Rosmarinus officinalis L. essential oil enhances salt stress tolerance of durum wheat seedlings through ROS detoxification and stimulation of antioxidant defense.

Salt-induced stress poses a significant barrier to agricultural productivity by impeding crop growth. Presently, environmentalists are dedicated to safeguarding food security by enhancing agricultural yields in challenging environments. Biostimulants play a crucial role in mitigating abiotic stresses in crop production, and among these, plant essential oils (EOs) stand out as organic substances with diverse biological effects on living organisms. Among the natural promoters of plant growth, Rosmarinus officinalis L. essential oil (RoEO) has gained considerable attention. Although the manifold effects of essential oils (EOs) on plant growth have been extensively demonstrated, their impact on salt stress tolerance in durum wheat seedlings remains unexplored. This investigation was undertaken to evaluate the biostimulatory capabilities of RoEO on the durum wheat cultivar "Mahmoudi." The effects of three RoEO concentrations (1, 2.5, and 5 ppm) on seed germination, growth establishment, and the induction of salt resistance under salinity conditions (150 mM NaCl) were tested. At 5 ppm, RoEO enhanced seedlings' tolerance to salinity by improving growth and reducing membrane deterioration and oxidative stress-induced damage. The expression profile analyses of seven stress-related genes (TdNHX1, TdSOS1, TdSOD, TdCAT, TdGA20-ox1, TdNRT2.1, and TdGS) using RT-qPCR showed enhancement of several important genes in durum wheat seedlings treated with 5 ppm RoEO, even under control conditions, which may be related to salt stress tolerance. The results indicate that the application of RoEO suggests a possible alternative strategy to increase salt tolerance in durum wheat seedlings towards better growth quality, thus increasing ROS scavenging and activation of antioxidant defense.

Development of a marker-free engineered durum wheat overexpressing Lobularia maritima GASA1 with improved drought tolerance.

Due to their fixed lifestyle, plants must adapt to abiotic or biotic stresses by orchestrating various responses, including protective and growth control measures. Growth arrest is provoked upon abiotic stress and can impair plant production. Members of the plant-specific GASA (gibberellic acid-stimulated Arabidopsis) gene family play crucial roles in phytohormone responses, abiotic and biotic stresses, and plant growth. Here, we recognized and examined the LmGASA1 gene from the halophyte plant Lobularia maritima and developed marker-free engineered durum wheat plants overexpressing the gene. The LmGASA1 transcript profile revealed that it's induced by stressful events as well as by phytohormones including GA3, MeJA, and ABA, suggesting that the LmGASA1 gene may contribute to these stress and hormone signal transduction pathways. Transient expression of GFP-LmGASA1 fusion in onion epidermal cells indicated that LmGASA1 is localized to the cell membrane. Further analysis showed that overexpression of LmGASA1 in durum wheat plants enhanced tolerance to drought stress compared with that in non-transgenic (NT) plants, imposing no yield penalty and enabling seed production even following drought stress at the vegetative stage. Altogether, our data indicate that LmGASA1 regulates both the scavenging capacity of the antioxidant enzymatic system and the activation of at least six stress-related genes that function as positive regulators of drought stress tolerance. LmGASA1 appears to be a novel gene useful for further functional analysis and potential engineering for drought stress tolerance in crops.

Biological Activity and Phytochemical Characteristics of Star Anise (Illicium verum) Essential Oil and Its Anti-Salmonella Activity on Sous Vide Pumpkin Model.

