Effects of salt stress and exogenous Ca2+ on Na+ compartmentalization,ion pump activities of tonoplast and plasma membrane in Nitraria tangutorum Bobr. leaves

Nitraria tangutorum Bobr. is a typical halophyte with superior tolerance to salinity. However, little is known about its physiological adaptation mechanisms to the salt environment. In the present study, N. tangutorum seedlings were treated with different concentrations of NaCl (100, 200, 300 and 400 mmol L^?1) combined with five levels of Ca²⁺ (0, 5, 10, 15 and 20 mmol L^?1) to investigate the effects of salt stress and exogenous Ca²⁺ on Na⁺ compartmentalization and ion pump activities of tonoplast and plasma membrane (PM) in leaves. Na⁺ and Ca²⁺ treatments increased the fresh weight and dry weight of N. tangutorum seedlings. The absorption of Na⁺ in roots, stems and leaves was substantially increased with the increases of NaCl concentration, and Na⁺ was mainly accumulated in leaves. Exogenous Ca²⁺ reduced Na⁺ accumulation in roots but promoted Na⁺ accumulation in leaves. The absorption and transportation of Ca²⁺ in N. tangutorum seedlings were inhibited under NaCl treatments. Exogenous Ca²⁺ promoted Ca²⁺ accumulation in the plant. Na⁺ contents in apoplast and symplast of leaves were also significantly increased, and symplast was the main part of Na⁺ intracellular compartmentalization. The tonoplast H⁺-ATPase and H⁺-PPase activities were significantly promoted under salt stress (NaCl concentrations ≤300 mmol L^?1). PM H⁺-ATPase activities gradually increased under salt stress (NaCl concentrations ≤200 mmol L^?1) followed by decreases with NaCl concentration increasing. The tonoplast H⁺-ATPase, H⁺-PPase and PM H⁺-ATPase activities increased first with the increasing exogenous Ca²⁺ concentration, reached the maximums at 15 mmol L^?1 Ca²⁺, and then decreased. The tonoplast and PM Ca²⁺-ATPase activities showed increasing trends with the increases of NaCl and Ca²⁺ concentration. These results suggested that certain concentrations of exogenous Ca²⁺ effectively enhanced ion pump activities of tonoplast and PM as well as promoted the intracellular Na⁺ compartmentalization to improve the salt tolerance of N. tangutorum.     

Enhanced tolerance to salinity following cellular acclimation to increasing NaCl levels in Medicago truncatula

Salinity is a major abiotic stress that limits plant productivity. Plants respond to salinity by switching on a coordinated set of physiological and molecular responses that can result in acclimation. Medicago truncatula is an important model legume species, thus understanding salt stress responses and acclimation in this species is of both fundamental and applied interest. The aim of this work was to test whether acclimation could enhance NaCl tolerance in calli of M. truncatula. A new protocol is described incorporating multi-step up acclimation over 0–350 mM exogenous NaCl. By the end of the experiment, calli were tolerant to 150 mM and competent for embryogenesis at 100 mM NaCl. Positive and negative linear relationships between Na⁺ and K⁺ uptake and exogenous NaCl concentration intercepted at 160 mM suggesting a Na⁺/K⁺ homeostasis. Proline level peaked at 100/150 mM whilst highest osmolarity and lowest water content occurred at 250/350 mM NaCl. The concentration of water soluble sugars was positively related to 0–250 mM NaCl whilst callus growth and embryogenesis occurred regardless of endoreduplication. Expression of genes linked to growth (WEE1), in vitro embryogenesis (SERK), salt tolerance (SOS1), proline synthesis (P5CS) and ploidy level (CCS52 and WEE1) peaked at 100/150 mM NaCl. Hence, these genes and various physiological traits except sugar levels, served as useful markers of NaCl tolerance. To our knowledge, this is the first report of a multi-step acclimation conferring tolerance to 150 mM NaCl in leaf-derived calli of M. truncatula.     

