Adaptational change in proline and water content of Staphylococcus aureus after alteration of environmental salt concentration

Adaptation of Staphylococcus aureus to a change in salinity was studied by estimating the intracellular content of water and proline after alteration of the salt concentration of the culture medium. The intracellular water content of S. aureus cultured in normal broth was 1.70 g/g (dry weight). After transfer to 1.8 M NaCl-containing broth, the water content decreased to 0.80 g/g (dry weight) within 1 min. After changing the salt concentration of the medium, intracellular free proline (assumed to be one of the osmoregulators in S. aureus) increased gradually from 0 to 1,400 mumol/g (dry weight) during 30 min of incubation at 37 degrees C. The water content rose to 0.88 g/g (dry weight) in 30 min. Proline was not taken up at 0 to 4 degrees C, suggesting that the process was one of active transport. The salt tolerance of S. aureus, therefore, appears to occur initially by dehydration of the cell after transfer from a medium of low salinity to one of high salinity and then by accumulation of proline, which carries water into the cell with it.     

<i>Azospirillum brasilense</i> and <i>Saccharomyces cerevisiae</i> as Alternative for Decrease the Effect of Salinity Stress in Tomato (<i>Lycopersicon esculentum</i>) Growth

The salinity stress is one of the most relevant abiotic stresses that affects the agricultural production. The present study was performed to study the improvement of the salt tolerance of tomato plants which is known for their susceptibility to salt stress. The present study aimed to assess to what extent strain Azospirillum brasilense (N040) and Saccharomyces cerevisiae improve the salt tolerance to tomato plants treated with different salt concentration. The inoculant strain A. brasilense (N040) was previously adapted to survive up to 7% NaCl in the basal media. A greenhouse experiment was conducted to evaluate the effect of this inoculation on growth parameter such as: plant height, root length, fresh and dry weight, fruits fresh weight, chlorophyll content, proline and total soluble sugar in tomato plants under salt stress condition. The results revealed that co-inoculation of Azospirillum brasilense (N040) and Saccharomyces cerevisiae significantly increased the level of proline (8.63 mg/g FW) and total soluble sugar (120 mg/g FW) of leaves under salinity condition comparing to non-inoculated plants (2.3 mg/g FW and 70 mg/g FW, respectively). Plants co-inoculated with adapted strain of A. brasilense and S. cerevisiae showed the highest significant (p < 0.01) increase in fruit yield (1166.6 g/plant), plant high (115 cm) and roots length (52.6) compared whit un-inoculated control plants (42 g/pant, 43.3 cm and 29.6 cm, respectively). In contrast, Na⁺ ion content was significantly decreased in the leaves of salt stressed plants treated with the A. brasilense (N040) and S. cerevisiae. Finally, the results showed that dual benefits provided by both A. brasilense (N040) and S. cerevisiae can provide a major way to improve tomato yields in saline soils.     

Influence of salinity on axillary bud cultures of six lowland tropical varieties of potato (Solanum tuberosum)

Axillary bud cultures of six low-land tropical varieties of potato Solanum tuberosum (L. T. 2, 5, 6, 7, 8 and 9) were studied for their response to salinity stress using crude sea salt. Concentrations of 0.8% and above severely inhibited the growth, while decreased growth was observed between 0.4% and 0.6% salt in all the varieties, with occasional shoot tip necrosis and compound leaf formation. A lower concentration of salt (0.2%) was beneficial and increased shoot weight in most varieties. Protein content showed a positive correlation with proline levels, indicating that proline accumulation was due to new synthesis rather than breakdown of existing proteins. Soluble carbohydrates increased in the stem with increasing salt. Roots showed an increase in sodium content while shoots showed increased potassium levels, with increasing salt concentrations. There was a positive correlation between dry weight yields and K/Na ratio. It is apparent that the synthesis of proline, accumulation of soluble carbohydrates as well as increase in potassium and sodium content were all used to adjust the physiology in response to salt stress by the different varieties of potato, with varying degrees, in the present study.     

