Understanding Saline and Osmotic Tolerance of Populus euphratica Suspended Cells

Tolerance of Populus euphratica suspended cells to ionic and osmotic stresses implemented respectively by NaCl and PEG (6000) was characterized by monitoring cell growth, morphological features, ion compartmentation and polypeptide patterns. The cells grew and proliferated when submitted to stresses of 137 mM NaCl or 250 g l⁻¹ PEG, and survived at 308 mM of NaCl, showing tolerance to saline and particularly osmotic stress. They were resistant to plasmolysis and had dense cytoplasms, large nuclei and nucleoli, and evident cytoplasmic strands under high saline and osmotic stress. The sequestration of Cl⁻ into the vacuoles was observed in the cells stressed with 137 and 223 mM NaCl. The cellular protein profile was modified by high salt and osmotic stress and showed 28 kDa polypeptides up-regulated by both NaCl and PEG, and 66 and 25 kDa polypeptides up-regulated only by high NaCl stress. The salt tolerance of P. euphratica cells might be related to their capacity of adapting to higher osmotic stress by maintaining cell integrity, sequestrating Cl⁻ into vacuoles and modulating polypeptides that reflect cellular metabolic adaptations.     

Functional expression of Sinorhizobium meliloti BetS, a high-affinity betaine transporter, in Bradyrhizobium japonicum USDA110

Among the Rhizobiaceae, Bradyrhizobium japonicum strain USDA110 appears to be extremely salt sensitive, and the presence of glycine betaine cannot restore its growth in medium with an increased osmolarity (E. Boncompagni, M. Osteras, M. C. Poggi, and D. Le Rudulier, Appl. Environ. Microbiol. 65:2072-2077, 1999). In order to improve the salt tolerance of B. japonicum, cells were transformed with the betS gene of Sinorhizobium meliloti. This gene encodes a major glycine betaine/proline betaine transporter from the betaine choline carnitine transporter family and is required for early osmotic adjustment. Whereas betaine transport was absent in the USDA110 strain, such transformation induced glycine betaine and proline betaine uptake in an osmotically dependent manner. Salt-treated transformed cells accumulated large amounts of glycine betaine, which was not catabolized. However, the accumulation was reversed through rapid efflux during osmotic downshock. An increased tolerance of transformant cells to a moderate NaCl concentration (80 mM) was also observed in the presence of glycine betaine or proline betaine, whereas the growth of the wild-type strain was totally abolished at 80 mM NaCl. Surprisingly, the deleterious effect due to a higher salt concentration (100 mM) could not be overcome by glycine betaine, despite a significant accumulation of this compound. Cell viability was not significantly affected in the presence of 100 mM NaCl, whereas 75% cell death occurred at 150 mM NaCl. The absence of a potential gene encoding Na(+)/H(+) antiporters in B. japonicum could explain its very high Na(+) sensitivity.     

Effects of iso-osmotic NaCl and mannitol on growth, proline content, and antioxidant defense in Mammillaria gracilis Pfeiff. in vitro-grown cultures

Effects of iso-osmotic concentrations of NaCl and mannitol were studied in Mammilaria gracilis (Cactaceae) in both calli and tumors grown in vitro. In both tissues, relative growth rates were reduced under osmotic stress, which were accompanied by a decrease in both tissue water and K⁺ content. However, growth was inhibited to a lesser extent after exposure to NaCl, when accumulation of Na⁺ ions was observed. In calli, only salinity increased proline content, whereas with tumors proline accumulated after both osmotic stresses. Osmotic stresses also induced oxidative damage in both cactus tissues, although higher oxidative injury was caused by mannitol in calli and by salt in tumors. Low iso-osmotic concentrations of NaCl (75 mM) and mannitol (150 mM) increased peroxidase, ascorbate peroxidase, and esterase activities, whereas elevated catalase activity was recorded only after mannitol treatment in both tissues. High osmotic stress generally decreased enzymatic activities. However, in calli, esterase activity increased in response to high salinity, whereas ascorbate peroxidase activity was enhanced after high mannitol stress. In conclusion, both in vitro-grown cactus tissues were found to be sensitive to osmotic stress caused by either mannitol or NaCl, but accumulation of Na⁺ ions in response to salt somewhat contributed to osmotic adjustment. However, more prominent oxidative damage induced by NaCl compared to mannitol in tumor could be related to ion toxicity. The mechanisms that mediate responses to salt- and mannitol-induced osmotic stresses differed and were dependent on tissue type.     

