Mahkota Dewa Subcritical Water Extraction Process: Experimental and Molecular Dynamics Simulation Study

Subcritical water extraction and molecular dynamics simulation were performed to investigate mangiferin and antioxidant extraction from Mahkota Dewa fruits at different operating temperatures. The mangiferin yield and antioxidant activity were analyzed using high‐performance liquid chromatography and 2‐diphenyl‐1‐picrylhydrazyl assay method, respectively. The diffusivity and intermolecular interactions were determined by mean squared displacement (MSD) and radial distribution function (RDF) analysis, respectively. The temperature exerts a momentous effect on mangiferin yield and antioxidant activity of the extract. The MSD result showed an increment in diffusivity coefficient of mangiferin with temperature. The RDF analysis revealed hydrogen bonding formations between mangiferin and water through O_H2O^??H_MR4(OH1) interaction that plays a role in the extraction process.     

Ethanol Extract of Maclura tricuspidata Fruit Protects SH-SY5Y Neuroblastoma Cells against H2O2-Induced Oxidative Damage via Inhibiting MAPK and NF-κB Signaling

Free radical generation and oxidative stress push forward an immense influence on the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. Maclura tricuspidata fruit (MT) contains many biologically active substances, including compounds with antioxidant properties. The current study aimed to investigate the neuroprotective effects of MT fruit on hydrogen peroxide (H₂O₂)-induced neurotoxicity in SH-SY5Y cells. SH-SY5Y cells were pretreated with MT, and cell damage was induced by H₂O₂. First, the chemical composition and free radical scavenging properties of MT were analyzed. MT attenuated oxidative stress-induced damage in cells based on the assessment of cell viability. The H₂O₂-induced toxicity caused by ROS production and lactate dehydrogenase (LDH) release was ameliorated by MT pretreatment. MT also promoted an increase in the expression of genes encoding the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT). MT pretreatment was associated with an increase in the expression of neuronal genes downregulated by H₂O₂. Mechanistically, MT dramatically suppressed H₂O₂-induced Bcl-2 downregulation, Bax upregulation, apoptotic factor caspase-3 activation, Mitogen-activated protein kinase (MAPK) (JNK, ERK, and p38), and Nuclear factor-κB (NF-κB) activation, thereby preventing H₂O₂-induced neurotoxicity. These results indicate that MT has protective effects against H₂O₂-induced oxidative damage in SH-SY5Y cells and can be used to prevent and protect against neurodegeneration.     

Mitochondrial Protection and Anti-aging Activity of Astragalus Polysaccharides and Their Potential Mechanism

The current study was performed to investigate mitochondrial protection and anti-aging activity of Astragalus polysaccharides (APS) and the potential underlying mechanism. Lipid peroxidation of liver and brain mitochondria was induced by Fe²⁺–Vit C in vitro. Thiobarbituric acid (TBA) colorimetry was used to measure the content of thiobarbituric acid reactive substances (TBARS). Mouse liver mitochondrial permeability transition (PT) was induced by calcium overload in vitro and spectrophotometry was used to measure it. The scavenging activities of APS on superoxide anion (O₂^•−) and hydroxyl radical (•OH), which were produced by reduced nicotinamide adenine dinucleotide (NADH)—N-Methylphenazonium methyl sulfate (PMS) and hydrogen peroxide (H₂O₂)–Fe²⁺ system respectively, were measured by 4-nitrobluetetrazolium chloride (NBT) reduction and Fenton reaction colorimetry respectively. The Na₂S₂O₃ titration method was used to measure the scavenging activities of APS on H₂O₂. APS could inhibit TBARS production, protect mitochondria from PT, and scavenge O₂^•−, •OH and H₂O₂ significantly in a concentration-dependent manner respectively. The back of the neck of mice was injected subcutaneously with D-galactose to induce aging at a dose of 100 mg/kg/d for seven weeks. Moreover, the activities of catalase (CAT), surperoxide dismutase (SOD) and glutathione peroxidase (GPx) and anti-hydroxyl radical which were assayed by using commercial monitoring kits were increased significantly in vivo by APS. According to this research, APS protects mitochondria by scavenging reactive oxygen species (ROS), inhibiting mitochondrial PT and increasing the activities of antioxidases. Therefore, APS has the effect of promoting health.     