Salinity adaptation of plasma membrane H+-ATPase in the salt marsh plant Spartina patens: ATP hydrolysis and enzyme kinetics

Spartina patens, an intertidal C4 grass, grows in the upper salt marsh and tolerates coastal seawater salinity. The regulation of ion movement across the plasma membrane (PM) for plant salt tolerance is thought to be achieved by an electrochemical gradient generated by plasma membrane H⁺-ATPase. In this study, the change of PM H⁺-ATPase in response to NaCl was characterized for S. patens callus. Callus was cultured for 10 weeks under salinity levels of 0 mM, 170 mM, 340 mM, and 510 mM NaCl. Plasma membrane was isolated from a Dextran/PEG aqueous polymer two-phase system and the purity was demonstrated with membrane enzyme markers. There was a significant increase (up to 2-3 fold) of PM H⁺-ATPase activity when callus was grown on media containing NaCl. The incremental activation of PM H⁺-ATPase activity would enable the cell to tolerate higher cytoplasmic NaCl concentrations. PM H⁺-ATPase appeared to have a higher Vmax and a lower substrate concentration (Km to reach Vmax. When growth medium salinity increased from 0 mM to 170 and 340 mM, the Vmax of H⁺-ATPase increased from 0.64 to 1.00 and 1.73, respectively, while the Km decreased from 3.58 to 2.07 and 2.44 mM, respectively. In vitro NaCl inhibition kinetic data revealed a pattern of non-competitive inhibition by NaCl on PM H⁺-ATPase. The response of PM H⁺-ATPase in S. patens callus suggests that this species has evolved mechanisms that can regulate this important enzyme when cells are exposed to NaCl.     

Growth,photosynthesis and H+-ATPase activity in two Jerusalem artichoke varieties under NaCl-induced stress

In order to evaluate differential growth, photosynthesis and H⁺-ATPase activity responses to salt-induced stress, two Jerusalem artichoke (Helianthus tuberosus L.) genotypes (Nanyu No. 1 and Qingyu No. 2) were used in sand-culture experiment with different concentrations of NaCl (0, 30, 60, 90, 120 and 150 mM). After 20 days of growth, the NaCl stress resulted in a decrease of biomass accumulation, relative leaf expansion rate and photosynthetic rate, but an increase of proline content in both genotypes. Compared with Qingyu No. 2, Nanyu No. 1 had lower biomass, photosynthetic rate, gas exchange and transpiration rate, but higher proline content, activities of plasma membrane H⁺-ATPase (PM H⁺-ATPase) and vacuolar membrane H⁺-ATPase (VM H⁺-ATPase). Hence, the NaCl adaptation strategy in Nanyu No. 1 was by lowering photosynthetic rate, stomatal conductance and transpiration rate while maintaining high H⁺-ATPase activities, whereas the adjustment of Qingyu No. 2 was by keeping much higher rate of proline accumulation and concentration of chlorophyll. The differences in salt tolerance showed that different adaptation mechanisms existed between cultivars of Jerusalem artichoke. The findings offered the possibility of selecting salt-tolerant genotypes of Jerusalem artichoke.     

NaCl-induced changes in vacuolar H<Superscript>+</Superscript>-ATPase expression and vacuolar membrane lipid composition of two shrub willow clones differing in their response to salinity

It has been suggested that vacuolar H⁺-ATPase (V-H⁺-ATPase) plays a pivotal role in salt stress, and salt stress could modulate the expression and enzyme activity of V-H⁺-ATPase. In this work, salt modulation of V-H⁺-ATPase and tonoplast fatty acid compositions were evaluated in two shrub willow clones differing in salt tolerance after 3, 6 and 12 days of treatment. The results showed that the activity of V-H⁺-ATPase was regulated in tissue and clone specifically under NaCl stress. In the leaves of salt-tolerant clone 2345, treatment with 100 mM NaCl increased V-H⁺-ATPase activity first and then decreased it at day 12, while V-H⁺-ATPase activity was stimulated in the roots by NaCl during the treatment time. In contrast, V-H⁺-ATPase activity reached the highest value at day 3 in the leaves of salt-sensitive clone 2367 and then it decreased. Accumulation of Na⁺ in the vacuole was observed in parallel with increase in V-H⁺-ATPase activity. Western blot and immunofluorescence analysis of V-H⁺-ATPase subunit E revealed that the protein content varied in parallel with V-H⁺-ATPase activity. Moreover, a decreased unsaturated fatty acids ratio to saturated ones together with an increased V-H⁺-ATPase activity was detected in the roots of salt-tolerant clone 2345 at day 12. Altogether, it suggested that the induction of V-H⁺-ATPase expression and increase in the saturation of tonoplast fatty acids as a homeostatic mechanism for shrub willow to cope with salt stress.     

