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.     

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.     

Genotypic variation in sulphur assimilation and metabolism of onion (Allium cepa L.). II: Characterisation of ATP sulphurylase activity

To investigate the regulation of sulphur (S)-assimilation in onion further at the biochemical level, the pungent cultivar W202A and the milder cultivar Texas Grano 438 PVP (TG) have been grown in S-sufficient (S⁺; 4 meq S⁻¹) or S-deficient (S⁻; 0.1 meq S⁻¹) growth conditions, and tissues excised at the seedling stage (pre-bulbing; ca. 10-weeks-old) and at the mature stage (bulbing; ca. 16-weeks-old). S-supply negatively influenced adenosine-5′-phosphosulphate (APS) reductase (APR) enzyme activity in both cultivars at bulbing only, and a higher abundance of APR was observed in both cultivars at bulbing in response to low S-supply. In contrast, S-supply significantly influenced ATP sulphurylase (ATPS) activity in leaf tissues of W202A only, and only at bulbing, while an increase in abundance in response to high S-supply was observed for both cultivars at bulbing. To investigate the regulation of the ATPS enzyme activity and accumulation further, activity was shown to decrease significantly in roots at bulbing in the S-deficient treatment in both cultivars, a difference that was only supported by western analyses in W202A. Phylogenetic analysis revealed that AcATPS1 groups in a broad monocot clade with the closest sequences identified in Sorghum bicolour, Zea mays and Oryza sativa, but with some support for a divergence of AcATPS1. Detection of ATPS in leaf extracts after two dimensional gel electrophoresis (2-DE) revealed that the protein may undergo post-translational modification with a differential pattern of ATPS accumulation detected in both cultivars over the developmental progression from the seedling to the bulbing stage. Treatment of leaf extracts of W202A to dephosphorylate proteins resulted in the loss of immuno-recognised ATPS spots after 2-DE separation, although enzyme activity was not influenced. These results are discussed in terms of the tiers of control that operate at the biochemical level in the reductive S-assimilation pathway in a S-accumulating species particularly during the high-S-demanding bulbing stage.     

Effects of Salinity on Metabolic Profiles, Gene Expressions, and Antioxidant Enzymes in Halophyte Suaeda salsa

Halophyte Suaeda salsa is native to the saline soil in the Yellow River Delta. Soil salinity can reduce plant productivity and therefore is the most important factor for the degradation of wetlands in the Yellow River Delta. In this work we characterized the salinity-induced effects in S. salsa in terms of metabolic profiling, antioxidant enzyme activities, and gene expression quantification. Our results showed that salinity inhibited plant growth of S. salsa and upregulated gene expression levels of myo-inositol-1-phosphate synthase (INPS), choline monooxygenase (CMO), betaine aldehyde dehydrogenase (BADH), and catalase (CAT), and elevated the activities of superoxide dismutase (SOD), peroxidase (POD), CAT, and glutathione peroxidase (GPx). The significant metabolic responses included the depleted amino acids malate, fumarate, choline, phosphocholine, and elevated betaine and allantoin in the aboveground part of S. salsa seedlings as well as depleted glucose and fructose and elevated proline, citrate, and sucrose in root tissues. Based on these significant biological markers, salinity treatments induced clear osmotic stress (for example, INPS, CMO, BADH, betaine, proline) and oxidative stress (for example, SOD, POD, CAT, GPx activities), disturbed protein biosynthesis/degradation (amino acids and total protein) and energy metabolism (for example, glucose, sucrose, citrate) in S. salsa.     

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.     

Stress tolerance and antioxidant enzymatic activities in the metabolisms of the reactive oxygen species in two intertidal red algae Grateloupia turuturu and Palmaria palmata

This investigation was designed to compare the differential stress tolerance in young thalli of two similar intertidal red seaweeds, Grateloupia turuturu Yamada and Palmaria palmata Kuntze, and to identify whether the invasive alga G. turuturu was more stress tolerant than P. palmata to cope with adverse environmental conditions. To do so, we measured the production of reactive oxygen caused by methyl viologen (MV) by assessing the oxidation of dichlorohydrofluorescein (DCFH) to dichlorofluorescein (DCF), the activities of reactive oxygen scavenging enzymes and the changes of the optimal fluorescence quantum yield (Fv/Fm) when the thalli of the two species were exposed to oxidative stresses caused by the addition of MV, H₂O₂, 3(3, 4-dichlorophenyl)-1,1-dimethyl urea (DCMU), heavy metal, changes of salinities, heat and freezing. Results demonstrated that the activities of superoxide dismutase (SOD) and peroxidase (POD) in G. turuturu were much higher than in P. palmata. Fv/Fm in G. turuturu was less sensitive than that in P. palmata to MV, H₂O₂, DCMU, heavy metal, salinity and heat stress, indicating that G. turuturu could be better acclimatized to changing environments and thus had a higher threshold for oxidative stress than P. palmata. G. turuturu was shown to be more sensitive to freezing treatment (− 20 °C), which explained why the appearance of G. turuturu was rarely reported in colder water environments.     

