Faiçal Brini

Plant dehydrins and stress tolerance: versatile proteins for complex mechanisms

Dehydrins (DHNs), or group 2 LEA (Late Embryogenesis Abundant) proteins, play a fundamental role in plant response and adaptation to abiotic stresses. They accumulate typically in maturing seeds or are induced in vegetative tissues following salinity, dehydration, cold, and freezing stress. The generally accepted classification of dehydrins is based on their structural features, such as the presence of conserved sequences, designated as Y, S, and K segments. The K segment representing a highly conserved 15 amino acid motif forming amphiphilic α-helix is especially important since it has been found in all dehydrins. Since more than 20 years, they are thought to play an important protective role during cellular dehydration but their precise function remains unclear. This review outlines the current status of the progress made towards the structural, physico-chemical and functional characterization of plant dehydrins and how these features could be exploited in improving stress tolerance in plants.

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.

Proteomic analysis of wheat embryos with 2-DE and liquid-phase chromatography (ProteomeLab PF-2D) - a wider perspective of the proteome.

Cereal embryos are a model system to study desiccation tolerance due to their ability to survive extreme water loss during late embryogenesis. To identify proteins accumulating in mature embryos which can be used as potential markers for dehydration tolerance, we compared the embryo proteome from two durum wheat genotypes (Triticum durum Desf.), Mahmoudi (salt and drought sensitive) and Om Rabia3 (salt and drought tolerant). Total protein extracts from wheat embryos were analyzed by using conventional 2-DE and ProteomeLab PF-2D. Analysis using different pH ranges showed that a larger number of fractions were solved by LC, than by conventional 2-DE at extreme technical pHs (pH 4.0-5.0 and pH 6.5-8.0). In contrast, at intermediate pHs (pH 5.0-6.5), resolution was better in 2-DE gels. The two techniques were used in parallel to analyze total protein extracts from embryos of the two wheat varieties. Several proteins belonging to the seed storage family, LEA-type/heat shock proteins, enzyme metabolism and radical scavengers were identified by analysis of trypsin digested peptides via mass spectrometry. These proteins accumulate in different amounts in embryos of tolerant and sensitive wheat varieties. The differences in expression pattern were further validated by enzyme activity, western blotting analysis and correlated with their corresponding mRNA expression by RT-PCR analyses for the corresponding protein. We suggest that the differential expression pattern could be used as a basis for a biochemical screen of tolerance/sensitivity to drought and salt stress in wheat embryos and germplasm.

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.AbstractnThe 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.

Wheat Dehydrin DHN-5 Exerts a Heat-Protective Effect on β-Glucosidase and Glucose Oxidase Activities

Group-2 late embryogenesis abundant (LEA) proteins, also known as dehydrins, are claimed to stabilize macromolecules against damage caused by freezing, dehydration, ionic or osmotic stresses. However, their precise function remains unknown. Here, we investigated the effect of wheat dehydrin (DHN-5) protein on the activity and thermostability of two distinct enzymes, β-glucosidase (bglG) and glucose oxidase/peroxidase (GOD/POD) in vitro. The purified DHN-5 protein had the capacity to preserve and stabilize the activity of bglG subjected to heat treatment. In addition, DHN-5 stabilized oxidizing enzymes, as it improved reliability in measuring glucose concentrations with a glucose oxidase/peroxidase (GOD/POD) kit while the temperature increased from 37 to 70 °C. All together the data presented provide evidence that DHN-5 is a dehydrin able to preserve enzyme activities in vitro from adverse effects induced by heating.

Physiological and molecular analyses of seedlings of two Tunisian durum wheat (Triticum turgidum L. subsp. Durum [Desf.]) varieties showing contrasting tolerance to salt stress

Salinity is one of the severest environmental stresses affecting plant productivity. In many plant species, salt sensitivity is associated with the accumulation of sodium (Na+) in photosynthetic tissues. Here, we provide the physiological and molecular analyses of seedlings of two Tunisian durum wheat genotypes (Triticum turgidum L. subsp. Durum [Desf.]), Mahmoudi (salt sensitive) and Om Rabia3 (salt tolerant). Na+ and K+ contents in leaf sheath from Om Rabia3 were significantly higher than those of Mahmoudi. However, the net uptake of Na+ from the soil occurred at similar rates in both varieties, suggesting that Om Rabia3 has much stronger ability to limit Na+ flux from roots to leaf blades. This mechanism could be explained by a capacity of Om Rabia3 to retain higher Na+ concentration in leaf sheath and unload less Na+ from the xylem to the upper shoots. When treated with 100xa0mM NaCl leaf sheaths of Om Rabia3 developed lower water potentials and a higher relative water contents than those of Mahmoudi. These features may arise from enhanced osmotic adjustment in Om Rabia3. Measurements of stomatal conductance, free proline and chlorophyll content also indicate that Om Rabia3 is better adapted to tolerate high salt than Mahmoudi. A correlation was obtained between the expression pattern of TaSOS1 (a plasma membrane Na+/H+ antiporter) in the roots and sheaths of both wheat varieties and the Na+ fluxes from roots to leaves. TaSOS1 transcript accumulated in Mahmoudi than in Om Rabia3, suggesting repression of TaSOS1 in the tolerant variety that reduces loading of Na+ to the upper shoots. These results help to design new genetic screens for salt tolerance in wheat.

