Salt Tolerance In Transgenic Tobacco Research Articles

Overexpression of BvNHX1, a novel tonoplast Na+/H+ antiporter gene from sugar beet (Beta vulgaris), confers enhanced salt tolerance in transgenic tobacco

Overexpression of BvNHX1, a novel tonoplast Na+/H+ antiporter gene from sugar beet (Beta vulgaris), confers enhanced salt tolerance in transgenic tobacco

The apple 14-3-3 gene MdGRF6 negatively regulates salt tolerance.

The 14-3-3 (GRF, general regulatory factor) regulatory proteins are highly conserved and are widely distributed throughout the eukaryotes. They are involved in the growth and development of organisms via target protein interactions. Although many plant 14-3-3 proteins were identified in response to stresses, little is known about their involvement in salt tolerance in apples. In our study, nineteen apple 14-3-3 proteins were cloned and identified. The transcript levels of Md14-3-3 genes were either up or down-regulated in response to salinity treatments. Specifically, the transcript level of MdGRF6 (a member of the Md14-3-3 genes family) decreased due to salt stress treatment. The phenotypes of transgenic tobacco lines and wild-type (WT) did not affect plant growth under normal conditions. However, the germination rate and salt tolerance of transgenic tobacco was lower compared to the WT. Transgenic tobacco demonstrated decreased salt tolerance. The transgenic apple calli overexpressing MdGRF6 exhibited greater sensitivity to salt stress compared to the WT plants, whereas the MdGRF6-RNAi transgenic apple calli improved salt stress tolerance. Moreover, the salt stress-related genes (MdSOS2, MdSOS3, MdNHX1, MdATK2/3, MdCBL-1, MdMYB46, MdWRKY30, and MdHB-7) were more strongly down-regulated in MdGRF6-OE transgenic apple calli lines than in the WT when subjected to salt stress treatment. Taken together, these results provide new insights into the roles of 14-3-3 protein MdGRF6 in modulating salt responses in plants.

Ectopic overexpression of mulberry MnT5H2 enhances melatonin production and salt tolerance in tobacco.

Soil salinization severely inhibits plant growth and has become one of the major limiting factors for global agricultural production. Melatonin (N-acetyl-5-methoxytryptamine) plays an important role in regulating plant growth and development and in responding to abiotic stresses. Tryptamine-5-hydroxylase (T5H) is an enzyme essential for the biosynthesis of melatonin in plants. Previous studies have identified the gene MnT5H for melatonin synthesis in mulberry (Morus notabilis), but the role of this gene in response to salinity stress in mulberry is remain unclear. In this study, we ectopically overexpressed MnT5H2 in tobacco (Nicotiana tabacum L.) and treated it with NaCl solutions. Compared to wild-type (WT), melatonin content was significantly increased in the overexpression-MnT5H2 tobacco. Under salt stress, the expression of NtCAT, NtSOD, and NtERD10C and activity of catalase (CAT), peroxidase (POD), and the content of proline (Pro) in the transgenic lines were significantly higher than that in WT. The Malondialdehyde (MDA) content in transgenic tobacco was significantly lower than that of WT. Furthermore, transgenic tobacco seedlings exhibited faster growth in media with NaCl. This study reveals the changes of melatonin and related substance content in MnT5H2-overexpressing tobacco ultimately lead to improve the salt tolerance of transgenic tobacco, and also provides a new target gene for breeding plant resistance to salt.

Characterization of the Poplar R2R3-MYB Gene Family and Over-Expression of PsnMYB108 Confers Salt Tolerance in Transgenic Tobacco.

The MYB, one of the largest transcription factor families in plants, is related to various biological processes. For an example, the R2R3-MYB family plays an important role in regulation of primary and secondary metabolism, plant growth and development, and responses to hormones and stresses. However, functional studies on the poplar R2R3-MYB genes are limited. In this study, we identified 207 poplar R2R3-MYB genes that are unevenly distributed on the 19 chromosomes of poplar, followed by characterization of their conserved domains. On the basis of phylogenetic analysis, these genes can be divided into 23 groups. Evidence from synteny analyses indicated that the poplar R2R3-MYB gene family is featured by tandem and segmental duplication events. On the basis of RNA-Seq data, we investigated salt responsive genes and explored their expression patterns. Furthermore, we cloned the PsnMYB108 gene from poplar, which is significantly up-regulated in roots and leaves in response to salt stress. To validate its function, we developed transgenic tobacco plants that over-express the PsnMYB108 gene. It appears that the transgenic lines are more tolerant to salt stress than the wild type does. Evidence from physiological analyses demonstrated that over-expression of PsnMYB108 may improve tobacco salt stress tolerance by increasing the reactive oxygen species scavenging ability and the accumulation of proline. These results laid the foundation for future analysis and functional studies of poplar R2R3-MYB family members, and revealed that PsnMYB108 plays an important role in improving plant salt stress tolerance.