Sodium and proline accumulation in Corynebacterium glutamicum as a response to an osmotic saline upshock

In order to understand the role of the medium osmolality on the metabolism of glumate-producing Corynebacterium glutamicum, effects of saline osmotic upshocks from 0.4 osnol. kg^–1 to 2 osmol. kg^–1 have been investigated on the growth kinetics and the intracellular content of the bacteria. Addition of a high concentration of NaCl after a few hours of batch culture results in a temporary interruption of the cellular growth. Cell growth resumes after about 1 h but at a specific rate that decreases with increasing medium osmolality. Investigation of the intracellular content showed, during the first 30 min following the shock, a rapid but transient influx of sodium ions. This was followed by a strong accumulation of proline, which rose from 5 to 110 mg/g dry weight at the end of the growth phase. A slight accumulation of intracellular glutamate from 60 to 75 mg/g dry weight was also observed. Accordingly, for Corynebacterium glutamicum an increased osmolality in the glutamate and proline synthesis during the growth phase.     

Effects of salt and proline on Medicago sativa callus

In this study, two cultivars of Medicago sativa (cv. Yazdi and cv. Hamedani) were used for callus production. Calluses were transferred to MS medium containing 0, 30, 60, 90, and 120 mM NaCl and 0, 5, 10 mM proline. After 4–5 weeks dry weight and intracellular free proline of the calluses were measured. The growth of callus in both cultivars decreased with increasing salt concentration. Addition of exogenous proline to the culture medium increased the dry weight and free proline content of callus. The difference between control and treated calluses with 10 mM exogenous proline in the medium was significant. The data obtained from experiments indicated that the responses of two Medicago cultivars was genotype dependent.     

Over-expression of Osmotin Induces Proline Accumulation and Confers Tolerance to Osmotic Stress in Transgenic Tobacco

Osmotin has been implicated in conferring tolerance to drought and salt stress in plants. We have over-expressed the osmotin gene under the control of constitutive CaMV 35S promoter in transgenic tobacco, and studied involvement of the protein in imparting tolerance to salinity and drought stress. The transgenic plants exhibited retarded leaf senescence and improved germination on a medium containing 200mM NaCl. Further, the transgenics maintained higher leaf relative water content (RWC), leaf photosynthesis and free proline content than the wild type plants during water stress and after recovery from stress. When subjected to salt stress (200mM NaCl), the transgenic plants accumulated significantly more proline than the wild type plants. These results suggest the involvement of the osmotin-induced increase in proline in imparting tolerance to salinity and drought stress in transgenic plants over-expressing the osmotin gene.     

Ion accumulation in the cell walls of rice plants growing under saline conditions: evidence for the Oertli hypothesis

Abstract. When plants of rice ( Oryza saliva L.) are subjected to mildly saline (50mol m⁻³ NaCl) conditions, the leaves show symptoms of water deficit, even though ion accumulation has been more than sufficient to adjust to the decrease in external water potential. After a few days of exposure to salt, there is a negative correlation, in a population of leaves, between the leaf water concentration (g water per g dry weight) and their sodium concentration (mmol Na per g dry weight). Ion concentrations in the cell walls and the cytoplasm of cells of plants grown in low salinity were measured by X-ray microanalysis. The NaCl concentration in solution in the apoplast was calculated to be around 600mol m⁻³ in leaves of plants whose roots were exposed to only 50 mol m⁻³ NaCl. This constitutes strong evidence that an important factor in salt damage in rice is dehydration due to the extracellular accumulation of salt as suggested in the Oertli hypothesis. The implication, that changes in tissue ion concentration and solute potentials equivalent to the external medium is not evidence of plant osmotic adjustment to salinity, is discussed.     