Callus Induction, Proliferation, and Plantlets Regeneration of Two Bread Wheat (Triticum aestivum L.) Genotypes under Saline and Heat Stress Conditions

Response of two genotypes of bread wheat (Triticum aestivum), Mahon-Demias (MD) and Hidhab (HD1220), to mature embryo culture, callus production, and in vitro salt and heat tolerance was evaluated. For assessment of genotypes to salt and heat tolerance, growing morphogenic calli were exposed to different concentrations of NaCl (0, 5, 10, and 15u2009g·L−1) and under different thermal stress intensities (25, 30, 35, and 40°C). Comparison of the two genotypes was reported for callus induction efficiency from mature embryo. While, for salt and heat tolerance, the proliferation efficiency, embryonic efficiency, and regeneration efficiency were used. The results show significant medium and genotype effects for the embryogenesis capacity of calluses induction and plantlets regeneration under saline and thermal stresses. Mahon-Demias showed good callus induction and ability to proliferate and regenerate seedling under heat and salt stress conditions compared to Hidhab. No sizeable differences were observed between the two genotypes at higher salt stress rates. This study will serve as a base line for in vitro screening of several elite wheat cultivars for their ability to induce callus and regenerate plants from mature embryos, and to start selection for tolerance to salinity.

Multiple abiotic stress tolerance of the transformants yeast cells and the transgenic Arabidopsis plants expressing a novel durum wheat catalase.

Catalases are reactive oxygen species scavenging enzymes involved in response to abiotic and biotic stresses. In this study, we described the isolation and functional characterization of a novel catalase from durum wheat, designed TdCAT1. Molecular Phylogeny analyses showed that wheat TdCAT1 exhibited high amino acids sequence identity to other plant catalases. Sequence homology analysis showed that TdCAT1 protein contained the putative calmodulin binding domain and a putative conserved internal peroxisomal targeting signal PTS1 motif around its C-terminus. Predicted three-dimensional structural model revealed the presence of four putative distinct structural regions which are the N-terminal arm, the β-barrel, the wrapping and the α-helical domains. TdCAT1 protein had the heme pocket that was composed by five essential residues. TdCAT1 gene expression analysis showed that this gene was induced by various abiotic stresses in durum wheat. The expression of TdCAT1 in yeast cells and Arabidopsis plants conferred tolerance to several abiotic stresses. Compared with the non-transformed plants, the transgenic lines maintained their growth and accumulated more proline under stress treatments. Furthermore, the amount of H2O2 was lower in transgenic lines, which was due to the high CAT and POD activities. Taken together, these data provide the evidence for the involvement of durum wheat catalase TdCAT1 in tolerance to multiple abiotic stresses in crop plants.

Identification and expression analysis of the NAC transcription factor family in durum wheat (Triticum turgidum L. ssp. durum)

The NAC (NAM, ATAF and CUC) proteins belong to one of the largest plant-specific transcription factor (TF) families and play important roles in plant development processes, response to biotic and abiotic cues and hormone signaling. Our analysis led to the identification of 168 NAC genes in durum wheat, including nine putative membrane-bound TFs and 48 homeologous genes pairs. Phylogenetic analyses of TtNACs along with their Arabidopsis, grape, barley and rice counterparts divided these proteins into 8 phylogenetic groups and allowed the identification of TtNAC-A7, TtNAC-B35, TtNAC-A68, TtNAC-B69 and TtNAC-A43 as homologs of OsNAC1, OsNAC8, OsNTL2, OsNTL5 and ANAC025/NTL14, respectively. In silico expression analysis, using RNA-seq data, revealed tissue-specific and stress responsive TtNAC genes. The expression of ten selected genes was analyzed under salt and drought stresses in two contrasting tolerance cultivars. This analysis is the first report of NAC gene family in durum wheat and will be useful for the identification and selection of candidate genes associated with stress tolerance.

Isolation and characterization of a differentially expressed sequence tag from Triticum durum salt-stressed roots

Abstract Stress responses in plants are important for environmental adaptation and are associated with rapid changes in gene expression. In order to investigate correlations between phenotypic adaptation to stress limitation and induced gene expression, we have studied a model system consisting of a salt-tolerant line (R1) and a salt-sensitive line (S1) of durum wheat ( Triticum durum Desf.) subjected to a severe short-term NaCl treatment (200xa0mM). A mRNA differential display was established for this system to allow the identification of cDNAs induced during salt stress. We cloned and sequenced a cDNA fragment corresponding to a gene that was differentially expressed in salt-stressed (R1) wheat seedlings. Based on the high similarity between the predicted translation product of the cDNA sequence and known members of the dehydrin protein family from wheat (groupxa02 LEA protein), the cDNA was identified as a member of this gene family. The gene was strongly and rapidly induced in roots and leaves of R1 plants but induction was delayed and transcripts accumulated to a low level in S1 plants.