Changes in water relation parameters under osmotic and salt stresses in maize and sorghum

A relatively drought tolerant cultivar of maize ( Zea mays L. cv. Pioneer 3950) and a drought tolerant line of sorghum ( Sorghum bicolor [L.] Moench cv. ICSV 112) were grown hydroponically for 11 days. Treatments for non-ionic osmotic and salt stresses were started at the 8th day by addition of polyethylene glycol 6000 and NaCl, respectively, at 200 mOsm equivalent concentrations in the presence or absence of 0. 1 μ M abscisic acid. Relative growth rate was depressed by both stress factors, more severely for maize than sorghum. Abscisic acid increased the growth rate and reverted the negative effect of NaCl in maize, while sorghum was only slightly affected. In general, sorghum had higher levels of K⁺ and lower levels of Na⁺ and the K⁺/Na⁺ ratio was further increased by abscisic acid treatment. From the pressure-volume curves, osmotic potential, the water potential at turgor loss point, bulk elastic modulus and the water saturation deficit at initial turgor loss were estimated. Most significantly, sorghum had a higher elastic modulus than maize and it decreased under osmotic treatment, while in maize it increased under NaCl stress. The results suggest that bulk tissue turgor was not limiting growth under these conditions and underscores the possible implications of changes in the elastic condition of the cell walls in stress responses.     

Osmoadaptation of Thiocapsa roseopersicina OP-1 in batch and continuous culture: Accumulation of K+ and sucrose in response to osmotic stress

Abstract Increasing growth medium NaCl concentration inhibited the growth of Thiocapsa roseopersicina OP-1 due to both an increase in the lag phase of the growth cycle and a reduction in specific growth rate. Addition of 0.05% w/v acetate to the growth medium stimulated growth at all NaCl concentrations, but this stimulation was greatest at supra-optimal NaCl concentrations. Optimal growth under all conditions tested in both batch and continuous culture was recorded at a salt concentration of 0.3 M NaCl. The intracellular concentrations of both K⁺ and sucrose increased linearly with increasing growth medium NaCl concentration indicating as osmoregulatory role for these solutes. Time courses of osmoadaptation in batch culture demonstrated a biphasic response to osmotic stress. The initial phase consisted of a rapid accumulation (within 30 min) of K⁺ from the growth medium. This was followed by a slower synthesis of sucrose which partially replaced intracellular K⁺ during the second phase of osmoadaptation.     

Osmotic adjustment and requirement for sodium in marine protist thraustochytrid

A non-invasive ion-selective microelectrode technique was used to elucidate the ionic mechanisms of osmotic adjustment in a marine protist thraustochytrid. Hypoosmotic stress caused significant efflux of Na⁺, Cl^– and K⁺ from thraustochytrid cells. Model calculations showed that almost complete osmotic adjustment was achieved within the first 30 min after stress onset. Of these, sodium was the major contributor (more than half of the total osmotic adjustment), with chloride being the second major contributor. The role of K⁺ in the process of osmotic adjustment was relatively small. Changes in Ca²⁺ and H⁺ flux were attributed to intracellular signalling. Ion flux data were confirmed by growth experiments. Thraustochytrium cells showed normal growth patterns even when grown in a sodium-free solution provided the medium osmolality was adjusted by mannitol to one of the seawater. That suggests that the requirement of sodium for thraustochytrid growth cycle is due to its role in cell osmotic adjustment rather than because of the direct Na⁺ involvement in cell metabolism. Altogether, these data demonstrate the evidence for turgor regulation in thraustochytrids and suggest that these cells may be grown in the absence of sodium providing that cell turgor is adjusted by some other means.     

