Improved yield,fruit quality,and salt resistance in tomato co-overexpressing LeNHX2 and SlSOS2 genes

The K⁺, Na⁺/H⁺ antiporter LeNHX2 and the regulatory kinase SlSOS2 are important determinants of salt tolerance in tomato plants and their fruit production ability. In this work, we have analyzed the effects of LeNHX2 and SlSOS2 co-overexpression on fruit production, quality in tomato plants (Solanum lycopersicum L. cv. MicroTom), and analyzed physiological parameters related to salt tolerance. Plants overexpressing LeNHX2, SlSOS2 or both were grown in greenhouse. They were treated with 125 mM NaCl or left untreated and their salt tolerance was analyzed in terms of plant biomass and fruit yield. Under NaCl cultivation conditions, transgenic tomato plants overexpressing either SlSOS2 or LeNHX2 or both grew better and showed a higher biomass compared to their wild-type plants. Proline, glucose and protein content in leaves as well as pH and total soluble solid (TSS) in fruits were analyzed. Our results indicate that salinity tolerance of transgenic lines is associated with an increased proline, glucose and protein content in leaves of plants grown either with or without NaCl. Salt treatment significantly reduced yield, pH and TSS in fruits of WT plants but increased yield, pH and TSS in fruits of transgenic plants, especially those overexpressing both LeNHX2 and SlSOS2. All these results indicate that the co-overexpression of LeNHX2 and SlSOS2 improve yield and fruit quality of tomato grown under saline conditions.     

Herbivore exposure alters ion fluxes and improves salt tolerance in a desert shrub

Plants have evolved complex mechanisms that allow them to withstand multiple environmental stresses, including biotic and abiotic stresses. Here, we investigated the interaction between herbivore exposure and salt stress of Ammopiptanthus nanus, a desert shrub. We found that jasmonic acid (JA) was involved in plant responses to both herbivore attack and salt stress, leading to an increased NaCl stress tolerance for herbivore-pretreated plants and increase in K⁺/Na⁺ ratio in roots. Further evidence revealed the mechanism by which herbivore improved plant NaCl tolerance. Herbivore pretreatment reduced K⁺ efflux and increased Na⁺ efflux in plants subjected to long-term, short-term, or transient NaCl stress. Moreover, herbivore pretreatment promoted H⁺ efflux by increasing plasma membrane H⁺-adenosine triphosphate (ATP)ase activity. This H⁺ efflux creates a transmembrane proton motive force that drives the Na⁺/H⁺ antiporter to expel excess Na⁺ into the external medium. In addition, high cytosolic Ca²⁺ was observed in the roots of herbivore-treated plants exposed to NaCl, and this effect may be regulated by H⁺-ATPase. Taken together, herbivore exposure enhance s A. nanus tolerance to salt stress by activating the JA-signalling pathway, increasing plasma membrane H ⁺ - ATPase activity, promoting cytosolic Ca²⁺ accumulation, and then restricting K⁺ leakage and reducing Na⁺ accumulation in the cytosol.     

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.     

ZxAKT1 is essential for K+ uptake and K+/Na+ homeostasis in the succulent xerophyte Zygophyllum xanthoxylum

