Drought Tolerance In Transgenic Tobacco Research Articles

Identification of PP2Cs in six rosaceae species highlights RcPP2C24 as a negative regulator in rose drought tolerance

Drought is a major environmental stress that limits plant growth, so it's important to identify drought-responsive genes to understand the mechanism of drought response and breed drought-tolerant roses. Protein phosphatase 2C (PP2C) plays a crucial role in plant abiotic stress response. In this study, we identified 412 putative PP2Cs from six Rosaceae species. These genes were divided into twelve clades, with clade A containing the largest number of PP2Cs (14.1%). Clade A PP2Cs are known for their important role in ABA-mediated drought stress response; therefore, the analysis focused on these specific genes. Conserved motif analysis revealed that clade A PP2Cs in these six Rosaceae species shared conserved C-terminal catalytic domains. Collinearity analysis indicated that segmental duplication events played a significant role in the evolution of clade A PP2Cs in Rosaceae. Analysis of the expression of 11 clade A RcPP2Cs showed that approximately 60% of these genes responded to drought, high temperature, and salt stress. Among them, RcPP2C24 exhibited the highest responsiveness to both drought and ABA. Furthermore, overexpression of RcPP2C24 significantly reduced drought tolerance in transgenic tobacco by increasing stomatal aperture after exposure to drought stress. The transient overexpression of RcPP2C24 weakened the dehydration tolerance of rose petal discs, while its silencing increased their dehydration tolerance. In summary, our study identified PP2Cs in six Rosaceae species and highlighted the negative role of RcPP2C24 on rose's drought tolerance by inhibiting stomatal closure. Our findings provide valuable insights into understanding the mechanism behind rose's response to drought.

A mulberry 9-cis-epoxycarotenoid dioxygenase gene MaNCED1 is involved in plant growth regulation and confers salt and drought tolerance in transgenic tobacco.

The phytohormone abscisic acid (ABA) is vital in regulating root elongation, seed germination, and abiotic stress responses in plants. Conversely, the mechanisms of ABA in mulberry root growth, seed germination, and abiotic stress responses are poorly understood. Here, we reported that exogenous ABA and drought treatment inhibited the growth of mulberry seedlings but significantly increased the ratio of root/stem. Inhibition of ABA synthesis by fluridone and sodium tungstate resulted in the decrease of root/stem ratio. We also showed that the expression of MaNCED1 in the root was strongly induced by drought and salt stress. Increasing the expression of MaNCED1 in tobacco using overexpression leads to increased root elongation and reduced seed germination. Compared with the wild type, the accumulation of H2O2 and MDA was reduced, while the POD activity and proline content was increased in the transgenic plants after drought and salt treatment. Further studies revealed increased resistance to drought and salt stress in MaNCED1 overexpressed tobaccos. Meanwhile, the auxin and ethylene signal pathway-related gene expression levels increased in MaNCED1 overexpressed tobaccos. This study demonstrated the roles of mulberry MaNCED1 in regulating plant development and abiotic stress responses. It gave further insights into the coordinated regulation of ABA, auxin, and ethylene in seed growth and germination.

Soybean GmGolS2-2 improves drought resistance of transgenic tobacco

Galactinol synthase (GolS) genes play important roles in plant response to abiotic stress. In this research, the plant expression vector of soybean GmGolS2-2 gene was constructed and transformed into tobacco to study the drought tolerance of transgenic tobacco. A GmGolS2-2 gene with 975 bp coding sequence was cloned from soybean leaves by reverse transcription-polymerase chain reaction (RT-PCR). GmGolS2-2 was linked to the plant expression vector pRI101 by restriction enzyme sites Nde Ⅰ and EcoR Ⅰ, and transformed into tobacco by leaf disc method. Genomic DNA PCR and real-time PCR showed that three GmGolS2-2 transgenic tobacco plants were obtained. The growth status of GmGolS2-2 transgenic tobacco under drought stress was better than that of wild-type tobacco. After drought stress treatment, the electrolyte leakage and malondialdehyde content of transgenic tobacco were lower than those of wild-type tobacco, but the proline content and soluble sugar content were higher than those of wild-type tobacco. The results of real-time PCR showed that the heterologous expression of GmGolS2-2 increased the expression of stress-related genes NtERD10C and NtAQP1 in transgenic tobacco. The above results indicated that GmGolS2-2 improved drought resistance of transgenic tobacco.