Illicium verum, commonly known as star anise, represents one of the notable botanical species and is recognized for its rich reservoir of diverse bioactive compounds. Beyond its culinary application as a spice, this plant has been extensively utilized in traditional medicine. Given the contemporary emphasis on incorporating natural resources into food production, particularly essential oils, to enhance sensory attributes and extend shelf life, our study seeks to elucidate the chemical composition and evaluate the antibacterial (in vitro, in situ) and insecticidal properties of Illicium verum essential oil (IVEO). Also, microbiological analyses of pumpkin sous vide treated with IVEO after inoculation of Salmonella enterica were evaluated after 1 and 7 days of study. GC/MS analysis revealed a significantly high amount of (E)-anethole (88.4%) in the investigated EO. The disc diffusion method shows that the antibacterial activity of the IVEO ranged from 5.33 (Streptococcus constellatus) to 10.33 mm (Citrobacter freundii). The lowest minimal inhibition concentration was found against E. coli and the minimum biofilm inhibition concertation was found against S. enterica. In the vapor phase, the best antimicrobial activity was found against E. coli in the pears model and against S. sonei in the beetroot model. The application of the sous vide method in combination with IVEO application decreased the number of microbial counts and eliminated the growth of S. enterica. The most isolated microbiota identified from the sous vide pumpkin were Bacillus amyloliquefaciens, B. cereus, B. licheniformis, and Ralstonia picketii. Modifications to the protein composition of biofilm-forming bacteria S. enterica were suggested by the MALDI TOF MS instigations. The IVEO showed insecticidal potential against Harmonia axyridis. Thanks to the properties of IVEO, our results suggest it can be used in the food industry as a natural supplement to extend the shelf life of foods and as a natural insecticide.

Changes in inoculated Salmonella enterica subsp. enterica Serovar Enteritidis and other microbiological qualities of vacuum-packed carrot slices after treatment with aqueous extract of Lobularia maritima.

After harvesting, pathogens can infect fresh vegetables in different ways. Pathogenic bacteria associated with fresh vegetables can cause widespread epidemics associated with foodborne illness. The aim of this study was to assess the microbiological quality of carrot slices after treatment with aqueous extracts of Lobularia maritima (AELm) at different concentrations AELm1 (10 mg/mL), AELm2 (5 mg/mL), AELm3 (2.5 mg/mL) and AELm4 (1.25 mg/mL), and Salmonella enterica subsp. enterica serovar Enteritidis, along with vacuum packaging and storage of carrots for 7 days at 4 °C. On days 1. and 7., total viable counts (TVC), and coliforms bacteria (CB), and Salmonella count were all analysed. Microorganisms that were obtained from carrots were identified using MALDI-TOF MS Biotyper Mass Spectrometry. The total viable, coliform bacteria and Salmonella counts were varied by the group of treatment. Higher counts were found in the control group on both days. The most isolated species of bacteria were Salmonella enterica and Pantoea agglomerans on the 1. day and Klebsiella oxytoca on the 7. day. The current study adds useful information for a better understanding of how Salmonella enterica reacts to the effect of AELm and its potential use as a sustainable washing method to eliminate bacteria from freshly cut carrots.

Citrus limon Essential Oil: Chemical Composition and Selected Biological Properties Focusing on the Antimicrobial (In Vitro, In Situ), Antibiofilm, Insecticidal Activity and Preservative Effect against Salmonella enterica Inoculated in Carrot.

New goals for industry and science have led to increased awareness of food safety and healthier living in the modern era. Here, one of the challenges in food quality assurance is the presence of pathogenic microorganisms. As planktonic cells can form biofilms and go into a sessile state, microorganisms are now more resistant to broad-spectrum antibiotics. Due to their proven antibacterial properties, essential oils represent a potential option to prevent food spoilage in the search for effective natural preservatives. In this study, the chemical profile of Citrus limon essential oil (CLEO) was evaluated. GC-MS analysis revealed that limonene (60.7%), ß-pinene (12.6%), and γ-terpinene (10.3%) are common constituents of CLEO, which prompted further research on antibacterial and antibiofilm properties. Minimum inhibitory concentration (MIC) values showed that CLEO generally exhibits acceptable antibacterial properties. In addition, in situ antimicrobial research revealed that vapour-phase CLEO can arrest the growth of Candida and Y. enterocolitica species on specific food models, indicating the potential of CLEO as a preservative. The antibiofilm properties of CLEO were evaluated by MIC assays, crystal violet assays, and MALDI-TOF MS analysis against S. enterica biofilm. The results of the MIC and crystal violet assays showed that CLEO has strong antibiofilm activity. In addition, the data obtained by MALDI-TOF MS investigation showed that CLEO altered the protein profiles of the bacteria studied on glass and stainless-steel surfaces. Our study also found a positive antimicrobial effect of CLEO against S. enterica. The anti-Salmonella activity of CLEO in vacuum-packed sous vide carrot samples was slightly stronger than in controls. These results highlight the advantages of the antibacterial and antibiofilm properties of CLEO, suggesting potential applications in food preservation.

