Ecophysiological and Biochemical Responses Depicting Seed Tolerance to Osmotic Stresses in Annual and Perennial Species of Halopeplis in a Frame of Global Warming.

Plant abundance and distribution are regulated by subtle changes in ecological factors, which are becoming more frequent under global climate change. Species with a higher tolerance to such changes, especially during early lifecycle stages, are highly likely to endure climate change. This study compared the germination adaptability of Halopeplis amplexicaulis and H. perfoliata, which differ in life-form and grow in different environments. Optimal conditions, tolerances and the biochemical responses of seeds to osmotic stresses were examined. Seeds of H. perfoliata germinated in a wider range of temperature regimes and were more tolerant to osmotic stresses than H. amplexicaulis seeds. Neither NaCl nor PEG treatment invoked the H2O2 content in germinating seeds of the tested species. Consequently, unaltered, or even decreased activities of H2O2 detoxification enzymes and non-enzymatic antioxidants were observed in germinating seeds in response to the aforementioned stresses. High and comparable levels of recovery from isotonic treatments, alongside a lack of substantial oxidative damage indicated that the osmotic stress, rather than the ionic toxicity, may be responsible for the germination inhibition. Hence, rainy periods, linked to water availability, may act as a key determinant for germination and H. perfoliata could be less affected by global warming owing to better germinability under high temperatures compared with H. amplexicaulis. Such studies involving biochemical analysis coupled with the germination ecology of congeneric species, which differ in life-form and occurrence are scarce, therefore are important in understanding the impacts of global changes on species abundance/distribution.

Combined Transcriptome and Proteome Analysis to Elucidate Salt Tolerance Strategies of the Halophyte Panicum antidotale Retz.

Panicum antidotale, a C4 monocot, has the potential to reclaim saline and drylands and to be utilized as fodder and forage. Its adaptability to survive saline stress has been proven with eco-physiological and biochemical studies. However, little is known about its molecular mechanisms of salt tolerance. In this study, an integrated transcriptome and proteome analysis approach, based on RNA sequencing and liquid chromatography tandem mass spectrometry (LC-MS/MS), was used to identify the said mechanisms. Plants were treated with control (0 mM), low (100 mM), and high (300 mM) sodium chloride (NaCl) treatments to distinguish beneficial and toxic pathways influencing plant biomass. The results indicated differential expression of 3,179 (1,126 upregulated/2,053 downregulated) and 2,172 (898 upregulated/1,274 downregulated) genes (DEGs), and 514 (269 upregulated/245 downregulated) and 836 (494 upregulated/392 downregulated) proteins (DEPs) at 100 and 300 mM NaCl, respectively. Among these, most upregulated genes and proteins were involved in salt resistance strategies such as proline biosynthesis, the antioxidant defense system, ion homeostasis, and sugar accumulation at low salinity levels. On the other hand, the expression of several genes and proteins involved in the respiratory process were downregulated, indicating the inability of plants to meet their energy demands at high salinity levels. Moreover, the impairments in photosynthesis were also evident with the reduced expression of genes regulating the structure of photosystems and increased expression of abscisic acid (ABA) mediated pathways which limits stomatal gas exchange. Similarly, the disturbance in fatty acid metabolism and activation of essential ion transport blockers damaged the integrity of the cell membrane, which was also evident with enhanced malondialdehyde (MDA). Overall, the analysis of pathways revealed that the plant optimal performance at low salinity was related to enhanced metabolism, antioxidative defense, cell growth, and signaling pathways, whereas high salinity inhibited biomass accumulation by altered expression of numerous genes involved in carbon metabolism, signaling, transcription, and translation. The data provided the first global analysis of the mechanisms imparting salt stress tolerance of any halophyte at transcriptome and proteome levels.

Effects of Salinity Stress on Chloroplast Structure and Function.

