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.

A novel PGPF Penicillium olsonii isolated from the rhizosphere of Aeluropus littoralis promotes plant growth, enhances salt stress tolerance, and reduces chemical fertilizers inputs in hydroponic system.

The hydroponic farming significantly enhances the yield and enables multiple cropping per year. These advantages can be improved by using plant growth-promoting fungi (PGPF) either under normal or stress conditions. In this study, the fungal strain (A3) isolated from the rhizosphere of the halophyte plant Aeluropus littoralis was identified as Penicillium olsonii based on sequence homology of its ITS region. The A3 fungus was shown to be halotolerant (up to 1 M NaCl) and its optimal growth was at 27°C, but inhibited at 40°C. In liquid culture medium, the A3 produced indole acetic acid (IAA) especially in the presence of L-tryptophan. Tobacco plants grown under hydroponic farming system were used to evaluate the promoting activity of the direct effect of A3 mycelium (DE) and the indirect effect (IDE) of its cell-free culture filtrate (A3CFF). The results showed that for the two conditions (DE or IDE) the tobacco seedlings exhibited significant increase in their height, leaf area, dry weight, and total chlorophyll content. Interestingly, the A3CFF (added to the MS liquid medium or to nutrient solution (NS), prepared from commercial fertilizers) induced significantly the growth parameters, the proline concentration, the catalase (CAT) and the superoxide dismutase (SOD) activities of tobacco plants. The A3CFF maintained its activity even after extended storage at 4°C for 1 year. Since the A3 is a halotolerant fungus, we tested its ability to alleviate salt stress effects. Indeed, when added at 1:50 dilution factor to NS in the presence of 250 mM NaCl, the A3CFF enhanced the plant salt tolerance by increasing the levels of total chlorophyll, proline, CAT, and SOD activities. In addition, the treated plants accumulated less Na+ in their roots but more K+ in their leaves. The A3CFF was also found to induce the expression of five salt stress related genes (NtSOS1, NtNHX1, NtHKT1, NtSOD, and NtCAT1). Finally, we proved that the A3CFF can reduce by half the chemical fertilizers inputs. Indeed, the tobacco plants grown in a hydroponic system using 0.5xNS supplemented with A3CFF (1:50) exhibited significantly higher growth than those grown in 0.5xNS or 1xNS. In an attempt to explain this mechanism, the expression profile of some growth related genes (nitrogen metabolism (NR1, NRT1), auxin (TRYP1, YUCCA6-like), and brassinosteroid (DET2, DWF4) biosynthesis) was performed. The results showed that all these genes were up-regulated following plant treatment with A3CFF. In summary the results revealed that the halotolerant fungus P. olsonii can stimulates tobacco plant growth, enhances its salt tolerance, and reduces by half the required chemical fertilizer inputs in a hydroponic farming system.

Analysis of Salinity Tolerance in Tomato Introgression Lines Based on Morpho-Physiological and Molecular Traits.

