Overexpression of SlSOS2 (SlCIPK24) confers salt tolerance to transgenic tomato

The Ca(2+)-dependent SOS pathway has emerged as a key mechanism in the homeostasis of Na(+) and K(+) under saline conditions. We have identified and functionally characterized the gene encoding the calcineurin-interacting protein kinase of the SOS pathway in tomato, SlSOS2. On the basis of protein sequence similarity and complementation studies in yeast and Arabidopsis, it can be concluded that SlSOS2 is the functional tomato homolog of Arabidopsis AtSOS2 and that SlSOS2 operates in a tomato SOS signal transduction pathway. The biotechnological potential of SlSOS2 to provide salt tolerance was evaluated by gene overexpression in tomato (Solanum lycopersicum L. cv. MicroTom). The better salt tolerance of transgenic plants relative to non-transformed tomato was shown by their faster relative growth rate, earlier flowering and higher fruit production when grown with NaCl. The increased salinity tolerance of SlSOS2-overexpressing plants was associated with higher sodium content in stems and leaves and with the induction and up-regulation of the plasma membrane Na(+)/H(+) (SlSOS1) and endosomal-vacuolar K(+), Na(+)/H(+) (LeNHX2 and LeNHX4) antiporters, responsible for Na(+) extrusion out of the root, active loading of Na(+) into the xylem, and Na(+) and K(+) compartmentalization.     

The K+/H+ antiporter LeNHX2 increases salt tolerance by improving K+ homeostasis in transgenic tomato

The endosomal LeNHX2 ion transporter exchanges H⁺ with K⁺ and, to lesser extent, Na⁺. Here, we investigated the response to NaCl supply and K⁺ deprivation in transgenic tomato (Solanum lycopersicum L.) overexpressing LeNHX2 and show that transformed tomato plants grew better in saline conditions than untransformed controls, whereas in the absence of K⁺ the opposite was found. Analysis of mineral composition showed a higher K⁺ content in roots, shoots and xylem sap of transgenic plants and no differences in Na⁺ content between transgenic and untransformed plants grown either in the presence or the absence of 120 mm NaCl. Transgenic plants showed higher Na⁺/H⁺ and, above all, K⁺/H⁺ transport activity in root intracellular membrane vesicles. Under K⁺ limiting conditions, transgenic plants enhanced root expression of the high‐affinity K⁺ uptake system HAK5 compared to untransformed controls. Furthermore, tomato overexpressing LeNHX2 showed twofold higher K⁺ depletion rates and half cytosolic K⁺ activity than untransformed controls. Under NaCl stress, transgenic plants showed higher uptake velocity for K⁺ and lower cytosolic K⁺ activity than untransformed plants. These results indicate the fundamental role of K⁺ homeostasis in the better performance of LeNHX2 overexpressing tomato under NaCl stress.     

Expression analysis of LeNHX1 gene in mycorrhizal tomato under salt stress

The plant growth, stem sap flow, Na⁺ and Cl^? content, and the expression of vacuolar Na⁺/H⁺ antiporter gene (LeNHX1) in the leaves and roots of tomato under different NaCl stresses (0.5% and 1%) were studied to analyze the effect of arbuscular mycorrhizal fungi (AMF) on Na⁺ and Cl^? accumulation and ion exchange. The results showed that arbuscular mycorrhizal (AM) plant growth and stem sap flow increased and salt tolerance improved, whereas Na⁺ and Cl^? accumulated. Na⁺ significantly decreased, and no significant decline was detected in Cl^? content after AMF inoculation compared with the non-AM plants. The LeNHX1 gene expression was induced in the AM and non-AM plants by NaCl stress. However, AMF did not improve the LeNHX1 level, and low expression was observed in the AM tomato. Hence, the mechanism that reduced the Na⁺ damage to tomato induced by AMF has little relation to LeNHX1, which can export Na⁺ from the cytosol to the vacuole across the tonoplast.     

