Effects of copper sulphate stress on the morphological and biochemical characteristics of Spinacia oleracea and Avena sativa.

Plants are subjected to various biotic and abiotic stresses that significantly impact their growth and productivity. To achieve balanced crop growth and yield, including for leafy vegetables, the continuous application of micronutrient is crucial. This study investigates the effects of different concentrations of copper sulphate (0, 75, 125, and 175 ppm) on the morphological and biochemical features of Spinacia oleracea and Avena sativa. Morphological parameters such as plant height, leaf area, root length, and fresh and dry weights were optimized at a concentration of 75 ppm copper sulfate. At this concentration, chlorophyll a & b levels increased significantly in Spinacia oleracea (462.9 and 249.8 𝜇𝑔/𝑔), and Avena sativa (404.7 and 437.63𝜇𝑔/𝑔). However, carotenoid content and sugar levels in Spinacia oleracea were negatively affected, while sugar content in Avena sativa increased at 125 ppm (941.6 µg/ml). Protein content increased in Spinacia oleracea (75 ppm, 180.3 µg/ml) but decreased in Avena sativa. Phenol content peaked in both plants at 75 ppm (362.2 and 244.5 µg/ml). Higher concentrations (175 ppm) of copper sulfate reduced plant productivity and health. Plants exposed to control and optimal concentrations (75 and 125 ppm) of copper sulpate exhibited the best health and growth compared to those subjected to higher concentrations. Maximum plant height, leaf area, root length, fresh and dry weights were observed at lower concentrations (75 and 125 ppm) of copper sulfate, while higher concentrations caused toxicity. Optimal copper sulfate levels enhanced chlorophyll a, chlorophyll b, total chlorophyll, protein, and phenol contents but inhibited sugar and carotenoid contents in both Spinacia oleracea and Avena sativa. Overall, increased copper sulfate treatment adversely affected the growth parameters and biochemical profiles of these plants.

Efficacy of Fe-Mg-bimetallic biochar in stabilization of multiple heavy metals-contaminated soil and attenuation of toxicity in spinach (Spinacia oleracea L.).

Globally, soil contamination with heavy metals (HMs) pose serious threats to soil health, crop productivity, and human health. The present investigation involved synthesis and analysis of biochar with bimetallic combination of iron and magnesium (Fe-Mg-BC). Our study evaluated how Fe-Mg-BC affects the absorption of cadmium (Cd), lead (Pb), and copper (Cu) in spinach (Spinacia oleracea L.) and remediation of soil contaminated with multiple HMs. Results demonstrated the successful loading of iron (Fe) and magnesium (Mg) onto pristine biochar (BC) derived from peanut shells. The addition of Fe-Mg-BC (3%) notably increased spinach biomass, enhancing photosynthesis, transpiration, stomatal conductance, and intercellular CO2 levels by 22%, 21%, 103%, and 15.3%, respectively. Compared to control, Fe-Mg-BC (3%) suppressed metal-induced oxidative stress by boosting levels of superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) in roots by 40.9%, 57%, 54.8 %, and in shoots by 55.5%, 65.5%, and 37.4% in shoots, respectively. The Fe-Mg-BC effectively reduced the uptake of Cd, Pb, and Cu in spinach tissues by transforming their bioavailable fractions to non-bioavailable forms. The Fe-Mg-BC (3%) significantly reduced the mobility of Cd, Pb and Cu in soil and limited the concentration of Cd, Pb, and Cu in plant roots by 34.1%, 79.2%, 47%, and shoots by 56.3%, 43.3%, and 54.1%, respectively, compared to control. These findings underscore the potential of Fe-Mg-BC as a promising amendment for reclaiming soils contaminated with variety of HMs, thereby making a significant contribution to the promotion of safer food production.

Effect of biochar amendment on bacterial community and their role in nutrient acquisition in spinach (Spinacia oleracea L.) grown under elevated CO2.

