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.

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.

Ectopic Expression of Aeluropus littoralis Plasma Membrane Protein Gene AlTMP1 Confers Abiotic Stress Tolerance in Transgenic Tobacco by Improving Water Status and Cation Homeostasis.

We report here the isolation and functional analysis of AlTMP1 gene encoding a member of the PMP3 protein family. In Aeluropus littoralis, AlTMP1 is highly induced by abscisic acid (ABA), cold, salt, and osmotic stresses. Transgenic tobacco expressing AlTMP1 exhibited enhanced tolerance to salt, osmotic, H2O2, heat and freezing stresses at the seedling stage. Under greenhouse conditions, the transgenic plants showed a higher level of tolerance to drought than to salinity. Noteworthy, AlTMP1 plants yielded two- and five-fold more seeds than non-transgenic plants (NT) under salt and drought stresses, respectively. The leaves of AlTMP1 plants accumulated lower Na⁺ but higher K⁺ and Ca2+ than those of NT plants. Tolerance to osmotic and salt stresses was associated with higher membrane stability, low electrolyte leakage, and improved water status. Finally, accumulation of AlTMP1 in tobacco altered the regulation of some stress-related genes in either a positive (NHX1, CAT1, APX1, and DREB1A) or negative (HKT1 and KT1) manner that could be related to the observed tolerance. These results suggest that AlTMP1 confers stress tolerance in tobacco through maintenance of ion homeostasis, increased membrane integrity, and water status. The observed tolerance may be due to a direct or indirect effect of AlTMP1 on the expression of stress-related genes which could stimulate an adaptive potential not present in NT plants.

The promoter of the AlSAP gene from the halophyte grass Aeluropus littoralis directs a stress-inducible expression pattern in transgenic rice plants.

KEY MESSAGE: When fused to " Pr AlSAP " promoter, transcripts of gusA exhibited similar accumulation patterns in transgenic rice as AlSAP transcripts in A. littoralis. Pr AlSAP can be used for engineering abiotic stress tolerance. We previously showed that ectopic expression of a stress-associated protein gene from Aeluropus littoralis (AlSAP) enhances tolerance to multiple abiotic stresses in tobacco, wheat and rice. The ortholog of AlSAP in rice is OsSAP9. Here, we demonstrate that AlSAP transcripts accumulate in Aeleuropus in response to multiple abiotic stresses and at a higher level in roots, while those of OsSAP9 are preferentially induced by cold and heat treatments and accumulate preferentially in leaves of rice. In silico analysis of the AlSAP promoter "Pr AlSAP " predicted several cis-acting elements responsible for gene regulation by dehydration, salt, heat, ABA, SA, wounding and tissue-specific expression. The Pr AlSAP promoter was fused to the gusA gene and used to produce transgenic rice plants. Transcripts of gusA exhibited similar accumulation patterns in transgenic rice as AlSAP transcripts in A. littoralis. Indeed, accumulation of gusA transcripts was higher in roots than in leaves and induced by salt, drought, cold and heat treatments. GUS activity was confirmed in roots, coleoptiles, leaves and glumes, but absent in the root cell elongation zone and in dry seeds. A wound treatment strongly induced GUS accumulation in leaves and imbibed seeds. Altogether, these results indicate that the regulatory regions of two ortholog genes "AlSAP" and "OsSAP9" have diverged in the specificity of the signals promoting their induction, but that the trans-acting elements allowing the correct spatiotemporal regulation and stress induction of Pr AlSAP exist in rice. Therefore, the AlSAP promoter appears to be an interesting candidate for engineering abiotic stress tolerance in cereals.

The newly assembled chloroplast genome of Aeluropus littoralis: molecular feature characterization and phylogenetic analysis with related species.