SUMO protease GmOTSa positively regulates drought tolerance in transgenic tobacco and soybean hairy roots

SUMO protease is important for regulating the balance of SUMOylation and deSUMOylation. Despite the investigations of several SUMO protease genes in model plants, to date, the biological functions of these regulatory enzymes in soybean have yet to be ascertained. In the present study, a SUMO protease GmOTSa was identified in soybean, and was localized in the nucleus with the bona fide SUMO protease activity both in vitro and in vivo. Further RTqPCR analysis showed that GmOTSa was expressed in various tissues and was highly induced by PEG treatment. We demonstrated that GmOTSa conferred drought tolerance in tobacco plants by increasing the germination rate, the fresh and dry weights upon water deficit, and improving the survival rate after rewatering. Overexpression of GmOTSa gene in tobacco lines and soybean hairy roots enhanced drought tolerance by positively regulating ROS scavenging, thus maintained lower ROS content. Moreover, less MDA content and higher total chlorophyll content as well as promoted closure of stomata were detected in the OE plants than in the WT plants in response to drought stress. Conversely, soybean hairy roots with simultaneous knockout of GmOTSa and the homologous gene GmOTSb using the CRISPRCas9 method exhibited reduced drought tolerance. Correspondingly, the expression of drought-related genes showed greater induction upon drought stress in the GmOTSa-overexpressed lines. Overall, these results indicated that GmOTSa may act as a positive regulator of drought responses and therefore could be considered a potential gene for the genetic improvement of drought tolerance.

Isolation, characterization and functional validation of a soybean transcription factor, GmDof4.2 improves drought tolerance in transgenic tobacco

Isolation, characterization and functional validation of a soybean transcription factor, GmDof4.2 improves drought tolerance in transgenic tobacco

Carex rigescens caffeic acid O-methyltransferase gene CrCOMT confer melatonin-mediated drought tolerance in transgenic tobacco.

Melatonin is an important, multifunctional protective agent against a variety of abiotic and biotic stressors in plants. Caffeic acid O-methyltransferase (COMT) catalyzes the last step of melatonin synthesis in plants and reportedly participates in the regulation of stress response and tolerance. However, few studies have reported its function in melatonin-mediated drought resistance. In this study, CrCOMT was identified and was strongly induced by drought stress in Carex rigescens. CrCOMT overexpression in transgenic tobacco increased tolerance to drought stress with high levels of seed germination, relative water content, and survival rates. CrCOMT overexpression in tobacco improved membrane stability, and plants exhibited lower relative electrolytic leakage and malondialdehyde content, as well as higher photochemical efficiency than the wildtype (WT) under drought stress. The transgenic plants also had higher levels of proline accumulation and antioxidant enzyme activity, which decreased oxidative stress damage due to reactive oxygen species (ROS) hyperaccumulation under drought stress. The transcription of drought stress response and ROS scavenging genes was significantly higher in the CrCOMT overexpression plants than in the WT plants. In addition, CrCOMT transgenic tobacco plants exhibited higher melatonin content under drought stress conditions. Exogenous melatonin was applied to C. rigescens under drought stress to confirm the function of melatonin in mediating drought tolerance; the relative water content and proline content were higher, and the relative electrolytic leakage was lower in melatonin-treated C. rigescens than in the untreated plants. In summary, these results show that CrCOMT plays a positive role in plant drought stress tolerance by regulating endogenous melatonin content.

