Uptake of sodium in protoplasts of salt-sensitive and salt-tolerant cultivars of rice, Oryza sativa L. determined by the fluorescent dye SBFI

In this study, the uptake of Na+ into the cytosol of rice (Oryza sativa L. cvs Pokkali and BRRI Dhan29) protoplasts was measured using the acetoxy methyl ester of the fluorescent sodium-binding benzofuran isopthalate, SBFI-AM, and fluorescence microscopy. By means of inhibitor analyses the mechanisms for uptake and sequestration of Na+ in the salt-sensitive indica rice cv. BRRI Dhan29 and in the salt-tolerant indica rice cv. Pokkali were detected. Less Na+ was taken up into the cytosol of Pokkali than into BRRI Dhan29. The results indicate that K+-selective channels do not contribute to the Na+ uptake in Pokkali, whereas they are the major pathways for Na+ uptake in BRRI Dhan29 along with non-selective cation channels. However, non-selective cation channels seem to be the main pathways for Na+ uptake in Pokkali. Protoplasts from Pokkali leaves took up Na+ only transiently in the presence of extracellular Na+ at 5-100 mM. Therefore, it is likely that the protoplasts have a mechanism for fast extrusion of Na+ out of the cytoplasm. Experiments with protoplasts pretreated with NH4NO3 and NH4VO3 suggest that the salt-tolerant Pokkali extrudes Na+ mainly into the vacuole. After cultivation of both cultivars in the presence of 10 or 50 mM NaCl for 72 h, the isolated protoplasts from Pokkali took up less Na+ than the control protoplasts. The results suggest that the salt-tolerance in Pokkali depends on reduced uptake through K+-selective channels and a fast extrusion of Na+ into the vacuoles.     

Expression levels of the Na + /K + transporter OsHKT2;1 and vacuolar Na + /H + exchanger OsNHX1 , Na enrichment,maintaining the photosynthetic abilities and growth performances of indica rice seedlings under salt stress

Salt affected soil inhibits plant growth, development and productivity, especially in case of rice crop. Ion homeostasis is a candidate defense mechanism in the salt tolerant plants or halophyte species, where the salt toxic ions are stored in the vacuoles. The aim of this investigation was to determine the OsNHX1 (a vacuolar Na+/H+ exchanger) and OsHKT2;1 (Na+/K+ transporter) regulation by salt stress (200 mM NaCl) in two rice cultivars, i.e. Pokkali (salt tolerant) and IR29 (salt susceptible), the accumulation of Na+ in the root and leaf tissues using CoroNa Green® staining dye and the associated physiological changes in test plants. Na+ content was largely increased in the root tissues of rice seedlings cv. Pokkali (15 min after salt stress) due to the higher expression of OsHKT2;1 gene (by 2.5 folds) in the root tissues. The expression of OsNHX1 gene in the leaf tissues was evidently increased in salt stressed seedlings of Pokkali, whereas it was unchanged in salt stressed seedlings of IR29. Na+ in the root tissues of both Pokkali and IR29 was enriched, when subjected to 200 mM NaCl for 12 h and easily detected in the leaf tissues of salt stressed plants exposed for 24 h, especially in cv. Pokkali. Moreover, the overexpression of OsNHX1 gene regulated the translocation of Na+ from root to leaf tissues, and compartmentation of Na+ into vacuoles, thereby maintaining the photosynthetic abilities in cv. Pokkali. Overall growth performance, maximum quantum yield (Fv/Fm), photon yield of PSII (ΦPSII) and net photosynthetic rate (Pn) was improved in salt stressed leaves of Pokkali than those in salt stressed IR29.     

