The heat-stable protein fraction from Opuntia ficus-indica seeds exhibits an enzyme protective effect against thermal denaturation and an antibacterial activity.

Desiccation tolerance in developing seeds occurs through several mechanisms among which, a common group of proteins named dehydrins has received considerable attention. So far, there is no information dealing with the accumulation of dehydrins in seeds of Opuntia ficus-indica. We have initiated here an extraction protocol based on two critical steps: heat and acid treatments, and the purity of this fraction was analyzed by FTIR spectroscopy. Western blot analysis of the heat-stable protein fraction (HSF) revealed two main bands of approximately 45 and 44 kDa, while three others of ∼40, 32, and 31 kDa were faintly visible, which were recognized by anti-dehydrin antibodies. This fraction exhibited a Cu2+ -dependent resistance to protease treatments. Next, we performed a series of assays to compare the functional properties of the HSF with those of the previously characterized wheat dehydrin (DHN-5). Antibacterial assays revealed that HSF exhibits only moderate antibacterial activities against gram-negative and gram-positive bacteria, with a minimum inhibition concentration ranging from 0.25 to 1 mg/ml. However, in vitro assays revealed that compared to DHN-5, HSF exhibits higher protective activities of the lactate dehydrogenase (LDH) when exposed to heat, freezing, and dehydration stresses. The protective role of HSF seems to be linked to its best ability to minimize protein aggregation.

Immunomodulatory effect of Linalool (Lin) against CCl4 -induced hepatotoxicity and oxidative damage in rats.

The current study explored the hepatoprotective and immunomodulatory effects of Linalool (Lin) against carbon tetrachloride (CCl4 )-induced toxicity in mice. Four study groups (n = 8 each) were used: (1) a negative control group and (2) a toxicity control group (single dose of CCl4 administered on day 14 as 1 mL/kg of CCL4 in 1% olive oil). Intraperitoneally (i.p.)), and two experimental groups where mice were treated with either (3) Lin (25 mg/kg b.w., orally, daily for 15 days) or (4) pretreated with Lin (25 mg/kg b.w., orally, daily for 14 days) and intoxicated with CCl4 (1 mL/kg of CCL4 in 1% olive oil. i.p.) on day 14. The levels of the anti-inflammatory cytokine interleukin 10 (IL-10), the proinflammatory cytokines TNF-α, IL-6, and TGF-1ß, and the histopathology of the liver were assessed. According to our findings, IL-10 concentrations were significantly increased in Lin-treated groups, while other cytokine levels were marked by a considerable decrease in the toxicity model group (CCl4 -treated group). Histopathological examinations of liver tissues showed that the Lin-treated groups had an almost normal structure. The current findings showed that Lin could inhibit CCl4 -induced liver injury in mice, which warrants further investigation of Lin as a potential protective and therapeutic agent against hepatotoxicity.

Lobularia maritima leave extract, a nutraceutical agent with antioxidant activity, protects against CCl4-induced liver injury in mice.

Lobularia maritima (Alyssum maritimum, Brassicaceae), commonly known as sweet alyssum, is an annual ornamental halophyte widely spread along the Tunisian seashore. Lobularia maritima leaf ethanol extract was tested in an experimental model of hepatotoxicity induced by carbon tetrachloride (CCl4). L. maritima extract was found to possess in vitro antioxidant activity by scavenging the DPPH radical (IC50= 45 µg/mL), reducing/chelating iron ions and inhibiting liver lipid peroxidation induced by FeSO4. The levels of total phenolics and flavonoids were 175 ± 2.66 mg GAE/g, and 35 ± 2.88 mg QE/g respectively. Moreover, HPLC analysis revealed six compounds, namely gallic, salicylic, ellagic and ferulic acids as well as catechin and quercetin. A mice model of acute liver injury was successfully established after a single intraperitoneal injection of CCl4, as evidenced by histological analysis, Masson trichrome and Sirius red staining. Compared with the CCl4 intoxicated group, the L. maritima treatment resulted to reduce the liver serum marker enzymes, lipid peroxidation and increased the activities of antioxidant enzymes with further amelioration in the oxidative stress. The present findings discover the therapeutic potentials of L. maritima empowered with promising natural leads for the treatment of oxidative stress associated health disorders by attenuating free radicals, inhibiting lipid peroxidation, and upregulating the tissue-specific antioxidant enzymes.

