The responses of the enzymes related with ascorbate–glutathione cycle during drought stress in apple leaves

To explore the significance of the ascorbate–glutathione cycle under drought stress, the leaves of 2-year-old potted apple (Malus domestica Borkh.) plants were used to investigate the changes of each component of the ascorbate–glutathione cycle as well as the gene expression of dehydroascorbate reductase (DHAR, EC 1.8.5.1), ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) under drought stress. The results showed that the malondialdehyde (MDA) and H₂O₂ concentrations in apple leaves increased during drought stress and began to decrease after re-watering. The contents of total ascorbate, reduced ascorbic acid (AsA), total glutathione and glutathione (GSH) were obviously upregulated in apple leaves when the soil water content was 40–45%. With further increase of the drought level, the contents of the antioxidants and especially redox state of AsA and GSH declined. However, levels of them increased again after re-watering. Moreover, drought stress induced significant increase of the activities of enzymes such as APX, scavenging H₂O₂, and also of monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), DHAR and GR used to regenerate AsA and GSH, especially when the soil water content was above 40–45%. During severe drought stress, activities of the enzymes were decreased and after re-watering increased again. Gene expression of cytoplasmic DHAR, cytoplasmic APX and cytoplasmic GR showed similar changes as the enzyme activities, respectively. The results suggest that the ascorbate–glutathione cycle is up-regulated in response to drought stress, but cannot be regulated at severe drought stress conditions.     

Enhanced activity of galactono-1,4-lactone dehydrogenase and ascorbate–glutathione cycle in mitochondria from complex III deficient Arabidopsis

The mitochondrial antioxidant homeostasis was investigated in Arabidopsis ppr40-1 mutant, which presents a block of electron flow at complex III. The activity of the ascorbate biosynthetic enzyme, l-galactono-1,4-lactone dehydrogenase (EC 1.3.2.3) (GLDH) was elevated in mitochondria isolated from mutant plants. In addition increased activities of the enzymes of Foyer–Halliwell–Asada cycle and elevated glutathione (GSH) level were observed in the mutant mitochondria. Lower ascorbate and ascorbate plus dehydroascorbate contents were detected at both cellular and mitochondrial level. Moreover, the more oxidized mitochondrial redox status of ascorbate in the ppr40-1 mutant indicated that neither the enhanced activity of GLDH nor Foyer–Halliwell–Asada cycle could compensate for the enhanced ascorbate consumption in the absence of a functional respiratory chain.     

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.     

Methyl jasmonate alleviates arsenic-induced oxidative damage and modulates the ascorbate–glutathione cycle in oilseed rape roots

Methyl jasmonate (MJ) is an important plant growth regulator, involves in various physiological processes of plants. In the present study, role of MJ in tolerance to oilseed rape (Brassica napus L.) roots under arsenic (As) stress was investigated. The treatments were comprised of three MJ doses (0, 0.1, and 1 µM) and two levels of As (0 and 200 µM). Arsenic stress resulted in oxidative damage as evidenced by decreased root growth and enhanced reactive oxygen species and lipid peroxidation. However, plants treated with MJ decreased the H₂O₂ and O₂ ^·? contents in roots and have higher antioxidant activities. Importantly, results showed that MJ enhanced the redox states of AsA and GSH, and the related enzymes involved in the AsA–GSH cycle. Moreover, MJ also induced the secondary metabolites related enzymes (PAL and PPO) activities, under As stress. PAL and PPO expression was further increased by MJ application in the roots of B. napus under As stress. MJ also reduced the total As content compared with As alone treated plants. These findings suggest the role of MJ in mitigation of the As-induced oxidative damage by regulating AsA and GSH redox states and by reducing As uptake in both cultivars.     

Changes in ascorbate–glutathione pathway enzymes in response to Mycosphaerella fragariae infection in selected strawberry genotypes

Ten strawberry genotypes, resistant and moderately resistant (Joliette, Seascape, Aromas, FIN005-55 and FIN005-50) and susceptible ones (FIN00132-8, FIN00134-11, FIN00132-14, FIN005-7 and Kent) were used to assess the role of the antioxidative defence system against Mycosphaerella fragariae infection. The pathogen-induced changes of hydrogen peroxide (H₂O₂) and antioxidant enzymes ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) involved in the ascorbate–glutathione (ASC–GSH) cycle were examined in leaves of the selected genotypes. A significant different response was observed among the genotypes. A marked increase in H₂O₂ content, APX, MDHAR, DHAR and GR activities were observed in resistant and moderately resistant genotypes after inoculation by M. fragariae. In contrast, weak changes were observed in susceptible genotypes for the aforementioned enzymes and compounds. It seems that resistant genotypes capable of overproducing H₂O₂ have a higher capacity to scavenge and reduce the injury to strawberry leaves by regulating the ASC–GSH cycle. The results may be useful in future breeding programmes to select those individuals with high scavenging properties to breed new resistant lines.     