The development of salt-tolerant tomato genotypes is a basic requirement to overcome the challenges of tomato production under salinity in the field or soil-free farming. Two groups of eight tomato introgression lines (ILs) each, were evaluated for salinity tolerance. Group-I and the group-II resulted from the following crosses respectively: Solanum lycopersicum cv-6203 × Solanum habrochaites and Solanum lycopersicum M82 × Solanum pennellii. Salt tolerance level was assessed based on a germination percentage under NaCl (0, 75, 100 mM) and in the vegetative stage using a hydroponic growing system (0, 120 mM NaCl). One line from group I (TA1648) and three lines from group II (IL2-1, IL2-3, and IL8-3) were shown to be salt-tolerant since their germination percentages were significantly higher at 75 and 100 mM NaCl than that of their respective cultivated parents cvE6203 and cvM82. Using the hydroponic system, IL TA1648 and IL 2-3 showed the highest value of plant growth traits and chlorophyll concentration. The expression level of eight salt-responsive genes in the leaves and roots of salt-tolerant ILs (TA1648 and IL 2-3) was estimated. Interestingly, SlSOS1, SlNHX2, SlNHX4, and SlERF4 genes were upregulated in leaves of both TA1648 and IL 2-3 genotypes under NaCl stress. While SlHKT1.1, SlNHX2, SlNHX4, and SlERF4 genes were upregulated under salt stress in the roots of both TA1648 and IL 2-3 genotypes. Furthermore, SlSOS2 and SlSOS3 genes were upregulated in TA1648 root and downregulated in IL 2-3. On the contrary, SlSOS1 and SlHKT1.2 genes were upregulated in the IL 2-3 root and downregulated in the TA1648 root. Monitoring of ILs revealed that some of them have inherited salt tolerance from S. habrochaites and S. pennellii genetic background. These ILs can be used in tomato breeding programs to develop salt-tolerant tomatoes or as rootstocks in grafting techniques under saline irrigation conditions.

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.

Overexpression of AlTMP2 gene from the halophyte grass Aeluropus littoralis in transgenic tobacco enhances tolerance to different abiotic stresses by improving membrane stability and deregulating some stress-related genes.

Herein, we report isolation of the AlTMP2 gene from the halophytic C4 grass Aeluropus littoralis. The subcellular localization suggested that AlTMP2 is a plasma membrane protein. In A. littoralis exposed to salt and osmotic stresses, the AlTMP2 gene was induced early and at a high rate, but was upregulated relatively later in response to abscisic acid and cold treatments. Expression of AlTMP2 in tobacco conferred improved tolerance against salinity, osmotic, H2O2, heat, and freezing stresses at the germination and seedling stages. Under control conditions, no growth or yield penalty were mentioned in transgenic plants due to the constitutive expression of AlTMP2. Interestingly, under greenhouse conditions, the seed yield of transgenic plants was significantly higher than that of non-transgenic (NT) plants grown under salt or drought stress. Furthermore, AlTMP2 plants had less electrolyte leakage, higher membrane stability, and lower Na+ and higher K+ accumulation than NT plants. Finally, six stress-related genes were shown to be deregulated in AlTMP2 plants relative to NT plants under both control and stress conditions. Collectively, these results indicate that AlTMP2 confers abiotic stress tolerance by improving ion homeostasis and membrane integrity, and by deregulating certain stress-related genes.

Identification of TIFY/JAZ family genes in Solanum lycopersicum and their regulation in response to abiotic stresses.

Plant phenotypic plasticity determines plant adaptation to changing environments and agricultural productivity. Phytohormones are essential plant signalling molecules regulating this plasticity through complex signalling networks. Jasmonates (JAs) are key phytohormones regulating many aspects of growth, development and defence responses. An important role of JAs in tolerance to abiotic stresses is also emerging. The expression of JAZ (JASMONATE-ZIM-DOMAIN PROTEIN) genes, encoding for the key repressors in the JA-pathway, is regulated by multiple abiotic stresses, suggesting a role for the JAZ proteins in response to these stresses. The JAZ proteins belong to the TIFY family, well described in many plant species. However, only the role of few tomato JAZ proteins in response to microbial infection has been analysed so far. Here, we identify the members of the tomato TIFY family, and characterize them phylogenetically. In addition, we analyse the transcriptional regulation of several SlJAZ in response to abiotic stresses and hormone treatments both in root and leaves to assess their specific expression in response to stresses. Most SlJAZ are JA-induced and responsive to one or more abiotic stresses, providing clues for functional analysis of JAZ genes in abiotic responses in tomato.

Expression of the Aeluropus littoralis AlSAP Gene Enhances Rice Yield under Field Drought at the Reproductive Stage.

