Regulation of ascorbate peroxidase activity in dark-grown radish cotyledons by a catalase inhibitor, 3-Amino-1,2,4-Triazole

The present study was performed to see the physiological role of cytosolic ascorbate peroxidase (APX) and its relationship to other enzymes involved in the H₂O₂ scavenging metabolism, and also to elucidate the regulation of APX expression in dark-grown radish (Raphanus sativus L. cv Taiwang) cotyledons. To do so, 3-amino-l,2,4-triazole (aminotriazole), a known specific inhibitor of catalase, was used to simulate a catalase-deficient phenomenon in cotyledons. Aminotriazole, in very low concetration (10^-4 M), inhibited remarkably the development of catalase activity in cotyledons during dark germination. This inhibition of catalase by aminotriazole, however, did not result in any significant changes in the growth response and the H₂O₂ level of developing cotyledons. In addition, the development of guaiacol peroxidase (GPX) activity was also not significantly affected. Unlike GPX, cytosolic APX activity was induced rapidly and reached a 1.7-fold increase in aminotriazole treated cotyledons at day 7 after germination. However,in vitro incubation of cytosolic APX preparation from cotyledons with aminotriazole did not result in any significant change in activity. One cytosolic APX isozyme (APXa) band involved in this APX activation was predominantly intensified in a native polyacrylamide gel by activity staining assay. This means that this APXa isozyme seems to play a key role in the expression of cytosolic APX activity. On the other hand, 2-day-old control seedlings treated with exogenous 1 mM H₂O₂ for 1 h showed a significant increase of cytosolic APX acitivity even in the absence of aminotriazole. Also, 2 μM cycloheximide treatment substantially inhibited the increase of APX activity due to aminotriazole. Based on these results, we suggest that a radish cytosolic APX could probably be substituted for catalase in H₂O₂ removal and that the expression of APX seems to be regulated by a change of endogenous H₂O₂ level which couples to APX protein synthesis in a translation stage in cotyledons.     

Effect of salinity on antioxidant enzymes in calli of the halophyte <Emphasis Type="Italic">Nitraria tangutorum</Emphasis> Bobr.

Nitraria tangutorum Bobr., a typical desert halophyte, plays an important ecological role because of its superior tolerance to severe drought and high salinity. Very little is known about the physiological adaptative mechanism of this species to environmental stresses. The aim of this study was to investigate the changes of antioxidant enzyme activities and the regulatory mechanism of ascorbate peroxidase (APX) activity in the calli from Nitraria tangutorum Bobr. after treatment with different NaCl concentrations. The activities of superoxide dismutase (SOD) and catalase (CAT) significantly increased in the calli treated with NaCl, while the peroxidase activity decreased. APX activity was also elevated significantly in response to NaCl, but the increase was partly abolished by H₂O₂ scavenger dimethylthiourea (DMTU). Furthermore, the excitatory effect of salinity on APX could be alleviated by the addition of exogenous CAT and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium, indicating that the modulation of the APX activity in Nitraria tangutorum Bobr. calli might be associated with NADPH oxidase-dependent H₂O₂ generation. Measurement and analysis using fluorescent dye 2′,7′-dichlorodihydrofluorescein diacetate showed the increase of H₂O₂ content in salinity-treated calli. The investigation of NADPH-dependent O₂⁻ production in plasma membrane (PM) vesicles isolated from Nitraria tangutorum Bobr. calli revealed that salinity treatment stimulated NADPH oxidase activity. In conclusion, these results suggest that the higher activities of antioxidant enzymes play an important role in the salt tolerance of Nitraria tangutorum Bobr. calli and that the extracellular production of H₂O₂, depending on the excitation of PM NADPH oxidase, is responsible for enhancing the APX activity in Nitraria tangutorum Bobr. calli under salinity stress.     