Differential expression of α-amylase genes in germinating rice and barley seeds

Steady-state levels of mRNA from individual -amylase genes were measured in the embryo and aleurone tissues of rice (Oryza sativa) and two varieties of barley (Hordeum vulgare L. cv. Himalaya and cv. Klages) during germination. Each member of the -amylase multigene families of rice and barley was differentially expressed in each tissue. In rice, -amylase genes displayed tissue-specific expression in which genes RAmy3B, RAmy3C, and RAmy3E were preferentially expressed in the aleurone layer, genes RAmy1A, RAmy1B and RAmy3D were expressed in both the embryo and aleurone, and genes RAmy3A and RAmy2A were not expressed in either tissue. Whenver two or more genes were expressed in any tissue, the rate of mRNA accumulation from each gene was unique. In contrast to rice, barley -amylase gene expression was not tissue-specific. Messenger RNAs encoding low- and high-pI -amylase isozymes were detectable in both the embryo and aleurone and accumulated at different rates in each tissue. In particular, peak levels of mRNA encoding high-pI -amylases always preceded those encoding low-pI -amylases. Two distinct differences in -amylase gene expression were observed between the two barley varieties. levels of high-pI -amylase mRNA peaked two days earlier in Klages embryos than in Himalaya embryos. Throughout six days of germination, Klages produced three times as much high-pI -amylase mRNA and nearly four times as much low-pI -amylase mRNA than the slower-germinating Himalaya variety.     

The dual location of β-oxidation enzymes in germinating pea cotyledons

-Oxidation enzymes were detected both in the mitochondria and microbodies of pea cotyledons. Intact mitochondria did not show -oxidation enzyme activity but in ruptured mitochondria this activity was high. It is apparent that the mitochondrial membrane barrier prevents rapid access of acyl-CoA substrates to matrix -oxidation sites. Removal of the membrane barrier permits rapid access of acyl-CoAs and these enzyme activities may then be measured.     

Seasonal changes in anthocyanin contents and in activities of xanthophyll and ascorbate–glutathione cycles in Sabina species derived from different environments

Qilian Juniper (Sabina przewalskii Kom.) and Chinese juniper [Sabina chinensis (Lin.) Ant.] are overwintering plants. S. przewalskii, a protected species in China, is distributed in subalpine and alpine area on the Qinghai-Tibet Plateau. S. chinensis is distributed in plain area. We investigated seasonal changes in photoprotective stress compounds such as anthocyanins, activities of three enzymes of ascorbate–glutathione pathway, as well as xanthophyll size and conversion in these species. Ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) levels were higher in the low-temperature months, which was associated with changes in anthocyanins and in de-epoxidation index [(A + Z)/(V + A + Z)]. Photochemical efficiency of PSII (Fv/Fm) was lower (<0.70) during winter and late autumn in both species. During the low-temperature months, S. przewalskii had higher levels of photoprotective stress compounds than S. chinensis. The results suggested that these two species possess cold-induced photoinhibition functions and show the inherent, season-dependent differences in the amounts of the stress-related compounds.     

Defying the activity–stability trade-off in enzymes: taking advantage of entropy to enhance activity and thermostability

The biotechnological applications of enzymes are limited due to the activity–stability trade-off, which implies that an increase in activity is accompanied by a concomitant decrease in protein stability. This premise is based on thermally adapted homologous enzymes where cold-adapted enzymes show high intrinsic activity linked to enhanced thermolability. In contrast, thermophilic enzymes show low activity around ambient temperatures. Nevertheless, genetically and chemically modified enzymes are beginning to show that the activity–stability trade-off can be overcome. In this review, the origin of the activity–stability trade-off, the thermodynamic basis for enhanced activity and stability, and various approaches for escaping the activity–stability trade-off are discussed. The role of entropy in enhancing both the activity and the stability of enzymes is highlighted with a special emphasis placed on the involvement of solvent water molecules. This review is concluded with suggestions for further research, which underscores the implications of these findings in the context of productivity curves, the Daniel–Danson equilibrium model, catalytic antibodies, and life on cold planets.     