Sodium sensing induces different changes in free cytosolic calcium concentration and pH in salt-tolerant and -sensitive rice (Oryza sativa) cultivars

Perception of salt stress in plant cells induces a change in the free cytosolic Ca²⁺, [Ca²⁺]_cyt, which transfers downstream reactions toward salt tolerance. Changes in cytosolic H⁺ concentration, [H⁺]_cyt, are closely linked to the [Ca²⁺]_cyt dynamics under various stress signals. In this study, salt‐induced changes in [Ca²⁺]_cyt, and [H⁺]_cyt and vacuolar [H⁺] concentrations were monitored in single protoplasts of rice (Oryza sativa L. indica cvs. Pokkali and BRRI Dhan29) by fluorescence microscopy. Changes in cytosolic [Ca²⁺] and [H⁺] were detected by use of the fluorescent dyes acetoxy methyl ester of calcium‐binding benzofuran and acetoxy methyl ester of 2′, 7′‐bis‐(2‐carboxyethyl)‐5‐(and‐6) carboxyfluorescein, respectively, and for vacuolar pH, fluorescent 6‐carboxyfluorescein and confocal microscopy were used. Addition of NaCl induced a higher increase in [Ca²⁺]_cyt in the salt‐tolerant cv. Pokkali than in the salt‐sensitive cv. BRRI Dhan29. From inhibitor studies, we conclude that the internal stores appear to be the major source for [Ca²⁺]_cyt increase in Pokkali, although the apoplast is more important in BRRI Dhan29. The [Ca²⁺]_cyt measurements in rice also suggest that Na⁺ should be sensed inside the cytosol, before any increase in [Ca²⁺]_cyt occurs. Moreover, our results with individual mesophyll protoplasts suggest that ionic stress causes an increase in [Ca²⁺]_cyt and that osmotic stress sharply decreases [Ca²⁺]_cyt in rice. The [pH]_cyt was differently shifted in the two rice cultivars in response to salt stress and may be coupled to different activities of the H⁺‐ATPases. The changes in vacuolar pH were correlated with the expressional analysis of rice vacuolar H⁺‐ATPase in these two rice cultivars.     

Effects of exogenous proline and glycinebetaine on the salt tolerance of rice cultivars

Salinity significantly increased trisodium-8-hydroxy-1,3,6-pyrenetrisulphonic acid (PTS) uptake and decreased the K(+)/Na(+) ratio in salt-sensitive rice (Nipponbare) but did not markedly in salt-tolerant rice (Pokkali). Proline and glycinebetaine (betaine) suppressed the increase in PTS uptake and the decrease in the K(+)/Na(+) ratio in Nipponbare, but did not affect PTS uptake or the K(+)/Na(+) ratio in Pokkali.     

Potassium nitrate application alleviates sodium chloride stress in winter wheat cultivars differing in salt tolerance

A sand culture experiment was conducted to answer the question whether or not exogenous KNO(3) can alleviate adverse effects of salt stress in winter wheat by monitoring plant growth, K(+)/Na(+) accumulation and the activity of some antioxidant enzymes. Seeds of two wheat cultivars (CVs), DK961 (salt-tolerant) and JN17 (salt-sensitive), were planted in sandboxes and controls germinated and raised with Hoagland nutrient solution (6 mM KNO(3), no NaCl). Experimental seeds were exposed to seven modified Hoagland solutions containing increased levels of KNO(3) (11, 16, 21 mM) or 100 mM NaCl in combination with the four KNO(3) concentrations (6, 11, 16 and 21 mM). Plants were harvested 30 d after imbibition, with controls approximately 22 cm in height. Both CVs showed significant reduction in plant height, root length and dry weight of shoots and roots under KNO(3) or NaCl stress. However, the combination of increased KNO(3) and NaCl alleviated symptoms of the individual salt stresses by improving growth of shoots and roots, reducing electrolyte leakage, malondialdehyde and soluble sugar contents and enhancing the activities of antioxidant enzymes. The salt-tolerant cultivar accumulated more K(+) in both shoots and roots compared with the higher Na(+) accumulation typical for the salt-sensitive cultivar. Soluble sugar content and activities of antioxidant enzymes were found to be more stable in the salt-tolerant cultivar. Our findings suggest that the optimal K(+)/Na(+) ratio of the nutrient solution should be 16:100 for both the salt-tolerant and the salt-sensitive cultivar under the experimental conditions used, and that the alleviation of NaCl stress symptoms through simultaneously applied elevated KNO(3) was more effective in the salt-tolerant than in the salt-sensitive cultivar.     