We evaluated the yields of Oryza sativa L. 'Nipponbare' rice lines expressing a gene encoding an A20/AN1 domain stress-associated protein, AlSAP, from the halophyte grass Aeluropus littoralis under the control of different promoters. Three independent field trials were conducted, with drought imposed at the reproductive stage. In all trials, the two transgenic lines, RN5 and RN6, consistently out-performed non-transgenic (NT) and wild-type (WT) controls, providing 50-90% increases in grain yield (GY). Enhancement of tillering and panicle fertility contributed to this improved GY under drought. In contrast with physiological records collected during previous greenhouse dry-down experiments, where drought was imposed at the early tillering stage, we did not observe significant differences in photosynthetic parameters, leaf water potential, or accumulation of antioxidants in flag leaves of AlSAP-lines subjected to drought at flowering. However, AlSAP expression alleviated leaf rolling and leaf drying induced by drought, resulting in increased accumulation of green biomass. Therefore, the observed enhanced performance of the AlSAP-lines subjected to drought at the reproductive stage can be tentatively ascribed to a primed status of the transgenic plants, resulting from a higher accumulation of biomass during vegetative growth, allowing reserve remobilization and maintenance of productive tillering and grain filling. Under irrigated conditions, the overall performance of AlSAP-lines was comparable with, or even significantly better than, the NT and WT controls. Thus, AlSAP expression inflicted no penalty on rice yields under optimal growth conditions. Our results support the use of AlSAP transgenics to reduce rice GY losses under drought conditions.

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.

Expression of the Aeluropus littoralis AlSAP gene in rice confers broad tolerance to abiotic stresses through maintenance of photosynthesis.

The expression of AlSAP, in rice cv. Nipponbare, enhances plant tolerance to cold, drought and salt stresses. AlSAP lines showed 100% survival rate and set seeds while control plants did not recover from the cold treatment. Under a severe drought stress treatment (fraction of transpirable soil water down to 0.1), AlSAP lines exhibited enhanced Transpiration Efficiency (TE) and maintained a high A (Assimilation rate) value (22 µmol·m(-2) s(-1) ) while these values dramatically decreased (A = 4 µmol·m(-2) s(-1) ) in control plants which were subsequently unable to recover from the stress. Of noteworthy is that AlSAP rice plants yielded a similar and a 60% seed set under control and stress conditions respectively, with regard to wild-type (WT) plants grown under control conditions. This indicates that AlSAP expression imposes no yield penalty and allows seed production even following a severe drought stress at the vegetative stage. Furthermore, AlSAP rice was shown to accumulate transcripts of a pilot set of eight stress-related genes at a significantly higher level than WT plants, both under control and stressed conditions. The results suggest that AlSAP expression generates stress tolerance in plants through maintenance of the photosynthetic apparatus integrity and by stimulating an endogenous adaptive potential which is not effectively accomplished in WT plants.

Promoter of the AlSAP gene from the halophyte grass Aeluropus littoralis directs developmental-regulated, stress-inducible, and organ-specific gene expression in transgenic tobacco.

In our recent published work it has been demonstrated that AlSAP, a gene encoding an A20/AN1 zinc-finger protein (stress-associated protein) of the C4 halophyte grass Aeluropus littoralis, is inducible by various abiotic stresses and by hormonal stimuli. To further investigate the regulation of the gene, a 586-bp genomic fragment upstream of the AlSAP translated sequence has been isolated, cloned, and designated as the "Pr ( AlSAP )" promoter. Sequence analysis of "Pr ( AlSAP )" revealed the presence of cis-regulatory elements which could be required for abiotic stress, abscisic acid (ABA), and salicylic acid (SA) responsiveness and for tissue-specific and vascular expression. The Pr ( AlSAP ) promoter was fused to the ß-glucuronidase (gusA) gene and the resulting construct transferred into tobacco. Histochemical assays of stably transformed tobacco plants showed that Pr ( AlSAP ) is active in this heterologous C3 system. While full-length gusA transcripts accumulated in whole 15, 30, and 45-day-old plants, GUS histochemical staining was only observed in leaves and stems of 45-day-old, or older, transgenic seedlings. Histological sections prepared at this stage revealed activity localized in leaf veins (phloem and bundle sheath) and stems (phloem and cortex) but not in roots. Furthermore, gusA transcripts accumulated in an age-dependent manner with a basipetal pattern in leaf and stem tissues throughout the plant. In flowers, GUS expression was detected in sepals only. The accumulation of gusA transcripts was up-regulated by salt, dehydration, ABA, and SA treatment. Altogether, these results show that, when used in a heterologous dicot system, Pr ( AlSAP ) is an age-dependent, abiotic-stress-inducible, organ-specific and tissue-specific promoter.

