Osmoregulation in the Extremely Euryhaline Marine Micro-Alga Chlorella autotrophica

Chlorella autotrophica (Clone 580) grows over the external salinity range of 1 to 400% artificial sea water (ASW), can photosynthesize over the range from 1 to 600% ASW, and survives the complete evaporation of seawater. The alga grown at high salinities shows an increase in cell volume and a small decrease in cell water content. Measurements of ion content were made by neutron activation analysis on cells washed in isoosmotic sorbitol solutions which contained a few millimolar of major ions to prevent ion leakage. Cells grown at various ASW concentrations contain large quantities of sodium, potassium, and chloride ions. Measurements of cations associated with cell wall and intracellular macromolecules were made to determine intracellular concentration of free ions. The proline content of cells increases in response to increases in external salinity. Cells in 300% ASW contain 1500 to 1600 millimolar proline.     

PROTEIN BIOSYNTHESIS IN SALT-SHOCKED CELLS OF STICHOCOCCUS BACILLARIS (CHLOROPHYCEAE)1

We have developed an in vivo¹⁴C-amino acid labelling procedure for monitoring protein synthesis in salt-shocked cells of Stichococcus bacillaris Naeg. This alga possesses an efficient transport system for the uptake of leucine, methionine, and phenylalanine and rapidly incorporates these amino acids into proteins. Of the three amino acids tested, ¹⁴C-phenylalanine is ideally suited for labelling proteins in S. bacillaris, as it establishes an early equilibrium between uptake and incorporation of the amino acid into proteins. The uptake of phenylalanine shows little inhibition following transfer of cells to higher salinities and is also not affected in short-term experiments by the presence of the protein inhibitors cycloheximide and chloramphenicol. While Stichococcus bacillaris grows slowly at salinities equal to, or higher than, 150% artificial seawater (ASW), it shows surprising rates of recovery of major physiological functions following considerable salt shocks. Cells transferred from 33 to 150% ASW show complete recovery of photosynthetic activity and protein synthesis within 10–15 min, and cell transferred from 33 to 300% ASW recover 50% of their capacity to synthesize proteins within. 1 h. Cytoplasmic and organellar protein synthesis appears to be equally sensitive to the effects of salt shocks according to studies with protein synthesis inhibitors.     

Pyrroline-5-Carboxylate Reductase in Chlorella autotrophica and Chlorella saccharophila in Relation to Osmoregulation

Pyrroline-5-carboxylate (P5C) reductase (EC 1.5.1.2), which catalyzes the reduction of P5C to proline, was partially purified from two Chlorella species; Chlorella autotrophica, a euryhaline marine alga that responds to increases in salinity by accumulating proline and ions, and Chlorella saccharophila, which does not accumulate proline for osmoregulation. From the elution profile of this enzyme from an anion exchange column in Tris-HCl buffer (pH 7.6), containing sorbitol and glycine betaine, it was shown that P5C reductase from C. autotrophica was a neutral protein whereas the enzyme from C. saccharophila was negatively charged. The kinetic mechanisms of the reductase was characteristic of a ping-pong mechanism with double competitive substrate inhibition. Both enzymes showed high specificity for NADH as cofactor. The affinities of the reductases for their substrates did not change when the cells were grown at different salinities. In both algae, the apparent K_m values of the reductase for P5C and NADH were 0.17 and 0.10 millimolar, respectively. A fourfold increase in maximal velocity of the reductase was observed when C. autotrophica was transferred from 50 to 150% artificial sea water. Even though the reductase was inhibited by NaCl, KCl, and proline, it still showed appreciable activity in the presence of these compounds at molar concentrations. A possible role for the regulation of proline synthesis at the step catalyzed by P5C reductase is discussed in relation to the specificity of P5C reductase for NADH and its responses to salt treatments.     

Transport and Assimilation of Nitrogen by Stichococcus bacillaris Grown in the Presence of Methionine Sulfoximine

Stichococcus bacillaris Naeg., a green soil alga, can grow in the presence of methionine sulfoximine (MSX), an inhibitor of glutamine synthetase, by maintaining a high level of NADPH-glutamate dehydrogenase activity. MSX-grown cells can utilize both NH₄⁺ and NO₃⁻ as nitrogen source for growth. [¹⁴C]Methylammonium is not metabolized by S. bacillaris, and is transported by a carrier system that obeys Michaelis Menten kinetics, and is insensitive to MSX.     