Global climate change is anticipated to shift the soil bacterial community structure and plant nutrient utilization. The use of biochar amendment can positively influence soil bacterial community structure, soil properties, and nutrient use efficiency of crops. However, little is known about the underlying mechanism and response of bacterial community structure to biochar amendment, and its role in nutrient enhancement in soil and plants under elevated CO2. Herein, the effect of biochar amendment (0, 0.5, 1.5%) on soil bacterial community structure, spinach growth, physiology, and soil and plant nutrient status were investigated under two CO2 concentrations (400 and 600 µmol mol-1). Findings showed that biochar application 1.5% (B.2.E) significantly increased the abundance of the bacterial community responsible for growth and nutrient uptake i.e. Firmicutes (42.25%) Bacteroidetes (10.46%), and Gemmatimonadetes (125.75%) as compared to respective control (CK.E) but interestingly abundance of proteobacteria decreased (9.18%) under elevated CO2. Furthermore, the soil available N, P, and K showed a significant increase in higher biochar-amended treatments under elevated CO2. Spinach plants exhibited a notable enhancement in growth and photosynthetic pigments when exposed to elevated CO2 levels and biochar, as compared to ambient CO2 conditions. However, there was variability observed in the leaf gas exchange attributes. Elevated CO2 reduced spinach roots and leaves nutrient concentration. In contrast, the biochar amendment (B2.E) enhanced root and shoot Zinc (494.99%-155.33%), magnesium (261.15%-183.37%), manganese (80.04%-152.86%), potassium (576.24%-355.17%), calcium (261.88%-165.65%), copper (325.42%-282.53%) and iron (717.63%-177.90%) concentration by influencing plant physiology and bacterial community. These findings provide insights into the interaction between plant and bacterial community under future agroecosystems in response to the addition of biochar contributing to a deeper understanding of ecological dynamics.

Microwave seed priming and ascorbic acid assisted phytoextraction of heavy metals from surgical industry effluents through spinach.

The prevalence of inorganic pollutants in the environment, including heavy metals (HMs), necessitates a sustainable and cost-effective solution to mitigate their impacts on the environment and living organisms. The present research aimed to assess the phytoextraction capability of spinach (Spinach oleracea L.), under the combined effects of ascorbic acid (AA) and microwave (MW) irradiation amendments, cultivated using surgical processing wastewater. In a preliminary study, spinach seeds were exposed to MW radiations at 2.45 GHz for different durations (15, 30, 45, 60, and 90 seconds). Maximum germination was observed after the 30 seconds of radiation exposure. Healthy spinach seeds treated with MW radiations for 30 s were cultivated in the sand for two weeks, after which juvenile plants were transferred to a hydroponic system. Surgical industry wastewater in different concentrations (25 %, 50 %, 75 %, 100 %) and AA (10 mM) were provided to both MW-treated and untreated plants. The results revealed that MW-treatment significantly enhanced the plant growth, biomass, antioxidant enzyme activities and photosynthetic pigments, while untreated plants exhibited increased reactive oxygen species (ROS) and electrolyte leakage (EL) compared with their controls. The addition of AA to both MW-treated and untreated plants improved their antioxidative defense capacity under HMs-induced stress. MW-treated spinach plants, under AA application, demonstrated relatively higher concentrations and accumulation of HMs including lead (Pb), cadmium (Cd) and nickel (Ni). Specifically, MW-treated plants with AA amendment showed a significant increase in Pb concentration by 188 % in leaves, Cd by 98 %, and Ni by 102 % in roots. Additionally, the accumulation of Ni increased by 174 % in leaves, Cd by 168 % in roots, and Pb by 185 % in the stem of spinach plant tissues compared to MW-untreated plants. These findings suggested that combining AA with MW irradiation of seeds could be a beneficial strategy for increasing the phytoextraction of HMs from wastewater and improving overall plant health undergoing HMs stress.

Impact of nitrogen fertilizer type and application rate on growth, nitrate accumulation, and postharvest quality of spinach.

