Molecular characterization and expression analysis of the Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger gene family in <Emphasis Type="Italic">Medicago truncatula</Emphasis>

One important mechanism plants use to cope with salinity is keeping the cytosolic Na⁺ concentration low by sequestering Na⁺ in vacuoles, a process facilitated by Na⁺/H⁺ exchangers (NHX). There are eight NHX genes (NHX1 through NHX8) identified and characterized in Arabidopsis thaliana. Bioinformatics analyses of the known Arabidopsis genes enabled us to identify six Medicago truncatula NHX genes (MtNHX1, MtNHX2, MtNHX3, MtNHX4, MtNHX6, and MtNHX7). Twelve transmembrane domains and an amiloride binding site were conserved in five out of six MtNHX proteins. Phylogenetic analysis involving A. thaliana, Glycine max, Phaseolus vulgaris, and M. truncatula revealed that each individual MtNHX class (class I: MtNHX1 through 4; class II: MtNHX6; class III: MtNHX7) falls under a separate clade. In a salinity-stress experiment, M. truncatula exhibited ~?20% reduction in biomass. In the salinity treatment, sodium contents increased by 178 and 75% in leaves and roots, respectively, and Cl^? contents increased by 152 and 162%, respectively. Na⁺ exclusion may be responsible for the relatively smaller increase in Na⁺ concentration in roots under salt stress as compared to Cl^?. Decline in tissue K⁺ concentration under salinity was not surprising as some antiporters play an important role in transporting both Na⁺ and K ⁺ . MtNHX1, MtNHX6, and MtNHX7 display high expression in roots and leaves. MtNHX3, MtNHX6, and MtNHX7 were induced in roots under salinity stress. Expression analysis results indicate that sequestering Na⁺ into vacuoles may not be the principal component trait of the salt tolerance mechanism in M. truncatula and other component traits may be pivotal.     

Co-expression of <Emphasis Type="Italic">AtNHX1</Emphasis> and <Emphasis Type="Italic">TsVP</Emphasis> improves the salt tolerance of transgenic cotton and increases seed cotton yield in a saline field

Salinity is one of the major abiotic stressors affecting cotton production. The AtNHX1 gene from Arabidopsis thaliana and the TsVP gene from Thellungiella halophila?were co-expressed in cotton (cv. GK35) to improve its salt tolerance. Cotton with overexpressed AtNHX1-TsVP genes had higher emergence rates and higher dry matter accumulation under salt stress in the greenhouse and better emergence rates and survival rates in a saline field compared to the WT. More importantly, the cotton with overexpressed AtNHX1-TsVP genes had higher seed cotton yield in the saline field. The growth of transgenic cotton with overexpression of the AtNHX1-TsVP genes may be related to the accumulation of Na⁺, K⁺ and Ca²⁺ in leaves under salt stress. The accumulation of these cations could improve the ability to maintain ion homeostasis and osmotic potential in plant cells under salt stress, thereby conferring cells with higher relative water content and maintaining higher carbon assimilation capacity. These results reveal that overexpression of AtNHX1-TsVP significantly enhances the tolerance of transgenic cotton to high salinity compared to WT. This study aids efforts of breeding salt-tolerant cotton to achieve the strategy of “westward, eastward, northward” in Chinese cotton production.     

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.     

Overexpression of <Emphasis Type="Italic">AlTMP2</Emphasis> gene from the halophyte grass <Emphasis Type="Italic">Aeluropus littoralis</Emphasis> in transgenic tobacco enhances tolerance to different abiotic stresses by improving membrane stability and deregulating some stress-related genes