Heat shock-mediated H2O2 accumulation and protection against Cd toxicity in rice seedlings

Rice (Oryza sativa L.) seedlings stressed with CdCl₂ (0.5 mM or 50 μM) showed typical Cd toxicity (leaf chlorosis, decrease in chlorophyll content, or increase in H₂O₂ and malondialdehyde contents). Rice seedlings pretreated with heat shock at 45°C (HS) for 2 or 3 h were protected against subsequent Cd stress. Rice seedlings pretreated with HS had similar Cd concentration in leaves caused by CdCl₂ as those non-HS. The content of H₂O₂ increased in leaves 1 h after HS exposure. However, APX and GR activities were higher in HS-treated leaves than their respective control, and it occurred after 2 h of HS treatment. Pretreatment of rice seedlings with H₂O₂ under non-HS conditions resulted in an increase in APX, GR, and CAT activities and protected rice seedlings from subsequent Cd stress. HS-induced H₂O₂ production and protection against subsequent Cd stress can be counteracted by imidazole, an inhibitor of NADPH oxidase complex. Results of the present study suggest that early accumulation of H₂O₂ during HS signals the increase in APX and GR activities, which in turn prevents rice seedlings from Cd-caused oxidative damage.     

Spermidine alleviates the growth of saline-stressed ginseng seedlings through antioxidative defense system

Protective effects of exogenous spermidine (Spd), activity of antioxygenic enzymes, and levels of free radicals in a well-known medicinal plant, Panax ginseng was examined. Seedlings grown in salinized nutrient solution (150 mM NaCl) for 7 d exhibited reduced relative water content, plant growth, increased free radicals, and showing elevated lipid peroxidation. Application of Spd (0.01, 0.1, and 1 mM) to the salinized nutrient solution showed increased plant growth by preventing chlorophyll degradation and increasing PA levels, as well as antioxidant enzymes such as CAT, APX, and GPX activity in the seedlings of ginseng. During salinity stress, Spd was effective for lowering the accumulation of putrescine (Put), with a significant increase in the spermidine (Spd) and spermine (Spm) levels in the ginseng seedlings. A decline in the Put level ran parallel to the higher accumulation of proline (Pro), and exogenous Spd also resulted in the alleviation of Pro content under salinity. Hydrogen peroxide (H₂O₂) and superoxide (O₂⁻) production rates were also reduced in stressed plants after Spd treatment. Furthermore, the combined effect of Spd and salt led to a significant increase in diamine oxidase (DAO), and subsequent decline in polyamine oxidase (PAO). These positive effects were observed in 0.1 and 1 mM Spd concentrations, but a lower concentration (0.01 mM) had a very limited effect. In summary, application of exogenous Spd could enhance salt tolerance of P. ginseng by enhancing the activities of enzyme scavenging system, which influence the intensity of oxidative stress.     

IBA-induced changes in antioxidant enzymes during adventitious rooting in mung bean seedlings: The role of H2O2

The changes in antioxidant enzyme activity during the induction of adventitious roots in mung bean seedlings treated with Indole-3-butyric acid (IBA), hydrogen peroxide (H₂O₂), ascorbic acid (ASA) and diphenylene iodonium (DPI) were investigated. As compared with the controls, treatments of seedlings with 10 μM IBA significantly decreased POD activity by 55% and 49.6% at 3 h and 12 h of incubation, respectively, and significantly increased by 49.8% at 36 h of incubation; treatments of seedlings with 10 mM H₂O₂ significantly decreased POD activity by 42%, 60%, 39% and 38% at 3 h, 12 h, 24 h and 48 h of incubation, respectively, the changes in POD activity were coincident with those in IBA-treated seedlings during the 0–12 h incubation period; treatments of seedlings with 2 mM ASA significantly decreased APX activities by 27% only at 3 h of incubation, the varying trend of POD activity was similar to incubation with water; 10 μM DPI treatments significantly decreased POD activity by 42%, 40%, 54% and 28% at 3 h, 6 h, 12 h and 48 h of treatment, respectively. CAT activities remained at relatively stable levels and no major changes occurred from 0 h to 48 h during the incubation phase of adventitious rooting. The results may imply that CAT, an H₂O₂-metabolizing enzyme, is inactivated by H₂O₂ during the formation of adventitious roots. As compared with the controls, IBA treatments significantly decreased APX activities by 48%, 53% and 66% at 3 h, 9 h and 12 h of treatment, respectively; H₂O₂ treatments significantly decreased APX activities by 59%, 51% and 57% at 3 h, 12 h and 36 h of incubation, respectively; ASA treatments significantly decreased APX activities by 37% only at 3 h of incubation; DPI treatments significantly decreased APX activities by 54%, 53% and 63% at 3 h, 6 h and 12 h of incubation, respectively, and significantly increased APX activity by 106% at 24 h. These results indicated that the influence of IBA, H₂O₂, ASA and DPI on the changes in APX activity were the same as on the changes in POD activity. Furthermore, similar trends in the changes of APX activity and POD activity were observed during the induction and initiation rooting phase. This finding implies that APX and POD serve the same functions, possibly related to the level of H₂O₂, during the formation of adventitious roots. The early decrease of POD and APX activities in the initiation phase of IBA- and H₂O₂-treated seedlings may be one mechanism underlying the IBA- and H₂O₂-mediated facilitation of adventitious rooting.     