The inward‐rectifying K⁺ channel AKT1 constitutes an important pathway for K⁺ acquisition in plant roots. In glycophytes, excessive accumulation of Na⁺ is accompanied by K⁺ deficiency under salt stress. However, in the succulent xerophyte Zygophyllum xanthoxylum, which exhibits excellent adaptability to adverse environments, K⁺ concentration remains at a relatively constant level despite increased levels of Na⁺ under salinity and drought conditions. In this study, the contribution of ZxAKT1 to maintaining K⁺ and Na⁺ homeostasis in Z. xanthoxylum was investigated. Expression of ZxAKT1 rescued the K⁺‐uptake‐defective phenotype of yeast strain CY162, suppressed the salt‐sensitive phenotype of yeast strain G19, and complemented the low‐K⁺‐sensitive phenotype of Arabidopsis akt1 mutant, indicating that ZxAKT1 functions as an inward‐rectifying K⁺ channel. ZxAKT1 was predominantly expressed in roots, and was induced under high concentrations of either KCl or NaCl. By using RNA interference technique, we found that ZxAKT1‐silenced plants exhibited stunted growth compared to wild‐type Z. xanthoxylum. Further experiments showed that ZxAKT1‐silenced plants exhibited a significant decline in net uptake of K⁺ and Na⁺, resulting in decreased concentrations of K⁺ and Na⁺, as compared to wild‐type Z. xanthoxylum grown under 50 mm NaCl. Compared with wild‐type, the expression levels of genes encoding several transporters/channels related to K⁺/Na⁺ homeostasis, including ZxSKOR, ZxNHX, ZxSOS1 and ZxHKT1;1, were reduced in various tissues of a ZxAKT1‐silenced line. These findings suggest that ZxAKT1 not only plays a crucial role in K⁺ uptake but also functions in modulating Na⁺ uptake and transport systems in Z. xanthoxylum, thereby affecting its normal growth.     

The AtNHX1 exchanger mediates potassium compartmentation in vacuoles of transgenic tomato

NHX‐type antiporters in the tonoplast have been reported to increase the salt tolerance of various plants species, and are thought to mediate the compartmentation of Na⁺ in vacuoles. However, all isoforms characterized so far catalyze both Na⁺/H⁺ and K⁺/H⁺ exchange. Here, we show that AtNHX1 has a critical involvement in the subcellular partitioning of K⁺, which in turn affects plant K⁺ nutrition and Na⁺ tolerance. Transgenic tomato plants overexpressing AtNHX1 had larger K⁺ vacuolar pools in all growth conditions tested, but no consistent enhancement of Na⁺ accumulation was observed under salt stress. Plants overexpressing AtNHX1 have a greater capacity to retain intracellular K⁺ and to withstand salt‐shock. Under K⁺‐limiting conditions, greater K⁺ compartmentation in the vacuole occurred at the expense of the cytosolic K⁺ pool, which was lower in transgenic plants. This caused the early activation of the high‐affinity K⁺ uptake system, enhanced K⁺ uptake by roots, and increased the K⁺ content in plant tissues and the xylem sap of transformed plants. Our results strongly suggest that NHX proteins are likely candidates for the H⁺‐linked K⁺ transport that is thought to facilitate active K⁺ uptake at the tonoplast, and the partitioning of K⁺ between vacuole and cytosol.     

Heterologous expression of Arabidopsis H+‐pyrophosphatase enhances salt tolerance in transgenic creeping bentgrass (Agrostis stolonifera L.)

The Arabidopsis vacuolar H⁺‐pyrophosphatase (AVP1), when over‐expressed in transgenic (TG) plants, regulates root and shoot development via facilitation of auxin flux, and enhances plant resistance to salt and drought stresses. Here, we report that TG perennial creeping bentgrass plants over‐expressing AVP1 exhibited improved resistance to salinity than wild‐type (WT) controls. Compared to WT plants, TGs grew well in the presence of 100 mm NaCl, and exhibited higher tolerance and faster recovery from damages from exposure to 200 and 300 mm NaCl. The improved performance of the TG plants was associated with higher relative water content (RWC), higher Na⁺ uptake and lower solute leakage in leaf tissues, and with higher concentrations of Na⁺, K⁺, Cl^‐ and total phosphorus in root tissues. Under salt stress, proline content was increased in both WT and TG plants, but more significantly in TGs. Moreover, TG plants exhibited greater biomass production than WT controls under both normal and elevated salinity conditions. When subjected to salt stress, fresh (FW) and dry weights (DW) of both leaves and roots decreased more significantly in WT than in TG plants. Our results demonstrated the great potential of genetic manipulation of vacuolar H⁺‐pyrophosphatase expression in TG perennial species for improvement of plant abiotic stress resistance.     

