Hydrogen peroxide and nitric oxide mediate K+/Na+ homeostasis and antioxidant defense in NaCl-stressed callus cells of two contrasting poplars

Using callus cells of a salt-tolerant Populus euphratica Oliver and a salt-sensitive P. popularis 35–44 (P. popularis), the effects of NaCl stress on hydrogen peroxide (H₂O₂) and nitric oxide (NO) production and the relevance to ionic homeostasis and antioxidant defense were investigated. Results show that P. euphratica exhibited a greater capacity to tolerate NaCl stress in terms of cell viability, membrane permeability and K⁺/Na⁺ relations. NaCl salinity (150 mM) caused a rapid increase of H₂O₂ and NO in P. euphratica cells, but not in P. popularis. Moreover, salinised P. euphratica cells retained a high and stable level of H₂O₂ and NO during the period of 24-h salt stress. Noteworthy, P. eupratica cells increased activities of superoxide dismutase, ascorbate peroxidase, catalase and glutathione reductase under salinity stress, but these antioxidant enzymes were significantly inhibited by the salt treatment in P. popularis cells. Pharmacological experiments proved that the NaCl-induced H₂O₂ and NO was interdependent and contributed to the mediation of K⁺/Na⁺ homeostasis and antioxidant defense in P. euphratica cells. Given these results, we conclude that the increased H₂O₂ and NO enable P. euphratica cells to regulate ionic and ROS (reactive oxygen species) homeostasis under salinity stress in the longer term.     

Effect of NaCl on leaf H+-ATPase and the relevance to salt tolerance in two contrasting poplar species

During a 30-day period of increasing salinity, we examined the effects of NaCl on leaf H⁺-ATPase and salinity tolerance in 1-year-old plants of Populus euphratica Oliv. (salt resistant) and P. popularis 35–44 (P. popularis) (salt sensitive). Electron probe X-ray microanalysis of leaf mesophyll revealed that P. euphratica had a higher ability to retain lower NaCl concentrations in the cytoplasm, as compared to P. popularis. The sustained activity of H⁺ pumps (by cytochemical staining) in salinised P. euphratica suggests a role in energising salt transport through the plasma membrane (PM) and tonoplast. Salt-induced alterations of leaf respiration, ATP content and expression of PM H⁺-ATPase were compared between the two species. Results show that P. euphratica retained a constant respiratory rate, ATP production and protein abundance of PM H⁺-ATPase (by Western blotting) in salt-stressed plants. P. euphratica was able to maintain a comparatively high capacity of ATP hydrolysis and H⁺ pumping during prolonged salt exposure. By contrast, the activity and expression of PM H⁺-ATPase were markedly decreased in P. popularis leaves in response to salt stress. Furthermore, NaCl-stressed P. popularis plants showed a marked decline of respiration (70%) and ATP production (66%) on day 30. We conclude that the inability of P. popularis to transport salt to the apoplast and vacuole was partly due to the decreased activity of H⁺ pumps. As a consequence, cytosolic ion concentrations were observed to be comparatively high for an extended period of time, so that cell metabolism, in particular respiration, was disrupted in P. popularis leaves.     

Extracellular ATP Signaling Is Mediated by H2O2 and Cytosolic Ca2+ in the Salt Response of Populus euphratica Cells

Extracellular ATP (eATP) has been implicated in mediating plant growth and antioxidant defense; however, it is largely unknown whether eATP might mediate salinity tolerance. We used confocal microscopy, a non-invasive vibrating ion-selective microelectrode, and quantitative real time PCR analysis to evaluate the physiological significance of eATP in the salt resistance of cell cultures derived from a salt-tolerant woody species, Populus euphratica. Application of NaCl (200 mM) shock induced a transient elevation in [eATP]. We investigated the effects of eATP by blocking P2 receptors with suramin and PPADS and applying an ATP trap system of hexokinase-glucose. We found that eATP regulated a wide range of cellular processes required for salt adaptation, including vacuolar Na⁺ compartmentation, Na⁺/H⁺ exchange across the plasma membrane (PM), K⁺ homeostasis, reactive oxygen species regulation, and salt-responsive expression of genes related to K⁺/Na⁺ homeostasis and PM repair. Furthermore, we found that the eATP signaling was mediated by H₂O₂ and cytosolic Ca²⁺ released in response to high salt in P. euphratica cells. We concluded that salt-induced eATP was sensed by purinoceptors in the PM, and this led to the induction of downstream signals, like H₂O₂ and cytosolic Ca²⁺, which are required for the up-regulation of genes linked to K⁺/Na⁺ homeostasis and PM repair. Consequently, the viability of P. euphratica cells was maintained during a prolonged period of salt stress.     

