Expression of an Arabidopsis vacuolar H+‐pyrophosphatase gene (AVP1) in cotton improves drought‐ and salt tolerance and increases fibre yield in the field conditions

The Arabidopsis gene AVP1 encodes a vacuolar pyrophosphatase that functions as a proton pump on the vacuolar membrane. Overexpression of AVP1 in Arabidopsis, tomato and rice enhances plant performance under salt and drought stress conditions, because up‐regulation of the type I H⁺‐PPase from Arabidopsis may result in a higher proton electrochemical gradient, which facilitates enhanced sequestering of ions and sugars into the vacuole, reducing water potential and resulting in increased drought‐ and salt tolerance when compared to wild‐type plants. Furthermore, overexpression of AVP1 stimulates auxin transport in the root system and leads to larger root systems, which helps transgenic plants absorb water more efficiently under drought conditions. Using the same approach, AVP1‐expressing cotton plants were created and tested for their performance under high‐salt and reduced irrigation conditions. The AVP1‐expressing cotton plants showed more vigorous growth than wild‐type plants in the presence of 200 mm NaCl under hydroponic growth conditions. The soil‐grown AVP1‐expressing cotton plants also displayed significantly improved tolerance to both drought and salt stresses in greenhouse conditions. Furthermore, the fibre yield of AVP1‐expressing cotton plants is at least 20% higher than that of wild‐type plants under dry‐land conditions in the field. This research indicates that AVP1 has the potential to be used for improving crop’s drought‐ and salt tolerance in areas where water and salinity are limiting factors for agricultural productivity.     

H+‐pyrophosphatase from Salicornia europaea confers tolerance to simultaneously occurring salt stress and nitrogen deficiency in Arabidopsis and wheat

High salinity and nitrogen (N) deficiency in soil are two key factors limiting crop productivity, and they usually occur simultaneously. Here we firstly found that H⁺‐PPase is involved in salt‐stimulated NO₃^? uptake in the euhalophyte Salicornia europaea. Then, two genes (named SeVP1 and SeVP2) encoding H⁺‐PPase from S. europaea were characterized. The expression of SeVP1 and SeVP2 was induced by salt stress and N starvation. Both SeVP1 or SeVP2 transgenic Arabidopsis and wheat plants outperformed the wild types (WTs) when high salt and low N occur simultaneously. The transgenic Arabidopsis plants maintained higher K⁺/Na⁺ ratio in leaves and exhibited increased NO₃^? uptake, inorganic pyrophosphate‐dependent vacuolar nitrate efflux and assimilation capacity under this double stresses. Furthermore, they had more soluble sugars in shoots and roots and less starch accumulation in shoots than WT. These performances can be explained by the up‐regulated expression of ion, nitrate and sugar transporter genes in transgenic plants. Taken together, our results suggest that up‐regulation of H⁺‐PPase favours the transport of photosynthates to root, which could promote root growth and integrate N and carbon metabolism in plant. This work provides potential strategies for improving crop yields challenged by increasing soil salinization and shrinking farmland.     

High salt‐treatment‐induced Na+ extrusion and low salt‐treatment‐induced Na+ accumulation in suspension‐cultured cells of the mangrove plant,Bruguiera sexangula

A suspension‐cultured cell strain of the mangrove plant (Bruguiera sexangula) was established from a callus culture and maintained in an amino acid medium in the absence of NaCl. NaCl non‐adapted cells were transferred to media containing 0–200 mm NaCl. The initial growth rate decreased gradually with increasing salt concentrations. However, at up to 150 mm NaCl, cell number growth at the highest point was almost the same as that at lower salt concentrations. Cells even continued to grow in the presence of 200 mm NaCl. Cells incubated in a medium containing 50 mm NaCl for 3 weeks accumulated Na⁺, while those incubated in 150 mm NaCl for 2 d showed only a transient increase in Na⁺ and Cl^– concentrations. In the latter treatment, the intracellular concentration of Na⁺ returned to the original low level within 2 weeks. It took a longer time for Cl^– to return to its original level. As a result, the Na⁺ and Cl^– concentrations in cells cultured with 50 mm NaCl were much larger than those in cells cultured with 150 mm NaCl. The intracellular distribution of ions after transfer to the medium containing 150 mm NaCl was analysed by isolating the vacuoles. Treatment with amiloride, an inhibitor of the Na⁺/H⁺ antiporter, suppressed the recovery of Na⁺ to the original level in the cells. Treatment with 150 mm NaCl for 3 d stimulated the activities of both the vanadate‐dependent H⁺‐ATPase and the Na⁺/H⁺ antiporter in the plasma membrane fraction.     

