Crosstalk between melatonin and nitric oxide restrains Cadmium-induced oxidative stress and enhances vinblastine biosynthesis in Catharanthus roseus (L) G Don.

KEY MESSAGE: Nitric oxide functions downstream of the melatonin in adjusting Cd-induced osmotic and oxidative stresses, upregulating the transcription of D4H and DAT genes, and increasing total alkaloid and vincristine contents. A few studies have investigated the relationship between melatonin (MT) and nitric oxide (NO) in regulating defensive responses. However, it is still unclear how MT and NO interact to regulate the biosynthesis of alkaloids and vincristine in leaves of Catharanthus roseus (L.) G. Don under Cd stress. Therefore, this context was explored in the present study. Results showed that Cd toxicity (200 µM) induced oxidative stress, decreased biomass, Chl a, and Chl b content, and increased the content of total alkaloid and vinblastine in the leaves. Application of both MT (100 µM) and sodium nitroprusside (200 µM SNP, as NO donor) enhanced endogenous NO content and accordingly increased metal tolerance index, the content of total alkaloid and vinblastine. It also upregulated the transcription of two respective genes (D4H and DAT) under non-stress and Cd stress conditions. Moreover, the MT and SNP treatments reduced the content of H2O2 and malondialdehyde, increased the activities of superoxide dismutase and ascorbate peroxidase, enhanced proline accumulation, and improved relative water content in leaves of Cd-exposed plants. The scavenging NO by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy l-3-oxide (cPTIO) averted the effects of MT on the content of total alkaloid and vinblastine and antioxidative responses. Still, the effects conferred by NO on attributes mentioned above were not significantly impaired by p-chlorophenylalanine (p-CPA as an inhibitor of MT biosynthesis). These findings and multivariate analyses indicate that MT motivated terpenoid indole alkaloid biosynthesis and mitigated Cd-induced oxidative stress in the leaves of periwinkle in a NO-dependent manner.

Synergistic effects of vermicompost and mycorrhizal inoculation on arsenic tolerance and phytostabilization in safflower (Carthamus tinctorius L.).

Arsenic (As) is a hazardous metalloid, and mycorrhizal inoculation and vermicompost amendment can influence As bioremediation. However, the studies concerning the sole and joint effects of arbuscular mycorrhizal fungi (AMF) and vermicompost on the phytoremediation efficacy are limited. In the present study at first, the impact of various levels of vermicompost (0, 2, 4, and 8% w/w) was investigated on As mobility in soil and safflower (Carthamus tinctorius L.) plants grown in soils of spiked with 0, 40, and 80 mg kg-1 As. Results revealed that with increasing dose of vermicompost, bioavailable As in soil decreased which resulted in a lower bioaccumulation factor and translocation factor (TF) and led to a significant increase of tolerance index (TI) and total chlorophyll content in plants. The highest effect on TI and total As accumulation per plant was obtained in the dosage of 8% vermicompost. Therefore, in the second experiment, the sole and joint effects of 8% vermicompost and inoculation with Rhizophagus intraradices were assessed on the tolerance and accumulation of As in safflower. The addition of vermicompost aggravated mycorrhizal colonization but did not significantly influence mycorrhizal dependency under As stress. The joint effects of AMF and vermicompost improved the dry weight of roots and shoots, increased P concentration and P:As ratio in shoots, reduced malondialdehyde content, and moderated ascorbate peroxidase activity in leaves of As-stressed plants. Interestingly, co-application of AMF and vermicompost more than their sole usage decreased As concentration in shoots and TF and more strongly increased total As accumulation per plant. These findings suggest that mycorrhizal inoculation and vermicompost have a synergistic effect on As tolerance and phytostabilization efficacy of safflower plants, and their combined application may be a new option to remediate As-contaminated soils.

Interactive effect of silicon and nitric oxide effectively contracts copper toxicity in Salvia officinalis L.

