The HAL1 function on Na+ homeostasis is maintained over time in salt-treated transgenic tomato plants, but the high reduction of Na+ in leaf is not associated with salt tolerance

To achieve a deeper knowledge on the function of HAL1 gene in tomato ( Solanum lycopersicum ) plants submitted to salt stress, in this study, we studied the growth and physiological responses to high salt stress of T3 transgenic plants (an azygous line without transgene and both homozygous and hemizygous lines for HAL1 ) proceeding from a primary transformant with a very high expression level of HAL1 gene. The homozygous plants for HAL1 gene did not increase their salt tolerance in spite of an earlier and higher reduction of the Na⁺ accumulation in leaves, being moreover the Na⁺ homeostasis maintained throughout the growth cycle. The greater ability of the homozygous line to regulate the Na⁺ transport to the shoot to long term was even shown in low accumulation of Na⁺ in fruits. By comparing the homozygous and hemizygous lines, a higher salt tolerance in the hemizygous line, with respect to the homozygous line, was observed on the basis of fruit yield. The Na⁺ homeostasis and osmotic homeostasis were also different in homozygous and hemizygous lines. Indeed, the Na⁺ accumulation rate in leaves was greater in hemizygous than in homozygous line after 35 days of 100 m M NaCl treatment and only at the end of growth cycle did the hemizygous line show leaf Na⁺ levels similar to those found in the homozygous line. With respect to the osmotic homeostasis, the main difference between lines was the different contribution of inorganic and organic solutes to the leaf osmotic balance. Taken together, these results suggest that the greater Na⁺ exclusion ability of the homozygous line overexpressing HAL1 induces a greater use of organic solutes for osmotic balance, which seems to have an energy cost and hence a growth penalty that reverts negatively on fruit yield.     

Antiporter Gene from Hordum brevisubulatum （Trin.） Link and Its Overexpression in Transgenic Tobaccos

A vacuolar Na^ /H^ antiporter cDNA gene was successfully isolated fromHordeum brevisubulatum (Trin.) Link using the rapid amplification ofcDNA ends (RACE) method. The gene was named HbNHXI and was found to consist of 1 916 bp encoding a predicted polypeptide of 540 amino acids with a conserved amiloride-binding domain. Phylogenetic tree analysis of the Na^ /H^ antiporters showed that the HbNHXI gene shares 55.3%-74.8% similarity with the vacuolar-type Na^ /H^ antiporters. Transgenic tobaccos that contain the HbNHXI gene, integrated by forward insertion into the tobacco genome, were obtained via Agrobacterium tumerfaciens and characterized for the determination of the concentration of Na^ and K^ ions, as well as proline, in the presence of 300 mmol/L NaCl. The T1 transgenic plants showed more tolerance to salt and drought than did wild-type plants. Our data suggest that overexpression of the HbNHXI gene could improve the tolerance of transgenic tobaccos to salt and drought through the function of the vacuolar Na^ /H^ antiporter.     

Genetic basis of sodium exclusion and sodium tolerance in yeast. A model for plants

A yeast strain carrying disruptions in TRK1 and ENA genes was very sensitive to Na⁺ because uptake discriminated poorly between K⁺ and Na⁺, and Na⁺ efflux was insignificant. Transformation with TRK1 and ENA1 restored discrimination, Na⁺ efflux and Na⁺ tolerance. Increasing external Ca²⁺ increased Na⁺ tolerance almost in the same proportion in TRK1 enal cells and in trkl ENAI cells, suggesting an unspecific effect of this cation. By using a vacuolar ATPase mutant, the role of the vacuole in Na⁺ tolerance was also demonstrated. The yeast model of Na⁺ exclusion and Na⁺ tolerance may be extended to plants.     

Changes in sodium and potassium in Nitellopsis cells treated with transient salt stress

Abstract. Nitellopsis cells grown in fresh water have a relatively low cytoplasmic Na⁺ (11 mol m⁻³) and high cytoplasmic K⁺ (90 mol m⁻³) content. A 30-min treatment with 100 mol m⁻³ external NaCl resulted in a high [Na⁺]_c (90 mol m⁻³) and a low [K⁺]_c (33 mol m⁻³), Subsequent addition of external Ca²⁺ (10 mol m⁻³) prevented Na⁺ influx and then [Na⁺]_c decreased slowly. Changes in [K⁺]_c were opposite to [Na⁺]_c. During the recovery time vacuolar Na⁺ increased, while vacuolar K⁺ decreased. Since all these processes proceeded also under ice-cold conditions, the restoration of original cytoplasmic ion compositions is suggested to be a passive nature. The notion that the passive movement of ions across the tonoplast can act as an effective and economic mechanism of salt tolerance under transient or under mild salt stress conditions is discussed.     