Reactive oxygen species, ascorbate–glutathione pool, and enzymes of their metabolism in drought-sensitive and tolerant indica rice (Oryza sativa L.) seedlings subjected to progressing levels of water deficit

Water deficit for rice is a worldwide concern, and to produce drought-tolerant varieties, it is essential to elucidate molecular mechanisms associated with water deficit tolerance. In the present study, we investigated the differential responses of nonenzymatic antioxidants ascorbate (AsA), glutathione (GSH), and their redox pool as well as activity levels of enzymes of ascorbate–glutathione cycle in seedlings of drought-sensitive rice (Oryza sativa L.) cv. Malviya-36 and drought-tolerant cv. Brown Gora subjected to water deficit treatment of ?1.0 and ?2.1 MPa for 24–72 h using PEG-6000 in sand cultures. Water deficit caused increased production of reactive oxygen species such as O₂?^?, H₂O₂, and HO? in the tissues, and the level of production was higher in the sensitive than the tolerant cultivar. Water deficit caused reduction in AsA and GSH and decline in their redox ratios (AsA/DHA and GSH/GSSG) with lesser decline in tolerant than the sensitive seedlings. With progressive level of water deficit, the activities of monodehydroascorbate reductase, dehydroascorbate reductase, ascorbate peroxidase (APX), and glutathione transferase increased in the seedlings of both rice cultivars, but the increased activity levels were higher in the seedlings of drought-tolerant cv. Brown Gora compared to the sensitive cv. Malviya-36. Greater accumulation of proline was observed in stressed seedlings of tolerant than the sensitive cultivar. In-gel activity staining of APX revealed varying numbers of their isoforms and their differential expression in sensitive and tolerant seedlings under water deficit. Results suggest that an enhanced oxidative stress tolerance by a well-coordinated cellular redox state of ascorbate and glutathione in reduced forms and induction of antioxidant defense system by elevated activity levels of enzymes of ascorbate–glutathione cycle is associated with water deficit tolerance in rice.     

Ascorbate and glutathione: guardians of the cell cycle,partners in crime?

Besides the implication of ascorbate and glutathione in the defence against oxidative stress, these two compounds are involved in plant growth and cell cycle control. Ascorbate metabolism is closely linked to the development of embryos and seedlings. Furthermore, ascorbate stimulates cell cycle activity in competent cells, while the oxidised form, dehydroascorbate, blocks normal cell cycle progression. Several possible mechanisms have been proposed to explain the effect of these compounds. The links between glutathione and the cell cycle are less clear. It has long been assumed that both compounds are closely linked by way of the Halliwell–Asada cycle. Any hypothesis concerning the pathways by which ascorbate or glutathione influence cell division, should take this connection into account. However, other mechanisms have been proposed for ascorbate-mediated cell cycle control, e.g. via the thioredoxin pathway.     

Geographie variation of α-amylase, β-amylase, β-glucanase,pullulanase and chitinase activity in germinating Hordeum spontaneum barley from Israel and Jordan

The enzyme activities of -amylase, -amylase, -glucanase, pullulanase and chitinase were determined in extracts of wild barley (Hordeum vulgare ssp. spontaneum) germinated for five days under axenic standard conditions. The material comprises 257 accessions, for 242 of which the botanical territory of origin in Israel or Jordan is known. The enzyme activities based on soluble protein in the extracts showed significant differences (P<0.001) among the eleven territories. The territorial moisture parameters mostly correlate with the enzyme activities. As determined by one gene or oligogenes, the significant territorial differences and the correlation with moisture are thought to reflect natural selection of genes responsible for favourable activity, or of genes linked to the enzyme coding loci, or in a coadaptive manner, of physiologically allied genes. Genes for high -glucanase activity at germination are probably coadaptive with genes for high -glucan content of the grain. The generally low starch content of wild barley grains probably makes any high -amylase activity unnecessary at the germination stage. An inverse relationship appears between -glucanase and chitinase activity; these two enzymes are also pathogenesis related proteins.     

Mapping of β-glucan content and β-glucanase activity loci in barley grain and malt

Genetic study of -glucan content and -glucanase activity has been facilitated by recent developments in quantitative trait loci (QTL) analysis. QTL for barley and malt -glucan content and for green and finished malt -glucanase activity were mapped using a 123-point molecular marker linkage map from the cross of Steptoe/Morex. Three QTL for barley -glucan, 6 QTL for malt -glucan, 3 QTL for -glucanase in green malt and 5 QTL for -glucanase in finished malt were detected by interval mapping procedures. The QTL with the largest effects on barley -glucan, malt glucan, green malt -glucanase and finished malt glucanase were identified on chromosomes 2,1,4 and 7, respectively. A genome map-based approach allows for dissection of relationships among barley and malt glucan content, green and finished malt -glucanase activity, and other malting quality parameters.     