Functional analysis of TmHKT1;4-A2 promoter through deletion analysis provides new insight into the regulatory mechanism underlying abiotic stress adaptation.

MAIN CONCLUSION: Bioinformatic, molecular, and biochemical analysis were performed to get more insight into the regulatory mechanism by which TmHKT1;4-A2 is regulated. HKT transporters from different plant species have been shown to play important role in plant response to salt. In previous work, TmHKT1;4-A2 gene from Triticum monococcum has been characterized as a major gene for Nax1 QTL (Tounsi et al. Plant Cell Physiol 57:2047-2057, 2016). So far, little is known about its regulatory mechanism. In this study, the promoter region of TmHKT1;4-A2 (1400 bp) was isolated and considered as the full-length promoter (PA2-1400). In silico analysis revealed the presence of important cis-acting elements related to abiotic stresses and phytohormones. Interestingly, our real-time RT-PCR analysis provided evidence that TmHKT1;4-A2 is regulated not only by salt stress but also by osmotic, heavy metal, oxidative, and hormones stresses. In transgenic Arabidopsis plants, TmHKT1;4-A2 is strongly active in vascular tissues of roots and leaves. Through 5'-end deletion analysis, we showed that PA2-1400 promoter is able to drive strong GUS activity under normal conditions and in response to different stresses compared to PA2-824 and PA2-366 promoters. These findings provide new information on the regulatory mechanism of TmHKT1;4-A2 and shed more light on its role under different stresses.

Novel non-specific lipid-transfer protein (TdLTP4) isolated from durum wheat: Antimicrobial activities and anti-inflammatory properties in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages.

Lipid transfer proteins (LTP) are members of the family of pathogenesis-related proteins (PR-14) that play a key role in plant defense mechanisms. In this study, a novel gene TdLTP4 encoding an antifungal protein from wheat (cv. Om Rabiaa) was cloned, overexpressed in Escherichia coli BL-21 (DE3) and enriched using ammonium sulfate fractionation. The TdLTP4 fusion protein was then tested against a panel of pathogens, food-borne and spoilage bacteria and fungi in order to evaluate the antimicrobial properties. TdLTP4 was applied to 0.5 µg/mL LPS-induced RAW 264.7 macrophages in vitro at different concentrations (5, 10, 20, 50 and 100 µg/mL). Levels of nitric oxide (NO), pro-inflammatory cytokines interleukin (IL)-1ß (IL-1 ß), interleukin (IL)-6 (IL-6), tumor necrosis factor (TNF-α) and anti-inflammatory cytokine IL-10 in the supernatant fraction were measured using enzyme-linked immunosorbent assay (ELISA). Expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were detected via Western blot. The inhibition zones and minimal inhibitory concentration (MIC) values of bacterial strains were in the range of 14-26 mm and 62.5-250 µg/mL, respectively. Moreover, a remarkable activity against several fungal strains was revealed. TdLTP4 (5-100 µg/mL) decreased the production of NO (IC50 = 4.32 µg/mL), IL-6 (IC50 = 11.52 µg/mL), IL-1ß (IC50 = 7.87 µg/mL) and TNF-α (IC50 = 8.66 µg/mL) by lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. TdLTP4 could modulate the macrophages inflammatory mode by causing reduction in iNOS and COX-2. According to these findings, TdLTP4 fusion protein could be used as natural anti-inflammatory and antimicrobial agent in food preservation and human health.

Differential regulation of the durum wheat Pathogenesis-related protein (PR1) by Calmodulin TdCaM1.3 protein.