Adaptational changes in Staphylococcus aureus MF31 grown above its maximum temperature when protected by NaCl: physiological studies

Staphylococcus aureus MF31 can grow at 46 degrees C, 2 degrees C above its normal maximum temperature of growth if 1 M NaCl is added to the medium. In the present work we show that monosodium glutamate, proline, threonine, aspartic acid, and betaine (in order of decreasing effectiveness) also enabled cells to grow at 46 degrees C. Cells grown at 46 degrees C in he presence of salt (protected or P cells) accumulated glutamate more rapidly than cells grown at 37 degrees C without salt (normal or N cells) and contained an increased amino acid pool. The principal constituents of this pool were dicarboxylic amino acids and proline. Turbidimetric evidence suggests that NaCl caused plasmolysis in S. aureus. The P cells, although grown in 1 M NaCl, had about the same Cl- and K+ content as the N cells grown without added NaCl. P cells had increased heat resistance but high concentrations of CaCl2 in the heating menstruum reduced their D55 value from a maximum of 214 min to less than 30 s. We suggest that growth at 46 degrees C in 1 M NaCl can be explained, in part at least, by the increased amino acid pool internal to the cell and the external osmotic support given by Cl- anions excluded by the cell.     

Proline transport in Staphylococcus aureus: a high-affinity system and a low-affinity system involved in osmoregulation.

L-Proline enhanced the growth of Staphylococcus aureus in high-osmotic-strength medium, i.e., it acted as an osmoprotectant. Study of the kinetics of L-[14C]proline uptake by S. aureus NCTC 8325 revealed high-affinity (Km = 1.7 microM; maximum rate of transport [Vmax] = 1.1 nmol/min/mg [dry weight]) and low-affinity (Km = 132 microM; Vmax = 22 nmol/min/mg [dry weight]) transport systems. Both systems were present in a proline prototrophic variant grown in the absence of proline, although the Vmax of the high-affinity system was three to five times higher than that of the high-affinity system in strain 8325. Both systems were dependent on Na+ for activity, and the high-affinity system was stimulated by lower concentrations of Na+ more than the low-affinity system. The proline transport activity of the low-affinity system was stimulated by increased osmotic strength. The high-affinity system was highly specific for L-proline, whereas the low-affinity system showed a broader substrate specificity. Glycine betaine did not compete with proline for uptake through either system. Inhibitor studies confirmed that proline uptake occurred via Na(+)-dependent systems and suggested the involvement of the proton motive force in creating an Na+ gradient. Hyperosmotic stress (upshock) of growing cultures led to a rapid and large uptake of L-[14C]proline that was not dependent on new protein synthesis. It is suggested that the low-affinity system is involved in adjusting to increased environmental osmolarity and that the high-affinity system may be involved in scavenging low concentrations of proline.     

Glycine betaine, carnitine, and choline enhance salinity tolerance and prevent the accumulation of sodium to a level inhibiting growth of Tetragenococcus halophila

Natural-abundance (13)C-nuclear magnetic resonance was used to probe the intracellular organic solute content of the moderately halophilic bacterium Tetragenococcus halophila. When grown in complex growth media supplemented or not with NaCl, T. halophila accumulates glycine betaine and carnitine. Unlike other moderate halophiles, T. halophila was not able to produce potent osmoprotectants (such as ectoines and glycine betaine) through de novo synthesis when cultured in defined medium under hyperosmotic constraint. Addition of 2 mM carnitine, glycine betaine, or choline to defined medium improved growth parameters, not only at high salinity (up to 2.5 M NaCl) but also in media lacking NaCl. These compounds were taken up when available in the surrounding medium. The transport activity occurred at low and high salinities and seems to be constitutive. Glycine betaine and carnitine were accumulated by T. halophila in an unmodified form, while exogenously provided choline led to an intracellular accumulation of glycine betaine. This is the first evidence of the existence of a choline-glycine betaine pathway in a lactic acid bacterium. An assay showed that the compatible solutes strikingly repressed the accumulation of glutamate and slightly increased the intracellular potassium level only at high salinity. Interestingly, osmoprotectant-treated cells were able to maintain the intracellular sodium concentration at a relatively constant level (200 to 300 nmol/mg [dry weight]), independent of the NaCl concentration of the medium. In contrast, in the absence of osmoprotectant, the intracellular sodium content increased sharply from 200 to 2,060 nmol/mg (dry weight) when the salinity of the medium was raised from 1 to 2 M. Indeed, the imported compatible solutes play an actual role in regulating the intracellular Na(+) content and confer a much higher salt tolerance to T. halophila.     

Osmotically regulated transport of proline by Lactobacillus acidophilus IFO 3532.