H2O2 and cytosolic Ca2+ signals triggered by the PM H+‐coupled transport system mediate K+/Na+ homeostasis in NaCl‐stressed Populus euphratica cells

Using confocal microscopy, X‐ray microanalysis and the scanning ion‐selective electrode technique, we investigated the signalling of H₂O₂, cytosolic Ca²⁺ ([Ca²⁺]_cyt) and the PM H⁺‐coupled transport system in K⁺/Na⁺ homeostasis control in NaCl‐stressed calluses of Populus euphratica. An obvious Na⁺/H⁺ antiport was seen in salinized cells; however, NaCl stress caused a net K⁺ efflux, because of the salt‐induced membrane depolarization. H₂O₂ levels, regulated upwards by salinity, contributed to ionic homeostasis, because H₂O₂ restrictions by DPI or DMTU caused enhanced K⁺ efflux and decreased Na⁺/H⁺ antiport activity. NaCl induced a net Ca²⁺ influx and a subsequent rise of [Ca²⁺]_cyt, which is involved in H₂O₂‐mediated K⁺/Na⁺ homeostasis in salinized P. euphratica cells. When callus cells were pretreated with inhibitors of the Na⁺/H⁺ antiport system, the NaCl‐induced elevation of H₂O₂ and [Ca²⁺]_cyt was correspondingly restricted, leading to a greater K⁺ efflux and a more pronounced reduction in Na⁺/H⁺ antiport activity. Results suggest that the PM H⁺‐coupled transport system mediates H⁺ translocation and triggers the stress signalling of H₂O₂ and Ca²⁺, which results in a K⁺/Na⁺ homeostasis via mediations of K⁺ channels and the Na⁺/H⁺ antiport system in the PM of NaCl‐stressed cells. Accordingly, a salt stress signalling pathway of P. euphratica cells is proposed.     

Leaf photosynthesis,fluorescence response to salinity and the relevance to chloroplast salt compartmentation and anti-oxidative stress in two poplars

In a 16-day study, the effect of increasing soil NaCl on leaf photosynthesis, chlorophyll a fluorescence, chloroplast ion compartmentation, variations of SOD (superoxide dismutase) and POD (peroxidase) isoenzymes and the relevance to salt resistance were investigated in seedlings of Populus euphratica Oliv. (P. euphratica) (salt-resistant) and rooted cuttings of P. “popularis 35–44” (P. popularis) (salt-sensitive). Initial salinity caused a rapid decline of net photosynthetic rate (Pn) and unit transpiration rate (TRN) in P. euphratica, resulting from the NaCl-induced stomatal closure. In a longer-term of salinity, CO₂ assimilation in P. popularis was severely reduced whereas stressed P. euphratica maintained a relatively higher and constant level of Pn. Pn–Ci curves showed that salt stress (12 days) reduced CO₂ saturation point (CSP), CO₂ saturated Pn (CSP _n ), and carboxylation efficiency (CE), but increased CO₂ compensation point (CCP) in the two genotypes. Similarly, salinity lowered light saturation point (LSP), light saturated Pn (LSP _n ), and apparent quantum yield (AQY) in both genotypes but the inhibitory effect of NaCl on light reaction was more pronounced in P. popularis, as compared to P. euphratica. Chlorophyll a fluorescence data indicated that a longer-term of salt stress (12 days) exhibited a marked influence on fluorescence parameters of P. popularis in both dark- and light-adapted states: (a) NaCl inhibited the maximal efficiency of PSII photochemistry (Fv/Fm) due to the salt-induced increase of Fo (the minimal fluorescence) and the marked decline of Fm (the maximal fluorescence); (b) salinity decreased coefficient of photochemical quenching (qP) but markedly elevated coefficient of nonphotochemical quenching (qN) in the light-adapted state. In contrast, there were no corresponding changes of chlorophyll a fluorescence in salinised P. euphratica. X-ray microanalysis results showed that salinity caused salt accumulation in the chloroplasts of P. popularis in which Na⁺ and Cl⁻ increased up to 42 and 221 mmol dm⁻³, respectively. Great buildup of Na⁺ and Cl⁻ in chloroplasts of P. popularis may exhibit direct and indirect restrictions on dark and light reactions. The activity of SOD isoenzymes (CuZn-SOD I and CuZn-SOD II) and POD isoenzymes in P. popularis decreased with increasing exposure period, and leaf malondialdehyde (MDA) content and membrane permeability (MP) increased correspondingly. In contrast to P. popularis, stressed P. euphratica maintained activity of SOD and POD isoenzymes and there was no significant increase of MDA and MP during the period of salt stress. In conclusion, P. euphratica plants exhibited a higher capacity to maintain the activity of anti-oxidant enzymes and restrict salt accumulation in the chloroplasts, the photosynthesis processes were less restricted consequently.     