In plants, pathogenesis-related 1 protein (PR1) is considered as important defense protein. The production and accumulation of PR proteins in plants are one of the important responses to several biotic and abiotic stresses. In this regard, PR1 gene was isolated from Triticum turgidum ssp durum and was named as TdPR1.2. The amino acid sequence of TdPR1.2 protein showed 100%, 97.13%, and 44.41% with known PR1 proteins isolated from Triticum aestivum TdPR1-18, PRB1.2 of Aegilops tauschii subsp. tauschii and Arabidopsis thaliana respectively. qRT-PCR showed that TdPR1.2 was induced specially in leaves of durum wheat treated with Salicylic acid for 48 h. Besides, bioinformatic analysis showed that the durum wheat TdPR1.2 harbors a calmodulin binding domain located in it's C-terminal part and that this domain is conserved among different PR1 proteins isolated so far. However, no information is available about the regulation of PR genes by calmodulin and Ca2+ complex (CaM/Ca2+). Here, we showed that TdPR1.2 gene exhibits an antibacterial effect as revealed by the in vitro tests against 8 different bacteria and against the fungi Septoria tritici. In addition, we demonstrate for the first time that PR1 proteins are able to bind to CaM in a Ca2+-dependent manner via a GST-Pull down assay. Finally, in presence of Mn2+ cations, CaM/Ca2+ complex stimulated the antimicrobial effect of TdPR1.2. Such effects were not reported so far, and raise a possible role for CaM/Ca2+ complex in the regulation of plant PRs during cellular response to external signals.

Role of jasmonic acid in plants: the molecular point of view.

KEY MESSAGE: Recent updates in JA biosynthesis, signaling pathways and the crosstalk between JA and others phytohormones in relation with plant responses to different stresses. In plants, the roles of phytohormone jasmonic acid (JA), amino acid conjugate (e.g., JA-Ile) and their derivative emerged in last decades as crucial signaling compounds implicated in stress defense and development in plants. JA has raised a great interest, and the number of researches on JA has increased rapidly highlighting the importance of this phytohormone in plant life. First, JA was considered as a stress hormone implicated in plant response to biotic stress (pathogens and herbivores) which confers resistance to biotrophic and hemibiotrophic pathogens contrarily to salicylic acid (SA) which is implicated in plant response to necrotrophic pathogens. JA is also implicated in plant responses to abiotic stress (such as soil salinity, wounding and UV). Moreover, some researchers have recently revealed that JA controls several physiological processes like root growth, growth of reproductive organs and, finally, plant senescence. JA is also involved in the biosynthesis of various metabolites (e.g., phytoalexins and terpenoids). In plants, JA signaling pathways are well studied in few plants essentially Arabidopsis thaliana, Nicotiana benthamiana, and Oryza sativa L. confirming the crucial role of this hormone in plants. In this review, we highlight the last foundlings about JA biosynthesis, JA signaling pathways and its implication in plant maturation and response to environmental constraints.

A novel Triticum durum Annexin 12 protein: Expression, purification and biological activities against Listeria monocytogenes growth in meat under refrigeration.

The present study was undertaken to investigate the expression, purification and biological activities of a novel Triticum durum Annexin 12 protein (TdAnn12). The findings indicated that the molecular weight of the purified TdAnn12 was estimated to 35 kDa. The purified TdAnn12 protein was modulated by, Methyl-jasmonate, and ethephon treatments. The purified TdAnn12 protein displayed good antimicrobial activities against 9 tested pathogenic bacteria. The antioxidant activities showed that TdAnn12 displayed an excellent DPPH scavenging ability with an IC50 of 8.33 µg/ml and a strong Beta-carotene bleaching inhibition after 120 min of incubation with an IC50 of 2 µg/ml the cytotoxic effects of the TdAnn 12 showed that HepG2 and MCF-7 were examined by MTT assay. The IC50 values were 250.35 and 400.25 µg/ml for HepG2 and MCF-7 cells, respectively. The inhibitory effects of this TdAnn12 was assessed in vivo against Listeria monocytogenes, inoculated in minced beef meat at 6.105 CFU/g amended with different concentrations of the purified TdAnn12 and stored at 4 °C for 21 days. Results showed an excellent inhibitory effect of TdAnn12 of this pathogenic bacterium at 4 °C. Overall, the TdAnn12 have potential application as active ingredients in food and pharmaceutical industry.

Identification and molecular characterization of allergenic non-specific lipid-transfer protein from durum wheat (Triticum turgidum).