We reported previously that, when exposed to high osmotic pressure, Lactobacillus acidophilus IFO 3532 cells accumulated N,N,N-trimethylglycine (glycine betaine), which serves as a compatible intracellular solute. When grown in medium with high osmotic pressure, these cells also accumulated one amino acid, proline. The uptake of [3H]proline by resting, glucose-energized cells was stimulated by increasing the osmotic pressure of the assay medium with 0.5 to 1.0 M KCl, 1.0 M NaCl, or 0.5 M sucrose. The accumulated [3H]proline was not metabolized further. In contrast, there was no osmotic stimulation of [3H]leucine uptake. The uptake of proline was activated rather than induced by exposure of the cells to high osmotic pressure. Only one proline transport system could be discerned from kinetics plots. The affinity of the carrier for proline remained constant over a range of osmotic pressures from 650 to 1,910 mosM (Kt, 7.8 to 15.5 mM). The Vmax, however, increased from 15 nmol/min/mg of dry weight in 0.5 M sucrose to 27 and 40 nmol/min/mg of dry weight in 0.5 M KCl and in 1.0 M KCl or NaCl, respectively. The efflux of proline from preloaded cells occurred rapidly when the osmotic pressure of the suspending buffer was lowered.     

Osmotically regulated transport of proline by Lactobacillus acidophilus IFO 3532.

We reported previously that, when exposed to high osmotic pressure, Lactobacillus acidophilus IFO 3532 cells accumulated N,N,N-trimethylglycine (glycine betaine), which serves as a compatible intracellular solute. When grown in medium with high osmotic pressure, these cells also accumulated one amino acid, proline. The uptake of [3H]proline by resting, glucose-energized cells was stimulated by increasing the osmotic pressure of the assay medium with 0.5 to 1.0 M KCl, 1.0 M NaCl, or 0.5 M sucrose. The accumulated [3H]proline was not metabolized further. In contrast, there was no osmotic stimulation of [3H]leucine uptake. The uptake of proline was activated rather than induced by exposure of the cells to high osmotic pressure. Only one proline transport system could be discerned from kinetics plots. The affinity of the carrier for proline remained constant over a range of osmotic pressures from 650 to 1,910 mosM (Kt, 7.8 to 15.5 mM). The Vmax, however, increased from 15 nmol/min/mg of dry weight in 0.5 M sucrose to 27 and 40 nmol/min/mg of dry weight in 0.5 M KCl and in 1.0 M KCl or NaCl, respectively. The efflux of proline from preloaded cells occurred rapidly when the osmotic pressure of the suspending buffer was lowered.     

Growth of Corynebacterium glutamicum in glucose-limited continuous cultures under high osmotic pressure. Influence of growth rate on the intracellular accumulation of proline,glutamate and trehalose

In order to determine the response of Corynebacterium glutamicum to osmotic stress under different growth conditions, the bacteria were grown in glucose-limited continuous cultures at osmotic pressures of 0.4–2.4 osmol kg^–1 by addition of NaCl to the culture medium. Steady-state continuous cultures were obtained for all investigated osmotic pressures. Increasing the medium osmolality resulted in a higher specific glucose-uptake rate, a lower glucose-to-biomass conversion yield, as well as important changes in the cellular content. A short-term response to the addition of NaCl to a continuous culture was the rapid but transient uptake of Na⁺ ions. At steady state a higher osmotic pressure resulted in a strong increase of the intracellular concentrations of proline, from 5 mg/g to 125 mg/g dry weight, and of trehalose from 20 mg/g to 60 mg/g dry weight. The level of glutamate, which was the dominant intracellular amino acid at low osmotic pressure at 55 mg/g dry weight, was not affected by the addition of NaCl. The influence of the specific growth rate, between 0.1 h^–1 and 0.4 h^–1, on the intracellular metabolite concentration was also determined. The level of proline was found to increase strongly with the growth rate, whereas the trehalose content decreased slightly and the glutamate content did not change. The observed net increase in accumulated metabolites may be related to a requirement of a higher turgor pressure for rapid cell growth.     

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.     