Excess copper (Cu) causes the toxic effects in plants and health hazards to humans. Therefore, in this study, the effect of sodium silicate (1 mM Si) and sodium nitroprusside (200 µM SNP as a releasing NO), was assessed on Cu tolerance in Salvia officinalis L. plants exposed to 400 µM CuSO4. Results revealed that the combined supplementation with Si and SNP rather than the single application of these chemicals lowered Cu concentrations and translocation factor and increased Mg, Zn, and Fe concentrations in roots and shoots. Furthermore, combined treatment more efficiently decreased electrolyte leakage enhanced the activities of POD and APX in the leaves and roots, and improved relative water content and the content of Chl. a and Chl. b in leaves and consequently further increased tolerance index. Silicon supply enhanced NO content and applying Si + SNP more than the treatment of Si alone increased Si concentrations in the roots and shoots under Cu stress. Therefore, the reciprocal interaction of Si and NO might enhance Cu tolerance in plants, and the combined application of Si and SNP might be a promising strategy to decrease heavy metal accumulation in medicinal plants grown in polluted lands.

In most studies, co-precipitation of silicon and heavy metals in medium has been suggested as a reason for reducing heavy metal uptake in plants. In this study, the impact of Si on NO generation and the role of NO signaling in regulating Cu uptake and translocation and defensive responses were assessed to clarify another mechanism of Si in inducing Cu tolerance in sage. Furthermore, the combined application of Si and SNP has been indicated as an innovative strategy to enhance Cu tolerance and decrease heavy metal accumulation in medicinal plants grown in polluted lands.

He-Ne Laser Enhances Seed Germination and Salt Acclimation in Salvia officinalis Seedlings in a Manner Dependent on Phytochrome and H2O2.

In the current study the role of H2O2 in He-Ne laser-induced effects on seed germination and post-germinative performance of Salvia officinalis seedlings was assessed under both non-stress and saline conditions. Salinity had adverse impacts on seed germination and root length and decreased seed germination tolerance index. Seed priming with H2O2 and He-Ne laser impacted the seed germination and vigoration in a dose-dependent manner. The optimal effects were gathered by energy dose of 6 J/cm2 laser and concentration of 5 mM H2O2. These pre-treatments enhanced seed germination due to increasing contents of total soluble and reducing sugars and the amylase activity in seeds and improved seedling performance under saline and non-saline conditions. Furthermore, Phy B transcripts were upregulated, salt-accrued oxidative stress was mitigated, and the activities of POD and CAT increased in seedlings primed with H2O2 and laser. Interestingly, applying diphenyleneiodonium (DPI as an inhibitor of NADPH oxidase activity) and N, N-dimethyl thiourea (DMTU as a H2O2 scavenger) arrested the upregulation of phy B gene and abolished stimulatory impact of laser priming on the aforementioned attributes under both non-stress and saline conditions. These novel findings suggest that H2O2 as a downstream signal modulates the impacts of He-Ne laser on seed germination, seedling performance and salt acclimation in sage seedlings, and likely phy B also is involved in these responses.

Silicon and nitric oxide synergistically modulate the production of essential oil and rosmarinic acid in Salvia officinalis under Cu stress.

The individual impact of silicon (Si) and nitric oxide (NO) on secondary metabolism in several plant species has been reported, but their combined effect has never been evaluated yet. Therefore, in this study, single and combined impacts of NO and Si on the biosynthesis of rosmarinic acid (RA) and essential oil (EO) content in leaves of Salvia officinalis were investigated under both non-stress and Cu stress conditions. The results indicated that high Cu concentration decreased biomass and the content of polyphenols, but elevated electrolyte leakage, while lower Cu concentrations, especially 200 µM Cu, increased the content of polyphenols, EO, and antioxidant capacity in leaves of S. officinalis. The foliar application of sodium silicate (1 mM Si) and sodium nitroprusside (200 µM SNP as a NO donor) alone and particularly in combination improved shoot dry biomass, restored chlorophyll and carotenoids, increased EO content, the amounts of flavonoids, and phenolic compounds especially RA, and enhanced antioxidant capacity in the leaves of S. officinalis under both non-stress and Cu stress conditions. Copper treatment increased NO content, upregulated expression of PAL, TAT, and RAS genes, and enhanced phenylalanine ammonia-lyase activity in the leaves, which were responsible for improving the production of phenolic compounds, particularly rosmarinic acid. Foliar spraying with Si and SNP intensified these attributes. All responses were more pronounced when NO and Si were simultaneously applied under Cu stress. These findings suggest that NO and Si synergistically modulate secondary metabolism through upregulation of related gene expression and enzyme activities under both non-stress and Cu stress conditions.

Melatonin improves the photosynthesis in Dracocephalum kotschyi under salinity stress in a Ca2+/CaM-dependent manner.