Background: A balanced supply of nitrogen is essential for spinach, supporting both optimal growth and appropriate nitrate (NO3 -) levels for improved storage quality. Thus, choosing the correct nitrogen fertilizer type and application rate is key for successful spinach cultivation. This study investigated the effects of different nitrogen (N) fertilizer type and application rates on the growth, nitrate content, and storage quality of spinach plants. Methods: Four fertilizer types were applied at five N doses (25, 50, 200, and 400 mg N kg-1) to plants grown in plastic pots at a greenhouse. The fertilizer types used in the experiment were ammonium sulphate (AS), slow-release ammonium sulphate (SRAS), calcium nitrate (CN), and yeast residue (YR). Spinach parameters like Soil Plant Analysis Development (SPAD) values (chlorophyll content), plant height, and fresh weight were measured. Nitrate content in leaves was analyzed after storage periods simulating post-harvest handling (0, 5, and 10 days). Results: The application of nitrogen fertilizer significantly influenced spinach growth parameters and nitrate content. The YRx400 treatment yielded the largest leaves (10.3 ± 0.5 cm long, 5.3 ± 0.2 cm wide). SPAD values increased with higher N doses for AS, SRAS, and CN fertilizers, with AS×400 (58.1 ± 0.8) and SRAS×400 (62.0 ± 5.8) reaching the highest values. YR treatments showed a moderate SPAD increase. Fresh weight response depended on fertilizer type, N dose, and storage period. While fresh weight increased in all fertilizers till 200 mg kg-1 dose, a decrease was observed at the highest dose for AS and CN. SRAS exhibited a more gradual increase in fresh weight with increasing nitrogen dose, without the negative impact seen at the highest dose in AS and CN. Nitrate content in spinach leaves varied by fertilizer type, dose, and storage day. CNx400 resulted in the highest NO3 - content (4,395 mg kg-1) at harvest (Day 0), exceeding the European Union's safety limit. This level decreased over 10 days of storage but remained above the limit for CN on Days 0 and 5. SRAS and YR fertilizers generally had lower NO3 - concentrations throughout the experiment. Storage at +4 °C significantly affected NO3 - content. While levels remained relatively stable during the first 5 days, a substantial decrease was observed by Day 10 for all fertilizers and doses, providing insights into the spinach's nitrate content over a 10-day storage period. Conclusion: For rapid early growth and potentially higher yields, AS may be suitable at moderate doses (200 mg kg-1). SRAS offers a more balanced approach, promoting sustained growth while potentially reducing NO3 - accumulation compared to AS. Yeast residue, with its slow nitrogen release and consistently low NO3 - levels, could be a viable option for organic spinach production.

Enhancing zinc biofortification and mitigating cadmium toxicity in soil-earthworm-spinach systems using different zinc sources.

Cadmium (Cd) pollution poses significant threats to soil organisms and human health by contaminating the food chain. This study aimed to assess the impact of various concentrations (50, 250, and 500 mg·kg-1) of zinc oxide nanoparticles (ZnO NPs), bulk ZnO, and ZnSO4 on morphological changes and toxic effects of Cd in the presence of earthworms and spinach. The results showed that Zn application markedly improved spinach growth parameters (such as fresh weight, plant height, root length, and root-specific surface area) and root morphology while significantly reducing Cd concentration and Cd bioconcentration factors (BCF-Cd) in spinach and earthworms, with ZnO NPs exhibiting the most pronounced effects. Earthworm, spinach root, and shoot Cd concentration decreased by 82.3 %, 77.0 %, and 75.6 %, respectively, compared to CK. Sequential-step extraction (BCR) analysis revealed a shift in soil Cd from stable to available forms, consistent with the available Cd (DTPA-Cd) results. All Zn treatments significantly reduced Cd accumulation, alleviated Cd-induced stress, and promoted spinach growth, with ZnO NPs demonstrating the highest Cd reduction and Zn bioaugmentation efficiencies compared to bulk ZnO and ZnSO4 at equivalent concentrations. Therefore, ZnO NPs offer a safer and more effective option for agricultural production and soil heavy metal pollution management than other Zn fertilizers.

Salt stress amelioration and nutrient strengthening in spinach (Spinacia oleracea L.) via biochar amendment and zinc fortification: seed priming versus foliar application.