Improved Drought and Salt Tolerance in Transgenic Nicotiana benthamiana by Overexpressing Sugarcane ScSEC14p Gene

Plant phosphatidylinositol transfer proteins (PITPs) are involved in several cellular processes such as lipid metabolism, signal transduction, osmotic regulation, protein transport and stress response. In this study, the effects of sugarcane PITP gene ScSEC14p (accession no. MH899751) on drought and salt tolerance of transgenic Nicotiana benthamiana were evaluated along with the underlying mechanisms. The results showed that ScSEC14p gene effectively improved drought and salt tolerance of transgenic lines of N. benthamiana, as indicated by the increased germination rate and superoxide dismutase levels, as well as the decreased malondialdehyde level. In addition, the enhanced tolerance was closely related to the upregulation of stress-related genes and key genes in phosphatidylinositol pathway. It is revealed that the overexpression of ScSEC14p gene resulted in improved drought and salt tolerance of transgenic tobacco and also elucidated the underlying regulation mechanism by ScSEC14p in sugarcane.

Over-Expression of LcPDS, LcZDS, and LcCRTISO, Genes From Wolfberry for Carotenoid Biosynthesis, Enhanced Carotenoid Accumulation, and Salt Tolerance in Tobacco.

It is of great importance to combine stress tolerance and plant quality for breeding research. In this study, the role of phytoene desaturase (PDS), ζ-carotene desaturase (ZDS) and carotene isomerase (CRTISO) in the carotenoid biosynthesis are correlated and compared. The three genes were derived from Lycium chinenses and involved in the desaturation of tetraterpene. Their over-expression significantly increased carotenoid accumulation and enhanced photosynthesis and salt tolerance in transgenic tobacco. Up-regulation of almost all the genes involved in the carotenoid biosynthesis pathway and only significant down-regulation of lycopene ε-cyclase (ε-LCY) gene were detected in those transgenic plants. Under salt stress, proline content, and activities of catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) were significantly increased, whereas malonaldehyde (MDA) and hydrogen peroxide (H2O2) accumulated less in the transgenic plants. The genes encoding ascorbate peroxidase (APX), CAT, POD, SOD, and pyrroline-5-carboxylate reductase (P5CR) were shown to responsive up-regulated significantly under the salt stress in the transgenic plants. This study indicated that LcPDS, LcZDS, and LcCRTISO have the potential to improve carotenoid content and salt tolerance in higher plant breeding.

A new Na+/H+ antiporter gene KvNHX1 isolated from the halophyte Kosteletzkya virginica improves salt tolerance in transgenic tobacco

Vacuolar Na+/H+ antiporters (NHXs) play an important role in plant salt tolerance and a large number of NHX genes have been identified in many plant species. In this study, we isolated and characterized a novel vacuolar-type KvNHX1 cDNA from the halophyte Kosteletzkya virginica. It is 2047 bp long, with an open reading frame of 1632 bp, encoding a protein of 543 amino acids with a theoretical molecular weight of 60.03 kDa. Bioinformatics and phylogenetic analysis showed that it was highly similar to the known vacuolar-type NHXs, especially with 92% identity with GhNHX1 of Gossypium hirsutum from the same Malvaceae family. Quantitative RT-PCR showed that the expression analysis of KvNHX1was significantly increased by NaCl, abscisic acid (ABA) and polyethylene glycol (PEG), but its expression levels and patterns differed under different abiotic stresses, implying that KvNHX1 might be involved in various abiotic stress responses. In addition, overexpression of KvNHX1 enhanced the salt tolerance in transgenic tobacco lines with better growth, more chlorophyll, higher antioxidant enzyme activities and osmoregulation capability by accumulating more proline, Na+ and K+ in the leaves than the wild type under salt stress. Our result suggested that KvNHX1 may have potential use for improving the salt tolerance of plants via genetic engineering.

The transcription factor FcWRKY40 of Fortunella crassifolia functions positively in salt tolerance through modulation of ion homeostasis and proline biosynthesis by directly regulating SOS2 and P5CS1 homologs.