盐胁迫下水稻叶绿体中Na+、Cl-积累导致叶片净光合速率下降

研究了0-200mmol/L的NaCl胁迫下耐盐性不同的水稻品种Pokkali(耐盐）和Peta(盐敏感）根系,叶片和叶绿体中Na^ ,K^ 和Cl^-含量的变化及其与叶片光合作用的关系。结果表明：随着NaCl胁迫时间和浓度的增加,供试2个品种在根,叶片和叶绿体中Na^ ,Cl^－含量增加,K^ 含量下降。耐盐品种体内Na^ ,Cl^－含量增加或K^ 含量减少的幅度小于盐敏感品种。在200mmol/L的NaCl胁迫下盐敏感品种根,叶片和叶绿体中的Na^ /K^ 分别是耐盐品种的208％,308％和297％。与Na^ 相比,耐盐品种根系对K^ 吸收和向叶片运输的选择性（SK,Na)较强。但在经过0,100和200mmol/L的NaCl处理后2个品种叶绿体中的Na^ /K^ 均高于叶片（SK,Na均小于1）。盐胁迫下水稻叶绿体中Na^ ,Cl^-含量和Na^ /K^ 与叶片净光合速度呈极显著负相关。     

HKT2;2/1, a K(+) -permeable transporter identified in a salt-tolerant rice cultivar through surveys of natural genetic polymorphism

We have investigated OsHKT2;1 natural variation in a collection of 49 cultivars with different levels of salt tolerance and geographical origins. The effect of identified polymorphism on OsHKT2;1 activity was analysed through heterologous expression of variants in Xenopus oocytes. OsHKT2;1 appeared to be a highly conserved protein with only five possible amino acid substitutions that have no substantial effect on functional properties. Our study, however, also identified a new HKT isoform, No-OsHKT2;2/1 in Nona Bokra, a highly salt-tolerant cultivar. No-OsHKT2;2/1 probably originated from a deletion in chromosome 6, producing a chimeric gene. Its 5' region corresponds to that of OsHKT2;2, whose full-length sequence is not present in Nipponbare but has been identified in Pokkali, a salt-tolerant rice cultivar. Its 3' region corresponds to that of OsHKT2;1. No-OsHKT2;2/1 is essentially expressed in roots and displays a significant level of expression at high Na(+) concentrations, in contrast to OsHKT2;1. Expressed in Xenopus oocytes or in Saccharomyces cerevisiae, No-OsHKT2;2/1 exhibited a strong permeability to Na(+) and K(+) , even at high external Na(+) concentrations, like OsHKT2;2, and in contrast to OsHKT2;1. Our results suggest that No-OsHKT2;2/1 can contribute to Nona Bokra salt tolerance by enabling root K(+) uptake under saline conditions.     

盐胁迫下水稻叶绿体中Na~ 、Cl~-积累导致叶片净光合速率下降

研究了 0～ 2 0 0mmol/L的NaCl胁迫下耐盐性不同的水稻品种Pokkali(耐盐 )和Peta(盐敏感 )根系、叶片和叶绿体中Na 、K 和Cl-含量的变化及其与叶片光合作用的关系。结果表明 :随着NaCl胁迫时间和浓度的增加 ,供试 2个品种在根、叶片和叶绿体中Na 、Cl-含量增加 ,K 含量下降。耐盐品种体内Na 、Cl-含量增加或K 含量减少的幅度小于盐敏感品种。在 2 0 0mmol/L的NaCl胁迫下盐敏感品种根、叶片和叶绿体中的Na /K 分别是耐盐品种的 2 0 8%、30 8%和 2 97%。与Na 相比 ,耐盐品种根系对K 的吸收和向叶片运输的选择性 (SK ,Na)较强。但在经过0、10 0和 2 0 0mmol/L的NaCl处理后 2个品种叶绿体中的Na /K 均高于叶片 (SK ,Na均小于 1)。盐胁迫下水稻叶绿体中Na 、Cl-含量和Na /K 与叶片净光合速率呈极显著负相关。     

Two types of HKT transporters with different properties of Na+ and K+ transport in Oryza sativa

