Co-expression of monodehydroascorbate reductase and dehydroascorbate reductase from Brassica rapa effectively confers tolerance to freezing-induced oxidative stress

Plants are exposed to various environmental stresses and have therefore developed antioxidant enzymes and molecules to protect their cellular components against toxicity derived from reactive oxygen species (ROS). Ascorbate is a very important antioxidant molecule in plants, and monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) and dehydroascorbate reductase (DHAR; EC 1.8.5.1) are essential to regeneration of ascorbate for maintenance of ROS scavenging ability. The MDHAR and DHAR genes from Brassica rapa were cloned, transgenic plants overexpressing either BrMDHAR and BrDHAR were established, and then, each transgenic plant was hybridized to examine the effects of co-expression of both genes conferring tolerance to freezing. Transgenic plants co-overexpressing BrMDHAR and BrDHAR showed activated expression of relative antioxidant enzymes, and enhanced levels of glutathione and phenolics under freezing condition. Then, these alteration caused by co-expression led to alleviated redox status and lipid peroxidation and consequently conferred improved tolerance against severe freezing stress compared to transgenic plants overexpressing single gene. The results of this study suggested that although each expression of BrMDHAR or BrDHAR was available to according tolerance to freezing, the simultaneous expression of two genes generated synergistic effects conferring improved tolerance more effectively even severe freezing.     

Effects of artificially enhanced levels of ascorbate and glutathione on the enzymes monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase in spinach (Spinacia oleracea)

Effect of high intracellular concentrations of the antioxidants ascorbate and glutathione on the extractable activity of the reducting enzymes dehydroascorbate reductase, monodehydroascorbate reductase, and glutathione reductase were investigated with spinach cells ( Spinacia oleracea ). An elevated ascorbate concentration was obtained by treatment with the ascorbate biosynthesis precursor L-galactono-1,4-lactone (GAL). To increase the intracellular level of glutathione, cells were treated with the 5-oxo-L-proline analog L-2-oxothiazolidin-4-carboxylate (OTC), or with the peroxidative herbicide acifluorfen (sodium 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid). Extractable monodehydroascorbate reductase activity increased in the presence of a high level of ascorbate or glutathione, and enzyme activity was at maximum when cells were treated with acifluorfen + OTC, or acifluorfen + GAL. Extractable dehydroascorbate reductase activity decreased when the intracellular concentration of glutathione was high and non-enzymatic reduction of dehydroascorbate by glutathione was the dominant reaction. Maximal decrease of enzyme activity was found in cells treated with acifluorfen + OTC. Extractable activity of glutathione reductase (GR) increased after treatment of cells with acifluorfen alone, or acifluorfen + OTC, but enzyme activity was unaffected by a high intracellular concentration of glutathione obtained by treatment of cells with OTC alone, or by treatment with acifluorfen + GAL. The degree of GR activation seemed to be controlled by several factors including inhibition by a high concentration of glutathione and possibly oxidative damage to the enzyme. Overall, the enzymes tested in this study, which provide the reduced forms of ascorbate and glutathione, were differently affected by high antioxidant levels.     

Chloroplasts and mitochondria have multiple heat tolerant isozymes of SOD and APX in leaf and inflorescence in Chenopodium album

Thermal stability of antioxidant defense enzymes superoxide dismutase (SOD, EC 1.15.1.1) and ascorbate peroxidase (APX, EC 1.11.1.11) was studied in chloroplasts and mitochondria of leaf and inflorescence in heat adaptive weed Chenopodium album. Leaf samples were taken in March (31 °C/14 °C) and young inflorescence (INF) was sampled at flowering in April (40 °C/21 °C). Leaf and INF chloroplast and mitochondrial fractions were subjected to elevated temperatures in vitro (5–100 °C) for 30′. SOD and APX showed activity even after boiling treatment in both chloroplast and mitochondria of leaf and INF. SOD was more heat stable than APX in both chloroplasts and mitochondria in both the tissues. Chloroplast contained more heat stable SOD and APX isozymes than mitochondria in both leaf and INF. To the best of our knowledge this is the first report showing presence of thermostable APX isozymes (100 °C for 30′) in chloroplasts and mitochondria in C. album. Heat stable isozymes of SOD and APX in chloroplasts and mitochondria in leaves and inflorescence may contribute to heat tolerance in C. album.     