Although some WRKYs have been characterized, regulatory roles of most WRKYs remain poorly understood. Herein, we elucidated function of FcWRKY40 from Fortunella crassifolia in salt tolerance via overexpression and virus-induced gene silencing (VIGS) and unraveled its target genes. Overexpression of FcWRKY40 enhanced salt tolerance in transgenic tobacco and lemon, while silencing of FcWRKY40 increased salt susceptibility. Homolog genes of Salt Overly Sensitive 2 (SOS2) and Δ-1-pyrroline-5-carboxylate synthetase 1 (P5CS1) were dramatically up-regulated in transgenic lemon but down-regulated in VIGS line. Consistently, transgenic lemon displayed lower Na+ and higher proline concentrations, whereas the silenced line accumulated more Na+ but less proline. Treatment of transgenic lemon with 24-epi-brassinolide compromised salt tolerance, while supply of exogenous proline partially restored salt tolerance of the VIGS line. FcWRKY40 specifically binds to and activates promoters of FcSOS2 and FcP5CS1. FcWRKY40 was up-regulated by ABA and salt, and confirmed as a target of ABA-responsive element binding factor 2 (FcABF2). Moreover, salt treatment up-regulated FcABF2 and FcP5CS1, and elevated proline concentrations. Taken together, our findings demonstrate that FcWRKY40 participates in the ABA signaling pathway and as a positive regulator functions in salt tolerance by regulating genes involved in ion homeostasis and proline biosynthesis.

Expression of TaGF14b, a 14-3-3 adaptor protein gene from wheat, enhances drought and salt tolerance in transgenic tobacco.

TaGF14b enhances tolerance to multiple stresses through ABA signaling pathway by altering physiological and biochemical processes, including ROS-scavenging system, stomatal closure, compatible osmolytes, and stress-related gene expressions in tobaccos. The 14-3-3 proteins are involved in plant growth, development, and in responding to abiotic stresses. However, the precise functions of 14-3-3s in responding to drought and salt stresses remained unclear, especially in wheat. In this study, a 14-3-3 gene from wheat, designated TaGF14b, was cloned and characterized. TaGF14b was upregulated by polyethylene glycol 6000, sodium chloride, hydrogen peroxide, and abscisic acid (ABA) treatments. Ectopic expression of TaGF14b in tobacco conferred enhanced tolerance to drought and salt stresses. Transgenic tobaccos had longer root, better growth status, and higher relative water content, survival rate, photosynthetic rate, and water use efficiency than control plants under drought and salt stresses. The contribution of TaGF14b to drought and salt tolerance relies on the regulations of ABA biosynthesis and ABA signaling, as well as stomatal closure and stress-related gene expressions. Moreover, TaGF14b expression could significantly enhance the reactive oxygen species (ROS) scavenging system to ameliorate oxidative damage to cells. In addition, TaGF14b increased tolerance to osmotic stress evoked by drought and salinity through modifying water conservation and compatible osmolytes in plants. In conclusion, TaGF14b enhances tolerance to multiple abiotic stresses through the ABA signaling pathway in transgenic tobaccos by altering physiological and biochemical processes.

An Alcohol Dehydrogenase Gene from Synechocystis sp. Confers Salt Tolerance in Transgenic Tobacco.

Synechocystis salt-responsive gene 1 (sysr1) was engineered for expression in higher plants, and gene construction was stably incorporated into tobacco plants. We investigated the role of Sysr1 [a member of the alcohol dehydrogenase (ADH) superfamily] by examining the salt tolerance of sysr1-overexpressing (sysr1-OX) tobacco plants using quantitative real-time polymerase chain reactions, gas chromatography-mass spectrometry, and bioassays. The sysr1-OX plants exhibited considerably increased ADH activity and tolerance to salt stress conditions. Additionally, the expression levels of several stress-responsive genes were upregulated. Moreover, airborne signals from salt-stressed sysr1-OX plants triggered salinity tolerance in neighboring wild-type (WT) plants. Therefore, Sysr1 enhanced the interconversion of aldehydes to alcohols, and this occurrence might affect the quality of green leaf volatiles (GLVs) in sysr1-OX plants. Actually, the Z-3-hexenol level was approximately twofold higher in sysr1-OX plants than in WT plants within 1–2 h of wounding. Furthermore, analyses of WT plants treated with vaporized GLVs indicated that Z-3-hexenol was a stronger inducer of stress-related gene expression and salt tolerance than E-2-hexenal. The results of the study suggested that increased C6 alcohol (Z-3-hexenol) induced the expression of resistance genes, thereby enhancing salt tolerance of transgenic plants. Our results revealed a role for ADH in salinity stress responses, and the results provided a genetic engineering strategy that could improve the salt tolerance of crops.