Molecular cloning and functional analysis of a Chrysanthemum vestitum GME homolog that enhances drought tolerance in transgenic tobacco

GDP-mannose 3, 5-epimerase (GME, EC 5.1.3.18), a key enzyme in the ascorbic acid synthesis pathway, catalyzes the conversion of GDP-D-mannose to GDP-l-galactose in higher plants. Here, a homolog of GME was isolated from Chrysanthemum vestitum. The cDNA sequence of CvGME was 1131 bp and contained a complete open reading frame encoding a protein comprising 376 amino acids. Quantitative real-time PCR analysis revealed that CvGME was most highly expressed in the stems and roots. Phylogenetic analysis showed that CvGME was closely related to LsGME from Lactuca sativa. Subcellular localization studies revealed that CvGME was localized in the nucleus. Heterologous expression of CvGME in transgenic tobacco plants increased the ascorbic acid content in the leaves. In addition, overexpression of CvGME reduced the malondialdehyde content and increased superoxide dismutase and peroxidase activity in tobacco leaves compared to those in the wild-type plants under drought stress conditions, explaining the increased drought tolerance of transgenic tobacco lines. These results suggest that CvGME can effectively enhance the tolerance of plants to drought by increasing the ascorbic acid content, which may help improve the drought tolerance of chrysanthemums through molecular breeding.

Overexpression of VyDOF8, a Chinese wild grapevine transcription factor gene, enhances drought tolerance in transgenic tobacco

DNA-binding one zinc finger (DOF) proteins play a key role in plant resistance to drought conditions. In this paper, a DOF gene (VyDOF8) identified from Chinese wild grapevine Vitis yeshanensis 'Yanshan', was significantly increased by salt, drought, and cold stresses. The VyDOF8 protein was localized to the nucleus and functioned as a transcription factor. VyDOF8-overexpressing (VyDOF8-OE) tobacco (Nicotiana benthamiana) exhibited a smaller percent water loss and advanced root development, and accumulated more chlorophyll and proline under drought conditions compared with the control, which suggested that overexpression of VyDOF8 might confer enhanced drought tolerance by increasing the abscisic acid content and promoting stress-responsive gene expression. Meanwhile, VyDOF8-OE plants also showed reduced malondialdehyde (MDA) and H2O2 contents by boosting key enzymatic antioxidant activities under drought conditions. These findings provide valuable information for functional analysis of DOF genes in grape.

Overexpression of the pitaya phosphoethanolamine N-methyltransferase gene (HpPEAMT) enhanced simulated drought stress in tobacco

Phosphoethanolamine N-methyltransferase (PEAMTase) catalyzes the methylation of phosphoethanolamine to produce phosphocholine and plays an important role in the abiotic stress response. Although the PEAMT genes has been isolated from many species other than pitaya, its role in the drought stress response has not yet been fully elucidated. In the present study, we isolated a 1485 bp cDNA fragment of HpPEAMT from pitaya (Hylocereus polyrhizus). Phylogenetic analysis showed that, during its evolution, HpPEAMT has shown a high degree of amino acid sequence similarity with the orthologous genes in Chenopodiaceae species. To further investigate the function of HpPEAMT, we generated transgenic tobacco plants overexpressing HpPEAMT, and the transgenic plants accumulated significantly more glycine betaine (GB) than did the wild type (WT). Drought tolerance trials indicated that, compared with those of the wild-type (WT) plants, the roots of the transgenic plants showed higher drought tolerance ability and exhibited improved drought tolerance. Further analysis revealed that overexpression of HpPEAM in Nicotiana tabacum resulted in upregulation of transcript levels of GB biosynthesis-related genes (NiBADH, NiCMO and NiSDC) in the leaves. Furthermore, compared with the wild-type plants, the transgenic tobacco plants displayed a significantly lower malondialdehyde (MDA) accumulation and higher activities of the superoxide dismutase (SOD) and peroxidase (POD) antioxidant enzymes under drought stress. Taken together, our results suggested that HpPEAMT enhanced the drought tolerance of transgenic tobacco.

Constitutive expression of a group 3 LEA protein from Medicago falcata (MfLEA3) increases cold and drought tolerance in transgenic tobacco.

