Effect of thiocyanate on the peroxidase and pseudocatalase activities of Leishmania major ascorbate peroxidase

We report here that the Leishmania major ascorbate peroxidase (LmAPX), having similarity with plant ascorbate peroxidase, catalyzes the oxidation of suboptimal concentration of ascorbate to monodehydroascorbate (MDA) at physiological pH in the presence of added H₂O₂ with concurrent evolution of O₂. This pseudocatalatic degradation of H₂O₂ to O₂ is solely dependent on ascorbate and is blocked by a spin trap, α-phenyl-n-tert-butyl nitrone (PBN), indicating the involvement of free radical species in the reaction process. LmAPX thus appears to catalyze ascorbate oxidation by its peroxidase activity, first generating MDA and H₂O with subsequent regeneration of ascorbate by the reduction of MDA with H₂O₂ evolving O₂ through the intermediate formation of O₂⁻. Interestingly, both peroxidase and ascorbate-dependent pseudocatalatic activity of LmAPX are reversibly inhibited by SCN⁻ in a concentration dependent manner. Spectral studies indicate that ascorbate cannot reduce LmAPX compound II to the native enzyme in presence of SCN⁻. Further kinetic studies indicate that SCN⁻ itself is not oxidized by LmAPX but inhibits both ascorbate and guaiacol oxidation, which suggests that SCN⁻ blocks initial peroxidase activity with ascorbate rather than subsequent nonenzymatic pseudocatalatic degradation of H₂O₂ to O₂. Binding studies by optical difference spectroscopy indicate that SCN⁻ binds LmAPX (Kd = 100 ± 10 mM) near the heme edge. Thus, unlike mammalian peroxidases, SCN⁻ acts as an inhibitor for Leishmania peroxidase to block ascorbate oxidation and subsequent pseudocatalase activity.     

Responses of antioxidant gene, protein and enzymes to salinity stress in two genotypes of perennial ryegrass (Lolium perenne) differing in salt tolerance

Salinity could damage cellular membranes through overproduction of reactive oxygen species (ROS), while antioxidant capacities play a vital role in protecting plants from salinity caused oxidative damages. The objective of this study was to investigate the toxic effect of salt on the antioxidant enzyme activities, isoforms and gene expressions in perennial ryegrass (Lolium perenne L.). Salt-tolerant ‘Quickstart II’ and salt-sensitive ‘DP1′ were subjected to 0 and 250 mM NaCl for 12 d. Salt stress increased the content of lipid peroxidation (MDA), electrolyte leakage (EL) and hydrogen peroxide (H₂O₂), to a greater extent in salt-sensitive genotype. Salt-stressed plant leaves exhibited a greater activity of superoxide dismutase (SOD, EC 1.15.1.1), peroxidase (POD, EC 1.11.1.7), ascorbate peroxidase (APX, EC 1.11.1.11) at 4 d after treatment (DAT), but a lower level of enzyme activity at 8 and 12 d, when compared to the control. Catalase (CAT, EC 1.11.1.6) activity was greater at 4 DAT and thereafter decreased in salt tolerant genotype relative to the control, whereas lower than the control during whole experiment period for salt-sensitive genotype. There were different patterns of five isoforms of SOD, POD and two isoforms of APX between two genotypes. Antioxidant gene expression was positively related to isoenzymatic and total enzymatic activities during 12-d salt-treated leaves of two genotypes, with a relatively higher level in salt-tolerant genotype. Thus, salt tolerance could be related to the constitutive/induced antioxidant gene, leading to more efficient enzyme stimulation and protection in perennial ryegrass.     

Characterization and expression of cytoplasmic copper/zinc superoxide dismutase (CuZn SOD) gene under temperature and hydrogen peroxide (H2O2) in rotifer Brachionus calyciflorus