Salinity is a growing problem affecting soils and agriculture in many parts of the world. The presence of salt in plant cells disrupts many basic metabolic processes, contributing to severe negative effects on plant development and growth. This review focuses on the effects of salinity on chloroplasts, including the structures and function of these organelles. Chloroplasts house various important biochemical reactions, including photosynthesis, most of which are considered essential for plant survival. Salinity can affect these reactions in a number of ways, for example, by changing the chloroplast size, number, lamellar organization, lipid and starch accumulation, and interfering with cross-membrane transportation. Research has shown that maintenance of the normal chloroplast physiology is necessary for the survival of the entire plant. Many plant species have evolved different mechanisms to withstand the harmful effects of salt-induced toxicity on their chloroplasts and its machinery. The differences depend on the plant species and growth stage and can be quite different between salt-sensitive (glycophyte) and salt-tolerant (halophyte) plants. Salt stress tolerance is a complex trait, and many aspects of salt tolerance in plants are not entirely clear yet. In this review, we discuss the different mechanisms of salt stress tolerance in plants with a special focus on chloroplast structure and its functions, including the underlying differences between glycophytes and halophytes.

Increasing salinity leads to differential growth and H2O2 homeostasis in plants produced from heteromorphic seeds of the succulent halophyte Arthrocnemum indicum.

Information about responses of plants grown from heteromorphic seeds is limited and inconclusive. This is especially true of subtropical halophytes where such studies have yet to be published. Therefore, growth, water-relations, and oxidative stress mitigation of plants germinated from the heteromorphic seeds of the succulent halophyte Arthrocnemum indicum under increasing (0, 300, and 900 mM NaCl) salinity were studied. Growth of plants from only small seeds was stimulated in moderate (300 mM NaCl) salinity. High (900 mM NaCl) salinity inhibited the growth of plants emerged from both small and large sized seeds. Plants germinating from both seed sizes demonstrated similar patterns of osmotic adjustment and did not develop signs of oxidative damage under increasing salinity. However, the magnitude of hydrogen peroxide and antioxidant responses differed between plant types. Under moderate salinity, plants from small seeds showed constitutive activities of most antioxidant enzymes (except superoxide dismutase) and levels of non-enzymatic antioxidants (except ascorbate). Conversely, a decline in activities of most antioxidant enzymes and levels of most non-enzymatic antioxidants occurred in plants from large seeds. While under high salinity, increased ascorbate peroxidase, glutathione, and polyphenol levels, along with unaffected ascorbate and superoxide dismutase levels, occurred in plants from small seeds. In plants from large seeds, there were increased ascorbate and polyphenol levels, but changes to the ascorbate peroxidase levels were not observed. These results thus indicate differential growth and hydrogen peroxide homeostasis in A. indicum plants emerged from heteromorphic seeds.

Salinity induced alterations in photosynthetic and oxidative regulation are ameliorated as a function of salt secretion.

Ion secretion facilitates recretohalophytes to tolerate saline and drought conditions but its relative contribution to the survival of many species remains poorly understood. Tamarix chinensis has high potential for restoration of saline deteriorated lands. The water management and high salt tolerance of the plant have highlighted the need to determine the strategies that govern these mechanisms. Here we report the selectivity of this halophyte to transport, utilize, and secrete different cations and anions under various NaCl (0, 100, 200 and 400 mM) concentrations. Plant growth, photosynthesis and antioxidant defense responses were also determined to relate them with the function of ion secretion. Results reflected two different sets of strategies adopted by plants to survive low and high salinities. Exposure to highly saline conditions caused reduction in photosynthesis due to stomatal and biochemical limitations. The decreased content of photosynthetic pigments exposed plants to excessive light energy that accelerated production of ROS (i.e., hydrogen peroxide H2O2) and caused damage to cellular membranes. The increased activities of anti-oxidative enzymes (superoxide-dismutase, catalase, ascorbate-peroxidase, and glutathione-reductase) were insufficient to detoxify H2O2. In contrast, plants treated with low salinity did not face stomatal limitations while the photosynthetic pigments increased. As no damage to membranes was detected, the increased content of H2O2 was postulated for its messenger role. The assimilation of essential nutrients was affected due to increased content of toxic ions (Na+ and Cl-) in the growing medium and within the plants. However, the ability to regulate K+ facilitated plants to improve water use efficiency under hyper-osmotic environment. The removal of toxic ions from the photosynthesizing tissues demands high energy, which was evident in the compromised growth of plants. This study offers a window to physiological mechanisms, e.g., potassium retention that ensure salt secretion as a beneficial strategy for prolonged survival of T. chinensis.