Enhanced Tonoplast H+-ATPase Activity and Superoxide Dismutase Activity in the Halophyte Suaeda salsa Containing High Level of Betacyanin

In this study, high-betacyanin Suaeda salsa seedlings were developed and used to explore whether the betacyanin accumulation is related to salinity tolerance in S. salsa. After 8 days of culture, betacyanin content decreased markedly in both high-betacyanin S. salsa seedlings and the control under nonsalt stress, but the decreases were suppressed by NaCl treatments. Betacyanin content in high-betacyanin seedlings was much higher than that in the control throughout the salt treatments. Growth of S. salsa plants was significantly promoted by NaCl treatments, and the fresh weight of high-betacyanin seedlings was much higher than that of the control when grown in 400 mmol L⁻¹ NaCl. Similar cell sap osmolarity and K⁺/Na⁺ ratios were observed in high-betacyanin seedlings and the control. No obvious differences in V-ATPase (tonoplast H⁺-ATPase) activity, leaf SOD (superoxide dismutase) activity, and total chloroplast SOD (including thylakoid-bound SOD and stroma SOD) activity were detected between high-betacyanin seedlings and the control under nonsalt stress conditions. However, V-ATPase hydrolytic activity increased dramatically in S. salsa seedlings when subjected to different levels of NaCl, and the increases in V-ATPase activity in high-betacyanin seedlings were much higher than that in the control. No clear pattern was observed for NaCl-dependent activity changes of P-ATPase (plasma membrane H⁺-ATPase) and V-PPase (tonoplast H⁺-pyrophosphatase). Similar changes were demonstrated in leaf SOD activity and chloroplast SOD activity under salt stress. Both leaf SOD activity and chloroplast SOD activity were markedly enhanced with the increase of NaCl or with time, especially thylakoid-bound SOD activity. Furthermore, the increases in chloroplast SOD activity and thylakoid-bound SOD activity were much higher in high-betacyanin seedlings than that in the control at different levels of NaCl treatment. The higher V-ATPase activity, chloroplastic SOD activity, and thylakoid-bound SOD activity demonstrated in high-betacyanin seedlings, but lower in the control, suggest that high-betacyanin S. salsa seedlings may have higher potential to be energized by the electrochemical gradient for ion uptake into the vacuole and to scavenge O₂^−• in situ produced in the chloroplasts, which may lead to higher salt tolerance than the control under salt stress. Thus, betacyanin may be involved in salt tolerance of S. salsa.     

Ethylene and nitric oxide are involved in maintaining ion homeostasis in <Emphasis Type="Italic">Arabidopsis</Emphasis> callus under salt stress

In the present study, the role of ethylene in nitric oxide (NO)-mediated protection by modulating ion homeostasis in Arabidopsis callus under salt stress was investigated. Results showed that the ethylene-insensitive mutant etr1-3 was more sensitive to salt stress than the wild type (WT). Under 100 mM NaCl, etr1-3 callus displayed a greater electrolyte leakage and Na⁺/K⁺ ratio but a lower plasma membrane (PM) H⁺-ATPase activity compared to WT callus. Application of exogenous 1-aminocyclopropane-1-carboxylic acid (ACC, an ethylene precursor) or sodium nitroprusside (SNP, a NO donor) alleviated NaCl-induced injury by maintaining a lower Na⁺/K⁺ ratio and an increased PM H⁺-ATPase activity in WT callus but not in etr1-3 callus. The SNP actions in NaCl stress were attenuated by a specific NO scavenger or an ethylene biosynthesis inhibitor in WT callus. Under 100 mM NaCl, the NO accumulation and ethylene emission appeared at early time, and NO production greatly stimulated ethylene emission in WT callus. In addition, ethylene induced the expression of PM H⁺-ATPase genes under salt stress. The recovery experiment showed that NaCl-induced injury was reversible, as signaled by the similar recovery of Na⁺/K⁺ ratio and PM H⁺-ATPase activity in WT callus. Taken together, the results indicate that ethylene and NO cooperate in stimulating PM H⁺-ATPase activity to modulate ion homeostasis for salt tolerance, and ethylene may be a part of the downstream signal molecular in NO action.     

