention of some types of cancer.

Involvement of the Plasma Membrane ATPase in the Osmoregulatory Mechanism of the Alga Dunaliella salina.

The unicellular halotolerant alga Dunaliella salina recovers normally from a hypertonic shock even when suspended in NaCl and buffer only. Furthermore, addition of Cu(2+), valinomycin and KCl, or permeable ions such as methyltriphenylphosphonium or thiocyanate, do not affect the recovery. However, treatment with two specific inhibitors of the plasma membrane adenosine triphosphatase (ATPase), diethylstilbestrol, or vanadate, fully inhibit the recovery. The inhibition is manifested by the inability of the cells to both synthesize glycerol and return to their original volume. The inhibitions are nonlethal, reversible and equally effective in the dark or the light. Since the plasma membrane ATPase is the only enzyme known to be inhibited by both diethylstilbestrol and vanadate, it is concluded that its activity is essential for the recovery of Dunaliella from a hypertonic shock. Mechanisms by which the plasma membrane ATPase may participate in the activation of glycerol production in the algae are discussed.

P and C-NMR Studies of the Phosphorus and Carbon Metabolites in the Halotolerant Alga, Dunaliella salina.

The intracellular phosphorus and carbon metabolites in the halotolerant alga Dunaliella salina adapted to different salinities were monitored in living cells by (31)P- and (13)C-nuclear magnetic resonance (NMR) spectroscopy. The (13)C-NMR studies showed that the composition of the visible intracellular carbon metabolites other than glycerol is not significantly affected by the salinity of the growth medium. The T(1) relaxation rates of the (13)C-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 (31)P-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 NaCl 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 (31)P-NMR spectra of cells adapted to high (>1.5 molar NaCl) salinites.

Partial Characterization of K and Ca Uptake Systems in the Halotolerant Alga Dunaliella salina.

The uptake of K(+) and Ca(2+) in Dunaliella salina is mediated by two distinct carriers: a K(+) carrier with a high selectivity against Na(+), Li(+), and choline(+) but not towards Rb(+), K(+), Cs(+), or NH(4) (+), and a Ca(2+) carrier with a high selectivity against Mg(2+). The latter is specifically blocked by La(3+) and by Cd(2+). Apparent K(m) values for K(+) and Ca(2+) uptake are 2.5 and 0.8 millimolar, respectively, and their maximal calculated fluxes are 22 and 0.8 nanomoles per square meter per second, respectively. Effects of permeable ions and ionophores on K(+) and Ca(2+) uptake suggest that the driving force for their uptake is the transmembrane electrical potential. Inhibitors of ATP production, typical inhibitors of plasma membrane H(+)-ATPases and protonionophores inhibit K(+) and Ca(2+) uptake and accelerate K(+) efflux. The results suggest that an H(+)-ATPase in the cell membrane provides the driving force for K(+) and Ca(2+) uptake. Efflux measurements from (86)Rb(+) and (45)Ca(2+) loaded cells suggest that part of the intracellular K(+) and most of the intracellular Ca(2+) is nonexchangeable with the extracellular pool. Correlations between phosphate and K(+) contents and the effect of phosphate on K(+) efflux suggest intracellular associations between K(+) and polyphosphates. On the basis of these results, it is suggested that: (a) K(+) and Ca(2+) uptake in D. salina is driven by the transmembrane electrical potential which is generated by the action of an H(+)-ATPase of the plasma membrane. (b) Part of the intracellular K(+) is associated with polyphosphate bodies, while most of the intracellular Ca(2+) is accumulated in intracellular organelles in the algal cells.

Properties of ATP-induced chlorophyll luminescence in chloroplasts.

1. The recently described reaction of ATP-induced luminescence is analyzed for its relation to other ATP-induced reactions such as ATP-driven transmembrane proton gradient formation and ATP-driven reverse electron flow. 2. In the absence of phenazine methosulfate ATP-induced luminescence is optimal while the main phase of ATP-driven reverse electron flow is eliminated. 3. DCMU which by itself causes a much smaller luminescence, inhibits the ATP-induced luminescence. 4. Nigericin plus valinomycin, but not each by itself, fully inhibit the ATP-induced luminescence. 5. The observations are interpreted as indicating that ATP stimulates luminescence by a 2-fold mechanism: (a) increasing the amount of the reducing primary electron acceptor of Photosystem II, Q, and (b) creating a transmembrane electrochemical potential which serves to decrease the activation energy required for the charge recombination reaction which leads to luminescence.

Properties of ATP-driven reverse electron flow in chloroplasts.

1. The reverse reactions induced by coupled ATP hydrolysis were studied in spinach chloroplasts by measurements of the ATP-induced increase in chlorophyll fluorescence reflecting reverse electron flow, and of the ATP-induced decrease in 9-aminoacridine fluorescence, representing formation of the transthylakoidal proton gradient (deltapH). ATP-induced reverse electron flow was kinetically analysed into three phases, of which only the second and third one were paralleled by corresponding phases in deltapH formation. The rapid first phase and formation of a deltapH occur also in the absence of the electron transfer mediator phenazine methosulfate. 2. The rate and extent of the reverse reactions were measured at temperatures in the range from 0 to 30 degrees C. The rate of formation of delta pH and of reverse electron flow were faster at high temperatures, but the maximal extent of delta pH and chlorophyll fluorescence increase were observed at the lowest temperature. Considering rate and extent of the ATP-stimulated reactions, a temperature optimum around 15 degrees C was found. Light activation of the ATPase occurred throughout the range studied. At 0 degrees C and in the presence of inorganic phosphate the activated state for ATPase was maintained for more than 10 min. 3. The ATP-induced rise in chlorophyll fluorescence yield was found to be of similar magnitude as the rise induced by 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea (DCMU), when both were measured with an extremely weak measuring beam. It is concluded, that both effects, although derived via distinctly different pathways, are limited by the same electron donating or electron accepting pool.