MfLEA3 is involved in protection of catalase activity and confers multiple abiotic stress tolerance. Late embryogenesis abundant (LEA) proteins are involved in plant growth, development and abiotic stress tolerance. A member of group 3 LEA proteins from Medicago sativa subsp. falcata (L.) Arcang, MfLEA3, was investigated in the study. MfLEA3 transcript was induced in response to cold, dehydration, and abscisic acid (ABA), while the cold-induced transcript of MfLEA3 was blocked by pretreatment with inhibitor of ABA synthesis. Constitutive expression of MfLEA3 led to enhanced tolerance to cold, drought, and high-light stress in transgenic tobacco plants. Compared to accumulated reactive oxygen species (ROS) in the wild-type in response to treatments with low temperature, drought, and high light, ROS were not accumulated in transgenic plants. Superoxide dismutase, catalase (CAT), and ascorbate-peroxidase activities were increased in all plants after treatments with the above stresses, while higher CAT activity was maintained in transgenic plants compared with wild-type. However, transcript level of CAT-encoding genes including CAT1, CAT2, and CAT3 showed no significant difference between transgenicplants and wild-type, indicating that the higher CAT activity was not associated with its gene expression. ABA sensitivity and transcripts of several ABA and stress-responsive genes showed no difference between transgenic plant and wild-type, indicating that ABA signaling was not affected by constitutive expression of MfLEA3. The results suggest that MfLEA3 may be involved in the protection of CAT activity and confers multiple abiotic stress tolerance.

Overexpression of Saussurea involucrata dehydrin gene SiDHN promotes cold and drought tolerance in transgenic tomato plants.

Dehydrins are late embryogenesis abundant proteins that help regulate abiotic stress responses in plants. Overexpression of the Saussurea involucrata dehydrin gene SiDHN has previously been shown to improve water-use efficiency and enhance cold and drought tolerance of transgenic tobacco. To understand the mechanism by which SiDHN exerts its protective function, we transformed the SiDHN gene into tomato plants (Solanum lycopersicum L.) and assessed their response to abiotic stress. We observed that in response to stresses, the SiDHN transgenic tomato plants had increased contents of chlorophyll a and b, carotenoid and relative water content compared with wild-type plants. They also had higher maximal photochemical efficiency of photosystem II and accumulated more proline and soluble sugar. Compared to those wild-type plants, malondialdehyde content and relative electron leakage in transgenic plants were not significantly increased, and H2O2 and O2- contents in transgenic tomato plants were significantly decreased. We further observed that the production of stress-related antioxidant enzymes, including superoxide dismutase, ascorbate peroxidase, peroxidase, and catalase, as well as pyrroline-5-carboxylate synthetase and lipid transfer protein 1, were up-regulated in the transgenic plants under cold and drought stress. Based on these observations, we conclude that overexpression of SiDHN gene can promote cold and drought tolerance of transgenic tomato plants by inhibiting cell membrane damage, protecting chloroplasts, and enhancing the reactive oxygen species scavenging capacity. The finding can be beneficial for the application of SiDHN gene in improving crop tolerance to abiotic stress and oxidative damage.

A novel cold-regulated protein isolated from Saussurea involucrata confers cold and drought tolerance in transgenic tobacco (Nicotiana tabacum)

Adverse environmental conditions, such as cold and drought, can inhibit plant growth, development, and productivity. The isolation and characterization of stress response genes from stress-tolerant plants can provide a better understanding of the underlying adaptive mechanisms. In this study, a novel cold-regulated gene, SikCOR413PM1, was isolated from Saussurea involucrata Kar. et Kir., which is a plant that survives at the high altitudes and in the low temperatures of alpine slopes in northwestern China. SikCOR413PM1 was induced in response to cold and drought in S. involucrata, and phylogenetic analysis revealed that the gene groups with a COR gene encoding a COR413PM protein family member. Subcellular localization of a SikCOR413PM1-green fluorescent fusion protein showed that SikCOR413PM1 was localized to the plasma membrane. A transgenic tobacco (Nicotiana tabacum) system was employed to investigate the possible role of SikCOR413PM1 in cold and drought tolerance. Analyses of growth, germination and survival rates, relative water content, malondialdehyde content, relative electrolyte leakage, and maximal photochemical efficiency of photosystem II showed that transgenic tobacco plants expressing SikCOR413PM1 were more tolerant to cold and drought stresses than WT plants. SikCOR413PM1 overexpression was also accompanied by constitutive activation of NtDREB1 and NtDREB3, two cold-responsive transcription factor genes, and NtERD10A and NtERD10B, two cold-induced genes. The expression levels of downstream transcription factor genes NtDREB3, NtERD10C, NtERD10D, and NtLEA5 were also induced in SikCOR413PM1-expressing transgenic plants under drought conditions. Our results suggest that the overexpression of SikCOR413PM1 induces changes in tobacco plants, and facilitates enhanced tolerance to cold and drought stresses.