Switchgrass PvDREB1C plays opposite roles in plant cold and salt tolerance in transgenic tobacco

BackgroundThe C-repeat-binding factors/DRE-binding factors (CBF/DREBs) comprise a key transcription factor family involved in plant stress tolerance. Yet, there is limited information about switchgrass DREB genes and their functional roles.ResultsIn this study, four cold-inducible PvDREB1s were identified from switchgrass (Panicum virgatum), among which PvDREB1C was the one responded to cold stress later than the other three PvDREB1s. Yet, ectopic overexpression of PvDREB1C led to significantly compromised, instead of improved cold tolerance in transgenic tobacco. On the other hand, PvDREB1C was transcriptionally down-regulated in response to salt stress, but overexpression of PvDREB1C improved plant salt tolerance in transgenic tobacco. The improved salt tolerance was associated with increased K+/Na+ ratio and Ca2+ content, higher cellular osmotic potential, and activation of stress-related functional genes in the leaves of transgenic plants under salt stress.ConclusionsThe current results implied that PvDREB1C played opposite roles in plant cold and salt tolerance. Although DREB1s were known as positive stress regulators, particular attentions shall be paid to their potential negative regulatory role(s).

Molecular characterization and functional analysis of a cation-chloride cotransporter gene from trifoliate orange (Poncirus trifoliata L.)

A cation-chloride cotransporter gene PtrCCC from trifoliate orange was identified, its ectopic expression confers salt tolerance in transgenic tobacco. Salinity is one of the most serious environmental stresses that limit crop growth. Cation-chloride cotransporters (CCCs) are membrane proteins that are involved in plant development and salt tolerance. Here we provide an insight into the molecular characterization and functional analysis of CCC-encoding gene (PtrCCC) from trifoliate orange (Poncirus trifoliata L.). The open-reading frame of PtrCCC is 2943 bp in length, which encodes a protein of 980 amino acids. Phylogenetic analysis revealed that PtrCCC is closely related to CCCs from its allied genera as well as woody perennials. Quantitative real-time PCR (qRT-PCR) analysis demonstrated that PtrCCC was expressed in all tested tissues, with higher expression level in root tip and leaf tip. Its expression was up-regulated in the leaves of trifoliate orange under the presence of KCl conditions. Ectopic expression of PtrCCC gene in tobacco exhibited an improvement in salt tolerance. The transgenic plants had higher dry biomass in the roots and shoots than that in wild type plants. Moreover, under KCl and NaCl + KCl stress conditions, the accumulation of shoot Cl− was remarkably decreased in the transgenic lines as compared with the wild type plants. Collectively, these results suggest that PtrCCC plays an important role in salt tolerance, which is partially attributed to its role in ions homeostasis.

The improvement of salt tolerance in transgenic tobacco by overexpression of wheat F-box gene TaFBA1

F-box protein is a major subunit of the Skp1-Cullin-F-box (SCF) complex. We previously isolated an F-box gene from wheat, TaFBA1, and here we show that overexpression of TaFBA1 in transgenic plants under salt stress increases germination rate, root elongation, and biomass accumulation compared with WT plants. Improvements in the photosynthetic rate and its corresponding parameters were also found in the transgenic plants. These results suggest that overexpression of TaFBA1 improves salt stress tolerance in transgenic tobacco. Further, the transgenic plants displayed less membrane damage, higher antioxidant enzyme activity, and less accumulation of ROS under salt stress. The transgenic plants also had lower Na+ content and higher K+ content than WT plants in leaves and roots. The activity of H+-ATPase on the plasma membrane in the transgenic plants was higher than in WT plants, and was accompanied by a net Na+ efflux. In the tonoplast, the activity levels of V-ATPase and PPase were also higher in the transgenic plants, thus helping to maximize intracellular Na+ compartmentalization. The expression of some stress-related genes was upregulated by salt stress. This suggests that the enhancement of plant salt stress tolerance may be associated with an improvement in antioxidative competition and Na+/K+ ion regionalization.