Mixture design of α-pinene, α-terpineol, and 1,8-cineole: A multiobjective response followed by chemometric approaches to optimize the antibacterial effect against various bacteria and antioxidant activity.

α-Pinene, α-terpineol, and 1,8-cineole are compounds naturally present in essential oils, although their amounts vary from oil to oil. Although several studies have reported their antibacterial and antioxidant effects, there are few reports on the synergistic or antagonistic effects of their combinations. The objective of this study was to investigate the combined antibacterial effect of these three compounds. To our knowledge, this is the first report on the prediction of their optimal combination using the mixture design approach. The experimental antibacterial activity of the α-pinene, α-terpineol, and 1,8-cineole mixtures depended on the proportion of each compound in the mixture and the target strain, with minimum inhibitory concentrations (MIC) ranging from 0.31 to 1.85 mg/mL. Using the increased simplex-centroid mixture design, the mixture containing 0.33% of each molecule proved to be the most effective against Bacillus cereus and had the lowest MIC values. In addition, α-pinene, α-terpineol, and 1,8-cineole showed significant antioxidant activity against 2,2-picryl-1-hydrazyl radical (DPPH), with IC50 values of 24.53 ± 0.05, 65.63 ± 0.71, and 63.58 ± 0.01 µg/mL, respectively. Statistical planning and the development of utility profiles of the substance mixtures can predict the optimal composition that will exhibit the highest antibacterial activity against B. cereus as well as antioxidant properties. Furthermore, the synergistic effect of the mixtures can contribute significantly to their successful use as natural preservatives in various applications.

Salvia officinalis L. and Salvia sclarea Essential Oils: Chemical Composition, Biological Activities and Preservative Effects against Listeria monocytogenes Inoculated into Minced Beef Meat.

In this study, Salvia officinalis L. and Salvia sclarea essential oils (EOs) were investigated using gas chromatography-mass spectrometry (GC-MS) to describe their chemical composition. The obtained results show, for both EOs, a profile rich in terpene metabolites, with monoterpenes predominating sesquiterpenes but with significant qualitative and quantitative differences. The main compound found in the Salvia officinalis EO (SOEO) was camphor (19.0%), while in Salvia sclarea EO (SCEO), it was linalyl acetate (59.3%). Subsequently, the in vitro antimicrobial activity of the EOs against eight pathogenic strains was evaluated. The disc diffusion method showed a significant lysis zone against Gram-positive bacteria. The minimum inhibitory concentrations (MICs) ranged from 3.7 mg/mL to 11.2 mg/mL, indicating that each EO has specific antimicrobial activity. Both EOs also showed significant antiradical activity against DPPH radicals and total antioxidant activity. In addition, the preservative effect of SOEO (9.2%) and SCEO (9.2%), alone or in combination, was tested in ground beef, and the inhibitory effect against Listeria monocytogenes inoculated into the raw ground beef during cold storage was evaluated. Although the effect of each individual EO improved the biochemical, microbiological, and sensory parameters of the samples, their combination was more effective and showed complete inhibition of L. monocytogenes after 7 days of storage at 4 °C. The results show that both EOs could be used as safe and natural preservatives in various food and/or pharmaceutical products.

GASA Proteins: Review of Their Functions in Plant Environmental Stress Tolerance.

Gibberellic acid-stimulated Arabidopsis (GASA) gene family is a class of functional cysteine-rich proteins characterized by an N-terminal signal peptide and a C-terminal-conserved GASA domain with 12 invariant cysteine (Cys) residues. GASA proteins are widely distributed among plant species, and the majority of them are involved in the signal transmission of plant hormones, the regulation of plant development and growth, and the responses to different environmental constraints. To date, their action mechanisms are not completely elucidated. This review reports an overview of the diversity, structure, and subcellular localization of GASA proteins, their involvement in hormone crosstalk and redox regulation during development, and plant responses to abiotic and biotic stresses. Knowledge of this complex regulation can be a contribution to promoting multiple abiotic stress tolerance with potential agricultural applications through the engineering of genes encoding GASA proteins and the production of transgenic plants.