BACKGROUND: Common wheat (Triticum aestivum) and durum wheat (T. turgidum) are both involved in Baker's asthma (BA) and food allergy (FA) including wheat-dependent exercise-induced asthma (WDEIA). However, allergens in durum wheat have not been described, and the over-expression of T. turgidum non-specific lipid-transfer protein (nsLTPs) is considered to increase resistance to phytopathogens. OBJECTIVE: To identify and assess the allergenicity of nsLTP from T. turgidum. METHODS: Recombinant T. turgidum nsLTP Tri tu 14 was generated and tested for structural integrity (circular dichroism-spectroscopy) and purity (SDS-PAGE). Thirty-two wheat allergic patients were enrolled: 20 Spanish patients (BA) with positive bronchial challenge to wheat flour, and 12 Italian patients (wheat FA/WDEIA) with positive double-blind placebo-controlled food challenge/open food challenge (OFC) to pasta. IgE values to wheat, Tri tu 14, Tri a 14 (T. aestivum) and Pru p 3 (P. persica) were determined by ImmunoCAP testing. Allergenic potency (in vitro mediator release) and IgE cross-reactivity were investigated. RESULTS: Tri tu 14 was found to share 49% and 52% amino acid identity with Tri a 14 and Pru p 3, respectively. Among 25 Tri a 14 CAP positive sera, 23 (92%) were reactive to wheat extract, 22 (88%) to Tri tu 14 and 20 (80%) to Pru p 3. The correlation between Tri a 14 and Tri tu 14 specific IgE levels was r = 0.97 (BA) and r = 0.93 (FA/WDEIA), respectively. FA/WDEIA patients showed higher specific IgE values to Tri tu 14 and Pru p 3 than BA patients. Tri tu 14 displayed allergenic activity by mediator release from effector cells and IgE cross-reactivity with Pru p 3. The degree of IgE cross-reactivity between the two wheat nsLTPs varied between individual patients. CONCLUSIONS AND CLINICAL RELEVANCE: Sensitization to Tri tu 14 likely appears to be more important in wheat FA/WDEIA than in BA. Over-expression of Tri tu 14 in wheat would represent a risk for patients with nsLTP-mediated FA.

Overexpression of a Wheat Aquaporin Gene, TdPIP2;1, Enhances Salt and Drought Tolerance in Transgenic Durum Wheat cv. Maali.

In this study, we generated transgenic durum wheat cv. Maali overexpressing the wheat plasma membrane aquaporin TdPIP2;1 gene under the control of PrTdPIP2;1 promoter or under the constitutive PrCaMV35S promoter. Histochemical analysis of the fusion PrTdPIP2;1::TdPIP2;1::GusA in wheat plants showed that the ß-glucuronidase (GUS) activity was detected in the leaves, stems and roots of stably transformed wheat T3 plants. Our results showed that transgenic wheat lines overexpressing the TdPIP2;1 gene exhibited improved germination rates and biomass production and retained low Na+ and high K+ concentrations in their shoots under high salt and osmotic stress conditions. In a long-term study under greenhouse conditions on salt or drought stress, transgenic TdPIP2;1 lines produced filled grains, whereas wild-type (WT) plants either died at the vegetative stage under salt stress or showed drastically reduced grain filling under drought stress. Performing real time RT-PCR experiments on wheat plants transformed with the fusion PrTdPIP2;1::GusA, we showed an increase in the accumulation of GusA transcripts in the roots of plants challenged with salt and drought stress. Study of the antioxidant defence system in transgenic wheat TdPIP2;1 lines showed that these lines induced the antioxidative enzymes Catalase (CAT) and Superoxide dismutase (SOD) activities more efficiently than the WT plants, which is associated with lower malondialdehyde and hydrogen peroxide contents. Taken together, these results indicate the high potential of the TdPIP2;1 gene for reducing water evaporation from leaves (water loss) in response to water deficit through the lowering of transpiration per unit leaf area (stomatal conductance) and engineering effective drought and salt tolerance in transgenic TdPIP2;1 lines.

Salinity Effects on Sugar Homeostasis and Vascular Anatomy in the Stem of the Arabidopsis Thaliana Inflorescence.

The regulation of sugar metabolism and partitioning plays an essential role for a plant's acclimation to its environment, with specific responses in autotrophic and heterotrophic organs. In this work, we analyzed the effects of high salinity on sugar partitioning and vascular anatomy within the floral stem. Stem sucrose and fructose content increased, while starch reduced, in contrast to the response observed in rosette leaves of the same plants. In the stem, the effects were associated with changes in the expression of SWEET and TMT2 genes encoding sugar transporters, SUSY1 encoding a sucrose synthase and several FRK encoding fructokinases. By contrast, the expression of SUC2, SWEET11 and SWEET12, encoding sugar transporters for phloem loading, remained unchanged in the stem. Both the anatomy of vascular tissues and the composition of xylem secondary cell walls were altered, suggesting that high salinity triggered major readjustments of sugar partitioning in this heterotrophic organ. There were changes in the composition of xylem cell walls, associated with the collapse and deformation of xylem vessels. The data are discussed regarding sugar partitioning and homeostasis of sugars in the vascular tissues of the stem.

