Quantification of Apoplastic Potassium Content by Elution Analysis of Leaf Lamina Tissue from Pea (Pisum sativum L. cv Argenteum)

K⁺ content and concentration within the apoplast of mesophyll tissue of pea (Pisum sativum L., cv Argenteum) leaflets were determined using an elution procedure. Following removal of the epidermis, a 1 centimeter (inside diameter) glass cylinder was attached to the exposed mesophyll tissue and filled with 5 millimolar CaCl₂ solution (1°C). From time-course curves of cumulative K⁺ diffusion from the tissue, the amount of K⁺ of extracellular origin was estimated. Apoplastic K⁺ contents for leaves from plants cultured in nutrient solution containing 2 or 10 millimolar K⁺ were found to range from 1 to 4.5 micromoles per gram fresh weight, comprising less than 3% of the total K⁺ content within the lamina tissue. Assuming an apoplastic solution volume of 0.04 to 0.1 milliliters per gram fresh weight and a Donnan cation exchange capacity of 2.63 micromoles per gram fresh weight (experimentally determined), the K⁺ concentration within apoplastic solution was estimated at 2.4 to 11.8 millimolar. Net movement of Rb⁺ label from the extracellular compartment within mesophyll tissue into the symplast was demonstrated by pulse-chase experiments. It was concluded that the mesophyll apoplast in pea has a relatively low capacitance as an ion reservoir. Apoplastic K⁺ content was found to be highly sensitive to changes in xylem solution concentration.     

Effects of ouabain and low temperature on the sodium efflux pump in excised corn roots

Ouabain (0.05 millimolar) and low temperature (4 C) both caused the tissue Na⁺ content of excised 5-day-old corn roots to increase, indicating that there is an inhibition of the Na⁺ efflux pump. Na⁺ efflux was measured utilizing three different methods. Each method gave similar results in terms of rate and ouabain sensitivity. With one of these methods, the compartmental efflux method, it was demonstrated that rates for Na⁺ efflux increase as the external Na⁺ concentration is increased; e.g. the efflux rates are 0.529, 1.78, and 3.64 microequivalents per gram fresh weight per hour for external NaCl concentrations of 1, 10, and 30 millimolar, respectively. The data indicate that the Na⁺ efflux pump is located in the plasmalemma of root cells.     

Increased Potassium Absorption Confers Resistance to Group IA Cations in Rubidium-Selected Suspension Cells of Brassica napus

Cell lines of suspension cultures of Brassica napus cv. Jet Neuf were identified for their ability to tolerate 100 millimolar Rb⁺, a level which was double the normally lethal concentration. Ten spontaneous isolates were obtained from approximately 5 × 10⁷ cells, one of which was reestablished as a cell suspension. This cell line, JL5, was also resistant to the other group IA cations— Li⁺, Na⁺, K⁺, and Cs⁺—and this trait was stable for at least 30 cell generations in the absence of Rb⁺ selection pressure. The growth characteristics were similar to those of sensitive cells under nonselective conditions. The selected JL5 cells were shown by analysis to have effected more net accumulation of K⁺ and Rb⁺ and less of Na⁺ than did the unselected cells. JL5 and unselected cells after 14 days of culture in basal medium contained 597.2 and 258.2 micromoles of K per gram dry weight, respectively. Michaelis-Menten kinetic analysis of K⁺ influx showed that JL5 possessed an elevated phase 1 V_max, but there was no alteration in its K_m. This is the first time that a plant mutation has been shown to have both increased influx and net absorption of a major essential cation.     

Potassium transport in two tomato species : lycopersicon esculentum and lycopersicon cheesmanii

