Habib Khoudi

Overexpression of wheat dehydrin DHN-5 enhances tolerance to salt and osmotic stress in Arabidopsis thaliana

Late Embryogenesis Abundant (LEA) proteins are associated with tolerance to water-related stress. A wheat (Triticum durum) group 2 LEA proteins, known also as dehydrin (DHN-5), has been previously shown to be induced by salt and abscisic acid (ABA). In this report, we analyze the effect of ectopic expression of Dhn-5 cDNA in Arabidopsis thaliana plants and their response to salt and osmotic stress. When compared to wild type plants, the Dhn-5 transgenic plants exhibited stronger growth under high concentrations of NaCl or under water deprivation, and showed a faster recovery from mannitol treatment. Leaf area and seed germination rate decreased much more in wild type than in transgenic plants subjected to salt stress. Moreover, the water potential was more negative in transgenic than in wild type plants. In addition, the transgenic plants have higher proline contents and lower water loss rate under water stress. Also, Na+ and K+ accumulate to higher contents in the leaves of the transgenic plants. Our data strongly support the hypothesis that Dhn-5, by its protective role, contributes to an improved tolerance to salt and drought stress through osmotic adjustment.

Expression of wheat Na+/H+ antiporter TNHXS1 and H+- pyrophosphatase TVP1 genes in tobacco from a bicistronic transcriptional unit improves salt tolerance

Abiotic stress tolerance of plants is a very complex trait and involves multiple physiological and biochemical processes. Thus, the improvement of plant stress tolerance should involve pyramiding of multiple genes. In the present study, we report the construction and application of a bicistronic system, involving the internal ribosome entry site (IRES) sequence from the 5′UTR of the heat-shock protein of tobacco gene NtHSF-1, to the improvement of salt tolerance in transgenic tobacco plants. Two genes from wheat encoding two important vacuolar ion transporters, Na+/H+ antiporter (TNHXS1) and H+-pyrophosphatase (TVP1), were linked via IRES to generate the bicistronic construct TNHXS1-IRES-TVP1. Molecular analysis of transgenic tobacco plants revealed the correct integration of the TNHXS1-IRES-TVP1construct into tobacco genome and the production of the full-length bicistronic mRNA from the 35S promoter. Ion transport analyses with tonoplast vesicles isolated from transgenic lines confirmed that single-transgenic lines TVP1cl19 and TNHXS1cl7 had greater H+-PPiase and Na+/H+ antiport activity, respectively, than the WT. Interestingly, the co-expression of TVP1 and TNHXS1 increased both Na+/H+ antiport and H+-PPiase activities and induced the H+ pumping activity of the endogenous V-ATPase. Transgenic tobacco plants expressing TNHXS1-IRES-TVP1 showed a better performance than either of the single gene-transformed lines and the wild type plants when subjected to salt treatment. In addition, the TNHXS1-IRES-TVP1 transgenic plants accumulated less Na+ and more K+ in their leaf tissue than did the wild type and the single gene-transformed lines. These results demonstrate that IRES system, described herein, can co-ordinate the expression of two important abiotic stress-tolerance genes and that this expression system is a valuable tool for obtaining transgenic plants with improved salt tolerance.

A constitutively active form of a durum wheat Na+/H+ antiporter SOS1 confers high salt tolerance to transgenic Arabidopsis

AbstractKey messageExpression of a truncated form of wheat TdSOS1 in Arabidopsis exhibited an improved salt tolerance. This finding provides new hints about this protein that can be considered as a salt tolerance determinant.Abstract The SOS signaling pathway has emerged as a key mechanism in preserving the homeostasis of Na+ and K+ under saline conditions. We have recently identified and functionally characterized, by complementation studies in yeast, the gene encoding the durum wheat plasma membrane Na+/H+ antiporter (TdSOS1). To extend these functional studies to the whole plant level, we complemented Arabidopsis sos1-1 mutant with wild-type TdSOS1 or with the hyperactive form TdSOS1∆972 and compared them to the Arabidopsis AtSOS1 protein. The Arabidopsis sos1-1 mutant is hypersensitive to both Na+ and Li+ ions. Compared with sos1-1 mutant transformed with the empty binary vector, seeds from TdSOS1 or TdSOS1∆972 transgenic plants had better germination under salt stress and more robust seedling growth in agar plates as well as in nutritive solution containing Na+ or Li+ salts. The root elongation of TdSOS1∆972 transgenic lines was higher than that of Arabidopsis sos1-1 mutant transformed with TdSOS1 or with the endogenous AtSOS1 gene. Under salt stress, TdSOS1∆972 transgenic lines showed greater water retention capacity and retained low Na+ and high K+ in their shoots and roots. Our data showed that the hyperactive form TdSOS1∆972 conferred a significant ionic stress tolerance to Arabidopsis plants and suggest that selection of hyperactive alleles of the SOS1 transport protein may pave the way for obtaining salt-tolerant crops.

Transgenic tobacco plants expressing ectopically wheat H⁺-pyrophosphatase (H⁺-PPase) gene TaVP1 show enhanced accumulation and tolerance to cadmium.

Cadmium (Cd) is considered an extremely significant pollutant due to its high toxicity to many organisms. Plants have evolved several mechanisms to cope with Cd, the most important of which is vacuolar sequestration. Cadmium can be directly transported into vacuoles by cations/H(+) exchangers, such as CAXs, which are energized by the pH gradient established by proton pumps. A cDNA (TaVP1) encoding wheat vacuolar H(+)-pyrophosphatase (V-H-PPase) was ectopically expressed in transgenic tobacco to evaluate whether this proton pump expression would enhance Cd tolerance and accumulation in planta. When TaVP1-expressing plants were exposed to various concentrations of Cd, they were found to be more tolerant to Cd compared to wild type plants. Cadmium accumulation in the plant biomass in transgenic plants was higher than that in wild type plants. To the best of our knowledge, this is the first report on the potential for enhancing proton pump expression as a strategy to improve Cd tolerance and accumulation in plants.