Soil salinity is a major nutritional challenge with poor agriculture production characterized by high sodium (Na+) ions in the soil. Zinc oxide nanoparticles (ZnO NPs) and biochar have received attention as a sustainable strategy to reduce biotic and abiotic stress. However, there is a lack of information regarding the incorporation of ZnO NPs with biochar to ameliorate the salinity stress (0, 50,100 mM). Therefore, the current study aimed to investigate the potentials of ZnO NPs application (priming and foliar) alone and with a combination of biochar on the growth and nutrient availability of spinach plants under salinity stress. Results demonstrated that salinity stress at a higher rate (100 mM) showed maximum growth retardation by inducing oxidative stress, resulted in reduced photosynthetic rate and nutrient availability. ZnO NPs (priming and foliar) alone enhanced growth, chlorophyll contents and gas exchange parameters by improving the antioxidant enzymes activity of spinach under salinity stress. While, a significant and more pronounced effect was observed at combined treatments of ZnO NPs with biochar amendment. More importantly, ZnO NPs foliar application with biochar significantly reduced the Na+ contents in root 57.69%, and leaves 61.27% of spinach as compared to the respective control. Furthermore, higher nutrient contents were also found at the combined treatment of ZnO NPs foliar application with biochar. Overall, ZnO NPs combined application with biochar proved to be an efficient and sustainable strategy to alleviate salinity stress and improve crop nutritional quality under salinity stress. We inferred that ZnO NPs foliar application with a combination of biochar is more effectual in improving crop nutritional status and salinity mitigation than priming treatments with a combination of biochar.

Effects of superoxide radical on photosynthesis and K+ and redox homeostasis in quinoa and spinach.

Methyl viologen (MV), also known as paraquat, is a widely used herbicide but has also been reported as highly toxic to different life forms. The mode of its operation is related to superoxide radical (O2.-) production and consequent oxidative damage. However, besides the damage to key macromolecules, reactive oxygen species (ROS; to which O2.- belongs) are also known as regulators of numerous ion transport systems located at cellular membranes. In this study, we used MV as a tool to probe the role of O2.- in regulating membrane-transport activity and systemic acquired tolerance in halophytic Chenopodium quinoa and glycophytic spinach plants. Both plant species showed growth reduction in terms of reduced shoot length, lower shoot fresh and dry weight, photosynthesis rate, and chlorophyll contents; however, quinoa showed less reduction in growth compared with spinach. This whole plant response was further examined by measuring the ion concentration, gene expression of ion transporters, activation of antioxidants, and osmolyte accumulation. We observed that at the mechanistic level, the differences in growth in response to MV were conferred by at least four complementary physiological mechanisms: (1) higher K+ loss from spinach leaves resulted from higher expression of MV-induced plasma membrane-based depolarization-activated K+ efflux GORK channel, (2) higher activation of high-affinity K+ uptake transporter HAK5 in quinoa, (3) higher antioxidant production and osmolyte accumulation in quinoa as compared with spinach, and (4) maintaining a higher rate of photosynthesis due to higher chlorophyll contents, and efficiency of photosystem II and reduced ROS and MDA contents. Obtained results also showed that MV induced O2.- significantly reduced N contents in both species but with more pronounced effects in glycophytic spinach. Taken together this study has shown the role of O2.- in regulating membrane ion transport and N metabolism in the leaves of halophyte vs. glycophyte in the context of oxidative stress tolerance.

Enhancing spinach (Spinacia oleracea L.) resilience in pesticide-contaminated soil: Role of pesticide-tolerant Ciceribacter azotifigens and Serratia marcescens in root architecture, leaf gas exchange attributes and antioxidant response restoration.

This study unveils the detoxification potential of insecticide-tolerant plant beneficial bacteria (PBB), i.e., Ciceribacter azotifigens SF1 and Serratia marcescens SRB1, in spinach treated with fipronil (FIP), profenofos (PF) and chlorantraniliprole (CLP) insecticides. Increasing insecticide doses (25-400 µg kg-1 soil) significantly curtailed germination attributes and growth of spinach cultivated at both bench-scale and in greenhouse experiments. Profenofos at 400 µg kg-1 exhibited maximum inhibitory effects and reduced germination by 55%; root and shoot length by 78% and 81%, respectively; dry matter accumulation in roots and shoots by 79% and 62%, respectively; leaf number by 87% and leaf area by 56%. Insecticide application caused morphological distortion in root tips/surfaces, increased levels of oxidative stress, and cell death in spinach. Application of insecticide-tolerant SF1 and SRB1 strains relieved insecticide pressure resulting in overall improvement in growth and physiology of spinach grown under insecticide stress. Ciceribacter azotifigens improved germination rate (10%); root biomass (53%); shoot biomass (25%); leaf area (10%); Chl-a (45%), Chl-b (36%) and carotenoid (48%) contents of spinach at 25 µg CLP kg-1 soil. PBB inoculation reinvigorated the stressed spinach and modulated the synthesis of phytochemicals, proline, malondialdehyde (MDA), superoxide anions (O2•-), and hydrogen peroxide (H2O2). Scanning electron microscopy (SEM) revealed recovery in root tip morphology and stomatal openings on abaxial leaf surfaces of PBB-inoculated spinach grown with insecticides. Ciceribacter azotifigens inoculation significantly increased intrinsic water use efficiency, transpiration rate, vapor pressure deficit, intracellular CO2 concentration, photosynthetic rate, and stomatal conductance in spinach exposed to 25 µg FIP kg-1. Also, C. azotifigens and S. marcescens modulated the antioxidant defense systems of insecticide-treated spinach. Bacterial strains were strongly colonized to root surfaces of insecticide-stressed spinach seedlings as revealed under SEM. The identification of insecticide-tolerant PBBs such as C. azotifigens and S. marcescens hold the potential for alleviating abiotic stress to spinach, thereby fostering enhanced and safe production within polluted agroecosystems.