Growth of Corynebacterium glutamicum in ammonium- and potassium-limited continuous cultures under high osmotic pressure

 In order to determine the possible effect of nutrient limitations on the response of Corynebacterium glutamicum to a saline osmotic up-shock, the bacteria were grown in continuous cultures, at osmotic pressures of 0.4 osmol/kg and 1.2 osmol/kg, under ammonia and potassium limitation. At the low osmolality of 0.4 osmol/kg, the glutamate and proline levels of 15 mg/g and 5 mg/g dry weight respectively were lower than previously reported in glucose-limited continuous cultures (50 mg/g and 10 mg/g dry weight respectively). On the other hand, the internal trehalose pool was much higher at 40 mg/g dry weight. When the medium osmolality was increased to 1.2 osmol/kg by NaCl addition, under ammonia limitation, the proline content rose from 5 mg/g to 20 mg/g dry weight and the trehalose content from 40 mg/g to 70 mg/g dry weight, whereas the intracellular pool of glutamate remained essentially constant. An increase in the internal sodium content was also observed. Similar results were found for the internal pool of glutamate, proline and trehalose when C. glutamicum was grown under potassium limitations at an osmolality of 1.2 osmol/kg. There were also higher levels of sodium ions, glutamine and alanine. According to the present results, whereas proline was previously reported to be the dominantly accumulated osmoprotectant in C. glutamicum grown under glucose limitations, under ammonia and potassium limitations trehalose represented the dominantly synthesized metabolite. Received: 19 December 1995/Received revision: 9 April 1996/Accepted: 15 April 1996     

咸水灌溉对沙土土壤盐分和胡杨生理生长的影响

通过咸水灌溉沙土土质生长的幼龄胡杨,分析了咸水灌溉沙土土壤盐分分布累积特点、盐分胁迫对胡杨的耗水生长关系、叶绿素、Pro、MDA的影响,结果表明:(1)在1.2—3 g/L范围内,微咸水灌溉沙土处于脱盐状态,6—12 g/L咸水灌溉使沙土积盐大增。在整个生长周期内,微咸水和咸水灌溉,0—200 cm内土体的总盐都呈累积趋势。(2)咸水灌溉胡杨,不同盐分处理的生长耗水关系可以用对数模型描述。(3)盐分胁迫下,胡杨叶片内叶绿素含量呈抛物线递减,Pro和MDA含量则呈现抛物线递增趋势。说明短期内咸水灌溉对土壤安全和胡杨的生长影响有限,可用咸水解决生态缺水现状,3种生理指标可用来衡量胡杨的盐胁迫程度,以此为指导提高人工造林的成活率。     

Adaptation versus shock response to polyethylene glycol-induced low water potential in cultured potato cells

We compared long-term adaptation versus short-term or shock response of potato ( Solanum tuberosum ) cells to polyethylene glycol (PEG)-induced low water potential. Potato cells, which were allowed to adapt gradually to a decreasing water potential, were able to grow actively in a medium containing 20% PEG. In contrast, no appreciable gain in dry weight was observed in potato cells shocked by abrupt transfer to the same medium. PEG-adapted cells were also salt-tolerant, as they were able to proliferate in a medium supplemented with 200 m M NaCl. No visible ultrastructural changes of mitochondria or proplastids were observed in adapted cells at values of low water potential (about −2.0 MPa), which caused membrane disruption and appearance of lipid droplets in unadapted cells. ABA cellular content increased 5-fold in PEG-shocked cells but no significant increase was found in PEG-adapted cells. The intracellular content of free proline increased 12.5 times over the basal level in PEG-adapted cells and 6.5 times in PEG-shocked cells. As shown by in vivo protein labeling, shock conditions strongly inhibited protein synthesis, which was completely recovered in PEG-adapted cells. Osmotin, a protein associated with salt adaptation in tobacco, was constitutively expressed at a high level in PEG-adapted cells and accumulated in PEG-shocked cells only three days after the transfer in a medium supplemented with 20% PEG. Proline and osmotin accumulation were coincident with the increase in cellular ABA content in PEG-shocked cells, but not in PEG-adapted cells. These data suggest that this hormone is mainly involved in shock response rather than long-term adaptation.     