Promoter of the TmHKT1;4-A1 gene of Triticum monococcum directs stress inducible, developmental regulated and organ specific gene expression in transgenic Arbidopsis thaliana.

HKT transporters which mediate Na+-specific transport or Na+-K+ co-transport, play an important role in protecting plants from salinity stress by preventing Na+-over-accumulation in leaves. In this work, a 1508-bp genomic fragment upstream of the TmHKT1;4-A1 translated sequence from Triticum monococcum has been isolated, cloned, and designated as the ''PrTmHKT1;4-A1'' promoter. Sequence analysis of ''PrTmHKT1;4-A1'' revealed the presence of cis-regulatory elements which could be required for abiotic stress and abscisic acid (ABA) responsiveness. The PrTmHKT1;4-A1 sequence was fused to the ß-glucuronidase gene and the resulting construct was transferred into Arabidopsis plants. Histochemical assays of stably transformed Arabidopsis plants showed that PrTmHKT1;4-A1 is active in this heterologous system. Under control conditions, GUS histochemical staining was observed significantly only in leaves of 20-day-old plants. Histological sections prepared at this stage and in leaves revealed activity localized in leaf veins (phloem and bundle sheath). In flowers, GUS activity was detected only in sepals. After 3 days of challenging the plants with salt, dehydration or ABA treatments, the PrTmHKT1;4-A1 transformed Arabidopsis plants showed a substantial increase in the GUS staining in leaves, compared to untransformed plants under the same conditions. Real time qPCR expression analysis of the uidA gene, showed that GusA transcripts were up-regulated by salt, dehydration, and ABA treatments. All together, these results showed that PrTmHKT1;4-A1 is an age-dependent, abiotic-stress-inducible, organ-specific and tissue-specific promoter in a heterologous dicot system.

Apigenin isolated from A. americana encodes Human and Aspergillus oryzae S2 α-amylase inhibitions: credible approach for antifungal and antidiabetic therapies.

Agave americana extract was analyzed by reverse phase HPLC for characterization. Among phenolic compounds identified, apigenin was observed to be present. The finding showed an inhibitory effect of apigenin towards Human and Aspergillus oryzae S2 α-amylases. Apigenin inhibition towards Human and A. oryzae α-amylase activities was observed to be competitive. IC50 and  % inhibition of apigenin for A. oryzae α-amylase were 3.98 and 1.65 fold higher than for Human α-amylase. The inhibition of the described biocatalyst activity was significantly lowered when apigenin was pre-incubated with starch. In addition to the catalytic residues, 44 amino acid residues were involved on A. oryzae α-amylase-apigenin interactions while only 11 amino acid residues were exposed for Human α-amylase-apigenin complex. The binding site of apigenin showed 76 polar contacts for A. oryzae S2 α-amylase against 44 interactions for Human α-amylase. The docking studies confirmed the mode of action of apigenin and strongly suggested a higher inhibitory activity towards fungal amylase which was experimentally exhibited. These findings provided a rational reason to establish apigenin capability as a therapeutic target for postprandial hyperglycaemia modulation and antifungal therapy.

Salt stress reveals differential physiological, biochemical and molecular responses in T. monococcum and T. durum wheat genotypes.