Enhanced V-ATPase activity contributes to the improved salt tolerance of transgenic tobacco plants overexpressing vacuolar Na(+)/H (+) antiporter AtNHX1

AtNHX1, a vacuolar Na⁺/H⁺ antiporter gene from Arabidopsis thaliana, was introduced into tobacco genome via Agrobacterium tumefaciens-mediated transformation to evaluate the role of vacuolar energy providers in plants salt stress response. Compared to the wild-type plants, over-expression of AtNHX1 increased salt tolerance in the transgenic tobacco plants, allowing higher germination rates of seeds and successful seedling establishment in the presence of toxic concentrations of NaCl. More importantly, the induced Na⁺/H⁺ exchange activity in the transgenic plants was closely correlated to the enhanced activity of vacuolar H⁺-ATPase (V-ATPase) when exposed to 200 mM NaCl. In addition, inhibition of V-ATPase activity led to the malfunction of Na⁺/H⁺ exchange activity, placing V-ATPase as the dominant energy provider for the vacuolar Na⁺/H⁺ antiporter AtNHX1. V-ATPase and vacuolar Na⁺/H⁺ antiporter thus function in an additive or synergistic way. Simultaneous overexpression of V-ATPase and vacuolar Na⁺/H⁺ antiporter might be appropriate for producing plants with a higher salt tolerance ability.     

Characterization of Na+ exclusion mechanisms of salt-tolerant reed plants in comparison with salt-sensitive rice plants

Salt-tolerant reed plants ( Phragmites communis Trinius) and salt-sensitive rice plants ( Oryza sativa L. cv. Kinmaze) were grown in salinized nutrient solutions up to 50 m M NaCl, and growth, Na⁺ contents and kinetics of ²²Na⁺ uptake and translocation were compared between the species to characterize the salt tolerance mechanisms operating in reed plants. When both plants were grown under the same salinity, Na⁺ contents of the shoots were lower in reed plants, although those of the roots were quite similar. The shoot base region of both species accumulated Na⁺ more than the leaf blades did. Sodium-22 uptake and pulse-chase experiments suggested that the lower Na⁺ transport rate from root to shoot could limit excessive Na⁺ accumulation in the reed shoot. There was a possibility that the apparently lower ²²Na⁺ transport rate to the shoot of reed plants was due to net downward Na⁺ transport from shoot base to root.     

Puccinellia tenuiflora maintains a low Na+ level under salinity by limiting unidirectional Na+ influx resulting in a high selectivity for K+ over Na+

Puccinellia tenuiflora is a useful monocotyledonous halophyte that might be used for improving salt tolerance of cereals. This current work has shown that P. tenuiflora has stronger selectivity for K⁺ over Na⁺ allowing it to maintain significantly lower tissue Na⁺ and higher K⁺ concentration than that of wheat under short- or long-term NaCl treatments. To assess the relative contribution of Na⁺ efflux and influx to net Na⁺ accumulation, unidirectional ²²Na⁺ fluxes in roots were carried out. It was firstly found that unidirectional ²²Na⁺ influx into root of P. tenuiflora was significantly lower (by 31–37%) than in wheat under 100 and 150 m m NaCl. P. tenuiflora had lower unidirectional Na⁺ efflux than wheat; the ratio of efflux to influx was similar between the two species. Leaf secretion of P. tenuiflora was also estimated, and found the loss of Na⁺ content from leaves to account for only 0.0006% of the whole plant Na⁺ content over 33 d of NaCl treatments. Therefore, it is proposed that neither unidirectional Na⁺ efflux of roots nor salt secretion by leaves, but restricting unidirectional Na⁺ influx into roots with a strong selectivity for K⁺ over Na⁺ seems likely to contribute to the salt tolerance of P. tenuiflora .     

