Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> and K<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporters AtNHX1 and AtNHX3 from <Emphasis Type="Italic">Arabidopsis</Emphasis> improve salt and drought tolerance in transgenic poplar

The tonoplast and plasma membrane localized sodium (potassium)/proton antiporters have been shown to play an important role in plant resistance to salt stress. In this study, AtNHX1 and AtNHX3, two tonoplast Na⁺(K⁺)/H⁺ antiporter encoding genes from Arabidopsis thaliana, were expressed in poplar to investigate their biological functions in the resistance to abiotic stresses in woody plants. Transgenic poplar plants expressing either gene exhibited increased resistance to both salt and water-deficit stresses. Compared to the wild type (WT) plants, transgenic plants accumulated more sodium and potassium ions in the presence of 100 mM NaCl and showed reduced electrolyte leakage in the leaves under water stress. Furthermore, the proton-translocating and cation-dependent H⁺ (Na⁺/H⁺ or K⁺/H⁺) exchange activities in the tonoplast vesicles isolated from the leaves of transgenic plants were higher than in those isolated from WT plants. Therefore, constitutive expression of either AtNHX1 or AtNHX3 genetically modified the salt and water stress tolerance of transgenic poplar plants, providing a potential tool for engineering tree species with enhanced resistance to multiple abitotic stresses.     

Overexpression of SeNHX1 improves both salt tolerance and disease resistance in tobacco

Recently, we found NHX1, the gene encoding a Na⁺/H⁺ exchanger, participated in plant disease defense. Although NHX1 has been confirmed to be involved in plant salt tolerance, whether the NHX1 transgenic plants exhibit both salt tolerance and disease resistance has not been investigated. The T1 progenies of Nicotiana tabacum L. lines expressing SeNHX1 (from Salicornia europaea) were generated for the present study. Compared with PBI-type control plants, SeNHX1 transgenic tobaccos exhibited more biomass, longer root length, and higher K⁺/Na⁺ ratio at post germination or seedling stage under NaCl treatment, indicating enhanced salt tolerance. The vacuolar H⁺ efflux in SeNHX1 transgenic tobacco was increased after treatment of NaCl with different concentration. Meanwhile, the SeNHX1 transgenic tobaccos showed smaller wilted spot area, less H₂O₂ accumulation in leaves after infection of Phytophthora parasitica var. nicotianae. Further investigation demonstrated a larger NAD(P)(H) pool in SeNHX1 transgenic tobacco. These evidences revealed that overexpression of SeNHX1 intensified the compartmentation of Na⁺ into vacuole under salt stress and improved the ability of eliminating ROS after pathogen attack, which then enhanced salt tolerance and disease resistance simultaneously in tobacco. Our findings indicate NHX1 has potential value in creating crops with both improved salt tolerance and disease resistance.     

Overexpression of NHX1s in transgenic Arabidopsis enhances photoprotection capacity in high salinity and drought conditions

In this study, we observed that transgenic plants overexpressing NHX1 from different organisms grew well in the presence of 200 mM NaCl and also under water deprivation, while the wild-type plants exhibited chlorosis and growth inhibition. The photosynthesis activity of five kinds of transgenic plants was higher than the wild-type plants. The leaf water potential was less negative for wild-type than for transgenic plants. Moreover, these transgenic plants accumulated more Na⁺ and K⁺ in their leaf tissue than the wild-type plants. The toxic effects of Na⁺ accumulation in the cytosol are reduced by its sequestration into the vacuole. In addition, the thermal dissipation and ROS scavenging components increased in all transgenic Arabidopsis plants compared with that in non-transgenics. The salt tolerance of transgenic plants was passed on to the offsprings to the T₅ generation. Furthermore, it should be noted that in transgenic Arabidopsis plants, overexpression of NHX1s from dicots showed higher salt and drought tolerance than that from wheat.     

