Dark induction of nitrate reductase in the halophilic alga Dunaliella salina

The effect of nitrogen starvation on the NO₃-dependent induction of nitrate reductase (NR) and nitrite reductases (NIR) has been investigated in the halophilic alga Dunaliella salina. When D. salina cells previously grown in a medium with NH ₄ ⁺ as the only nitrogen source (NH ₄ ⁺ -cells) were transferred into NO ₃ ^? medium, NR was induced in the light. In contrast, when cells previously grown in N-free medium were transferred into a medium containing NO ₃ ^? , NR was induced in light or in darkness. Nitrate-dependent NR induction, in darkness, in D. salina cells previously grown at a photon flux density of 500 umol · m^?2 s^?1 was observed after 4 h preculture in N-free medium, whilst in cells grown at 100 umol · m^?2 s^?1 NR induction was observed after 7–8 h. An inhibitor of mRNA synthesis (6-methylpurine) did not inhibit NO ₃ ^? -induced NR synthesis when the cells, previously grown in NH ₄ ⁺ medium, were transferred into NO ₃ ^? medium (at time 0 h) after 4-h-N starvation. However, when 6-methylpurine was added simultaneously with the transfer of the cells from NH ₄ ⁺ to NO ₃ ^? medium (at time 0 h), NO ₃ ^? induced NR synthesis was completely inhibited. The activity of NIR decreased in N-starved cells and the addition of NO ₃ ^? to those cells greatly stimulated NIR activity in the light. The ability to induce NR in darkness was observed when glutamine synthetase activity reached its maximal level during N starvation. Although cells grown in NO ₃ ^? medium exhibited high NR activity, only 0.33% of the total NR was found in intact chloroplasts. We suggest that the ability, to induce NR in darkness is dependent on the level of N starvation, and that NR in D. salina is located in the cytosol. Light seems to play an indirect regulatory role on NO ₃ ^? uptake and NR induction due to the expression of NR and NO ₃ ^? -transporter mRNAs.     

The role of intracellular orthophosphate in triggering osmoregulation in the alga Dunaliella salina

A new hypothesis is presented for the mechanism of metabolic response during osmoregulation in the alga Dunaliella salina. We propose that the osmotic response is initiated by differential volume changes of the cytoplasm and the chloroplast (observed using the electron microscope) which alter the cytoplasmic orthophosphate concentration. This triggers a flow through the Pi/triose-phosphate shuttle, activating chloroplast enzymes in the direction of either starch or glycerol synthesis. The Pi-dependent response was investigated in vivo using NMR. The rates of glycerol synthesis or elimination following osmotic shocks were modulated by the intracellular Pi level as predicted by the hypothesis.     

The photosynthetic induction response in wheat leaves: net CO2 uptake,enzyme activation,and leaf metabolites