Genetic analysis of Na+ and K+ concentrations in leaf and stem as physiological components of salt tolerance in Tomato

The sodium and potassium concentrations in leaf and stem have been genetically studied as physiological components of the vegetative and reproductive development in two populations of F₈ lines, derived from a salt sensitive genotype of Solanum lycopersicum cv. Cerasiforme, as female parent, and two salt tolerant lines, as male parents, from S. pimpinellifolium, the P population (142 lines), and S. cheesmaniae, the C population (116 lines). Genetic parameters of ten traits under salinity and five of them under control conditions were studied by ANOVA, correlation, principal component and QTL analysis to understand the global response of the plant. Two linkage maps including some tomato flowering time and salt tolerance candidate genes encoding for SlSOS1, SlSOS2, SlSOS3, LeNHX1, LeNHX3, were used for the QTL detection. Thirteen and 20 QTLs were detected under salinity in the P and C populations, respectively, and four under control conditions. Highly significant and contributing QTLs (over 40%) for the concentrations of Na⁺ and K⁺ in stems and leaves have been detected on chromosome 7 in both the populations. This is the only genomic position where the concentration QTLs for both the cations locate together. The proportion of QTLs significantly affected by salinity was larger in the P population (64.3%, including all QTLs detected under control) than in the C population (21.4%), where the estimated genetic component of variance was larger for most traits. A highly significant association between the leaf area and fruit yield under salinity was found only in the C population, which is supported by the location of QTLs for these traits in a common region of chromososome C1. As far as breeding for salt tolerance is concerned, only two sodium QTLs (lnc1.1 and lnc8.1) map in genomic regions of C1 and C8 where fruit yield QTLs are also located but in both the cases the profitable allele corresponds to the salt sensitive, cultivated species. One of those QTLs, lnc1.1 might involve LeNHX3.     

Effect of water stress on yield and nutrition quality of tomato plant overexpressing StAPX

We investigated the effect of water stress on yield and quality of tomato plants overexpressing Solanum lycopersicum thylakoid-bound ascorbate peroxidase gene (StAPX). APX activity, hydrogen peroxide content, net photosynthetic rate of tomato leaves, and yield and nutrition quality of tomato fruits were measured under soil moisture 70, 60, and 50 % of full field capacity. Results show that the capability of APX for scavenging hydrogen peroxide induced by water stress was higher in the transgenic than the wild type (WT) plants. The yield of fruits of the transgenic tomato plants was higher than that of WT plants under water stress and the fruit nutrition quality was not different. These results indicate that overexpression of StAPX might improve water stress tolerance in the transgenic tomato plants.     

Overexpression of GlyI and GlyII genes in transgenic tomato (Solanum lycopersicum Mill.) plants confers salt tolerance by decreasing oxidative stress

The glyoxalase system plays an important role in various physiological processes in plants, including salt stress tolerance. We report the effects of overexpressing glyoxalase I and glyoxalase II genes in transgenic tomato (Solanum lycopersicum Mill.) cv. Ailsa Craig. Stable expression of both transgenes was detected in the transformed tomato plants under salt stress. The transgenic lines overexpressing GlyI and GlyII under a high NaCl concentration (800 mM) showed reduced lipid peroxidation and the production of H₂O₂ in leaf tissues. A greater decrease in the chlorophyll a+b content in wild-type (WT) compared with transgenic lines was also observed. These results suggest that the over expression of two genes, GlyI and GlyII, may enhance salt stress tolerance by decreasing oxidative stress in transformed tomato plants. This work will help our understanding of the putative role of the glyoxalase system in the tolerance to abiotic stress in tomato plants.     

Ectopic expression of wheat expansin gene TaEXPA2 improved the salt tolerance of transgenic tobacco by regulating Na+/K+ and antioxidant competence

High salinity is one of the most serious environmental stresses that limit crop growth. Expansins are cell wall proteins that regulate plant development and abiotic stress tolerance by mediating cell wall expansion. We studied the function of a wheat expansin gene, TaEXPA2, in salt stress tolerance by overexpressing it in tobacco. Overexpression of TaEXPA2 enhanced the salt stress tolerance of transgenic tobacco plants as indicated by the presence of higher germination rates, longer root length, more lateral roots, higher survival rates and more green leaves under salt stress than in the wild type (WT). Further, when leaf disks of WT plants were incubated in cell wall protein extracts from the transgenic tobacco plants, their chlorophyll content was higher under salt stress, and this improvement from TaEXPA2 overexpression in transgenic tobacco was inhibited by TaEXPA2 protein antibody. The water status of transgenic tobacco plants was improved, perhaps by the accumulation of osmolytes such as proline and soluble sugar. TaEXPA2‐overexpressing tobacco lines exhibited lower Na⁺ but higher K⁺ accumulation than WT plants. Antioxidant competence increased in the transgenic plants because of the increased activity of antioxidant enzymes. TaEXPA2 protein abundance in wheat was induced by NaCl, and ABA signaling was involved. Gene expression regulation was involved in the enhanced salt stress tolerance of the TaEXPA2 transgenic plants. Our results suggest that TaEXPA2 overexpression confers salt stress tolerance on the transgenic plants, and this is associated with improved water status, Na⁺/K⁺ homeostasis, and antioxidant competence. ABA signaling participates in TaEXPA2‐regulated salt stress tolerance.     