Hyperactive mutant of a wheat plasma membrane Na+/H+ antiporter improves the growth and salt tolerance of transgenic tobacco

Wheat SOS1 (TaSOS1) activity could be relieved upon deletion of the C-terminal 168 residues (the auto-inhibitory domain). This truncated form of wheat SOS1 (TaSOS1-974) was shown to increase compensation (compared to wild-type TaSOS1) for the salt sensitivity of a yeast mutant strain, AXT3K, via increased Na+ transportation out of cells during salinity stress. Expression of the plasma membrane proteins TaSOS1-974 or TaSOS1 improved the growth of transgenic tobacco plants compared with wild-type plants under normal conditions. However, plants expressing TaSOS1-974 grew better than TaSOS1-transformed plants. Upon salinity stress, Na+ efflux and K+ influx rates in the roots of transgenic plants expressing TaSOS1-974 or TaSOS1 were greater than those of wild-type plants. Furthermore, compared to TaSOS1-transgenic plants, TaSOS1-974-expressing roots showed faster Na+ efflux and K+ influx, resulting in less Na+ and more K+ accumulation in TaSOS1-974-transgenic plants compared to TaSOS1-transgenic and wild-type plants. TaSOS1-974-expressing plants had the lowest MDA content and electrolyte leakage among all tested plants, indicating that TaSOS1-974 might protect the plasma membrane against oxidative damage generated by salt stress. Overall, TaSOS1-974 conferred higher salt tolerance in transgenic plants compared to TaSOS1. Consistent with this result, transgenic plants expressing TaSOS1-974 showed a better growth performance than TaSOS1-expressing and wild-type plants under saline conditions.

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.

Over-expression of poplar transcription factor ERF76 gene confers salt tolerance in transgenic tobacco

Ethylene response factors (ERFs) belong to a large plant-specific transcription factor family, which play a significant role in plant development and stress responses. Poplar ERF76 gene, a member of ERF TF family, can be up-regulated in response to salt stress, osmotic stress, and ABA treatment. The ERF76 protein was confirmed to be targeted preferentially in the nucleus of onion cell by particle bombardment. In order to understand the functions of ERF76 gene in salt stress response, we conducted temporal and spatial expression analysis of ERF76 gene in poplar. Then the ERF76 cDNA fragment containing an ORF was cloned from di-haploid Populus simonii×P. nigra and transferred into tobacco (Nicotiana tobacum) genome by Agrobacterium-mediated leaf disc method. Under salt stress, transgenic tobacco over-expressing ERF76 gene showed a significant increase in seed germination rate, plant height, root length, and fresh weight, as well as in relative water content (RWC), superoxide dismutase (SOD) activity, peroxidase (POD) activity, and proline content, compared to control tobacco lines. In contrast, transgenic tobacco lines displayed a decrease in malondialdehyde (MDA) accumulation, relative electrical conductivity (REC) and reactive oxygen species (ROS) accumulation in response to salt stress, compared to control tobacco lines. Over all, the results indicated that ERF76 gene plays a critical role in salt tolerance in transgenic tobacco.

Overexpression of IrlVHA-c, A Vacuolar-Type H+-ATPase c Subunit Gene from Iris lactea, Enhances Salt Tolerance in Tobacco

Vacuolar-type H+-ATPase (V-ATPase), a multi-subunit endomembrane proton pump, plays an important role in plant growth and response to environmental stresses. In the present study, transgenic tobacco that overexpressed the V-ATPase c subunit gene from Iris lactea (IrlVHA-c) was used to determine the function of IrlVHA-c. Quantitative PCR analysis showed that IrlVHA-c expression was induced by salt stress in I. lactea roots and leaves. Subcellular localization of green fluorescent protein (GFP) as marker combined with FM4-64 staining showed that the IrlVHA-c-GFP was localized to the endosomal compartment in tobacco cells. Compared with the wild-type, the IrlVHA-c transgenic tobacco plants exhibited greater seed germination rates, root length, fresh weight, and higher relative water content (RWC) of leaves under salt stress. Furthermore, the IrlVHA-c transgenic tobacco leaves have lower stomatal densities and larger stomatal apertures than wild-type. Under salt stress, superoxide dismutase (SOD) activity in the transgenic tobacco was significantly enhanced. Moreover, the level of malondialdehyde (MDA) in the transgenic tobacco was significantly lower than that in wild-type plants under salt stress. Taken together, these results suggested that the IrlVHA-c plays an important role in salt tolerance in transgenic tobacco by influencing stomatal movement and physiological changes.

