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.     

Cloning and Functional Characterization of a Novel Glutathione <Emphasis Type="Italic">S</Emphasis>-Transferase Gene from <Emphasis Type="Italic">Limonium bicolor</Emphasis>

Plant glutathione S-transferases (GSTs) are involved in protecting plants against both diverse biotic and abiotic stresses. In the present study, a novel GST gene (LbGST1) was cloned from Limonium bicolor (Bunge) Kuntze (Plumbaginaceae). To characterize its function in salt tolerance, tobacco lines transformed with LbGST1 were generated. Compared with wild-type (WT) tobacco, transgenic plants overexpressing LbGST1 exhibited both GST and glutathione peroxidase activities. Moreover, superoxide dismutase, peroxidase (POD), and catalase activities in transgenic plants were significantly higher than those in WT plants, particularly when grown under conditions of salt stress. Similarly, levels of proline in transgenic plants were also higher than those in WT plants grown under NaCl stress conditions. Whereas, Malondialdehyde contents in transgenic plants were lower than those in WT plants under NaCl conditions. Furthermore, Na⁺ content in transgenic plants was lower than that in WT plants under these stress conditions. Subcellular localization analysis revealed that the LbGST1 protein was localized in the nucleus. These results suggested that overexpression of LbGST1 gene can affect many physiological processes associated with plant salt tolerance. Therefore, we hypothesize that LbGST1 gene can mediate many physiological pathways that enhance stress resistance in plants.     

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.     

Ovexpression of a Vacuolar H<Superscript>+</Superscript>-ATPase c Subunit Gene Mediates Physiological Changes Leading to Enhanced Salt Tolerance in Transgenic Tobacco

H⁺-ATPase subunit c (VHA-c) is involved in the adaptation to environmental stresses, including salt, drought, and heavy metals. However, it remains unclear whether VHA-c can induce a physiological response related to stress tolerance. To investigate this possibility, we generated transgenic tobacco lines overexpressing a V-ATPase subunit c (LbVHA-c1) gene from Limonium bicolor (Bunge) Kuntze. Compared with wild-type (WT) tobacco, superoxide dismutase (SOD) and peroxidase (POD) activities in the transgenic plants were significantly enhanced under salt stress conditions. The level of malondialdehyde (MDA) in the transgenic plants was significantly lower than that in WT plants grown under salt stress conditions. Moreover, the transgenic plants displayed obviously better growth than the WT plants under salt stress. These results suggest that LbVHA-c1 may confer stress tolerance through enhancing POD and SOD activities, and by protecting membranes from damage by decreasing lipid peroxidation under salt stress.     

Co‐expression of tonoplast Cation/H+ antiporter and H+‐pyrophosphatase from xerophyte Zygophyllum xanthoxylum improves alfalfa plant growth under salinity,drought and field conditions

Salinity and drought are major environmental factors limiting the growth and productivity of alfalfa worldwide as this economically important legume forage is sensitive to these kinds of abiotic stress. In this study, transgenic alfalfa lines expressing both tonoplast NXH and H⁺‐PPase genes, ZxNHX and ZxVP1‐1 from the xerophyte Zygophyllum xanthoxylum L., were produced via Agrobacterium tumefaciens‐mediated transformation. Compared with wild‐type (WT) plants, transgenic alfalfa plants co‐expressing ZxNHX and ZxVP1‐1 grew better with greater plant height and dry mass under normal or stress conditions (NaCl or water‐deficit) in the greenhouse. The growth performance of transgenic alfalfa plants was associated with more Na⁺, K⁺ and Ca²⁺ accumulation in leaves and roots, as a result of co‐expression of ZxNHX and ZxVP1‐1. Cation accumulation contributed to maintaining intracellular ions homoeostasis and osmoregulation of plants and thus conferred higher leaf relative water content and greater photosynthesis capacity in transgenic plants compared to WT when subjected to NaCl or water‐deficit stress. Furthermore, the transgenic alfalfa co‐expressing ZxNHX and ZxVP1‐1 also grew faster than WT plants under field conditions, and most importantly, exhibited enhanced photosynthesis capacity by maintaining higher net photosynthetic rate, stomatal conductance, and water‐use efficiency than WT plants. Our results indicate that co‐expression of tonoplast NHX and H⁺‐PPase genes from a xerophyte significantly improved the growth of alfalfa, and enhanced its tolerance to high salinity and drought. This study laid a solid basis for reclaiming and restoring saline and arid marginal lands as well as improving forage yield in northern China.     