Proline overproduction in cells of the green alga Nannochloris bacillaris resistant to azetidine-2-carboxylic acid

Abstract Cells of N. bacillaris have been selected that are resistant to the toxic proline analogue azetidine-2-carboxylic acid (A2C) in 7% artificial seawater (ASW). This phenotype is stable in the absence of selection pressure. A2C resistance at low salinity was demonstrated to be due to an overproduction of proline in these cells, while levels of other amino acids were unaffected. Both wild-type and A2C-resistant cells respond to growth in high salinity media (100% ASW, 200% ASW) by accumulation of proline, but proline levels at all salinities are higher in the A2C-resistant cells than in the wild-type. Proline overproduction in the A2C-resislant cells did not affect fluctuations in the levels of other salinity-dependent solutes, such as homarine. A mutant with this level of specificity over a wide range of water potentials has not been reported for other plants and algae. Both the wild-type and A2C-resistant cells were able to grow over the entire salinity range tested (7%-300% ASW). However, the A2C-resistant cells showed a lower division rate than the wild-type in 300% ASW, and yield of A2C-resislant cells was lower than yield of wild-type cells at the salinity extremes (7% ASW, 300% ASW). The response or wild-type and A2C-resistant cells to rapid increases in salinity were similar for both growth and photosynthesis. The presence of a constitutive high level of proline in the A2C-resistant cell line did not confer any obvious increased tolerance to salinity shocks, indicating that there are other important factors in the biochemical adaptation to salinity in these cells.     

Nitrogen Metabolism of the Marine Microalga Chlorella autotrophica

The levels of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in Chlorella autotrophica (clone 580) are strongly regulated by the nitrogen source and salt concentration of the medium. GS is present at high levels in NO₃⁻-grown cells, and at maximum levels in nitrogen-starved cells. However, the levels of GS in these cells are somewhat decreased by increasing salinity. Cells growing on NH₄⁺ have high NADPH-GDH activity, the levels of which increase with increasing NH₄⁺ supply, while GS decreases to a very low level under these conditions. Salinity intensifies the induction of NADPH-GDH activity in NH₄⁺-grown cells. The levels of NADH-GDH are low in this alga, but present under all growth conditions. Methionine sulfoximine (MSX) has little effect on growth and nitrogen assimilation of the alga in the presence of NH₄⁺.     

The Role of Glycerol and Inorganic Ions in Osmoregulatory Responses of the Euryhaline Flagellate Chlamydomonas pulsatilla Wollenweber

The green euryhaline flagellate Chlamydomonas pulsatilla Wollenweber, isolated from a coastal marine environment, was grown exponentially over the salinity range of 10 to 200% artificial seawater (ASW). The cellular volume and aqueous space of the alga, measured by [¹⁴C] mannitol and ³H₂O tracer analyses of centrifuged cell pellets, ranged between 2.3 and 3.1 picoliters and between 1.5 and 2.1 picoliters, respectively. The nonaqueous space determined in those analyses (28-35%) was consistent with the cell composition of the alga. The glycerol content of the alga increased almost linearly with increasing salinity; its contribution to intracellular osmolality at 200% ASW was about 57%. The contribution of amino acids and soluble carbohydrates to the cell osmotic balance was small. Intracellular ion concentrations determined by analyzing centrifuged cell pellets of known [¹⁴C]mannitol space by atomic absorption spectrophotometry, and by neutron activation analyses of washed cells were similar. At 10% ASW, potassium and magnesium were the major cations, and chloride and phosphate were the major anions. The sodium and chloride content of the alga increased with increasing salinity; at 200% ASW the intracellular concentration of both sodium and chloride was about 400 millimolar. The intracellular osmolality (π_int) matched closely the external osmolality (π_ext) over the entire salinity range except at 10% ASW where π_int exceeded π_ext by 120 to 270 milliosmoles per kilogram H₂O.     