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.     

Adaptation of the tonoplast V-type H+-ATPase of Mesembryanthemum crystallinum to salt stress, C3–CAM transition and plant age

Plants of the facultative halophyte and CAM species Mesembryanthemum crystallinum L. (Aizoaceae) were stressed for 8 d with 400 mol m⁻³ NaCl in the root medium. NaCl was then removed from the substratum, and the plants were watered again with NaCl-free solution. A second set of plants was maintained as controls. A small degree of CAM, as indicated by day-night changes in malate levels, was expressed during ageing of the plants. Salinity-stress-dependent CAM induction was reversible by the removal of salt, as indicated by similar Δ malate levels in previously salt-stressed plants and in non-stressed plants on day 19 of the experiment. Tonoplast vesicles were isolated from leaves during the time-course of stress application, stress removal and ageing. Parameters of the tonoplast H⁺-ATPase were correlated to the application of salinity, the expression of CAM and ageing. It was concluded, first, that a pronounced increase in the amount of tonoplast H⁺-ATPase is related to salinity per se and a smaller increase to ageing; secondly, that there is an increase in the specific activity of the enzyme related to ageing; thirdly, that the induction of two new polypeptides with molecular masses of 32 and 28 kDa is correlated in time with the expression of CAM, and, fourthly, that the two new polypeptides are part of the tonoplast H⁺-ATPase holoenzyme.     

Humic acid differentially improves nitrate kinetics under low‐ and high‐affinity systems and alters the expression of plasma membrane H+‐ATPases and nitrate transporters in rice

Humic acids (HAs) have a major effect on nutrient uptake, metabolism, growth and development in plants. Here, we evaluated the effect of HA pretreatment applied with a nutrient solution on the uptake kinetics of nitrate nitrogen (N‐NO₃^?) and the metabolism of nitrogen (N) in rice under conditions of high and low NO₃^? supply. In addition, the kinetic parameters of NO₃^? uptake, N metabolites, and nitrate transporters (NRTs) and the plasma membrane (PM) H⁺‐ATPase gene expression were examined. The plants were grown in a growth chamber with modified Hoagland and Arnon solution until 21 days after germination (DAG), and they were then transferred to a solution without N for 48 h and then to another solution without N and with and without the addition of HAs for another 48 h. After this period of N deprivation, the plants received new nutrient solutions containing 0.2 and 2.0 mM N‐NO₃^?. Treatment of rice plants with HA promoted the induction of the genes OsNRT2.1‐2.2/OsNAR2.1 and some isoforms PM H⁺‐ATPase in roots. The application of HAs differentially modified the parameters of the uptake kinetics of NO₃^? under both concentrations. When grown with 0.2 mM NO₃^?, the plants pretreated with HA had lower K_m and C_min values as well as a higher V_max/K_m ratio. When grown with 2 mM NO₃^?, the plants pretreated with HA had a higher V_max value, a greater root and shoot mass, and a lower root/shoot ratio. The N fractions were also altered by pretreatment with HA, and a greater accumulation of NO₃^? and N‐amino was observed in the roots and shoots, respectively, of plants pretreated with HA. The results suggest that pretreatment with HA modifies root morphology and gene expression of PM H⁺‐ATPases and NO₃^? transporters, resulting in a greater efficiency of NO₃^? acquisition by high‐ and low‐affinity systems.     