Aeluropus littoralis, a halophyte grass, is widely distributed from the Mediterranean to the Indian subcontinent through the Mongolian Gobi. This model halophyte has garnered increasing attention owing to its use as forage and its high tolerance to environmental stressors. The chloroplast genomes of many plants have been extensively examined for molecular, phylogenetic and transplastomic applications. However, no published research on the A. littoralis chloroplast (cp) genome was discovered. Here, the entire chloroplast genome of A. littoralis was assembled implementing accurate long-read sequences. The entire chloroplast genome, with an estimated length of 135,532 bp (GC content: 38.2%), has a quadripartite architecture and includes a pair of inverted repeat (IR) regions, IRa and IRb (21,012 bp each), separated by a large and a small single-copy regions (80,823 and 12,685 bp, respectively). The features of A. littoralis consist of 133 genes that synthesize 87 peptides, 38 transfer RNAs, and 8 ribosomal RNAs. Of these genes, 86 were unique, whereas 19 were duplicated in IR regions. Additionally, a total of forty-six simple sequence repeats, categorized into 32-mono, four-di, two-tri, and eight-tetranucleotides, were discovered. Furthermore, ten sets of repeats greater than 20 bp were located primarily in the LSC region. Evolutionary analysis based on chloroplast sequence data revealed that A. littoralis with A. lagopoides and A. sinensis belong to the Aeluropodinae subtribe, which is a sister to the Eleusininae in the tribe Cynodonteae and the subfamily Chloridoideae. This subfamily belongs to the PACMAD clade, which contains the majority of the C4 photosynthetic plants in the Poaceae. The newly constructed A. littoralis cp genome offers valuable knowledge for DNA barcoding, phylogenetic, transplastomic research, and other biological studies.

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.

Aeluropus littoralis stress-associated protein promotes water deficit resilience in engineered durum wheat.

Global climate change-related water deficit negatively affect the growth, development and yield performance of multiple cereal crops, including durum wheat. Therefore, the improvement of water-deficit stress tolerance in durum wheat varieties in arid and semiarid areas has become imperative for food security. Herein, we evaluated the water deficiency resilience potential of two marker-free transgenic durum wheat lines (AlSAP-lines: K9.3 and K21.3) under well-watered and water-deficit stress conditions at both physiological and agronomic levels. These two lines overexpressed the AlSAP gene, isolated from the halophyte grass Aeluropus littoralis, encoding a stress-associated zinc finger protein containing the A20/AN1 domains. Under well-watered conditions, the wild-type (WT) and both AlSAP-lines displayed comparable performance concerning all the evaluated parameters. Ectopic transgene expression exerted no adverse effects on growth and yield performance of the durum wheat plants. Under water-deficit conditions, no significant differences in the plant height, leaf number, spike length, and spikelet number were observed between AlSAP-lines and WT plants. However, compared to WT, the AlSAP-lines exhibited greater dry matter production, greater flag leaf area, improved net photosynthetic rate, stomatal conductance, and water use efficiency. Notably, the AlSAP-lines displayed 25 % higher grain yield (GY) than the WT plants under water-deficit conditions. The RT-qPCR-based selected stress-related gene (TdDREB1, TdLEA, TdAPX1, and TdBlt101-2) expression analyses indicated stress-related genes enhancement in AlSAP-durum wheat plants under both well-watered and water-deficit conditions, potentially related to the water-deficit resilience. Collectively, our findings support that the ectopic AlSAP expression in durum wheat lines enhances water-deficit resilience ability, thereby potentially compensate for the GY loss in arid and semi-arid regions.

Ectopic Expression of a Wheat R2R3-Type MYB Gene in Transgenic Tobacco Enhances Osmotic Stress Tolerance via Maintaining ROS Balance and Improving Root System Architecture.

Water scarcity is a critical cause of plant yield loss and decreased quality. Manipulation of root system architecture to minimize the impact of water scarcity stresses may greatly contribute towards an improved distribution of roots in the soil and enhanced water and nutrient uptake abilities. In this study, we explored the potential of TaMYB20 gene, a wheat gene belonging to the R2R3-MYB transcription factor family, to improve root system architecture in transgenic tobacco plants. The full-length TaMYB20 gene was isolated from Triticum aestivum.cv. Sakha94 and used to produce genetically engineered tobacco plants. The transgenic plants exhibited enhanced tolerance to extended osmotic stress and were able to maintain their root system architecture traits, including total root length (TRL), lateral root number (LRN), root surface area (RSa), and root volume (RV), while the wild-type plants failed to maintain the same traits. The transgenic lines presented greater relative water content in their roots associated with decreased ion leakage. The oxidative stress resulted in the loss of mitochondrial membrane integrity in the wild-type (WT) plants due to the overproduction of reactive oxygen species (ROS) in the root cells, while the transgenic lines were able to scavenge the excess ROS under stressful conditions through the activation of the redox system. Finally, we found that the steady-state levels of three PIN gene transcripts were greater in the TaMYB20-transgenic lines compared to the wild-type tobacco. Taken together, these findings confirm that TaMYB20 is a potentially useful gene candidate for engineering drought tolerance in cultivated plants.

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.

Differential Morpho-Physiological and Biochemical Responses of Duckweed Clones from Saudi Arabia to Salinity.