RP-HPLC-based phytochemical analysis and diverse pharmacological evaluation of Quercus floribunda Lindl. ex A. Camus nuts extracts.

Quercus floribunda Lindl. ex A. Camus nuts have important folklore uses, assessed for underexplored biological potential. Nuts galls or cores and coats were utilized for the preparation of extracts using 14 solvent systems. Antioxidant, antimicrobial, cytotoxic and enzyme inhibition assays along with phytochemical profiling was carried out. Distilled water cores extract demonstrated maximum percent yield, phenolics content and total antioxidant capacity. Methanol: ethyl acetate cores extract showed maximum flavonoids content, total reducing power and protein kinase inhibition. Highest percentage radical scavenging and brine shrimp lethality was revealed by acetone: distilled water cores extract. Ethyl acetate cores extract indicated maximum α-amylase inhibition. Methanol: water coats extract showed substantial leishmanial growth inhibition. n-Hexane and chloroform coats extracts showed maximum cytotoxicity against HepG2 and THP-1 cell lines, respectively. Polyphenols quantified through RP-HPLC analysis were quercetin, pyrocatechol, gallic acid, catechin and chlorogenic acid ranging from 0.003 ± 0.001 to 1.785 ± 0.5 µg/mg extract.

Low Salinity Improves Photosynthetic Performance in Panicum antidotale Under Drought Stress.

Salinity and drought are two often simultaneously occurring abiotic stresses that limit the production of food crops worldwide. This study aimed to distinguish between the separate and combined impacts of drought and salinity on the plant response. Panicum antidotale was cultivated in a greenhouse under the following growth conditions: control, 100 mM NaCl (100) and 300 mM NaCl (300) salinity, drought (D; 30% irrigation), and two combinations of salinity and drought (100 + D and 300 + D). The growth response was as follows: 0 ≈ 100 > 100 + D > > D ≈ 300 ≈ 300 + D. Growth correlated directly with photosynthesis. The net photosynthesis, stomatal conductance, intercellular CO2, transpiration, ribulose 1,5-bisphosphate carboxylase (Rubisco), ribulose 1,5-bisphosphate (RuBP) regeneration, and triose phosphate utilization protein (e.g., phosphoenolpyruvate carboxylase) were highest in the control and declined most at 300 + D, while 100 + D performed significantly better as compared to drought. Maximum and actual photosystem II (PSII) efficiencies, along with photochemical quenching during light harvesting, resemble the plant growth and contemporary CO2/H2O gas exchange parameters in the given treatments. Plant improves water use efficiency under salt and drought treatments, which reflects the high water conservation ability of Panicum. Our findings indicate that the combination of low salinity with drought was able to minimize the deleterious effects of drought alone on growth, chlorophyll content, cell integrity, photosynthesis, leaf water potential, and water deficit. This synergetic effect demonstrates the positive role of Na+ and Cl- in carbon assimilation and osmotic adjustment. In contrast, the combination of high salinity and drought enforced the negative response of plants in comparison to single stress, demonstrating the antagonistic impact of water availability and ion toxicity.

Salt induced modulations in antioxidative defense system of Desmostachya bipinnata.