Overexpression of a maize SUMO conjugating enzyme gene (ZmSCE1e) increases Sumoylation levels and enhances salt and drought tolerance in transgenic tobacco

As an essential regulatory process of post-translational modifications, Sumoylation has been shown to play a central role in stress responses in higher plants. However, the mechanisms underlying the involvement of the Sumoylation in stress responses in crops are largely unknown. In this study, a putative SUMO conjugating enzyme ortholog from Zea mays (ZmSCE1e) was isolated. Sequence alignments and phylogenetic analysis showed that ZmSCE1e possesses a central active domain similar to known SCE1 proteins, but is the cereal-specific isoform.The transcript levels of ZmSCE1e were markedly up-regulated by salt or drought stress. Over-expression of ZmSCE1e in tobacco plants increased levels of SUMO conjugates and enhanced their tolerances to salt and drought stresses. ZmSCE1e-transgenic plants showed higher activities of key antioxidant enzymes but lower hydrogen peroxide (H2O2) and malondialdehyde (MDA) accumulations under salt or drought stress. Furthermore, expression of several stress defense genes was significantly elevated as revealed by qPCR in the ZmSCE1e-transgenic lines. Together, these data have demonstrated that ZmSCE1e improved salt and drought tolerance likely by modulating Sumoylation levels, antioxidant capability, and stress defense gene expression in transgenic plants. This study may facilitate our understanding of the biological roles of SCE-mediated Sumoylation under stress conditions in higher plants and accelerate genetic improvement of crop plants tolerant to environmental stresses.

An apple transcription factor, MdDREB76, confers salt and drought tolerance in transgenic tobacco by activating the expression of stress-responsive genes.

An apple gene, MdDREB76 encodes a functional transcription factor and imparts salinity and drought stress endurance to transgenic tobacco by activating expression of stress-responsive genes. The dehydration-responsive element (DRE)-binding protein (DREB) transcription factors are well known to be involved in regulating abiotic stress-mediated gene expression in plants. In this study, MdDREB76 gene was isolated from apple (Malus x domestica), which encodes a functional transcription factor protein. Overexpression of MdDREB76 in tobacco conferred salt and drought stress tolerance to transgenic lines by inducing antioxidant enzymes, such as superoxide dismutase, ascorbate peroxidase and catalase. The higher membrane stability index, relative water content, proline, total soluble sugar content and lesser H2O2content, electrolyte leakage and lipid peroxidation in transgenics support the improved physiological status of transgenic plants as compared to WT plants under salinity and drought stresses. The MdDREB76 overexpression upregulated the expression of stress-responsive genes that provide salinity and drought stress endurance to the plants. Compared to WT plants, transgenic lines exhibited healthy growth and higher yield under stress conditions. The present study reports MdDREB76 as a key regulator that switches on the battery of downstream genes which impart salt and osmotic stress endurance to the transgenic plants and can be used for genetic engineering of crop plants to combat salinity and drought stresses.

Overexpression of the Maize Sulfite Oxidase Increases Sulfate and GSH Levels and Enhances Drought Tolerance in Transgenic Tobacco.

