RBOH-dependent signaling is involved in He-Ne laser-induced salt tolerance and production of rosmarinic acid and carnosol in Salvia officinalis.

BACKGROUND: In the past two decades, the impacts of Helium-Neon (He-Ne) laser on stress resistance and secondary metabolism in plants have been studied, but the signaling pathway which by laser regulates this process remains unclear. Therefore, the current study sought to explore the role of RBOH-dependent signaling in He-Ne laser-induced salt tolerance and elicitation of secondary metabolism in Salvia officinalis. Seeds were primed with He-Ne laser (6 J cm- 2) and peroxide hydrogen (H2O2, 5 mM) and 15-old-day plants were exposed to two salinity levels (0, 75 mM NaCl). RESULTS: Salt stress reduced growth parameters, chlorophyll content and relative water content (RWC) and increased malodialdehyde (MDA) and H2O2 contents in leaves of 45-old-day plants. After 48 h of salt exposure, higher transcription levels of RBOH (encoding NADPH oxidase), PAL (phenylalanine ammonia-lyase), and RAS (rosmarinic acid synthase) were recorded in leaves of plants grown from seeds primed with He-Ne laser and/or H2O2. Despite laser up-regulated RBOH gene in the early hours of exposing to salinity, H2O2 and MDA contents were lower in leaves of these plants after 30 days. Seed pretreatment with He-Ne laser and/or H2O2 augmented the accumulation of anthocyanins, total phenol, carnasol, and rosmarinic acid and increased total antioxidant capacity under non-saline and more extensively at saline conditions. Indeed, these treatments improved RWC, and K+/Na+ ratio, enhanced the activities of superoxide dismutase and ascorbate peroxidase and proline accumulation, and significantly decreased membrane injury and H2O2 content in leaves of 45-old-day plants under salt stress. However, applying diphenylene iodonium (DPI as an inhibitor of NADPH oxidase) and N, N-dimethyl thiourea (DMTU as a H2O2 scavenger) after laser priming reversed the aforementioned effects which in turn resulted in the loss of laser-induced salt tolerance and secondary metabolism. CONCLUSIONS: These findings for the first time deciphered that laser can induce a transient RBOH-dependent H2O2 burst, which might act as a downstream signal to promote secondary metabolism and salt stress alleviation in S. officinalis plants.

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.