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.     

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 Involvement of Wheat F-Box Protein Gene TaFBA1 in the Oxidative Stress Tolerance of Plants

As one of the largest gene families, F-box domain proteins have been found to play important roles in abiotic stress responses via the ubiquitin pathway. TaFBA1 encodes a homologous F-box protein contained in E3 ubiquitin ligases. In our previous study, we found that the overexpression of TaFBA1 enhanced drought tolerance in transgenic plants. To investigate the mechanisms involved, in this study, we investigated the tolerance of the transgenic plants to oxidative stress. Methyl viologen was used to induce oxidative stress conditions. Real-time PCR and western blot analysis revealed that TaFBA1 expression was up-regulated by oxidative stress treatments. Under oxidative stress conditions, the transgenic tobacco plants showed a higher germination rate, higher root length and less growth inhibition than wild type (WT). The enhanced oxidative stress tolerance of the transgenic plants was also indicated by lower reactive oxygen species (ROS) accumulation, malondialdehyde (MDA) content and cell membrane damage under oxidative stress compared with WT. Higher activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POD), were observed in the transgenic plants than those in WT, which may be related to the upregulated expression of some antioxidant genes via the overexpression of TaFBA1. In others, some stress responsive elements were found in the promoter region of TaFBA1, and TaFBA1 was located in the nucleus, cytoplasm and plasma membrane. These results suggest that TaFBA1 plays an important role in the oxidative stress tolerance of plants. This is important for understanding the functions of F-box proteins in plants’ tolerance to multiple stress conditions.     

Overexpression of the betaine aldehyde dehydrogenase gene from Atriplex hortensis enhances salt tolerance in the transgenic trifoliate orange (Poncirus trifoliata L. Raf.)

Trifoliate orange (Poncirus trifoliata L. Raf.), a rootstock widely used for citrus species, is salt-sensitive. Worldwide, salinity is a major abiotic stress affecting citrus growth and yield. Glycinebetaine (GB) is an important osmoprotectant involved in responses to salt stress. However, current evidence regarding the effect of salt stress on GB accumulation in trifoliate orange is limited, and the GB synthesis gene has not yet been shown to confer enhanced salt stress tolerance to this species in a transgenic context. In the current study, we first examined the change in GB level of trifoliate orange seedlings exposed to salt stress, and found that salt increased endogenous GB level in a concentration-dependent manner. A betaine aldehyde dehydrogenase gene (AhBADH) cloned from Atriplex hortensis was introduced into the trifoliate orange by means of Agrobacterium-mediated transformation. RT-PCR analysis on three selected transgenic lines showed that the AhBADH gene was overexpressed in each of them. GB levels in these lines were also higher than those in untransformed wild-type (WT) plants. In the transgenic lines, exposure to 200 mM NaCl resulted in significantly less serious leaf burning and defoliation, lower MDA accumulation, and higher chlorophyll contents than those in the WT plants. Moreover, when exposed to salt, shoots of transgenic plants contained lower levels of Na⁺ and Cl⁻, higher levels of K⁺, and a higher K/Na ratio, while the same was true for the roots in most cases. Taken together, the data suggest that overexpression of the AhBADH gene in transgenic trifoliate orange enhanced salt stress tolerance. This may be correlated with the low levels of lipid peroxidation, protection of the photosynthetic machinery, and increase in K⁺ uptake.     

<Emphasis Type="Italic">OsNHX2</Emphasis>, an Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene,can enhance salt tolerance in rice plants through more effective accumulation of toxic Na<Superscript>+</Superscript> in leaf mesophyll and bundle sheath cells

The Na⁺/H⁺ antiporters play an important role in salt tolerance in plants. However, the functions of OsNHXs in rice except OsNHX1 have not been well studied. Using the gain- and loss-of-function strategies, we studied the potential role of OsNHX2 in salt tolerance in rice. Overexpression of OsNHX2 (OsNHX2-OE) in rice showed the significant tolerance to salt stress than wild-type plants and OsNHX2 knockdown transgenic plants (OsNHX2-KD). Under salt treatments of 300-mM NaCl for 5 days, the plant fresh weights, relative water percentages, shoot heights, Na⁺ contents, K⁺ contents, and K⁺/Na⁺ ratios in leaves of OsNHX2-OE transgenic plants were higher than those in wild-type plants, while no differences were detected in roots. K⁺/Na⁺ ratios in rice leaf mesophyll cells and bundle sheath cells were higher in OsNHX2-OE transgenic plants than in wild-type plants and OsNHX2-KD transgenic plants. Our data indicate that OsNHX2 plays an important role in salt stress based on leaf mesophyll cells and bundle sheath cells and can be served in genetically engineering crop plants with enhanced salt tolerance.     

A Medicago truncatula EF-Hand Family Gene,MtCaMP1, Is Involved in Drought and Salt Stress Tolerance

Background

Calcium-binding proteins that contain EF-hand motifs have been reported to play important roles in transduction of signals associated with biotic and abiotic stresses. To functionally characterize gens of EF-hand family in response to abiotic stress, an MtCaMP1 gene belonging to EF-hand family from legume model plant Medicago truncatula was isolated and its function in response to drought and salt stress was investigated by expressing MtCaMP1 in Arabidopsis.