Enhanced salt tolerance of transgenic tobacco expressing a wheat salt tolerance gene

Soil salinity is one of the most important limiting factors of agricultural productivity in the world. The Triticum aestivum salt tolerance-related gene (TaSTRG) possesses a functional response to salt and drought stress conditions. A variety of stress factors, such as salt, drought, abscisic acid, and cold, may induce the expression of TaSTRG in wheat. In this study, the TaSTRG gene was transferred to tobacco via Agrobacterium-mediated transformation. Overexpression of TaSTRG in transgenic tobacco plants indicated higher salt tolerance and mediated more vigorous growth than in wild-type plants. Under salt stress conditions, the transgenic tobacco plants had higher germination and survival rates and longer root length than the control plants. Under salt treatments (200-250 mM), TaSTRG-overexpressing tobacco plants accumulated a higher amount of proline and had significantly lower malondialdehyde content than wild-type plants. Furthermore, transgene inheritance followed Mendelian laws, indicating the stability of TaSTRG in transgenic tobacco plants. These results indicated that the wheat TaSTRG gene plays an important role in responding to salt stress.

MsZEP, a novel zeaxanthin epoxidase gene from alfalfa (Medicago sativa), confers drought and salt tolerance in transgenic tobacco.

The zeaxanthin epoxidase gene ( MsZEP ) was cloned and characterized from alfalfa and validated for its function of tolerance toward drought and salt stresses by heterologous expression in Nicotiana tabacum. Zeaxanthin epoxidase (ZEP) plays important roles in plant response to various environment stresses due to its functions in ABA biosynthetic and the xanthophyll cycle. To understand the expression characteristics and the biological functions of ZEP in alfalfa (Medicago sativa), a novel gene, designated as MsZEP (KM044311), was cloned, characterized and overexpressed in Nicotiana tabacum. The open reading frame of MsZEP contains 1992bp nucleotides and encodes a 663-amino acid polypeptide. Amino acid sequence alignment indicated that deduced MsZEP protein was highly homologous to other plant ZEP sequences. Phylogenetic analysis showed that MsZEP was grouped into a branch with other legume plants. Real-time quantitative PCR revealed that MsZEP gene expression was clearly tissue-specific, and the expression levels were higher in green tissues (leaves and stems) than in roots. MsZEP expression decreased in shoots under drought, cold, heat and ABA treatment, while the expression levels in roots showed different trends. Besides, the results showed that nodules could up-regulate the MsZEP expression under non-stressful conditions and in the earlier stage of different abiotic stress. Heterologous expression of the MsZEP gene in N. tabacum could confer tolerance to drought and salt stress by affecting various physiological pathways, ABA levels and stress-responsive genes expression. Taken together, these results suggested that the MsZEP gene may be involved in alfalfa responses to different abiotic stresses and nodules, and could enhance drought and salt tolerance of transgenic tobacco by heterologous expression.

LchERF, a novel ethylene-responsive transcription factor from Lycium chinense, confers salt tolerance in transgenic tobacco.

An ERF gene, LchERF , was cloned from L. chinense for the first time. Overexpression of LchERF conferred salt stress tolerance in transgenic tobacco lines during seed germination and vegetative growth. Ethylene-responsive transcription factors (ERFs) play important roles in tolerance to biotic and abiotic stresses by regulating the expression of stress-responsive genes. Although the ERF proteins involved in defense responses against biotic stresses have been extensively documented, the mechanisms by which ERF subfamily genes regulate plant responses to abiotic stresses are largely unknown. In this study, a novel ethylene-responsive transcription factor, named LchERF, was isolated from Lycium chinense (a salinity-resistant plant). Analysis of the LchERF-deduced protein sequence showed that it had a typical AP2/ERF domain and belonged to the B-3 subgroup of the ERF subfamily. The expression of LchERF was found to be tissue specific in L. chinense under normal conditions. Upon treatment with NaCl, polyethylene glycol (PEG) or ethephon (ET), transcript levels of LchERF rapidly increased in L. chinense. Overexpression of LchERF conferred salt stress tolerance in transgenic tobacco during seed germination and vegetative growth. Compared with control lines, LchERF-overexpressing plants showed higher chlorophyll and proline contents, and were associated with lower H2O2 content under salt stress. Overall, our results demonstrate that LchERF might play an important role in the regulation of plant responses to abiotic stresses and mediate various physiological pathways that enhance salt stress tolerance in plants.