It is thought that Na+ and K+ homeostasis is crucial for salt-tolerance in plants. To better understand the Na+ and K+ homeostasis in important crop rice (Oryza sativa L.), a cDNA homologous to the wheat HKT1 encoding K+-Na+ symporter was isolated from japonica rice, cv Nipponbare (Ni-OsHKT1). We also isolated two cDNAs homologous to Ni-OsHKT1 from salt-tolerant indica rice, cv Pokkali (Po-OsHKT1, Po-OsHKT2). The predicted amino acid sequence of Ni-OsHKT1 shares 100% identity with Po-OsHKT1 and 91% identity with Po-OsHKT2, and they are 66-67% identical to wheat HKT1. Low-K+ conditions (less than 3 mM) induced the expression of all three OsHKT genes in roots, but mRNA accumulation was inhibited by the presence of 30 mM Na+. We further characterized the ion-transport properties of OsHKT1 and OsHKT2 using an expression system in the heterologous cells, yeast and Xenopus oocytes. OsHKT2 was capable of completely rescuing a K+-uptake deficiency mutation in yeast, whereas OsHKT1 was not under K+-limiting conditions. When OsHKTs were expressed in Na+-sensitive yeast, OsHKT1 rendered the cells more Na+-sensitive than did OsHKT2 in high NaCl conditions. The electrophysiological experiments for OsHKT1 expressed in Xenopus oocytes revealed that external Na+, but not K+, shifted the reversal potential toward depolarization. In contrast, for OsHKT2 either Na+ or K+ in the external solution shifted the reversal potential toward depolarization under the mixed Na+ and K+ containing solutions. These results suggest that two isoforms of HKT transporters, a Na+ transporter (OsHKT1) and a Na+- and K+-coupled transporter (OsHKT2), may act harmoniously in the salt tolerant indica rice.     

Comparative lipid peroxidation,antioxidant defense systems and proline content in roots of two rice cultivars differing in salt tolerance

The changes in the activity of antioxidant enzymes such as superoxide dismutase (SOD: EC 1.15.1.1), catalase (CAT: EC 1.11.1.6), peroxidase (POX: EC 1.11.1.7), ascorbate peroxidase (APOX: EC 1.11.1.11) and glutathione reductase (GR: EC 1.6.4.2), free proline content, and the rate of lipid peroxidation level in terms of malondialdehyde (MDA) in roots of two rice cultivars (cvs.) differing in salt tolerance were investigated. Plants were subjected to three salt treatments, 0, 60, and 120 mol m⁻³ NaCl for 7 days. The results showed that activated oxygen species may play a role in cellular toxicity of NaCl and indicated differences in activation of antioxidant defense systems between the two cvs. The roots of both cultivars showed a decrease in GR activity with increase in salinity. CAT and APOX activities increased with increasing salt stress in roots of salt-tolerant cultivar Pokkali but decreased and showed no change, respectively, in roots of IR-28 cultivar. POX activity decreased with increasing NaCl concentrations in salt-tolerant Pokkali but increased in IR-28. SOD activity showed no change in roots of both cultivars under increasing salinity. MDA level in the roots increased under salt stress in sensitive IR-28 but showed no change in Pokkali. IR-28 produced higher amount of proline under salt stress than in Pokkali. Increasing NaCl concentration caused a reduction in root fresh weight of Pokkali and root dry weight of IR-28. The results indicate that improved tolerance to salt stress in root tissues of rice plants may be accomplished by increased capacity of antioxidative system.     

Isolation and characterization of plasma membrane Na(+)/H(+) antiporter genes from salt-sensitive and salt-tolerant reed plants

We isolated cDNAs for Na(+)/H(+) antiporter genes (PhaNHA1s) from salt-sensitive and salt-tolerant reed plants. A phylogenetic analysis and localization analysis using yeast strains expressing PhaNHA1-GFP protein showed that PhaNHA1s were plasma membrane Na(+)/H(+) antiporters. Yeast strains expressing PhaNHA1 from salt-tolerant reed plants (PhaNHA1-n) grew well than yeast strains expressing PhaNHA1 from salt-sensitive reed plants (PhaNHA1-u) in the presence of 100mM NaCl. Furthermore, Na(+) contents of yeast cells expressing PhaNHA1-n were less than half of those of yeast cells expressing PhaNHA1-u. These results suggest that PhaNHA1-n is more efficient at excluding Na(+) from the cells than PhaNHA1-u.     