The commercial tomato, lycopersicon esculentum Mill. cv Walter, and its wild relative, Lycopersicon cheesmanii ssp. minor (Hook.) C.H. Mull., were grown for 30 days under controlled conditions and in solution culture varying in its content of Na⁺ and K⁺. Subsequently, ⁸⁶Rb-labeled K⁺ uptake and distribution were studied. From all solutions, `Walter' consistently absorbed <sup>86</sup>Rb-labeled K<sup>+</sup> at a higher rate in micromoles per gram fresh weight per 30 minutes than L. cheesmanii. L. cheesmanii distributed a greater proportion of the absorbed K<sup>+</sup> from its root to its shoot. When 0.6 millimolar NaNO<sub>3</sub> replaced 0.6 millimolar KNO<sub>3</sub> in the pretreatment solution, intact plants of both genotypes followed a similar pattern as when they were pretreated with K<sup>+</sup> only, but a greater percentage of the absorbed K<sup>+</sup> remained in the roots. Leaf slices of L. cheesmanii plants deprived of K<sup>+</sup> for 6 days showed a greater rate of K<sup>+</sup> uptake than did slices from `Walter' plants pretreated the same way. Stem slices of L. cheesmanii, however, had a lower uptake rate than did those of `Walter'. Both leaf and stem slices of `Walter' plants, pretreated 6 days with 0.6 millimolar NaNO₃ substituting for 0.6 millimolar KNO₃ in their growth medium, had greater rates of ⁸⁶Rb-labeled K⁺ uptake from 0.5 and 20 millimolar KCl solutions than did slices of L. cheesmanii. These marked differences in patterns of ion uptake and translocation indicate that these genotypes of tomato have evolved different mechanisms to deal with K⁺ and Na⁺ in their environments.     

Effect of Glyoxylate on the Sensitivity of Net Photosynthesis to Oxygen (the Warburg Effect) in Tobacco

The addition of glyoxylate to tobacco (Nicotiana tabacum) leaf discs inhibited glycolate synthesis and photorespiration and increased net photosynthetic ¹⁴CO₂ fixation. This inhibition of photorespiration was investigated further by studying the effect of glyoxylate on the stimulation of photosynthesis that occurs when the atmospheric O₂ level was decreased from 21 to 3% (the Warburg effect). The Warburg effect is usually ascribed to the increased glycolate synthesis and metabolism that occurs at higher O₂ concentrations. Photosynthesis in control discs increased from 59.1 to 94.7 micromoles of CO₂ per gram fresh weight per hour (a 60% increase) when the O₂ level was lowered from 21 to 3%, while the rate for discs floated on 15 millimolar glyoxylate increased only from 82.0 to 99.7 micromoles of CO₂ per gram fresh weight per hour (a 22% increase). The decrease in the O₂ sensitivity of photosynthesis in the presence of glyoxylate was explained by changes in the rate of glycolate synthesis under the same conditions.

The rate of metabolism of the added glyoxylate by tobacco leaf discs was about 1.35 micromoles per gram fresh weight per hour and was not dependent on the O₂ concentration in the atmosphere. This rate of metabolism is about 10% the amount of stimulation in the rate of CO₂ fixation caused by the glyoxylate treatment on a molar carbon basis. Glyoxylate (10 millimolar) had no effect on the carboxylase/oxygenase activity of isolated ribulose diphosphate carboxylase. Although the biochemical mechanism by which glyoxylate inhibits glycolate synthesis and photorespiration and thereby decreases the Warburg effect is still uncertain, these results show that cellular metabolites can regulate the extent of the Warburg effect.

     

The Hypersensitive Reaction of Tobacco to Pseudomonas syringae pv. pisi: Activation of a Plasmalemma K/H Exchange Mechanism

Net electrolyte efflux from suspension-cultured tobacco cells undergoing the hypersensitive reaction to Pseudomonas syringae pv. pisi resulted from a specific efflux of K⁺ which was accompanied by an equimolar net influx of H⁺. These fluxes began 60 to 90 minutes after inoculation of tobacco cells with bacteria, reached maximum rates of 6 to 9 micromoles per gram fresh weight tobacco cells per hour within 2.5 to 3 hours, and dropped below 4 micromoles per gram per hour within 5 hours. Tobacco cells lost approximately 35% of total K⁺ during this period, and average cellular pH declined by approximately 0.75 pH unit. These events were accompanied by a 30% decrease in cellular ATP. K⁺ and H⁺ fluxes were inhibited by the protonophore (p-trifluoromethoxy)carbonyl cyanide phenylhydrazone and by increasing the K⁺ concentration of the external solution. Tobacco leaf discs inoculated with the bacterium also exhibited a specific net K⁺ efflux and H⁺ influx. These results suggest that induction of the hypersensitive reaction in tobacco proceeds through the activation of a passive plasmalemma K⁺/H⁺ exchange mechanism. It is hypothesized that activation of this exchange is a major contributing factor in hypersensitive plant cell death.     