Exogenous curcumin mitigates As stress in spinach plants: A biochemical and metabolomics investigation.

The spinach (S. oleracea L.) was used as a model plant to investigate As toxicity on physio-biochemical processes, exploring the potential mitigation effect of curcumin (Cur) applied exogenously at three concentrations (1, 10, and 20 µM Cur). The employment of Cur significantly mitigated As-induced stress in spinach photosynthetic performance (Fv/Fm, Fo/Fm, and Fv/Fo). Moreover, the co-incubation of Cur with As improved physiological processes mainly associated with plant water systems affected by As stress by recovering the leaf's relative water content (RWC) and osmotic potential (ψπ) nearly to the control level and increasing the transpiration rate (E; 39-59%), stomatal conductivity (gs; 86-116%), and carbon assimilation rate (A; 84-121%) compared to As stressed plants. The beneficial effect of Cur in coping with As-induced stress was also assessed at the plant's oxidative level by reducing oxidative stress biomarkers (H2O2 and MDA) and increasing non-enzymatic antioxidant capacity. Untargeted metabolomics analysis was adopted to investigate the main processes affected by As and Cur application. A multifactorial ANOVA discrimination model (AMOPLS-DA) and canonical correlation analysis (rCCA) were employed to identify relevant metabolic changes and biomarkers associated with Cur and As treatments. The results highlighted that Cur significantly determined the accumulation of glucosinolates, phenolic compounds, and an increase in glutathione redox cycle activities, suggesting an overall elicitation of plant secondary metabolisms. Specifically, the correlation analysis reported a strong and positive correlation between (+)-dihydrokaempferol, L-phenylalanine (precursor of phenolic compounds), and serotonin-related metabolites with antioxidant activities (ABTS and DPPH), suggesting the involvement of Cur application in promoting a cross-talk between ROS signaling and phytohormones, especially melatonin and serotonin, working coordinately to alleviate As-induced oxidative stress. The modulation of plant metabolism was also observed at the level of amino acids, fatty acids, and secondary metabolites synthesis, including N-containing compounds, terpenes, and phenylpropanoids to cooperate with As-induced stress response.

The mechanism behind tenuazonic acid-mediated inhibition of plant plasma membrane H+-ATPase and plant growth.

The increasing prevalence of herbicide-resistant weeds has led to a search for new herbicides that target plant growth processes differing from those targeted by current herbicides. In recent years, some studies have explored the use of natural compounds from microorganisms as potential new herbicides. We previously demonstrated that tenuazonic acid (TeA) from the phytopathogenic fungus Stemphylium loti inhibits the plant plasma membrane (PM) H+-ATPase, representing a new target for herbicides. In this study, we further investigated the mechanism by which TeA inhibits PM H+-ATPase and the effect of the toxin on plant growth using Arabidopsis thaliana. We also studied the biochemical effects of TeA on the PM H+-ATPases from spinach (Spinacia oleracea) and A. thaliana (AHA2) by examining PM H+-ATPase activity under different conditions and in different mutants. Treatment with 200 µM TeA-induced cell necrosis in larger plants and treatment with 10 µM TeA almost completely inhibited cell elongation and root growth in seedlings. We show that the isoleucine backbone of TeA is essential for inhibiting the ATPase activity of the PM H+-ATPase. Additionally, this inhibition depends on the C-terminal domain of AHA2, and TeA binding to PM H+-ATPase requires the Regulatory Region I of the C-terminal domain in AHA2. TeA likely has a higher binding affinity toward PM H+-ATPase than the phytotoxin fusicoccin. Finally, our findings show that TeA retains the H+-ATPase in an inhibited state, suggesting that it could act as a lead compound for creating new herbicides targeting the PM H+-ATPase.