Oxygen-Dependent Exclusion of Sodium Ions from Shoots by Roots of Zea mays (cv Pioneer 3906) in Relation to Salinity Damage

Using radio-tracers, we measured Na⁺ and K⁺ accumulation in roots and transport to shoots in Zea mays (cv Pioneer 3906) as a function of NaCl concentration and O₂ partial pressure in the nutrient solution. Under fully aerobic conditions, roots partially excluded Na⁺ from the shoots over a wide range of NaCl concentration (0.2-200 millimolar). With root anoxia, the exclusion mechanism broke down so that much greater amounts of Na⁺ reached the shoots, with simultaneous inhibition of K⁺ transport. The ratio Na⁺/K⁺ entering the shoot consequently increased 90 to 200 times. Increases in Na⁺ transport were first detected when the O₂ partial pressure was reduced from ambient (21% v/v) to 15%, whereas K⁺ transport was not inhibited until O₂ concentrations were <5%. Since soil O₂ deficiency can often accompany high salinity in irrigation agriculture, failure of the Na⁺ exclusion mechanism may be a contributory factor in salinity damage of salt-sensitive glycophytes.     

The over-expression of Chrysanthemum crassum CcSOS1 improves the salinity tolerance of chrysanthemum

Soil salinity represents a major constraint on plant growth. Here, we report that the over-expression of the Chrysanthemum crassum plasma membrane Na⁺/H⁺ antiporter gene CcSOS1, driven by the CaMV 35S promoter, improved the salinity tolerance of chrysanthemum ‘Jinba’. In salinity-stressed transgenic plants, both the proportion of the leaf area suffering damage and the electrical conductivity of the leaf were lower in the transgenic lines than in salinity-stressed wild type plants. After a 6 day exposure to 200 mM NaCl, the leaf content of both chlorophyll (a+b) and proline was higher in the transgenic than in the wild type plants. The activity of both superoxide dismutase and peroxidase was higher in the transgenic than in the wild type plants throughout the period of NaCl stress. The transgenic plants had a stronger control over the ingress of Na⁺ into the plant, particularly with respect to the youngest leaves, and so maintained a more favorable K⁺/Na⁺ ratio. The result suggests that a possible strategy for improving the salinity tolerance of chrysanthemum could target the restriction of Na⁺ accumulation. This study is the first to report the transgenic expression of a Na⁺ efflux carrier in chrysanthemum.     

Ecosystem changes in Galápagos highlands by the invasive tree Cinchona pubescens

Background and aims

Salinity is an increasing problem for agricultural production worldwide. Understanding how Na⁺ enters plants is important if reducing Na⁺ influx, a key component of the regulation of Na⁺ accumulation in plants and improving salt tolerance of crop plants, is to be achieved. Our previous work indicated that two distinct low-affinity Na⁺ uptake pathways exist in the halophyte Suaeda maritima. Here, we report the external NaCl concentration at which uptake switches from pathway 1 to pathway 2 and the kinetics of the interaction between external K⁺ concentration and Na⁺ uptake and accumulation in S. maritima in order to determine the roles of K⁺ transporters or channels in low-affinity Na⁺ uptake.