L'induction du rythme endogène de sporulation chez Aspergillus niger: Rõles du rapport glucose/potassium et de micro-éléments

In Aspergillus niger Van Tieghem cultivated on a synthetic medium, the induction of an endogenous rhythm of sporulation and its perpetuation depend on the glucose K⁺ balance in the medium, an excess of one of them suppressing the oscillations. In its inducing effect on the rhythm K⁺ is partially replaced by Rb⁺, but not by Na⁺, Li⁺ or Cs⁺. While the glucose K⁺ balance is favourable for the manifestation of the rhythm, the addition of increasing levels of Na⁺, Li⁺ or Cs⁺ do not modify the period length. Nevertheless, at 0.3 M of Na⁺ or Li⁺ and 0.03 M of Cs⁺ rhythm disappears. The amplitude of oscillations depends on the level of the micro-elements furnished, especially on Mn²⁺. EDTA (1 × 10^?3M) inhibits the rhythm.     

Steady state proline levels in salt-shocked barley leaves

Excised barley (Hordeum vulgare var Larker) leaves were treated with salt solutions or wilted. After the treatment period, the leaves were allowed to recover in a 50 millimolar sucrose and 1 millimolar glutamate solution, and proline, Na⁺, and K⁺ were measured at intervals. Na⁺ and K⁺ concentrations stayed at a constant high level after the salt treatments, and proline increased to a steady state concentration in response. The relationship between the maximum rate of proline accumulation and the Na⁺ concentration reached in each experiment was linear. The final steady state proline concentration reached was also directly proportional to the Na⁺ concentration. For a given Na⁺ concentration in the leaves, the steady state proline level was greater when 410 millimolar NaCl was added to the leaves than when 205 millimolar NaCl was added. These results are consistent with proline acting as a compatible cytoplasmic solute, balancing an accumulation of salts outside of the cytoplasm.

In contrast to the proline levels in salt-shocked leaves, the concentrations in wilted leaves decreased to near control levels within 24 hours of relief of stress.

     

The effect of sodium chloride on solute potential and proline accumulation in soybean leaves

Two cultivars of soybean (Glycine max [L.] Merr.) were grown in solution with up to 100 millimolar NaCl. Leaf solute potential was −1.1 to −1.2 megapascals in both cultivars without NaCl. At 100 millimolar NaCl leaf solute potential was −3.1 to −3.5 megapascals in Bragg and −1.7 megapascals in Ransom. The decrease in solute potential was essentially proportional to the concentration of NaCl. In both salt susceptible Bragg and salt semitolerant Ransom, leaf proline was no more than 0.4 micromole per gram fresh weight at or below 20 millimolar NaCl. At 40 and 60 millimolar NaCl, Bragg leaf proline levels were near 1.2 and 1.9 micromoles per gram fresh weight, respectively. Proline did not exceed 0.5 micromole per gram fresh weight in Ransom even at 100 millimolar NaCl. Proline accumulated in Bragg only after stress was severe enough to induce injury; therefore proline accumulation is not a sensitive indicator of salt stress in soybean plants.     

Sucrose Phosphate Synthase, Sucrose Synthase, and Invertase Activities in Developing Fruit of Lycopersicon esculentum Mill. and the Sucrose Accumulating Lycopersicon hirsutum Humb. and Bonpl

The green-fruited Lycopersicon hirsutum Humb. and Bonpl. accumulated sucrose to concentrations of about 118 micromoles per gram fresh weight during the final stages of development. In comparison, Lycopersicon esculentum Mill. cultivars contained less than 15 micromoles per gram fresh weight of sucrose at the ripe stage. Glucose and fructose levels remained relatively constant throughout development in L. hirsutum at 22 to 50 micromoles per gram fresh weight each. Starch content was low even at early stages of development, and declined further with development. Soluble acid invertase (EC 3.2. 1.26) activity declined concomitant with the rise in sucrose content. Acid invertase activity, which was solubilized in 1 molar NaCl (presumably cell-wall bound), remained constant throughout development (about 3 micromoles of reducing sugars (per gram fresh weight) per hour. Sucrose phosphate synthase (EC 2.4.1.14) activity was present at about 5 micromoles of sucrose (per gram fresh weight) per hour even at early stages of development, and increased sharply to about 40 micromoles of sucrose (per gram fresh weight) per hour at the final stages of development studied, parallel to the rise in sucrose content. In comparison, sucrose phosphate synthase activity in L. esculentum remained low throughout development. The possible roles of the sucrose metabolizing enzymes in determining sucrose accumulation are discussed.     