Salinity response of a freshwater charophyte, Chara vulgaris

Abstract. Chara vulgaris L. growing in an oligohaline lake was adapted to laboratory conditions and subjected to long-term salinity treatments ranging from 0 to 350 mol m ³ NaCl added to the lake water (40–680 mosmol kg ¹). Osmotic potential and concentration of the main osmotically active solutes (K⁺, Na⁺, Mg²⁺, Cl and sucrose) in the vacuolar sap of the central internodal cells were estimated. C. vulgaris did regulate turgor but incompletely. Turgor decreased from 335 mosmol kg ¹ under control conditions to 52–111 mosmol kg ¹ at 350 mol m ³ NaCl. The enhancement of πⁱ was achieved by increase in both ions and sucrose. Sterile and fertile plants differed in their response to osmotic stress. In sterile plants, the ions accounted for about 87% of the vacuolar osmotic potential. The increase of πⁱ under osmotic stress was exclusively due to an accumulation of Na⁺ and Cl^-. In fertile plants, sucrose accounted for about 35% of πⁱ and ions for about 51% Under osmotic stress, sucrose content increased together with the ionic content of Na⁺ and Cl^-.     

Potassium dependence and phytoplankton ecology: an experimental study

SUMMARY 1. Unialgal cultures of three species common in the freshwater phytoplankton were used to test limitation of specific growth rate and final yield in defined media of low K⁺ concentration (range <0.3–6 μmol L⁻¹ or mmol m⁻³).
2. Growth rate of the diatom Asterionella formosa was independent of K⁺ concentration above 0.7 μmol L⁻¹. Final yield was dependent on initial concentration when accompanied by K⁺ depletion below this concentration, but not by lesser depletion with more residual K⁺. Analyses of particulate K in the biomass indicated a mean final cell content of 2.8 μmol K 10⁻⁸ cells, approximately 1.0% of the organic dry weight.
3. Less detailed work with the diatom Diatoma elongatum showed no dependence of growth rate or final yield upon the initial K⁺ concentration in the range 0.8–3.2 μmol L⁻¹. The phytoflagellate Plagioselmis nannoplanctica suffered net mortality in the lowest concentration tested, 0.8 μmol L⁻¹.
4. Comparison with the range of K⁺ concentration in natural fresh waters, including a depletion induced by an aquatic macrophyte, suggests that K⁺ is unlikely to limit growth of phytoplankton. Nevertheless, there can be correlation of K⁺ with lake trophy.     

Screening plants for salt tolerance by measuring K+ flux: a case study for barley

Development of salt-tolerant genotypes is central both to remediation of salinity-affected land and to meet increasing global food demand, which has been driving expansion of cropping into marginal areas. The bottleneck of any breeding programme is the lack of a reliable screening technique. This study tested the hypothesis that the ability of plants to retain K⁺ under saline conditions is central to their salt tolerance. Using seven barley cultivars contrasting in salt tolerance (CM72, Numar, ZUG293, ZUG95, Franklin, Gairdner, ZUG403), a comprehensive study was undertaken of whole-plant (growth rate, biomass, net CO₂ assimilation, chlorophyll fluorescence, root and leaf elemental and water content) and cellular (net fluxes of H⁺, K⁺, Na⁺ and Ca²⁺) responses to various concentrations of NaCl (20–320 m m ). Na⁺ selective microelectrodes were found to be unsuitable for screening purposes because of non-ideal selectivity of the commercially available Na⁺ LIX. At the same time, our results show very strong negative correlation between the magnitude of K⁺ efflux from the root and salt tolerance of a particular cultivar. K⁺ efflux from the mature root zone of intact 3-day-old seedlings following 40 min pretreatment with 80 m m NaCl was found to be a reliable screening indicator for salinity tolerance in barley. As a faster and more cost-effective alternative to microelectrode measurements, a procedure was developed enabling rapid screening of large numbers of seedlings, based on amount of K⁺ leaked from plant roots after exposure to NaCl.     

Water efflux from the surface of field-grown grass roots. Observations by cryo-scanning electron microscopy

The objective of this study was to compare whole plant growth and physiological responses to salt stress of two Acacia nilotica subspecies (ssp. cupressiformis and ssp. tomentosa ). Salt stress was induced by adding NaCl at different concentrations to the nutrient solution: 0, 75, 100 and 200 m M . After one month under such stress, plants were still healthy and actively growing in both subspecies up to 100 m M NaCl. Water potential (Ψ) and osmotic potential (π) decreased with salinity and the lower π enabled the plants to maintain turgor. Höfler diagrams confirmed that osmotic adjustment had occurred under all treatments. Furthermore, the point of zero turgor occurred at a higher relative water content. An increase in the elastic modulus (ɛ) was observed under stress (low elasticity of the cell wall). Both osmotic adjustment and a high ɛ modified the capacity of both subspecies to maintain a positive water balance. Accumulation of ions (Na⁺, K⁺ and Cl⁻) and proline could explain such osmotic adjustment. Acacia nilotica ssp. cupressiformis showed a higher absorption of K⁺ than ssp. tomentosa up to 100 m M NaCl treatment.     