Effects of NaCl and Mycorrhizal Fungi on Antioxidative Enzymes in Soybean

The effects of different concentrations of NaCl on the activities of antioxidative enzymes in the shoots and roots of soybean (Glycine max [L.] Merr cv. Pershing) inoculated or not with an arbuscular mycorrhizal fungus, Glomus etunicatum Becker & Gerdemann, were studied. Furthermore, the effect of salt acclimated mycorrhizal fungi on the antioxidative enzymes in soybean plants grown under salt stress (100 mM NaCl) was investigated. Activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were increased in the shoots of both mycorrhizal (M) and nonmycorrhizal (NM) plants grown under NaCl salinity. Salinity increased SOD activity in the roots of M and NM plants, but had no effect on CAT and polyphenol oxidase activities in the roots. M plants had greater SOD, POD and ascorbate peroxidase activity under salinity. Under salt stress, soybean plants inoculated with salt pre-treated mycorrhizal fungi showed increased SOD and POD activity in shoots, relative to those inoculated with the non pre-treated fungi.     

Seasonal variations in the chemical composition and dry matter degradability of exclosure forages in the semi-arid region of northern Ethiopia

The present study was conduced at two sites (Tembien and Wukro) in the semi-arid region of Tigray in northern Ethiopia to investigate the seasonal dynamics in the chemical composition and dry matter digestibility of grass and browse species of exclosures. The browse species studied in Tembien and Wukro had a mean crude protein (CP) value of 166 and 117 g/kg dry matter (DM), respectively. The mean in vitro dry matter digestibility (IVDMD) coefficient and predicted metabolizable energy (ME) density of the browse species were 0.72 and 9.83 MJ/kg DM, respectively at Tembien, 0.62 and 8.38 MJ/kg DM, respectively, at Wukro. Neutral detergent fibre (NDF) and acid detergent fibre (ADF) values of the browse species varied from 192 to 437 and 127 to 391 g/kg DM, respectively. Acid detergent lignin (ADL) values ranged from 36 to 190 g/kg DM. The mean CP of the grass species in Tembien and Wukro during the long rainy season was 76 and 73 g/kg DM, respectively and values declined below a critical maintenance level during the dry and short rain seasons. Mean IVDMD and ME values for the two sites were 0.41 and 0.47, and 5.38 and 6.11 MJ/kg DM, respectively. The NDF, ADF, and lignin values of the grass species were generally above 700, 400, and 70 g/kg DM, respectively. The CP, IVDMD and ME values of the mixed grass samples differed (P<0.05) among harvesting months and values ranged from 20 to 103 g/kg DM, 0.47 to 0.72 MJ/kg DM, and 6.16 to 9.91 MJ/kg DM, respectively. The browse species could be used as useful dry season protein supplements to the N deficient native grass species. Especial emphasis should be given to propagate Maerua angolensis and Cadaba farinosa at community nursery sites. Harvesting in September, rather than the current extended harvest period that took place in October and November, can considerably improve the feeding value of native grass hay for smallholder ruminant production systems.     

cDNA-AFLP analysis of salt-inducible genes expression in Chrysanthemum lavandulifolium under salt treatment

Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino is a halophyte species that belongs to the Asteraceae family, and the genus Chrysanthemum. It is one of the ancestors of C. × morifolium Ramatella. Understanding the tolerance mechanism associated with salt stress in C. lavandulifolium could provide important information for explaining the salt tolerance of higher plants and could also help enhancing breeding programs of cultivated Chrysanthemum. In this study, cDNA amplified fragment length polymorphism (cDNA-AFLP) was used to detect differential gene expression in leaves of C. lavandulifolium in response to NaCl treatment. The determination of membrane permeablility, peroxidase activity (POD), malon-dialdehyde (MDA), as well as proline and leaf chlorophyll contents under different NaCl concentrations showed that a 200 mM NaCl treatment was an optimal condition for the cDNA-AFLP experiment. Using this concentration during different times (0, 3 h, 12 h, 24 h and 48 h), we obtained 1930 cDNA fragments using 64 primers. After sequencing 234 randomly chosen cDNA clones and BLASTx analyzing, we got 129 expressed sequence tags (ESTs) which had no significant homology with other sequences, 85 ESTs were homologous to genes with known functions, whereas the rest of ESTs showed homology to unclassified or putative proteins. 25 ESTs that were similar to known functional genes involved in several abiotic and biotic stresses were confirmed by semi-quantitative RT-PCR and qRT-PCR. The expression patterns of these salt-responsive genes not only responded to salt stress but also to plant hormones, such as abscisic acid (ABA), and to other abiotic stresses such as drought and cold. These results indicate an extensive cross-talk among several stresses. Our results provide interesting information for further understanding the molecular mechanisms of salt tolerance in C. lavandulifolium.     