Effect of Methionine Sulfoximine on the Accumulation of Ammonia in C(3) and C(4) Leaves : The Relationship between NH(3) Accumulation and Photorespiratory Activity

Additions of methionine sulfoximine (MSX), an inhibitor of glutamine synthetase (GS), result in an increase in NH₃ in seedling leaves of C₃ (wheat [Triticum aestivum cv. Kolibri] and barley [Hordeum vulgare var Perth]) and C₄ (corn [Zea mays W6A × W182E] and sorghum [Sorghum Vulgare var MK300]) plants. NH₃ accumulation is higher in C₃ (about 17.8 micromoles per gram fresh weight per hour) than in C₄ (about 4.7 micromoles) leaves. Under ideal conditions, when photosynthesis is not yet inhibited by the accumulation of NH₃, the rate of NH₃ accumulation is about 16% of the apparent rate of photosynthesis. A maximum accumulation of NH₃ was elicited by 2.5 millimolar MSX and was essentially independent of the addition of NO₃⁻ during either the growth or experimental period. When O₂ levels in the air were reduced to 2%, MSX resulted in some accumulation of NH₃ (6.0 micromoles per gram fresh weight per hour). At these levels of NH₃, there was no significant inhibition of rates of CO₂ fixation. There was also a minor, but significant, accumulation of NH₃ in corn roots treated with MSX. Inhibitors of photorespiration (isonicotinic hydrazide, 70 millimolar; 2-pyridylhydroxymethanesulfonic acid, 20 millimolar) or transaminase reactions (aminooxyacetate, 1 millimolar) inhibited the accumulation of NH₃ in both C₃ and C₄ leaves. These results support the hypothesis that GS is important in the assimilation of NH₃ in leaves and that the glycine-serine conversion is a major source of that NH₃.     

The Second Sodium Site in the Dopamine Transporter Controls Cation Permeation and Is Regulated by Chloride

The dopamine transporter (DAT) belongs to the family of neurotransmitter:sodium symporters and controls dopamine (DA) homeostasis by mediating Na⁺- and Cl⁻-dependent reuptake of DA. Here we used two-electrode voltage clamp measurements in Xenopus oocytes together with targeted mutagenesis to investigate the mechanistic relationship between DAT ion binding sites and transporter conductances. In Li⁺, DAT displayed a cocaine-sensitive cation leak current ∼10-fold larger than the substrate-induced current in Na⁺. Mutation of Na⁺ coordinating residues in the first (Na1) and second (Na2) binding sites suggested that the Li⁺ leak depends on Li⁺ interaction with Na2 rather than Na1. DA caused a marked inhibition of the Li⁺ leak, consistent with the ability of the substrate to interact with the Li⁺-occupied state of the transporter. The leak current in Li⁺ was also potently inhibited by low millimolar concentrations of Na⁺, which according to our mutational data conceivably depended on high affinity binding to Na1. The Li⁺ leak was further regulated by Cl⁻ that most likely increases Li⁺ permeation by allosterically lowering Na2 affinity. Interestingly, mutational lowering of Na2 affinity by substituting Asp-420 with asparagine dramatically increased cation permeability in Na⁺ to a level higher than seen in Li⁺. In addition to reveal a functional link between the bound Cl⁻ and the cation bound in the Na2 site, the data support a key role of Na2 in determining cation permeability of the transporter and thereby possibly in regulating the opening probability of the inner gate.     

Influence of Protein Synthesis on NO(3) Reduction, NH(4) Accumulation, and Amide Synthesis in Suspension Cultures of Paul's Scarlet Rose

Changes in the concentrations of NH₄⁺ and amides during the growth of suspension cultures of rose (Rosa cv. Paul's Scarlet) cells were examined. When cells were grown in medium possessing only NO₃⁻ as a nitrogen source, the concentrations of NH₄⁺ and amides increased to 4.0 × 10⁻¹ and 5.9 micromoles per gram fresh weight, respectively. The amounts of both constituents declined during the later stages of growth. When a trace amount of NH₄⁺ was added to the NO₃⁻ base starting medium, the concentration of NH₄⁺ in the cells was increased to 7.0 × 10⁻¹ micromoles per gram fresh weight.     

