Enhanced salinity tolerance and improved yield properties in Bangladeshi rice Binnatoa through <Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated transformation of <Emphasis Type="Italic">PgNHX1</Emphasis> from <Emphasis Type="Italic">Pennisetum glaucum</Emphasis>

Rice yield is severely affected by high-salt concentration in the vicinity of the plant. In an effort to engineer rice for improved salt tolerance Agrobacterium-mediated transformation of rice cv. Binnatoa was accomplished with the Pennisetum glaucum vacuolar Na⁺/H⁺ antiporter gene (PgNHX1) under the constitutive CaMV35S promoter. For the molecular analysis of putative transgenic plants, PCR and RT-PCR were performed. Transgenic rice plants expressing PgNHX1 showed better physiological status and completed their life cycle by setting flowers and seeds in salt stress, while wild-type plants exhibited rapid chlorosis and growth inhibition. Moreover, transgenic rice plants produced higher grain yields than wild-type plants under salt stress. Assessment of the salinity tolerance of the transgenic plants at seedling and reproductive stages demonstrated the potential of PgNHX1 for imparting enhanced salt tolerance capabilities and improved yield.     

<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.     

Enhanced Expression of <Emphasis Type="Italic">AtNHX1</Emphasis>, in Transgenic Groundnut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.) Improves Salt and Drought Tolerence

Salinity and drought are main threat to agriculture productivity, to avoid further losses it is necessary to improve the genetic material of crops against these stresses In this present study, AtNHX1, a vacuolar type Na⁺/H⁺ antiporter gene driven by 35S promoter was introduced into groundnut using Agrobacterium tumefaciens transformation system. The stable integration of the AtNHX1 gene was confirmed by polymerase chain reaction (PCR) and southern blot analysis. It was found that transgenic plants having AtNHX1 gene are more resistant to high concentration of salt and water deprivation than the wild type plants. Salt and proline level in the leaves of the transgenic plants were also much higher than that of wild type plants. The results showed that overexpression of AtNHX1 gene not only improved salt tolerance but also drought tolerance in transgenic groundnut. Our results suggest that these plants could be cultivated in salt and drought-affected soils.     

Ectopic expression of PgRab7 in rice plants (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) results in differential tolerance at the vegetative and seed setting stage during salinity and drought stress

In this work, we have overexpressed a vesicle trafficking protein, Rab7, from a stress-tolerant plant, Pennisetum glaucum, in a high-yielding but stress-sensitive rice variety Pusa Basmati-1 (PB-1). The transgenic rice plants were tested for tolerance against salinity and drought stress. The transgenic plants showed considerable tolerance at the vegetative stage against both salinity (200 mM NaCl) and drought stress (up to 12 days after withdrawing water). The protection against salt and drought stress may be by regulating Na⁺ ion homeostasis, as the transgenic plants showed altered expression of multiple transporter genes, including OsNHX1, OsNHX2, OsSOS1, OsVHA, and OsGLRs. In addition, decreased generation and maintenance of lesser reactive oxygen species (ROS), with maintenance of chloroplast grana and photosynthetic machinery was observed. When evaluated for reproductive growth, 89–96 % of seed setting was maintained in transgenic plants during drought stress; however, under salt stress, a 33–53 % decrease in seed setting was observed. These results indicate that PgRab7 overexpression in rice confers differential tolerance at the seed setting stage during salinity and drought stress and could be a favored target for raising drought-tolerant crops.     

Overexpression of a <Emphasis Type="Italic">Malus</Emphasis> vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene (<Emphasis Type="Italic">MdNHX1</Emphasis>) in apple rootstock M.26 and its influence on salt tolerance

Soil salinity is a major factor limiting apple production in some areas. Tonoplast Na⁺/H⁺ antiporters play a critical role in salt tolerance. Here, we isolated MdNHX1, a vacuolar Na⁺/H⁺ antiporter from Luo-2, a salt-tolerant rootstock of apple (Malus × domestica Borkh.), and introduced it into apple rootstock M.26 by Agrobacterium-mediated transformation. PCR and DNA gel blot analyses confirmed successful integration of MdNHX1. RT-PCR analysis indicated that the gene was highly expressed in transgenic plants, but the degree of this expression varied among lines. Its overexpression conferred high tolerance to salt stress. Analysis of ion contents showed that, when exposed to salinity stress, the transgenics compartmentalized more Na⁺ in the roots and also maintained a relatively high K⁺/Na⁺ ratio in the leaves compared with non-transformed plants. Under normal conditions, however, amounts of potassium and sodium did not differ significantly between transgenic and control plants.     