Effect of zinc nanoparticles seed priming and foliar application on the growth and physio-biochemical indices of spinach (Spinacia oleracea L.) under salt stress.

Salt stress is the major risk to the seed germination and plant growth via affecting physiological and biochemical activities in plants. Zinc nanoparticles (ZnNPs) are emerged as a key agent in regulating the tolerance mechanism in plants under environmental stresses. However, the tolerance mechanisms which are regulated by ZnNPs in plants are still not fully understood. Therefore, the observation was planned to explore the role of ZnNPs (applied as priming and foliar) in reducing the harmful influence of sodium chloride (NaCl) stress on the development of spinach (Spinacia oleracea L.) plants. Varying concentrations of ZnNPs (0.1%, 0.2% & 0.3%) were employed to the spinach as seed priming and foliar, under control as well as salt stress environment. The alleviation of stress was observed in ZnNPs-applied spinach plants grown under salt stress, with a reduced rise in the concentration hydrogen peroxide, melondialdehyde and anthocyanin contents. A clear decline in soluble proteins, chlorophyll contents, ascorbic acid, sugars, and total phenolic contents was observed in stressed conditions. Exogenous ZnNPs suppressed the NaCl generated reduction in biochemical traits, and progress of spinach plants. However, ZnNPs spray at 0.3% followed by priming was the most prominent treatment in the accumulation of osmolytes and the production of antioxidant molecules in plants.

Photosynthesis-Inhibiting Activity of N-(Disubstituted-phenyl)-3-hydroxynaphthalene-2-carboxamides.

A set of twenty-four 3-hydroxynaphthalene-2-carboxanilides, disubstituted on the anilide ring by combinations of methoxy/methyl/fluoro/chloro/bromo and ditrifluoromethyl groups at different positions, was prepared. The compounds were tested for their ability to inhibit photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. N-(3,5-Difluorophenyl)-, N-(3,5-dimethylphenyl)-, N-(2,5-difluorophenyl)- and N-(2,5-dimethylphenyl)-3-hydroxynaphthalene-2-carboxamides showed the highest PET-inhibiting activity (IC50 ~ 10 µM) within the series. These compounds were able to inhibit PET in photosystem II. It has been found that PET-inhibiting activity strongly depends on the position of the individual substituents on the anilide ring and on the lipophilicity of the compounds. The electron-withdrawing properties of the substituents contribute towards the PET activity of these compounds.

Singlet oxygen damages the function of Photosystem II in isolated thylakoids and in the green alga Chlorella sorokiniana.

Singlet oxygen (1O2) is an important damaging agent, which is produced during illumination by the interaction of the triplet excited state pigment molecules with molecular oxygen. In cells of photosynthetic organisms 1O2 is formed primarily in chlorophyll containing complexes, and damages pigments, lipids, proteins and other cellular constituents in their environment. A useful approach to study the physiological role of 1O2 is the utilization of external photosensitizers. In the present study, we employed a multiwell plate-based screening method in combination with chlorophyll fluorescence imaging to characterize the effect of externally produced 1O2 on the photosynthetic activity of isolated thylakoid membranes and intact Chlorella sorokiniana cells. The results show that the external 1O2 produced by the photosensitization reactions of Rose Bengal damages Photosystem II both in isolated thylakoid membranes and in intact cells in a concentration dependent manner indicating that 1O2 plays a significant role in photodamage of Photosystem II.

Photophysics of DFHBI bound to RNA aptamer Baby Spinach.