Assessing the structure–activity relationships of fluorotelomer unsaturated acids and aldehydes with glutathione

Fluorotelomer alcohols (FTOHs) have been shown to degrade via abiotic and biotic mechanisms to perfluorocarboxylates (PFCAs) which are environmentally persistent and bioaccumulate in humans and wildlife depending on their chain length. Fluorotelomer unsaturated aldehydes (FTUALs) and acids (FTUCAs) are intermediate metabolites that form from the degradation of FTOHs. Their potential for toxicity is not yet defined and may be more significant compared to PFCAs. Past studies have shown that these intermediates form adducts with glutathione (GSH). The purpose of this study was to further assess the reactivity of these intermediate compounds. In vitro experiments were carried out in an aqueous buffer system (pH 7.4) where FTUCAs and FTUALs of varying chain lengths were reacted with GSH. To quantify the reactivity of FTUCAs and FTUALs, unreacted free GSH was derivatized with 5,5′-dithiobis(2-nitrobenzoic acid), its absorbance measured at 412 nm, and the percentage of unconjugated free GSH evaluated over time. EC₅₀ values were obtained for the reactions of GSH with acrolein and methyl methacrylate to assess the accuracy of the method, as well as for acrylic acid, FTUCAs, and FTUALs. The results of this study indicated that α,β-unsaturated aldehydes are comparatively the most reactive and reaction with GSH may be influenced by the length of the fluorinated tail. This is the first study to examine the relationship of FTUCAs and FTUALs with biological nucleophiles by quantifying their intrinsic reactivity.     

Abscisic acid enhances tolerance to spring freeze stress and regulates the expression of ascorbate–glutathione biosynthesis-related genes and stress-responsive genes in common wheat

Molecular Breeding - Hybrid sterility is a major obstacle to the development of superior inter-subspecific hybrids between indica and japonica subspecies of Asian-cultivated rice. To overcome...     

Spatial distribution of β-amylase in germinating barley seeds based on the avidin-biotin-peroxidase complex method

Summary In comparison with two wild type barley cultivars, Sundance and Bomi, biochemical data show that the high-lysine mutant Hiproly contains abundant amounts of lysine-rich -amylase, whereas mutant Risø 1508, also a high-lysine mutant, contains negligible amounts of this enzyme. Immunocytochemical studies of germinating barley seeds, using both mono- and polyclonal antibodies to -amylase, support the biochemical findings of enzyme abundance in developing seeds. Three immunostaining methods for localization of -amylase were tested; of these, the avidin-biotin-peroxidase complex method, a relatively new procedure for study of plant tissues, is by far the most sensitive. -Amylase occurs predominantly in cytoplasm of the endosperm, with a minor, but previously unknown localization in the aleurone of the mid-region of the seeds. A spatial distribution of -amylase is seen. Endosperm in the upper and lower regions has the greatest amount of -amylase, with the amount decreasing toward the mid-region. In the mid-region, a limited aleurone localization of -amylase is found in all four barley strains. The function of this aleurone localization is unclear. In the endosperm, the abundance of -amylase appears to be inversely correlated with number of starch grains per unit area in Hiproly but not in Risø 1508, yet the rate of germination of the two mutants is essentially identical. Whether -amylase has a role in starch metabolism in these germinating barley seeds is unclear.Abbreviations Ab antibodies - ABC avidin-biotin-peroxidase complex - a -Amylase - BSA bovine serum albumin - DAB diaminobenzidine tetrahydrochloride dihydrate - FAA formalin: glacial acetic acid: ethyl alcohol - FITC fluorescein isothiocyanate - mAb mouse monoclonal antibodies - pAb rabbit polyclonal antibodies - PAP peroxidase-anti-peroxidase - PBS phosphate buffer saline - R-1508 Risø 1508     

β-galactosidase activity in the germinating seeds of Vigna sinensis

The β-galactosidase activity in cotyledons of Vigna sinensis increases during seed germination and is inhibited by cycloheximide. The increasing activity may be due to the de novo synthesis of enzyme protein. The enzyme has been partially purified by gel filtration and characterized with respect to some biochemical parameters. The optimum pH and optimum temperature are 4.5 and 55°, respectively and the enzymes follows typical Michaelis kinetics with K_m and V_max of 4.5 x 10^?4 M and 2.0 x 10^?5 mol/hr respectively. K_i for galactose and lactose are 4.5 and 220 mM, respectively. The energy of activation of the enzyme for p-nitrophenyl β-D-galactoside is 9.83 kcal/mol. The apparent relative MW of the enzyme as determined by gel filtration was 56000.