This study investigated: (1) the effects of various concentrations of melatonin (MT) and Ca2+; and (2) the impact of crosstalk between these signal molecules on photosynthesis and salt tolerance of Dracocephalum kotschyi Boiss. Results indicated that 5mM CaCl2, as well as 100µM MT were the best concentrations for increasing shoot dry weight, leaf area, SPAD index, maximum quantum efficiency of photosystem II (Fv/Fm), and decreasing malondialdehyde content under salinity stress. The impact of MT on growth and photosynthesis was closely linked to its effect on enhancing antioxidant enzyme activities in leaves. Application of p-chlorophenylalanine, as an inhibitor of MT biosynthesis, negated the impacts of MT on the aforementioned attributes. Salinity and MT boosted cytosolic Ca2+ concentration. Exogenous MT, as well as Ca2+, enhanced tolerance index, membrane stability, leaf area, the content of chlorophyll (Chl) a, Chl b, and carotenoids (Car), Fv/Fm, and stomatal conductance under salinity stress. These impacts of MT were eliminated by applying a calmodulin antagonist, a Ca2+ chelator and a Ca2+ channel blocker. These novel findings indicate that the MT-induced effects on photosynthetic parameters and salt-evoked oxidative stress were mediated through calcium/calmodulin (Ca2+/CaM) signalling.

Silicon- induced nitric oxide burst modulates systemic defensive responses of Salvia officinalis under copper toxicity.

In this study, the role of nitric oxide (NO) burst in modulating Si-induced defensive responses in leaves and roots of Salvia officinalis under copper (Cu) stress were investigated. The result showed that 400 µM Cu markedly reduced shoot dry weight, but increased electrolyte leakage (EL) in leaves and both Si and sodium nitroprusside (SNP as the NO donor) improved these attributes in a dose-dependent manner. Interestingly, Cu toxicity systemically boosted a NO burst in both roots and shoots and applying Si and SNP markedly intensified it. The application of Si and SNP alone as well as their combination improved growth parameters and systemically alleviated Cu-induced lipid peroxidation and H2O2 accumulation through lowering Cu accumulation, increasing proline content, enhancing the activities of catalase (CAT) and superoxide dismutase (SOD) in both roots and leaves and up-regulating expression of SOD gene in leaves of S. officinalis. NO generation was substantially arrested and the responses induced by Si were significantly suppressed by pretreatment with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy l-3-oxide (cPTIO) as a NO scavenger, Nx-Nitro- L-arginine methyl ester hydrochloride (L-NAME) as a nitric oxide synthase inhibitor, and tungstate as a nitrate reductase inhibitor. These novel results indicate that Si can induce Cu tolerance through triggering NO generation which systemically modulates defensive reactions in both roots and leaves of Salvia officinalis.

Calcium signaling confers nickel tolerance in Cucurbita pepo L.

Excessive nickel (Ni) accumulation in edible parts of the plants has become a serious challenge for food security over the past few decades. Therefore, in this study, the role of calcium (Ca2+) signaling in imparting Ni tolerance was investigated in zucchini (Cucurbita pepo L. cv Courgette d'Italie). Exposure of zucchini seedlings to Hogland solution containing 0.28 mmol L-1 Ni(NO3)2 reduced plant growth, the content of chlorophyll and carotenoids and the relative water content (RWC) in leaves, increased Ni accumulation that was accompanied to depletion of the essential bivalent cations and induced oxidative stress and proline accumulation in both shoots and roots. Pretreatment with the nutrient solution containing 15 mmol L-1 calcium chloride (CaCl2), significantly improved zucchini growth and photosynthetic pigment contents and maintained RWC in leaves under both control and Ni stress conditions. Pretreatment with CaCl2 reduced Ni accumulation, modified cation homeostasis, increased the activities of peroxidase and catalase enzymes and lowered Ni-induced accumulation of hydrogen peroxide, malondialdehyde and proline in leaves and roots. Pre-exposure of root with Ca2+ chelator (ethylene glycol tetraacetic acid) and plasma membrane Ca2+ channel blocker (lanthanum chloride) impaired impact of Ca2+ on the aforementioned attributes. Outcomes of this study not only highlight the signaling role of Ca2+ in regulating defensive responses but also suggest an eco- friendly approach for reducing the Ni contamination in plants that ensure food safety.

Crosstalk between melatonin and Ca2+/CaM evokes systemic salt tolerance in Dracocephalum kotschyi.