Methods

Na⁺ influx, Na⁺ and K⁺ accumulations in S. maritima exposed to various concentrations of NaCl (0–200 mM) were analyzed in the absence and presence of the inhibitors TEA and Ba⁺ (5 mM TEA or 3 mM Ba²⁺) or KCl (0, 10 or 50 mM).

Results

Our earlier proposal was confirmed and extended that there are two distinct low-affinity Na⁺ uptake pathways in S. maritima: pathway 1 might be mediated by a HKT-type transporter under low salinity conditions and pathway 2 by an AKT1-type channel or a KUP/HAK/KT type transporter under high salinity conditions. The external NaCl concentration at which two distinct low-affinity Na⁺ uptake switches from pathway 1 to pathway 2, the ‘turning point’, is between 90 and 95 mM. Over a short period (12 h) of Na⁺ and K⁺ treatments, a low concentration of K⁺ (10 mM) facilitated Na⁺ uptake by S. maritima under high salinity (100–200 mM NaCl), whether or not the plants had been subjected to a longer (3 d) period of K⁺ starvation. The kinetics suggests that low concentration of K⁺ (10 mM) might activate AKT1-type channels or KUP/HAK/KT-type transporters under high salinity (100–200 mM NaCl).

Conclusions

The turning-point of external NaCl concentrations for the two low-affinity Na⁺ uptake pathways in Suaeda maritima is between 90 and 95 mM. A low concentration of K⁺ (10 mM) might activate AKT1 or KUP/HAK/KT and facilitate Na⁺ uptake under high salinity (100–200 mM NaCl). The kinetics of K⁺ on Na⁺ uptake and accumulation in S maritima are also consistent with there being two low-affinity Na⁺ uptake pathways.     

Role of Tonoplast Proton Pumps and Na+/H+ Antiport System in Salt Tolerance of Populus euphratica Oliv.

Populus euphratica has been used as a plant model to study resistance against salt and osmotic stresses, with recent studies having characterized the tonoplast and the plasma membrane ATPases, and two Na⁺/H⁺ antiporters, homologs of the Arabidopsis tonoplast AtNHX1, were published in databases. In the present work we show that P. euphratica suspension-cultured cells are highly tolerant to high salinity, being able to grow with up to 150 mM NaCl in the culture medium without substantial modification of the final population size when compared to the control cells in the absence of salt. At a salt concentration of 300 mM, cells were unable to grow but remained highly viable up to 17 days after subculture. The addition of a 1-M-NaCl pulse to unadapted cells did not promote a significant loss in cell viability within 48 h. In tonoplast vesicles purified from cells cultivated in the absence of salt and from salt-stressed cells, vacuolar H⁺-pyrophosphatase (V-H⁺-PPase) seemed to be the primary tonoplast proton pump; however, there appears to be a decrease in V-H⁺-PPase activity with exposure to NaCl, in contrast to the sodium-induced increase in the activity of vacuolar H⁺-ATPase (V-H⁺-ATPase). Despite reports that in P. euphratica there is no significant difference in the concentration of Na⁺ in the different cell compartments under NaCl stress, in the present study, confocal and epifluorescence microscopic observations using a Na⁺-sensitive probe showed that suspension-cultured cells subject to a salt pulse accumulated Na⁺ in the vacuole when compared with control cells. Concordantly, a tonoplast Na⁺/H⁺ exchange system is described whose activity is upregulated by salt and, indirectly, by a salt-mediated increase of V-H⁺-ATPase activity.     