Polyamine Oxidase Triggers H2O2-Mediated Spermidine Improved Oxidative Stress Tolerance of Tomato Seedlings Subjected to Saline-Alkaline Stress

Saline-alkaline stress is one of several major abiotic stresses in crop production. Exogenous spermidine (Spd) can effectively increase tomato saline-alkaline stress resistance by relieving membrane lipid peroxidation damage. However, the mechanism through which exogenous Spd pre-treatment triggers the tomato antioxidant system to resist saline-alkaline stress remains unclear. Whether H₂O₂ and polyamine oxidase (PAO) are involved in Spd-induced tomato saline-alkaline stress tolerance needs to be determined. Here, we investigated the role of PAO and H₂O₂ in exogenous Spd-induced tolerance of tomato to saline-alkaline stress. Results showed that Spd application increased the expression and activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), and the ratio of reduced ascorbate (AsA) and glutathione (GSH) contents under saline-alkaline stress condition. Exogenous Spd treatment triggered endogenous H₂O₂ levels, SlPAO4 gene expression, as well as PAO activity under normal conditions. Inhibiting endogenous PAO activity by 1,8-diaminooctane (1,8-DO, an inhibitor of polyamine oxidase) significantly reduced H₂O₂ levels in the later stage. Moreover, inhibiting endogenous PAO or silencing the SlPAO4 gene increased the peroxidation damage of tomato leaves under saline-alkaline stress. These findings indicated that exogenous Spd treatment stimulated SlPAO4 gene expression and increased PAO activity, which mediated the elevation of H₂O₂ level under normal conditions. Consequently, the downstream antioxidant system was activated to eliminate excessive ROS accumulation and relieve membrane lipid peroxidation damage and growth inhibition under saline-alkaline stress. In conclusion, PAO triggered H₂O₂-mediated Spd-induced increase in the tolerance of tomato to saline-alkaline stress.     

Cloning,Characterization and Expression Pattern Analysis of a Cytosolic Copper/Zinc Superoxide Dismutase (SaCSD1) in a Highly Salt Tolerant Mangrove (Sonneratia alba)

Mangroves are critical marine resources for their remarkable ability to tolerate seawater. Antioxidant enzymes play an especially significant role in eliminating reactive oxygen species and conferring abiotic stress tolerance. In this study, a cytosolic copper/zinc superoxide dismutase (SaCSD1) cDNA of Sonneratia alba, a mangrove species with high salt tolerance, was successfully cloned and then expressed in Escherichia coli Rosetta-gami (designated as SaCSD1). SaCSD1 comprised a complete open reading frame (ORF) of 459 bp which encoded a protein of 152 amino acids. Its mature protein is predicted to be 15.32 kDa and the deduced isoelectric point is 5.78. SaCSD1 has high sequence similarity (85%–90%) with the superoxide dismutase (CSD) of some other plant species. SaCSD1 was expressed with 30.6% yield regarding total protein content after being introduced into the pET-15b (Sma I) vector for expression in Rosetta-gami and being induced with IPTG. After affinity chromatography on Ni-NTA, recombinant SaCSD1 was obtained with 3.2-fold purification and a specific activity of 2200 U/mg. SaCSD1 showed good activity as well as stability in the ranges of pH between 3 and 7 and temperature between 25 and 55 °C. The activity of recombinant SaCSD1 was stable in 0.25 M NaCl, Dimethyl Sulphoxide (DMSO), glycerol, and chloroform, and was reduced to a great extent in β-mercaptoethanol, sodium dodecyl sulfate (SDS), H₂O_2, and phenol. Moreover, the SaCSD1 protein was very susceptive to pepsin digestion. Real-time Quantitative Polymerase Chain Reaction (PCR) assay demonstrated that SaCSD1 was expressed in leaf, stem, flower, and fruit organs, with the highest expression in fruits. Under 0.25 M and 0.5 M salt stress, the expression of SaCSD1 was down-regulated in roots, but up-regulated in leaves.     