Constitutive accumulation of zeaxanthin in tomato alleviates salt stress-induced photoinhibition and photooxidation

Zeaxanthin (Z) has a role in the dissipation of excess excitation energy by participating in non‐photochemical quenching (NPQ) and is essential in protecting the chloroplast from photooxidative damage. To investigate the physiological effects and functional mechanism of constitutive accumulation of Z in the tomato at salt stress‐induced photoinhibition and photooxidation, antisense‐mediated suppression of zeaxanthin epoxidase transgenic plants and the wild‐type (WT) tomato were used. The ratio of Z/(V + A + Z) and (Z + 0.5A)/(V + A + Z) in antisense transgenic plants were maintained at a higher level than in WT plants under salt stress, but the value of NPQ in WT and transgenic plants was not significantly different under salt stress. However, the maximal photochemical efficiency of PSII (Fv/Fm) and the net photosynthetic rate (Pn) in transgenic plants decreased more slowly under salt stress. Furthermore, transgenic plants showed lower level of hydrogen peroxide (H₂O₂), superoxide anion radical (O₂^??) and ion leakage, lower malondialdehyde content. Compared with WT, the content of D1 protein decreased slightly in transgenic plants under salt stress. Our results suggested that the constitutive accumulation of Z in transgenic tomatoes can alleviate salt stress‐induced photoinhibition because of the antioxidant role of Z in the scavenging quenching of singlet oxygen and/or free radicals in the lipid phase of the membrane.     

Expression of yeast <Emphasis Type="Italic">Hem1</Emphasis> controlled by <Emphasis Type="Italic">Arabidopsis HemA1</Emphasis> promoter enhances leaf photosynthesis in transgenic tobacco

A gene encoding aminolevulinate synthase (ALA-S) in yeast (Saccharomyces cerevisiae YHem1) was introduced into the genome of tobacco (Nicoliana tabacum) under the control of Arabidopsis thaliana HemA1 gene promoter (AtHemA1 P). All transgenic lines transcribed the YHem1 gene, especially under light condition. The capacity to synthesize ALA and therefore chlorophyll was increased in transformed plants. Determination of gas exchange suggested that transgenic plants had significantly higher level of net photosynthetic rate (P _n ), stomatal conductance (G _s ) and transpiration rate (T _r ), compared to the wild type (WT). Analysis with a modulated chlorophyll fluorometer demonstrated that the genetic transformation also caused a significant increase in photochemical efficiency of PSII (F^￠_v /F^￠_m F^{\prime}_{v} /F^{\prime}_{m} ), actual photochemical efficiency (Ф _PSII ), photochemical quenching (qP), electron transfer rate (ETR) and the energy proportion in photochemistry (Pc), but decrease in proportion in heat dissipation (Hd). Chlorophyll-a fast fluorescence measurement and JIP-test indicated that photosynthetic performance index on cross section basis (PI _CS ) and electron transport flux (ET _o /CS) of transgenic tobacco were increased remarkably. And the probability that a trapped exciton can move a electron into the electron transport chain beyond Q _A ⁻ (Ψ _o ) and the density of active reaction centers (RC/CS) were also increased obviously in transgenic tobacco. These results imply that transgenic tobacco plants expressing YHem1 gene had higher photosynthetic capacity and energy conversion efficiency than the WT plants.     

Co-expression of the Arabidopsis SOS genes enhances salt tolerance in transgenic tall fescue (Festuca arundinacea Schreb.)

Crop productivity is greatly affected by soil salinity; therefore, improvement in salinity tolerance of crops is a major goal in salt-tolerant breeding. The Salt Overly Sensitive (SOS) signal-transduction pathway plays a key role in ion homeostasis and salt tolerance in plants. Here, we report that overexpression of Arabidopsis thaliana SOS1+SOS2+SOS3 genes enhanced salt tolerance in tall fescue. The transgenic plants displayed superior growth and accumulated less Na⁺ and more K⁺ in roots after 350 mM NaCl treatment. Moreover, Na⁺ enflux, K⁺ influx, and Ca²⁺ influx were higher in the transgenic plants than in the wild-type plants. The activities of the enzyme superoxide dismutase, peroxidase, catalase, and proline content in the transgenic plants were significantly increased; however, the malondialdehyde content decreased in transgenic plants compared to the controls. These results suggested that co-expression of A. thaliana SOS1+SOS2+SOS3 genes enhanced the salt tolerance in transgenic tall fescue.     