The photosynthetic induction response was studied in whole leaves of wheat (Triticum aestivum L.) following 5-min, 30-min and 10-h dark periods. After the 5-min dark treatment there was a rapid burst in the rate of photosynthesis upon illumination (half of maximum after 30s), followed by a slight decrease after 1.5 more min and then a gradual rise to the maximum rate. During this initial burst in photosynthesis, there was a rapid rise in the level of 3-phosphoglycerate (PGA) and a high PGA/triose-phosphate (triose-P) ratio was obtained. In addition, after the 5-min dark treatment, ribulose-1,5-bisphosphate carboxylase (Rubisco, EC 4.1.1.39), ribulose-5-phosphate kinase (EC 2.7.1.19) and chloroplastic fructose-1,6-bisphosphatase (EC 3.1.3.11) maintained a relatively high state of activation, and maximum activation occurred within 1 min of illumination. The results indicate there is a high capacity for CO₂ fixation in the cycle upon illumination but attaining maximum rates requires an increase in the ribulose-1,5-bisphosphate (RuBP) pool (adjustment in triose-P utilization for carbohydrate synthesis versus RuBP synthesis). With both the 30-min and 10-h dark pretreatments there was only a slight rise in photosynthesis upon illumination, followed by a lag, then a gradual increase to steady-state (half-maximum rate after 6 min). In contrast to the 5-min dark treatment, the level of PGA was low and actually decreased initially, whereas the level of RuBP increased and was high during induction, indicating that Rubisco is limiting. This regulation via the carboxylase was not reflected in the initial extractable activity, which reached a maximum by 1 min after illumination. The light activation of chloroplastic fructose-1,6-bisphosphatase in leaves darkened for 30 min and 10 h prior to illumination was relatively slow (reaching a maximum after 8 min). However, this was not considered to limit carbon flux through the carbon-fixation cycle during induction since RuBP was not limiting. When photosynthesis approached the maximum steady-state rate, a high PGA/triose-P ratio and a high PGA/RuBP ratio were obtained. This may allow a high rate of photosynthesis by producing a favorable mass-action ratio for the reductive phase (the conversion of PGA to triose phosphate) while stimulating starch and sucrose synthesis.Abbreviations Chl chlorophyll - FBP fructose-1,6-bisphosphate - FBPase fructose-1,6-bisphosphatase - Fru6P fructose-6-phosphate - Glc6P glucose-6-phosphate - PGA 3-phosphoglycerate - Pi inoganic phosphate - Rubisco RuBP carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - Ru5P ribulose-5-phosphate - triose-P triose phosphates (dihydroxyacetone phosphate+glyceraldehyde-3-phosphate)     

Lipid profile of the halophilic alga,Dunaliella salina

The lipid composition comprised more than 50% of the cellular organic material. Greater than 30% of the total lipid composition consisted of hydrocarbons. The aliphatic hydrocarbons consisted of C-17 and C-19 saturated and unsaturated ones, some of them with an internal methyl branch. The remaining lipids consisted of a large quantity of pigmented hydrocarbons together with at least six sterol derivatives, six phospholipids, two glycolipids, one sulfolipid and numerous other components that were not fully identified. This wall-less alga is an immensely rich source of a wide variety of lipids.     

Control analysis of photosynthetic CO2 fixation

The potential of control analysis to aid our understanding of regulation and control of photosynthetic carbon metabolism is investigated. Methods of metabolic control analysis are used to determine flux control coefficients of photosynthetic reactions from enzyme elasticities. Equations expressing control coefficients symbolically by enzyme elasticities are derived, and general properties of these expressions are analysed. Suggestions for experimental determination of flux control coefficients from enzyme elasticities are given. A simplified model of the Calvin-Benson cycle is used to illustrate interrelations between patterns of photosynthetic metabolites and that of control coefficients.Abbreviations GAPDH glyceraldehyde phosphate dehydrogenase - PGA 3-phosphoglycerate - PGK 3-phosphoglycerate kinase - Pi inorganic phosphate - PRK phosphoribulokinase - RuBP ribulose-1,5-bisphosphate(_{total, free}) - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - Ru5P ribulose-5-phosphate     