In this study, the role of calcium/calmodulin (Ca2+/CaM) and melatonin (Mel) as two signal molecules in inducing systemic salt tolerance of Dracocephalum kotschyi Boiss. was investigated. Salinity stress (100 mM NaCl) reduced plant growth and induced ionic, osmotic, and oxidative damages in D. kotschyi leaves. Detection of cytosolic free Ca2+ ([Ca2+]cyt) by the Fura-2 method and the measurement of endogenous Mel by GC-MS demonstrated that salinity induced Ca2+ burst and increased endogenous Mel content in D. kotschyi leaves. Root pretreatment with 5 mM Ca2+ or 100 µM Mel recovered plant growth, reduced leaf electrolytic leakage, H2O2, and MDA contents and improved membrane integrity not only at the application site (roots), but also at the untreated distal parts (leaves) under salt stress. Rhizospheric treatment with Mel and Ca2+ triggered systemic tolerance in D. kotschyi, as judged from improving RWC, increasing proline content, modulating Na+, K+, and Ca2+ homeostasis, and enhancing the activities of SOD, CAT, APX, and POD in the leaves of salt-stressed plants. Mel augmented [Ca2+]cyt, but the rhizospheric application of Ca2+ antagonists impaired the latter responses. Furthermore, root pretreatment with Ca2+ increased Mel content, but the application of p-chlorophenylalanine (as an inhibitor of Mel biosynthesis) decreased the above attributes in the leaves of Ca2+-treated plants, leading to an arrest in the Ca2+-induced systemic salt tolerance. These novel results suggest that interaction of Ca2+/CaM and Mel is involved in overcoming salt-induced ionic, osmotic, and oxidative damages and Ca2+ and Mel may act as long-distance signals for inducing systemic salt tolerance in D. kotschyi.

Melatonin and calcium modulate the production of rosmarinic acid, luteolin, and apigenin in Dracocephalum kotschyi under salinity stress.

Melatonin (Mel) and calcium (Ca2+) have a regulatory role in the induction of specialized metabolites production and defensive responses against stresses. Therefore, in this study, the effects of Mel and Ca2+ and the possible relationship between them in the increase of the production of phenolic compounds in Dracocephalum kotschyi Boiss. under both control and salinity stress conditions were investigated. The results showed that 75 mM NaCl reduced shoot dry biomass but elevated H2O2 content, electrolyte leakage (EL) level, total phenolic and flavonoid contents (TPC and TFC), and DPPH scavenging capacity. Salinity stress also upregulated gene expression of phenylalanine ammonia-lyase (PAL) and rosmarinic acid synthase (RAS), as well as the activities of PAL and tyrosine ammonia-lyase (TAL) enzymes. Pre-treatment of the plants with CaCl2 and Mel affected these attributes in a dose-dependent manner. Application of 5 mM Ca2+ and 100 µM Mel improved shoot dry biomass and reduced the level of EL and H2O2 content but enhanced TPC and TFC, DPPH scavenging capacity, PAL and TAL activities, PAL and RAS transcripts, and content of rosmarinic acid (RA), luteolin flavone (LF) and apigenin flavone (AF) under salinity stress. Pre-treatment of D. kotschyi with lanthanum chloride (LaCl3) as a plasma membrane channel blocker, ethylene glycol tetra-acetic acid (EGTA) as a Ca2+ chelator and trifluoperazine (TFP) as a calmodulin (CaM) antagonist, impaired Mel effects on the above attributes under salinity stress. In contrast, pre-treatment with p-chlorophenylalanine (p-CPA), as an inhibitor of Mel biosynthesis, did not impair the impacts of Ca2+ on the production of phenolic compounds in salt-exposed plants. These results suggested that the effect of Mel on the induction of phenolic compounds production requires the influx of extracellular Ca2+ into the cells and is dependent on Ca2+/CaM signaling.

Impact of chitosan on nickel bioavailability in soil, the accumulation and tolerance of nickel in Calendula tripterocarpa.