Sulfite oxidase (SO) plays a pivotal role in sulfite metabolism. In our previous study, sulfite-oxidizing function of the SO from Zea mays (ZmSO) was characterized. To date, the knowledge of ZmSO’s involvement in abiotic stress response is scarce. In this study, we aimed to investigate the role of ZmSO in drought stress. The transcript levels of ZmSO were relatively high in leaves and immature embryos of maize plants, and were up-regulated markedly by PEG-induced water stress. Overexpression of ZmSO improved drought tolerance in tobacco. ZmSO-overexpressing transgenic plants showed higher sulfate and glutathione (GSH) levels but lower hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents under drought stress, indicating that ZmSO confers drought tolerance by enhancing GSH-dependent antioxidant system that scavenged ROS and reduced membrane injury. In addition, the transgenic plants exhibited more increased stomatal response than the wild-type (WT) to water deficit. Interestingly, application of exogenous GSH effectively alleviated growth inhibition in both WT and transgenic plants under drought conditions. qPCR analysis revealed that the expression of several sulfur metabolism-related genes was significantly elevated in the ZmSO-overexpressing lines. Taken together, these results imply that ZmSO confers enhanced drought tolerance in transgenic tobacco plants possibly through affecting stomatal regulation, GSH-dependent antioxidant system, and sulfur metabolism-related gene expression. ZmSO could be exploited for developing drought-tolerant maize varieties in molecular breeding.

AlSRG1, a novel gene encoding an RRM-type RNA-binding protein (RBP) from Aeluropus littoralis, confers salt and drought tolerance in transgenic tobacco

We characterized a novel stress tolerance gene from Aeluropus littoralis encoding a protein designed as AlSRG1 (A. littoralisStress-Related Gene 1). Phylogenetic and conserved domain prediction identified AlSRG1 as an uncharacterized protein of unknown function. The conserved domain database revealed that AlSRG1 contains an RNA-recognition motif (RRM). This domain was highlighted in AlSRG1 primary, secondary and tertiary structures with the two conserved motifs RNP1 and RNP2 being part of it. These two conserved motifs contain the active site for RNA-binding and a 3D-model of conserved domain (AlSRG1p) was built from known X-ray structures and sequence alignments. The analysis with RT-qPCR revealed higher AlSRG1 transcript accumulation under salt and osmotic stresses. Overexpression of AlSRG1 in transgenic tobacco plants improved both salt and osmotic tolerance and was accompanied by higher seedling survival rates, better growth parameters and ROS accumulation than non-transgenic plants (NT). Furthermore, the transgenic tobacco plants were able to complete their life cycle and to produce viable seeds while the NT plants died at the vegetative stage. The AlSRG1 transgenic lines compared to NT tobacco, showed a significantly lower water loss rate in senescent-basal leaves. AlSRG1 overexpression also enhanced the transcript levels of ROS-scavenging genes and some stress-related transcription factors under salt and osmotic stresses. Taken together, these results show that AlSRG1 may play an important positive modulation role in abiotic stress tolerance.

Overexpression of a tomato carotenoid ε-hydroxylase gene (SlLUT1) improved the drought tolerance of transgenic tobacco

Drought stress is a considerable environmental factor that restrains photosynthesis. Lutein, the most prolific carotenoid in plant photosynthetic tissues, plays vital roles in the light-harvesting complexes. However, its biological functions under abiotic stresses remain unclear. In our research, transgenic tobacco plants were utilized to investigate the function of the tomato chloroplast-targeted carotenoid epsilon-ring hydroxylase (SlLUT1) in drought stress tolerance. The analysis of SlLUT1-pro-LUC and qRT-PCR showed that drought stress induced SlLUT1 expression. Transgenic tobacco plants exhibit higher lutein content than wild-type (WT) tobacco. Under drought stress, transgenic plants overexpressing SlLUT1 showed better growth performance, higher chlorophyll and relative water contents and more intact chloroplast and PSII supercomplex structures than WT tobacco. The Fv/Fm, Pn, NPQ, and content of D1 protein in transgenic plants were higher than those in WT plants under drought stress. The accumulation of H2O2 and O2− decreased in transgenic tobacco plants. Moreover, transgenic plants exhibited lower MDA accumulation and REL. These results indicate that overexpression of SlLUT1 enhances tolerance to drought stress by maintaining photosynthesis and scavenging ROS in transgenic tobacco.