This study addressed the interactions between salt stress and the antioxidant responses of a halophytic grass, Desmostachya bipinnata. Plants were grown in a semi-hydroponic system and treated with different NaCl concentrations (0 mM, 100 mM, 400 mM) for a month. ROS degradation enzyme activities were stimulated by addition of NaCl. Synthesis of antioxidant compounds, such as phenols, was enhanced in the presence of NaCl leading to accumulation of these compounds under moderate salinity. However, when the ROS production rate exceeded the capacity of enzyme-controlled degradation, antioxidant compounds were consumed and oxidative damage was indicated by significant levels of hydrogen peroxide at high salinity. The cellular concentration of salicylic acid increased upon salt stress, but since no direct interaction with ROS was detected, a messenger function may be postulated. High salinity treatment caused a significant decrease of plant growth parameters, whereas treatment with moderate salinity resulted in optimal growth. The activity and abundance of superoxide dismutase (SOD) increased with salinity, but the abundance of SOD isoforms was differentially affected, depending on the NaCl concentration applied. Detoxification of hydrogen peroxide (H2O2) was executed by catalase and guaiacol peroxidase at moderate salinity, whereas the enzymes detoxifying H2O2 through the ascorbate/glutathione cycle dominated at high salinity. The redox status of glutathione was impaired at moderate salinity, whereas the levels of both ascorbate and glutathione significantly decreased only at high salinity. Apparently, the maximal activation of enzyme-controlled ROS degradation was insufficient in comparison to the ROS production at high salinity. As a result, ROS-induced damage could not be prevented, if the applied stress exceeded a critical value in D. bipinnata plants.

Identification and evolutionary characterization of salt-responsive transcription factors in the succulent halophyte Suaeda fruticosa.

Transcription factors are key regulatory elements that affect gene expression in response to specific signals, including environmental stresses such as salinity. Halophytes are specialized plants that have the ability to complete their life cycle in saline environments. In this study we have identified and characterized the evolutionary relationships of putative transcription factors (TF) in an obligate succulent halophyte, Suaeda fruticosa, that are involved in conferring salt tolerance. Using RNA-seq data we have analyzed the expression patterns of certain TF families, predicted protein-protein interactions, and analyzed evolutionary trajectories to elucidate their possible roles in salt tolerance. We have detected the top differentially expressed (DE) transcription factor families (MYB, CAMTA, MADS-box and bZIP) that show the most pronounced response to salinity. The majority of DE genes in the four aforementioned TF families cluster together on TF phylogenetic trees, which suggests common evolutionary origins and trajectories. This research represents the first comprehensive TF study of a leaf succulent halophyte including their evolutionary relationships with TFs in other halophyte and salt-senstive plants. These findings provide a foundation for understanding the function of salt-responsive transcription factors in salt tolerance and associated gene regulation in plants.

Heteromorphic seeds of coastal halophytes Arthrocnemum macrostachyum and A. indicum display differential patterns of hydrogen peroxide accumulation, lipid peroxidation and antioxidant activities under increasing salinity.

Reactive oxygen species homeostasis during germination of heteromorphic seeds is not fully understood. This study elucidates changes in levels of hydrogen peroxide (H2O2), malondialdehyde (MDA) and enzymatic antioxidants in heteromorphic seeds of contrasting congeneric halophytes Arthrocnemum macrostachyum (C3 perennial) and A. indicum (C4 perennial) during germination under increasing salinity. There was no dormancy in A. macrostachyum (black and brown) and A. indicum (large and small) seeds. Seeds of A. macrostachyum displayed greater salinity tolerance compared to A. indicum seeds. Under non-saline conditions, large A. indicum seeds and brown A. macrostachyum seeds showed slightly higher germination than their respective counterparts. H2O2 content of black compared to brown A. macrostachyum seeds increased with salinity and that of small compared to large A. indicum seeds increased only in 400 mM NaCl. High catalase and ascorbate peroxidase with constitutive superoxide dismutase levels coincided with unaltered MDA in black A. macrostachyum seeds under salinity. Whereas, there was a decline in most antioxidant enzyme activities alongside low/unchanged H2O2 in the brown A. macrostachyum seeds under salinity. Unaltered H2O2 and MDA with low/unchanged antioxidant enzyme activities in large A. indicum seeds under salinity occurred. Unchanged enzyme activities alongside a rise in H2O2 and MDA levels were observed in the small A. indicum seeds under salinity. These data hence highlight differential H2O2 homeostasis strategies in the heteromorphic seeds of the test species.