Excessive heavy metals in medicinal plants cause critical health issues to humans. Therefore, in the present study, the effect of soil amendment with chitosan (0, 0.125, 0.25, 0.5, and 1%) on bioavailability and tolerance of nickel in Calendula tripterocarpa grown in a soil spiked with Ni (100 and 150 mg/kg soil) was investigated. The results showed that Ni toxicity significantly reduced plant growth and content of chlorophyll a, b but increased carotenoid levels, lipid peroxidation, and catalase (CAT) and superoxide dismutase (SOD) activities in roots and shoots. The Ni bioaccumulation was significantly higher in shoots than roots. The soil amendment with chitosan reduced Ni bioavailability in soil, as well as lowered the biological accumulation of Ni in roots and shoots, and Ni transfer to leaves. The chitosan application also increased growth parameters and levels of chlorophyll a, b and carotenoids under both normal and Ni stress conditions. Furthermore, chitosan reduced the level of malondialdehyde and the activities of SOD and CAT in roots and shoots under Ni stress. In conclusion, results indicated that chitosan through lowering bioavailability of Ni in soils can remarkably relieve adverse effects of Ni toxicity in C. tripterocarpa.

Melatonin and nitric oxide enhance cadmium tolerance and phytoremediation efficiency in Catharanthus roseus (L.) G. Don.

In this study, a pot experiment was performed to evaluate the effects of foliar spray with sodium nitroprusside (200 µM SNP) and melatonin (100 µM) singly and in combination on tolerance and accumulation of cadmium (Cd) in Catharanthus roseus (L.) G. Don plants exposed to different levels of cadmium (0, 50, 100, and 200 mg Cd kg-1 soil). The results showed that 50 mg kg-1 Cd had no significant effect on the fresh and dry weight of roots and shoots and content of chlorophyll (Chl) a and b, but the higher levels of Cd (100 and 200 mg kg-1) significantly reduced these attributes and induced an increase in the level of leaf electrolyte leakage and disrupted nutrient homeostasis. The activities of catalase (CAT) and peroxidase (POD) in leaves were increased under lower Cd concentrations (50 and 100 mg kg-1) but decreased under 200 mg kg-1 Cd. However, foliar spray with melatonin and/or SNP increased shoot biomass and the content of Chl a and b, augmented activities of POD and CAT, lowered electrolyte leakage (EL), and improved essential cations homeostasis in leaves. Cadmium content in shoots of C. roseus was less than roots and TF (transfer factor) was < 1. Interestingly, foliar spray with SNP and/or melatonin increased Cd accumulation and bioconcentration factor (BCF) in both roots and shoots and elevated the Cd transport from roots to shoot, as TF values increased in these treatments. The co-application of melatonin and SNP further than their separate usage augmented Cd tolerance through increasing activities of antioxidant enzymes and regulating mineral homeostasis in C. roseus. Furthermore, co-treatment of SNP and melatonin increased Cd phytoremediation efficiency in C. roseus through increasing biomass and elevating uptake and translocation of Cd from root to shoot.

Seed priming with H2S and Ca2+ trigger signal memory that induces cross-adaptation against nickel stress in zucchini seedlings.

In this study, the effect of seed priming with sodium hydro sulfide (NaHS) and CaCl2 as well as the possible relationship between them in inducing post-germinative cross-adaptation in zucchini seedlings (cv Courgette d'Italie) were investigated. Results showed that Ni toxicity reduced plant growth and photosynthetic pigments, decreased the content of ascorbate (AsA) and total thiols, increased hydrogen peroxide (H2O2) content and electrolyte leakage (EL), up-regulated the transcription levels of Ca2+-dependent protein kinase (CDPK) and phytochelatin (PCs) genes and elevated H2S content in leaves of zucchini seedlings. Individual or combined seed priming with Ca2+ and NaHS improved the content of photosynthetic pigments and seedling biomass, reduced H2O2 content and EL, increased the content of AsA and total thiols, decreased ascorbate peroxidase activity and enhanced glutathione reductase activity in leaves. These findings suggest the last time effect of seed priming with Ca2+ and NaHS on inducing cross-adaptation in seedlings under Ni stress. H2S accumulation and other responses induced with Ca2+ in leaves were weakened with hypotaurine (HT as H2S scavenger), denoting seed priming with Ca2+ established cross-adaptation in a H2S-dependent manner. Seed priming with NaHS amplified CDPK transcripts in leaves of seedlings and seed priming with ethylene glycol tetraacetic acid (as Ca2+ chelator), lanthanum chloride and verapamil (as plasma membrane channel blockers) reduced transcript levels of CDPK and PCs genes and reversed impacts of seed priming with NaHS. These results indicated that the cross-adaptation induced with NaHS is mediated through Ca2+ signaling. Overall our findings suggest that two-side cross-talk between Ca2+ and H2S is involved in the acquisition of a signal memory in seed embryo cells which can be employed upon a later Ni-exposure and more strongly enhance AsA-GSH cycle, redox homeostasis and phytochelatin transcripts in leaves of zucchini seedlings grown from primed seeds.