Salt stress responses implicate a complex mechanism and differ from plant species to another. In this study, we analyzed the physiological, biochemical and molecular responses to salt stress of the diploid wheat (T. monococcum) and compared to the tetraploid wheat (T. durum). Our results showed that the diploid wheat cultivar (cv. Turkey) is relatively tolerant to different salt stress conditions than the tetraploid wheat cultivar (cv. Om Rabia3). This tolerance was manifested by significant germination, plant growth and uptake of water generating cell turgor and development. Moreover, total chlorophyll content was higher in the diploid wheat than that in the tetraploid wheat. The Na+ content in leaf blade of the cv. Om Rabia3 was significantly higher than that of the cv. Turkey, suggesting that the diploid cultivar accumulates less toxic sodium in the photosynthetic tissues. This mechanism could be explained by the recirculation of the toxic ions Na+ into the xylem sap by SOS1 protein, which coordinates with HKT-like proteins to reduce the accumulation of Na+ ions in leaf blade. Interestingly, the expression of the three genes SOS1, HKT and NHX was enhanced under salinity especially in leaf blade of the cv. Turkey. Moreover, this wheat cultivar induced the antioxidative enzymes CAT and SOD activity more efficiently than the other cultivar.

Characterization of Two HKT1;4 Transporters from Triticum monococcum to Elucidate the Determinants of the Wheat Salt Tolerance Nax1 QTL.

TmHKT1;4-A1 and TmHKT1;4-A2 are two Na+ transporter genes that have been identified as associated with the salt tolerance Nax1 locus found in a durum wheat (Triticum turgidum L. subsp. durum) line issued from a cross with T. monococcum. In the present study, we were interested in getting clues on the molecular mechanisms underpinning this salt tolerance quantitative trait locus (QTL). By analyzing the phylogenetic relationships between wheat and T. monococcum HKT1;4-type genes, we found that durum and bread wheat genomes possess a close homolog of TmHKT1;4-A1, but no functional close homolog of TmHKT1;4-A2. Furthermore, performing real-time reverse transcription-PCR experiments, we showed that TmHKT1;4-A1 and TmHKT1;4-A2 are similarly expressed in the leaves but that TmHKT1;4-A2 is more strongly expressed in the roots, which would enable it to contribute more to the prevention of Na+ transfer to the shoots upon salt stress. We also functionally characterized the TmHKT1;4-A1 and TmHKT1;4-A2 transporters by expressing them in Xenopus oocytes. The two transporters displayed close functional properties (high Na+/K+ selectivity, low affinity for Na+, stimulation by external K+ of Na+ transport), but differed in some quantitative parameters: Na+ affinity was 3-fold lower and the maximal inward conductance was 3-fold higher in TmHKT1;4-A2 than in TmHKT1;4-A1. The conductance of TmHKT1;4-A2 at high Na+ concentration (>10 mM) was also shown to be higher than that of the two durum wheat HKT1;4-type transporters so far characterized. Altogether, these data support the hypothesis that TmHKT1;4-A2 is responsible for the Nax1 trait and provide new insight into the understanding of this QTL.

Vitamins for enhancing plant resistance.

MAIN CONCLUSION: This paper provides an overview on vitamins with inducing activities in plants, the molecular and cellular mechanisms implicated, and the hormonal signalling-network regulating this process. Moreover, it reports how vitamins might be part of the molecular events linked to induced resistance by the conventional elicitors. Induced resistance (IR), exploiting the plant innate-defense system is a sustainable strategy for plant disease control. In the last decade, vitamins have been proven to act as inducers of disease resistance, and these findings have received an important attention owing to their safety and cost effectiveness. Vitamins, including thiamine (TH, vitamin B1), riboflavin (RF, vitamin B2), menadione sodium bisulfite (MSB, vitamin K3), Para-aminobenzoic acid (PABA, vitamin Bx), and folic acid (FA, vitamin B9) provided an efficient protection against a wide range of pathogens through the modulation of specific host-defense facets. However, other vitamins, such as ascorbic acid (AA, vitamin C) and tocopherols (vitamin E), have been shown to be a part of the molecular mechanisms associated to IR. The present review is the first to summarize what vitamins are acting as inducers of disease resistance in plants and how could they be modulated by the conventional elicitors. Thus, this report provides an overview on the protective abilities of vitamins and the molecular and cellular mechanisms underlying their activities. Moreover, it describes the hormonal-signalling network regulating vitamin-signal transduction during IR. Finally, a biochemical model describing how vitamins are involved in the establishment of IR process is discussed.

Durum wheat dehydrin (DHN-5) confers salinity tolerance to transgenic Arabidopsis plants through the regulation of proline metabolism and ROS scavenging system.