Overexpression of Rice CBS Domain Containing Protein Improves Salinity, Oxidative, and Heavy Metal Tolerance in Transgenic Tobacco

We have recently identified and classified a cystathionine ??-synthase domain containing protein family in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa L.). Based on the microarray and MPSS data, we have suggested their involvement in stress tolerance. In this study, we have characterized a rice protein of unknown function, OsCBSX4. This gene was found to be upregulated under high salinity, heavy metal, and oxidative stresses at seedling stage. Transgenic tobacco plants overexpressing OsCBSX4 exhibited improved tolerance toward salinity, heavy metal, and oxidative stress. This enhanced stress tolerance in transgenic plants could directly be correlated with higher accumulation of OsCBSX4 protein. Transgenic plants could grow and set seeds under continuous presence of 150?mM NaCl. The total seed yield in WT plants was reduced by 80%, while in transgenic plants, it was reduced only by 15?C17%. The transgenic plants accumulated less Na⁺, especially in seeds and maintained higher net photosynthesis rate and Fv/Fm than WT plants under NaCl stress. Transgenic seedlings also accumulated significantly less H₂O₂ as compared to WT under salinity, heavy metal, and oxidative stress. OsCBSX4 overexpressing transgenic plants exhibit higher abiotic stress tolerance than WT plants suggesting its role in abiotic stress tolerance in plants.     

Overexpression of AaPal,a peptidoglycan-associated lipoprotein from Alkalomonas amylolytica,improves salt and alkaline tolerance of Escherichia coli and Arabidopsis thaliana

The outer membrane lipoprotein, Pal, plays a major role maintaining the integrity of outer membrane and cell morphology in Gram-negative bacteria. Here, we represent A novel role of AaPal in tolerance to salt and alkaline stresses. The cell density of Escherichia coli expressing AaPal was approx. three times as that of control strain when grown in the presence of 1 M NaCl or at pH 9.0 for 14 h, and transgenic Arabidopsis thaliana grew taller and stronger than wild-type plants when subjected to 200 mM NaCl or pH 9.0 stress. This tolerance was attributed to higher concentrations of K⁺ and lower concentrations of Na⁺ in the transgenic organism. Our study provides a potential use of AaPal in the improvement of salt and alkaline tolerance in bacteria and plants.     

The effects of calcium sulphate on growth,membrane stability and nutrient uptake of tomato plants grown under salt stress

A pot experiment was carried out with tomato (Lycopersicon esculentum Mill.) cv. “Target F1” in a mixture of peat, perlite, and sand (1:1:1) to investigate the effects of supplementary calcium sulphate on plants grown at high NaCl concentration (75 mM). The treatments were: (i) control (C), nutrient solution alone; (ii) salt treatment (C + S), 75 mM NaCl; (iii) salt plus calcium treatment 1 (C + S + Ca1), 75 mM NaCl plus additional mixture of 2.5 mM CaSO₄ in nutrient solution; (iv) salt plus calcium treatment 2 (C + S + Ca2), 75 mM NaCl plus additional mixture of 5 mM CaSO₄ in nutrient solution. The plants grown under salt stress produced low dry matter, fruit weight, and relative water content than those grown in standard nutrient solution. Supplemental calcium sulphate added to nutrient solution containing salt significantly improved growth and physiological variables affected by salt stress (e.g. plant growth, fruit yield, and membrane permeability) and also increased leaf K⁺, Ca²⁺, and N in tomato plants. The effects of supplemental CaSO₄ in maintaining membrane permeability, increasing concentrations of Ca²⁺, N, and K⁺ and reducing concentration of Na⁺ (because of cation competition in root zone) in leaves could offer an economical and simple solution to tomato crop production problems caused by high salinity.     