Arabidopsis sos1 mutant in a salt-tolerant accession revealed an importance of salt acclimation ability in plant salt tolerance

An analysis of the salinity tolerance of 354 Arabidopsis thaliana accessions showed that some accessions were more tolerant to salt shock than the reference accession, Col-0, when transferred from 0 to 225 mM NaCl. In addition, several accessions, including Zu-0, showed marked acquired salt tolerance after exposure to moderate salt stress. It is likely therefore that Arabidopsis plants have at least two types of tolerance, salt shock tolerance and acquired salt tolerance. To evaluate a role of well-known salt shock tolerant gene SOS1 in acquired salt tolerance, we isolated a sos1 mutant from ion-beam-mutagenized Zu-0 seedlings. The mutant showed severe growth inhibition under salt shock stress owing to a single base deletion in the SOS1 gene and was even more salt sensitive than Col-0. Nevertheless, it was able to survive after acclimation on 100 mM NaCl for 7 d followed by 750 mM sorbitol for 20 d, whereas Col-0 became chlorotic under the same conditions. We propose that genes for salt acclimation ability are different from genes for salt shock tolerance and play an important role in the acquisition of salt or osmotic tolerance.     

Circadian stress tolerance in adult Caenorhabditis elegans

Circadian rhythms control several behaviors through neural networks, hormones and gene expression. One of these outputs in invertebrates, vertebrates and plants is the stress resistance behavior. In this work, we studied the circadian variation in abiotic stress resistance of adult C. elegans as well as the genetic mechanisms that underlie such behavior. Measuring the stress resistance by tap response behavior we found a rhythm in response to osmotic (NaCl LC(50) = 340 mM) and oxidative (H(2)O(2) LC(50) = 50 mM) shocks, with a minimum at ZT0 (i.e., lights off) and ZT12 (lights on), respectively. In addition, the expression of glutathione peroxidase (C11E4.1) and glycerol-3-phosphate dehydrogenase (gpdh-1) (genes related to the control of stress responses) also showed a circadian fluctuation in basal levels with a peak at night. Moreover, in the mutant osr-1 (AM1 strain), a negative regulator of the gpdh-1 pathway, the osmotic resistance rhythms were masked at 350 mM but reappeared when the strain was treated with a higher NaCl concentration. This work demonstrates for the first time that in the adult nematode, C. elegans stress responses vary daily, and provides evidence of an underlying rhythmic gene expression that governs these behaviors.     

Rapid determination of the damage to photosynthesis caused by salt and osmotic stresses using delayed fluorescence of chloroplasts

Chloroplasts are one of the most susceptible systems to salt and osmotic stresses. Based on quantitative measurements of delayed fluorescence (DF) of the chloroplasts, we have investigated the damage to photosynthesis caused by these two kinds of stresses in Arabidopsis seedlings by using a custom-built multi-channel biosensor. Results showed that the DF intensity and net photosynthesis rate (Pn) decreased in a similar way with increasing NaCl or sorbitol concentration. Incubation of the seedlings in 200 mM NaCl induced a rapid and reversible decline and subsequent slow and irreversible loss in both the DF intensity and Pn. The rapid decline was dominantly related to osmotic stress, whereas the slow declines in the DF intensity and Pn were specific to ionic stress and could be reversed to a similar extent by a Na+-channel blocker. The DF intensity and Pn also exhibited a similar response to irradiation light under NaCl or sorbitol stress. All results indicated that the DF intensity correlated well with Pn under salt and osmotic stresses. We thus conclude that DF is an excellent marker for detecting the damage to photosynthesis caused by these two stresses. The mechanism of the correlation between the DF intensity and Pn under salt and osmotic stresses was also analyzed in theory and investigated with experiments by measuring intercellular CO2 concetration (Ci), stomatal conductance (Gs), chlorophyll fluorescence parameter, and chlorophyll content. This proposed DF technique holds the potential to be a useful means for analyzing the dynamics of salt and osmotic stresses in vivo and elucidating the mechanism by which plants respond to stress.     