Long-term changes in macrophyte vegetation after reduction of fish stock in a shallow lake

Starting in the middle of the 1970s, submerged macrophytes began to disappear from shallow Lake Warniak due to feeding pressure by grass carp (Ctenopharyngodon idella). In the middle of the 1980s, the lake was stocked with seston-feeding silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis). By 1993, the biomass of silverhead carp and bighead carp had declined. This allowed charophytes to recolonize the bottom of the lake. The main charophyte species at this time were Chara globularis and Chara rudis. Since then, five other stonewort species have been found: Chara contraria, C. filiformis, C. tomentosa, C. aspera and Nitellopsis obtusa. Seventeen species of aquatic angiosperms have also been found. There were distinct changes in the relative abundance and spatial distribution of particular species. C. rudis developed most intensely in the shallow parts of the lake near the southern and western shores. C. globularis gradually took over the deeper central part of the lake. In 2001, C. rudis began to retreat again. The relative abundance and spatial distribution of charophytes was correlated to water clarity (r = 0.87, p < 0.05), total phosphorus level (r = −0.78; p < 0.05), and chlorophyll a content (r = −0.79; p < 0.05).     

Kinetic study of the plastoquinone pool availability correlated with H2O2 release in seawater and antioxidant responses in the red alga Kappaphycus alvarezii exposed to single or combined high light, chilling and chemical stresses

Under biotic/abiotic stresses, the red alga Kappaphycus alvarezii reportedly releases massive amounts of H₂O₂ into the surrounding seawater. As an essential redox signal, the role of chloroplast-originated H₂O₂ in the orchestration of overall antioxidant responses in algal species has thus been questioned. This work purported to study the kinetic decay profiles of the redox-sensitive plastoquinone pool correlated to H₂O₂ release in seawater, parameters of oxidative lesions and antioxidant enzyme activities in the red alga Kappaphycus alvarezii under the single or combined effects of high light, low temperature, and sub-lethal doses of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), which are inhibitors of the thylakoid electron transport system. Within 24 h, high light and chilling stresses distinctly affected the availability of the PQ pool for photosynthesis, following Gaussian and exponential kinetic profiles, respectively, whereas combined stimuli were mostly reflected in exponential decays. No significant correlation was found in a comparison of the PQ pool levels after 24 h with either catalase (CAT) or ascorbate peroxidase (APX) activities, although the H₂O₂ concentration in seawater (R = 0.673), total superoxide dismutase activity (R = 0.689), and particularly indexes of protein (R = 0.869) and lipid oxidation (R = 0.864), were moderately correlated. These data suggest that the release of H₂O₂ from plastids into seawater possibly impaired efficient and immediate responses of pivotal H₂O₂-scavenging activities of CAT and APX in the red alga K. alvarezii, culminating in short-term exacerbated levels of protein and lipid oxidation. These facts provided a molecular basis for the recognized limited resistance of the red alga K. alvarezii under unfavorable conditions, especially under chilling stress.     

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.     

Antioxidant metabolism of grapefruit infected with Xanthomonas axonopodis pv. citri

Grapefruit is one of the most susceptible citrus genotypes to Asiatic Citrus Canker, caused by Xanthomonas axonopodis pv. citri (Xac), that can cause severe losses in citrus yield and quality. Although much is known about citrus response to Xac, little is known of the role of antioxidant metabolism. Grapefruit leaves were artificially injected with a strain of Xac obtained from a commercial grove in Florida and components of oxidative metabolism were measured. Symptoms observed included water soaking (2 dai; days after inoculation), raised and ruptured epidermis (6-8 dai), formation of necrotic lesions (16 dai), and leaf abscission (21 dai). The Xac population increased to a maximum (≈10⁹ CFU/cm²) 8 dai and then declined to ≈10⁷ CFU/cm² by 20 dai. Lipid peroxidation was higher in infected leaves than uninoculated controls from 4 to 21 dai indicating greater oxidative stress. H₂O₂ concentration demonstrated a biphasic pattern with peak concentrations at 4 and 13 dai and minimum concentrations that were lower than the controls at 10 and 20 dai. The H₂O₂ concentration somewhat corresponded with superoxide dismutase (SOD) activity, which generates H₂O₂ via dismutase of superoxide ions. Total SOD activity in Xac-infected leaves increased to a maximum at 4 dai, the day of highest H₂O₂ concentration, and then declined and remained at or below controls. Mn-SOD and Fe-SOD activities both increased to maximum activities at 4 dai. Mn-SOD had four isoforms in Xac-infected leaves but only three in the controls. Fe-SOD had three isoforms in both infected and control plants. Suppression of H₂O₂ in Xac-infected leaves also corresponded to higher activities of the H₂O₂ catabolising enzymes catalase (CAT), ascorbate peroxidase (APOD), and peroxidase (POD). Two additional CAT isoforms were detected in infected leaves and not the controls. Three POD isoforms were detected in both control and infected leaves. Previous research has shown that Xac is sensitive to intraplant H₂O₂ concentration, however, the pattern of Xac in this study did not correspond to H₂O₂ concentration, which initially increased due to enhanced SOD activity, but was later suppressed apparently with the aid of peroxidases. In conclusion, Xac infection altered H₂O₂ metabolism in grapefruit leaves by changes in the activities and isoforms of SODs, CATs, PODs and APOD.