Recent Advances in the Incorporation of Polysaccharides with Antioxidant and Antibacterial Functions to Preserve the Quality and Shelf Life of Meat Products.

Meat and meat products are susceptible to various types of natural processes such as oxidative degradation due to their high content of protein and essential amino acids. However, finding solutions to maintain the nutritional and sensory quality of meat and meat products is unavoidable. Hence, there is a pressing need to investigate alternatives to synthetic preservatives, focusing on active biomolecules of natural provenance. Polysaccharides are natural polymers of various sources that exhibit antibacterial and antioxidant properties via a variety of mechanisms, owing to their diversity and structural variation. For this reason, these biomolecules are widely studied in order to improve texture, inhibit the growth of pathogens, and improve the oxidative stability and sensory characteristics of meat products. However, the literature has not addressed their biological activity in meat and meat products. This review summarizes the various sources of polysaccharides, their antioxidant and antibacterial activities (mainly against pathogenic food strains), and their use as natural preservatives to replace synthetic additives in meat and meat products. Special attention is given to the use of polysaccharides to improve the nutritional value of meat, resulting in more nutrient-rich meat products with higher polysaccharide content and less salt, nitrites/nitrates, and cholesterol.

Biopreservative Effect of the Tunisian Halophyte Lobularia maritima Flavonoid Fraction, Used Alone and in Combination with Linalool in Stored Minced Beef Meat.

In the present study, Lobularia maritima (Lm) flavonoid extract (LmFV) was characterized by HPLC analyses and five compounds were detected. Further, to describe the chemical content of the matrix, GC-MS analyses after silylation were performed; the obtained results showed the presence of a large number of components belonging to several chemical classes, mostly sugar alcohols, sugars, fatty acids, and terpenes. Firstly, the antibacterial activities of this fraction and linalool (Lin) were evaluated against eight foodborne pathogenic strains with MIC values between 2.3 and 5.8 mg/mL and 0.23 and 0.7 mg/mL, respectively. Then, the antioxidant activity of both was evaluated by the DPPH antiradical test and the phosphomolybdenum test. Furthermore, the biopreservative effect of LmFV alone and in combination with Lin on minced beef stored at 4 °C for 14 days was evaluated using microbiological and physiochemical tests. LmFV at 4.6% alone significantly reduced microbial spoilage in ground meat (p < 0.05). The combination of LmFV (4.6%) and Lin (0.46%) was more effective than LmFV alone in inhibiting bacterial contamination, reducing TBARS values and the risk of bacterial contamination, and reducing the accumulation of Met myoglobin (MetMb). This combination, therefore, extends the shelf life of the product by about 10 days. Based on these microbiological results and physicochemical parameters, it can be stated that the addition of Lin potentiates the flavonoid fraction of L. maritima more strongly against the deterioration of meat quality by significantly improving its biopreservative effect as a natural conservative.

Functional Characterization of Lobularia maritima LmTrxh2 Gene Involved in Cold Tolerance in Tobacco through Alleviation of ROS Damage to the Plasma Membrane.