Overexpression of a <Emphasis Type="Italic">Panax ginseng</Emphasis> tonoplast aquaporin alters salt tolerance,drought tolerance and cold acclimation ability in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> plants

Water movement across cellular membranes is regulated largely by a family of water channel proteins called aquaporins (AQPs). Since several abiotic stresses such as, drought, salinity and freezing, manifest themselves via altering water status of plant cells and are linked by the fact that they all result in cellular dehydration, we overexpressed an AQP (tonoplast intrinsic protein) from Panax ginseng, PgTIP1, in transgenic Arabidopsis thaliana plants to test its role in plant’s response to drought, salinity and cold acclimation (induced freezing tolerance). Under favorable conditions, PgTIP1 overexpression significantly increased plant growth as determined by the biomass production, and leaf and root morphology. PgTIP1 overexpression had beneficial effect on salt-stress tolerance as indicated by superior growth status and seed germination of transgenic plants under salt stress; shoots of salt-stressed transgenic plants also accumulated greater amounts of Na⁺ compared to wild-type plants. Whereas PgTIP1 overexpression diminished the water-deficit tolerance of plants grown in shallow (10 cm deep) pots, the transgenic plants were significantly more tolerant to water stress when grown in 45 cm deep pots. The rationale for this contrasting response, apparently, comes from the differences in the root morphology and leaf water channel activity (speed of dehydration/rehydration) between the transgenic and wild-type plants. Plants overexpressed with PgTIP1 exhibited lower (relative to wild-type control) cold acclimation ability; however, this response was independent of cold-regulated gene expression. Our results demonstrate a significant function of PgTIP1 in growth and development of plant cells, and suggest that the water movement across tonoplast (via AQP) represents a rate-limiting factor for plant vigor under favorable growth conditions and also significantly affect responses of plant to drought, salt and cold stresses.     

Evaluation of salinity tolerance and analysis of allelic function of <Emphasis Type="Italic">HvHKT1</Emphasis> and <Emphasis Type="Italic">HvHKT2</Emphasis> in Tibetan wild barley

Tibetan wild barley is rich in genetic diversity with potential allelic variation useful for salinity-tolerant improvement of the crop. The objectives of this study were to evaluate salinity tolerance and analysis of the allelic function of HvHKT1 and HvHKT2 in Tibetan wild barley. Salinity tolerance of 189 Tibetan wild barley accessions was evaluated in terms of reduced dry biomass under salinity stress. In addition, Na⁺ and K⁺ concentrations of 48 representative accessions differing in salinity tolerance were determined. Furthermore, the allelic and functional diversity of HvHKT1 and HvHKT2 was determined by association analysis as well as gene expression assay. There was a wide variation among wild barley genotypes in salt tolerance, with some accessions being higher in tolerance than cultivated barley CM 72, and salinity tolerance was significantly associated with K⁺/Na⁺ ratio. Association analysis revealed that HvHKT1 and HvHKT2 mainly control Na⁺ and K⁺ transporting under salinity stress, respectively, which was validated by further analysis of gene expression. The present results indicated that Tibetan wild barley offers elite alleles of HvHKT1 and HvHKT2 conferring salinity tolerance.     

Molecular Cloning and Functional Analysis of a Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Antiporter Gene <Emphasis Type="Italic">ThNHX1</Emphasis> from a Halophytic Plant <Emphasis Type="Italic">Thellungiella halophila</Emphasis>

Na⁺/H⁺ exchanger catalyzes the countertransport of Na⁺ and H⁺ across membranes. Using the rapid amplification of cDNA ends method, a Na⁺/H⁺ antiporter gene (ThNHX1) was isolated from a halophytic plant, salt cress (Thellungiella halophila). The deduced amino acid sequence contained 545 amino acid residues with a conserved amiloride-binding domain (⁸⁷LFFIYLLPPI⁹⁶) and shared more than 94% identity with that of AtNHX1 from Arabidopsis thaliana. The ThNHX1 mRNA level was upregulated by salt and other stresses (abscisic acid, polyethylene glycol, and high temperature). This gene partially complemented the Na⁺/Li⁺-sensitive phenotype of a yeast mutant that was deficient in the endosomal–vacuolar Na⁺/H⁺ antiporter ScNHX1. Overexpression of ThNHX1 in Arabidopsis increased salt tolerance of transgenic plants compared with the wild-type plants. In addition, the silencing of ThNHX1 gene in T. halophila caused the transgenic plants to be more salt and osmotic sensitive than wild-type plant. Together, these results suggest that ThNHX1 may function as a tonoplast Na⁺/H⁺ antiporter and play an important role in salt tolerance of T. halophila. Chunxia Wu, Xiuhua Gao, and Xiangqiang Kong contributed equally to this work.     