Correlation of decreased calcium contents with proline accumulation in the marine green macroalga Ulva fasciata exposed to elevated NaCl contents in seawater

The involvement of Na⁺, K⁺, Cl^- or Ca²⁺ in the regulation of salinity stress-induced proline accumulation via the inhibition of the activity of proline dehydrogenase (PDH; EC 1.4.3.1), a catabolic enzyme of proline, was investigated in the marine green macroalga Ulva fasciata Delile. After 6 h of exposure to elevated artificial seawater (ASW) salinity, adjusted either by increasing the NaCl content in 30 ASW (a change in ion ratio) or by concentrating ASW (a constant ion ratio), the contents of Na⁺, K⁺ and Cl^- linearly accumulated with increasing salinity from 30-90 (parts per thousand); the accumulation pattern of each ion was similar between the two treatments. An increase in NaCl content in ASW induced proline accumulation, but decreased both the PDH activity and the total water-soluble Ca²⁺ contents, while concentrated ASW had no effect. As compared to a constant value at 30, both the contents of total and water-soluble CA²⁺ and the activity of PDH decreased 1 h after exposure to 60 (adjusted by increasing NaCl content in 30 ASW) and concomitantly the content of seawater Ca²⁺ increased, while proline accumulated after 3 h. The addition of 15 mM ethylene glycol-bis-(2-aminoethyl ether) N,N,N-tetraacetic acid (EGTA) in 60 ASW (adjusted by increasing the NaCl content in 30 ASW) enhanced both the proline accumulation and the decrease in the content of total and water-soluble cellular Ca²⁺ and the activity of PDH; the effects of EGTA were reversed by 10 mM CaSO4. These results indicate that a loss of cellular Ca²⁺ is associated with the NaCl induction of proline accumulation via an inhibition of PDH activity in U. fasciata.     

NITROGEN METABOLISM AND AMINO ACID NUTRITION IN THE SOIL ALGA STICHOCOCCUS BACILLARIS (CHLOROPHYCEAE)1

Nitrate-grown cells of Stichococcus bacillaris Naeg. (UTEX 314) contained much higher activities of glutamine synthetase (GS) and NADPH-glutamate dehydrogenase (GDH) than ammonium-grown cells. Methylamine, a non-metabolizable ammonium analog, caused a decrease in GS activity in nitrate-grown cells suggesting that GS is regulated by the size of the endogenous ammonium pool. The decrease in GS observed in methylammonium-loaded nitrate-grown cells was accompanied by an increase in NADPH-GDH activity. Stichococcus bacillaris can be grown in the presence of methionine sulfoximine (MSX), a potent inhibitor of GS. However, only a fraction of a control cell population showed a requirement for glutamine or arginine for growth following MSX addition. Fully adapted MSX-grown cells were indistinguishable from control cells in their ability to photosynthesize and utilize amino acids as nitrogen sources. Alanine, arginine, asparagine, glutamine, glycine and proline were good nitrogen sources, and maximum capacity for amino acid transport was developed in cells grown on these amino acids. Compared to nitrate-grown cells the activity of GS in ammo acid-grown cells was low, whereas NADPH-GDH was very active. The activity of NADH-GDH in amino acid-grown cells was highest under heterotrophic conditions.     

Application of zeolite improves water and nitrogen use efficiency while increasing essential oil yield and quality of Salvia officinalis under water-deficit stress

Soil moisture and nitrogen (N) are two of the most important factors affecting the production of medicinal plants. So, the management strategy of these factors is critical and to be identified. In order to study the application of zeolite (Z) (0 and 10 ton ha^?1) in S. officinalis culture medium under different irrigation regimes (30 % depletion of available soil water (ASW)) and 60 % depletion of ASW) and N (0, 75 and 150 kg N ha^?1) a split-factorial experiment was carried out with three replicates in 2018. The highest fresh and dry weight were achieved at irrigation after 30 % depletion of ASW while using 150 kg N ha^?1 and 10 ton Z ha^?1. Maximum water use efficiency (WUE) (22.10 g.L^-1) was obtained after 60 % depletion of ASW and 150 kg N ha^?1 and 10 ton Z ha^?1. Besides, the maximum nitrogen use efficiency (NUE) was obtained after 60 % depletion of ASW and 75 kg N ha^?1 and 10 ton Z ha^?1 (14.25 kg.kg^-1N). Maximum essential oil (EO) content (1.06%) and cis-Thujone were obtained from plants subjected to 60 % depletion of ASW and, application of 75 kg N ha^?1 and 10 ton Z ha^?1. Applying Z with N, in different irrigation regimes did improve soil conditions for achieving higher, WUE and NUE, increased the EO content and yield while decreasing the negative effects from water-deficit stress and has provided a direction towards a stable system.     