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.     

The K<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter <Emphasis Type="Italic">AhNHX1</Emphasis> improved tobacco tolerance to NaCl stress by enhancing K<Superscript>+</Superscript> retention

High salinity is the one of important factors limiting plant growth and crop production. Many NHX-type antiporters have been reported to catalyze K⁺/H⁺ exchange to mediate salt stress. This study shows that an NHX gene from Arachis hypogaea L. has an important role in K⁺ uptake and transport, which affects K⁺ accumulation and plant salt tolerance. When overexpressing AhNHX1, the growth of tobacco seedlings is improved with longer roots and a higher fresh weight than the wild type (WT) under NaCl treatment. Meanwhile, when exposed to NaCl stress, the transgenic seedlings had higher K⁺/H⁺ antiporter activity and their roots got more K⁺ uptake. NaCl stress could induce higher K⁺ accumulation in the roots, stems, and leaves of transgenic tobacco seedlings but not Na⁺ accumulation, thus, leading to a higher K⁺/Na⁺ ratio in the transgenic seedlings. Additionally, the AKT1, HAK1, SKOR, and KEA genes, which are involved in K⁺ uptake or transport, were induced by NaCl stress and kept higher expression levels in transgenic seedlings than in WT seedlings. The H⁺-ATPase and H⁺-PPase activities were also higher in transgenic seedlings than in the WT seedlings under NaCl stress. Simultaneously, overexpression of AhNHX1 increased the relative distribution of K⁺ in the aerial parts of the seedlings under NaCl stress. These results showed that AhNHX1 catalyzed the K⁺/H⁺ antiporter and enhanced tobacco tolerance to salt stress by increasing K⁺ uptake and transport.     

RETRACTED ARTICLE: Analysis of the physiological mechanism of salt-tolerant transgenic rice carrying a vacuolar Na+/H+ antiporter gene from Suaeda salsa

Salt stress is one of the most serious factors limiting the productivity of agricultural crops. Increasing evidence has demonstrated that vacuolar Na⁺/H⁺ antiporters play a crucial role in plant salt tolerance. In the present study, we expressed the Suaeda salsa vacuolar Na⁺/H⁺ antiporter SsNHX1 in transgenic rice to investigate whether this can increase the salt tolerance of rice, and to study how overexpression of this gene affected other salt-tolerant mechanisms. It was found that transgenic rice plants showed markedly enhanced tolerance to salt stress and to water deprivation compared with non-transgenic controls upon salt stress imposition under outdoor conditions. Measurements of ion levels indicated that K⁺, Ca²⁺ and Mg²⁺ contents were all higher in transgenic plants than in non-transformed controls. Furthermore, shoot V-ATPase hydrolytic activity was dramatically increased in transgenics compared to that of non-transformed controls under salt stress conditions. Physiological analysis also showed that the photosynthetic activity of the transformed plants was higher whereas the same plants had reduced reactive oxygen species generation. In addition, the soluble sugar content increased in the transgenics compared with that in non-transgenics. These results imply that up-regulation of a vacuolar Na⁺/H⁺ antiporter gene in transgenic rice might cause pleiotropic up-regulation of other salt-resistance-related mechanisms to improve salt tolerance.Fengyun Zhao and Zenglan Wang contributed equally to this work.     

Lack of immunological cross reactivity between the transport enzymes (Na+ + K+)-ATPase and (K+ + H+)-ATPase

Goat antisera against (Na⁺ + K⁺)-ATPase and its isolated subunits and against (K⁺ + H⁺)-ATPase have been prepared in order to test for immune cross-reactivity between the two enzymes, whose catalytic subunits show great chemical similarity. None of the (Na⁺ + K⁺)-ATPase antisera cross-reacted with (K⁺ + H⁺)-ATPase or inhibited its enzyme activity. The same was true for the (K⁺ + H⁺)-ATPase antiserum with regard to (Na⁺ + K⁺)-ATPase and its subunits and its enzyme activity. So not withstanding the chemical similarity of their subunits, there is no immunological cross-reactivity between these two plasma membrane ATPases.Number LIII in the series Studies on (Na⁺ + K⁺)-Activated ATPase.     