Overexpression of Ferredoxin,PETF, Enhances Tolerance to Heat Stress in Chlamydomonas reinhardtii

Reactive oxygen species (ROS) produced by plants in adverse environments can cause damage to organelles and trigger cell death. Removal of excess ROS can be achieved through the ascorbate scavenger pathway to prevent plant cell death. The amount of this scavenger can be regulated by ferredoxin (FDX). Chloroplastic FDXs are electron transfer proteins that perform in distributing photosynthetic reducing power. In this study, we demonstrate that overexpression of the endogenous photosynthetic FDX gene, PETF, in Chlamydomonas reinhardtii could raise the level of reduced ascorbate and diminish H₂O₂ levels under normal growth conditions. Furthermore, the overexpressing PETF transgenic Chlamydomonas lines produced low levels of H₂O₂ and exhibited protective effects that were observed through decreased chlorophyll degradation and increased cell survival under heat-stress conditions. The findings of this study suggest that overexpression of PETF can increase the efficiency of ROS scavenging in chloroplasts to confer heat tolerance. The roles of PETF in the downregulation of the ROS level offer a method for potentially improving the tolerance of crops against heat stress.     

Characterization of CaHsp70-1, a Pepper Heat-Shock Protein Gene in Response to Heat Stress and Some Regulation Exogenous Substances in Capsicum annuum L.

Pepper (Capsicum annuum L.) is sensitive to heat stress (HS). Heat shock proteins 70 (Hsp70s) play a crucial role in protecting plant cells against HS and control varies characters in different plants. However, CaHsp70-1 gene was not well characterized in pepper. In this study, CaHsp70-1 was cloned from the pepper thermotolerant line R9, which encoded a protein of 652 amino acids, with a molecular weight of 71.54 kDa and an isoelectric point of 5.20. CaHsp70-1 belongs to the cytosolic Hsp70 subgroup, and best matched with tomato SlHsp70. CaHsp70-1 was highly induced in root, stem, leaf and flower in R9 with HS treatment (40 °C for 2 h). In both thermosensitive line B6 and thermotolerant line R9, CaHsp70-1 significantly increased after 0.5 h of HS (40 °C), and maintained in a higher level after 4 h HS. The expression of CaHsp70-1 induced by CaCl₂, H₂O₂ and putrescine (Put) under HS were difference between B6 and R9 lines. The different expression patterns may be related to the differences in promoters of CaHsp70-1 from the two lines. These results suggest that CaHsp70-1 as a member of cytosolic Hsp70 subgroup, may be involved in HS defense response via a signal transduction pathway contained Ca²⁺, H₂O₂ and Put.     

Silencing the SpMPK1, SpMPK2, and SpMPK3 Genes in Tomato Reduces Abscisic Acid—Mediated Drought Tolerance

Drought is a major threat to agriculture production worldwide. Mitogen-activated protein kinases (MAPKs) play a pivotal role in sensing and converting stress signals into appropriate responses so that plants can adapt and survive. To examine the function of MAPKs in the drought tolerance of tomato plants, we silenced the SpMPK1, SpMPK2, and SpMPK3 genes in wild-type plants using the virus-induced gene silencing (VIGS) method. The results indicate that silencing the individual genes or co-silencing SpMPK1, SpMPK2, and SpMPK3 reduced the drought tolerance of tomato plants by varying degrees. Co-silencing SpMPK1 and SpMPK2 impaired abscisic acid (ABA)-induced and hydrogen peroxide (H₂O₂)-induced stomatal closure and enhanced ABA-induced H₂O₂ production. Similar results were observed when silencing SpMPK3 alone, but not when SpMPK1 and SpMPK2 were individually silenced. These data suggest that the functions of SpMPK1 and SpMPK2 are redundant, and they overlap with that of SpMPK3 in drought stress signaling pathways. In addition, we found that SpMPK3 may regulate H₂O₂ levels by mediating the expression of CAT1. Hence, SpMPK1, SpMPK2, and SpMPK3 may play crucial roles in enhancing tomato plants’ drought tolerance by influencing stomatal activity and H₂O₂ production via the ABA-H₂O₂ pathway.     