Low-CO2-inducible protein synthesis in the green alga Dunaliella tertiolecta

In the green marine alga Dunaliella tertiolecta, a CO₂-concentrating mechanism is induced when the cells are grown under low-CO₂ conditions (0.03% CO₂). To identify proteins induced under low-CO₂ conditions the cells were labelled with ³⁵SO₄ ^2–, and seven polypeptides with molecular weights of 45, 47, 49, 55, 60, 68 and 100 kDa were detected. The induction of these polypeptides was observed when cells grown in high CO₂ (5% CO₂ in air) were switched to low CO₂, but only while the cultures were growing in light. Immunoblot analysis of total cell protein against pea chloroplastic carbonic anhydrase polyclonal antibodies showed immunoreactive 30-kDa bands in both high- and low-CO₂-grown cells and an aditional 49-kDa band exclusively in low-CO₂-grown cells. The 30-kDa protein was shown to be located in the chloroplast. Western blot analysis of the plasmamembrane fraction against corn plasma-membrane AT-Pase polyclonal antibodies showed 60-kDa bands in both high- and low-CO₂ cell types as well as an immunoreactive 100-kDa band occurring only in low-CO₂-grown cells. These results suggest that there are two distinct forms of both carbonic anhydrase and plasma-membrane ATPase, and that one form of each of them can be regulated by the CO₂ concentration.Abbreviations CA carbonic anhydrase - DIC dissolved inorganic carbon (CO₂+ HCO₃ ^–) - CCM CO₂-concentrating mechanism - low CO₂ air containing 0.03% CO₂ - high CO₂ air supplemented with 5% CO₂ (v/v) We thank Prof. John Coleman for providing antibodies raised against pea chloroplast CA, Dr. James V. Moroney for providing antibodies raised against the 37-kDa periplasmic carbonic anhydrase of CO₂ Chlamydomonas reinhardtii, and Prof. Leonard T. Robert for a gift of corn plasma-membrane 100-kDa ATPase antibodies. We thank Dr. Jeanine Olsen (University of Groningen, the Netherlands) for style comments. This work was supported by the Institute Tecnológico de Canarias (Spain).     

The role of pH in the regulation of carbon fixation in the chloroplast stroma. Studies on CO2 fixation in the light and dark.

1. The pH in the stroma and in the thylakoid space has been measured in a number of chloroplast preparations in the dark and in the light at 20 degrees C. Illumination causes a decrease of the pH in the thylakoid space by 1.5 and an increase of the pH in the stroma by almost 1 pH unit. 2. CO2 fixation is shown to be strongly dependent on the pH in the stroma. The pH optimum was 8.1, with almost zero activity below pH 7.3.Phosphoglycerate reduction, which is a partial reaction of CO2 fixation, shows very little pH dependency. 3. Low concentrations of the uncoupler m-chlorocarbonylcyanide phenylhydrazone (CCCP) inhibit CO2 fixation without affecting phosphoglycerate reduction. This inhibition of CO2 fixation appears to be caused by reversal of light induced alkalisation in the stroma by CCCP. 4. Methylamine has a very different effect compared to CCCP. Increasing concentrations of methylamine inhibit CO2 fixation and phosphoglycerate reduction to the same extent. The light induced alkalisation of the stroma appears not to be significantly inhibited by methylamine, but the protons in the thylakoid space are neutralized. The inhibition of CO2 fixation by higher concentrations of methylamine is explained by an inhibition of photophosphorylation. It appears that methylamine does not abolish proton transport. 5. It is shown that intact chloroplasts are able to fix CO2 in the dark, yielding 3-phosphoglycerate. This requires the addition of dihydroxyacetone phosphate as precursor of ribulosemonophosphate and also to supply ATP, and the addition of oxaloacetate for reoxidation of the NADPH in the stroma. 6. Dark CO2 fixation in the presence of dihydroxyacetone phosphate and oxaloacetate has the same pH dependency as CO2 fixation in the light. This demonstrates that CO2 fixation in the dark is not possible, unless the pH in the medium is artificially raised to pH 8.8.     

Salt induction of fatty acid elongase and membrane lipid modifications in the extreme halotolerant alga Dunaliella salina

In studies of the outstanding salt tolerance of the unicellular green alga Dunaliella salina, we isolated a cDNA for a salt-inducible mRNA encoding a protein homologous to plant beta-ketoacyl-coenzyme A (CoA) synthases (Kcs). These microsomal enzymes catalyze the condensation of malonyl-CoA with acyl-CoA, the first and rate-limiting step in fatty acid elongation. Kcs activity, localized to a D. salina microsomal fraction, increased in cells transferred from 0.5 to 3.5 M NaCl, as did the level of the kcs mRNA. The function of the kcs gene product was directly demonstrated by the condensing activity exhibited by Escherichia coli cells expressing the kcs cDNA. The effect of salinity on kcs expression in D. salina suggested the possibility that salt adaptation entailed modifications in the fatty acid composition of algal membranes. Lipid analyses indicated that microsomes, but not plasma membranes or thylakoids, from cells grown in 3.5 M NaCl contained a considerably higher ratio of C18 (mostly unsaturated) to C16 (mostly saturated) fatty acids compared with cells grown in 0.5 M salt. Thus, the salt-inducible Kcs, jointly with fatty acid desaturases, may play a role in adapting intracellular membrane compartments to function in the high internal glycerol concentrations balancing the external osmotic pressure.     