Functional Characterization of a Wheat NHX Antiporter Gene TaNHX2 That Encodes a K+/H+ Exchanger

The subcellular localization of a wheat NHX antiporter, TaNHX2, was studied in Arabidopsis protoplasts, and its function was evaluated using Saccharomyces cerevisiae as a heterologous expression system. Fluorescence patterns of TaNHX2-GFP fusion protein in Arabidopsis cells indicated that TaNHX2 localized at endomembranes. TaNHX2 has significant sequence homology to NHX sodium exchangers from Arabidopsis, is abundant in roots and leaves and is induced by salt or dehydration treatments. Western blot analysis showed that TaNHX2 could be expressed in transgenic yeast cells. Expressed TaNHX2 protein suppressed the salt sensitivity of a yeast mutant strain by increasing its K⁺ content when exposed to salt stress. TaNHX2 also increased the tolerance of the strain to potassium stress. However, the expression of TaNHX2 did not affect the sodium concentration in transgenic cells. Western blot analysis for tonoplast proteins indicated that the TaNHX2 protein localized at the tonoplast of transgenic yeast cells. The tonoplast vesicles from transgenic yeast cells displayed enhanced K⁺/H⁺ exchange activity but very little Na^+/H⁺ exchange compared with controls transformed with the empty vector; Na⁺/H⁺ exchange was not detected with concentrations of less than 37.5 mM Na⁺ in the reaction medium. Our data suggest that TaNHX2 is a endomembrane-bound protein and may primarily function as a K⁺/H⁺ antiporter, which is involved in cellular pH regulation and potassium nutrition under normal conditions. Under saline conditions, the protein mediates resistance to salt stress through the intracellular compartmentalization of potassium to regulate cellular pH and K⁺ homeostasis.     

Overexpression of Arabidopsis thaliana Na+/H+ antiporter gene enhanced salt resistance in transgenic poplar (Populus?×?euramericana ‘Neva’)

Salinity is a major abiotic stress factor limiting plant growth and productivity. One possible method to enhance plant salt-resistance is to compartmentalize sodium ions away from the cytosol. In the present work, a vacuolar Na⁺/H⁺ antiporter gene AtNHX1 from Arabidopsis thaliana, was transferred into Populus × euramericana ‘Neva’ by Agrobacterium tumefaciens in order to enhance poplar salt-resistance. The results showed that the transgenic poplar were more resistant to NaCl than the wild-type (WT) in greenhouse condition. Compared with the WT, plant growth and photosynthetic capacity of the transgenic plants were enhanced, and the transgenic plants accumulated more Na⁺ and K⁺ in roots and leaves under the same NaCl condition, whereas malondialdehyde and relative electrical conductivity were lower. All of these properties of the transgenic poplar were likely to be a consequence of the overexpression of AtNHX1 caused Na⁺ sequestration in the vacuoles and improved K⁺ absorption, thus reducing their toxic effects. These results indicated overexpression of the AtNHX1 enhanced salt-resistance of poplar, and AtNHX1 played an important role in the compartmentation of Na⁺ into the vacuoles. Therefore, this study provides an effective way for improving salt resistance in trees.     

Protective effect of exogenous polyamines on root tonoplast function against salt stress in barley seedlings

Salinity stress is one of the most serious factors limiting the productivity of agricultural crops. A possible survival strategy of plants under saline conditions is to sequester excess Na⁺ in the vacuole by vacuolar Na⁺/H⁺ antiport using a pH gradient generated by H⁺-ATPasc (EC 3.6.1.35) and H⁺-Pyrophosphatase (H⁺-PPase; EC 3.6.1.1) to maintain a higher K⁺/Na⁺ ratio in cytoplasm. The effect of exogenously applied polyamines (PAs) in stabilizing root tonoplast integrity and function against salt stress in the barley (Hordeum vulgare L.) seedlings was investigated. The NaCl-induced reductions in the contents of phospholipids and PAs in tonoplast vesicles isolated from barely seedling roots, as well as the activities of H⁺-ATPase, H⁺-PPase and vacuolar Na⁺/H⁺ antiport were all partially restored by the application of 0.5 mM putrescine and 0.5 mM spermidine, especially the former. The above results indicated that one of the mechanisms involved in attenuating salt injury in barley seedlings by exogenous PAs application was to maintain tonoplast integrity and function under saline conditions. Moreover, the possible mechanism involved in counteracting detrimental effects of salt on the barley seedlings by the application of exogenous PAs was discussed.     