Overexpression of wheat Na+/H+ antiporter TNHX1 and H+-pyrophosphatase TVP1 improve salt- and drought-stress tolerance in Arabidopsis thaliana plants

Transgenic Arabidopsis plants overexpressing the wheat vacuolarNa⁺/H⁺ antiporter TNHX1 and H⁺-PPase TVP1 are much more resistantto high concentrations of NaCl and to water deprivation thanthe wild-type strains. These transgenic plants grow well inthe presence of 200 mM NaCl and also under a water-deprivationregime, while wild-type plants exhibit chlorosis and growthinhibition. Leaf area decreased much more in wild-type thanin transgenic plants subjected to salt or drought stress. Theleaf water potential was less negative for wild-type than fortransgenic plants. This could be due to an enhanced osmoticadjustment in the transgenic plants. Moreover, these transgenicplants accumulate more Na⁺ and K⁺ in their leaf tissue thanthe wild-type plants. The toxic effect of Na⁺ accumulation inthe cytosol is reduced by its sequestration into the vacuole.The rate of water loss under drought or salt stress was higherin wild-type than transgenic plants. Increased vacuolar soluteaccumulation and water retention could confer the phenotypeof salt and drought tolerance of the transgenic plants. Overexpressionof the isolated genes from wheat in Arabidopsis thaliana plantsis worthwhile to elucidate the contribution of these proteinsto the tolerance mechanism to salt and drought. Adopting a similarstrategy could be one way of developing transgenic staple cropswith improved tolerance to these important abiotic stresses. Key words: H⁺-pyrophosphatase, Na⁺/H⁺ antiporter, salt and drought tolerance, sodium sequestration, transgenic Arabidopsis plants     

Taurine Stimulation of Isolated Hamster Brain Na+, K+-ATPase: Activation Kinetics and Chemical Specificity

Epileptic foci are associated with locally reduced taurine (2-aminoethanesulfonic acid) concentration and Na⁺, K⁺-ATPase (EC 3.6.1.3) specific activity. Topically applied and intraperitoneally administered taurine can prevent the development and/or spread of foci in many animal models. Taurine has been implicated as a possible cytosolic modulator of monovalent ion distribution, cytosolic “free” calcium activity, and neuronal excitability. Taurine may act in part by modulating Na⁺, K⁺-ATPase activity of neuronal and glial cells. We characterized the requirements for in vitro modulation of Na⁺, K⁺-ATPase by taurine. Normal whole brain homogenate Na⁺, K⁺-ATPase activity is 5.1 ± 0.4 (4) μmol P_i± h^?1± mg^?1 Lowry protein. Partial purification of the plasma membrane fraction to remove cytosolic proteins and extrinsic proteins and to uncouple cholinergic receptors yields a membrane-bound Na⁺, K⁺-ATPase activity of 204.6 ± 5.8 (4) mol P_i± h^?1± mg^?1 Lowry protein. Taurine activates the Na⁺, K⁺-ATPase at all levels of purification. The concentration dependence of activation follows normal saturation kinetics (K_1/2= 39 mM taurine, activation maximum =+87%). The activation exhibits chemical specificity among the taurine analogues and metabolites: taurine = isethionic acid > hypotaurine > no activation =β-alanine = methionine = choline = leucine. Taurine can act as an endogenous activator/modulator of Na⁺, K⁺-ATPase. Its action is mediated by a membrane-bound protein.     