Cold stress is a key environmental factor affecting plant growth and development, crop productivity, and geographic distribution. Thioredoxins (Trxs) are small proteins that are ubiquitously expressed in all organisms and implicated in several cellular processes, including redox reactions. However, their role in the regulation of cold stress in the halophyte plant Lobularia maritima remains unknown. We recently showed that overexpression of LmTrxh2, which is the gene that encodes the h-type Trx protein previously isolated from L. maritima, led to an enhanced tolerance to salt and osmotic stress in transgenic tobacco. This study functionally characterized the LmTrxh2 gene via its overexpression in tobacco and explored its cold tolerance mechanisms. Results of the RT-qPCR and western blot analyses indicated differential temporal and spatial regulation of LmTrxh2 in L. maritima under cold stress at 4 °C. LmTrxh2 overexpression enhanced the cold tolerance of transgenic tobacco, as evidenced by increased germination rate, fresh weight and catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD) activities; reduced malondialdehyde levels, membrane leakage, superoxide anion (O2-), and hydrogen peroxide (H2O2) levels; and higher retention of chlorophyll than in non-transgenic plants (NT). Furthermore, the transcript levels of reactive oxygen species (ROS)-related genes (NtSOD and NtCAT1), stress-responsive late embryogenis abundant protein 5 (NtLEA5), early response to dehydration 10C (NtERD10C), DRE-binding proteins 1A (NtDREB1A), and cold-responsive (COR) genes (NtCOR15A, NtCOR47, and NtKIN1) were upregulated in transgenic lines compared with those in NT plants under cold stress, indicating that LmTrxh2 conferred cold stress tolerance by enhancing the ROS scavenging ability of plants, thus enabling them to maintain membrane integrity. These results suggest that LmTrxh2 promotes cold tolerance in tobacco and provide new insight into the improvement of cold-stress resistance to cold stress in non-halophyte plants and crops.

Genome-wide characterization and expression profiling of GASA gene family in Triticum turgidum ssp. durum (desf.) husn. (Durum wheat) unveils its involvement in environmental stress responses.

Family members within the plant-specific gibberellic acid-stimulated Arabidopsis (GASA) gene serve a crucial role in plant growth and development, particularly in flower induction and seed development. Through a genome-wide analysis of Triticum turgidum ssp. Durum (durum wheat), we identified 19 GASA genes, designated as TdGASA1‒19. Moreover, the chromosomal locations, exon-intron distribution and the physiochemical properties of these genes were determined and the subcellular localization of their encoded proteins was estimated. Analyses of their domain structure, motif arrangements, and phylogeny revealed four distinct groups that share a conserved GASA domain. Additionally, a real-time q-PCR analysis revealed differential expression patterns of TdGASA genes in various tissues (including leaves, roots, stems, and seeds) and in response to salinity, osmotic stress, and treatment with exogenous phytohormones (abscisic and gibberellic acid), implying that these genes may play a role in the growth, development, and stress responses of Triticum turgidum. Heterologous expression of TdGASA1, TdGASA4, TdGASA14, and TdGASA19 in Saccharomyces cerevisiae improved its tolerance to salt, osmotic, oxidative, and heat stresses, which suggests the involvement of these genes in abiotic stress tolerance mechanisms. The present study is the first to identify and analyze the expression profile of T. turgidum GASA genes, therefore offering novel insights for their further functional characterization, which may serve as a novel resource for molecular breeding of durum wheat.

Evaluation of Halophyte Biopotential as an Unused Natural Resource: The Case of Lobularia maritima.

Halophytes are plant species widely distributed in saline habitats, such as beaches, postindustrial wastelands, irrigated lands, salt flats, and others. Excessive salt level, known to limit plant growth, is not harmful to halophytes, which have developed a variety of defense mechanisms allowing them to colonize harsh environments. Plants under stress are known to respond with several morpho-anatomical adaptations, but also to enhance the production of secondary metabolites to better cope with difficult conditions. Owing to these adaptations, halophytes are an interesting group of undemanding plants with a high potential for application in the food and pharmaceutical industries. Therefore, this review aims to present the characteristics of halophytes, describe changes in their gene expression, and discuss their synthesized metabolites of pharmacognostic and pharmacological significance. Lobularia maritima is characterized as a widely spread halophyte that has been shown to exhibit various pharmacological properties in vitro and in vivo. It is concluded that halophytes may become important sources of natural products for the treatment of various ailments and for supplementing the human diet with necessary non-nutrients and minerals. However, extensive studies are needed to deepen the knowledge of their biological potential in vivo, so that they can be introduced to the pharmaceutical and food industries.

The Essential Oil of Tunisian Halophyte Lobularia maritima: A Natural Food Preservative Agent of Ground Beef Meat.

