Differential Salt Tolerance Strategies in Three Halophytes from the Same Ecological Habitat: Augmentation of Antioxidant Enzymes and Compounds.
ABSTRACT: Understanding the salt tolerance mechanism in obligate halophytes provides valuable information for conservation and re-habitation of saline areas. Here, we investigated the responses of three obligate halophytes namely Arthrocnemum macrostachyum, Sarcocornia fruticosa and Salicornia europaea to salt stress (0, 100, 200, 400 and 600 mM NaCl) during their vegetative growth with regard to biomass, ions contents (Na+, K+ and Ca+2), chlorophyll contents, carotenoids, phenolic compounds, flavonoids, and superoxide dismutase, peroxidase and esterase activities. S. europaea showed the lowest biomass, root K+ content, Chl a/b ratio, and carotenoids under salinity. This reduction of biomass is concomitant with the increase in proline contents and peroxidase activity. On the other hand, the promotion of growth under low salinity and maintenance under high salinity (200 and 400 Mm NaCl) in A. Macrostachyum and S. fruticosa are accompanied by an increase in Chl a/b ratio, carotenoids, phenolics contents, and esterase activity. Proline content was decreased under high salinity (400 and 600 mM NaCl) in both species compared to S. europaea, while peroxidase showed the lowest activity in both plants under all salt levels except under 600 mM NaCl in Arthrocnemum macrostachyum compared to S. europaea. These results suggest two differential strategies; (1) the salt tolerance is due to activation of antioxidant enzymes and biosynthesis of proline in S. europaea, (2) the salt tolerance in A. macrostachyum, S. fruticosa are due to rearrangement of chlorophyll ratio and biosynthesis of antioxidant compounds (carotenoids, phenolics and flavonoids) which their cost seem to need less energy than activation of antioxidant enzymes. The differential behavior in halophytes of the same habitat confirms that the tolerance mechanism in halophytes is species-specific which provides new insight about the restoration strategy of saline areas.
Project description:Halophytes have been characterized as a potential resource for fiber, food, fodder, and bioactive compounds. Proximate composition, bioactive compounds, and antioxidant activity of five wild dominant halophytes (<i>Arthrocnemum</i><i>macrostachyum</i>, <i>Halocnemum</i><i>strobilaceum</i>, <i>Limoniastrum</i><i>monopetalum</i>, <i>Limoniastrum</i><i>pruinosum</i>, and <i>Tamarix nilotica</i>) naturally growing along the Nile Delta coast were assessed. The soil supporting these halophytes was sandy to sand-silty, alkaline, with low organic carbon, and relatively high CaCO<sub>3</sub>. <i>H. strobilaceum</i> attained the highest moisture content, ash, crude fiber, lipids, and total soluble sugars. <i>L. monopetalum</i> showed the highest content of crude protein (18.00%), while <i>T. nilotica</i> had the highest content of total carbohydrates. The studied halophytes can be ranked according to their nutritive value as follows: <i>H.</i><i>strobilaceum</i> > <i>L.</i><i>monopetalum</i> > <i>A.</i><i>macrostachyum</i> > <i>L.</i><i>pruinosum</i> > <i>T. nilotica</i>. <i>A. macrostachyum</i> attained the highest amount of Na<sup>+</sup>, K<sup>+</sup>, Ca<sup>2+</sup>, and Mg<sup>2+</sup>. <i>A. macrostachyum</i> showed a high content of phenolic compounds, while <i>H.</i><i>strobilaceum</i> was rich in tannins and saponin contents. The MeOH extract of <i>A. macrostachyum</i> and <i>H. strobilaceum</i> exhibited substantial antioxidant activity. The present results showed that the studied halophytes could be considered as candidates for forage production or used as green eco-friendly natural resources for bioactive compounds.