Improved drought and salt stress tolerance in transgenic tobacco overexpressing a novel A20/AN1 zinc-finger "AlSAP" gene isolated from the halophyte grass Aeluropus littoralis.

We describe here the isolation of a novel gene, designated AlSAP, from A. littoralis in a first step to exploit the potential of this halophyte grass as a genetic resource to improve salt and drought tolerance in plants and, particularly, in cereals. The Aeluropus genome contains a single AlSAP gene which has an intron at its 5'UTR. Sequence homology analysis showed that the AlSAP protein is characterized by the presence of two conserved zinc-finger domains A20 and AN1. AlSAP is induced not only by various abiotic stresses such as salt, osmotic, heat and cold but, also by abscisic acid (ABA) and salicylic acid (SA). Tobacco plants expressing the AlSAP gene under the control of the duplicated CaMV35S promoter exhibited an enhanced tolerance to abiotic stresses such as salinity (350 mM NaCl), drought (soil Relative Water Content (RWC) = 25%), heat (55 degrees C for 2.5 h) and freezing (-20 degrees C for 3 h). Moreover, under high salt and drought conditions, the transgenic plants were able to complete their life cycle and to produce viable seeds while the wild-type plants died at the vegetative stage. Measurements of the leaf RWC and of the root and leaf endogenous Na(+) and K(+) levels in AlSAP transgenic lines compared to wild-type tobacco, showed an evident lower water loss rate and a higher Na(+) accumulation in senescent-basal leaves, respectively. Finally, we found that the steady state levels of transcripts of eight stress-related genes were higher in AlSAP transgenic lines than in wild-type tobacco. Taken together, these results show that AlSAP is a potentially useful candidate gene for engineering drought and salt tolerance in cultivated plants.

Identification and sequencing of ESTs from the halophyte grass Aeluropus littoralis.

Aeluropus littoralis (Gouan) Parl. is a C4 perennial halophyte monocotyledonous plant belonging to the same family as wheat. Growing as weed in dry salty areas or marshes, it is salt-secreting, rhizomatous and is used as forage. It is diploid (2n=2X=14) and has a relative small genome of around 342 Mb. A. littoralis is highly salt-tolerant since this plant has the ability to secrete salt. Thus, A. littoralis has the potential to become an important genetic resource for biotechnological strategies to improve salt and drought tolerance in economically important crops such as wheat. We have constructed SSH (Suppression Subtractive Hybridization) cDNA libraries from root (RSD45) and leaf (LSD45) tissues of 45 days old plants grown in the presence of 300 mM NaCl. We have also constructed full-length cDNA library from 15 days old salt stressed (300 mM NaCl) roots (RSTL15). Sequencing revealed 25 and 42 independent transcripts from the RSD45 and LSD45 cDNA libraries respectively, in both cases this was less than 25% of the clones sequenced. In contrast, 425 (60%) of the clones from the RSTL15 library revealed independent transcripts. After comparison with protein databases using BlastX, 335 (68%) ESTs (Expressed Sequence Tag) were classified into putative known functions and unclassified proteins, 59 (12%) have homology only to unidentified homologous sequences. A total of 98 (20%) of the ESTs have no homologies to known sequences in the protein databases which can be considered as novel.

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

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