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.     

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.     

Rubidium uptake is increased in shore crabs, Carcinus maenas, acclimated to dilute seawater

In dilute seawater, Carcinus maenas hyperosmoregulates by actively absorbing Na, K, and Cl. Here we characterize K uptake using a novel technique. Rb was used as a tracer for K transport, and hemolymph Rb levels were measured using cation chromatography. Hemolymph Rb was detectable at 0.1 mmol L(-1), which enabled determination of initial rate of Rb uptake. Crabs maintained for 3 wk in dilute artificial seawater (35% ASW crabs) maintained Na and K above the level of the external media and had elevated Na-K-ATPase activity in the posterior gills. In assay conditions matched to 100% ASW, Rb uptake was the same in 35% ASW crabs (0.45+/-0.04 micromol g(-1) h(-1)) and in crabs acclimated to normal seawater (100% ASW crabs, 0.49+/-0.05 micromol g(-1) h(-1)). In assay conditions matched to 35% ASW, Rb uptake was greater in 35% ASW crabs (0.28+/-0.03 micromol g(-1) h(-1)) compared with 100% ASW crabs (0.10+/-0.04 micromol g(-1) h(-1)). Low external [Rb] or reduced salinity were found to contribute independently to the difference between 100% ASW and 35% ASW crabs. Thus, whole-body Rb uptake in crabs can be measured by cation chromatography, and Rb uptake is greater in 35% ASW crabs than in 100% ASW crabs.     

Effects of NaCl on growth, ion accumulation, protein, proline contents and antioxidant enzymes activity in callus cultures of Jatropha curcas

Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel crop. The effect of NaCl stress on growth, ion accumulation, contents of protein, proline, and antioxidant enzymes activity in callus cultures of J. curcas was investigated. Exposure of callus to NaCl decreased growth in a concentration dependent manner. NaCl treated callus accumulated Na and declined in K, Ca and Mg contents. Na/K ratio increased steadily as a function of external NaCl treatment. NaCl induced significant differences in quality and quantity of proteins, whereas, proline accumulation remained more or less constant with treatment. NaCl stress enhanced the activity of superoxide dismutase (SOD; E.C. 1.15.1.1) and peroxidase (POX; E.C. 1.11.1.7). Further in the isoenzyme studies, four SOD isoenzymes (SOD 1, 2, 3, and 4) and two POX isoenzymes (POX 1 and 2) were detected with the treatment. NaCl strongly induced activity of SOD 4 isoenzyme in 40, 60, 80 mM and POX 2 isoenzyme in 40 and 80 mM NaCl concentrations. Increase in antioxidant enzymes activity could be a response to cellular damage induced by NaCl. This increase could not stop the deleterious effects of NaCl, but it reduced stress severity and thus allowed cell growth to occur.     

钙在无花果细胞盐诱导脯氨酸积累中的作用

接种于含ＮａＣｌ 培养基的无花果愈伤组织细胞生长极显著受抑,Ｎａ＋ 含量增加,Ｋ／Ｎａ 比值下降,游离脯氨酸积累。培养基中添加一定量ＣａＣｌ２ 不仅在一定程度上缓解盐分对生长的抑制作用,增加Ｋ＋／Ｎａ ＋ 比,而且明显促进游离脯氨酸积累。如果在添加钙的同时再添加细胞钙调素活性抑制剂盐酸氯丙嗪（ＣＰＺ） 或盐酸三氟拉嗪（ＴＦＰ） ,均使钙促进的脯氨酸积累受到明显抑制,表明盐胁迫诱导的脯氨酸积累可能涉及细胞ＣａＣａＭ系统。     

Effects of salinity on the growth rate,carrageenan yield,and cellular structure of <Emphasis Type="Italic">Kappaphycus alvarezii</Emphasis> (Rhodophyta,Gigartinales) cultured in vitro

Kappaphycus alvarezii was cultured in vitro under salinities ranging from 15 to 55 psu for 35 days to determine the differential effect on growth rate, carrageenan yield, and cellular structure. Plants kept in 15 psu died after 3 days, while plants cultured in 55 psu presented low growth rates during the entire experimental period (0.28% day⁻¹). Plants cultured in 25, 35, and 45 psu showed growth rates normally associated with this species (between 3% and 4% day⁻¹) and similar cellular morphology. Carrageenan yield was significantly higher in plants cultured in 25 psu in relation to the other treatments. As observed by light microscopy, plants cultured in 15 psu showed cellular turgidity and increased cell wall thickness, both consequences of hyposalinity. Chloroplasts and other membranous organelles underwent rupture and considerable disorganization in ultrastructure. Although branches from the 55 psu samples showed plasmolysis, cells were able to maintain chloroplast integrity, despite their rudimentary features. In high salinities, great concentrations of floridean starch grains were observed in subcortical cells, indicating their probable participation in osmoregulation. Based on these results, we defined the range of 25 to 45 psu as the limits of saline tolerance for K. alvarezii. While new field studies are required to confirm these results, it can be concluded that new sites, such as inactive or abandoned shrimp tanks with salinities up to 25 psu, could be considered for commercial farming.     