Project description:Improvement of crop production is needed to feed the growing world population as the amount and quality of agricultural land decreases and soil salinity increases. This has stimulated research on salt tolerance in plants. Most crops tolerate a limited amount of salt to survive and produce biomass, while halophytes (salt-tolerant plants) have the ability to grow with saline water utilizing specific biochemical mechanisms. However, little is known about the genes involved in salt tolerance. We have characterized the transcriptome of Suaeda fruticosa, a halophyte that has the ability to sequester salts in its leaves. Suaeda fruticosa is an annual shrub in the family Chenopodiaceae found in coastal and inland regions of Pakistan and Mediterranean shores. This plant is an obligate halophyte that grows optimally from 200-400 mM NaCl and can grow at up to 1000 mM NaCl. High throughput sequencing technology was performed to provide understanding of genes involved in the salt tolerance mechanism. De novo assembly of the transcriptome and analysis has allowed identification of differentially expressed and unique genes present in this non-conventional crop.Twelve sequencing libraries prepared from control (0 mM NaCl treated) and optimum (300 mM NaCl treated) plants were sequenced using Illumina Hiseq 2000 to investigate differential gene expression between shoots and roots of Suaeda fruticosa. The transcriptome was assembled de novo using Velvet and Oases k-45 and clustered using CDHIT-EST. There are 54,526 unigenes; among these 475 genes are downregulated and 44 are upregulated when samples from plants grown under optimal salt are compared with those grown without salt. BLAST analysis identified the differentially expressed genes, which were categorized in gene ontology terms and their pathways.This work has identified potential genes involved in salt tolerance in Suaeda fruticosa, and has provided an outline of tools to use for de novo transcriptome analysis. The assemblies that were used provide coverage of a considerable proportion of the transcriptome, which allows analysis of differential gene expression and identification of genes that may be involved in salt tolerance. The transcriptome may serve as a reference sequence for study of other succulent halophytes.
Project description:<h4>Background</h4>Halophytes such as Salicornia europaea have evolved to exhibit unique mechanisms controlled by complex networks and regulated by numerous genes and interactions to adapt to habitats with high salinity. However, these mechanisms remain unknown.<h4>Methods</h4>To investigate the mechanism by which halophytes tolerate salt based on changes in the whole transcriptome, we performed transcriptome sequencing and functional annotation by database search. Using the unigene database, we conducted digital gene expression analysis of S. europaea at various time points after these materials were treated with NaCl. We also quantified ion uptakes. Gene functional enrichment analysis was performed to determine the important pathways involved in this process.<h4>Results</h4>A total of 57,151 unigenes with lengths of >300 bp were assembled, in which 57.5% of these unigenes were functionally annotated. Differentially expressed genes indicated that cell wall metabolism and lignin biosynthetic pathways were significantly enriched in S. europaea to promote the development of the xylem under saline conditions. This result is consistent with the increase in sodium uptake as ions pass through the xylem. Given that PSII efficiency remained unaltered, salt treatment activated the expression of electron transfer-related genes encoded by the chloroplast chromosome. Chlorophyll biosynthesis was also inhibited, indicating the energy-efficient state of the electron transfer system of S. europaea.<h4>Conclusions</h4>The key function of adjusting important primary metabolic pathways in salt adaption was identified by analyzing the changes in the transcriptome of S. europaea. These pathways could involve unique salt tolerance mechanisms in halophytes. This study also provided information as the basis of future investigations on salt response genes in S. europaea. Ample gene resources were also provided to improve the genes responsible for the salt tolerance ability of crops.
Project description:Salinity is a critical environmental factor that adversely affects crop productivity. Halophytes have evolved various mechanisms to adapt to saline environments. Salicornia europaea L. is one of the most salt-tolerant plant species. It does not have special salt-secreting structures like a salt gland or salt bladder, and is therefore a good model for studying the common mechanisms underlying plant salt tolerance. To identify candidate genes encoding key proteins in the mediation of salt tolerance in S. europaea, we performed a functional screen of a cDNA library in yeast. The library was screened for genes that allowed the yeast to grow in the presence of 1.3 M NaCl. We obtained three full-length S. europaea genes that confer salt tolerance. The genes are predicted to encode (1) a novel protein highly homologous to thaumatin-like proteins, (2) a novel coiled-coil protein of unknown function, and (3) a novel short peptide of 32 residues. Exogenous application of a synthetic peptide corresponding to the 32 residues improved salt tolerance of Arabidopsis. The approach described in this report provides a rapid assay system for large-scale screening of S. europaea genes involved in salt stress tolerance and supports the identification of genes responsible for such mechanisms. These genes may be useful candidates for improving crop salt tolerance by genetic transformation.