Salinity affects the morphological, physiological, and biochemical characteristics of several plant species. The current study was conducted to investigate differential salt tolerance potentials among ten duckweed clones under different salt-stress conditions. Morphological and physiological parameters, including fronds length, fronds number, root length, root number, Na+/K+, chlorophyll, proline contents, and fresh harvest weight, were recorded for each of the ten duckweed clones collected from different Saudi Arabia regions. Additionally, the expression patterns of seven salt-related genes were monitored in a salt-tolerant duckweed genotype. The results show that the Madinah-2 (Spirodela polyryiza) and Al-Qassim (Landoltia punctata) clones presented higher performances for all the tested morphological and physiological parameters compared to other genotypes under salt-stress conditions. At concentrations greater than 150 mM NaCl, these aforementioned traits were affected for all the genotypes tested, except Madinah-2 (S. polyryiza) and Al-Qassim (L. punctata) clones, both of which exhibited high tolerance behavior under high salt conditions (200 mM and 250 mM NaCl). The principal component analysis (PCA) showed that the first five principal components accounted for 94.8% of the total variance among the studied traits. Morphological and physiological traits are the major portions of PC1. Moreover, the expression pattern analysis of NHX, BZIP, ST, and KTrans transcript revealed their upregulation in the Al-Qassim clone under salt-stress conditions, suggesting that these genes play a role in this clone's tolerance to salt-induced stress. Overall, this study indicates that the Al-Qassim clone could be used in a brackish-water duckweed-based treatment program with a simultaneous provision of valuable plant biomass.

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.

Identification of Wheat Ideotype under Multiple Abiotic Stresses and Complex Environmental Interplays by Multivariate Analysis Techniques.

Multiple abiotic stresses negatively impact wheat production all over the world. We need to increase productivity by 60% to provide food security to the world population of 9.6 billion by 2050; it is surely time to develop stress-tolerant genotypes with a thorough comprehension of the genetic basis and the plant's capacity to tolerate these stresses and complex environmental reactions. To approach these goals, we used multivariate analysis techniques, the additive main effects and multiplicative interaction (AMMI) model for prediction, linear discriminant analysis (LDA) to enhance the reliability of the classification, multi-trait genotype-ideotype distance index (MGIDI) to detect the ideotype, and the weighted average of absolute scores (WAASB) index to recognize genotypes with stability that are highly productive. Six tolerance multi-indices were used to test twenty wheat genotypes grown under multiple abiotic stresses. The AMMI model showed varying differences with performance indices, which disagreed with the trait and genotype differences used. The G01, G12, G16, and G02 were selected as the appropriate and stable genotypes using the MGIDI with the six tolerance multi-indices. The biplot features the genotypes (G01, G03, G11, G16, G17, G18, and G20) that were most stable and had high tolerance across the environments. The pooled analyses (LDA, MGIDI, and WAASB) showed genotype G01 as the most stable candidate. The genotype (G01) is considered a novel genetic resource for improving productivity and stabilizing wheat programs under multiple abiotic stresses. Hence, these techniques, if used in an integrated manner, strongly support the plant breeders in multi-environment trials.

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.

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 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.

Harnessing the Rhizosphere of the Halophyte Grass Aeluropus littoralis for Halophilic Plant-Growth-Promoting Fungi and Evaluation of Their Biostimulant Activities.