Alterations in HO-1 expression, heme oxygenase activity and endogenous NO homeostasis modulate antioxidant responses of Brassica nigra against nano silver toxicity.

Silver nanoparticles (AgNPs) are one of the most widely-used nanomaterials, which are toxic and can cause physiological disorders in plants. The aim of the present study was to investigate whether a possible signaling link between heme oxygenase (HO) and nitric oxide (NO) is implicated in alleviating the toxicity of AgNPs as well as AgNO3. The results showed that exposure to 400 mg L-1 of AgNPs or AgNO3 reduced the chlorophyll content and the growth parameters in Brassica nigra. Data on Ag accumulation as well as the evaluation of lipid peroxidation and the H2O2 content in roots and shoots revealed that AgNP exerted more toxicity than AgNO3. Applying AgNP and AgNO3, respectively, increased HO transcripts by 87.5 and 37.3% and elevated the endogenous NO content 51.8 and 28.5%. The application of both hematin (as an inducer of HO) and sodium nitroprusside (SNP, as a NO donor) reversed the chlorosis and improved plant growth under AgNP and Ag+ ions stresses. Hematin decreased Ag accumulation in plants, indicating that this compound triggered an avoidance mechanism. Hematin and SNP enhanced the activities of antioxidant enzymes and proline accumulation, in parallel to increasing HO transcripts and NO release levels in the roots. ZnPPIX, as the inhibitor of HO, and cPTIO, as the specific NO scavenger, differentially blocked these effects. These findings revealed for the first time that HO might confer an increased tolerance to AgNP by activating the antioxidant systems, which was partially mediated by NO signal.

Role of two-sided crosstalk between NO and H2S on improvement of mineral homeostasis and antioxidative defense in Sesamum indicum under lead stress.

H2S and NO are two important gasotransmitters that modulate stress responses in plants. There are the contradictory data on crosstalk between NO and H2S in the studies. Hence, in the present study, the role of interplay between NO and H2S was assessed on the Pb tolerance of Sesamum indicum using pharmacological and biochemical approaches. Results revealed that Pb stress reduced the plant growth and the content of photosynthetic pigments and Fv/Fm ratio, increased the lipid peroxidation and the H2O2 content, elevated the endogenous contents of nitric oxide (NO), H2S and enhanced the activities of antioxidant enzymes (except APX). Additionally, concentrations of most mineral ions (K, P, Mg, Fe, Mn and Zn) in both shoots and roots decreased. Pb accumulation in roots was more than it in shoots. Both sodium hydrosulfide (NaHS as a donor of H2S) and sodium nitroprusside (SNP as an NO donor) improved the plant growth, the chlorophyll and carotenoid contents and PSII efficiency, reduced oxidative damage, increased the activities of antioxidant enzymes and reduced the proline content in Pb-stressed plants. Furthermore, both NaHS and SNP significantly restricted the uptake and translocation of Pb, thereby minimizing antagonistic effects of Pb on essential mineral contents in sesame plants. NaHS increased the NO generation and many NaHS-induced responses were completely reversed by cPTIO, as the specific NO scavenger. Applying SNP also enhanced H2S release levels in roots of Pb-stressed plants and only some NO-driven effects were partially weakened by hypotuarine (HT), as the scavenger of H2S.These findings proposed for the first time that two-sided interplay between H2S and NO might confer an increased tolerance to Pb stress via activating the antioxidant systems, reducing the uptake and translocation of Pb, and harmonizing the balance of mineral nutrient.

Osmopriming-induced salt tolerance during seed germination of alfalfa most likely mediates through H2O2 signaling and upregulation of heme oxygenase.