Alpha-D-mannopyranosyl-(1-->2)-alpha-D-glucopyranosyl-(1-->2)-glycerate in the thermophilic bacterium Petrotoga miotherma--structure, cellular content and function

The intracellular accumulation of low molecular mass organic compounds in response to stressful conditions was investigated in the thermophilic bacterium Petrotoga miotherma, a member of the order Thermotogales. This led to the discovery of a new solute, whose structure was established as alpha-D-mannopyranosyl-(1-->2)-alpha-D-glucopyranosyl-(1-->2)-glycerate (MGG) by MMR spectroscopy and MS. Under optimum growth conditions (3% NaCl; 55 degrees C), MGG was the major solute [up to 0.6 micromol.(mg protein)(-1)]; alpha-glutamate and proline were also present but in minor amounts [below 0.08 micromol.(mg protein)(-1)]. The level of MGG increased notably with the salinity of the growth medium up to the optimum NaCl concentration. At higher NaCl concentrations, however, the level of MGG decreased, whereas the levels of proline and alpha-glutamate increased about five-fold and 10-fold, respectively. MGG plays a role during low-level osmotic adaptation of Petrotoga miotherma, whereas alpha-glutamate and, to a lesser extent, proline are used for osmoprotection under salt stress. MGG is not part of the cell strategy for coping with heat or oxidative stress. Nevertheless, MGG was an efficient protector of pig heart malate dehydrogenase against heat inactivation and freeze-drying, although mannosylglycerate was better. This is the first report on the occurrence of MGG in living systems.     

Salinity tolerance of Avicennia marina (Forssk.) Vierh. from Gujarat coasts of India

Greenhouse experiments were conducted to assess the effects of soil salinity on emergence, growth, water status, proline content and mineral accumulation of seedlings of Avicennia marina (Forssk.) Vierh. NaCl was added to the soil and salinity was maintained at 0.2, 2.5, 5.1, 7.7, 10.3, 12.6, 15.4, 17.9, 20.5, 23.0, 25.6 and 28.2 psu. A negative relationship between seedling emergence and salt concentration was obtained. Nevertheless, this mangrove is highly salt tolerant during germination. Growth of seedlings was significantly promoted by low salinity and optimum growth was obtained at 15.4 psu. Higher salinities inhibited plant growth. Growth and dry matter accumulation in tissues followed the same optimum curve. Water potential of tissues became significantly more negative with increasing salinity, and proline content significantly increased. Moreover, water potential and proline content of tissues displayed an S-curve with the inflection point below ∼10 psu. The concentration of Na in tissues increased significantly, whereas K, Ca, Mg, N and P content decreased.     

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.     

NaCl effects on proline metabolism in rice (Oryza sativa) seedlings

Salt-stress effects on osmotic adjustment, ion and proline concentrations as well as proline metabolizing enzyme activities were studied in two rice ( Oryza sativa L.) cultivars differing in salinity resistance: I Kong Pao (IKP; salt-sensitive) and Nona Bokra (salt-resistant). The salt-sensitive cultivar exposed to 50 and 100 m M NaCl in nutritive solution for 3 and 10 days accumulated higher levels of sodium and proline than the salt-resistant cultivar and displayed lower levels of osmotic adjustment. Proline accumulation was not related to proteolysis and could not be explained by stress-induced modifications in Δ¹-pyrroline-5-carboxylate reductase (P5CR; EC 1.5.1.2) or proline dehydrogenase (PDH; EC 1.5.1.2) activities recorded in vitro. The extracted ornithine Δ -aminotransferase (OAT; EC 2.6.1.13) activity was increased by salt stress in the salt-sensitive cultivar only. In both genotypes, salt stress induced an increase in the aminating activity of root glutamate dehydrogenase (GDH; EC 1.4.1.2) while deaminating activity was reduced in the leaves of the salt-sensitive cultivar. The total extracted glutamine synthetase activity (GS; EC 6.3.1.2) was reduced in response to salinity but NaCl had contrasting effects on GS1 and GS2 isoforms in salt-sensitive IKP. Salinity increased the activity of ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) extracted from leaves of both genotypes and increased the activity of NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) in the salt-sensitive cultivar. It is suggested that proline accumulation is a symptom of salt-stress injury in rice and that its accumulation in salt-sensitive plants results from an increase in OAT activity and an increase in the endogenous pool of its precursor glutamate. The physiological significance of the recorded changes are analyzed in relation to the functions of these enzymes in plant metabolism.     