Nox Gene Expression and Cytochemical Localization of Hydrogen Peroxide in Polyporus umbellatus Sclerotial Formation

The effect of temperature shift on Polyporus umbellatus sclerotial development was investigated. Micromorphology of the sclerotia was observed by using scanning electron microscopy (SEM). The cytochemical localization of H₂O₂ expressed as CeCl₃ deposition at the subcellular level was observed by using transmission electron microscopy (TEM). Nox gene expression in sclerotia and mycelia was detected by quantitative real-time PCR (qRT-PCR) analysis. In addition, superoxide dismutase (SOD) and catalase (CAT) specific activities increased during sclerotial development and decreased after the antioxidant diphenyleneiodonium (DPI) was used. Results indicated that the temperature shift treatment induced P. umbellatus sclerotial formation. Compared with the mycelia, the Nox gene was respectively upregulated by 10.577-, 30.984- and 25.469-fold in the sclerotia of SI, SD and SM stages respectively. During the sclerotial formation, H₂O₂ accumulation was observed in the cell walls or around the organelle membranes of the mycelial cells. The antioxidant DPI decreased the generation of H₂O₂ in mycelial cells. The specific activity of SOD and CAT levels was decreased significantly by DPI. The activity of the two antioxidant enzymes in the mycelia increased much more during sclerotial formation (p < 0.05). Oxidative stress was closely associated with sclerotial development in P. umbellatus induced by temperature shift treatment.     

Response of Different Genotypes of Faba Bean Plant to Drought Stress

Drought stress is one of the major abiotic stresses that are a threat to crop production worldwide. Drought stress impairs the plants growth and yield. Therefore, the aim of the present experiment was to select the tolerant genotype/s on the basis of moprpho-physiological and biochemical characteristics of 10 Vicia faba genotypes (Zafar 1, Zafar 2, Shebam, Makamora, Espan, Giza Blanka, Giza 3, C4, C5 and G853) under drought stress. We studied the effect of different levels of drought stress i.e., (i) normal irrigation (ii) mild stress (iii) moderate stress, and (iv) severe stress on plant height (PH) plant⁻¹, fresh weight (FW) and dry weight (DW) plant⁻¹, area leaf⁻¹, leaf relative water content (RWC), proline (Pro) content, total chlorophyll (Total Chl) content, electrolyte leakage (EL), malondialdehyde (MDA), hydrogen peroxide (H₂O₂) content, and activities of catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) of genotypes of faba bean. Drought stress reduced all growth parameters and Total Chl content of all genotypes. However, the deteriorating effect of drought stress on the growth performance of genotypes “C5” and “Zafar 1” were relatively low due to its better antioxidant enzymes activities (CAT, POD and SOD), and accumulation of Pro and Total Chl, and leaf RWC. In the study, genotype “C5” and “Zafar 1” were found to be relatively tolerant to drought stress and genotypes “G853” and “C4” were sensitive to drought stress.     

Lithium insertion/deinsertion properties of new layered vanadium oxides obtained by oxidation of the precursor H2V3O8

The electrochemical behavior of the three new vanadium oxides M_yH_1−yV₃^VO₈ (M=Li, Na, K; y=0.6-0.9) towards lithium insertion was studied. For each compound, the main insertion phenomena, and the lithium amount which can be inserted, were determined from galvanostatic studies performed at different rates. In order to better explain the differences between the compounds, the galvanostatic intermittent titration technique was used and chronoamperometry experiments were performed. Cyclability studies have shown that the performances of M_0.6H_0.4V₃O₈ (M=Li, K) are not better than those of H₂V₃O₈. On the contrary, Na_0.9H_0.1V₃O₈ presents very interesting lithium insertion properties, with a specific capacity of 215 mAh g⁻¹ in the 1.5-4 V voltage range at C/10, and a capacity loss at the 40th cycle remaining smaller than 2.5%. Na_0.9H_0.1V₃O₈ is therefore a good candidate as positive electrode material for rechargeable lithium batteries.     