Lipid composition of the plasma-membrane of the halotolerant alga,Dunaliella salina

The major lipids of the isolated plasma-membrane of the halotolerant alga Dunaliella salina are diacylglyceroltrimethylhomoserine (DGTS, 23.5%), sterol peroxides (7-dehydroporiferasterol peroxide and ergosterol peroxide, 22%), phosphatidylcholine (13%) and phosphatidylethanolamine (11%). Free sterols comprised 5% of the lipids and contained predominantly 7-dehydroporiferasterol and ergosterol. The major fatty acids of the plasma-membrane were palmitic (31%), oleic (13%), linoleic (20%) and γ-linolenic (17%) acids. In constrast to the whole cells, the plasma-membrane contained less (11%) α-linolenic acid and no 16-carbon unsaturated fatty acids. Sterol peroxides were identified by ¹H-NMR and ¹³C-NMR spectroscopy, mass spectrometry, and by comparison on thin-layer chromatography to the product of ergosterol photooxygenation. We believe that this is the first report on the occurrence of sterol peroxides as major constituents of a biological membrane. It is suggested that they may play a role in the unusual membrane-permeability properties of the plasma-membrane of Dunaliella.     

Active uptake of CO2 during photosynthesis in the green alga Eremosphaera viridis is mediated by a CO2-ATPase

Mass spectrometry was used to investigate the uptake of CO₂ in Eremosphaera viridis DeBary. Upon illumination, cells preincubated at pH 7.5 with 100 M dissolved inorganic carbon (DIC) rapidly depleted almost all the free CO₂ from the medium. Rapid equilibrium between HCO ₃ ^- and CO₂ occurred upon addition of bovine carbonic anhydrase (CA) to the medium, showing that CO₂ depletion resulted from a selective uptake of CO₂ rather than an uptake of all inorganic carbon species. Glycolaldehyde (10 mM) completely inhibited CO₂ fixation but had little effect on CO₂ transport. Transfer of glycolaldehyde-treated cells to the dark caused a rapid efflux of CO₂ from the unfixed intracellular DIC pool which was found to be at least threeto sixfold higher in concentration than that of the external medium. These results indicate that E. viridis actively transports CO₂ against a concentration gradient. No external CA was detected in these cells either by potentiometric or mass-spectrometric assay. In the absence of external CA, the rate of photosynthetic O₂ evolution in the pH range 7.5 to 8.0 did not exceed the calculated rate of CO₂ supply, indicating a limited capacity for HCO₂ uptake in these cells. Electrophysiological measurements indicate that CO₂ uptake is electrically silent and thus is not a consequence of H⁺-CO₂ symport activity. Microsomal membranes isolated from Eremosphaera showed ATPase activity which was enhanced by CO₂. These results indicate that active CO₂ uptake is mediated by an ATPase.Abbreviations BTP 1,3-bis[tris(hydroximethyl)-methylamino]-propane - CA carbonic anhydrase - Chl chlorophyll - DIC dissolved inorganic carbon - [¹⁴C]DMO 5,5-dimethyl-[2-¹⁴C]-oxaz-didine-2,4-dione - WA Wilbur-Anderson units This work was supported by grants to B.C. and R.R.L. from the Natural Sciences and Engineering Research Council of Canada. We thank the Department of Biology, Queen's University, Kingston, Ontario for the use of the mass-spectrometer facility. We are indebted to A.G. Miller for his expert advice on operating the mass spectrometer and to Ms. Shahebina Samji for running the Bradford assays.     