Salinity improves chilling resistance in Suaeda salsa

Suaeda salsa L., a C3 euhalophytic herb, is native to saline soils, demonstrates high resistance to salinity stress. The effect of chilling stress on S. salsa under high salinity, particularly the change in unsaturated fatty acid content within membrane lipids, has not been investigated. After a 12 h chilling treatment (4 °C) performed under low irradiance (100 μmol m^?2 s^?1), the chlorophyll contents, maximal photochemical efficiency of photosystem II (F _v/F _m) and actual PSII efficiency (ΦPSII) were determined. These measurements were significantly decreased in S. salsa leaves in the absence of salt treatment yet there were no significant changes with a 200 mM NaCl treatment. Chlorophyll contents, F _v/F _m and ΦPSII in S. salsa under 200 mM NaCl were higher than those without salt treatment. The unsaturated fatty acid content and the double bond index (DBI) of major membrane lipids of monogalactosyldiacylglycerols, digalactosyldiacylglycerols (DGDG), sulphoquinovosyldiacylglycerols and phosphatidylglycerols (PG) significantly increased following the chilling treatment (4 °C) (with 12 h of low irradiance and 200 mM of NaCl). The DBI of DGDG and PG was decreased in the absence of the salt treatment. These results suggest that in the euhalophyte S. salsa, a 200 mM NaCl treatment increases chilling tolerance under conditions of low irradiance (100 μmol m^?2 s^?1).     

Increased cucumber salt tolerance by grafting on pumpkin rootstock and after application of calcium

Self-grafted and pumpkin rootstock-grafted cucumber plants were subjected to the following four treatments: 1) aerated nutrient solution alone (control), 2) nutrient solution with 10 mM Ca(NO₃)₂ (Ca), 3) nutrient solution with 90 mM NaCl (NaCl), and 4) nutrient solution with 90 mM NaCl + 10 mM Ca(NO₃)₂ (NaCl+Ca). The NaCl treatment decreased the plant dry mass and content of Ca²⁺ and K⁺ but increased the Na⁺ content in roots and shoots. Smaller changes were observed in pumpkin rootstock-grafted plants. Supplementary Ca(NO₃)₂ ameliorated the negative effects of NaCl on plant dry mass, relative growth rate (RGR), as well as Ca²⁺, K⁺, and Na⁺ content especially for pumpkin rootstock-grafted plants. Supplementary Ca(NO₃)₂ distinctly stimulated the plasma membrane (PM) H⁺-ATPase activity which supplies the energy to remove excess Na⁺ from the cells. The expressions of gene encoding PM H⁺-ATPases (PMA) and gene encoding a PM Na⁺/H⁺ antiporter (SOS1) were up-regulated when Ca(NO₃)₂ was applied. The pumpkin rootstock-grafted plants had higher PM H⁺-ATPase activity as well as higher PMA and SOS1 expressions than the self-grafted plants under NaCl + Ca treatment. Therefore, the addition of Ca²⁺ in combination with pumpkin rootstock grafting is a powerful way to increase cucumber salt tolerance.     

Assessment of salt tolerance of Nasturtium officinale R. Br. using physiological and biochemical parameters

Nasturtium officinale R. Br. seedlings were treated with a range of NaCl concentrations (0, 50, 100 and 150 mM) for 21 days after seedling emergence. Physiological analysis based on growth and mineral nutrition, showed a substantial decrease in leaf dry matter with 150 mM NaCl treatment. The growth decrease was correlated with nutritional imbalance and a reduction in potassium accumulation and transport to the leaves. At the same time, we noted an increase in leaf sodium and chloride accumulation and transport. Salt tolerance of N. officinale under 100 mM NaCl was associated with osmotic adjustment via Na⁺ and Cl^? and the maintenance of high K⁺/Na⁺ selectivity. Salt decreased carotenoid content more than chlorophylls and also disturbed membrane integrity by increasing malondialdehyde content and electrolyte leakage. At 150 mM NaCl, an increase in antioxidant enzyme-specific activities for superoxide dismutase, catalase and guaiacol peroxidase occurred in concert with a decrease in ascorbic acid, polyphenol, tannin and flavonoid content. These results indicate that N. officinale can maintain growth and natural antioxidant defense compounds such as, vitamin C, carotenoids, and polyphenols, when cultivated in 100 mM NaCl, but not at higher salt levels.     