Salinity response of a freshwater charophyte, Chara vulgaris

Abstract. Chara vulgaris L. growing in an oligohaline lake was adapted to laboratory conditions and subjected to long-term salinity treatments ranging from 0 to 350 mol m ³ NaCl added to the lake water (40–680 mosmol kg ¹). Osmotic potential and concentration of the main osmotically active solutes (K⁺, Na⁺, Mg²⁺, Cl and sucrose) in the vacuolar sap of the central internodal cells were estimated. C. vulgaris did regulate turgor but incompletely. Turgor decreased from 335 mosmol kg ¹ under control conditions to 52–111 mosmol kg ¹ at 350 mol m ³ NaCl. The enhancement of πⁱ was achieved by increase in both ions and sucrose. Sterile and fertile plants differed in their response to osmotic stress. In sterile plants, the ions accounted for about 87% of the vacuolar osmotic potential. The increase of πⁱ under osmotic stress was exclusively due to an accumulation of Na⁺ and Cl^-. In fertile plants, sucrose accounted for about 35% of πⁱ and ions for about 51% Under osmotic stress, sucrose content increased together with the ionic content of Na⁺ and Cl^-.     

Role of sodium in the ABA‐mediated long‐term growth response of bean to salt stress

Long‐term salt effects on plant growth have often been related to direct ion toxicity due to the accumulation of high ion concentrations in plant tissue. This work examines the relative importance of endogenous ABA, as well as Na⁺ and Cl⁻ toxicity, in the inhibition of leaf growth and photosynthesis, in bean plants grown at 1, 25, 50 and 75 m M NaCl until the fruit‐bearing stage. All salt‐treated plants showed very high leaf Cl⁻ concentrations, with little difference between plants exposed to 50 or 75 m M NaCl. The 25 and 50 mM salt‐treated plants were able to successfully exclude Na⁺ from their leaves, and only suffered an initial decline in the rate of leaf growth. Plants exposed to 75 m M NaCl showed an increase in Na⁺ leaf concentrations with an accompanying decrease in growth and photosynthesis as salt exposure progressed. A high correlation was found between leaf Na⁺ and leaf growth. Leaf ABA significantly increased with salt supply, and was highly correlated with both leaf Na⁺ and leaf growth. Our results suggest that in bean plants under long‐term salt stress, leaf ABA may participate in the regulation of leaf growth, and leaf Na⁺ would be at least partly responsible for increased ABA levels.     

Expression of an Arabidopsis sodium/proton antiporter gene (AtNHX1) in peanut to improve salt tolerance

Salinity is a major environmental stress that affects agricultural productivity worldwide. One approach to improving salt tolerance in crops is through high expression of the Arabidopsis gene AtNHX1, which encodes a vacuolar sodium/proton antiporter that sequesters excess sodium ion into the large intracellular vacuole. Sequestering cytosolic sodium into the vacuoles of plant cells leads to a low level of sodium in cytosol, which minimizes the sodium toxicity and injury to important enzymes in cytosol. In the meantime, the accumulation of sodium in vacuoles restores the correct osmolarity to the intracellular milieu, which favors water uptake by plant root cells and improves water retention in tissues under soils that are high in salt. To improve the yield and quality of peanut under high salt conditions, AtNHX1 was introduced into peanut plants through Agrobacterium-mediated transformation. The AtNHX1-expressing peanut plants displayed increased tolerance of salt at levels up to 150 mM NaCl. When compared to wild-type plants, AtNHX1-expressing peanut plants suffered less damage, produced more biomass, contained more chlorophyll, and maintained higher photosynthetic rates under salt conditions. These data indicate that AtNHX1 can be used to enhance salt tolerance in peanut.     

Limiting cytosolic Na+ confers salt tolerance to rice cells in culture: a two-photon microscopy study of SBFI-loaded cells

Rice ( Oryza sativa L.), a staple food in Asia, is very sensitive to soil salinity. However, intraspecific variations exist, with the coastal cultivar Pokkali tolerating even brackish water. This study explores cellular mechanisms that contribute to salt tolerance in rice. It is widely accepted that limiting cytosolic Na⁺ should improve the survival of plants subjected to saline stress. However, an understanding of the mechanisms by which Na⁺ levels are controlled in relatively tolerant cultivars requires monitoring cytosolic Na⁺ non-invasively and in real time, which is technically challenging. We have used two-photon excitation for the ratiometric estimation of cytosolic Na⁺ in cultured cells using sodium-binding benzofuran isophthalate. Pokkali cells maintained low cytosolic Na⁺ (approximately 25 m M ), and a viability of over 85% under high salinity , while Jaya cells were unable to maintain low cytosolic Na⁺ and suffered decreased viability even at moderate saline stress. Here we show that the permeability of the Pokkali plasma membrane to Na⁺ is significantly lower than that of Jaya, to the extent that it is comparable with permeabilities reported for halophytes. Pokkali effectively sequesters Na⁺ in intracellular compartments utilizing a Ca²⁺-regulated transport system(s). Together these cellular mechanisms allow Pokkali to maintain low cytosolic Na⁺ up to a stress of 250 m M NaCl. The findings demonstrate that differences in survival between these contrasting varieties of rice are mainly because of differences in membrane transport mechanisms and thus have significance in crop improvement.     