Overexpression of AtNHX5 improves tolerance to both salt and water stress in rice (Oryza sativa L.)

Overexpression of NHX genes has been previously shown to improve salt tolerance of transgenic plants. In this study, transgenic rice plants overexpressing AtNHX5 showed not only high salt tolerance, but also high drought tolerance. Measurements of ion levels indicated that Na⁺ and K⁺ contents were all higher in AtNHX5 overexpressing shoots than in wild type (WT) shoots in high saline conditions. After exposure to water deficiency and salt stress, the WT plants all died, while the AtNHX5 overexpressing rice plants had a higher survival rate, dry weight, leaf water content, and leaf chlorophyll contents, accumulated more proline, and had less membrane damage than the WT plants. In addition, seeds of both transgenic and WT plants germinated on 1/2 MS medium supplemented with 250 mM mannitol, but overexpression of AtNHX5 improved the shoot growth of the seedlings. Taken together, the results indicate that AtNHX5 gene could enhance the tolerance of rice plants to multiple environmental stresses by promoting the accumulation of more effective osmolytes (ions or proline) to counter the osmotic stress caused by abiotic factors.     

Overexpression of a wheat Na+/H+ antiporter gene (TaNHX2) enhances tolerance to salt stress in transgenic tomato plants (Solanum lycopersicum L.)

Soil salinity is a major environmental stress limiting plant productivity. Vacuole Na⁺/H⁺ antiporters play important roles for the survival of plants under salt stress conditions. We have developed salt stress tolerant transgenic tomato plants (Solanum lycopersicum cv. PED) by overexpression of the wheat Na⁺/H⁺ antiporter gene TaNHX2 using Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBin438 that contains the TaNHX2 gene under the control of double CaMV 35S promoter and npt II as a selectable marker. PCR and Southern blot analysis confirmed that TaNHX2 gene has been integrated and expressed in the T₁ generation transgenic tomato plants. When TaNHX2 expressing plants were exposed to 100 or 150 mM NaCl, they were found to be more tolerant to salt stress compared to wild type plants. Biochemical analyses also showed that transgenic plants have substantial amount of relative water content and chlorophyll content under salt stress conditions compared to wild type plants. The relative water content in transgenic and wild type plants ranged from 68 to 75 % and 46–73 % and chlorophyll content fall in between 1.8 to 2.4 mg/g fw and 1.0 to 2.4 mg/g fw, respectively, in all stress conditions. In the present study, we observed a better germination rate of T₁ transgenic seeds under salt stress conditions compared with wild type plants. Our results indicated that TaNHX2-transgenic tomato plants coped better with salt stress than wild type plants.     

Isolated Thellungiella shoots do not require roots to survive NaCl and Na2SO4 salt stresses

Shoots of Thellungiella derived by micropropagation were used to estimate the plants'' salt tolerance and ability to regulate Na⁺ uptake. Two species with differing salt tolerances were studied: Thellungiella salsuginea (halophilla), which is less tolerant, and Thellungiella botschantzevii, which is more tolerant. Although the shoots of neither ecotype survived at 700 mM NaCl or 200 mM Na₂SO₄, micropropagated shoots of T. botschantzevii were more tolerant to Na₂SO₄ (10–100 mM) and NaCl (100–300 mM). In the absence of roots, Na₂SO₄ salinity reduced shoot growth more dramatically than NaCl salinity. Plantlets of both species were able to adapt to salt stress even when they did not form roots. First, there was no significant correlation between Na⁺ accumulation in shoots and Na⁺ concentration in the growth media. Second, K⁺ concentrations in the shoots exposed to different salt concentrations were maintained at equivalent levels to control plants grown in medium without NaCl or Na₂SO₄. These results suggest that isolated shoots of Thellungiella possess their own mechanisms for enabling salt tolerance, which contribute to salt tolerance in intact plants.Key words: Thellungiella salsuginea, Thellungiella botschantzevii, salt tolerance, isolated shoots, growth, rhizogenesis, ion accumulation     

Co-expression of the Arabidopsis SOS genes enhances salt tolerance in transgenic tall fescue (Festuca arundinacea Schreb.)