Physiological responses to NaCl stress in three wild species of potato in vitro

In this study, responses of wild species of potato to NaCl stress were investigated in vitro. In S. stoloniferum and S. bulbosum, length of the shoot, fresh and dry weight, photosynthetic pigments, K⁺ concentration, K⁺/Na⁺ ratio, ascorbate pool, anthocyanin, and phenolic and flavonoid compounds were decreased in response to salinity. In these species, salinity increased the level of Na⁺, lipid peroxidation, proline and ion leakage percentage. In S. acaule, the length of the shoot, and fresh and dry weight were not affected by salinity. Photosynthetic pigments, Na⁺ concentration, proline, flavonoid and phenolic compounds quantities were increased and K⁺/Na⁺ ratio were decreased. K⁺ concentration, lipid peroxidation, ascorbate pool, anthocyanin and ion leakage were not changed by NaCl stress. Superoxide dismutase, guaiacol peroxidase, ascorbate peroxidase and catalase activities were increased in all species. The results suggest that the non-enzymatic antioxidant capacity in S. acaule (salt tolerant) is more important than the enzymatic antioxidant capacity in comparison with the other species.     

Saccharomyces bacillaris is not a synonym of Candida stellata: reinstatement as Starmerella bacillaris comb. nov.

Torulopsis bacillaris (Kroemer and Krumbholz) Lodder (basionym Saccharomyces bacillaris Kroemer and Krumbholz) was frequently detected in oenological works on yeast ecology conducted in the mid-1950s in different wine regions of the world, before its unification with Torulopsis stellata (Kroemer and Krumbholz) Lodder. Most of the phenotypic characteristics pointed out for T. bacillaris are currently attributed to Candida zemplinina Sipiczki. In the present work isoenzyme profiles and rDNA restriction profiles of the neotype of S. bacillaris from two yeast culture collections (CBS 843 and PYCC 3044) and of the type strain of C. zemplinina (CBS 9494) were determined and similar profiles were detected. Moreover, the sequences of the D1/D2 region of the 26S rRNA gene of the three strains were 100 % identical. Different profiles were observed for the type strain of C. stellata (CBS 157) both for isoenzyme and rDNA restriction analysis and only 91 % similarity was found between the D1/D2 sequence of this strain and that of the neotype of S. bacillaris. In view of the newly obtained data and the fact that all above-mentioned species belong to the Starmerella clade, only distantly related to Candida tropicalis (the type species of the genus), S. bacillaris is hereby reinstated as Starmerella bacillaris comb. nov., with C. zemplinina as an obligate synonym.     

Relation between Paramylum Content and the Length of the Lag Period of Chlorophyll Synthesis during Greening of Dark-grown Euglena gracilis

Euglena cells, strains Z and bacillaris, were grown in the dark under various nutritional deficiencies. After 3 days of nondivision, cells were transferred to the light, and the following parameters were measured: the paramylum content at the time of illumination (zero time), the rate of paramylum consumption during the first 10 hours of greening, and the length of the lag phase of chlorophyll synthesis. Similar results were obtained with both strains and can be summarized as follows. (a) The use of various nutritional deficiencies allows the control, to a certain extent, of the amount of paramylum present at zero time. (b) The rate of paramylum consumption is proportional to the cellular paramylum content for values in excess of 50 picograms/cell. (c) The length of the lag phase increases rapidly when the cellular content of paramylum decreases below 50 picograms. This period can be greatly diminished by the addition of an exogenous organíc carbon source. (d) The amount of paramylum (rate of paramylum consumption × length of lag phase) consumed during the lag phase is around 5 to 10 picograms/cell for cells which contain less than 50 picograms of paramylum/cell. It increases when the cellular paramylum content increases, this increment being more rapid for bacillaris than for Z cells.     