Superoxide dismutase (SOD, EC 1.15.1.1) is an important antioxidant enzyme that protects organs from damage by reactive oxygen species (ROS). We cloned cDNA encoding SOD activated with copper/zinc (CuZn SOD) from the rotifer Brachionus calyciflorus Pallas. The full-length cDNA of CuZn SOD was 692 bp and had a 465 bp open reading frame encoding 154 amino acids. The deduced amino acid sequence of B. calyciflorus CuZn SOD showed 63.87%, 60.00%, 59.74% and 48.89% similarity with the CuZn SOD of the Ctenopharyn godonidella, Schistosoma japonicum, Drosophila melanogaster and Caenorhabditis elegans, respectively. The phylogenetic tree constructed based on the amino acid sequences of CuZn SODs from B. calyciflorus and other organisms revealed that rotifer is closely related to nematode. Analysis of the expression of CuZn SOD under different temperatures (15, 30 and 37 °C) revealed that its expression was enhanced 4.2-fold (p < 0.001) at 30 °C after 2 h, however, the lower temperature (15 °C) promoted CuZn SOD transiently (4.1-fold, p < 0.001) and then the expression of CuZn SOD decreased to normal level (p > 0.05). When exposed to H₂O₂ (0.1 mM), CuZn SOD, manganese superoxide dismutase (Mn SOD) and catalase (CAT) gene were upregulated, and in addition, the mRNA expression of CuZn SOD gene was induced instantaneously after exposure to vitamin E. It indicates that the CuZn SOD gene would be an important gene in response to oxidative and temperature stress.     

Cytokinin oxidase/dehydrogenase overexpression modifies antioxidant defense against heat,drought and their combination in Nicotiana tabacum plants

Cytokinins (CKs) as well as the antioxidant enzyme system (AES) play important roles in plant stress responses. The expression and activity of antioxidant enzymes (AE) were determined in drought, heat and combination of both stresses, comparing the response of tobacco plants overexpressing the main cytokinin degrading enzyme, cytokinin oxidase/dehydrogenase, under the control of root-specific WRKY6 promoter (W6:CKX1 plants) or constitutive promoter (35S:CKX1 plants) and the corresponding wild-type (WT). Expression levels as well as activities of cytosolic ascorbate peroxidase, catalase 3, and cytosolic superoxide dismutase were low under optimal conditions and increased after heat and combined stress in all genotypes. Unlike catalase 3, two other peroxisomal enzymes, catalase 1 and catalase 2, were transcribed extensively under control conditions. Heat stress, in contrast to drought or combined stress, increased catalase 1 and reduced catalase 2 expression in WT and W6:CKX1 plants. In 35S:CKX1, catalase 1 expression was enhanced by heat or drought, but not under combined stress conditions. Mitochondrial superoxide dismutase expression was generally higher in 35S:CKX1 plants than in WT. Genes encoding for chloroplastic AEs, stromatal ascorbate peroxidase, thylakoidal ascorbate peroxidase and chloroplastic superoxide dismutase, were strongly transcribed under control conditions. All stresses down-regulated their expression in WT and W6:CKX1, whereas more stress-tolerant 35S:CKX1 plants maintained high expression during drought and heat. The achieved data show that the effect of down-regulation of CK levels on AES may be mediated by altered habit, resulting in improved stress tolerance, which is associated with diminished stress impact on photosynthesis, and changes in source/sink relations.     

Cinnamic acid pretreatment mitigates chilling stress of cucumber leaves through altering antioxidant enzyme activity

To elucidate the physiological mechanism of chilling stress mitigated by cinnamic acid (CA) pretreatment, a cucumber variety (Cucumis sativus cv. Jinchun no. 4) was pretreated with 50 μM CA for 2 d and was then cultivated at two temperatures (15/8 and 25/18 °C) for 1 d. We investigated whether exogenous CA could protect cucumber plantlets from chilling stress (15/8 °C) and examined whether the protective effect was associated with the regulation of antioxidant enzymes and lipid peroxidation. At 2 d, exogenous CA did not influence plant growth, but induced the activities of some antioxidant enzymes, including superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), guaiacol peroxidase (GPX, EC 1.11.1.7), glutathione peroxidase (GSH-Px, EC 1.6.4.2) and ascorbate peroxidase (APX, EC 1.11.1.11) in cucumber leaves, and it also elevated the contents of reduced glutathione (GSH) and ascorbate (AsA). When CA was rinsed and the CA-pretreated seedlings were exposed to different temperatures, the antioxidant activities in leaves at 3 d had undergone additional change. Chilling increased the activities of CAT, GSH-PX, APX, GSH and AsA in leaves, but the combination of CA pretreatment and chilling enhanced the antioxidant activities even more. Moreover, chilling inhibited plant growth and increased the contents of malonaldehyde (MDA), superoxide radical (O₂⁻) and hydrogen peroxide (H₂O₂) in cucumber leaves, and the stress resulted in 87.5% of the second leaves being withered. When CA pretreatment was combined with the chilling stress, we observed alleviated growth inhibition and decreased contents of MDA, H₂O₂ and O₂⁻ in comparison to non-pretreated stressed plants, and found that the withered leaves occurred at a rate of 25.0%. We propose that CA pretreatment increases antioxidant enzyme activities in chilling-stressed leaves and decreases lipid peroxidation to some extent, enhancing the tolerance of cucumber leaves to chilling stress.     