Salt tolerance of a cash crop halophyte Suaeda fruticosa: biochemical responses to salt and exogenous chemical treatments

Suaeda fruticosa Forssk is a leaf succulent obligate halophyte that produces numerous seeds under saline conditions. Seeds are a good source of high quality edible oil and leaves are capable of removing substantial amount of salt from the saline soil besides many other economic usages. Little is known about the biochemical basis of salt tolerance in this species. We studied some biochemical responses of S. fruticosa to different exogenous treatments under non-saline (0 mM), moderate (300 mM) or high (600 mM) NaCl levels. Eight-week-old seedlings were sprayed twice a week with distilled water, hydrogen peroxide (H₂O₂, 100 μM), glycine betaine (GB, 10 mM), or ascorbic acid (AsA, 20 mM) for 30 days. At moderate (300 mM) NaCl, leaf Na⁺, Ca²⁺ and osmolality increased, along with unchanged ROS and antioxidant enzyme activities, possibly causing a better plant growth. Plants grew slowly at 600 mM NaCl to avoid leaf Na⁺ buildup relative to those at 300 mM NaCl. Exogenous application of distilled water and H₂O₂ improved ROS scavenging mechanisms, although growth was unaffected. ASA and GB alleviated salt-induced growth inhibition at 600 mM NaCl through enhancing the antioxidant defense system and osmotic and ion homeostasis, respectively.     

Glucose-6-phosphate dehydrogenase-dependent hydrogen peroxide production is involved in the regulation of plasma membrane H+-ATPase and Na+/H+ antiporter protein in salt-stressed callus from Carex moorcroftii

Glucose‐6‐phosphate dehydrogenase (G6PDH) is important for the activation of plant resistance to environmental stresses, and ion homeostasis is the physiological foundation for living cells. In this study, we investigated G6PDH roles in modulating ion homeostasis under salt stress in Carex moorcroftii callus. G6PDH activity increased to its maximum in 100 mM NaCl treatment and decreased with further increased NaCl concentrations. K⁺/Na⁺ ratio in 100 mM NaCl treatment did not exhibit significant difference compared with the control; however, in 300 mM NaCl treatment, it decreased. Low‐concentration NaCl (100 mM) stimulated plasma membrane (PM) H⁺‐ATPase and NADPH oxidase activities as well as Na⁺/H⁺ antiporter protein expression, whereas high‐concentration NaCl (300 mM) decreased their activity and expression. When G6PDH activity and expression were reduced by glycerol treatments, PM H⁺‐ATPase and NADPH oxidase activities, Na⁺/H⁺ antiporter protein level and K⁺/Na⁺ ratio dramatically decreased. Simultaneously, NaCl‐induced hydrogen peroxide (H₂O₂) accumulation was abolished. Exogenous application of H₂O₂ increased G6PDH, PM H⁺‐ATPase and NADPH oxidase activities, Na⁺/H⁺ antiporter protein expression and K⁺/Na⁺ ratio in the control and glycerol treatments. Diphenylene iodonium (DPI), the NADPH oxidase inhibitor, which counteracted NaCl‐induced H₂O₂ accumulation, decreased G6PDH, PM H⁺‐ATPase and NADPH oxidase activities, Na⁺/H⁺ antiporter protein level and K⁺/Na⁺ ratio. Western blot result showed that G6PDH expression was stimulated by NaCl and H₂O₂, and blocked by DPI. Taken together, G6PDH is involved in H₂O₂ accumulation under salt stress. H₂O₂, as a signal, upregulated PM H⁺‐ATPase activity and Na⁺/H⁺ antiporter protein level, which subsequently resulted in the enhanced K⁺/Na⁺ ratio. G6PDH played a central role in the process.     

Overexpression of a poplar two-pore K+ channel enhances salinity tolerance in tobacco cells