Hydroponic systems have gained interest and are increasingly used in hot and dry desert areas. Numbers of benefits are offered by hydroponic systems such as the ability to save water, enhance nutrients use efficiency, easy environmental control, and prevention of soil-borne diseases. However, the high consumption of chemical fertilizers for nutrient solution and the sensitivity of closed hydroponic systems to salinity are issues that need solutions. Thus, the main goal of our research activities is to isolate plant growth promoting fungi in order to develop sustainable hydroponic systems. We are working on isolating and testing the possibility to incorporate the cell-free filtrate (CFF) of plant growth promoting fungi (PGPF) in the composition of the nutrient solution. In this work, we isolated six strains of PGPF from the rhizosphere of the halophyte grass Aeluropus littoralis. Phylogenetic analyses of DNA sequences amplified by ITS1 and ITS4 primers identified the isolated fungi as: Byssochlamys spectabilis, Chaetomium globosum, Cephalotheca foveolata, Penicillium melinii, Alternaria tenuissima, and Nigrospora chinensis. The promoting of vigor in tobacco seedlings was used as criteria to evaluate the biostimulant activity of these fungi by adding either their mycelia (DE: direct effect) or their cell-free filtrates (CFF: indirect effect) to the plant-growth media. The best significant growth stimulation was obtained with plants treated by B. spectabilis. However, only the CFFs of Byssochlamys spectabilis (A5.1) and Penicillium melinii (A8) when added at a dilution factor of 1/50 to half-strength nutritive solution (0.5NS) resulted in significant improvement of all assessed growth parameters. Indeed, the A5.1CFF and A8CFF in 0.5NS induced a significant better increase in the biomass production when compared to NS or 0.5NS alone. All fungi produced indole acetic acid in the CFFs, which could be one of the key factors explaining their biostimulant activities. Furthermore, six genes involved in nitrogen-metabolism (NR1 and NRT1), auxin biosynthesis (Tryp1 and YUCCA6-like), and brassinosteroid biosynthesis (DET2 and DWF4) were shown to be induced in roots or leaves following treatment of plants with the all CFFs. This work opens up a prospect to study in deep the biostimulant activity of PGPFs and their applications to decrease the requirement of chemical fertilizers in the hydroponic growing systems.

A Lobularia maritima LmSAP protein modulates gibberellic acid homeostasis via its A20 domain under abiotic stress conditions.

Stress-associated proteins (SAPs) are favorable targets to improve stress tolerance in plants, owing to their roles in developmental processes and stress responses. However, the role of SAPs and the molecular mechanisms by which they regulate plant stress responses remain poorly understood. Previously, it was reported that LmSAP expression was upregulated by various abiotic stressors in Lobularia maritima, and that transgenic tobacco lines with constitutively expressed LmSAPΔA20 and LmSAPΔA20-ΔAN1 showed dwarf phenotypes due to the deficiency of cell elongation under salt and osmotic stresses. In this study, we examined the function of A20 domain in the GA pathway in response to abiotic stresses. Transient expression of acGFP-LmSAPΔA20 and acGFP-LmSAPΔA20-ΔAN1 in onion epidermal cells demonstrated that these fused proteins were localized in the nucleo-cytoplasm. However, the truncated form acGFP-LmSAPΔAN1 was localized in the nucleus. Moreover, comparison of native and truncated LmSAP showed dramatic structural changes caused by the deletion of the A20 domain, leading to loss of function and localization. Interestingly, overexpression LmSAP and truncated LmSAPΔAN1 led to up-regulation of GA biosynthetic genes and increased total gibberellins (GAs) content, corresponding with accelerated development in transgenic tobacco plants. Moreover, the dwarf phenotype of the transgenic lines that express LmSAPΔA20 and LmSAPΔA20-ΔAN1 under stress conditions was fully restored by the application of exogenous GA3. These findings improve our understanding of the role of LmSAP in regulating GA homeostasis, which is important for regulating plant development under abiotic stress conditions.

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.

Morphological and Genetic Diversity within Salt Tolerance Detection in Eighteen Wheat Genotypes.

Salinity is a major obstacle to wheat production worldwide. Salt-affected soils could be used by improving salt-tolerant genotypes depending upon the genetic variation and salt stress response of adapted and donor wheat germplasm. We used a comprehensive set of morpho-physiological and biochemical parameters and simple sequence repeat (SSR) marker technique with multivariate analysis to accurately demonstrate the phenotypic and genetic variation of 18 wheat genotypes under salinity stress. All genotypes were evaluated without NaCl as a control and with 150 mM NaCl, until the onset of symptoms of death in the sensitive plant (after 43 days of salinity treatment). The results showed that the relative change of the genetic variation was high for all parameters, heritability (> 60%), and genetic gain (> 20%). Stepwise regression analysis, noting the importance of the root dry matter, relative turgidity, and their respective contributions to the shoot dry matter, indicated their relevance in improving and evaluating the salt-tolerant genotypes of breeding programs. The relative change of the genotypes in terms of the relative turgidity and shoot dry matter during salt stress was verified using clustering methods. For cluster analysis, the genotypes were classified into three groups: tolerant, intermediate, and sensitive, representing five, six, and seven genotypes, respectively. The morphological and genetic distances were significantly correlated based on the Mantel test. Of the 23 SSR markers that showed polymorphism, 17 were associated with almost all examined parameters. Therefore, based on the observed molecular marker-phenotypic trait association, the markers were highly useful in detecting tolerant and sensitive genotypes. Thus, it considers a helpful tool for salt tolerance through marker-assisted selection.

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.