The role of salinity in seed maturation of the euhalophyte Suaeda salsa

Controlled conditions were used to investigate how salinity maintains the salt tolerance of seeds and seedlings of the euhalophyte Suaeda salsa. Seeds were harvested from S. salsa plants that had been treated with 1 or 500 mM NaCl for 113 days in a glasshouse. The results showed that high salinity (500 mM NaCl) increased chlorophyll concentration and oxygen production in embryos of maturing seeds. At 500 mM NaCl, the phosphatidylglycerol and sulfoquinovosyldiacylglycerol levels and the digalactosyldiacylglycerol/monogalactosyldiacylglycerol ratio were higher in young seedlings derived from seeds whose source plants were cultured in 500 mM rather than in 1 mM NaCl. When seeds were incubated with 600 mM NaCl, the conductivity and malondialdehyde concentration in the embryos was greater if the source plants had been cultured in 1 mM rather than in 500 mM NaCl. The opposite pattern was evident for seedling survival and shoot weight. In conclusion, salinity during seed maturation may increase the salt tolerance of seeds and seedlings by increasing the oxygen production in the embryos of the maturing seeds and by changing the lipid composition of membranes in the seedlings.     

Expression of a Suaeda salsa Vacuolar H+/Ca2+ Transporter Gene in Arabidopsis Contributes to Physiological Changes in Salinity

A cDNA (SsCAX1) encoding a tonoplast-localised Ca²⁺/H⁺ exchanger was isolated from a C₃ halophyte Suaeda salsa (L.). To clarify the role of SsCAX1 in plant salt tolerance, Arabidopsis plants expressing SsCAX1 were treated with NaCl. Transgenic Arabidopsis plants displayed decreased salt tolerance. Although Na⁺ content was close to wild-type plants, transgenic plants accumulated more Ca²⁺ and retained less K⁺ in leaves than the wild-type plants in salinity. Furthermore, transgenic lines held higher leaf membrane leakage than wild-type lines under NaCl treatment. In addition, transgenic plants showed a 23% increase in vacuolar H⁺-ATPase activity compared with wild-type plants in normal condition. But the leaf V-H⁺-ATPase activity had subtle changes in transgenic plants, while significantly increased in wild-type plants under saline condition. These results suggested that regulated expression of Ca²⁺/H⁺ antiport was critical for maintenance of cation homeostasis and activity of V-H⁺-ATPase under saline condition.     

Hydrogen sulfide is involved in maintaining ion homeostasis via regulating plasma membrane Na+/H+ antiporter system in the hydrogen peroxide-dependent manner in salt-stress Arabidopsis thaliana root

Hydrogen sulfide (H₂S) and hydrogen peroxide (H₂O₂) function as the signaling molecules in plants responding to salt stresses. The present study presents a signaling network involving H₂S and H₂O₂ in salt resistance pathway of the Arabidopsis root. Arabidopsis roots were sensitive to 100 mM NaCl treatment, which displayed a great increase in electrolyte leakage (EL) and Na⁺/K⁺ ratio under salt stress. The treatment of H₂S donors sodium hydrosulfide (NaHS) enhanced the salt tolerance by maintaining a lower Na⁺/K⁺ ratio. In addition, the inhibition of root growth under salt stress was removed by H₂S. Further studies indicated that H₂O₂ was involved in H₂S-induced salt tolerance pathway. H₂S induced the production of the endogenous H₂O₂ via regulating the activities of glucose-6-phosphate dehydrogenase (G6PDH) and plasma membrane (PM) NADPH oxidase, with the treatment with dimethylthiourea (DMTU, an ROS scavenger), diphenylene iodonium (DPI, a PM NADPH oxidase inhibitor), or glycerol (G6PDH inhibitor) removing the effect of H₂S. Treatment with amiloride (an inhibitor of PM Na⁺/H⁺ antiporter) and vanadate (an inhibitor of PM H⁺-ATPase) also inhibited the activity of H₂S on Na⁺/K⁺ ratio. Through an analysis of quantitative real-time polymerase chain reaction and Western blot, we found that H₂S promoted the genes expression and the phosphorylation level of PM H⁺-ATPase and Na⁺/H⁺ antiporter protein level. However, when the endogenous H₂O₂ level was inhibited by DPI or DMTU, the effect of H₂S on the PM Na⁺/H⁺ antiporter system was removed. Taken together, H₂S maintains ion homeostasis in the H₂O₂-dependent manner in salt-stress Arabidopsis root.     