Evidence for a role of salicylic acid in the oxidative damage generated by NaCl and osmotic stress in Arabidopsis seedlings.

Previous studies have shown that salicylic acid (SA) is an essential component of the plant resistance to pathogens. We now show that SA plays a role in the plant response to adverse environmental conditions, such as salt and osmotic stresses. We have studied the responses of wild-type Arabidopsis and an SA-deficient transgenic line expressing a salicylate hydroxylase (NahG) gene to different abiotic stress conditions. Wild-type plants germinated under moderate light conditions in media supplemented with 100 mM NaCl or 270 mM mannitol showed extensive necrosis in the shoot. In contrast, NahG plants germinated under the same conditions remained green and developed true leaves. The lack of necrosis observed in NahG seedlings under the same conditions suggests that SA potentiates the generation of reactive oxygen species in photosynthetic tissues during salt and osmotic stresses. This hypothesis is supported by the following observations. First, the herbicide methyl viologen, a generator of superoxide radical during photosynthesis, produced a necrotic phenotype only in wild-type plants. Second, the presence of reactive oxygen-scavenging compounds in the germination media reversed the wild-type necrotic phenotype seen under salt and osmotic stress. Third, a greater increase in the oxidized state of the glutathione pool under NaCl stress was observed in wild-type seedlings compared with NahG seedlings. Fourth, greater oxidative damage occurred in wild-type seedlings compared with NahG seedlings under NaCl stress as measured by lipid peroxidation. Our data support a model for SA potentiating the stress response of the germinating Arabidopsis seedling.     

Cell surface characteristics of two halotolerant strains of Sinorhizobium meliloti

The halotolerant Sinorhizobium meliloti strain Rmd201 and its variant Rmd201 a were examined for their cell surface properties. The variant strain formed rough colonies and was found to be more hydrophobic. Growth of the variant strain was not affected appreciably when NaCl concentration of the medium was increased from 2 mM to 700 mM. Exopolysaccharide (EPS) and the lipopolysaccharide (LPS) content of the variant strain was found to be 7 and 14 times less, respectively, than the parent strain. However, enhanced synthesis of high molecular weight LPS bands were observed in SDS-PAGE analysis in the variant strain when the NaCl concentration was raised from 2 mM to 700 mM. Ribose and glucosamine were present in the variant LPS only. Mannose appeared as a major LPS constituent of the variant when grown in high salt containing medium. All these cell surface characteristics indicated that there were significant differences between the halotolerant strains of S. meliloti. The changes in the cell surface of the variant strain indicated the possible mutation in the gene(s) of cell surface polysaccharide biosynthesis.     

Silicon increases salt tolerance by influencing the two-phase growth response to salinity in wheat (Triticum aestivum L.)

Salt usually stresses plants in two ways, osmotic stress and ion toxicity. Plant responds to salinity in two distinct phases through time. It is known that silicon (Si) could alleviate salt stress by decreasing the Na⁺ accumulated in the leaf. In order to determine the function of Si in the two-phase growth response (osmotic and ion toxicity) to salinity, we selected the wheat cultivar “Changwu 134” out of 10 wheat cultivars, and confirmed that it responds to salinity in two distinct phases through time. The fresh weight, leaf area, and leaf Na⁺ concentration were measured during 31 days of 120 mM NaCl supplemented with 1 mM Si treatment. The results revealed that the growth of plants under salinity conditions both with and without Si application were in accordance with the two-phase growth model. Si alleviated the salt stress in the both two-phase growth, but the alleviative effects were more pronounced in the osmotic stress phase than ion toxicity phase. These results clearly showed that Si can enhance plant salt tolerance by alleviating the salt-induced osmotic stress.     