Studies on antioxidant, hepatoprotective, and vasculoprotective potential of Viola odorata and Wrightia tinctoria.

We have reported the antidyslipidemic, antihypertensive, and Ca++ channel blocking activities of Viola odorata (VO) and Wrightia tinctoria (WT). This study extends our understanding of their therapeutic potential by exploring the effects on biomarkers of hepatic and vascular dysfunction together with phytochemical standardization and antioxidant potential. Total phenolic compounds, total flavonoids content, and proanthocyanins of the methanolic extracts were identified using HPLC. Antioxidant capacity was measured using the in vitro assays. Two studies of 6-week duration were conducted on a high-fat diet rat model to test the leaves and seed extracts of VO and WT (300 and 600 mg/kg) for their effect on biomarkers for hepatic and vascular dysfunction. The HPLC analysis showed high contents of total phenolic compounds, total flavonoids content, and proanthocyanins along with distinctive phenolic composition. Both extracts exhibited significant antioxidant potential in 1,1-diphenyl-2-picrylhydrazyl, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), fluorescence recovery after photobleaching, and total antioxidant capacity (TAC) assays, comparable with synthetic standard antioxidants. The in vivo studies indicated a significant reduction in the high-fat-diet-induced rise in serum uric acid, phosphorus, aspartate aminotransferase, alanine aminotransferase, and gamma-glutamyl transferase. This study indicates the potential of VO and WT to protect from vascular and hepatic damage and an antioxidant effect, thus making these herbs strong candidates for managing cardiometabolic disorders.

Differential protein expression reveals salt tolerance mechanisms of Desmostachya bipinnata at moderate and high levels of salinity.

A proteomics approach was used to investigate salt tolerance mechanisms of Desmostachya bipinnata (L.) Stapf. Plants were subjected to 0mM (control), 100mM (moderate) and 400mM (high) NaCl. Proteins were separated by two-dimensional gel electrophoresis and identified with available databases. Optimal plant fresh weight was found at moderate salinity but declined at high salinity. Water potential, osmotic potential, Na+/K+ ratio, leaf electrolyte leakage, sugars and proline were altered at high salinity. However, water potential, proline content and electrolyte leakage were maintained at moderate salinity; Na+ and K+ concentrations increased, whereas sugars and osmotic potential decreased. Comparative proteome analysis revealed 103 salt responsive proteins. At moderate salinity, most of the proteins involved in energy metabolism, transport, antioxidative defence and cell growth were either unchanged or increased. Proteins related to amino-acid metabolism were decreased while those associated with secondary metabolism were accumulated. At high salinity, amino-acid metabolism and dehydration responses were evident; proteins of energy metabolism, transport and stress defence were downregulated. These results suggest that an efficient defence system, improved transport of water and metabolites, increased cell wall lignification and regulation of energy and carbohydrate metabolism allowed better potential for plant growth under moderately saline conditions.

Polarity based characterization of biologically active extracts of Ajuga bracteosa Wall. ex Benth. and RP-HPLC analysis.