Isolation and characterization of MbWRKY2 gene involved in enhanced drought tolerance in transgenic tobacco

ABSTRACTPlant-specific WRKY transcription factors are widely involved in abiotic stress responses. In this study, a WRKY gene was isolated from Malus baccata (L.) Borkh and designated it as MbWRKY2. Subcellular localization showed that MbWRKY2 was localized in the nucleus. The expression levels of MbWRKY2 were up-regulated by abiotic stresses in M. baccata. When MbWRKY2 was introduced into tobacco, it improved drought stress tolerance in transgenic plants. The transgenic tobaccos had the higher contents of chlorophyl, proline, relative water content, AsA, and GSH, increased activities of CAT, APX, SOD, and POD, and decreased levels of MDA, H2O2, and electrolyte leakage than wild-type, especially when dealt with dehydration treatment. Moreover, the MbWRKY2-OE plants enhanced the expression of oxidative stress response and stress-related genes involved in osmotic adjustment and membrane protection. These results suggest that MbWRKY2 gene plays a positive regulatory role in drought stress response.

CaMKK1 from Chenopodium album positively regulates salt and drought tolerance in transgenic tobacco

Plants encounter various abiotic stresses during the whole life cycle. The mitogen-activated protein kinase kinase (MAPKK) acts as the convergent point of MAPK cascade in stress signaling pathway, which has not widely been studied so far. In the present study, we isolated a group A MAPKK gene (named as CaMKK1) from Chenopodium album, a salt-tolerant plant species in Chenopodiaceae. Quantitative RT-PCR analysis indicates that CaMKK1 was significantly up-regulated and displayed a fast and transient activation, which was similar with MAPK expression pattern in C. album under salt and drought stress. Ectopic expression of CaMKK1 in Escherichia coli BL21 (DE3) strains showed that overexpression of CaMKK1 could increase the salt and drought tolerance to bacterium. Overexpression of CaMKK1 in transgenic tobacco exhibited attenuated salt and PEG sensitivity by means of improving germination percentage and seedling growth. Furthermore, transgenic tobacco lines significantly accumulated organic osmoprotectants, and increased antioxidant enzyme activities which effectively relieved the accumulated reactive oxygen species under stress; the water loss rate was reduced and net photosynthetic rate was significantly increased in transgenic lines compared to non-transgenic plant. The more worth mentioning is that the transcripts of downstream transcription factors (NtDREB2, NtDREB3, NtDREB4) were quickly and dramatically accumulated (from 15 to 224 folds) in transgenic lines under stress. In conclusion, our results indicate that CaMKK1 is a positive regulator in response to salt and drought stress in plants, which should provide new data for further analyzing the function of plant MAPK pathway.

A ThDREB gene from Tamarix hispida improved the salt and drought tolerance of transgenic tobacco and T. hispida

Dehydration-responsive element-binding (DREB) transcription factors are important abiotic stress tolerance related genes, and some reports on the roles of DREB have primarily addressed herbal plants. To explore the abiotic stress tolerance role of DREB (ThDREB) from Tamarix hispida, a ThDREB gene with a complete ORF of 783 bp that encodes a 28.74 kDa protein with 260 amino acids, was isolated and functionally annotated. ThDREB expression was highly induced by NaCl, PEG, NaHCO3 and CdCl2 treatments, and the highest expression level (369.2-fold of control) was found for the roots that were under NaCl stress for 6 h. The tobacco plants that were transformed by ThDREB were conferred with higher germination rates, fresh weights and root lengths than the wild type (WT) tobacco plants under NaCl and mannitol treatments. The total chlorophyll content (tcc), superoxide dismutase (SOD) and peroxidase (POD) activities were also higher in the transgenic lines in comparison with the WT, and the malondialdehyde (MDA) and H2O2 content, electrolyte leakage (EL) rate and ROS as tracked by staining were generated to a lesser degree in ThDREB transgenic plants than in the WT under NaCl and mannitol stress. Furthermore, the transient overexpression analysis of ThDREB in T. hispida also improved plant salt and drought tolerance in comparison with the empty vector-transformed lines. Our results indicated that ThDREB expression could effectively improve tolerance to salt and drought stress by enhancing the antioxidase activity that keeps the ROS at a low accumulation level and makes them easy to scavenge.