Accumulation of Amino Acids in Rhizobium sp. Strain WR1001 in Response to Sodium Chloride Salinity

Rhizobium sp. strain WR1001, isolated from the Sonoran Desert by Eskew and Ting, was found to be able to grow in defined medium containing NaCl up to 500 mM, a concentration approaching that of sea water. Therefore, it is a valuable strain for studying the biochemical basis of salt tolerance. Intracellular free glutamate was found to increase rapidly in response to osmotic stress by NaCl. It accounted for 88% of the amino acid pool when the bacterium was grown in 500 mM NaCl. The role of glutamate dehydrogenase in glutamate biosynthesis was examined in several Rhizobium strains. Both NADH- and NADPH-dependent glutamate dehydrogenase activities in various Rhizobium strains were observed. The range of activity differed considerably depending on the particular strain. KCl (500 mM) did not stimulate glutamate dehydrogenase activity, as reported in a number of bacterial strains by Measures. The low activity of glutamate dehydrogenase in Rhizobium sp. strain WR1001 apparently cannot fulfill a biosynthetic function of glutamate formation in response to medium NaCl concentrations.     

Genetic analysis of involvement of ETR1 in plant response to salt and osmotic stress

The involvement of ethylene and ethylene receptor Ethylene Response 1 (ETR1) in plant stress responses has been highlighted. However, the physiological processes involved remain unclear. In this study, we have investigated the physiological response of two alleles etr1-1 and etr1-7 mutants during germination and post-germination seedling development in response to salt and osmotic stress. The etr1-1 mutants showed increased sensitivity to osmotic (200 mM or higher mannitol) and salt stress (50 mM NaCl or higher) during germination and seedling development, whereas the etr1-7 mutants displayed enhanced tolerance to the severe stresses (500 mM mannitol or 200 mM NaCl). These results provide physiological and genetic evidence that ethylene receptor ETR1 modulates plant response to abiotic stress. Furthermore, the etr1-1 and etr1-7 mutants showed different responses to exogenous abscisic acid (ABA) inhibition. The etr1-1 mutants were more sensitive to ABA than the wild type during germination, and young seedling development. In sharp contrast, the etr1-7 mutants showed enhanced insensitivity to ABA treatment (>1 μM ABA) in post-germination development including root elongation and greening of cotyledons of the treated seedlings, although the germination was not greatly altered at the tested doses of ABA. The results suggest that ETR1-modulated stress response may mediate ABA. Youning Wang and Tao Wang contributed equally to this report.     

等渗Ca(NO3)2和NaCl胁迫对黄瓜幼苗生长及渗透调节物质含量的影响

采用营养液培养方法,以耐盐性较弱的‘津春2号’黄瓜品种为试材,研究了等渗Ca(NO3)2和NaCl胁迫对黄瓜幼苗生长、根系电解质渗透率、根系活力、Na+和K+含量及渗透调节物质含量的影响。结果显示:(1)在84mmol.L-1 NaCl和56mmol.L-1 Ca(NO3)2等渗胁迫下,黄瓜幼苗鲜重和干重均显著下降,且NaCl处理下降的幅度大于等渗Ca(NO3)2处理。(2)NaCl主要通过对黄瓜根系的伤害来抑制植株生长,表现为根系活力下降、根系质膜透性增大、Na+大量积累、K+含量显著下降、Na+/K+明显上升,最终导致根冠比下降;而Ca(NO3)2处理对根系质膜透性、K+含量、Na+/K+的影响均小于NaCl胁迫,且根系活力和根冠比上升,但Ca(NO3)2胁迫后叶片含水量和渗透调节能力均小于NaCl胁迫。(3)NaCl胁迫条件下,黄瓜幼苗内渗透调节物质以可溶性糖为主,而Ca(NO3)2胁迫以可溶性蛋白为主。研究表明,NaCl胁迫对黄瓜幼苗的伤害大于等渗Ca(NO3)2,NaCl主要通过破坏根系质膜结构影响植株生长,而Ca(NO3)2主要通过引起地上部生理干旱来影响植株生长。     