Screening plants for salt tolerance by measuring K+ flux: a case study for barley

Development of salt-tolerant genotypes is central both to remediation of salinity-affected land and to meet increasing global food demand, which has been driving expansion of cropping into marginal areas. The bottleneck of any breeding programme is the lack of a reliable screening technique. This study tested the hypothesis that the ability of plants to retain K⁺ under saline conditions is central to their salt tolerance. Using seven barley cultivars contrasting in salt tolerance (CM72, Numar, ZUG293, ZUG95, Franklin, Gairdner, ZUG403), a comprehensive study was undertaken of whole-plant (growth rate, biomass, net CO₂ assimilation, chlorophyll fluorescence, root and leaf elemental and water content) and cellular (net fluxes of H⁺, K⁺, Na⁺ and Ca²⁺) responses to various concentrations of NaCl (20–320 m m ). Na⁺ selective microelectrodes were found to be unsuitable for screening purposes because of non-ideal selectivity of the commercially available Na⁺ LIX. At the same time, our results show very strong negative correlation between the magnitude of K⁺ efflux from the root and salt tolerance of a particular cultivar. K⁺ efflux from the mature root zone of intact 3-day-old seedlings following 40 min pretreatment with 80 m m NaCl was found to be a reliable screening indicator for salinity tolerance in barley. As a faster and more cost-effective alternative to microelectrode measurements, a procedure was developed enabling rapid screening of large numbers of seedlings, based on amount of K⁺ leaked from plant roots after exposure to NaCl.     

Na+/H+ antiport activity in tonoplast vesicles from roots of the salt-tolerant Plantago maritima and the salt-sensitive Plantago media

Plantago species differ in their strategy towards salt stress, a major difference being the uptake and distribution of Na⁺ ions. A salt-sensitive ( Plantago media L.) and a salt-tolerant ( P. maritima L.) species were compared with respect to Na⁺/H⁺ antiport activities at the tonoplast. After exposure of the plants to 50 m M NaCl for 6 days isolated tonoplast vesicles of P. maritima showed Na⁺/H⁺ antiport activity with saturation kinetics and a K_m of 2.4 m M Na⁺, NaCl-grown P. media and the control plants of both species showed no antiport activity. Selectivity of the antiport system for Na⁺ was high and was determined by adding different chloride salts after formation of a Δ pH in the vesicles. Specific tonoplast ATPase activities were similar in the two species and did not alter after exposure to NaCl stress.     

Effects of salinity and phosphate on ion distribution in lupin leaflets

Lupin ( Lupinus luteus L. cv. Weiko III) were grown in nutrient solution over a range of inorganic phosphate (P_i) concentrations, with or without 50 m M NaCl. Plants with high P_i (2 m M ) and salt showed progressive leaf necrosis and had higher concentrations of total phosphate than plants grown with high P_i alone. Most of the extra total phosphate in salt treated plants was in the P_i form. P_i supply did not influence Na⁺, K⁺ or Cl⁻ concentrations in epidermal vacuoles or mesophyll cells. However, epidermal vacuoles accumulated more monovalent cations (Na⁺ and K⁺) than Cl⁻, and in vacuoles of plants grown with 0.1 m M P_i additional P_i was accumulated, possibly to maintain charge balance. Plants grown with 2 m M P_i did not accumulate additional P_i in epidermal vacuoles, but showed higher phosphorus levels in cell walls. It is suggested that at moderate phosphorus concentrations P_i plays a role in epidermal osmotic adjustment, possibly explaining the beneficial role of additional phosphorus on salt stressed plants. At high P_i supply with salt, P_i does not contribute to osmotic adjustment and instead accumulates in cell walls. However, the cause of leaf damage under conditions of high phosphorus supply and salinity is still not entirely clear.