Exploring the genomes: From Arabidopsis to crops

Model systems have played a crucial role for understanding biological processes at genetic, molecular and systems levels. Arabidopsis thaliana is one of the best studied model species for higher plants. Large genomic resources and mutant collections made Arabidopsis an excellent source for functional and comparative genomics. Rice and Brachypodium have a great potential to become model systems for grasses. Given the agronomic importance of grass crops, it is an attractive strategy to apply knowledge from Arabidopsis to grasses. Despite many efforts successful reports are sparse. Knowledge transfer should generally work best between orthologous genes that share functionality and a common ancestor. In higher plants, however, recent genome projects revealed an active and rapid evolution of genome structure, which challenges the concept of one-to-one orthologous mates between two species. In this study, we estimated on the example of protein families that are involved in redox related processes, the impact of gene expansions on the success rate for a knowledge transfer from Arabidopsis to the grass species rice, sorghum and Brachypodium. The sparse synteny between dicot and monocot plants due to frequent rearrangements, translocations and gene losses strongly impairs and reduces the number of orthologs detectable by positional conservation. To address the limitations of sparse synteny and expanded gene families, we applied for the detection of orthologs in this study orthoMCL, a sequence-based approach that allows to group closely related paralogs into one orthologous gene cluster. For a total of 49 out of 170 Arabidopsis genes we could identify conserved copy numbers between the dicot model and the grass annotations whereas approximately one third (34.7%, 59 genes) of the selected Arabidopsis genes lack an assignment to any of the grass genome annotations. The remaining 62 Arabidopsis genes represent groups that are considerably biased in their copy numbers between Arabidopsis and all or most of the three grass genomes.     

The effects of short-term selenium stress on Polish and Finnish wheat seedlings-EPR, enzymatic and fluorescence studies

Biochemical analyses of antioxidant content were compared with measurements of fluorescence and electron paramagnetic resonance (EPR) to examine the alteration of radicals in wheat seedlings exposed to 2 days of selenium stress. Two genotypes of Polish and one of Finnish wheat, differing in their tolerance to long-term stress treatment, were cultured under hydroponic conditions to achieve the phase of 3-leave seedlings. Afterwards, selenium (sodium selenate, 100 μM concentration) was added to the media. After Se-treatment, all varieties showed an increase in carbohydrates (soluble and starch), ascorbate and glutathione content in comparison to non-stressed plants. These changes were more visible in Finnish wheat. On the basis of lipid peroxidation measurements, Finnish wheat was recognized as the genotype more sensitive to short-term Se-stress than the Polish varieties. The antioxidant enzyme activities (superoxide dismutase, ascorbate peroxidase and glutathione reductase) increased in Polish genotypes, whereas they decreased in Finnish wheat plants cultured on Se media. The action of reactive oxygen species in short-term action of Se stress was confirmed by the reduction of PSII and PSI system activities (measured by fluorescence parameters and EPR, respectively). EPR studies showed changes in redox status (especially connected with Mn(II)/Mn(III), and semiquinone/quinone ratios) in wheat cell after Se treatment. The involvement of the carbohydrate molecules as electron traps in production of long-lived radicals is postulated.     

The cellular redox state as a modulator in cadmium and copper responses in Arabidopsis thaliana seedlings

The cellular redox state is an important determinant of metal phytotoxicity. In this study we investigated the influence of cadmium (Cd) and copper (Cu) stress on the cellular redox balance in relation to oxidative signalling and damage in Arabidopsis thaliana. Both metals were easily taken up by the roots, but the translocation to the aboveground parts was restricted to Cd stress. In the roots, Cu directly induced an oxidative burst, whereas enzymatic ROS (reactive oxygen species) production via NADPH oxidases seems important in oxidative stress caused by Cd. Furthermore, in the roots, the glutathione metabolism plays a crucial role in controlling the gene regulation of the antioxidative defence mechanism under Cd stress. Metal-specific alterations were also noticed with regard to the microRNA regulation of CuZnSOD gene expression in both roots and leaves. The appearance of lipid peroxidation is dual: it can be an indication of oxidative damage as well as an indication of oxidative signalling as lipoxygenases are induced after metal exposure and are initial enzymes in oxylipin biosynthesis.In conclusion, the metal-induced cellular redox imbalance is strongly dependent on the chemical properties of the metal and the plant organ considered. The stress intensity determines its involvement in downstream responses in relation to oxidative damage or signalling.     