Methodology/Principal Findings

Transgenic Arabidopsis seedlings expressing MtCaMP1exhibited higher survival rate than wild-type seedlings under drought and salt stress, suggesting that expression of MtCaMP1 confers tolerance of Arabidopsis to drought and salt stress. The transgenic plants accumulated greater amounts of Pro due to up-regulation of P5CS1 and down-regulation of ProDH than wild-type plants under drought stress. There was a less accumulation of Na⁺ in the transgenic plants than in WT plants due to reduced up-regulation of AtHKT1 and enhanced regulation of AtNHX1 in the transgenic plants compared to WT plants under salt stress. There was a reduced accumulation of H₂O₂ and malondialdehyde in the transgenic plants than in WT plants under both drought and salt stress.

Conclusions/Significance

The expression of MtCaMP1 in Arabidopsis enhanced tolerance of the transgenic plants to drought and salt stress by effective osmo-regulation due to greater accumulation of Pro and by minimizing toxic Na⁺ accumulation, respectively. The enhanced accumulation of Pro and reduced accumulation of Na⁺ under drought and salt stress would protect plants from water default and Na⁺ toxicity, and alleviate the associated oxidative stress. These findings demonstrate that MtCaMP1 encodes a stress-responsive EF-hand protein that plays a regulatory role in response of plants to drought and salt stress.     

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 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.     

Marker-free transgenic durum wheat cv. Karim expressing the AlSAP gene exhibits a high level of tolerance to salinity and dehydration stresses

We have recently isolated the AlSAP (stress-associated protein) gene from the halophyte grass Aeluropus littoralis and demonstrated that AlSAP expression improves tolerance to continuous salt and drought stresses in transgenic tobacco. To extend these findings to an important crop, we generated marker-free transgenic durum wheat plants of the commercial cv. Karim expressing the AlSAP gene. The integration and expression of AlSAP in T3 homozygous plants were ascertained by Southern, Northern and Western blotting respectively. AlSAP wheat lines exhibited improved germination rates and biomass production under severe salinity and osmotic stress conditions. Following a long-term salt or drought stress greenhouse trial, AlSAP lines produced normally filled grains whereas wild-type (WT) plants either died at the vegetative stage under salt stress or showed markedly reduced grain filling under drought stress. Measurements of the RWC (relative water content) and endogenous Na⁺ and K⁺ levels in leaves of AlSAP plants, showed a lower water loss rate and a higher Na⁺ accumulation in senescent-basal leaves, respectively, compared to those of WT plants. Taken together, these results extend to cereals the high potential of the AlSAP gene for engineering effective drought and salt tolerance.     

Overexpression of Rice CBS Domain Containing Protein Improves Salinity, Oxidative, and Heavy Metal Tolerance in Transgenic Tobacco

We have recently identified and classified a cystathionine ??-synthase domain containing protein family in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa L.). Based on the microarray and MPSS data, we have suggested their involvement in stress tolerance. In this study, we have characterized a rice protein of unknown function, OsCBSX4. This gene was found to be upregulated under high salinity, heavy metal, and oxidative stresses at seedling stage. Transgenic tobacco plants overexpressing OsCBSX4 exhibited improved tolerance toward salinity, heavy metal, and oxidative stress. This enhanced stress tolerance in transgenic plants could directly be correlated with higher accumulation of OsCBSX4 protein. Transgenic plants could grow and set seeds under continuous presence of 150?mM NaCl. The total seed yield in WT plants was reduced by 80%, while in transgenic plants, it was reduced only by 15?C17%. The transgenic plants accumulated less Na⁺, especially in seeds and maintained higher net photosynthesis rate and Fv/Fm than WT plants under NaCl stress. Transgenic seedlings also accumulated significantly less H₂O₂ as compared to WT under salinity, heavy metal, and oxidative stress. OsCBSX4 overexpressing transgenic plants exhibit higher abiotic stress tolerance than WT plants suggesting its role in abiotic stress tolerance in plants.     

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.     

Overexpression of Tamarix albiflonum TaMnSOD increases drought tolerance in transgenic cotton

Drought is a major environmental stress that limits cotton (Gossypium hirsutum L.) production worldwide. TaMnSOD plays a crucial role as a peroxidation scavenger. In this study, TaMnSOD cDNA of Tamarix albiflonum was overexpressed in the cotton cultivar fy11 by Agrobacterium tumefaciens-mediated transformation. The transformed plants were assessed by gDNA PCR, RT-PCR and DNA gel blot analysis. The physiological and biochemical characters of two independent transgenic lines and control plants were tested and compared, and the morphological traits (biomass, root and lateral root length, leaf number) were also detected after recovery from water-withholding stress. When water was withheld from pot-grown 6-week-old seedlings for 18 days (watering to 8 % of field capacity), transgenic cotton plants accumulated more proline and soluble sugar than wild-type plants (WT). The activity of antioxidant enzymes such as superoxide dismutase and peroxidase was enhanced in transgenic plants under drought stress. Cell membrane integrity was also considerably improved under water stress, as indicated by reduced malondialdehyde content relative to control plants. Furthermore, net photosynthesis, stomatal conductance and transpiration rate were increased in transgenic plants compared with wild type. Transgenic cotton showed increases in biomass as well as root and leaf systems compared with WT after 2 weeks recovery from stress. These results suggest that TaMnSOD transgenic cotton plants acquired improved drought tolerance through enhanced development of the root and leaf system and the regulation of superoxide scavenging.