H2O2 Functions as a Downstream Signal of IAA to Mediate H2S-Induced Chilling Tolerance in Cucumber

Hydrogen sulfide (H₂S) plays a crucial role in regulating chilling tolerance. However, the role of hydrogen peroxide (H₂O₂) and auxin in H₂S-induced signal transduction in the chilling stress response of plants was unclear. In this study, 1.0 mM exogenous H₂O₂ and 75 μM indole-3-acetic acid (IAA) significantly improved the chilling tolerance of cucumber seedlings, as demonstrated by the mild plant chilling injury symptoms, lower chilling injury index (CI), electrolyte leakage (EL), and malondialdehyde content (MDA) as well as higher levels of photosynthesis and cold-responsive genes under chilling stress. IAA-induced chilling tolerance was weakened by N, N′-dimethylthiourea (DMTU, a scavenger of H₂O₂), but the polar transport inhibitor of IAA (1-naphthylphthalamic acid, NPA) did not affect H₂O₂-induced mitigation of chilling stress. IAA significantly enhanced endogenous H₂O₂ synthesis, but H₂O₂ had minimal effects on endogenous IAA content in cucumber seedlings. In addition, the H₂O₂ scavenger DMTU, inhibitor of H₂O₂ synthesis (diphenyleneiodonium chloride, DPI), and IAA polar transport inhibitor NPA reduced H₂S-induced chilling tolerance. Sodium hydrosulfide (NaHS) increased H₂O₂ and IAA levels, flavin monooxygenase (FMO) activity, and respiratory burst oxidase homolog (RBOH1) and FMO-like protein (YUCCA2) mRNA levels in cucumber seedlings. DMTU, DPI, and NPA diminished NaHS-induced H₂O₂ production, but DMTU and DPI did not affect IAA levels induced by NaHS during chilling stress. Taken together, the present data indicate that H₂O₂ as a downstream signal of IAA mediates H₂S-induced chilling tolerance in cucumber seedlings.     

Direct electrochemistry of Cytochrome c on natural nano-attapulgite clay modified electrode and its electrocatalytic reduction for H2O2

Natural nano-structural attapulgite clay was purified by mechanical stirring with the aid of ultrasonic wave and its structure and morphology was investigated by XRD and transmission electron microscopy (TEM). Cytochrome c was immobilized on attapulgite modified glassy carbon electrode. The interaction between Cytochrome c and attapulgite clay was examined by using UV-vis spectroscopy and electrochemical methods. The direct electron transfer of the immobilized Cytochrome c exhibited a pair of redox peaks with formal potential (E0′) of about 17 mV (versus SCE) in 0.1 mol/L, pH 7.0, PBS. The electrode reaction showed a surface-controlled process with the apparent heterogeneous electron transfer rate constant (k_s) of 7.05 s⁻¹ and charge-transfer coefficient (α) of 0.49. Cytochrome c immobilized on the attapulgite modified electrode exhibits a remarkable electrocatalytic activity for the reduction of hydrogen peroxide (H₂O₂). The calculated apparent Michaelis-Menten constant was 470 μmol/L, indicating a high catalytic activity of Cytochrome c immobilized on attapulgite modified electrode to the reduction of H₂O₂. Based on these, a third generation of reagentless biosensor can be constructed for the determination of H₂O₂.     

Discovery and Characterization of Two Novel Salt-Tolerance Genes in Puccinellia tenuiflora

Puccinellia tenuiflora is a monocotyledonous halophyte that is able to survive in extreme saline soil environments at an alkaline pH range of 9–10. In this study, we transformed full-length cDNAs of P. tenuiflora into Saccharomyces cerevisiae by using the full-length cDNA over-expressing gene-hunting system to identify novel salt-tolerance genes. In all, 32 yeast clones overexpressing P. tenuiflora cDNA were obtained by screening under NaCl stress conditions; of these, 31 clones showed stronger tolerance to NaCl and were amplified using polymerase chain reaction (PCR) and sequenced. Four novel genes encoding proteins with unknown function were identified; these genes had no homology with genes from higher plants. Of the four isolated genes, two that encoded proteins with two transmembrane domains showed the strongest resistance to 1.3 M NaCl. RT-PCR and northern blot analysis of P. tenuiflora cultured cells confirmed the endogenous NaCl-induced expression of the two proteins. Both of the proteins conferred better tolerance in yeasts to high salt, alkaline and osmotic conditions, some heavy metals and H₂O₂ stress. Thus, we inferred that the two novel proteins might alleviate oxidative and other stresses in P. tenuiflora.     