The effects of NaCl on growth,water relations,osmolytes and ion content in <Emphasis Type="Italic">Kochia prostrata</Emphasis>

The effects of NaCl (0, 50, 100, 150 and 200 mM) on growth, water relations, glycinebetaine, free proline, ion contents, stomata number and size of Kochia prostrata (L.) Schard were determined. Shoot and root fresh and dry matter, root and shoot length, relative growth rate, net assimilation rate, relative water content, water use efficiency, soluble sugars and glycinebetaine contents were not changed at low NaCl concentrations, but they were significantly decreased at 200 mM NaCl. The K⁺, Mg²⁺ and Ca²⁺ contents, water potential, chlorophyll a+b and carotenoides contents, and stomata number and size were reduced already at low concentrations of NaCl. In contrast, the Na⁺, Cl^– and proline contents increased several times with increasing NaCl concentration. Kochia prostrata is a salt tolerant species, the optimal growth of this plant occurred up to 150 mM NaCl. The mechanisms of salt tolerance in the plant may be balance among ion accumulation and production of glycinebetaine, proline, soluble sugars for maintenance of pressure potential.     

Effect of sodium and calcium chlorides,abscisic acid and proline on callus cultures ofArachis hypogaea L.

Callus cultures ofArachis hypogaea L. cv. JL-24 adapted to 200 mM NaCl (otherwise lethal to cells) were used for the study. Calli grew slowly when transferred to 250 mM NaCl, but the growth was enhanced when ABA was included in the medium. ABA induced increase in growth of callus was not accompanied by corresponding increase in internal free proline levels. 0.5 mM of CaCl₂ ameliorated the negative effect of NaCl indicating that cells require a specific Ca²⁺/Na⁺ ratio for their growth. Proline content also increased at this ratio thereby suggesting that increase in growth at 0.5 mM Ca²⁺ may be due to an increase in proline content. However, exogenous proline did not increase the growth of callus (adapted to 200 mM), and higher concentrations even inhibited the growth. This shows that proline is not required for growth or adaptation of cells to salt stress, but is produced as a consequence of stress.     

Osmotic and Ionic Regulation of North American Zebra Mussels (Dreissena polymorpha)

SYNOPSIS. Unlike other freshwater bivalves that survive formonths in deionized water, Dreissena polymorpha requires minimalconcentrations of Na, K, Mg, and Cl in the bathing medium forlong-term survival. Although ion transport rates are higherin D. polymorpha compared to other freshwater bivalves, theytend to have lower blood solute concentrations. D. polymorphahas an unusually "leaky" epithelium with a high paracellularpermeability to solutes. Thus, even with high transport rates,it may not be possible for zebra mussels to retain higher bloodsolutes because of the extensive passive loss of ions. Undera hyperosmotic stress, D. polymorpha will rapidly osmoconform(about 12 hr) due primarily to the diffusion of solutes andpartially to the osmotic loss of water. D. polymorpha is notcapable of surviving an imbalance of Na/K in the external medium.In the absence of K the cells will tend to lose volume to achieveisosmotic balance with the blood, but the animals usually diewithin a few days. If D. polymorpha is exposed to excess K inthe environment (1 mM), they will accumulate K in the blood.If the K enters the cells, cellular volume would expand dueto increase in osmolyte concentration, yet, if K remains inthe blood, there will be an electrochemical imbalance. In eithercase, the animal cannot survive much longer than a day. WhenNa and K are present in the medium in a balanced combinationapproximated by artificial seawater (ASW), D. polymorpha willsurvive an acute transfer to 100 mosm ASW indefinitely (months).Our preliminary studies have shown that D. polymorpha will toleratestep-wise acclimation to solutions >250 mosm provided thechanges in salinity do not exceed 50–100 mosm. Freshwaterbivalves, unlike the marine bivalves, have limited free aminoacids in their body fluids and must rely on inorganic ions forosmotic regulation. The free amino acids serve as an importantosmolyte buffer for volume regulation when an animal experiencesan environment of changing salinity. The inability of Dreissena,and perhaps other freshwater bivalves, to tolerate hyperosmoticallyinduced dehydration may be due, in part, to the inability toaccumulate or retain sufficient intracellular K to facilitateregulatory volume adjustments.     