Improved salt tolerance in tobacco plants by co-transformation of a betaine synthesis gene <Emphasis Type="Italic">BADH</Emphasis> and a vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene <Emphasis Type="Italic">SeNHX1</Emphasis>

Three types of transgenic tobacco plants were acquired by separate transformation or co-transformation of a vacuolar Na⁺/H⁺ antiporter gene, SeNHX1, and a betaine synthesis gene, BADH. When exposed to 200 mM NaCl, the dual gene-transformed plants displayed greater accumulation of betaine and Na⁺ than their wild-type counterparts. Photosynthetic rate and photosystem II activity in the transgenic plants were less affected by salt stress than wild-type plants. Transgenic plants exhibited a greater increase in osmotic pressure than wild-type plants when exposed to NaCl. More importantly, the dual gene transformed plants accumulated higher biomass than either of the single transgenic plants under salt stress. Taken together, these findings indicate that simultaneous transformation of BADH and SeNHX1 genes into tobacco plants can enable plants to accumulate betaine and Na⁺, thus conferring them more tolerance to salinity than either of the single gene transformed plants or wild-type tobacco plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Increased tolerance to salt stress in the phosphate-accumulating <Emphasis Type="Italic">Arabidopsis</Emphasis> mutants <Emphasis Type="Italic">siz1</Emphasis> and <Emphasis Type="Italic">pho2</Emphasis>

High salinity is an environmental factor that inhibits plant growth and development, leading to large losses in crop yields. We report here that mutations in SIZ1 or PHO2, which cause more accumulation of phosphate compared with the wild type, enhance tolerance to salt stress. The siz1 and pho2 mutations reduce the uptake and accumulation of Na⁺. These mutations are also able to suppress the Na⁺ hypersensitivity of the sos3-1 mutant, and genetic analyses suggest that SIZ1 and SOS3 or PHO2 and SOS3 have an additive effect on the response to salt stress. Furthermore, the siz1 mutation cannot suppress the Li⁺ hypersensitivity of the sos3-1 mutant. These results indicate that the phosphate-accumulating mutants siz1 and pho2 reduce the uptake and accumulation of Na⁺, leading to enhanced salt tolerance, and that, genetically, SIZ1 and PHO2 are likely independent of SOS3-dependent salt signaling.     

Overexpression of <Emphasis Type="Italic">Zm-HINT1</Emphasis> Confers Salt and Drought Tolerance in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Histidine triad nucleotide-binding protein 1 (HINT1) is highly conserved in many species and plays important roles in various biological processes. However, little is known about the responses of HINT1 to abiotic stress in plants. Salt and drought stress are major limiting factors for plant growth and development, and their negative effects on crop productivity may threaten the world’s food supply. Previously, we identified a maize gene, Zm-HINT1, which encodes a 138-amino-acid protein containing conserved domains including the HIT motif, helical regions, and β-strands. Here, we demonstrate that overexpression of Zm-HINT1 in Arabidopsis confers salt and drought tolerance to plants. Zm-HINT1 significantly regulated Na⁺ and K⁺ accumulation in plants under salt stress. The improve tolerance characteristics of Arabidopsis plants that were overexpressing Zm-HINT1 led to increased survival rates after salt and drought treatments. Compared with control plants, those plants that overexpressed Zm-HINT1 showed increased proline content and superoxide dismutase activity, as well as lower malondialdehyde and hydrogen peroxide accumulation under salt and drought treatments. The expression patterns of stress-responsive genes in Arabidopsis plants that overexpressed Zm-HINT1 significantly differed from those in control lines. Taken together, these results suggest that Zm-HINT1 has potential applications in breeding and genetic engineering strategies that are designed to produce new crop varieties with improved salt and drought tolerance.     