Characterization of Growth, Water Relations, and Proline Accumulation in Sodium Sulfate Tolerant Callus of Brassica napus L. cv Westar (Canola)

Unselected and sodium sulfate tolerant callus cultures of Brassica napus L. cv Westar were grown on media supplemented with mannitol, NaCl, or Na₂SO₄. In all cases, growth of tolerant callus, measured on a fresh weight or dry weight basis, was greater than that of unselected callus, which was also subject to necrosis on high levels of salt. Tissue water potential became more negative in both unselected and tolerant callus grown in the presence of mannitol or Na₂SO₄. Water potentials in unselected callus were more negative than those of the tolerant tissues; but over a range of Na₂SO₄ concentrations both cultures displayed osmotic adjustment, maintaining relatively constant turgor. Proline accumulation in both unselected and tolerant callus was low (15 to 20 micromoles per gram dry weight) in the absence of stress, but increased on media supplemented with mannitol, NaCl, or Na₂SO₄. Increases in proline concentration were approximately linear in tolerant callus, reaching a maximum of 130 to 175 micromoles per gram dry weight. In unselected callus, concentrations were higher, reaching 390 to 520 micromoles per gram dry weight. Proline accumulation was correlated with inhibition of growth, and there was a negative correlation between proline concentration and culture age for tolerant callus.     

Ureide metabolism in leaves of nitrogen-fixing soybean plants

In leaf pieces from nodulated soybean (Glycine max [L] Merr cv Maple Arrow) plants, [¹⁴C]urea-dependent NH₃ and ¹⁴CO₂ production in the dark showed an approximately 2:1 stoichiometry and was decreased to less than 11% of the control (12-19 micromoles NH₃ per gram fresh weight per hour) in the presence of 50 millimolar acetohydroxamate, a urease inhibitor. NH₃ and CO₂ production from the utilization of [2-¹⁴C] allantoin also exhibited a 2:1 stoichiometry and was reduced to a similar extent by the presence of acetohydroxamate with a concomitant accumulation of urea which entirely accounted for the loss in NH₃ production. The almost complete sensitivity of NH₃ and CO₂ production from allantoin and urea metabolism to acetohydroxamate, together with the observed stoichiometry, indicated a path of ureide assimilation (2.0 micromoles per gram leaf fresh weight per hour) via allantoate, ureidoglycolate, and glyoxylate with the production of two urea molecules yielding, in turn, four molecules of NH₃ and two molecules of CO₂.     

Proline is not the primary determinant of chilling tolerance induced by mannitol or abscisic Acid in regenerable maize callus cultures

Chilling sensitive regenerable maize (Zea mays L.) callus cultures can be induced to survive prolonged exposure to 4°C by treatments with mannitol, abscisic acid (ABA), and/or high levels of proline. Maize callus with a free proline content of about 122 micromoles/grain fresh weight survived longer exposures to 4°C than did callus with a free proline content of about 68 micromoles/grain fresh weight. The addition of 0.53 molar mannitol or 0.1 millimolar ABA to culture medium produced a free proline content in maize callus of about 136 and 145 micromoles/grain fresh weight, respectively, if the medium contained 12 millimolar proline or about 36 and 1 micromoles/grain fresh weight, respectively, if no proline was in the medium. Although these mannitol and ABA treatments produced drastically different free proline levels in maize callus, callus grown on these media survived longer exposures to 4°C than did maize callus grown on any proline treatment alone. Thus, the internal free proline level of treated callus is not the primary factor conferring chilling tolerance on these tissues.     

Pathways of Nitrogen Assimilation in Cowpea Nodules Studied using N(2) and Allopurinol

In the presence of 0.5 millimolar allopurinol (4-hydroxypyrazolo [3,4-d]pyrimidine), an inhibitor of NAD:xanthine oxidoreductase (EC 1.2.3.2), intact attached nodules of cowpea (Vigna unguiculata L. Walp. cv Vita 3) formed [¹⁵N]xanthine from ¹⁵N₂ at rates equivalent to those of ureide synthesis, confirming the direct assimilation of fixed nitrogen into purines. Xanthine accumulated in nodules and was exported in increasing amounts in xylem of allopurinol-treated plants. Other intermediates of purine oxidation, de novo purine synthesis, and ammonia assimilation did not increase and, over the time course of experiments (4 hours), allopurinol had no effect on nitrogenase (EC 1.7.99.2) activity. Negligible ¹⁵N-labeling of asparagine from ¹⁵N₂ was observed, suggesting that the significant pool (up to 14 micromoles per gram of nodule fresh weight) of this amide in cowpea nodules was not formed directly from fixation but may have accumulated as a consequence of phloem delivery.     