Effects of NaCl stress on photochemical activity and thylakoid membrane polypeptide composition of a salt-tolerant and a salt-sensitive rice cultivar

NaCl stress (200 mM) inhibited the electron transport activity of photosystem 2 (PS2) more than that of PS1. The degree of electron transport activity inhibition was lower in the salt-tolerant cultivar Pokkali than in the salt-sensitive cultivar Peta. The polypeptide composition of the thylakoid membrane and PS2 particles did not change after NaCl treatment but there was a difference in polypeptide compositions of thylakoid membrane and PS2 particles between the two cultivars. PS2 particles of cv. Pokkali contained more 33-kDa and 43-kDa polypeptides than cv. Peta. Additionally, PS2 particles after NaCl treatment showed deficiency of 23-kDa outside polypeptides of PS2.     

Paxillus involutus Strains MAJ and NAU mediate K(+)/Na(+) homeostasis in ectomycorrhizal Populus x canescens under sodium chloride stress

Salt-induced fluxes of H(+), Na(+), K(+), and Ca(2+) were investigated in ectomycorrhizal (EM) associations formed by Paxillus involutus (strains MAJ and NAU) with the salt-sensitive poplar hybrid Populus × canescens. A scanning ion-selective electrode technique was used to measure flux profiles in non-EM roots and axenically grown EM cultures of the two P. involutus isolates to identify whether the major alterations detected in EM roots were promoted by the fungal partner. EM plants exhibited a more pronounced ability to maintain K(+)/Na(+) homeostasis under salt stress. The influx of Na(+) was reduced after short-term (50 mm NaCl, 24 h) and long-term (50 mm NaCl, 7 d) exposure to salt stress in mycorrhizal roots, especially in NAU associations. Flux data for P. involutus and susceptibility to Na(+)-transport inhibitors indicated that fungal colonization contributed to active Na(+) extrusion and H(+) uptake in the salinized roots of P. × canescens. Moreover, EM plants retained the ability to reduce the salt-induced K(+) efflux, especially under long-term salinity. Our study suggests that P. involutus assists in maintaining K(+) homeostasis by delivering this nutrient to host plants and slowing the loss of K(+) under salt stress. EM P. × canescens plants exhibited an enhanced Ca(2+) uptake ability, whereas short-term and long-term treatments caused a marked Ca(2+) efflux from mycorrhizal roots, especially from NAU-colonized roots. We suggest that the release of additional Ca(2+) mediated K(+)/Na(+) homeostasis in EM plants under salt stress.     

Yeast plasma membrane Ena1p ATPase alters alkali-cation homeostasis and confers increased salt tolerance in tobacco cultured cells

In plants, the plasma membrane Na(+)/H(+) antiporter is the only key enzyme that extrudes cytosolic Na(+) and contributes to salt tolerance. But in fungi, the plasma membrane Na(+)/H(+) antiporter and Na(+)-ATPase are known to be key enzymes for salt tolerance. Saccharomyces cerevisiae Ena1p ATPase encoded by the ENA1/PMR2A gene is primarily responsible for Na(+) and Li(+) efflux across the plasma membrane during salt stress and for K(+) efflux at high pH and high K(+). To test if the yeast ATPase would improve salt tolerance in plants, we expressed a triple hemagglutinin (HA)-tagged Ena1p (Ena1p-3HA) in cultured tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 (BY2) cells. The Ena1p-3HA proteins were correctly localized to the plasma membrane of transgenic BY2 cells and conferred increased NaCl and LiCl tolerance to the cells. Under moderate salt stress conditions, the Ena1p-3HA-expressing BY2 clones accumulated lower levels of Na(+) and Li(+) than nonexpressing BY2 clones. Moreover, the Ena1p-3HA expressing BY2 clones accumulated lower levels of K(+) than nonexpressing cells under no-stress conditions. These results suggest that the yeast Ena1p can also function as an alkali-cation (Na(+), Li(+), and K(+)) ATPase and alter alkali-cation homeostasis in plant cells. We conclude that, even with K(+)-ATPase activity, Na(+)-ATPase activity of the yeast Ena1p confers increased salt tolerance to plant cells during salt stress.     