MAIN CONCLUSION: The wheat dehydrin (DHN-5) gives birth to salinity tolerance to transgenic Arabidopsis plants by the regulation of proline metabolism and the ROS scavenging system. Dehydrins (DHNs) are involved in plant abiotic stress tolerance. In this study, we reported that salt tolerance of transgenic Arabidopsis plants overexpressing durum wheat dehydrin (DHN-5) was closely related to the activation of the proline metabolism enzyme (P5CS) and some antioxidant biocatalysts. Indeed, DHN-5 improved P5CS activity in the transgenic plants generating a significant proline accumulation. Moreover, salt tolerance of Arabidopsis transgenic plants was accompanied by an excellent activation of antioxidant enzymes like catalase (CAT), superoxide dismutase (SOD) and peroxide dismutase (POD) and generation of a lower level of hydrogen peroxide (H2O2) in leaves compared to the wild-type plants. The enzyme activities were enhanced in these transgenic plants in the presence of exogenous proline. Nevertheless, proline accumulation was slightly reduced in transgenic plants promoting chlorophyll levels. All these results suggest the crucial role of DHN-5 in response to salt stress through the activation of enzymes implicated in proline metabolism and in ROS scavenging enzymes.

Functional characterization in Xenopus oocytes of Na+ transport systems from durum wheat reveals diversity among two HKT1;4 transporters.

Plant tolerance to salinity constraint involves complex and integrated functions including control of Na(+) uptake, translocation, and compartmentalization. Several members of the high-affinity K(+) transporter (HKT) family, which comprises plasma-membrane transporters permeable to K(+) and Na(+) or to Na(+) only, have been shown to play major roles in plant Na(+) and K(+) homeostasis. Among them, HKT1;4 has been identified as corresponding to a quantitative trait locus (QTL) of salt tolerance in wheat but was not functionally characterized. Here, we isolated two HKT1;4-type cDNAs from a salt-tolerant durum wheat (Triticum turgidum L. subsp. durum) cultivar, Om Rabia3, and investigated the functional properties of the encoded transporters using a two-electrode voltage-clamp technique, after expression in Xenopus oocytes. Both transporters displayed high selectivity for Na(+), their permeability to other monovalent cations (K(+), Li(+), Cs(+), and Rb(+)) being ten times lower than that to Na(+). Both TdHKT1;4-1 and TdHKT1;4-2 transported Na(+) with low affinity, although the half-saturation of the conductance was observed at a Na(+) concentration four times lower in TdHKT1;4-1 than in TdHKT1;4-2. External K(+) did not inhibit Na(+) transport through these transporters. Quinine slightly inhibited TdHKT1;4-2 but not TdHKT1;4-1. Overall, these data identified TdHKT1;4 transporters as new Na(+)-selective transporters within the HKT family, displaying their own functional features. Furthermore, they showed that important differences in affinity exist among durum wheat HKT1;4 transporters. This suggests that the salt tolerance QTL involving HKT1;4 may be at least in part explained by functional variability among wheat HKT1;4-type transporters.

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

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.

The thioredoxin h-type TdTrxh2 protein of durum wheat confers abiotic stress tolerance of the transformant Arabidopsis plants through its protective role and the regulation of redox homoeostasis.

The thioredoxins (Trxs) are ubiquitous and they play a crucial role in various biological processes like growth and stress response. Although the functions of Trxs proteins are described in several previous reports, the function of the isoform Trxh2 of durum wheat (Triticum durum L.), designated as TdTrxh2, in abiotic stress response still unknown. Thus, we aimed in this study the functional characterization of TdTrxh2 through its expression in yeast cells and Arabidopsis plants. Sequence analysis revealed that TdTrxh2 protein shared the conserved redox site with the other Trxh from other plant species. Under various abiotic stresses, TdTrxh2 was up-regulated in leaves and roots of durum wheat. Interestingly, we demonstrated that TdTrxh2 exhibit protective effect on LDH activity against various treatments. Besides, the expression of TdTrxh2 in yeast cells conferred their tolerance to multiple stresses. Moreover, transgenic Arabidopsis expressing TdTrxh2 showed tolerance phenotype to several abiotic stresses. This tolerance was illustrated by high rate of proline accumulation, root proliferation, low accumulation of reactive oxygen species like H2O2 and O2·-, and high antioxidant CAT and POD enzymes activities. All these findings suggested that TdTrxh2 promotes abiotic stress tolerance through the redox homoeostasis regulation and its protective role.