Overexpression of <Emphasis Type="Italic">Thellungiella halophila</Emphasis> H<Superscript>+</Superscript>-PPase (<Emphasis Type="Italic">TsVP</Emphasis>) in cotton enhances drought stress resistance of plants

An H⁺-PPase gene, TsVP from Thellungiella halophila, was transferred into two cotton (Gossypium hirsutum) varieties (Lumianyan19 and Lumianyan 21) and southern and northern blotting analysis showed the foreign gene was integrated into the cotton genome and expressed. The measurement of isolated vacuolar membrane vesicles demonstrated that the transgenic plants had higher V–H⁺-PPase activity compared with wild-type plants (WT). Overexpressing TsVP in cotton improved shoot and root growth, and transgenic plants were much more resistant to osmotic/drought stress than the WT. Under drought stress conditions, transgenic plants had higher chlorophyll content, improved photosynthesis, higher relative water content of leaves and less cell membrane damage than WT. We ascribe these properties to improved root development and the lower solute potential resulting from higher solute content such as soluble sugars and free amino acids in the transgenic plants. In this study, the average seed cotton yields of transgenic plants from Lumianyan 19 and Lumianyan 21 were significantly increased compared with those of WT after exposing to drought stress for 21 days at flowering stage. The average seed cotton yields were 51 and 40% higher than in their WT counterparts, respectively. This study benefits efforts to improve cotton yields in arid and semiarid regions.     

Molecular characterization and functional analysis of a vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene (<Emphasis Type="Italic">HcNHX1</Emphasis>) from <Emphasis Type="Italic">Halostachys caspica</Emphasis>

According to sequences of several vacuolar Na⁺/H⁺ antiporter genes from Xinjiang halophytic plants, a new vacuolar Na⁺/H⁺ antiporter gene (HcNHX1) from the halophyte Halostachys caspica was obtained by RACE and RT-PCR using primers corresponding to conserved regions of the coding sequences. The obtained HcNHX1 cDNA was 1,983 bp and contained a 1,656 bp open reading frame encoding a deduced protein of 551 amino acid residues. The deduced amino acid sequence showed high identity with other NHX1 we have cloned previously from halophyte in Xinjiang desert area. The phylogenetic analysis showed that HcNHX1 formed a clade with NHX homologs of Chenopodiaceae. Expression profiles under salt treatment and ABA induction were investigated, and the results revealed that expression of HcNHX1 was induced by NaCl and ABA. To compare the degree of salt tolerance, we over-expressed HcNHX1 in Arabidopsis. Two transgenic lines grew more vigorously than the wild type (WT) under salt stress. The analysis of ion contents indicated that under salt stress, the transgenic plants compartmentalized more Na⁺ in the leaves compared with wild-type plants. Together, these results suggest that the products of the novel gene HcNHX1 from halophyte Halostachys caspica is a functional tonoplast Na⁺/H⁺ antiporter.     

Contribution of <Emphasis Type="Italic">Glomus intraradices</Emphasis> inoculation to nutrient acquisition and mitigation of ionic imbalance in NaCl-stressed <Emphasis Type="Italic">Trigonella foenum-graecum</Emphasis>

The study aimed to investigate the effects of an AM fungus (Glomus intraradices Schenck and Smith) on mineral acquisition in fenugreek (Trigonella foenum-graecum) plants under different levels of salinity. Mycorrhizal (M) and non-mycorrhizal (NM) fenugreek plants were subjected to four levels of NaCl salinity (0, 50, 100, and 200 mM NaCl). Plant tissues were analyzed for different mineral nutrients. Leaf senescence (chlorophyll concentration and membrane permeability) and lipid peroxidation were also assessed. Under salt stress, M plants showed better growth, lower leaf senescence, and decreased lipid peroxidation as compared to NM plants. Salt stress adversely affected root nodulation and uptake of NPK. This effect was attenuated in mycorrhizal plants. Presence of the AM fungus prevented excess uptake of Na⁺ with increase in NaCl in the soil. It also imparted a regulatory effect on the translocation of Na⁺ ions to shoots thereby maintaining lower Na⁺ shoot:root ratios as compared to NM plants. Mycorrhizal colonization helped the host plant to overcome Na⁺-induced Ca²⁺ and K⁺ deficiencies. M plants maintained favorable K⁺:Na⁺, Ca²⁺:Na⁺, and Ca²⁺:Mg²⁺ ratios in their tissues. Concentrations of Cu, Fe, and Zn²⁺ decreased with increase in intensity of salinity stress. However, at each NaCl level, M plants had higher concentration of Cu, Fe, Mn²⁺, and Zn²⁺ as compared to NM plants. M plants showed reduced electrolyte leakage in leaves as compared to NM plants. The study suggests that AM fungi contribute to alleviation of salt stress by mitigation of NaCl-induced ionic imbalance thus maintaining a favorable nutrient profile and integrity of the plasma membrane.     