Comparison of Salt-Induced Osmotic Adjustment and Trigonelline Accumulation in Two Soybean Cultivars

21-d-old seedlings of the soybean (Glycine max) cvs. Essex and Forrest were treated with NaCl in a step-wise manner over 9 d (3 d 30 mM, 3 d 70 mM, and 3 d 100 mM) and maintained under 100 mM NaCl for an additional 14 d. During salt treatment, osmotic potential decreased more in cv. Forrest relative to cv. Essex. In non-stressed leaf tissue, cv. Forrest contained more trigonelline (TRG) relative to cv. Essex. During salt treatment, TRG amounts increased in cv. Forrest but were unchanged in cv. Essex. Both cvs. osmotically adjusted in response to salt stress; the maximal osmotic adjustment was 0.80 and 0.18 MPa in cv. Forrest and cv. Essex, respectively. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effet du Chlorure de Sodium sur la Croissance et le Potentiel Osmotique de Cals Normaux et Habitués de Betterave Sucrière

NaCl (0 to 274 mM) was added to the culture media of normal and habituated (auxin and cytokinin independent) sugarbeet calli and its effect on growth (estimated by the increase of dry and organic matters), water content and osmotic potential was tested. Growth of normal callus was stimulated by 68 mM NaCl after a lag period of two weeks. This callus tolerated up to 137 mM NaCl without growth reduction and maintained its hydric status by readjustment of its osmotic potential in 24 h. NaCl quantities under 34 mM stimulated growth of the habituated callus from the 3rd day on; higher NaCl concentrations (68 to 274 mM) inhibited growth or were lethal. NaCl sensitivity of this habituated callus was not due to its inability to adjust its osmotic potential: this adjustment occurred from the 4th h of culture whatever the media. From the 3rd day on, however, this callus presented a water deficit which depended on NaCl concentration. It is suggested that the lowering of osmotic potential corresponds to an important water loss in relation to changes in membrane permeability. This study finally shows that mechanisms of salt tolerance may have developed at the cellular level. Lower growth and lower salt tolerance of the habituated callus need further investigation in relation to cell structure and hormone autonomy.      

Short-term in vitro storage of t Miscanthus x ogiformis Honda 'Giganteus' as affected by medium composition,temperature, and photon flux density

Miscanthus x ogiformis Honda 'Giganteus' shoot cultures were stored in vitro on proliferation or rooting medium for up to 27 weeks at temperatures of 8, 12, 16, or 20 °C and photosynthetic photon flux densities of 5, 10, or 20 μmol m⁻² s⁻¹. Plants survived storage much better on rooting medium than on proliferation medium. Plants stored on rooting medium for 1 week survived well when survival was assessed immediately after storage or after 14 days of acclimatization, but had the lowest survival 28 days after transplantation. With increasing storage period on rooting medium increasing survival was found 28 days after transplantation. This was probably a result of the development of rhizomes and/or roots during storage. Best survival was observed at 20 μmol m⁻² s⁻¹ and a temperature of 8-16 °C. Increasing the temperature to 25 °C during the last week of storage improved survival considerably. Root formation was slow at 8 °C, but after 27 weeks of storage the rooting percentage was the same at all storage temperatures. An increasing number of shoots per plant 28 days after transplantation was found with increasing PPFD during storage.Miscanthus shoot cultures can be stored in vitro for at least 27 weeks with limited losses when stored on rooting medium at 20 μmol m⁻² s⁻¹, a temperature of 16 °C, and given a 1-week end-of-storage treatment of 25 °C. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.