This study was directed towards the investigation of the chemical composition and antimicrobial properties of the essential oil of Tunisian halophyte Lobularia maritime (LmEO). The antibacterial effects against major food-borne pathogenic and food spoilage bacteria were tested using the well diffusion method, followed by the determination of the minimum inhibitory (MIC) and bactericidal (MBC) concentrations. The essential oil has shown strong antimicrobial activity against eight pathogenic strains, which was attributed mostly to predominant constituents of the essential oil: benzyl alcohol, linalool, terpien-4-ol and globulol, as well as to synergistic effects of its major and minor constituents. Considering strong antimicrobial effects of the tested essential oil, it was further tested as a natural alternative to food preservatives, using minced beef meat as a model system. Minced beef meat was spiked with 0.019, 0.038, and 0.076% of the essential oil and stored during 14 days at 4 °C, monitoring its microbiological, physicochemical, and sensory properties. Chemical analyses revealed that meat treated with 0.076% of LmEO at underwent a significant decrease (p < 0.05) in primary and secondary lipid oxidation and reduced metmyoglobin accumulation compared with control samples. Furthermore, microflora proliferation in the meat model system spiked with 0.076% of LmEO was significantly (p < 0.05) reduced in comparison to control. In addition, two multivariate exploratory techniques, namely principal component analysis (PCA) and hierarchical analysis (HCA), were applied to the obtained data sets to describe the relationship between the main characteristics of the meat samples with and without essential oil addition. The chemometric approach highlighted the relationships between meat quality parameters. Overall, results indicated that the essential oil of Lobularia maritima deserves to be considered as a natural preservative in the meat industry.

Antioxidant and hepatoprotective effects of novel heteropolysaccharide isolated from Lobularia maritima on CCl4-induced liver injury in rats.

The aim of the present study was to investigate the extraction and the characterization of a novel heteropolysaccharide from Tunisian halophyte Lobularia maritima (LmPS). We were also interested in its antioxidant, anti-inflammatory, and hepatoprotective effects on carbon tetrachloride (CCl4)-induced liver injury in rats. LmPS physicochemical properties were evaluated by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), thermogravimetric analysis (TGA), and UV absorption. According to TLC and HPLC results, LmPS was a heteropolysaccharide composed of glucose, galactose, and xylose. Its molecular weight was 130.62 kDa. This heteropolysaccharide was characterized by a significant antioxidant potential and was efficient against oxidative stress and CCL4-induced hepatotoxicity in rat Wistar models (n = 8) treated with a single dose of LmPS 250 mg/kg of body weight. This was evidenced by a significant increase in serum marker enzymes specially aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH). The cytokines released after stimulation of rats with LmPS showed high anti-inflammatory profiles with an increased rate of interleukine-10 (IL-10) with 0.03 pg/mL compared to animals treated only with CCl4. On the contrary, we noticed a decrease of the other cytokines (tumor necrosis factor α: TNF-α, interleukine-6: IL-6, transforming growth factor beta 1: TGF-ß1) with average concentration values of <0.2, 0.1, and 0.04 pg/mL, respectively. Besides, histopathological examinations revealed that CCl4 causes acute liver damage, characterized by extensive hepatocellular necrosis, vacuolization, and inflammatory cell infiltration, as well as DNA fragmentation. LmPS administration at a dose of 250 mg/kg resulted in a significant hepatoprotection, evidenced by a reduction of CCl4-induced oxidative damage for all tested markers. These findings eagerly confirmed that LmPS was effective in the protection against CCl4-induced hepatotoxicity and genotoxicity. It, therefore, suggested a potential therapeutic use of this polysaccharide as an alternative medicine for patients with acute liver diseases.

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.

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.

The durum wheat annexin, TdAnn6, improves salt and osmotic stress tolerance in Arabidopsis via modulation of antioxidant machinery.