Populus euphratica is a plant model intensively studied for elucidating physiological and molecular mechanisms of salt tolerance in woody species. Several studies have shown that vacuolar potassium (K⁺) ion channels of the two-pore K⁺ (TPK) family play an important role in maintaining K⁺ homeostasis. Here, we cloned a putative TPK channel gene from P. euphratica, termed PeTPK. Sequence analysis of PeTPK1 identified the universal K-channel-specific pore signature, TXGYGD. Over-expression of PeTPK1 in tobacco BY-2 cells improved salt tolerance, but did not enhance tolerance to hyperosmotic stress caused by mannitol (200?C600?mM). After 3?weeks of NaCl stress (100 and 150?mM), PeTPK1-transgenic cells had higher fresh and dry weights than wild-type cells. Salt treatment caused significantly higher Na⁺ accumulation and K⁺ loss in wild-type cells compared to transgenic cells. During short-term salt stress (100?mM NaCl, 24-h), PeTPK1-transgenic cells showed higher cell viability and reduced membrane permeabilization compared to wild-type cells. Scanning ion-selective electrode data revealed that salt-shock elicited a significantly higher transient K⁺ efflux from PeTPK1-transgenic callus cells and protoplasts compared to that observed in wild-type cells and protoplasts. We concluded that salt tolerance in P. euphratica is most likely mediated through PeTPK1. We propose that, under salt stress, PeTPK1 functions as an outward-rectifying, K⁺ efflux channel in the vacuole that transfers K⁺ to the cytosol to maintain K⁺ homeostasis.     

Different mechanisms of ion homeostasis are dominant in the recretohalophyte <Emphasis Type="Italic">Tamarix ramosissima</Emphasis> under different soil salinity

Total ion (Na⁺, K⁺, Ca²⁺, SO₄ ^2? and Cl^?) accumulation by plants, ion contents in plant tissues and ion secretion by salt glands on the surface of shoots of Tamarix ramosissima adapted to different soil salinity, namely low (0.06 mmol Na⁺/g soil), moderate (3.14–4.85 mmol Na⁺/g soil) and strong (7.56 mmol Na⁺/g soil) were analyzed. There are two stages of interrelated and complementary regulation of ion homeostasis in whole T. ramosissima plants: (1) regulation of ion influx into the plant from the soil and (2) changing the secretion efficiency of salt glands on shoots. The secretion efficiency of salt glands was appraised by the ratio of ion secretion to tissue ion content. Independent of soil salinity, the accumulation of K⁺ and Ca²⁺ was higher than the contents of these ions in the soil. Furthermore, the accumulation of K⁺, Ca²⁺ and SO₄ ^2? ions by plants was maintained within a narrow range of values. Under low soil salinity, Na⁺ was accumulated, whereas under moderate and strong salinity, the influxes of Na⁺ were limited. However, under strong salinity, the accumulation of Na⁺ was threefold higher than that under low soil salinity. This led to a change in the Na⁺/K⁺ ratio (tenfold), an increase in the activity of salt glands (tenfold) and a reduction in plant growth (fivefold). An apparently high Na⁺/K⁺ ratio was the main factor determining over-active functioning of salt glands under strong salinity. Principal component analysis showed that K⁺ ions played a key role in ion homeostasis at all levels of salinity. Ca²⁺ played a significant role at low salinity, whereas Cl^? and interrelated regulatory components (K⁺ and proline) played a role under strong salinity. Proline, despite its low concentration under strong salinity, was involved in the regulation of secretion by salt glands. Different stages and mechanisms of ion homeostasis were dominant in T. ramosissima plants adapted to different levels of salinity. These mechanisms facilitated the accumulation of Na⁺ in plants under low soil salinity, the limitation of Na⁺ under moderate salinity and the over-activation of Na⁺ secretion by salt glands under strong salinity, which are all necessary for maintaining ion homeostasis and water potential in the whole plant.     

Functional Analysis of the Na+,K+/H+ Antiporter PeNHX3 from the Tree Halophyte Populus euphratica in Yeast by Model-Guided Mutagenesis

Na⁺,K⁺/H⁺ antiporters are H⁺-coupled cotransporters that are crucial for cellular homeostasis. Populus euphratica, a well-known tree halophyte, contains six Na⁺/H⁺ antiporter genes (PeNHX1-6) that have been shown to function in salt tolerance. However, the catalytic mechanisms governing their ion transport remain largely unknown. Using the crystal structure of the Na⁺/H⁺ antiporter from the Escherichia coli (EcNhaA) as a template, we built the three-dimensional structure of PeNHX3 from P. euphratica. The PeNHX3 model displays the typical TM4-TM11 assembly that is critical for ion binding and translocation. The PeNHX3 structure follows the ‘positive-inside’ rule and exhibits a typical physicochemical property of the transporter proteins. Four conserved residues, including Tyr149, Asn187, Asp188, and Arg356, are indentified in the TM4-TM11 assembly region of PeNHX3. Mutagenesis analysis showed that these reserved residues were essential for the function of PeNHX3: Asn187 and Asp188 (forming a ND motif) controlled ion binding and translocation, and Tyr149 and Arg356 compensated helix dipoles in the TM4-TM11 assembly. PeNHX3 mediated Na⁺, K⁺ and Li⁺ transport in a yeast growth assay. Domain-switch analysis shows that TM11 is crucial to Li⁺ transport. The novel features of PeNHX3 in ion binding and translocation are discussed.     