Outward-rectifying K+ channel activities regulate cell elongation and cell division of tobacco BY-2 cells

Potassium ions (K⁺) are required for plant growth and development, including cell division and cell elongation/expansion, which are mediated by the K⁺ transport system. In this study, we investigated the role of K⁺ in cell division using tobacco BY-2 protoplast cultures. Gene expression analysis revealed induction of the Shaker -like outward K⁺ channel gene, NTORK1 , under cell-division conditions, whereas the inward K⁺ channel genes NKT1 and NtKC1 were induced under both cell-elongation and cell-division conditions. Repression of NTORK1 gene expression by expression of its antisense construct repressed cell division but accelerated cell elongation even under conditions promoting cell division. A decrease in the K⁺ content of cells and cellular osmotic pressure in dividing cells suggested that an increase in cell osmotic pressure by K⁺ uptake is not required for cell division. In contrast, K⁺ depletion, which reduced cell-division activity, decreased cytoplasmic pH as monitored using a fluorescent pH indicator, SNARF-1. Application of K⁺ or the cytoplasmic alkalizing reagent (NH₄)₂SO₄ increased cytoplasmic pH and suppressed the reduction in cell-division activity. These results suggest that the K⁺ taken up into cells is used to regulate cytoplasmic pH during cell division.     

Physiological effects of Na2SO4 and NaCl on callus cultures of Brassica campestris (Chinese cabbage)

Hypocotyl-derived callus cultures of Brassica campestris L. ssp. pekinensis cv. Kim-jung (Chinese cabbage) were grown on Murashige and Skoog medium containing no additional salt, NaCl or Na₂SO₄. Na₂SO₄ was more than twice as inhibitory in comparison to the same concentration of NaCl when growth and fresh:dry weight ratios of established callus were measured. Levels of protein, starch, sucrose and α-amino nitrogen were not significantly altered in salt-grown callus. Concentrations of reducing sugars and chlorophyll were 2–3 times greater in callus grown on either salt. Proline concentration increased 15–20 fold on the highest levels of salt. Final concentrations (reached in 20–24 days) were closely correlated to the initial Na⁺ concentration of the medium, regardless of salt type. The osmotic potential in callus transferred to NaCl or Na₂SO₄ reached a maximum negative value after 16 days. For both salts, subsequent increases were correlated to increases in fresh:dry weight and growth. On both salts, turgor remained relatively constant (0. 6–0.75 MPa). Changes in Na⁺, K⁺, Mg²⁺ and Ca²⁺ content were correlated to initial Na⁺ concentration in the medium, not salt type. Accumulation of Na⁺ was accompanied by loss of K⁺ and Mg²⁺. Six to seven times less sulfate was measured in callus grown on Na₂SO₄ than chloride in callus grown on similar concentrations of NaCl.     

Puccinellia tenuiflora maintains a low Na+ level under salinity by limiting unidirectional Na+ influx resulting in a high selectivity for K+ over Na+

Puccinellia tenuiflora is a useful monocotyledonous halophyte that might be used for improving salt tolerance of cereals. This current work has shown that P. tenuiflora has stronger selectivity for K⁺ over Na⁺ allowing it to maintain significantly lower tissue Na⁺ and higher K⁺ concentration than that of wheat under short- or long-term NaCl treatments. To assess the relative contribution of Na⁺ efflux and influx to net Na⁺ accumulation, unidirectional ²²Na⁺ fluxes in roots were carried out. It was firstly found that unidirectional ²²Na⁺ influx into root of P. tenuiflora was significantly lower (by 31–37%) than in wheat under 100 and 150 m m NaCl. P. tenuiflora had lower unidirectional Na⁺ efflux than wheat; the ratio of efflux to influx was similar between the two species. Leaf secretion of P. tenuiflora was also estimated, and found the loss of Na⁺ content from leaves to account for only 0.0006% of the whole plant Na⁺ content over 33 d of NaCl treatments. Therefore, it is proposed that neither unidirectional Na⁺ efflux of roots nor salt secretion by leaves, but restricting unidirectional Na⁺ influx into roots with a strong selectivity for K⁺ over Na⁺ seems likely to contribute to the salt tolerance of P. tenuiflora .     