BACKGROUND: The concept of botanical therapeutics has revitalized due to wide importance of plant derived pharmaceuticals. Therefore, the ameliorative characteristics of Ajuga bracteosa were studied. METHODS: Total phenolic content, flavonoid content, antioxidant capacity, reducing power and free-radical scavenging activity were determined colorimetrically. Specific polyphenols were quantified by RP-HPLC analysis. Preliminary cytotoxicity was tested using brine shrimp lethality assay while antiproliferative activity against THP-1 and Hep-G2 cell lines was determined by MTT and SRB protocols respectively. Antileishmanial potential was assessed via MTT colorimetric method. To investigate antidiabetic prospect, α-amylase inhibition assay was adopted whereas disc diffusion method was used to detect likely protein kinase inhibitory, antibacterial and antifungal activities. RESULTS: Among fifteen different extracts, maximum total phenolic content (10.75 ± 0.70 µg GAE/mg DW), total reducing power (23.90 ± 0.70 µg AAE/mg DW) and total antioxidant capacity (11.30 ± 0.80 µg AAE/mg DW) were exhibited by methanol extract with superlative percent extract recovery (17.50 ± 0.80% w/w). Chloroform-methanol extract demonstrated maximum flavonoid content (4.10 ± 0.40 µg QE/mg DW) and ethanol extract exhibited greatest radical scavenging activity (IC50 14.40 ± 0.20 µg/ml). RP-HPLC based quantification confirmed polyphenols such as pyrocatechol, gallic acid, resorcinol, catechin, chlorogenic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, vanillic acid, coumarin, sinapinic acid, trans-cinnamic acid, rutin, quercetin and kaempferol. The brine shrimp lethality assay ranked 78.60% extracts as cytotoxic (LC50 ≤ 250 µg/ml) whereas significant THP-1 inhibition was shown by methanol-acetone extract (IC50 4.70 ± 0.43 µg/ml). The antiproliferative activity against Hep-G2 hepatoma cancer cell line was demonstrated by n-hexane, ethylacetate and methanol-distilled water (IC50 8.65-8.95 µg/ml) extracts. Methanol extract displayed prominent protein kinase inhibitory activity (MIC 12.5 µg/disc) while n-hexane extract revealed remarkable antileishmanial activity (IC50 4.69 ± 0.01 µg/ml). The antidiabetic potential was confirmed by n-hexane extract (44.70 ± 0.30% α-amylase inhibition at 200 µg/ml concentration) while a moderate antibacterial and antifungal activities were unveiled. CONCLUSION: The variation in biological spectrum resulted due to use of multiple solvent systems for extraction. We also deduce that the valuable information gathered can be utilized for discovery of anticancer, antileishmanial, antioxidant and antidiabetic bioactive lead candidates.

Polarity directed optimization of phytochemical and in vitro biological potential of an indigenous folklore: Quercus dilatata Lindl. ex Royle.

BACKGROUND: Plants have served either as a natural templates for the development of new chemicals or a phytomedicine since antiquity. Therefore, the present study was aimed to appraise the polarity directed antioxidant, cytotoxic, protein kinase inhibitory, antileishmanial and glucose modulatory attributes of a Himalayan medicinal plant- Quercus dilatata. METHODS: Total phenolic and flavonoid contents were determined colorimetrically and various polyphenols were identified by RP-HPLC analysis. Brine shrimp lethality, SRB and MTT assays were employed to test cytotoxicity against Artemia salina and human cancer cell lines respectively. Antileishmanial activity was determined using standard MTT protocol. Glucose modulation was assessed by α-amylase inhibition assay while disc diffusion assay was used to establish protein kinase inhibitory and antifungal spectrum. RESULTS: Among 14 extracts of aerial parts, distilled water-acetone extract demonstrated maximum extract recovery (10.52% w/w), phenolic content (21.37 ± 0.21 µg GAE/mg dry weight (DW)), total antioxidant capacity (4.81 ± 0.98 µg AAE/mg DW) and reducing power potential (20.03 ± 2.4 µg/mg DW). On the other hand, Distilled water extract proficiently extracted flavonoid content (4.78 ± 0.51 µg QE/mg DW). RP-HPLC analysis revealed the presence of significant amounts of phenolic metabolites (0.049 to 15.336 µg/mg extract) including, pyrocatechol, gallic acid, catechin, chlorogenic acid, p-coumaric acid, ferulic acid and quercetin. Highest free radical scavenging capacity was found in Methanol-Ethyl acetate extract (IC50 8.1 ± 0.5 µg/ml). In the brine shrimp toxicity assay, most of the tested extracts (57%) showed high cytotoxicity. Among these, Chloroform-Methanol extract had highest cytotoxicity against THP-1 cell line (IC50 3.88 ± 0.53 µg/ml). About 50% of the extracts were found to be moderately antiproliferative against Hep G2 cell line. Methanol extract exhibited considerable protein kinase inhibitory activity against Streptomyces 85E strain (28 ± 0.35 mm bald phenotype at 100 µg/disc; MIC = 12.5 µg/ disc) while, Chloroform extract displayed maximum antidiabetic activity (α-amylase inhibition of 21.61 ± 1.53% at 200 µg/ml concentration). The highest antileishmanial potential was found in Ethyl acetate-Acetone extract (12.91 ± 0.02% at 100 µg/ml concentration), while, Q. dilatata extracts also showed a moderate antifungal activity. CONCLUSION: This study proposes that multiple-solvent system is a crucial variable to elucidate pharmacological potential of Q. dilatata and the results of the present findings prospects its potential as a resource for the discovery of novel anticancer, antidiabetic, antileishmanial and antioxidant agents.