Photosynthesis is improved by exogenous calcium in heat-stressed tobacco plants

Effects of exogenous calcium chloride (CaCl₂) (20 mM) on photosynthetic gas exchange, photosystem II photochemistry, and the activities of antioxidant enzymes in tobacco plants under high temperature stress (43 °C for 2 h) were investigated. Heat stress resulted in a decrease in net photosynthetic rate (P_n), stomatal conductance as well as the apparent quantum yield (AQY) and carboxylation efficiency (CE) of photosynthesis. Heat stress also caused a decrease of the maximal photochemical efficiency of primary photochemistry (F_v/F_m). On the other hand, CaCl₂ application improved P_n, AQY, and CE as well as F_v/F_m under high temperature stress. Heat stress reduced the activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), peroxidase (POD), whereas the activities of these enzymes either decreased less or increased in plants pretreated with CaCl₂; glutathione reductase (GR) activity increased under high temperature, and it increased more in plants pretreated with CaCl₂. There was an obvious accumulation of H₂O₂ and O₂⁻ under high temperature, but CaCl₂ application decreased the contents of H₂O₂ and O₂⁻ under heat stress conditions. Heat stress induced the level of heat shock protein 70 (HSP70), while CaCl₂ pretreatment enhanced it. These results suggested that photosynthesis was improved by CaCl₂ application in heat-stressed plants and such an improvement was associated with an improvement in stomatal conductance and the thermostability of oxygen-evolving complex (OEC), which might be due to less accumulation of reactive oxygen species.     

Two rice cytosolic ascorbate peroxidases differentially improve salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

In order to determine the different roles of rice (Oryza sativa L.) cytosolic ascorbate peroxidases (OsAPXa and OsAPXb, GenBank accession nos. D45423 and AB053297, respectively) under salt stress, transgenic Arabidopsis plants over-expressing OsAPXa or OsAPXb were generated, and they all exhibited increased tolerance to salt stress compared to wild-type plants. Moreover, transgenic lines over-expressing OsAPXb showed higher salt tolerance than OsAPXa transgenic lines as indicated by root length and total chlorophyll content. In addition to ascorbate peroxidase (APX) activity, antioxidant enzyme activities of catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR), which are also involved in the salt tolerance process, and the content of H₂O₂ were also assayed in both transgenic and wild-type plants. The results showed that the overproduction of OsAPXb enhanced and maintained APX activity to a much higher degree than OsAPXa in transgenic Arabidopsis during treatment with different concentrations of NaCl, enhanced the active oxygen scavenging system, and protected plants from salt stress by equilibrating H₂O₂ metabolism. Our findings suggest that the rice cytosolic OsAPXb gene has a more functional role than OsAPXa in the improvement of salt tolerance in transgenic plants. Zhenqiang Lu and Dali Liu contributed equally.     

Ectopic over-expression of peroxisomal ascorbate peroxidase (SbpAPX) gene confers salt stress tolerance in transgenic peanut (Arachis hypogaea)

Peroxisomal ascorbate peroxidase gene (SbpAPX) of an extreme halophyte Salicornia brachiata imparts abiotic stress endurance and plays a key role in the protection against oxidative stress. The cloned SbpAPX gene was transformed to local variety of peanut and about 100 transgenic plants were developed using optimized in vitro regeneration and Agrobacterium mediated genetic transformation method. The T₀ transgenic plants were confirmed for the gene integration; grown under controlled condition in containment green house facility; seeds were harvested and T₁ plants were raised. Transgenic plants (T₁) were further confirmed by PCR using gene specific primers and histochemical GUS assay. About 40 transgenic plants (T₁) were selected randomly and subjected for salt stress tolerance study. Transgenic plants remained green however non-transgenic plants showed bleaching and yellowish leaves under salt stress conditions. Under stress condition, transgenic plants continued normal growth and completed their life cycle. Transgenic peanut plants exhibited adequate tolerance under salt stress condition and thus could be explored for the cultivation in salt affected areas for the sustainable agriculture.     