The discovery of the GFP-type dye DFHBI that becomes fluorescent upon binding to an RNA aptamer, termed Spinach, led to the development of a variety of fluorogenic RNA systems that enable genetic encoding of living cells. In view of increasing interest in small RNA aptamers and the scarcity of their photophysical characterisation, this paper is a model study on Baby Spinach, a truncated Spinach aptamer with half its sequence. Fluorescence and fluorescence excitation spectra of DFHBI complexes of Spinach and Baby Spinach are known to be similar. Surprisingly, a significant divergence between absorption and fluorescence excitation spectra of the DFHBI/RNA complex was observed on conditions of saturation at large excess of RNA over DFHBI. Since absorption spectra were not reported for any Spinach-type aptamer, this effect is new. Quantitative modelling of the absorption spectrum based on competing dark and fluorescent binding sites could explain it. However, following reasoning of fluorescence lifetimes of bound DFHBI, femtosecond-fluorescence lifetime profiles would be more supportive of the notion that the abnormal absorption spectrum is largely caused by trans-isomers formed  within the cis-bound DFHBI/RNA complex. Independent of the origin, the unexpected discrepancy between absorption and fluorescence excitation spectra allows for easily accessed screening and insight into the efficiency of a fluorogenic dye/RNA system.

Effect of the suspension of Ag-incorporated TiO2 nanoparticles (Ag-TiO2 NPs) on certain growth, physiology and phytotoxicity parameters in spinach seedlings.

In this work, the effect of the inoculation of silver-incorporated titanium dioxide nanoparticles (Ag-TiO2 NPs) in spinach seeds was evaluated on certain growth, physiology and phytotoxicity parameters of the plants. This is an important crop for human consumption with high nutritional value due to their low calorie and fat content, providing various vitamins and minerals, especially iron. These NPs were obtained by means of the sol-gel method and heat treatment; the resulting powder material was characterized using X-ray diffraction and scanning electron microscopy and the influence of these NPs on plants was measured by estimating the germination rate, monitoring morphological parameters and evaluating phytotoxicity. The photosynthetic activity of the spinach plants was estimated through the quantification of the Ratio of Oxygen Evolution (ROE) by the photoacoustic technique. Samples of TiO2 powder with particle size between 9 and 43 nm were used to quantify the germination rate, which served to determine a narrower size range between 7 and 26 nm in the experiments with Ag-TiO2 NPs; the presence of Ag in TiO2 powder samples was confirmed by energy-dispersive X-ray spectroscopy. The analysis of variance showed that the dependent variable (plant growth) could be affected by the evaluated factors (concentration and size) with significant differences. The statistical trend indicated that the application of the Ag-TiO2 NPs suspension of lowest concentration and smallest particle size could be a promoting agent of the growth and development of these plants. The inoculation with NPs of 8.3 nm size and lowest concentration was related to the highest average ROE value, 24.6 ± 0.2%, while the control group was 20.2 ± 0.2%. The positive effect of the Ag-TiO2 NPs treatment could be associated to the generation of reactive oxygen species, antimicrobial activity, increased biochemical attributes, enzymatic activity or improvements in water absorption.

Role of sulfur in combating arsenic stress through upregulation of important proteins, and in-silico analysis to study the interaction between phosphate transporter (PHO1), arsenic and phosphate in spinach.

An adequate amount of Sulfur (S) is essential for proper plant growth and defence against abiotic stresses including metals and metalloids. Arsenic (As) contamination is increasing in agricultural soils rapidly due to anthropogenic activities. Sulfur deficiency and arsenic stress could be more harmful than these individual stresses alone. To understand the impact of S-deficiency and arsenic (31 ppm Na3AsO4 of soil) on ecophysiology, growth, inorganic phosphate level, and proteomic profile of spinach, the present study was conducted. Interaction of arsenic with phosphate transporters, phytochelatins, and glutathione was also analyzed in silico. Comparative 2D MS/MS proteomics helped in the identification of important proteins which might be the key players under S-deficiency and As stress. Upregulation and downregulation of 36 and 21 proteins under As stress; 19 and 36 proteins under S-deficiency; 38 and 31 proteins under combined stress, respectively was observed. A total, 87 proteins subjected to identification via MS/MS ion search were found to be associated with important plant functions. PHO1 abundance was highly influenced by As stress; hence an in-silico homology modeling based molecular docking was performed which indicated high interaction between PHO1 and As/phosphate. Varied proximity of arsenic with phosphate transporters, phytochelatin, and glutathione revealed these components as a potential target of As toxicity/detoxification in Spinach, reflecting sulfur as an important criterion for arsenic tolerance.