Effects of silicon on H+-ATPase and H+-PPase activity,fatty acid composition and fluidity of tonoplast vesicles from roots of salt-stressed barley (Hordeum vulgare L.)

We investigated the effects of silicon (Si) on time-dependent changes in root tonoplast H⁺-ATPase and H⁺-PPase activities, membrane fatty acid compositions and tonoplast fluidity in two barley (Hordeum vulgare L.) cultivars differing in salt tolerance. Plants were grown in NaCl-free (control) and NaCl-supplied (60 and 120 mM, respectively) nutrient solutions with or without 1.0 mM Si. Plant roots were harvested to isolate tonoplast vesicles for assay of H⁺-ATPase and H⁺-PPase activities at days 2, 4, and 6 after treatment in the first experiment and for analysis of membrane fatty acid composition and fluidity at day 4 after treatment in the second experiment. The results showed that tonoplast H⁺-ATPase and H⁺-PPase activities in roots of salt-treated plants increased at day 2, which was more obvious at 60 mM NaCl in the salt-tolerant cultivar than in the salt-sensitive cultivar, and then decreased at day 4 and onward. These enzyme activities decreased consistently from days 2 to 6 for treatment with 120 mM NaCl. However, inclusion of 1.0 mM Si significantly enhanced both H⁺-ATPase and H⁺-PPase activities in roots of salt stressed barley, which was irrespective of NaCl level or cultivar used. The ratio of unsaturated to saturated fatty acids (U/S) increased under salt stress for both cultivars. Addition of Si to salt treatment increased the ratio of U/S in salt-tolerant cultivar but it did not in salt-sensitive cultivar compared to non-Si-amended salt treatment. Salt treatment decreased tonoplast fluidity of roots of barley significantly compared with control treatment. However, root tonoplast fluidity was significantly lower in the Si-amended salt treatment than in the non-Si-amended salt treatment. These results were in line with the previous findings that Si could help increase antioxidative defense and reduce membrane lipid oxidative damage in barley under salt stress. The possible mechanisms involved in Si-enhanced salt tolerance were discussed with respect to cell membrane integrity, stability and function in barley.     

Nitrogen deprivation induces cross-tolerance of Poa annua callus to salt stress

Alternative respiration pathway (AP) is an important pathway which can be induced by environment stresses in plants. In the present study, we show a new mechanism involving the AP in nitrogen deprivation-induced tolerance of Poa annua callus to salt stress. The AP capacity markedly increased under a 600 mM NaCl treatment or nitrogen deprivation pretreatment and reached a maximum under the nitrogen deprivation pretreatment combined with the NaCl treatment (–N+NaCl). Malondialdehyde (MDA) and H₂O₂ content and Na⁺/K⁺ ratio significantly increased under the 600 mM NaCl treatment but less under the–N+NaCl treatment. Moreover, both the nitrogen deprivation and the NaCl stress stimulated the plasma membrane (PM) H⁺-ATPase activity and increased pyruvate content. The maximal stimulating effect was found under the–N+NaCl treatment. When the AP capacity was reduced by salicylhydroxamic acid (SHAM, an inhibitor of AP), content of MDA and H₂O₂ and Na⁺/K⁺ ratio dramatically increased, whereas PM H⁺-ATPase activity decreased. Moreover, exogenous application of pyruvate produced a similar effect as the nitrogen deprivation pretreatment. The effects of SHAM on the Poa annua callus were counteracted by catalase (a H₂O₂ scavenger) and diphenylene iodonium (a plasma membrane NADPH oxidase inhibitor). Taken together, our results suggest that the nitrogen deprivation enhanced the capacity of AP by increasing pyruvate content, which in turn prevented the Poa annua callus from salt-induced oxidative damages and Na⁺ over-uptake.     