Sodium and chloride transport in soft water and hard water acclimated zebrafish (Danio rerio)

While the zebrafish is commonly used for studies of developmental biology and toxicology, very little is known about their osmoregulatory physiology. The present investigation of Na(+) and Cl(-) transport revealed that the zebrafish is able to tolerate extremely low ambient ion concentrations and that this is achieved at least in part by a greatly enhanced apparent uptake capacity and affinity for both ions. Zebrafish maintain plasma and whole body electrolyte concentrations similar to most other freshwater teleosts even in deionized water containing only 35 microM NaCl, i.e soft water. We recorded an extremely low transport affinity constant (K(m)) of 8+/-1 microM for the active uptake of Cl(-) in soft water acclimated fish, while other transport kinetic parameters were in agreement with reports for other freshwater organisms. While both Na(+) and Cl(-) uptake in soft water clearly depends on apical proton pump activity, changes in abundance and possibly localization of this protein did not appear to contribute to soft water acclimation. Active Cl(-) uptake was strongly dependent on branchial carbonic anhydrase (CA) activity regardless of water type, while the response of Na(+) transport to a CA inhibitor was more variable. Differential response of Na(+) uptake to amiloride depending on acclimation medium suggests that different Na(+) transport mechanisms are employed by zebrafish acclimated to soft and hard water.     

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.     

Effect of salt stress on physiological and antioxidative responses in two species of <Emphasis Type="Italic">Salicornia</Emphasis> (<Emphasis Type="Italic">S. persica</Emphasis> and <Emphasis Type="Italic">S. europaea</Emphasis>)

The effects of salt stress on growth parameters, free proline content, ion accumulation, lipid peroxidation, and several antioxidative enzymes activities were investigated in S. persica and S. europaea. The seedlings were grown for 2 months in half-strength Hoagland solution and treated with different concentrations of NaCl (0, 85, 170, 340, and 510 mM) for 21 days. The fresh and dry weights of both species increased significantly at 85 and 170 mM NaCl and decreased at higher concentrations. Salinity increased proline content in both the species as compared to that of control. Sodium (Na⁺) content in roots and shoots increased, whereas K⁺ and P_i content in both organs decreased. At all NaCl concentrations, the total amounts of Na⁺ and K⁺ were higher in shoots than in roots. Malondialdehyde (MDA) content declined at moderate NaCl concentrations (85 and 170 mM) and increased at higher levels. With increased salinity, superoxide dismutase (SOD), catalase (CAT), and guaiacol peroxidase (GPX) activities also increased gradually in both species. In addition, it seems that GPX, CAT, and SOD activities play an essential protective role in the scavenging reactive oxygen species (ROS) in both species. Native polyacrylamide gel electrophoresis (PAGE) indicated different isoform profiles between S. persica and S. europaea concerning antioxidant enzymes. These results showed that S. persica exhibits a better protection mechanism against oxidative damage and it is more salt-tolerant than S. europaea possibly by maintaining and/or increasing growth parameters, ion accumulation, and antioxidant enzyme activities.     

Research note: Induction of gamete discharge by hypertonic treatment in the green alga Bryopsis plumosa (Caulerpales,Chlorophyta)

Brief treatment with hypertonic solutions induced gamete discharge from gametangia in the coenocytic green alga Bryopsis plumosa (Hudson) C. Agardh. It is known that gamete discharge in this alga is triggered by a change from darkness to light. However, in this study mature gametangia, incubated for 30–120 s in artificial seawater (ASW) with an additional 0.4–0.6 M NaCl and then transferred into pure ASW, discharged gametes in darkness. The treatment did not affect the motility of the gametes. Addition of sucrose (1.0–1.2 M) to ASW also induced gamete discharge in darkness. Similar results were obtained by adding KCl (0.4 M) or mannitol (1.2 M) to ASW. Continuous incubation of gametangia in such hypertonic solutions also induced gamete discharge but led to a delay and a reduction in the rate of gamete discharge, and a loss of gamete motility. In gametangia treated with the hypertonic solutions, as well as in those exposed to light, shortening of the gametangial length was observed before gamete discharge.     