CAX‐ing a wide net: Cation/H+ transporters in metal remediation and abiotic stress signalling

Cation/proton exchangers (CAXs) are a class of secondary energised ion transporter that are being implicated in an increasing range of cellular and physiological functions. CAXs are primarily Ca²⁺ efflux transporters that mediate the sequestration of Ca²⁺ from the cytosol, usually into the vacuole. Some CAX isoforms have broad substrate specificity, providing the ability to transport trace metal ions such as Mn²⁺ and Cd²⁺, as well as Ca²⁺. In recent years, genomic analyses have begun to uncover the expansion of CAXs within the green lineage and their presence within non‐plant species. Although there appears to be significant conservation in tertiary structure of CAX proteins, there is diversity in function of CAXs between species and individual isoforms. For example, in halophytic plants, CAXs have been recruited to play a role in salt tolerance, while in metal hyperaccumulator plants CAXs are implicated in cadmium transport and tolerance. CAX proteins are involved in various abiotic stress response pathways, in some cases as a modulator of cytosolic Ca²⁺ signalling, but in some situations there is evidence of CAXs acting as a pH regulator. The metal transport and abiotic stress tolerance functions of CAXs make them attractive targets for biotechnology, whether to provide mineral nutrient biofortification or toxic metal bioremediation. The study of non‐plant CAXs may also provide insight into both conserved and novel transport mechanisms and functions.     

In vitro assessment of the growth and plasma membrane H+‐ATPase inhibitory activity of ebselen and structurally related selenium‐ and sulfur‐containing compounds in Candida albicans

Ebselen (EB, compound 1) is an investigational organoselenium compound that reduces fungal growth, in part, through inhibition of the fungal plasma membrane H⁺‐ATPase (Pma1p). In the present study, the growth inhibitory activity of EB and of five structural analogs was assessed in a fluconazole (FLU)‐resistant strain of Candida albicans (S2). While none of the compounds were more effective than EB at inhibiting fungal growth (IC₅₀ ～ 18 μM), two compounds, compounds 5 and 6, were similar in potency. Medium acidification assays performed with S2 yeast cells revealed that compounds 4 and 6, but not compounds 2, 3, or 5, exerted an inhibitory activity comparable to EB (IC₅₀ ～ 14 μM). Using a partially purified Pma1p preparation obtained from S2 yeast cells, EB and all the analogs demonstrated a similar inhibitory activity. Taken together, these results indicate that EB analogs are worth exploring further for use as growth inhibitors of FLU‐resistant fungi.     

The combination of arbuscular mycorrhizal fungi inoculation (Glomus versiforme) and 28‐homobrassinolide spraying intervals improves growth by enhancing photosynthesis,nutrient absorption,and antioxidant system in cucumber (Cucumis sativus L.) under salinity

Salinity is one of the major obstacles in the agriculture industry causing huge losses in productivity. Several strategies such as plant growth regulators with arbuscular mycorrhizal fungi (AMF) have been used to decrease the negative effects of salt stress. In our experiment, 28‐homobrassinolide (HBL) with spraying intervals was combined with AMF (Glomus versiforme) in cucumber cultivars Jinyou 1^# (salt sensitive) and (Changchun mici, in short, CCMC, salt tolerant) under NaCl (100 mmol/L). Studies have documented that the foliar application of HBL and AMF colonization can enhance tolerance to plants under stress conditions. However, the mechanism of the HBL spraying intervals after 15 and 30 days in combination with AMF in cucumber under salt stress is still unknown. Our results revealed that the HBL spraying interval after 15 days in combination with AMF resulted in improved growth, photosynthesis, and decreased sodium toxicity under NaCl. Moreover, the antioxidant enzymes SOD (superoxide dismutase; EC 1.15.1.1) and POD activity (peroxidase; EC 1.11.1.7) showed a gradual increase after every 10 days, while the CAT (catalase; EC 1.11.1.6) increased after 30 days of salt treatments in both cultivars. This research suggests that the enhanced tolerance to salinity was mainly related to elevated levels of antioxidant enzymes and lower uptake of Na⁺, which lowers the risk of ion toxicity and decreases cell membrane damage.