Differences in photosynthetic syndromes of four halophytic marsh grasses in Pakistan.

Salt-tolerant grasses of warm sub-tropical ecosystems differ in their distribution patterns with respect to salinity and moisture regimes. Experiments were conducted on CO2 fixation and light harvesting processes of four halophytic C4 grasses grown under different levels of salinity (0, 200 and 400 mM NaCl) under ambient environmental conditions. Two species were from a high saline coastal marsh (Aeluropus lagopoides and Sporobolus tremulus) and two were from a moderate saline sub-coastal draw-down tidal marsh (Paspalum paspalodes and Paspalidium geminatum). Analyses of the carbon isotope ratios of leaf biomass in plants indicated that carbon assimilation was occurring by C4 photosynthesis in all species during growth under varying levels of salinity. In the coastal species, with increasing salinity, there was a parallel decrease in rates of CO2 fixation (A), transpiration (E) and stomatal conductance (g s), with no effect on water use efficiency (WUE). These species were adapted for photoprotection by an increase in the Mehler reaction with an increase in activity of PSII/CO2 fixed accompanied by high levels of antioxidant enzymes, superoxide dismutase and ascorbate peroxidase. The sub-coastal species P. paspalodes and P. geminatum had high levels of carotenoid pigments and non-photochemical quenching by the xanthophyll cycle.

Transcriptome assembly, profiling and differential gene expression analysis of the halophyte Suaeda fruticosa provides insights into salt tolerance.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Effects of salinity and ascorbic acid on growth, water status and antioxidant system in a perennial halophyte.

Salinity causes oxidative stress in plants by enhancing production of reactive oxygen species, so that an efficient antioxidant system, of which ascorbic acid (AsA) is a key component, is an essential requirement of tolerance. However, antioxidant responses of plants to salinity vary considerably among species. Limonium stocksii is a sub-tropical halophyte found in the coastal marshes from Gujarat (India) to Karachi (Pakistan) but little information exists on its salt resistance. In order to investigate the role of AsA in tolerance, 2-month-old plants were treated with 0 (control), 300 (moderate) and 600 (high) mM NaCl for 30 days with or without exogenous application of AsA (20 mM) or distilled water. Shoot growth of unsprayed plants at moderate salinity was similar to that of controls while at high salinity growth was inhibited substantially. Sap osmolality, AsA concentrations and activities of AsA-dependant antioxidant enzymes increased with increasing salinity. Water spray resulted in some improvement in growth, indicating that the growth promotion by exogenous treatments could partly be attributed to water. However, exogenous application of AsA on plants grown under saline conditions improved growth and AsA dependent antioxidant enzymes more than the water control treatment. Our data show that AsA-dependent antioxidant enzymes play an important role in salinity tolerance of L. stocksii.

Excreting and non-excreting grasses exhibit different salt resistance strategies.