Herein, we report isolation of the AlTMP2 gene from the halophytic C4 grass Aeluropus littoralis. The subcellular localization suggested that AlTMP2 is a plasma membrane protein. In A. littoralis exposed to salt and osmotic stresses, the AlTMP2 gene was induced early and at a high rate, but was upregulated relatively later in response to abscisic acid and cold treatments. Expression of AlTMP2 in tobacco conferred improved tolerance against salinity, osmotic, H₂O₂, heat, and freezing stresses at the germination and seedling stages. Under control conditions, no growth or yield penalty were mentioned in transgenic plants due to the constitutive expression of AlTMP2. Interestingly, under greenhouse conditions, the seed yield of transgenic plants was significantly higher than that of non-transgenic (NT) plants grown under salt or drought stress. Furthermore, AlTMP2 plants had less electrolyte leakage, higher membrane stability, and lower Na⁺ and higher K⁺ accumulation than NT plants. Finally, six stress-related genes were shown to be deregulated in AlTMP2 plants relative to NT plants under both control and stress conditions. Collectively, these results indicate that AlTMP2 confers abiotic stress tolerance by improving ion homeostasis and membrane integrity, and by deregulating certain stress-related genes.     

Characterizing the growth of <Emphasis Type="Italic">Sarcoptes scabiei</Emphasis> infrapopulations

The plant stress hypothesis posits that a herbivore’s reproductive success increases when it feeds on stressed plants, while the plant vigor hypothesis predicts that a herbivore preferentially feeds on more vigorous plants. We examined these opposing hypotheses by growing spider mites (Tetranychus urticae) on the leaves of stressed and healthy (vigorous) cucumber plants. Host plants were grown under controlled conditions at low, moderate, and high concentrations of NaCl (to induce salinity stress), at low, moderate, and high fertilizer concentrations (to support growth), and without these additions (control). The effects of these treatments were evaluated by measuring fresh and dry plant biomass, carotenoid and chlorophyll content, antioxidant enzyme activity, and concentrations of PO₄^3?, K⁺, and Na⁺ in plant tissues. The addition of low concentrations of fertilizer increased dry mass, protein, and carotenoid content relative to controls, suggesting a beneficial effect on plants. The highest NaCl treatment (2560 mg L^?1) resulted in increased Na⁺ and protein content relative to control plants, as well as reduced PO₄^3?, K⁺, and chlorophyll levels and reduced catalase and ascorbate peroxidase enzyme activity levels. Analysis of life table data of T. urticae mites raised on leaves from the aforementioned plant groups showed the intrinsic rate of increase (r) for mites was 0.167 day^?1 in control specimens, 0.125 day^?1 for mites reared on plants treated with a moderate concentration of fertilizer (10 mL L^?1), and was highest (0.241 day^?1) on plants grown under moderate salinity conditions (1920 mg L^?1 NaCl). Reproductive success of T. urticae did not differ on plants watered with a moderate concentration of NaCl or a high concentration of fertilizer. The moderately-stressed plants formed a favorable environment for the development and reproduction of spider mites, supporting the plant stress hypothesis.     

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.     

Functional Identification of H<Superscript>+</Superscript>-ATPase and Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Antiporter in the Plasma Membrane Isolated from the Root Cells of Salt-Accumulating Halophyte <Emphasis Type="Italic">Suaeda altissima</Emphasis>

A membrane fraction enriched in plasma membrane (PM) vesicles was isolated from the root cells of a salt-accumulating halophyte Suaeda altissima (L.) Pall. by means of centrifugation in discontinuous sucrose density gradient. The PM vesicles were capable of generating ΔpH at their membrane and the transmembrane electric potential difference (Δψ). These quantities were measured with optical probes, acridine orange and oxonol VI, sensitive to ΔpH and Δψ, respectively. The ATP-dependent generation of ΔpH was sensitive to vanadate, an inhibitor of P-type ATPases. The results contain evidence for the functioning of H⁺-ATPase in the PM of the root cells of S. altissima. The addition of Na⁺ and Li⁺ ions to the outer medium resulted in dissipation of ΔpH preformed by the H⁺-ATPase, which indicates the presence in PM of the functionally active Na⁺/H⁺ antiporter. The results are discussed with regard to involvement of the Na⁺/H⁺ antiporter and the PM H⁺-ATPase in loading Na⁺ ions into the xylem of S. altissima roots.     