Silicon-mediated alleviation of Mn toxicity in Cucumis sativus in relation to activities of superoxide dismutase and ascorbate peroxidase

The effects of exogenous silicon (Si) on plant growth, activities of superoxide dismutase (SOD), guaiacol peroxidase (GPX), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), glutathione reductase (GR) and catalase, and concentrations of ascorbate and glutathione were investigated in cucumber (Cucumis sativus L.) plants treated with excess manganese (Mn) (600 microM). Compared with the treatment of normal Mn (10 microM), excess Mn significantly increased H2O2 concentration and lipid peroxidation indicated by accumulation of thiobarbituric acid reactive substances. The leaves showed apparent symptoms of Mn toxicity and the plant growth was significantly inhibited by excess Mn. The addition of Si significantly decreased lipid peroxidation caused by excess Mn, inhibited the appearance of Mn toxicity symptoms, and improved plant growth. This alleviation of Mn toxicity by Si was related to a significant increase in the activities of SOD, APX, DHAR and GR and the concentrations of ascorbate and glutathione.     

Salicylic acid alleviates decreases in photosynthesis under salt stress by enhancing nitrogen and sulfur assimilation and antioxidant metabolism differentially in two mungbean cultivars

Salicylic acid (SA) is known to affect photosynthesis under normal conditions and induces tolerance in plants to biotic and abiotic stresses through influencing physiological processes. In this study, physiological processes were compared in salt-tolerant (Pusa Vishal) and salt-sensitive (T44) cultivars of mungbean and examined how much these processes were induced by SA treatment to alleviate decrease in photosynthesis under salt stress. Cultivar T44 accumulated higher leaf Na⁺ and Cl⁻ content and exhibited greater oxidative stress than Pusa Vishal. Activity of antioxidant enzymes, ascorbate peroxidase (APX) and glutathione reductase (GR) was greater in Pusa Vishal than T44. Contrarily, activity of superoxide dismutase (SOD) was greater in T44. The greater accumulation of leaf nitrogen and sulfur through higher activity of their assimilating enzymes, nitrate reductase (NR) and ATP-sulfurylase (ATPS) increased reduced glutathione (GSH) content more conspicuously in Pusa Vishal than T44. Application of 0.5 mM SA increased nitrogen and sulfur assimilation, GSH content and activity of APX and GR. This resulted in the increase in photosynthesis under non-saline condition and alleviated the decrease in photosynthesis under salt stress. It also helped in restricting Na⁺ and Cl⁻ content in leaf, and maintaining higher efficiency of PSII, photosynthetic N-use efficiency (NUE) and water relations in Pusa Vishal. However, application of 1.0 mM SA resulted in inhibitory effects. The effect of SA was more pronounced in Pusa Vishal than T44. These results indicate that SA application alleviates the salt-induced decrease in photosynthesis mainly through inducing the activity of NR and ATPS, and increasing antioxidant metabolism to a greater extent in Pusa Vishal than T44.     

Production of a thermostable archaeal superoxide reductase in plant cells

Pyrococcus furiosus superoxide reductase (SOR) is a thermostable archaeal enzyme that reduces superoxide without producing oxygen. When produced as a fusion protein with the green fluorescent protein in plant cells, P. furiosus SOR is located in the cytosol and nucleus. The recombinant SOR enzyme retains its function and heat stability when assayed in vitro. Importantly, expressing SOR in plant cells enhances their survival at high temperature indicating that it functions in vivo. The archaeal SOR provides a novel mechanism to reduce superoxide and demonstrates the potential for using archaeal genes to alter eukaryotic metabolism.     

Adaptation strategies of two closely related Desmodesmus armatus (green alga) strains contained different amounts of cadmium: A study with light-induced synchronized cultures of algae