Crop productivity is greatly affected by soil salinity; therefore, improvement in salinity tolerance of crops is a major goal in salt-tolerant breeding. The Salt Overly Sensitive (SOS) signal-transduction pathway plays a key role in ion homeostasis and salt tolerance in plants. Here, we report that overexpression of Arabidopsis thaliana SOS1+SOS2+SOS3 genes enhanced salt tolerance in tall fescue. The transgenic plants displayed superior growth and accumulated less Na⁺ and more K⁺ in roots after 350 mM NaCl treatment. Moreover, Na⁺ enflux, K⁺ influx, and Ca²⁺ influx were higher in the transgenic plants than in the wild-type plants. The activities of the enzyme superoxide dismutase, peroxidase, catalase, and proline content in the transgenic plants were significantly increased; however, the malondialdehyde content decreased in transgenic plants compared to the controls. These results suggested that co-expression of A. thaliana SOS1+SOS2+SOS3 genes enhanced the salt tolerance in transgenic tall fescue.     

Influence of exogenous application of glycinebetaine on antioxidative system and growth of salt-stressed soybean seedlings (Glycine max L.)

Glycinebetaine is one of the most competitive compounds which play an important role in salt stress in plants. In this study, the enhanced salt tolerance in soybean (Glycine max L.) by exogenous application of glycinebetaine was evaluated. To improve salt tolerance at the seedling stage, GB was applied in four different concentrations (0, 5, 25 and 50 mM) as a pre-sowing seed treatment. Salinity stress in the form of a final concentration of 150 mM sodium chloride (NaCl) over a 15 day period drastically affected the plants as indicated by increased proline, MDA and Na⁺ content of soybean plants. In contrast, supplementation with 50 mM GB improved growth of soybean plants under NaCl as evidenced by a decrease in proline, MDA and Na⁺ content of soybean plants. Further analysis showed that treatments with GB, resulted in increasing of CAT and SOD activity of soybean seedlings in salt stress. We propose that the role of GB in increasing tolerance to salinity stress in soybean may result from either its antioxidant capacity by direct scavenging of H₂O₂ or its role in activating CAT activity which is mandatory in scavenging H₂O₂.     

<Emphasis Type="Italic">OsNHX2</Emphasis>, an Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene,can enhance salt tolerance in rice plants through more effective accumulation of toxic Na<Superscript>+</Superscript> in leaf mesophyll and bundle sheath cells

The Na⁺/H⁺ antiporters play an important role in salt tolerance in plants. However, the functions of OsNHXs in rice except OsNHX1 have not been well studied. Using the gain- and loss-of-function strategies, we studied the potential role of OsNHX2 in salt tolerance in rice. Overexpression of OsNHX2 (OsNHX2-OE) in rice showed the significant tolerance to salt stress than wild-type plants and OsNHX2 knockdown transgenic plants (OsNHX2-KD). Under salt treatments of 300-mM NaCl for 5 days, the plant fresh weights, relative water percentages, shoot heights, Na⁺ contents, K⁺ contents, and K⁺/Na⁺ ratios in leaves of OsNHX2-OE transgenic plants were higher than those in wild-type plants, while no differences were detected in roots. K⁺/Na⁺ ratios in rice leaf mesophyll cells and bundle sheath cells were higher in OsNHX2-OE transgenic plants than in wild-type plants and OsNHX2-KD transgenic plants. Our data indicate that OsNHX2 plays an important role in salt stress based on leaf mesophyll cells and bundle sheath cells and can be served in genetically engineering crop plants with enhanced salt tolerance.     

Expressing the yeast HAL1 gene in tomato increases fruit yield and enhances K+/Na+ selectivity under salt stress

The yeast HAL1 gene facilitates K⁺/Na⁺ selectivity and salt tolerance of cells. Ectopic expression of HAL1 in transgenic tomato (Lycopersicon esculentum Mill.) plants minimized the reduction in fruit production caused by salt stress. Maintenance of fruit production by transgenic plants was correlated with enhanced growth under salt stress of calli derived from the plants. The HAL1 transgene enhanced water and K⁺ contents in both leaf calli and leaves in the presence of salt, which indicates that HAL1 functions in plants using a similar mechanism to that in yeast, namely by facilitating K⁺/Na⁺ selectivity under salt stress.