Enhanced salinity stress tolerance in transgenic chilli pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) plants overexpressing the wheat antiporter (<Emphasis Type="Italic">TaNHX2</Emphasis>) gene

Transgenic chilli pepper (Capsicum annuum L.) plants tolerant to salinity stress were produced by introducing the wheat Na⁺/H⁺ antiporter gene (TaNHX2) via Agrobacterium-mediated transformation. Cotyledonary explants were infected with Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pBin438 that contains a wheat antiporter (TaNHX2) gene driven by the double CaMV 35S promoter and NPT II gene as a selectable marker. PCR and semiquantitative RT-PCR analysis confirmed that the TaNHX2 gene had been integrated and expressed in the T₁ generation of transgenic pepper plants as compared to the non-transformed plants. Southern blot analysis further verified the integration and presence of TaNHX2 gene in the genome of chilli pepper plants. Biochemical assays of these transgenic plants revealed enhanced levels of proline, chlorophyll, superoxide dismutase, ascorbate peroxidase, relative water content, and reduced levels of hydrogen peroxide (H₂O₂), malondialdehyde compared to wild-type plants under salt stress conditions. The present investigation clearly showed that overexpression of the TaNHX2 gene enhanced salt stress tolerance in transgenic chilli pepper plants.     

Cell ultrastructure and fatty acid composition of lipids in vegetative organs of <Emphasis Type="Italic">Chenopodium album</Emphasis> L. under salt stress conditions

White goosefoot plants (Chenopodium album L. of the family Chenopodiaceae) grown at various NaCl concentrations (3–350 mM) in the nutrient solution were used to study the cell ultrastructure as well as the qualitative and quantitative composition of fatty acids in the lipids of vegetative organs. In addition, the biomass of Ch. album vegetative organs, the water content, and the concentrations of K⁺, Na⁺, and Cl^– were determined. The growth rates of plants raised at NaCl concentrations up to 200–250 mM were the same as for the control plants grown at 3 mM NaCl; the growth parameters remained rather high even at NaCl concentrations of 300–350 mM. The water content in Ch. album organs remained high at all NaCl concentrations tested. Analysis of the ionic status of Ch. album revealed a comparatively high K⁺ content in plant organs. At low NaCl concentrations in the nutrient solution, K⁺ ions were the dominant contributors to the osmolarity (the total concentration of osmotically active substances) and, consequently, to the lowered cell water potential in leaves and roots. As the concentration of NaCl was increased, the plant organs accumulated larger amounts of Na⁺ and Cl^–, and the contribution of these ion species to osmolarity became increasingly noticeable. At 300–350 mM NaCl the contribution of Na⁺ and Cl^– to osmolarity was comparable to that of K⁺. An electron microscopy study of Ch. album cells revealed that, apart from the usual response to salinity manifested in typical ultrastructural changes of chloroplasts, mitochondria, and the cytosol, the salinity response comprised the enhanced formation of endocytic structures and exosomes and stimulation of autophagy. It is supposed that activation of these processes is related to the removal from the cytoplasm of toxic substances and the cell structures impaired by salt stress conditions. The qualitative and quantitative composition of fatty acids in the lipids of Ch. album organs was hardly affected by NaCl level. These findings are consistent with the high salt tolerance of Ch. album, manifested specifically in retention of growth functions under wide-range variations of NaCl concentration in the nutrient solution and in maintenance of K⁺, Na⁺, and Cl^– content in organs at a constant level characteristic of untreated plants.     

Construction of flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>-overproducing strains of the thermotolerant methylotrophic yeast <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> (<Emphasis Type="Italic">Pichia angusta</Emphasis>)

L-Lactate cytochrome c oxidoreductase (flavocytochrome b ₂, FC b ₂) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker’s yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b ₂ producers with over-expression of the H. polymorpha CYB2 gene, encoding FC b ₂. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (grc1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b ₂ activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b ₂ producer characterized by a sixfold increased (to 3 μmol min^?1 mg^?1 protein in cell-free extract) activity of the enzyme.     