TdAnn6 is a gene encoding an annexin protein in durum wheat (Triticum durum). The function of TdAnn6 in plant response to stress is not yet clearly understood. Here, we isolated TdAnn6 and characterized it in genetically modified Arabidopsis thaliana. Expressing TdAnn6 in Arabidopsis coincided with an improvement in stress tolerance at germination and seedling stages. In addition, TdAnn6-expressing seedling antioxidant activities were improved with lower level of malondialdehyde, and enhanced transcript levels of six stress-related genes during salt/osmotic stresses. Under greenhouse conditions, the TdAnn6 plants exhibited increased tolerance to salt or drought stress. To deepen our understanding of TdAnn6 function, we isolated a 1515-bp genomic fragment upstream of its coding sequence, designated as PrTdAnn6. The PrTdAnn6 promoter was fused to the ß-glucuronidase reporter gene and transferred to Arabidopsis. By histochemical GUS staining, GUS activity was detected in the roots, leaves, and floral organs, but no activity was detected in the seeds. Furthermore, we noticed a high stimulation of promoter activity when A. thaliana seedlings were exposed to NaCl, mannitol, ABA, GA, and cold conditions. This cross-talk between tissue-specific expression and exogenous stress stimulation may provide additional layers of regulation for salt and osmotic stress responses in crops.

Characterization of a novel LmSAP gene promoter from Lobularia maritima: Tissue specificity and environmental stress responsiveness.

Halophyte Lobularia maritima LmSAP encodes an A20AN1 zinc-finger stress-associated protein which expression is up-regulated by abiotic stresses and heavy metals in transgenic tobacco. To deepen our understanding of LmSAP function, we isolated a 1,147 bp genomic fragment upstream of LmSAP coding sequence designated as PrLmSAP. In silico analyses of PrLmSAP revealed the presence of consensus CAAT and TATA boxes and cis-regulatory elements required for abiotic stress, phytohormones, pathogen, and wound responses, and also for tissue-specific expression. The PrLmSAP sequence was fused to the ß-glucuronidase (gusA) reporter gene and transferred to rice. Histochemical GUS staining showed a pattern of tissue-specific expression in transgenic rice, with staining observed in roots, coleoptiles, leaves, stems and floral organs but not in seeds or in the root elongation zone. Wounding strongly stimulated GUS accumulation in leaves and stems. Interestingly, we observed a high stimulation of the promoter activity when rice seedlings were exposed to NaCl, PEG, ABA, MeJA, GA, cold, and heavy metals (Al3+, Cd2+, Cu2+ and Zn2+). These results suggest that the LmSAP promoter can be a convenient tool for stress-inducible gene expression and is a potential candidate for crop genetic engineering.

Early effects of salt stress on the physiological and oxidative status of the halophyte Lobularia maritima.

Soil salinity is an abiotic stress that reduces agricultural productivity. For decades, halophytes have been studied to elucidate the physiological and biochemical processes involved in alleviating cellular ionic imbalance and conferring salt tolerance. Recently, several interesting genes with proven influence on salt tolerance were isolated from the Mediterranean halophyte Lobularia maritima (L.) Desv. A better understanding of salt response in this species is needed to exploit its potential as a source of stress-related genes. We report the characterisation of L. maritima's response to increasing NaCl concentrations (100-400 mM) at the physiological, biochemical and molecular levels. L. maritima growth was unaffected by salinity up to 100 mM NaCl and it was able to survive at 400 mM NaCl without exhibiting visual symptoms of damage. Lobularia maritima showed a Na+ and K+ accumulation pattern typical of a salt-includer halophyte, with higher contents of Na+ in the leaves and K+ in the roots of salt-treated plants. The expression profiles of NHX1, SOS1, HKT1, KT1 and VHA-E1 in salt-treated plants matched this Na+ and K+ accumulation pattern, suggesting an important role for these transporters in the regulation of ion homeostasis in leaves and roots of L. maritima. A concomitant stimulation in phenolic biosynthesis and antioxidant enzyme activity was observed under moderate salinity, suggesting a potential link between the production of polyphenolic antioxidants and protection against salt stress in L. maritima. Our findings indicate that the halophyte L. maritima can rapidly develop physiological and antioxidant mechanisms to adapt to salt and manage oxidative stress.