Kinetics of net H+, Ca2+, K+, Na+, NH+4, and Cl- fluxes associated with post-chilling recovery of plasma membrane transporters in Zea mays leaf and root tissues

Although temperature-induced changes in membrane structure and activity seem to be central to chilling stress perception, the specific details of temperature effects on plant nutrient acquisition remain obscure. In this study, we have undertaken a comparative study of low temperature effects on the activity of plasma membrane transporters of different ions in corn ( Zea mays L.) leaf and root tissues by non-invasive measurements of net ion fluxes using ion-selective microelectrode (the MIFE) technique. Kinetics of net H⁺, Ca²⁺, K⁺, Na⁺,     and Cl^– fluxes were measured as plant tissues recovered after short-term (3 h) chilling stress. The major findings can be summarized as follows: (1) The critical temperatures, under which the recovery of the activity of plasma membrane transporters took place, were found to be the same for all ions measured and are likely to be associated with the phase transition of membrane lipids. (2) The most pronounced was the reduction in net uptake of K⁺ and     (3) Chilling treatment caused a significant net influx of Cl^– and Na⁺ in the leaf tissue. (4) For the same species, the critical temperatures for membrane-transport processes in roots were 2–2.5°C lower than in leaves. Possible physiological significance of these findings is discussed.     

Electrical signalling and cytokinins mediate effects of light and root cutting on ion uptake in intact plants

Nutrient acquisition in the mature root zone is under systemic control by the shoot and the root tip. In maize, exposure of the shoot to light induces short-term (within 1–2 min) effects on net K⁺ and H⁺ transport at the root surface. H⁺ efflux decreased (from −18 to −12 nmol m⁻² s⁻¹) and K⁺ uptake (∼2 nmol m⁻² s⁻¹) reverted to efflux (∼−3 nmol m⁻² s⁻¹). Xylem probing revealed that the trans-root (electrical) potential drop between xylem vessels and an external electrode responded within seconds to a stepwise increase in light intensity; xylem pressure started to decrease after a ∼3 min delay, favouring electrical as opposed to hydraulic signalling. Cutting of maize and barley roots at the base reduced H⁺ efflux and stopped K⁺ influx in low-salt medium; xylem pressure rapidly increased to atmospheric levels. With 100 m m NaCl added to the bath, the pressure jump upon cutting was more dramatic, but fluxes remained unaffected, providing further evidence against hydraulic regulation of ion uptake. Following excision of the apical part of barley roots, influx changed to large efflux (−50 nmol m⁻² s⁻¹). Kinetin (2–4  µ m ), a synthetic cytokinin, reversed this effect. Regulation of ion transport by root-tip-synthesized cytokinins is discussed.     

Synergistic activation of plasma membrane H+– ATPase in Arabidopsis thaliana cells by turgor decrease and by fusicoccin

The regulation of the H⁺-ATPase of plasma membrane is a crucial point in the integration of transport processes at this membrane. In this work the regulation of H⁺-ATPase activity induced by changes in turgor pressure was investigated and compared with the stimulating effect of fusicoccin (FC). The exposure of cultured cells of Arabidopsis thaliana L. (ecotype Landsberg 310–14-2) to media containing mannitol (0. 15 or 0. 3 M ) or polyethylene glycol 6000 (PEG) (15. 6% or 22% w/v) resulted in a decrease in the turgor pressure of the cells and in a strong stimulation of H⁺ extrusion in the incubation medium. The osmotica-induced H⁺ extrusion was (1) inhibited by the inhibitor of plasma membrane H⁺-ATPase, erythrosin B (EB), (2) dependent on the external K⁺ concentration, (3) associated with a net K⁺ influx, and (4) lead to an increase of cellular malate content. These results show that the reduction of external osmotic potential stimulates the activity of plasma membrane H⁺-ATPase
The effect of mannitol was only partially inhibited by treatments with cycloheximide (CH) and cordycepin, which block protein and mRNA synthesis, respectively. All the effects of osmotica were qualitatively and quantitatively similar to those induced by 5 μ M FC. However, when FC and mannitol (or PEG) were fed together, their effects on H⁺ extrusion appeared synergistic, irrespective of whether FC was present at suboptimal or optimal concentrations. This behaviour suggests that the modes of action of FC and of the osmotica on H⁺-ATPase activity differ at least in some step(s)     