During the Desmodesmus armatus cell cycle, 8-celled coenobia of 276-4d strain accumulated a much lower amounts of cadmium than unicells of B1-76 strain. Cadmium reduced growth and photosynthesis in the cells of strain B1-76, but not those of 276-4d strain. Cells of 276-4d strain revealed a higher activity of superoxide dismutase (SOD) isoforms, in particular the activity and protein content of Fe-SOD. Cu/Zn-SOD was earlier and much stronger induced by cadmium in 276-4d than in B1-76 strain, whereas Fe- and Mn-SOD activity and Fe-SOD synthesis were induced only in 276-4d strain. Cadmium did not affect the heat shock protein 70 synthesis in B1-76 strain, but significantly stimulated this process in 276-4d strain. The level of glutathione increased 30-fold during cell development of Cd-exposed 276-4d strain, while in B1-76 it increased about 12 timed. Matured cells of both strains exposed to cadmium produced comparable amounts of phytochelatins and other thiol peptides, but their production in young cells of B1-76 strain was much higher than in 276-4d strain. In conclusion, a complex of internal detoxification mechanisms appeared to be more efficient in cells of 276-4d strain than B1-76 one.     

A drought-sensitive barley variety displays oxidative stress and strongly increased contents in low-molecular weight antioxidant compounds during water deficit compared to a tolerant variety

Barley displays a great genetic diversity, constituting a valuable source to delineate the responses of contrasted genotypes to environmental constraints. Here, we investigated the level of oxidative stress and the participation of antioxidant systems in two barley genotypes: Express, a variety known to be sensitive to drought, and Saïda, an Algerian landrace selected for its tolerance to water deficit. Soil-grown 15-day-old plants were subjected to water deficit for 8 days and then rewatered. We observed that upon water stress Express exhibits compared to Saïda accelerated wilting and a higher level of oxidative stress evaluated by HPLC measurements of lipid peroxidation and by imaging techniques. In parallel, Express plants also display lower levels of catalase and superoxide dismutase activity. No great difference was observed regarding peroxiredoxins and methionine sulfoxide reductases, enzymes detoxifying peroxides and repairing oxidized proteins, respectively. In contrast, upon water stress and recovery, much higher contents and oxidation ratios of glutathione and ascorbate were measured in Express compared to Saïda. Express also shows during water deficit greater increases in the pools of lipophilic antioxidants like xantophyll carotenoids and α-tocopherol. Altogether, these data show that the differential behavior of the two genotypes involves distinct responses regarding antioxidant mechanisms. Indeed, the drought sensitivity of Express compared with Saïda is associated with oxidative damage and a lower enzymatic ROS-scavenging capacity, but in parallel with a much stronger enhancement of most mechanisms involving low-molecular weight antioxidant compounds.     

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.     

Reduced mitochondrial and ascorbate–glutathione activity after artificial ageing in soybean seed

The effect of artificial ageing on the relationship between mitochondrial activities and the antioxidant system was studied in soybean seeds (Glycine max L. cv. Zhongdou No. 27). Ageing seeds for 18 d and 41 d at 40 °C reduced germination from 99% to 52% and 0%, respectively. In comparison to the control, malondialdehyde content and leachate conductivity in aged seeds increased and were associated with membrane damage. Transmission electron microscopy and Percoll density gradient centrifugation showed that aged seeds mainly contained poorly developed mitochondria in which respiration and marker enzymes activities were significantly reduced. Heavy mitochondria isolated from the interface of the 21% and 40% Percoll were analyzed. Mitochondrial antioxidant enzymes activities including superoxide dismutase, ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase, and dehydroascorbate reductase were significantly reduced in aged seeds. A decrease in total ascorbic acid (ASC) and glutathione (GSH) content as well as the reduced/oxidized ratio of ASC and GSH in mitochondria with prolonged ageing showed that artificial ageing reduced ASC–GSH cycle activity. These results suggested an elevated reactive oxygen species (ROS) level in the aged seeds, which was confirmed by measurements of superoxide radical and hydrogen peroxide levels. We conclude that mitochondrial dysfunction in artificially aged seeds is due to retarded mitochondrial and ASC-GSH cycle activity and elevated ROS accumulation.     

Dissociation of superoxide production by mitochondrial complex I from NAD(P)H redox state

The relationship between the rate of superoxide production by complex I and NAD(P)H redox state was investigated in rat skeletal muscle mitochondria. A high rate of superoxide production was observed during succinate oxidation; the rate during pyruvate oxidation was over fourfold lower. However, the NAD(P)H pool was significantly less reduced during succinate oxidation than during pyruvate oxidation. We conclude that there is no unique relationship between superoxide production by complex I and the reduction state of the NAD(P)H pool. Our data suggest that less than 10% of the superoxide originates from the flavin site during reverse electron transport from succinate.     

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.