Physiological control of arginine decarboxylase activity in k-deficient oat shoots

The effect of K-deficiency on the putrescine biosynthetic enzyme, arginine decarboxylase (ADC), was investigated by growing oat (Avena sativa L. var Victory) plants on a low-K, but otherwise complete nutrient medium in washed quartz sand for up to 18 days. Enzyme activity rose as the concentration of KCl was dropped to 0.6 millimolar or below. However, growth was not inhibited significantly at 0.6 millimolar KCl. ADC activity increased in the whole shoot of K-deficient oats throughout the period of 6 to 18 days, but remained constant in normal plants. At 18 days, ADC activity in entire K-deficient shoots was 6 times greater than in normal shoots, while in the first (oldest) leaf, ADC specific activity increased to more than 30 times the specific activity in the first leaf of normal plants. This effect was due to a moderate rise in total ADC activity in the first leaf between 6 and 18 days, accompanied by a significant decline in protein content. Replacing K⁺ with Na⁺ or Li⁺ significantly inhibited the increase in ADC activity in K-deficient oats, while Rb⁺ depressed the specific activity to a level below that in normal plants. An alternative putrescine biosynthetic enzyme, ornithine decarboxylase, was also examined. The specific activity of a pelletable form of the enzyme was increased 2-fold in the shoots of K-deficient oats.     

Leaf k interaction with water stress inhibition of nonstomatal-controlled photosynthesis

The relationship between leaf K⁺ concentration, in vitro dehydration, and nonstomatal-controlled photosynthesis was investigated using leaf slices that were vacuum infiltrated with media containing varying sorbitol concentrations. The leaf slices were from plants either supplied with complete or K⁺-deficient medium throughout a 35-day growth period. During this time, leaf K⁺ concentration, water potential, osmotic potential, and turgor pressure were monitored. Leaf K⁺ concentration averaged 239 micomoles per gram (fresh weight) in control plants, and dropped to 74.3 micromoles per gram (fresh weight) in K⁺-deficient plants. Less negative osmotic potentials and resultant turgor loss in K⁺-deficient plants indicated that the osmotically active pool of cellular K⁺ was lower in those plants.

The decrease in leaf K⁺ concentration enhanced the dehydration inhibition of photosynthesis. For example, increasing sorbitol from 0.33 to 0.5 molar during incubation inhibited photosynthesis in the controls by 14% or less. This same protocol resulted in an inhibition of photosynthesis by as much as 41% in K⁺-deficient tissue. In contrast to the data obtained with leaf slices, dehydration inhibition of isolated chloroplast photosynthesis was not affected by K⁺ status of parent plant material. These data are consistent with the hypothesis that one effect of leaf K⁺ deficiencies on photosynthetic response to dehydration may be mediated by extra-choloroplastic factors.

Ammonium ions, which facilitate stromal alkalinization, reversed the increased sensitivity of K⁺-deficient leaf slice photosynthesis to cell dehydration. However, NH₄⁺ had no effect on photosynthesis of K⁺-deficient leaf slices under nonhypertonic conditions. These data suggest that endogenous extra-chloroplastic K⁺ may modulate dehydration inhibition of photosynthesis, possibly by facilitating stromal alkalinization.

     

Solute Accumulation in Tobacco Cells Adapted to NaCl

Cells of Nicotiana tabacum L. var Wisconsin 38 adapted to NaCl (up to 428 millimolar) which have undergone extensive osmotic adjustment accumulated Na⁺ and Cl⁻ as principal solutes for this adjustment. Although the intracellular concentrations of Na⁺ and Cl⁻ correlated well with the level of adaptation, these ions apparently did not contribute to the osmotic adjustment which occurred during a culture growth cycle, because the concentrations of Na⁺ and Cl⁻ did not increase during the period of most active osmotic adjustment. The average intracellular concentrations of soluble sugars and total free amino acids increased as a function of the level of adaptation; however, the levels of these solutes did not approach those observed for Na⁺ and Cl⁻. The concentration of proline was positively correlated with cell osmotic potential, accumulating to an average concentration of 129 millimolar in cells adapted to 428 millimolar NaCl and representing about 80% of the total free amino acid pool as compared to an average of 0.29 millimolar and about 4% of the pool in unadapted cells. These results indicate that although Na⁺ and Cl⁻ are principal components of osmotic adjustment, organic solutes also may make significant contributions.