Accumulation of carbohydrate by Escherichia coli B/r/1 during growth at low water activity

The carbohydrate content of Escherichia coli B/r/1, grown in a glucose or arabinose-limited salts medium in a chemostat, increased by a factor of 2–4 when the water activity (a_w) of the medium was reduced to 0.986 by addition of NaCl, KCl or sucrose. The biomass decreased by 30–45%. The sucrose system resulted in the lowest biomass and carbohydrate content. The monosaccharide part of the accumulated carbohydrate consisted of glucose or glucose and arabinose in the cultures fed glucose and arabinose, respectively, and accounted for 50% or more of the total carbohydrate in the NaCl and KCl systems and 16.79% in the sucrose system. In addition, the K⁺ content depended on the solute and related inversely to the monosaccharide content, being highest in the sucrose system. The combined molarity of the monosaccharide and K⁺ was deduced to be far in excess of that required for osmotic equilibration of the cultures, especially in the sucrose system. These observations are discussed in the context of osmoregulation, the effects of solutes on glucose metabolism and the morphological changes that occur in cultures at low a_w.     

Salt tolerance of the reed plant Phragmites communis

Reed plants ( Phragmites communis Trinius) were grown at NaCl concentrations up to 500 m M and their growth, mineral contents and leaf blade osmotic potential were determined. Addition of NaCl up to 300 m M did not affect growth significantly. Sucrose, Cl^-and Na⁺ concentrations in the shoots increased with the salinity of the medium and the shoot water content decreased. K⁺ always contributed most to the leaf osmotic potential. Even in the presence of 250 m M NaCl in the rooting medium, the leaf blade contained only 50 mM Na⁺, suggesting that the plants have an efficient mechanism for Na⁺ exclusion. ²²Na⁺ uptake experiments suggested that the retranslo-cation of absorbed Na⁺ from shoots to the rooting medium lowered the uptake of Na⁺.     

Multiphasic osmotic adjustment in a euryhaline cyanobacterium

Abstract Transfer of Synechocystis PCC6714 from a freshwater medium to a saline medium caused the cells to shrink; rapid entry of NaCl resulted in a partial recovery of cellular volume within 2 min. Active extrusion of internal Na⁺ in exchange for extracellular K⁺ then occurred (within 20 min). Finally, the low- M _r carbohydrates sucrose and glucosylglycerol were accumulated and internal KC1 levels declined. In long-term growth experiments, the relative importance of sucrose as a component of the low- M _r organic solute fraction decreased and glucosylglycerol became the single most important intracellular solute. These observations demonstrate that several inorganic and organic solutes are involved in osmotic adjustment in this cyanobacterium, with sequential changes in the relative importance of each solute following transfer to a saline medium.     

Aquaporin functionality in relation to H+-ATPase activity in root cells of Capsicum annuum grown under salinity

As water and nutrient uptake should be related in the response of plants to salinity, the aim of this paper is to establish whether or not aquaporin functionality is related to H⁺-ATPase activity in root cells of pepper ( Capsicum annuum L.) plants. Thus, H⁺-ATPase activity was measured in plasma membrane vesicles isolated from roots and aquaporin functionality was measured using a cell pressure probe in intact roots. Salinity was applied as 60 m M NaCl or 60 m M KCl, to determine which ion (Na⁺, K⁺ or Cl⁻) is producing the effects. We also investigated whether the effects of both salts were ameliorated by Ca²⁺. Similar results were obtained for cell hydraulic conductivity, Lp_c, and H⁺-ATPase activity, large reductions in the presence at NaCl or KCl and an ameliorative effect of Ca²⁺. However, fusicoccin (an activator of H⁺-ATPase) did not alter osmotic water permeability of protoplasts isolated from roots. Addition of Hg²⁺ inhibited both ATPase and aquaporins, but ATPase also contains Hg-binding sites. Therefore, the results indicate that H⁺-ATPase and aquaporin activities may not be related in pepper plants.     