A salt-inducible chloroplastic monodehydroascorbate reductase from halophyte Avicennia marina confers salt stress tolerance on transgenic plants

Plant growth and productivity are adversely affected by various abiotic stress factors. In our previous study, we used Avicennia marina, a halophytic mangrove, as a model plant system for isolating genes functioning in salt stress tolerance. A large scale random EST sequencing from a salt stressed leaf tissue cDNA library of one month old A. marina plants resulted in identification of a clone showing maximum homology to Monodehydroascorbate reductase (Am-MDAR). MDAR plays a key role in regeneration of ascorbate from monodehydroascorbate for ROS scavenging. In this paper, we report the cellular localization and the ability to confer salt stress tolerance in transgenic tobacco of this salt inducible Am-MDAR. A transit peptide at the N-terminal region of Am-MDAR suggested that it encodes a chloroplastic isoform. The chloroplastic localization was confirmed by stable transformation and expression of the Am-MDAR-GFP fusion protein in tobacco. Transgenic tobacco plants overexpressing Am-MDAR survived better under conditions of salt stress compared to untransformed control plants. Assays of enzymes involved in ascorbate–glutathione cycle revealed an enhanced activity of MDAR and ascorbate peroxidase whereas the activity of dehyroascorbate reductase was reduced under salt stressed and unstressed conditions in Am-MDAR transgenic lines. The transgenic lines showed an enhanced redox state of ascorbate and reduced levels of malondialdehyde indicating its enhanced tolerance to oxidative stress. The results of our studies could be used as a starting point for genetic engineering of economically important plants tolerant to salt stress.     

Salt tolerance and activity of antioxidative enzymes of transgenic finger millet overexpressing a vacuolar H-pyrophosphatase gene (SbVPPase) from Sorghum bicolor

A vacuolar proton pyrophosphatase cDNA clone was isolated from Sorghum bicolor (SbVPPase) using end-to-end gene-specific primer amplification. It showed 80–90% homology at the nucleotide and 85–95% homology at the amino acid level with other VPPases. The gene was introduced into expression vector pCAMBIA1301 under the control of the cauliflower mosaic virus 35S (CaMV35S) promoter and transformed into Agrobacterium tumifaciens strain LBA4404 to infect embryogenic calli of finger millet (Eleusine coracana). Successful transfer of SbVPPase was confirmed by a GUS histochemical assay and PCR analysis. Both, controls and transgenic plants were subjected to 100 and 200 mM NaCl and certain biochemical and physiological parameters were studied. Relative water content (RWC), plant height, leaf expansion, finger length and width and grain weight were severely reduced (50–70%), and the flowering period was delayed by 20% in control plants compared to transgenic plants under salinity stress. With increasing salt stress, the proline and chlorophyll contents as well as the enzyme activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and glutathione reductase (GR) increased by 25–100% in transgenics, while malondialdehyde (MDA) showed a 2–4-fold decrease. The increased activities of antioxidant enzymes and the reduction in the MDA content suggest efficient scavenging of reactive oxygen species (ROS) in transgenics and, as a consequence, probably alleviation of salt stress. Also, the leaf tissues of the transgenics accumulated 1.5–2.5-fold higher Na⁺ and 0.4–0.8-fold higher K⁺ levels. Together, these results clearly demonstrate that overexpression of SbVPPase in transgenic finger millet enhances the plant's performance under salt stress.     

Molecular characterization of two glutathione peroxidase genes of Panax ginseng and their expression analysis against environmental stresses

Glutathione peroxidases (GPXs) are a group of enzymes that protect cells against oxidative damage generated by reactive oxygen species (ROS). GPX catalyzes the reduction of hydrogen peroxide (H₂O₂) or organic hydroperoxides to water or alcohols by reduced glutathione. The presence of GPXs in plants has been reported by several groups, but the roles of individual members of this family in a single plant species have not been studied. Two GPX cDNAs were isolated and characterized from the embryogenic callus of Panax ginseng. The two cDNAs had an open reading frame (ORF) of 723 and 681 bp with a deduced amino acid sequence of 240 and 226 residues, respectively. The calculated molecular mass of the matured proteins are approximately 26.4 kDa or 25.7 kDa with a predicated isoelectric point of 9.16 or 6.11, respectively. The two PgGPXs were elevated strongly by salt stress and chilling stress in a ginseng seedling. In addition, the two PgGPXs showed different responses against biotic stress. The positive responses of PgGPX to the environmental stimuli suggested that ginseng GPX may help to protect against environmental stresses.     