The combination of traits that makes a plant successful under saline conditions varies with the type of plant and its interaction with the environmental conditions. Knowledge about the contribution of these traits towards salt resistance in grasses has great potential for improving the salt resistance of conventional crops. We attempted to identify differential adaptive response patterns of salt-excreting versus non-excreting grasses. More specifically, we studied the growth, osmotic, ionic and nutrient (carbon/nitrogen) relations of two salt-excreting (Aeluropus lagopoides and Sporobolus tremulus) and two non-excreting (Paspalum paspalodes and Paspalidium geminatum) perennial C4 grasses under non-saline and saline (0, 200 and 400 mM NaCl) conditions. Growth and relative growth rate decreased under saline conditions in the order P. geminatum > S. tremulus = A. lagopoides > P. paspalodes. The root-to-shoot biomass allocation was unaffected in salt-excreting grasses, increased in P. paspalodes but decreased in P. geminatum. Salt-excreting grasses had a higher shoot/root Na(+) ratio than non-excreting grasses. K(+), Ca(2+) and Mg(2+) homoeostasis remained undisturbed among test grasses possibly through improved ion selectivity with rising substrate salinity. Salt-excreting grasses increased leaf succulence, decreased ψs and xylem pressure potential, and accumulated proline and glycinebetaine with increasing salinity. Higher salt resistance of P. paspalodes could be attributed to lower Na(+) uptake, higher nitrogen-use efficiency and higher water-use efficiency among the test species. However, P. geminatum was unable to cope with salt-induced physiological drought. More information is required to adequately document the differential strategies of salt resistance in salt-excreting and non-excreting grasses.

The influence of genes regulating transmembrane transport of Na+ on the salt resistance of Aeluropus lagopoides.

Plantlets of Aeluropus lagopoides (Linn.) Trin. Ex Thw. were grown at different NaCl concentrations (26, 167, 373 and 747mM) for 3, 7 and 15 days; their growth, osmotic adjustment, gas exchange, ion compartmentalisation and expression of various genes related to Na+ flux was studied. Plantlets showed optimal growth in non-saline (control; 26mM NaCl) solutions, whereas CO2/H2O gas exchange, leaf water concentration and water use efficiency decreased under all salinity treatments, accompanied by increased leaf senescence, root ash, sodium content and leaf osmolality. A decrease in malondialdehyde (MDA) content with time was correlated with Na+ accumulation in the leaf apoplast and a concomitant increase in Na+ secretion rate. A. lagopoides accumulated a higher concentration of Na+ in root than in leaf vacuoles, corresponding with higher expression of V-NHX and lower expression of PM-NHX in root than leaf tissue. It appears that V-ATPase plays a vital role during Na+ transport by producing an electromotive force, driving ion transport. Leaf calcium increased with increasing salinity, with more rapid accumulation at high salinity than at low salinity, indicating a possible involvement of Ca2+ in maintaining K+:Na+ ratio. Our results suggest that A. lagopoides successfully compartmentalised Na+ at salinities up to 373mM NaCl by upregulating the gene expression of membrane linked transport proteins (V-NHX and PM-NHX). At higher salinity (747mM NaCl), a reduction in the expression of V-NHX and PM-NHX in leaves without any change in the rate of salt secretion, is a possible cause of the toxicity of NaCl.

Dormancy and germination responses of halophyte seeds to the application of ethylene.

Ethylene is invariably produced during seed germination but its role in regulating seed dormancy and germination is poorly understood. Seeds of 22 halophytic species having different life forms - salt secreting dicots, salt secreting monocots, stem succulents and leaf succulents were germinated in Petri dishes kept in a growth chamber set at 20/30 degrees C (night/day) temperature and a 12 hr light period. Sodium chloride and ethephon were added to the medium from the beginning of the experiment. Seed germination was recorded every other day for twenty days. Application of ethylene did not have any significant effect on releasing seeds from innate dormancy. However, it appeared to have a role in alleviation of salinity effects which varied from negative in certain species to almost complete alleviation of high salinity effects in others. Our data indicates that ethylene appears to have little role in breaking innate seed dormancy however, in most halophyte seeds studied, application of ethylene alleviate the salinity effect to various degrees. Halophyte seeds which could germinate under saline conditions approaching twice the salinity of seawater may offer clues to understand management of seed germination under highly saline conditions.