Increase in Salt Tolerance of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> by <Emphasis Type="Italic">TaDi19</Emphasis>

The gene expression profile chip of salt-resistant wheat mutant RH8706-49 under salt stress was investigated. The overall length of the cDNA sequence of the probe was obtained using electronic cloning and RT-PCR. An unknown gene induced by salt was obtained, cloned, and named TaDi19 (Triticum aestivum drought-induced protein). No related report or research on the protein is available. qPCR analysis showed that gene expression was induced by many stresses, such as salt. Arabidopsis thaliana was genetically transferred using the overexpressing gene, which increased its salt tolerance. After salt stress, the transgenic plant demonstrated better physiological indicators (higher Ca²⁺ and lower Na⁺) than those of the wild-type plant. Results of non-invasive micro-test technology indicate that TaDi19-overexpressing A. thaliana significantly effluxed Na⁺ after salt treatment, whereas the wild-type plant influxed Na⁺. Chelating extracellular Ca²⁺ resulted in insignificant differences in salt tolerance between overexpressing and wild-type A. thaliana. Subcellular localization showed that the gene encoding protein was mainly located in the cell membrane and nucleus. TaDi19 was overexpressed in wild-type A. thaliana, and the transgenic lines were more salt-tolerant than the control A. thaliana. Thus, the wheat gene TaDi19 could increase the salt tolerance of A. thaliana.     

<Emphasis Type="Italic">Piriformospora indica</Emphasis>-mediated salinity tolerance in <Emphasis Type="Italic">Aloe vera</Emphasis> plantlets

Piriformospora indica, a root endophytic fungus, has been reported to promote growth of many plants under normal condition and allow the plants to survive under stress conditions. However, its impact on an important medicinal plant Aloe vera L. has not been well studied. Therefore, this study was undertaken to investigate the effect of P. indica on salinity stress tolerance of A. vera plant. P. indica inoculated and non-inoculated A. vera plantlets were subjected to four levels of salinity treatment- 0, 100, 200 and 300 mM NaCl. The salinity stress decreased the ability of the fungus to colonize roots of A. vera but the interaction of A. vera with P. indica resulted in an overall increase in plant biomass and greater shoot and root length as well as number of shoots and roots. The photosynthetic pigment (Chl a, Chl b and total Chl) and gel content were significantly higher for the fungus inoculated A. vera plantlets, at respective salinity concentrations. Furthermore, the inoculated plantlets had higher phenol, flavonoid, flavonol, aloin contents and radical scavenging activity at all salinity concentrations. The higher phenolic and flavonoid content may help the plants ameliorate oxidative stress resulting from high salinity.     

Physiological adaptation and gene expression analysis of <Emphasis Type="Italic">Casuarina equisetifolia</Emphasis> under salt stress

Casuarina equisetifolia is widely planted in coastal areas of tropical and subtropical regions as windbreaks or to stabilize dunes against wind erosion due to its high salt tolerance and nitrogen-fixing ability. To investigate the mechanisms responsible for its salt tolerance, we examined growth, mineral composition, expression of genes for sodium (Na⁺) and potassium (K⁺) transport proteins, and antioxidant responses under NaCl treatments. Increasing NaCl concentrations inhibited lateral root elongation and decreased plant height, length of internodes, and numbers of branches and twigs. The Na⁺ content significantly increased whereas the K⁺ content significantly decreased in both shoots and roots with increasing external NaCl concentration, resulting in a significant increase in Na⁺/K⁺ ratio. Most of the Na⁺/H⁺ antiporter genes (NHXs) were obviously upregulated in roots after 24 and 168 h of salt stress, and NHX7 was especially induced after 168 h. Almost all salt overly sensitive (SOS) genes were induced after 168-h treatment. Additionally, activities of superoxide dismutase, glutathione peroxidase, and catalase were significantly changed in shoots and roots under salt stress. Hence, we conclude that salinity tolerance of C. equisetifolia mainly relied on sequestering excess Na⁺ into vacuoles and on induced expression of NHX and SOS genes in roots and thus the maintenance of sufficient K⁺ content in shoots.