Homologous expression of γ-glutamylcysteine synthetase increases grain yield and tolerance of transgenic rice plants to environmental stresses

Various environmental stresses induce reactive oxygen species (ROS), causing deleterious effects on plant cells. Glutathione (GSH), a critical antioxidant, is used to combat ROS. GSH is produced by γ-glutamylcysteine synthetase (γ-ECS) and glutathione synthetase (GS). To evaluate the functional roles of the Oryza sativa L. Japonica cv. Ilmi ECS (OsECS) gene, we generated transgenic rice plants overexpressing OsECS under the control of an inducible promoter (Rab21). When grown under saline conditions (100 mM) for 4 weeks, 2-independent transgenic (TGR1 and TGR2) rice plants remained bright green in comparison to control wild-type (WT) rice plants. TGR1 and TGR2 rice plants also showed a higher GSH/GSSG ratio than did WT rice plants in the presence of 100 mM NaCl, which led to enhanced redox homeostasis. TGR1 and TGR2 rice plants also showed lower ion leakage and higher chlorophyll-fluorescence when exposed to 10 μM methyl viologen (MV). Furthermore, the TGR1 and TGR2 rice seeds had approximately 1.5-fold higher germination rates in the presence of 200 mM salt. Under paddy field conditions, OsECS-overexpression in transgenic rice plants increased rice grain yield (TGW) and improved biomass. Overall, our results show that OsECS overexpression in transgenic rice increases tolerance and germination rate in the presence of abiotic stress by improving redox homeostasis via an enhanced GSH pool. Our findings suggest that increases in grain yield by OsECS overexpression could improve crop yields under natural environmental conditions.     

The inductive responses of the antioxidant enzymes by salt stress in the rice (Oryza sativa L.)

To access contributions of inductive responses of the antioxidant enzymes in the resistance to salt stress, activities of the enzymes were determined in the rice (Oryza sativaL. cv. Dongjin) plant. In the leaves of the rice plant, salt stress preferentially enhanced the content of H₂O₂ as well as the activities of the superoxide dismutase (SOD), ascorbate peroxidase (APX), and peroxidase specific to guaiacol, whereas it induced the decrease of catalase activity. On the other hand, salt stress had little effect on the activity levels of glutathione reductase (GR). In order to analyze the changes of antioxidant enzyme isoforms against salt stress, plant extracts were subjected to native PAGE. Leaves of the rice plant had two isoforms of Mn-SOD and five isoforms of Cu/Zn-SOD. Fe-SOD isoform was not observed in the activity gels. Expression of Cu/Zn-1, -2, and Mn-SOD-2 isoforms was preferentially enhanced by salt stress. Seven APX isoforms were presented in the leaves of the rice plants. The intensities of APX-4 to -7 were enhanced by salt stress, whereas those of APX-1 to -3 were minimally in changed response to salt stress. There were seven GR isoforms in the leaves of rice plants. Levels of activity for most GR isoforms did not change in the stressed plants compared to the control plants. On the other hand, the levels of activity for most antioxidant enzymes changed little in the roots of stressed plants compared to the control plants. These results collectively suggest that SOD leads to the overproduction of hydrogen peroxide in the leaves of rice plants subjected to salt stress: The overproduction of hydrogen peroxide functions as the signal of salt stress, which induces the induction of specific APX isoforms but not specific GR isoforms under catalase deactivation.     

Molecular cloning and characterization of the glutathione reductase gene from Stipa purpurea

Reactive oxygen species (ROS) are a key factor in abiotic stresses; excess ROS is harmful to plants. Glutathione reductase (GR) plays an important role in scavenging ROS in plants. Here, a GR gene, named SpGR, was cloned from Stipa purpurea and characterized. The full-length open reading frame was 1497 bp, encoding 498 amino acids. Subcellular localization analysis indicated that SpGR was localized to both the plasma membrane and nucleus. The expression of SpGR was induced by cold, salt, and drought stresses. Functional analysis indicated that ectopic expression of SpGR in Arabidopsis thaliana resulted in greater tolerance to salt stress than that of wild-type plants, but no difference under cold or drought treatments. The results of GR activity and GSSG and GSH content analyses suggested that, under salt stress, transgenic plants produced more GR to reduce GSSG to GSH for scavenging ROS than wild-type plants. Therefore, SpGR may be a candidate gene for plants to resist abiotic stress.