Salt, nutrient uptake and transport, and ABA of Populus euphratica; a hybrid in response to increasing soil NaCl

The uptake and transport of salt ions (Na⁺, Cl^-), macronutrients (K⁺, Ca²⁺, Mg²⁺) and abscisic acid (ABA) response to increasing soil salinity were examined in 2-year-old seedlings of Populus euphratica and a hybrid, P. talassica Kom 2 (P. euphratica + Salix alba L.). Leaf burn symptoms appeared in the hybrid after 8 days of exposure to salinity when soil NaCl concentration increased to 206 mM, whereas P. euphratica exhibited leaf damage after day 21 when soil NaCl exceeded 354 mM. Leaf necrosis was the result of excess salt accumulation since the injury followed an abrupt increase of endogenous salt levels. Compared with the hybrid, P. euphratica exhibited a greater capacity to exclude salt ions from leaves under increasing salinity, especially Cl^-. Salt treatment altered nutrient balance of the hybrid, leaf K⁺, Ca²⁺ and Mg²⁺ concentrations significantly declined and the same trends were observed in roots with the exception of K⁺. Although K⁺ levels decreased in salinised P. euphratica, increasing salinity did not affect the levels of Ca²⁺ and Mg²⁺ in leaves, but did increase the uptake of these nutrients when salt stress was initiated. NaCl-induced increase of ABA concentration in xylem sap [ABA] was observed in the two tested genotypes, however xylem [ABA] increased more rapidly in P. euphratica and a fivefold increase of xylem [ABA] was recorded after the first day of exposure to salt stress. Therefore, we conclude that the increase of Ca²⁺ uptake may be associated with the rise of ABA, and thus contributes to membrane integrity maintenance, which enables P. euphratica to regulate uptake and transport of salt ions under high levels of external salinity in the longer term.     

Manganese-induced salt stress tolerance in rice seedlings: regulation of ion homeostasis,antioxidant defense and glyoxalase systems

Hydroponically grown 12-day-old rice (Oryza sativa L. cv. BRRI dhan47) seedlings were exposed to 150 mM NaCl alone and combined with 0.5 mM MnSO₄. Salt stress resulted in disruption of ion homeostasis by Na⁺ influx and K⁺ efflux. Higher accumulation of Na⁺ and water imbalance under salinity caused osmotic stress, chlorosis, and growth inhibition. Salt-induced ionic toxicity and osmotic stress consequently resulted in oxidative stress by disrupting the antioxidant defense and glyoxalase systems through overproduction of reactive oxygen species (ROS) and methylglyoxal (MG), respectively. The salt-induced damage increased with the increasing duration of stress. However, exogenous application of manganese (Mn) helped the plants to partially recover from the inhibited growth and chlorosis by improving ionic and osmotic homeostasis through decreasing Na⁺ influx and increasing water status, respectively. Exogenous application of Mn increased ROS detoxification by increasing the content of the phenolic compounds, flavonoids, and ascorbate (AsA), and increasing the activities of monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), superoxide dismutase (SOD), and catalase (CAT) in the salt-treated seedlings. Supplemental Mn also reinforced MG detoxification by increasing the activities of glyoxalase I (Gly I) and glyoxalase II (Gly II) in the salt-affected seedlings. Thus, exogenous application of Mn conferred salt-stress tolerance through the coordinated action of ion homeostasis and the antioxidant defense and glyoxalase systems in the salt-affected seedlings.