AtNHX3 is a vacuolar K+/H+ antiporter required for low‐potassium tolerance in Arabidopsis thaliana

Three vacuolar cation/H⁺ antiporters, AtNHX1 (At5g27150), 2 (At3g05030) and 5 (At1g54370), have been characterized as functional Na⁺/H⁺ antiporters in Arabidopsis. However, the physiological functions of AtNHX3 (At5g55470) still remain unclear. In this study, the physiological functions of AtNHX3 were studied using T‐DNA insertion mutant and 35S‐driven AtNHX3 over‐expression Arabidopsis plants. RT‐PCR analyses revealed that AtNHX3 is highly expressed in germinating seeds, flowers and siliques. Experiments with AtNHX3::YFP fusion protein in tobacco protoplasts indicated that AtNHX3 is mainly localized to vacuolar membrane, with a minor localization to pre‐vacuolar compartments (PVCs) and endoplasmic reticulum (ER). Seedlings of null nhx3 mutants were hypersensitive to K⁺‐deficient conditions. Expression of AtNHX3 complemented the sensitivity to K⁺ deficiency in nhx3 seedlings. Tonoplast vesicles isolated from transgenic plants over‐expressing AtNHX3 displayed significantly higher K⁺/H⁺ exchange rates than those isolated from wild‐type plants. Furthermore, nhx3 seeds accumulated less K⁺ and more Na⁺ when both wild‐type and nhx3 were grown under normal growth condition. The overall results indicate that AtNHX3 encodes a K⁺/H⁺ antiporter required for low‐potassium tolerance during germination and early seedling development, and may function in K⁺ utilization and ion homeostasis in Arabidopsis.     

Arabidopsis NHX Transporters: Sodium and Potassium Antiport Mythology and Sequestration During Ionic Stress

A crucial prerequisite for plant growth and survival under high salinity is maintenance of Na⁺ and K⁺ balance. Accumulation of Na⁺ and K⁺ in high concentration in the cytosol reduces crop yield. To cope with such imbalance ionic conditions, plants use a number of transporters to maintain Na⁺ and K⁺ homoeostasis inside the cell and regulate plant growth and development. This cation and pH homoeostasis is regulated by monovalent cation/proton antiporters (CPA) that fall in two categories, the CPA1 family that includes Na⁺/H⁺ NHX antiporters, and the CPA2 family that includes Cation/H⁺ (CHX) and K⁺ efflux antiporters (KEA). In this review we highlighted the role of NHX-antiporters in regulation of Na⁺ and K⁺ balance. NHX proteins are required for accurate K⁺ compartmentation. They mediate K⁺ specific vacuolar sequestration, pH adjustment, turgor and osmotic regulation, and play a unique role in stomatal movement and cell expansion.     

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

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

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.     

Cover Caption: Subcellular Localization of AaNhaD

The AaNhaD gene from soda lake Alkalimonas amylolytica encodes a Na⁺/H⁺ antiporter that plays a crucial role in the bacterium's resistance to salt/alkali stresses. Zhong et al. (pp. 412–421) reported that AaNhaD functions as a pH‐dependent tonoplast Na⁺/H⁺ antiporter in plant cells and is able to enhance the salinity/alkalinity tolerance in transgenic tobacco BY‐2 cells and plants. The cover picture illustrates that AaNhaD proteins are located primarily in the vacuole membranes in a BY‐2 cell.     

Role of Tonoplast Proton Pumps and Na+/H+ Antiport System in Salt Tolerance of Populus euphratica Oliv.

Populus euphratica has been used as a plant model to study resistance against salt and osmotic stresses, with recent studies having characterized the tonoplast and the plasma membrane ATPases, and two Na⁺/H⁺ antiporters, homologs of the Arabidopsis tonoplast AtNHX1, were published in databases. In the present work we show that P. euphratica suspension-cultured cells are highly tolerant to high salinity, being able to grow with up to 150 mM NaCl in the culture medium without substantial modification of the final population size when compared to the control cells in the absence of salt. At a salt concentration of 300 mM, cells were unable to grow but remained highly viable up to 17 days after subculture. The addition of a 1-M-NaCl pulse to unadapted cells did not promote a significant loss in cell viability within 48 h. In tonoplast vesicles purified from cells cultivated in the absence of salt and from salt-stressed cells, vacuolar H⁺-pyrophosphatase (V-H⁺-PPase) seemed to be the primary tonoplast proton pump; however, there appears to be a decrease in V-H⁺-PPase activity with exposure to NaCl, in contrast to the sodium-induced increase in the activity of vacuolar H⁺-ATPase (V-H⁺-ATPase). Despite reports that in P. euphratica there is no significant difference in the concentration of Na⁺ in the different cell compartments under NaCl stress, in the present study, confocal and epifluorescence microscopic observations using a Na⁺-sensitive probe showed that suspension-cultured cells subject to a salt pulse accumulated Na⁺ in the vacuole when compared with control cells. Concordantly, a tonoplast Na⁺/H⁺ exchange system is described whose activity is upregulated by salt and, indirectly, by a salt-mediated increase of V-H⁺-ATPase activity.