Endothelin 1 Stimulates Na+,K+-ATPase and Na+-K+-Cl− Cotransport Through ETA Receptors and Protein Kinase C-Dependent Pathway in Cerebral Capillary Endothelium

Abstract: The effect of endothelins (ET-1 and ET-3) on ⁸⁶Rb⁺ uptake as a measure of K⁺ uptake was investigated in cultured rat brain capillary endothelium. ET-1 or ET-3 dose-dependently enhanced K⁺ uptake (EC₅₀ = 0.60 ± 0.15 and 21.5 ± 4.1 nM, respectively), which was inhibited by the selective ET_A receptor antagonist BQ 123 (cyclo-d -Trp-d -Asp-Pro-d -Val-Leu). Neither the selective ET_B agonists IRL 1620 [N-succinyl-(Glu⁹,-Ala^11,15)-ET-1] and sarafotoxin S6c, nor the ET_B receptor antagonist IRL 1038 [(Cys¹¹,Cys¹⁵)-ET-1] had any effect on K⁺ uptake. Ouabain (inhibitor of Na⁺,K⁺-ATPase) and bumetanide (inhibitor of Na⁺-K⁺-Cl^? cotransport) reduced (up to 40% and up to 70%, respectively) the ET-1-stimulated K⁺ uptake. Complete inhibition was seen with both agents. Phorbol 12-myristate 13-acetate (PMA), activator of protein kinase C (PKC), stimulated Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport. ET-1-but not PMA-stimulated K⁺ uptake was inhibited by 5-(N-ethyl-N-isopropyl)amiloride (inhibitor of Na⁺/H⁺ exchange system), suggesting a linkage of Na⁺/H⁺ exchange with ET-1-stimulated Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport activity that is not mediated by PKC.     

SOS1 gene overexpression increased salt tolerance in transgenic tobacco by maintaining a higher K(+)/Na(+) ratio

Crop productivity is greatly affected by soil salinity, so improvement in salinity tolerance of crops is a major objective of many studies. We overexpressed the Arabidopsis thaliana SOS1 gene, which encodes a plasma membrane Na⁺/H⁺ antiporter, in tobacco (Nicotiana tabacum cv. Xanthi-nc). Compared with nontransgenic plants, seeds from transgenic tobacco had better germination under 120 mM (mmol L⁻¹) NaCl stress; chlorophyll loss in the transgenic seedlings treated with 360 mM NaCl was less; transgenic tobacco showed superior growth after irrigation with NaCl solutions; and transgenic seedlings with 150 mM NaCl stress accumulated less Na⁺ and more K⁺. In addition, roots of SOS1-overexpressing seedlings lost less K⁺ instantaneously in response to 50 mM NaCl than control plants. These results showed that the A. thaliana SOS1 gene potentially can improve the salt tolerance of other plant species.     

Overexpression of a novel soybean gene modulating Na+ and K+ transport enhances salt tolerance in transgenic tobacco plants

Salt is an important factor affecting the growth and development of soybean in saline soil. In this study, a novel soybean gene encoding a transporter (GmHKT1) was identified and its function analyzed using transgenic plants. GmHKT1 encoded a protein of 419 amino acids, with a potential molecular mass of 47.06 kDa and a predicted pI value of 8.59. Comparison of the genomic and cDNA sequences of GmHKT1 identified no intron. The deduced amino acid sequence of GmHKT1 showed 38–49% identity with other plant HKT‐like sequences. RT‐PCR analysis showed that the expression of GmHKT1 was upregulated by salt stress (150 mM NaCl) in roots and leaves but not in stems. Overexpression of GmHKT1 significantly enhanced the tolerance of transgenic tobacco plants to salt stress, compared with non‐transgenic plants. To investigate the role of GmHKT1 in K⁺ and Na⁺ transport, we compared K⁺ and Na⁺ accumulation in roots and shoots of wild‐type and transgenic tobacco plants. The results suggested that GmHKT1 is a transporter that affected K⁺ and Na⁺ transport in roots and shoots, and regulated Na⁺/K⁺ homeostasis in these organs. Our findings suggest that GmHKT1 plays an important role in response to salt stress and would be useful in engineering crop plants for enhanced tolerance to salt stress.