Project description:Comparative studies on the responses to salt stress of taxonomically related taxa should help to elucidate relevant mechanisms of stress tolerance in plants. We have applied this strategy to three Plantago species adapted to different natural habitats, P. crassifolia and P. coronopus-both halophytes-and P. major, considered as salt-sensitive since it is never found in natural saline habitats. Growth inhibition measurements in controlled salt treatments indicated, however, that P. major is quite resistant to salt stress, although less than its halophytic congeners. The contents of monovalent ions and specific osmolytes were determined in plant leaves after four-week salt treatments. Salt-treated plants of the three taxa accumulated Na+ and Cl- in response to increasing external NaCl concentrations, to a lesser extent in P. major than in the halophytes; the latter species also showed higher ion contents in the non-stressed plants. In the halophytes, K+ concentration decreased at moderate salinity levels, to increase again under high salt conditions, whereas in P. major K+ contents were reduced only above 400 mM NaCl. Sorbitol contents augmented in all plants, roughly in parallel with increasing salinity, but the relative increments and the absolute values reached did not differ much in the three taxa. On the contrary, a strong (relative) accumulation of proline in response to high salt concentrations (600-800 mM NaCl) was observed in the halophytes, but not in P. major. These results indicate that the responses to salt stress triggered specifically in the halophytes, and therefore the most relevant for tolerance in the genus Plantago are: a higher efficiency in the transport of toxic ions to the leaves, the capacity to use inorganic ions as osmotica, even under low salinity conditions, and the activation, in response to very high salt concentrations, of proline accumulation and K+ transport to the leaves of the plants.
Project description:The genus <i>Plantago</i> is particularly interesting for studying the mechanisms of salt tolerance in plants, as it includes both halophytes and glycophytes, as well as species adapted to xeric environments. In this study, the salt stress responses of two halophytes, <i>P. crassifolia</i> and <i>P. coronopus,</i> were compared with those of two glycophytes, <i>P. ovata</i> and <i>P. afra</i>. Plants obtained by seed germination of the four species, collected in different regions of Tunisia, were subjected to increasing salinity treatments for one month under greenhouse conditions. Morphological traits and biochemical parameters, such as ion accumulation and the leaf contents of photosynthetic pigments, osmolytes, oxidative stress markers and antioxidant metabolites, were measured after the treatments. Salt-induced growth inhibition was more pronounced in <i>P. afra</i>, and only plants subjected to the lowest applied NaCl concentration (200 mM) survived until the end of the treatments. The biochemical responses were different in the two groups of plants; the halophytes accumulated higher Na<sup>+</sup> and proline concentrations, whereas MDA levels in their leaves decreased, indicating a lower level of oxidative stress. Overall, the results showed that <i>P. coronopus</i> and <i>P. crassifolia</i> are the most tolerant to salt stress, and <i>P. afra</i> is the most susceptible of the four species. <i>Plantago ovata</i> is also quite resistant, apparently by using specific mechanisms of tolerance that are more efficient than in the halophytes, such as a less pronounced inhibition of photosynthesis, the accumulation of higher levels of Cl<sup>-</sup> ions in the leaves, or the activation of K<sup>+</sup> uptake and transport to the aerial part under high salinity conditions.
Project description:In their natural habitats, different mechanisms may contribute to the tolerance of halophytes to high soil salinity and other abiotic stresses, but their relative contribution and ecological relevance, for a given species, remain largely unknown. We studied the responses to changing environmental conditions of five halophytes (Sarcocornia fruticosa, Inula crithmoides, Plantago crassifolia, Juncus maritimus and J. acutus) in a Mediterranean salt marsh, from summer 2009 to autumn 2010. A principal component analysis was used to correlate soil and climatic data with changes in the plants' contents of chemical markers associated with stress responses: ions, osmolytes, malondialdehyde (MDA, a marker of oxidative stress) and antioxidant systems. Stress tolerance in S. fruticosa, I. crithmoides and P. crassifolia (all succulent dicots) seemed to depend mostly on the transport of ions to aerial parts and the biosynthesis of specific osmolytes, whereas both Juncus species (monocots) were able to avoid accumulation of toxic ions, maintaining relatively high K(+)/Na(+) ratios. For the most salt-tolerant taxa (S. fruticosa and I. crithmoides), seasonal variations of Na(+), Cl(-), K(+) and glycine betaine, their major osmolyte, did not correlate with environmental parameters associated with salt or water stress, suggesting that their tolerance mechanisms are constitutive and relatively independent of external conditions, although they could be mediated by changes in the subcellular compartmentalization of ions and compatible osmolytes. Proline levels were too low in all the species to possibly have any effect on osmotic adjustment. However-except for P. crassifolia-proline may play a role in stress tolerance based on its 'osmoprotectant' functions. No correlation was observed between the degree of environmental stress and the levels of MDA or enzymatic and non-enzymatic antioxidants, indicating that the investigated halophytes are not subjected to oxidative stress under natural conditions and do not, therefore, need to activate antioxidant defence mechanisms.