A Novel Thylakoid Ascorbate Peroxidase from Jatrophacurcas Enhances Salt Tolerance in Transgenic Tobacco

Ascorbate peroxidase (APX) plays an important role in the metabolism of hydrogen peroxide in higher plants. In the present study, a novel APX gene (JctAPX) was cloned from Jatropha curcas L. The deduced amino acid sequence was similar to that of APX of some other plant species. JctAPX has a chloroplast transit peptide and was localized to the chloroplasts by analysis with a JctAPX-green fluorescent protein (GFP) fusion protein. Quantitative polymerase chain reaction (qPCR) analysis showed that JctAPX was constitutively expressed in different tissues from J. curcas and was upregulated by NaCl stress. To characterize its function in salt tolerance, the construct p35S: JctAPX was created and successfully introduced into tobacco by Agrobacterium-mediated transformation. Compared with wild type (WT), the transgenic plants exhibited no morphological abnormalities in the no-stress condition. However, under 200 mM NaCl treatment, JctAPX over-expressing plants showed increased tolerance to salt during seedling establishment and growth. In addition, the transgenic lines showed higher chlorophyll content and APX activity, which resulted in lower H₂O₂ content than WT when subjected to 400 mM NaCl stress. These results suggest that the increased APX activity in the chloroplasts from transformed plants increased salt tolerance by enhancing reactive oxygen species (ROS)-scavenging capacity under short-term NaCl stress conditions.     

Phenolic Composition and Antioxidant Properties of Different Peach [Prunus persica (L.) Batsch] Cultivars in China

China is an important centre of diversity for Prunus persica. In the present study, 17 Chinese peach cultivars were evaluated for phenolic content and antioxidant activity. Neochlorogenic acid (NCHA), chlorogenic acid (CHA), procyanidin B1 (B1), catechin (CAT), cyanidin-3-O-glucoside (C3G), quercetin-3-O-galactoside (Q3GAL), quercetin-3-O-glucoside (Q3GLU), quercetin-3-O-rutinoside (Q3R), and kaempferol-3-O-rutinoside (K3R) were identified and quantified. CHA and CAT were the predominant components in both the peel and pulp of this fruit. In general, peel extracts showed higher antioxidant activities than the pulp counterparts, consistent with the observed higher phenolic content. The melting peach cultivar “Xinyu” showed the highest antioxidant potency composite (APC) index. The principal component analysis (PCA) of peel phenolics showed a clear distinction between the melting peach and nectarine. Overall, peach cultivars rich in hydroxycinnamates and flavan-3-ols showed relatively higher antioxidant activities and might be excellent sources of phytochemicals and natural antioxidants.     

The Metallothionein Gene,TaMT3, from Tamarix androssowii Confers Cd2+ Tolerance in Tobacco

Cadmium (Cd) is a nonessential microelement and low concentration Cd²⁺ has strong toxicity to plant growth. Plant metallothioneins, a class of low molecular, cystein(Cys)-rich and heavy-metal binding proteins, play an important role in both metal chaperoning and scavenging of reactive oxygen species (ROS) with their large number of cysteine residues and therefore, protect plants from oxidative damage. In this study, a metallothionein gene, TaMT3, isolated from Tamarix androssowii was transformed into tobacco (Nicotiana tobacum) through Agrobacterium-mediated leaf disc method, and correctly expressed under the control of 35S promoter. Under Cd²⁺ stress, the transgenic tobacco showed significant increases of superoxide dismutase (SOD) activity and chlorophyll concentration, but decreases of peroxidase (POD) activity and malondialdehyde (MDA) accumulation when compared to the non-transgenic tobacco. Vigorous growth of transgenic tobacco was observed at the early development stages, resulting in plant height and fresh weight were significantly larger than those of the non-transgenic tobacco under Cd²⁺ stress. These results demonstrated that the expression of the exogenous TaMT3 gene increased the ability of ROS cleaning-up, indicating a stronger tolerance to Cd²⁺ stress.