Changes in potassium fluxes in cells of carrot storage tissue related to turgor pressure

The effect of increasing external osmotic pressure on potassium fluxes in aged and fresh-cut discs of Daucus carota L. storage tissue was investigated. An increase of the external osmotic pressure by 5 bars of mannitol solution increased the rate of K⁺ net uptake of aged discs to 180% of their control rate. At 3°C and in 0.1 m M azide, in which a net efflux of potassium was observed, the mannitol treatment caused a reduction in the net efflux. In fresh-cut discs, in which the capability of net influx was rather low and a substantial net release of potassium was noted, the increase in the external osmotic pressure by mannitol caused a 70% inhibition in the net efflux. This effect was also observed at 3°C.
Measurements of separate fluxes confirmed the assumption that the mannitol treatment brought about two distinct effects on K⁺ fluxes: (a) raised the metabolically-dependent influx and (b) lowered the membrane permeability-dependent efflux. When a permeating solute (ethylene glycol) was used instead of mannitol, no effect on K⁺ flux was detectable. Reasons are given for relating the observed changes in K⁺ fluxes to the reduction in turgor pressure of the cells.     

Arabidopsis mutants with reduced response to NaCl and osmotic stress

We isolated 6 mutant lines of Arabidopsis thaliana that expressed reduced sensitivity to salt and osmotic stress during germination. All 6 lines cum recessive mutations in a single gene, designated reduced salt sensitivity (rss), linked to the ADH marker on chromosome 1. The rss mutants are less sensitive than wild type for NaCl and osmotic stress inhibition of germination, tolerating approximately 150 mM higher concentrations of NaCl and about 250 mM higher concentrations of sorbitol in the media. Germination assays on media containing various salts indicate that the rss mutations reduce sensitivity lo Na⁺ and Rh⁺ but also, to a much lesser degree, to K⁺ and Cs^s+. However, the rss mutation does not improve plant growth when plantlets are transferred to high salt or high osmotic pressure media after germination. The rss plantlets accumulate praline to a significantly lesser degree than wild type when they are exposed to either salt or osmotic stress. Thus, the rss mutants differ from wild type both at germination and during vegetative growth indicating that the rss mutations are pleiotropic and might affect perception of solutes or some aspect of stress-induced signaling. The rss mutations do not alter ABA sensitivity and therefore probably do not affect ABA-mediated signaling.     

Potassium Fluxes on Hyperosmotic Shock and the Effect of Phenol and Bronopol (2-bromo-2-nitropropan-1,3-diol) on Deplasmolysis of Pseudomonas aeruginosa

Potassium fluxes and the effect of phenol and bronopol on deplasmolysis of Pseudomonas aeruginosa were followed in sucrose and glycerol plasmolysing systems.
In sucrose, K⁺ uptake related to the solute concentration. Proline increased the rate and overall K⁺ uptake, the latter by a factor of three. It was concluded that there was no rigid maximum in the accumulation of intracellular K⁺ as long as intracellular neutrality in electrical charges was maintained.
In glycerol, K⁺ uptake was parallel with glycerol penetration. The process was reversed, however, on equilibration of glycerol. This suggested that glycerol inhibited K⁺ retention against a concentration gradient rather than that K⁺ was excluded as a consequence of the osmotic established steady state. This view was enforced by the fact that the reversal of K⁺ uptake occurred in 20 and 30% glycerol but not in 10%.
Phenol and bronopol did not affect deplasmolysis in glycerol significantly, although some effect on K⁺ uptake and glycerol permeability could be seen. In the sucrose system, phenol acted according to its mode of action generally accepted, i.e. inhibiting deplasmolysis at low and allowing solute penetration at higher concentrations, whereas very high concentrations caused coagulation of the cytoplasm. Bronopol inhibited deplasmolysis, except at very low concentrations. Proline did not prevent the inhibition of deplasmolysis in either of the solute systems, except at the very low bronopol concentrations where the deplasmolysis rate only was affected.