The salt relations of marine and halophilic species of the unicellular green alga,Dunaliella

1. Comparisons were made of the effects of salt on the exponential growth rates of two unicellular algae,Dunaliella tertiolecta (marine) andDunaliella viridis (halophilic).
2. The algae contained glycerol in amounts which varied directly with the salt concentration of the growth media. The highest measured glycerol content ofD. tertiolecta was approximately equivalent to 1.4 molal and occurred in algae grown in 1.36 M sodium chloride. The highest glycerol content measured inD. viridis was approximately equivalent to 4.4 molal and occurred in algae grown in 4.25 M sodium chloride. Lower concentrations of free glucose, which varied inversely with extracellular salt concentration, were also detected.
3. It is inferred that Na⁺ is effectively excluded from the two algae. There was some evidence of a moderate uptake of K⁺.
4. Comparisons were made of erude preparations of the glucose-6-phosphate dehydrogenase and an NADP-specific glycerol dehydrogenase from each species and of the effects of salt and glycerol on the activities of these enzymes. It is concluded that the different salt tolerances of the two algae cannot be explained by generalized differences between their enzyme proteins.
5. Although intracellular glycerol must necessarily contribute to the osmotic status of the algae, its primary function in influencing their salt relations is considered to be that of a compatible solute, whereby glycerol maintains enzyme activity under conditions of high extracellular salt concentration and hence low (thermodynamic) water activity.

     

Non-photochemical quenching of chlorophyll fluorescence in the green alga Dunaliella

The relaxation of the non-photochemical quenching of chlorophyll fluorescence has been investigated in cells of the green alga Dunaliella following illumination. The relaxation after the addition of DCMU or darkening was strongly biphasic. The uncoupler NH₄Cl induced rapid relaxation of both phases, which were therefore both energy-dependent quenching, qE. The proportion of the slow phase of qE increased at increasing light intensity. In the presence of the inhibitors rotenone and antimycin the slow phase of qE was stabilised for in excess of 15 min. NaN₃ inhibited the relaxation of almost all the qE. The implications of these results are discussed in terms of the interpretation of the non-photochemical quenching of chlorophyll fluorescence in vivo and the mechanism of qE.Abbreviations PS II Photosystem II - qQ photochemical quenching of chlorophyll fluorescence - qNP non-photochemical quenching of chlorophyll fluorescence - qE energy-dependent quenching of chlorophyll fluorescence - F _m maximum level of chlorophyll fluorescence for dark adapted cells - F _m level of fluorescence at any time when qQ is zero     

The role of temperature in the regulation of the circadian rhythm of CO2 fixation in Bryophyllum fedtschenkoi

Detached leaves of Bryophyllum fedtschenkoi Hamet et Perrier kept in normal air show a single period of net CO₂ fixation on transfer to constant darkness at temperatures in the range 0–25 °C. The duration of this initial fixation period is largely independent of temperature in the range 5–20 °C, but lengthens very markedly at temperatures below 4 °C, and is reduced at temperatures above 25 °C. The onset of net fixation of CO₂ on transfer of leaves to constant darkness is immediate at low temperatures, but is delayed as the temperature is increased. The ambient temperature also determines whether or not a circadian rhythm of CO₂ exchange occurs. The rhythm begins to appear at about 20 °C, is most evident at 30 °C and becomes less distinct at 35 °C. The occurrence of a distinct circadian rhythm in CO₂ output at 30° C in the absence of a detectable rhythm in PEPCase kinase activity shows that the kinase rhythm is not a mandatory requirement for the rhythm of PEPCase activity. However, when it occurs, the kinase rhythm undoubtedly amplifies the PEPCase rhythm.Abbreviation PEPCase phosphoenolpyruvate carboxylase We thank the Agricultural and Food Research Council for financial support for this work.