Effects of abscisic acid and proline on adaptation of tobacco callus cultures to salinity and osmotic shock

Tobacco callus ( Nicotina tabacum cv. Badischer Geudertheimer) took up sorbitol rapidly and without a lag period from media with up to 0.7 M of the polyol. Accumulation of proline was greatly enhanced under these conditions and was proportional to the absorbed sorbitol, while the viability of the callus cultures was quite low after a few hours of incubation. Under moderate conditions (0.1 M sorbitol) as well as under severe osmotic shock (0.7 M sorbitol), the cells adapted by adjusting the sorbitol/proline ratio to ca 3. NaCl (0.1 M ) had the same effect as sorbitol (0.7 M ) on the survival rate, but only slightly affected proline synthesis in the first hours of incubation. Addition of 10⁷ or 10 ⁵ M abscisic acid (ABA) did not increase the proline content, but 10 ⁷ M ABA delayed the deleterious effect of NaCl and improved the state of the cells. No influence of abscisic acid during the incubation with sorbitol could be detected. Two different strategies for the adjustment of tobacco callus to salinity and sorbitol are suggested: Non-ionic stress is controlled by the accumulation of proline, whereas ABA could be involved in the adaptation to ionic stress.     

Peroxidase activities of two rice cultivars differing in salinity tolerance as affected by proline and NaCl

Proline content, ion accumulation, cell wall and soluble peroxidase activities were determined in control and salt-treated calli (150 nM NaCl) and whole plants (30 mM NaCl) of two rice cultivars (salt sensitive cv. IKP and salt tolerant cv. Aiwu). Under salinity, the highest accumulation of Na⁺, Cl^? and proline occurred in calli, roots and younger leaves of cv. IKP, coupled with the highest decrease in K⁺ content; accumulations of Na⁺ and Cl^? were restricted to older leaves in cv. Aiwu. Relative growth rates of calli and roots or shoots from both cultivars were not linked to peroxidase activities. High concentrations (1 M) of exogenously applied glycerol did not inhibitin vitro activities of soluble peroxidase extracted from control and salt-treated calli or plants. Conversely, 35–55% (in cv. IKP) or 60–80% (in cv. Aiwu) of soluble peroxidase activities were found in presence of isosmotic proline concentration. There were no differences between proline and glycerol effects onin vitro cell wall peroxidase activities.     

Gene expression profiles and intracellular contents of stress protectants in Saccharomyces cerevisiae under ethanol and sorbitol stresses

In response to osmotic stress, proline is accumulated in many bacterial and plant cells. During various stresses, the yeast Saccharomyces cerevisiae induces glycerol or trehalose synthesis, but the fluctuations in gene expression and intracellular levels of proline in yeast are not yet well understood. We previously found that proline protects yeast cells from damage by freezing, oxidative, or ethanol stress. In this study, we examined the relationships between the gene expression profiles and intracellular contents of glycerol, trehalose, and proline under stress conditions. When yeast cells were exposed to 1 M sorbitol stress, the expression of GPD1 encoding glycerol-3-phosphate dehydrogenase is induced, leading to glycerol accumulation. In contrast, in the presence of 9% ethanol, the rapid induction of TPS2 encoding trehalose-6-phosphate phosphatase resulted in trehalose accumulation. We found that intracellular proline levels did not increase immediately after addition of sorbitol or ethanol. However, the expressions of genes involved in proline synthesis and degradation did not change during exposure to these stresses. It appears that the elevated proline levels are due primarily to an increase in proline uptake from a nutrient medium caused by the induction of PUT4. These results suggest that S. cerevisiae cells do not accumulate proline in response to sorbitol or ethanol stress different from other organisms.     