Effect of salinity on chlorophyll and proline contents in three aquatic macrophytes

The role of proline in imparting tolerance to salinity was investigated in Hydrilla verticillata, Najas indica and Najas gramenia. The plants were exposed to different concentrations of NaCl and artificial sea water (SWS) separately. The chlorophyll (Chl) a/b ratio decreased significantly in all the three plant species in both NaCl and SWS treatments, comparatively more in former than the latter. NaCl resulted in drastic decrease in this ratio in salt sensitive H. verticillata and N. indica, but in somewhat lesser decrease in salt resistant N. gramenia. Proline content increased at both NaCl and SWS treatments, especially at the latter. However, in H. verticillata proline content at 1.5 and 2.5 % NaCl decreased. It was concluded that proline cannot be used as a biochemical marker of salt tolerance in aquatic plants, however, the decrease in Chl a/b ratio in response to NaCl may be used as an index of salt sensitivity in this ecological group of plants. This revised version was published online in August 2006 with corrections to the Cover Date.     

Ionic and acid-base consequences of exposure to increased salinity in the zebra mussel, Dreissena polymorpha

Dreissena polymorpha, an invasive freshwater bivalve, displays physiological characteristics that reflect its ancestry in brackish water, yet it has limited ability to withstand modest increases in salinity. We examined changes in hemolymph ion concentrations and acid-base variables in mussels transferred to and incubated in 10% artificial seawater (ASW) for 7 days and then returned to pondwater (PW) for a further 7 days. Hemolymph was sampled (10 animals per sample period) every 4 h for the first 24-h incubation and at 72 h and 168 h for both the transfer to 10% ASW and the transfer back to PW. The initial response to transfer to 10% ASW was a rapid attainment of an apparent isoosmotic steady state, with most hemolymph ion concentrations rising and attaining steady state within 12 h. Hemolymph magnesium rose more slowly, and hemolymph calcium declined despite an increase in its concentration in the bathing medium. Hemolymph pH rose significantly during the first 24 h, from 7.96 to 8.25, as a result of increases in bicarbonate; pH subsequently returned to normal through increases in PCO2. When animals were returned to PW after 7 days' incubation in ASW, the response of the major hemolymph ions was largely the reverse of that effected by the transfer to ASW. Hemolymph pH was not altered significantly until after 72 h in PW, when declines in bicarbonate lowered the pH to 7.73. Strong ion difference (SID) was related significantly to hemolymph pH. Hemolymph calcium and magnesium showed a reciprocal relationship throughout both transfer and incubation. Solubility interactions between sulfate and calcium and magnesium may be important in determining calcium availability in solution. The Na/K ratio in hemolymph was maintained within relatively narrow bounds throughout the procedure and may contribute to the mussels' ability to volume-regulate during an osmotic challenge. Overall, the responses of D. polymorpha to modest changes in salinity were largely the result of passive processes.     

Comparison between a Stable NaCl-Selected Nicotiana Cell Line and the Wild Type : K, Na, and Proline Pools as a Function of Salinity

An NaCl-resistant line has been developed from suspension-cultured tobacco cells (Nicotiana tabacum/gossii) by stepwise increases in the NaCl concentration in the medium. Resistance showed stability through at least 24 generations in the absence of added NaCl.

Above an external NaCl concentration of 35 millimolar, proline concentration in the selected cells rose steeply with external NaCl, particularly so above 100 millimolar NaCl. Proline accumulation in the wild type was far slighter. Selected cells which had been grown for 24 generations in the absence of added NaCl accumulated proline strongly on re-exposure to NaCl medium, indicating stability of this character. Proline accumulation was fully reversible with a half-time of about 6 hours. When selected cells were transferred sequentially to lower and lower NaCl concentrations, their proline content fell to the level corresponding to the new NaCl concentration. The NaCl-selected cells responded to water stress (i.e. added mannitol) by accumulating markedly more proline than did the wild type.

The addition of Ca²⁺ to the growing and rinsing media minimized Na⁺ and K⁺ binding in the Donnan free space of cell walls and thus allowed assessment of intracellular Na⁺ and K⁺. In both cell types, internal Na⁺ content rose steadily as a function of external NaCl concentration. In the course of 7 days in NaCl media, the wild type cells lost a considerable part of their K⁺ content, the extent of the loss increasing with rise in external NaCl concentration. The selected cells, by contrast, lost no K⁺ at external NaCl concentrations below 50 millimolar external NaCl, and at higher concentrations lost less than the wild type.