Binary mixture of nanoparticles in sewage sludge: Impact on spinach growth.

Present study carried out pot experiments and evaluated effects of single and binary mixture of nanoparticles (exposed via sludge as soil conditioner) on spinach plant. Exposure of Ag2O nanoparticles (NPs) (1 and 10 mg/kg soil-sludge) did not show significant reduction in plant as compared to control. On the other hand, TiO2 NPs (exposed as single and in binary mixture) resulted in significant increase in root length (29% and 37%) and fresh weight (60% and 48%) at highest exposure concentration. Total chlorophyll content decreased for Ag2O and binary mixture (7% and 4%, respectively) and increased for TiO2 (5%) at 10 mg/kg soil-sludge. The toxic interaction between Ag2O and TiO2 NPs was additive at both exposure concentrations. Ag2O NPs had higher tendency of root surface adsorption than TiO2 NPs. Metal content in spinach leaves at highest exposure concentration was Ag: 2.6 ± 0.55 mg/g plant biomass(for Ag2O NPs) and 1.02 ± 0.32 mg/g plant biomass (for Ag2O + TiO2 NPs) and for Ti: 1.12 ± 0.78 (for TiO2 NPs) mg/g plant biomass and 0.58 ± 0.41 mg/g (for Ag2O + TiO2 NPs). The inadvertent ingestion of NPs- contaminated spinach resulted in projected daily intake (DI) of Ag and Ti for different age-mass classes (child to adult) exceeding the oral reference dose for toxicity during oral ingestion. In conclusion, we report no acute toxicity of single and binary mixture of NPs to spinach but significant accumulation of Ag and Ti metals in spinach leaves. There are high chances that ingestion of spinach grown in such environment might lead to human health risks.

Potential role of compost mixed biochar with rhizobacteria in mitigating lead toxicity in spinach.

Consumption of heavy metals, especially lead (Pb) contaminated food is a serious threat to human health. Higher Pb uptake by the plant affects the quality, growth and yield of crops. However, inoculation of plant growth-promoting rhizobacteria (PGPR) along with a mixture of organic amendments and biochar could be an effective way to overcome the problem of Pb toxicity. That's why current pot experiment was conducted to investigate the effect of compost mixed biochar (CB) and ACC deaminase producing PGPR on growth and yield of spinach plants under artificially induced Pb toxicity. Six different treatments i.e., control, Alcaligenes faecalis (PGPR1), Bacillus amyloliquefaciens (PGPR2), compost + biochar (CB), PGPR1 + CB and PGPR2 + CB were applied under 250 mg Pb kg-1 soil. Results showed that inoculation of PGPRs (Alcaligenes faecalis and Bacillus amyloliquefaciens) alone and along with CB significantly enhanced root fresh (47%) and dry weight (31%), potassium concentration (11%) in the spinach plant. Whereas, CB + Bacillus amyloliquefaciens significantly decreased (43%) the concentration of Pb in the spinach root over control. In conclusion, CB + Bacillus amyloliquefaciens has the potential to mitigate the Pb induced toxicity in the spinach. The obtained result can be further used in the planning and execution of rhizobacteria and compost mixed biochar-based soil amendment.

Combined effects of ultrasound and aqueous chlorine dioxide treatments on nitrate content during storage and postharvest storage quality of spinach (Spinacia oleracea L.).

The objectives of this study were to optimize the condition of ultrasonic treatment combined with aqueous chlorine dioxide (ClO2) on nitrate content of spinach by response surface methodology (RSM), and determine the effectiveness of ultrasound (US) and ClO2 alone and in combination, on spinach postharvest quality during 7 days' storage period. The optimal treatment parameters obtained were ultrasonic power (300 W), ClO2 concentration (50 ppm), treatment time (4 min). The combined treatments significantly reduced the nitrate content and maintained better storage quality in terms of total soluble solids (TSS) and ascorbic acid content compared with the individual treatment or untreated. For Chlorophyll content, the combined treatment was significantly higher than the control and ClO2 treatment, but lower than ultrasonic treatment. The results demonstrated that US combined with ClO2 are promising alternatives for the reduction of nitrate content, as well as preserving the quality of stored leafy vegetables.