The cotton GhNHX1 gene encoding a novel putative tonoplast Na(+)/H(+) antiporter plays an important role in salt stress

A cDNA clone was isolated from cotton (Gossypium hirsutum) cDNA library and characterized with regard to its sequence, regulation in response to salt stress and functions in yeast mutants and transgenic tobacco plants. The clone, designated as GhNHX1, contains 2485 nucleotides with an open reading frame of 1629 nucleotides, and the deduced amino acid sequence showed high identities with other plant vacuolar-type Na(+)/H(+) antiporters. Northern blot analysis indicated that the mRNA accumulation of GhNHX1 was strongly induced by salt stress and abscisic acid in cotton seedlings. The expression of GhNHX1 in yeast Na(+)/H(+) antiporter mutant showed function complementation. The transgenic tobacco plants overexpressing GhNHX1 also had higher salt tolerance than the wild-type plants. The salt-induced mRNA level of GhNHX1 was 3 and 7 times higher in the salt-tolerant cotton cultivar ZM3 than those in the salt-sensitive cotton cultivars ZMS17 and ZMS12, respectively. Together, these results suggest that the products of the novel gene, GhNHX1, function as a tonoplast Na(+)/H(+) antiporter and play an important role in salt tolerance of cotton.     

Effect of salt stress on antioxidant defense systems,lipid peroxidation,and chlorophyll content in green bean

Salt stress-induced changes in antioxidant enzymes, such as catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR), total chlorophyll content, and lipid peroxidation measured as malondialdehyde (MDA) content, in leaves of a green bean genotype Gevas sirsk 57 (GS57) and cv. Fransiz 4F-89 differing in salt tolerance were investigated. Plants were subjected to three salt treatments (0, 50, and 100 mM NaCl) under controlled climatic conditions for 7 days. The salt-sensitive cv. 4F-89 exhibited a decrease in GR activity at all salt treatments, but the salt-tolerant genotype GS57 showed only a slight decrease in GR under 50 mM salt treatment and an increase under 100 mM salt treatment. CAT and APX activities increased with increasing salt stress in both varieties. CAT and APX activities were higher in the salt-tolerant GS57 than salt-ensitive cv. 4F-89. The two varieties showed an increase in MDA content with an increase in salinity, but the increase in sensitive cv. 4F-89 under salt stress was higher than that in salt-tolerant GS57 genotype. The increasing NaCl concentration caused a reduction in the chlorophyll content in cv. 4F-89 but not in GS57.     

The Rice Monovalent Cation Transporter OsHKT2;4: Revisited Ionic Selectivity

The family of plant membrane transporters named HKT (for high-affinity K(+) transporters) can be subdivided into subfamilies 1 and 2, which, respectively, comprise Na(+)-selective transporters and transporters able to function as Na(+)-K(+) symporters, at least when expressed in yeast (Saccharomyces cerevisiae) or Xenopus oocytes. Surprisingly, a subfamily 2 member from rice (Oryza sativa), OsHKT2;4, has been proposed to form cation/K(+) channels or transporters permeable to Ca(2+) when expressed in Xenopus oocytes. Here, OsHKT2;4 functional properties were reassessed in Xenopus oocytes. A Ca(2+) permeability through OsHKT2;4 was not detected, even at very low external K(+) concentration, as shown by highly negative OsHKT2;4 zero-current potential in high Ca(2+) conditions and lack of sensitivity of OsHKT2;4 zero-current potential and conductance to external Ca(2+). The Ca(2+) permeability previously attributed to OsHKT2;4 probably resulted from activation of an endogenous oocyte conductance. OsHKT2;4 displayed a high permeability to K(+) compared with that to Na(+) (permeability sequence: K(+) > Rb(+) ≈ Cs(+) > Na(+) ≈ Li(+) ≈ NH(4)(+)). Examination of OsHKT2;4 current sensitivity to external pH suggested that H(+) is not significantly permeant through OsHKT2;4 in most physiological ionic conditions. Further analyses in media containing both Na(+) and K(+) indicated that OsHKT2;4 functions as K(+)-selective transporter at low external Na(+), but transports also Na(+) at high (>10 mm) Na(+) concentrations. These data identify OsHKT2;4 as a new functional type in the K(+) and Na(+)-permeable HKT transporter subfamily. Furthermore, the high permeability to K(+) in OsHKT2;4 supports the hypothesis that this system is dedicated to K(+) transport in the plant.     