The effect of salt stress and abscisic acid on proline production,chlorophyll content and growth of <Emphasis Type="Italic">in vitro</Emphasis> propagated shoots of <Emphasis Type="Italic">Eucalyptus camaldulensis</Emphasis>

Three clones, selected for their variation in salt tolerance, were examined regarding their growth and physiological responses on exposure to salt (NaCl) and abscisic acid (ABA) in vitro. The shoot proline levels significantly increased in two salt tolerant clones when exposed to 100 mM NaCl in the shoot multiplication medium. In contrast, proline in a salt sensitive clone did not change in comparison to the control treatment. When 10 M ABA was included in the medium all clones had an increase in proline regardless of whether they were salt tolerant or salt sensitive, linking proline production to the stress hormone ABA. Callus production was so variable that it was not possible to produce callus of consistent texture, colour and growth for all three clones. For the two clones where consistent growth was achievable, both the salt tolerant and salt sensitive clones increased proline production when exposed to salt. This response, however, was greater in the salt tolerant clone. Other parameters examined were growth (dry weight) and shoot chlorophyll content. These characteristics did not correlate with the salt tolerance of the clones, with similar weights being produced on non salt and salt media and similar chlorophyll in both salt sensitive and salt tolerant clones regardless of the medium in which they were grown. The production of proline is considered with regard to selection for differences in salt tolerance in vitro.     

Osmolyte accumulation in different rape genotypes under sodium chloride salinity

Physiological mechanisms of two rape (Brassica napus L.) genotype adaptation to chlorine salinity were investigated. The plants of two cultivars (Olga and Westar) differing in salt tolerance were grown in the pots filled with Perlite on the Hoagland and Snyder’s medium under controlled conditions. At a stage of 3–4 true leaves, the plants experienced 7-day-long salinity induced by a single addition of NaCl to the nutrient medium in order to attain desired final salt concentration (from 50 to 400 mM). The obtained results showed that a greater salt tolerance of cv. Olga plants (as compared with cv. Westar) could be accounted for by a capability of their root cells to uptake water under high salinity (300–400 mM NaCl), which is evident from a greater content of water in the tissues of cv. Olga. This was ensured by a sharp fall of the osmotic potential of the cellular contents (down to −2.3 MPa) at a low water potential of nutrient solution owing to more active uptake of Na⁺ (57–61 μeq/g fr wt) and K⁺ (210–270 μeq/g fr wt) as well as active accumulation of proline (30–50 μmol/g fr wt). The latter is caused by a reduced activity of proline dehydrogenase and retarded degradation of this osmolyte. It is important that, in contrast to less tolerant genotype, the rape plants of salt-resistant cultivar were able to maintain the K⁺/Na⁺ ratio at a rather high level at salinity of different degree, which made it possible to preserve ionic homeostasis under adverse conditions. Original Russian Text ? A.M. Mokhamed, G.N. Raldugina, V.P. Kholodova, Vl.V. Kuznetsov, 2006, published in Fiziologiya Rastenii, 2006, Vol. 53, No. 5, pp. 732–738.     

Proline accumulation and the adaptation of cultured plant cells to water stress

The transfer of cultured tomato cells (Lycopersicon esculentum cv VFNT-Cherry) to a low water potential environment resulted in an increased dry weight to fresh weight ratio accompanied by a rapid accumulation of proline. Proline content continued to increase as osmotic adjustment and growth occurred. The initial increase in proline concentration was accompanied by a drop in turgor. However, proline levels continued to increase with a gain in turgor during osmotic adjustment. Thus, the accumulation of proline depended not only on cell water potential, or on the initial loss of turgor but more closely on cell osmotic potential. The ultimate level of proline depended on the level of adaptation. Proline levels remained high after more than 100 cell generations in low water potential media, but declined rapidly after transfer to media with a less negative water potential. Addition of exogenous proline to the medium during water stress and during osmotic downshock alleviated the normally resulting inhibition of growth. The results suggest a positive role for proline accumulation in adaptation of cells to changing external water potentials.