The present study showed that osmopriming or pretreatment with low H2O2 doses (2 mM) for 6 h alleviated salt-reduced seed germination. The NADPH oxidase activity was the main source, and superoxide dismutase (SOD) activity might be a secondary source of H2O2 generation during osmopriming or H2O2 pretreatment. Hematin pretreatment similar to osmopriming improved salt-reduced seed germination that was coincident with the enhancement of heme oxygenase (HO) activity. The semi-quantitative RT-PCR confirmed that osmopriming or H2O2 pretreatment was able to upregulate heme oxygenase HO-1 transcription, while the application of N,N-dimethyl thiourea (DMTU as trap of endogenous H2O2) and diphenyleneiodonium (DPI as inhibitor of NADPHox) not only blocked the upregulation of HO but also reversed the osmopriming-induced salt attenuation. The addition of CO-saturated aqueous rescued the inhibitory effect of DMTU and DPI on seed germination and α-amylase activity during osmopriming or H2O2 pretreatment, but H2O2 could not reverse the inhibitory effect of ZnPPIX (as HO inhibitor) or Hb (as CO scavenger) that indicates that the CO acts downstream of H2O2 in priming-driven salt acclimation. The antioxidant enzymes and proline synthesis were upregulated in roots of seedlings grown from primed seeds, and these responses were reversed by adding DMTU, ZnPPIX, and Hb during osmopriming. These findings for the first time suggest that H2O2 signaling and upregulation of heme oxygenase play a crucial role in priming-driven salt tolerance.

Synthesis, characterization and biocompatibility of silver nanoparticles synthesized from Nigella sativa leaf extract in comparison with chemical silver nanoparticles.

Despite the development potential in the field of nanotechnology, there is a concern about possible effects of nanoparticles on the environment and human health. In this study, silver nanoparticles (AgNPs) were synthesized by 'green' and 'chemical' methods. In the wet-chemistry method, sodium borohydrate, sodium citrate and silver nitrate were used as raw materials. Leaf extract of Nigella sativa was used as reducing as well as capping agent to reduce silver nitrate in the green synthesis method. In addition, toxic responses of both synthesized AgNPs were monitored on bone-building stem cells of mice as well as seed germination and seedling growth of six different plants (Lolium, wheat, bean and common vetch, lettuce and canola). In both synthesis methods, the colorless reaction mixtures turned brown and UV-visible spectra confirmed the presence of silver nanoparticles. Scanning electron microscope (SEM) observations revealed the predominance of silver nanosized crystallites and fourier transform infra-red spectroscopy (FTIR) indicated the role of different functional groups in the synthetic process. MTT assay showed cell viability of bone-building stem cells of mice was further in the green AgNPs synthesized using black cumin extract than chemical AgNPs. IC50 (inhibitory concentrations) values for seed germination, root and shoot length for 6 plants in green AgNPs exposures were higher than the chemical AgNPs. These results suggest that cytotoxicity and phytotoxicity of the green synthesized AgNPs were significantly less than wet-chemistry synthesized ones. This study indicated an economical, simple and efficient ecofriendly technique using leaves of N. sativa for synthesis of AgNPs and confirmed that green AgNPs are safer than chemically-synthesized AgNPs.

Role of hematin and sodium nitroprusside in regulating Brassica nigra seed germination under nanosilver and silver nitrate stresses.

Silver nanoparticles (AgNPs) are one of the most widely used nanomaterials, although the mechanisms of AgNP toxicity in terrestrial plants is still unclear. We compared the toxic effects of AgNPs and AgNO3 on Brassica nigra seed germination at physiological and molecular levels. Both AgNPs and AgNO3 inhibited seed germination, lipase activity, soluble and reducing sugar contents in germinating seeds and seedlings. These reductions were more pronounced in AgNP treatments than AgNO3 treatments. Application of 200-400mg/L both AgNPs and AgNO3 increased transcription of heme oxygenase-1. However, at 800, 1600 mg/L, AgNPs or AgNO3 suppressed HO-1 expression. At 400mg/L, AgNPs or AgNO3-induced inhibitory effects on seed germination and were ameliorated by the HO-1 inducer, hematin, or NO donor, sodium nitroprusside (SNP). Additionally, 4 µM hematin and 400 µM SNP were able to markedly boost the HO/NO system. However, the addition of the HO-1 inhibitor (ZnPPIX) or the specific scavenger of NO (cPTIO) not only reversed the protective effects conferred by hematin, but also blocked the up-regulation of HO activity. In addition, hematin-drived NO production in B. niger seeds under AgNPs was confirmed. Our results at physiological and molecular levels suggested that AgNPs were more toxic than AgNO3. Based on these results, for the first time, we suggest that endogenous HO is needed to alleviate AgNPs-induced germination inhibition, which might have a possible interaction with NO.