NADPH oxidase AtrbohD and AtrbohF function in ROS-dependent regulation of Na⁺/K⁺homeostasis in Arabidopsis under salt stress

Maintaining cellular Na(+)/K(+) homeostasis is pivotal for plant survival in saline environments. However, knowledge about the molecular regulatory mechanisms of Na(+)/K(+) homeostasis in plants under salt stress is largely lacking. In this report, the Arabidopsis double mutants atrbohD1/F1 and atrbohD2/F2, in which the AtrbohD and AtrbohF genes are disrupted and generation of reactive oxygen species (ROS) is pronouncedly inhibited, were found to be much more sensitive to NaCl treatments than wild-type (WT) and the single null mutant atrbohD1 and atrbohF1 plants. Furthermore, the two double mutant seedlings had significantly higher Na(+) contents, lower K(+) contents, and resultant greater Na(+)/K(+) ratios than the WT, atrbohD1, and atrbohF1 under salt stress. Exogenous H(2)O(2) can partially reverse the increased effects of NaCl on Na(+)/K(+) ratios in the double mutant plants. Pre-treatments with diphenylene iodonium chloride, a widely used inhibitor of NADPH oxidase, clearly enhanced the Na(+)/K(+) ratios in WT seedlings under salt stress. Moreover, NaCl-inhibited inward K(+) currents were arrested, and NaCl-promoted increases in cytosolic Ca(2+) and plasma membrane Ca(2+) influx currents were markedly attenuated in atrbohD1/F1 plants. No significant differences in the sensitivity to osmotic or oxidative stress among the WT, atrbohD1, atrbohF1, atrbohD1/F1, and atrbohD2/F2 were observed. Taken together, these results strongly suggest that ROS produced by both AtrbohD and AtrbohF function as signal molecules to regulate Na(+)/K(+) homeostasis, thus improving the salt tolerance of Arabidopsis.     

Salinity tolerance and antioxidant status in cotton cultures

This investigation focuses upon cell growth and antioxidant status in cultured cells of cotton (Gossypium herbaceum) cvs. Dhumad (salt-tolerant, TOL), H-14 (medium salt-tolerant, MED), and RAhs-2 (salt-sensitive, SEN) exposed to saline stress (50-200 mM NaCl). Mean (+/- SEM) callus fresh weight (f.wt.) and dry weight (d.wt.) gains were significantly (p <.05) greater on Murashige and Skoog (MS) [1]-based medium with 50 mM NaCl for the TOL cv. (62% and 16%, respectively) over NaCl-free controls (2020 +/- 45 and 166 +/- 4 mg, respectively); comparable differences were not observed for the MED cv. A significant (p <.05) decrease in mean f.wt. occurred with the SEN cv. exposed to 50 mM NaCl. For all cvs., there were (p <.05) reductions in mean f.wts. in medium with >or=100 mM NaCl. At 200 mM NaCl, mean f.wt. decreases were 52% (TOL), 89% (MED), and 91% (SEN), respectively. A strong correlation existed between antioxidant status and growth of cells with NaCl. Superoxide dismutase and glutathione reductase activities increased with increasing salinity in the TOL cv. to maximum values of 26.3 +/- 1.1 U mg(-1) protein and 1.05 +/- 0.01 AB(340 nm) min(-1) mg(-1) protein, respectively, at 150 mM NaCl; for the MED and SEN cvs., there were no changes in activities of these enzymes between control and salt treatments. Catalase activity decreased progressively with increasing salt concentration in all cvs. except for SEN with 100 mM NaCl, where mean catalase activity (1.75 +/- 0.04 AB(240 nm) min(-1) mg(-1) protein) was greater (p <.05) than control (1.13 +/- 0.08). Overall, cultured cotton cells provide an experimental system for investigating the role of antioxidants in salt tolerance at the cellular level.