Overexpression of the PtSOS2 gene improves tolerance to salt stress in transgenic poplar plants

In higher plants, the salt overly sensitive (SOS) signalling pathway plays a crucial role in maintaining ion homoeostasis and conferring salt tolerance under salinity condition. Previously, we functionally characterized the conserved SOS pathway in the woody plant Populus trichocarpa. In this study, we demonstrate that overexpression of the constitutively active form of PtSOS2 (PtSOS2TD), one of the key components of this pathway, significantly increased salt tolerance in aspen hybrid clone Shanxin Yang (Populus davidiana × Populus bolleana). Compared to the wild‐type control, transgenic plants constitutively expressing PtSOS2TD exhibited more vigorous growth and produced greater biomass in the presence of high concentrations of NaCl. The improved salt tolerance was associated with a decreased Na⁺ accumulation in the leaves of transgenic plants. Further analyses revealed that plasma membrane Na⁺/H⁺ exchange activity and Na⁺ efflux in transgenic plants were significantly higher than those in the wild‐type plants. Moreover, transgenic plants showed improved capacity in scavenging reactive oxygen species (ROS) generated by salt stress. Taken together, our results suggest that PtSOS2 could serve as an ideal target gene to genetically engineer salt‐tolerant trees.     

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.     

The <Emphasis Type="Italic">SlCMO</Emphasis> Gene Driven by Its Own Promoter Enhances Salt Tolerance of Transgenic Tomato Without Affecting Growth and Yield

Choline monooxygenase (CMO) is a key enzyme involved in betaine synthesis and our preliminary work has shown that the SlCMO gene promoter (pC5: ??267 to +?128 base pair), cloned from Suaeda liaotungensis, is salt-inducible. In the present study, pC5-SlCMO was transferred into tomato (Solanum lycopersicon L. ‘Micro-Tom’) plants via Agrobacterium mediation. Homozygous transgenic plants were selected using quantitative real-time polymerase chain reaction. The expression of SlCMO in pC5-SlCMO transgenic plants was induced by salinity. Under salt tolerance, betaine content, chlorophyll content, and net photosynthetic rate were higher in transgenic plants than in wild-type (WT) plants. Proline content was lower in transgenic plants than in WT plants. Under normal conditions, seed germination, length of the whole plant, dry weight, and fruit products of transgenic plants were the same as in WT plants. These results demonstrated that the pC5 promoter can drive increased expression of SlCMO in transgenic tomato plants under salt stress and increase salt tolerance without affecting plant growth and yield.     

A novel wheat α‐amylase inhibitor gene,TaHPS, significantly improves the salt and drought tolerance of transgenic Arabidopsis

On the basis of microarray analyses of the salt‐tolerant wheat mutant RH8706‐49, a previously unreported salt‐induced gene, designated as TaHPS [Triticum aestivum hypothetical (HPS)‐like protein], was cloned. Real‐time quantitative polymerase chain reaction analyses showed that expression of the gene was induced by abscisic acid, salt and drought. The encoded protein was found to be localized mainly in the plasma membranes. Transgenic Arabidopsis plants overexpressing TaHPS were more tolerant to salt and drought stresses than non‐transgenic wild‐type (WT) plants. Under salt stress, the root cells of the transgenic plants secreted more Na⁺ and guard cells took up more Ca²⁺ ions. Compared with wild‐type plants, TaHPS‐expressing transgenic plants showed significantly lower amylase activity and glucose and malic acid levels. Our results showed that the expression of TaHPS inhibited amylase activity, which subsequently led to a closure of stomatal apertures and thus improved plant tolerance to salt and drought.     