Project description:The halophytes have evolved several strategies to survive in saline environments; however, an additional support from their associated microbiota helps combat adverse conditions. Hence, our driving interests to investigate the endophytic bacterial community richness, diversity, and composition associated to roots of Salicornia europaea from two test sites with different origins of soil salinity. We assumed that salinity will have a negative effect on the diversity of endophytes but simultaneously will permit the high occurrence of halophylic bacteria. Further, to establish the role of the host and its external environment in determining the endophytic diversity, we analyzed the physico-chemical parameters of root zone soil and the concentration of salt ions in the plant roots. The results based on the Miseq Illumina sequencing approach revealed a higher number of endophytic bacterial OTUs at naturally saline test site with a higher level of soil salinity. Proteobacteria and Bacteriodetes were the dominant endophytic phyla at both analyzed sites; additionally, the high occurrence of Planctomycetes and Acidobacteria at more saline site and the occurrence of Firmicutes, Actinobacteria, and Chloroflexi at less saline site were recorded. The salinity in the root zone soil was crucial in structuring the endophytic community of S. europaea, and the significant prevalence of representatives from the phyla Deltaproteobacteria, Acidobacteria, Caldithrix, Fibrobacteres, and Verrucomicrobia at the more saline test site suggest domination of halophylic bacteria with potential role in mitigation of salt stress of halophytes.
Project description:In the face of climate change, water deficit and increasing soil salinity pose an even greater challenge to olive cultivation in the Mediterranean basin. Due to its tolerance to abiotic stresses, wild olive (<i>Olea europaea</i> subsp. <i>europaea</i> var. <i>sylvestris</i>) presents a good candidate in breeding climate-resilient olive varieties. In this study, the early response of the native Croatian wild olive genotype (WOG) to salinity was evaluated and compared with that of well-known cultivars (cv.) Leccino and Koroneiki. Potted olive plants were exposed either to 150 mM NaCl or 300 mM mannitol for 3 weeks to distinguish between the osmotic and ionic components of salt stress. To determine the impact of the plant age on salinity, 1-, 2-, and 3-year-old WOG plants were used in the study. The growth parameters of both the cultivars and WOG of different ages decreased in response to the mannitol treatment. In contrast to cv. Leccino, the NaCl treatment did not significantly affect the growth of cv. Koroneiki or WOG of any age. The contents of Na<sup>+</sup> and Cl<sup>-</sup> were considerably higher in the salt-treated WOG, regardless of age, compared with the cultivars. However, while both treatments significantly reduced the K<sup>+</sup> content of cv. Koroneiki, that nutrient was not significantly affected in either cv. Leccino or WOG. Unlike the cultivars and older WOG, the NaCl treatment caused a significant decline of photosynthetic pigments in the 1-year-old WOG. The cultivars and WOG of different ages experienced a similar drop in the chlorophyll <i>a</i> content under the isotonic mannitol treatment. The absence of lipid peroxidation, modulation of superoxide dismutase, and guaiacol peroxidase activity were noted in all WOG ages under both stressors. These data suggest that WOG resilience to salinity is associated with its large leaf capacity for Na<sup>+</sup> and Cl<sup>-</sup> accumulation, K<sup>+</sup> retention, and its adaptable antioxidative mechanisms. The results are promising with regard to obtaining a new olive cultivar with better resilience to soil salinity.
Project description:Salinization of agricultural land is a devastating phenomenon which will affect future food security. Understanding how plants survive and thrive in response to salinity is therefore critical to potentiate tolerance traits in crop species. The halophyte Salicornia europaea has been used as model system for this purpose. High salinity causes NH4+ accumulation in plant tissues and consequent toxicity symptoms that may further exacerbate those caused by NaCl. In this experiment we exposed Salicornia plants to five concentrations of NaCl (0, 1, 10, 50 and 200 mM) in combination with two concentrations of NH4Cl (1 and 50 mM). We confirmed the euhalophytic behavior of Salicornia that grew better at 200 vs. 0 mM NaCl in terms of both fresh (+34%) and dry (+46%) weights. Addition of 50 mM NH4Cl to the growth medium caused a general growth reduction, which was likely caused by NH4+ accumulation and toxicity in roots and shoots. When plants were exposed to high NH4Cl, high salinity reduced roots NH4+ concentration (-50%) compared to 0 mM NaCl. This correlates with the activation of the NH4+ assimilation enzymes, glutamine synthetase and glutamate dehydrogenase, and the growth inhibition was partially recovered. We argue that NH4+ detoxification is an important trait under high salinity that may differentiate halophytes from glycophytes and we present a possible model for NH4+ detoxification in response to salinity.