Improved salt tolerance in a wheat stay-green mutant <Emphasis Type="Italic">tasg1</Emphasis>

Salt stress inhibited the growth of both tasg1 and wild-type (WT) wheat seedlings, but the inhibition in tasg1 plants was relatively weaker than that of WT. Compared to the WT, the chlorophyll content, thylakoid membrane polypeptides, Hill reaction activity, actual photochemical efficiency of PSII (ΦPSII), and Mg²⁺- and Ca²⁺-ATPase activities were higher in tasg1 under salt stress. At the same time, the photosynthetic activity of the tasg1 was significantly higher than that of WT. In addition, tasg1 plants displayed relatively less accumulation of reactive oxygen species and oxidative damage accompanied by higher activity of some antioxidant enzymes, and the up-regulation of antioxidant genes further demonstrated the improvement of antioxidant activity in tasg1 under salt stress. Furthermore, tasg1 plants also showed relatively weaker Na⁺ fluorescence and lower Na⁺ content, but relatively higher content of K⁺ in their roots and shoots, and then, the roots of tasg1 plants enhanced net outward Na⁺ flux and a correspondingly increased net inward K⁺ flux during salt stress. This might be associated with the relatively higher activity of H⁺-ATPase in tasg1 plants. These results suggest that the improved antioxidant competence and Na⁺/K⁺ ion homeostasis play an important role in the enhanced salinity tolerance of tasg1 plants.     

Over-expression of a vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene improves salt tolerance in an upland rice

To develop a salt-tolerant upland rice cultivar (Oryza sativa L.), OsNHX1, a vacuolar-type Na⁺/H⁺ antiporter gene from rice was transferred into the genome of an upland rice cultivar (IRAT109), using an Agrobacterium-mediated method. Seven independent transgenic calli lines were identified by polymerase chain reaction (PCR) analysis. These 35S::OsNHX1 transgenic plants displayed a little accelerated growth during seedling stage but showed delayed flowering time and a slight growth retardation phenotype during late vegetative stage, suggesting that the OsNHX1 has a novel function in plant development. Northern and western blot analyses showed that the expression levels of OsNHX1 mRNA and protein in the leaves of three independent transgenic plant lines were significantly higher than in the leaves of wild type (WT) plants. T₂ generation plants exhibited increased salt tolerance, showing delayed appearance and development of damage or death caused by salt stress, as well as improved recovery upon removal from this condition. Several physiological traits, such as increased Na⁺ content, and decreased osmotic potential in transgenic plants grown in high saline concentrations, further indicated that the transgenic plants had enhanced salt tolerance. Our results suggest the potential use of these transgenic plants for further agricultural applications in saline soil.     

Contributions of inorganic ions,soluble carbohydrates,and multiatomic alcohols to water homeostasis in <Emphasis Type="Italic">Artemisia lerchiana</Emphasis> and <Emphasis Type="Italic">A. pauciflora</Emphasis>

Two morphological forms of wormwood Artemisia lerchiana (f. erecta and f. nutans) and A. pauciflora Web. (morphological form erecta) were grown on sand culture at a range of NaCl concentrations in the nutrient medium and then assayed for Na⁺, K⁺, and Cl^? content in various organs. In addition, the content of mono-, di-, and trisaccharides and multiatomic alcohols (mannitol and glycerol); water content; and organ biomass were determined. All plants examined showed high NaCl tolerance, comparable to that of halophytes. They were able to maintain high tissue hydration under conditions of salinity-induced growth suppression. The intracellular osmotic pressure in wormwood organs was mainly determined by the presence of Na⁺, K⁺, and Cl^?, as well as by mono-, di-, and trisaccharides, mannitol, and glycerol. The high content of Na⁺ and Cl^? in wormwood organs was also observed in the absence of salinity, which implies the ability of these organs to absorb ions from diluted NaCl solutions and accumulate ions in cells of their tissues. With the increase in salinity, the content of Na⁺ and Cl^? in roots and leaves increased to even higher levels. It is concluded that the ability of wormwood plants to absorb and accumulate inorganic ions provides for sustainable high intracellular osmotic pressure and, accordingly, low water potential under drought and salinity conditions. Growing plants under high salinity lowered the content of monosaccharides in parallel with accumulation of the trisaccharide raffinose. It is supposed that soluble carbohydrates and multiatomic alcohols are not only significant for osmoregulation but also perform a protective function in wormwood plants. The lower osmotic pressure in root cells compared to that in leaf cells of all plants examined was mainly due to the gradient distribution of K⁺ and Cl^? between roots and leaves. The two Artemisia species and two morphological forms of A. lerchiana did not differ appreciably in the ways of water balance regulation. It is found that different morphologies of two A. lerchiana forms are unrelated to variations in intracellular osmotic and turgor pressures.