Over-expression of the Arabidopsis Na+/H+ antiporter gene in Populus deltoides CL?×?P. euramericana CL “NL895” enhances its salt tolerance

Soil salinity is a serious problem worldwide. It is necessary to improve the salt tolerance of plants to avoid the progressive deterioration of saline soil. We showed that the over-expression of AtNHX1 improves salt tolerance in a transgenic poplar (Populus deltoides CL × P. euramericana CL “NL895”) under mannose selection. Four transgenic poplar plants were obtained. Southern blot analysis showed that the pmi gene had integrated into the genome of the poplar. RT-PCR confirmed that AtNHX1 could be expressed normally in the transgenic plants. When tested for salt tolerance by NaCl stress, we measured a 100% increase in Na⁺ content in the three transgenic lines (T18, T50, T98) significantly higher than the 33% increase seen in wild-type plants. The chlorophyll content of the transgenic plants was not altered significantly, while the chlorophyll content in the control plants showed a small decrease. MDA content was decreased in the transgenic plants. These results show that the AtNHX1 gene may enhance salt tolerance due to increased vacuolar compartmentalization of sodium ions.     

Exogenously applied salicylic acid maintains redox homeostasis in salt-stressed <Emphasis Type="Italic">Arabidopsis gr1</Emphasis> mutants expressing cytosolic roGFP1

Exogenous salicylic acid (SA) can be used for chemical hardening to alleviate oxidative stress in plants exposed to salinity. The treatment of 5-week-old Arabidopsis thaliana plants with increasing doses of SA alters the ascorbate (ASC) and glutathione (GSH) pools, and modulates their redox status and the activity of several antioxidant enzymes, such as ascorbate peroxidase (APX) and glutathione reductase (GR). To investigate the role of GR in the maintenance of cytoplasmic redox homeostasis after hardening by SA, wild type (WT) and gr1 mutant plants, expressing the cytoplasmic redox-sensitive green fluorescent protein (c-roGFP1), were pre-treated with 10^?7 and 10^?5 M SA for 2 weeks and subsequently exposed to 100 mM NaCl. The redox status of the salt-stressed WT plants became more oxidized, which was prevented by pretreatment with 10^?5 M SA. The gr1 mutants showed more positive redox potential than WT plants, which could be reversed by treatment with 10^?5 M SA. In mutants, the increased GSH levels may have compensated for the deleterious effect of GR deficiency and stabilized the redox potential in plants exposed to salinity. The ASC regeneration in WT plants shifted from the GSH-dependent dehydroascorbate reductase (DHAR) reaction to the NAD(P)H-dependent monodehydroascorbate reductase (MDHAR) activity during chemical hardening, which contributed to the preservation of the GSH pool in plants under salt stress. Our results suggest that the maintenance of GSH levels and redox homeostasis by SA-mediated hardening play a major role in priming and defending against salt stress.     

Development and evaluation of novel salt‐tolerant Eucalyptus trees by molecular breeding using an RNA‐Binding‐Protein gene derived from common ice plant (Mesembryanthemum crystallinum L.)

The breeding of plantation forestry trees for the possible afforestation of marginal land would be one approach to addressing global warming issues. Here, we developed novel transgenic Eucalyptus trees (Eucalyptus camaldulensis Dehnh.) harbouring an RNA‐Binding‐Protein (McRBP) gene derived from a halophyte plant, common ice plant (Mesembryanthemum crystallinum L.). We conducted screened‐house trials of the transgenic Eucalyptus using two different stringency salinity stress conditions to evaluate the plants’ acute and chronic salt stress tolerances. Treatment with 400 mM NaCl, as the high‐stringency salinity stress, resulted in soil electrical conductivity (EC) levels >20 mS/cm within 4 weeks. With the 400 mM NaCl treatment, >70% of the transgenic plants were intact, whereas >40% of the non‐transgenic plants were withered. Treatment with 70 mM NaCl, as the moderate‐stringency salinity stress, resulted in soil EC levels of approx. 9 mS/cm after 2 months, and these salinity levels were maintained for the next 4 months. All plants regardless of transgenic or non‐transgenic status survived the 70 mM NaCl treatment, but after 6‐month treatment the transgenic plants showed significantly higher growth and quantum yield of photosynthesis levels compared to the non‐transgenic plants. In addition, the salt accumulation in the leaves of the transgenic plants was 30% lower than that of non‐transgenic plants after 15‐week moderate salt stress treatment. There results suggest that McRBP expression in the transgenic Eucalyptus enhances their salt tolerance both acutely and chronically.