Enhanced drought and salt tolerance by expression of AtGSK1 gene in poplar

A GSK3/shaggy-like kinase (AtGSK1) has been implicated in the regulation of drought and salt tolerance. We transferred AtGSK1 from Arabidopsis thaliana to a hybrid poplar (Populus alba × P. tremula var. grandulosa) to determine the effect of the transgene expression in the transgenic trees. The results from northern blot and RT-PCR analyses showed that the expression level varied among the transgenic lines. During their culture on tissue culture media, the transgenic poplars formed vigorous growing roots even in the presence of 125 mM NaCl and callus in the presence of 150 mM NaCl. When the transgenic poplars were growing in pots and provided with NaCl solution, they stayed much healthier than did nontransgenic poplars, showing higher rates of photosynthetic rates, stomatal conductance, and evaporation rates under the stress. Whereas the total level of leaf Na⁺ level increased dramatically in transgenic poplars under severe saline conditions (150 mM NaCl), that of leaf K⁺ decreased in the same plants under the same conditions. Total root Na⁺ level increased in nontransgenic poplars under severe saline conditions. In contrast, total root K⁺ level decreased in the same plants under the same conditions. The chloride content and relative electrical conductivity of the transgenic poplars after salt stress treatment were lower than those of nontransgenic poplars. The transgenic poplars were also tolerant to up to 20 % PEG remaining significantly healthy when compared with nontransgenic poplars with necrosis and chlorosis symptoms. Another dramatic feature of the transgenic poplars was wilting tolerance for prolonged drought treatment up to 2 weeks. The results provide evidence that the expression of AtGSK1 gene conferred drought and salt tolerance in the transgenic poplars.     

Physiological and proteomic analysis of salinity tolerance of the halotolerant cyanobacterium <Emphasis Type="Italic">Anabaena</Emphasis> sp

The halotolerant cyanobacterium Anabaena sp was grown under NaCl concentration of 0, 170 and 515 mM and physiological and proteomic analysis was performed. At 515 mM NaCl the cyanobacterium showed reduced photosynthetic activities and significant increase in soluble sugar content, proline and SOD activity. On the other hand Anabaena sp grown at 170 mM NaCl showed optimal growth, photosynthetic activities and comparatively low soluble sugar content, proline accumulation and SOD activity. The intracellular Na⁺ content of the cells increased both at 170 and 515 mM NaCl. In contrast, the K⁺ content of the cyanobacterium Anabaena sp remained stable in response to growth at identical concentration of NaCl. While cells grown at 170 mM NaCl showed highest intracellular K⁺/Na⁺ ratio, salinity level of 515 mM NaCl resulted in reduced ratio of K⁺/Na⁺. Proteomic analysis revealed 50 salt-responsive proteins in the cyanobacterium Anabaena sp under salt treatment compared with control. Ten protein spots were subjected to MALDI-TOF–MS/MS analysis and the identified proteins are involved in photosynthesis, protein folding, cell organization and energy metabolism. Differential expression of proteins related to photosynthesis, energy metabolism was observed in Anabaena sp grown at 170 mM NaCl. At 170 mM NaCl increased expression of photosynthesis related proteins and effective osmotic adjustment through increased antioxidant enzymes and modulation of intracellular ions contributed to better salinity tolerance and optimal growth. On the contrary, increased intracellular Na⁺ content coupled with down regulation of photosynthetic and energy related proteins resulted in reduced growth at 515 mM NaCl. Therefore reduced growth at 515 mM NaCl could be due to accumulation of Na⁺ ions and requirement to maintain higher organic osmolytes and antioxidants which is energy intensive. The results thus show that the basis of salt tolerance is different when the halotolerant cyanobacterium Anabaena sp is grown under low and high salinity levels.     

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.