Free proline levels in ulva (chlorophyta) in response to hypersalinity: elevated nacl in seawater versus concentrated seawater (Note)

Changes in free proline concentrations following exposure to elevated NaCl in 35 psu artificial seawater (ASW) (an imbalance in ion ratio) or concentrated ASW (constant ion ratio) were investigated in the intertidal green alga Ulva fasciata Delile. When exposed to 55 psu achieved by adding NaCl to 35 psu ASW, free proline concentrations increased at hour 3 and reached a plateau after 12 h, whereas free proline concentrations in response to 55 psu achieved by concentrating ASW were constant during the treatment period. It is likely that an imbalance in ion ratio is a cue for the induction of a moderate accumulation of free proline in U. fasciata .     

Elucidation of physiological and biochemical mechanisms of an endemic halophyte Centaurea tuzgoluensis under salt stress

In this study, physiological and biochemical responses of Centaurea tuzgoluensis, a Turkish endemic halophyte, to salinity were studied. Therefore, the changes in shoot growth, leaf relative water content (RWC), ion concentrations, lipid peroxidation, hydroxyl (OH^.) radical scavenging activity, proline (Pro) content, and antioxidant system [superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR)] were investigated. The 60 days (d) old C. tuzgoluensis seedlings were subjected to 0, 150 and 300 mM NaCl for 7 d and 14 d. The relative shoot growth was generally did not change in the 150 mM NaCl, but reduced with 300 mM NaCl stress at 7 d and 14 d. RWC was higher in 150 mM NaCl-treated leaves than that of 300 mM NaCl. Salinity decreased K⁺/Na⁺ ratio, but increased Na⁺, Cl^?, Ca⁺² and Na⁺/Cl^? ratio in the leaves. On the other hand, it did not change or increase the K⁺ content at 150 and 300 mM NaCl, respectively. MDA content in the 150 and 300 mM NaCl-treated leaves remained close to control at 7 d. This was related to enhanced activities of SOD, CAT, APX and GR enzymes, and their isoenzymes especially Fe-SOD in the leaves. On the other hand, the higher sensitivity to 300 mM NaCl at 14 d was associated with inadequate increase in antioxidant enzymes and the decreased OH radical scavenging activity. All these results suggest that C. tuzgoluensis has different antioxidant metabolisms between short- (7 d) and long-term (14 d) salt treatments and salinity tolerance of C. tuzgoluensis might be closely related to increased capacity of antioxidative system to scavenge reactive oxygen species (ROS) and accumulation of osmoprotectant proline under salinity conditions.     

Effects of salinity and nitrogen on growth,ion relations and proline accumulation in Triglochin bulbosa

The effects of salinity and nitrogen on growth, ion relations and prolineaccumulation in the monocotyledonous halophyte, Triglochin bulbosa,was investigated in hydroponic culture over 5 months. The experimentaldesign was a 3 × 3 factorial with three salinity treatments (0, 150 and 300 mol m^-3 NaCl) and three levels of N (5, 10 and 20 gml^-1 N as NaNO₃). Total and root dry biomass accumulationwere significantly affected by salinity, but not by N or N × salinityinteraction. Increase in NaCl from 0 to 150 mol m^-3 had no effecton total or root dry biomass, while further increase in salinity to 300mol m^-3 significantly reduced biomass by 21% and 25%respectively. Shoot dry biomass, which was significantly affected by N andnot by salinity, increased with increase in N from 5 to 10 gml^-1. Ion concentrations in roots and shoots were significantlyaffected by salinity, but not by N or N × salinity interaction. Theconcentration of Na⁺ and Cl^- in roots and shoots increasedprogressively with an increase in salinity, while that of K⁺ decreased. Under non-saline conditions, Na⁺/K⁺ ratios were low (0.41to 0.44) and increased significantly with an increase in salinity in both rootsand shoots. Shoot sap osmotic potentials decreased progressively with anincrease in salinity. Increase in N in the hydroponic solution from 5 to20 g ml^-1 significantly increased root and shoot N by 66%and 41% respectively. Tissue concentrations of proline were significantlyaffected by salinity and substrate N but not by N × salinity interaction. Theconcentration of proline in roots and shoots increased significantly by334% and 48%, respectively, with an increase in salinity from 0 to 300mol m^-3 NaCl. Increase in substrate N from 5 to 20 g ml^-1 significantly increased proline in roots and shoots by 66% and41% respectively. The significance of substrate N on the accumulationof proline is discussed in relation to salt tolerance.