Optimization of regeneration and transformation parameters in tomato and improvement of its salinity and drought tolerance

As part of our efforts to improve tomato tolerance to abiotic stress, we have undertaken this study to introduce two candidate genes encoding: a sodium antiporter and a vacuolar pyrophosphatase, previously shown to enhance drought and salt tolerance in transgenic Arabidopsis plants. First, we evaluated the potential of primary leaves from three to four week-old in vitro-grown tomato seedlings as alternative explants to cotyledons for tomato transformation. Our results demonstrated that primary leaves are three times more efficient then cotyledons in terms of regeneration percentage, productivity, and transformation frequencies independently of the medium and genetic construct used. Second, primary leaves were used to introduce the genes of interest using Agrobacterium-mediated transformation. Many transgenic tomato plants were easily recovered. The presence of the transgenes and their expression were confirmed by PCR and RT-PCR analysis. The transformation frequencies for primary leaf explants ranged from 4 to 10% depending on the genetic construct used. The time required from inoculation of primary leaves with Agrobacterium cells to transfer of transgenic tomato plants to soil was only 2 months compared to 3 to 4 months using standard tomato transformation protocols. The transgenic tomato plants obtained in the current study were more tolerant to salinity and drought stress than their wild-type counterparts.

Isolation and molecular characterization of a novel WIN1/SHN1 ethylene-responsive transcription factor TdSHN1 from durum wheat (Triticum turgidum. L. subsp. durum)

Over the last decade, APETALA2/Ethylene Responsive Factor (AP2/ERF) proteins have become the subject of intensive research activity due to their involvement in a variety of biological processes. This research led to the identification of AP2/ERF genes in many species; however, little is known about these genes in durum wheat, one of the most important cereal crops in the world. In this study, a new member of the AP2/ERF transcription factor family, designated TdSHN1, was isolated from durum wheat using thermal asymetric interlaced PCR (TAIL-PCR) method. Protein sequence analysis showed that TdSHN1 contained an AP2/ERF domain of 63 amino acids and a putative nuclear localization signal (NLS). Phylogenetic analysis showed that TdSHN1 belongs to a group Va protein in the ERF subfamily which contains the Arabidopsis ERF proteins (SHN1, SHN2, and SHN3). Expression of TdSHN1 was strongly induced by salt, drought, abscisic acid (ABA), and cold. In planta, TdSHN1 protein was able to activate the transcription of GUS reporter gene driven by the GCC box and DRE element sequences. In addition, TdSHN1 was targeted to the nucleus when transiently expressed in tobacco epidermal cells. In transgenic yeast, overexpression of TdSHN1 increased tolerance to multiple abiotic stresses. Taken together, the results showed that TdSHN1 encodes an abiotic stress-inducible, transcription factor which confers abiotic stress tolerance in yeast. TdSHN1 is therefore a promising candidate for improvement of biotic and abiotic stress tolerance in wheat as well as other crops.

Ethylene Response Factors (ERF) are differentially regulated by different abiotic stress types in tomato plants

Plants are sessile organisms, hence to face environmental constrains they developed strategies that rely on the activation of stress-response genes under the control of specific transcription factors. The plant hormone ethylene mediates physiological, developmental and stress responses through the activation of Ethylene Response Factors (ERFs) which belong to a large multigene family of transcription factors. While an increasing number of studies supports the involvement of ERFs in abiotic stress responses, so far the specific role of ERF family members in different abiotic stress conditions remains unexplored. The present work investigates the expression profile of a set of ERFs, representative of different ERF types, in tomato plants subjected to cold, heat, salt, drought and flooding conditions. The study revealed that a group of ERFs is preferentially associated with cold and heat stress responses while another set is expressed in response to salt, water and flooding stresses. Transactivation assays indicated that ERFs can regulate the expression of abiotic stress genes regardless of whether or not they harbor conserved GCC or DRE cis-elements in their promoter region. The outcome of the study provides clue on which ERFs should be targeted when aiming to improve adaptation to a particular stress type.

Molecular cloning and characterization of novel WIN1/SHN1 ethylene responsive transcription factor HvSHN1 in barley (Hordeum vulgare L.)

Barley (Hordeum vulgare L.) is the fourth major cereal crop and shows high adaptive capabilities to diverse environments. Thus, it might represent a potential reservoir of novel genes to improve abiotic stress tolerance. In this study, a novel AP2/ERF transcription factor gene designated as HvSHN1 was isolated from barley. Protein sequence analysis showed that the HvSHN1 protein contained a nuclear localization signal and the conserved AP2/ERF domain. Phylogenetic analysis showed that HvSHN1 belongs to the group Va protein in the ERF subfamily which contains the Arabidopsis genes (SHN1, 2 and 3) and the wheat gene TdSHN1 with which it has 94.7% protein sequence identity. Expression profile analysis revealed that HvSHN1 is strongly induced by heat, cold, salt and drought. Transient expression using tobacco BY-2 protoplast coupled to confocal microscopy analysis revealed that HvSHN1 is exclusively targeted to the nucleus. Interestingly, when constitutively expressed in transgenic tobacco, HvSHN1 up-regulated stress responsive genes known to harbor GCC or DRE motif in their promoter regions. Therefore, HvSHN1 might represent a potential candidate for improvement of abiotic stress tolerance in economically important crops.