The involvement of hydrogen peroxide in abscisic acid-induced activities of ascorbate peroxidase and glutathione reductase in rice roots

We have monitored the changes in antioxidant enzyme activities and H₂O₂ concentrations in roots of rice (Oryza sativa L., cv. Taichung Native 1) seedlings treated with exogenous abscisic acid(ABA). Decrease in superoxide dismutase (SOD) and catalase (CAT) activities was observed in rice roots in the presence of ABA. However, ascorbate peroxide (APX) and glutathione reductase (GR) activities were increased after the ABA treatment. ABA treatment resulted in an increase in H₂O₂ concentrations in rice roots. Pre-treatment with dimethylthiourea, a chemical trap for H₂O₂, and diphenyleneiodonium chloride (DPI), a well known inhibitor of NADPH oxidase, inhibited ABA-induced accumulation of H₂O₂ and ABA-induced activities of APX and GR. ABA-induced accumulation of H₂O₂ was found to be prior to ABA-induced activities of APX and GR. Our results suggest that H₂O₂ is involved in ABA-induced APX and GR activities in rice roots.     

Changes in antioxidants and kinetics of glutathione-S-transferase of maize in response to isoproturon treatment

Abstract

Isoproturon at the recommended field dose (RFD) significantly reduced fresh and dry weights of shoots and roots as well as chlorophyll and carotenoid contents of 10-day-old maize seedlings during the following 20 days. The higher the herbicide dose, the greater the reduction. Meanwhile, ascorbate (AsA) and reduced glutathione (GSH) increased in leaves for only the first few days. Similar increases in activities of superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX) and ascorbate peroxidase (APX) were detected. Low doses caused general increases while high doses induced diminutions; however, CAT and APX activities were inhibited by all doses. Nevertheless, H₂O₂ was significantly accumulated throughout the experiment; the magnitude of accumulation increased with time and herbicide dose. On the contrary, there were significant inhibitions in activities of the glutathione S-transferase (GST) isoforms (GST_(CDNB), GST_(ALA), or GST_(MET)) with no variation in GST_(ATR); the inhibition was greater with increasing isoproturon doses. These findings suggest the occurrence of an oxidative stress induced by isoproturon, a state that prolonged with increasing herbicide dose and/or treatment time. Moreover, V _max of GST was lowered by isoproturon, whereas K _m was unchanged, indicating that the herbicide is a competitive inhibitor of GST.     

Salicylic acid-mediated hydrogen peroxide accumulation and protection against Cd toxicity in rice leaves

The role of H₂O₂ in salicylic acid (SA)-induced protection of rice leaves against subsequent Cd toxicity was investigated. SA pretreatment resulted in an increase in the contents of endogenous SA, as judged by the expression of OsWRKY45 (a SA responsive gene), and H₂O₂ in rice leaves. Diphenyleneiodonium (DPI) and imidazole (IMD), inhibitors of NADPH oxidase, prevented SA-increased H₂O₂ production, suggesting that NADPH oxidase is a H₂O₂-generating enzyme in SA-pretreated rice leaves. DPI and IMD also inhibited SA-increased activities of superoxide dismutase (SOD), ascorbate peroixdase (APX), and glutathione reductase (GR) activities, but had no effect on SA-increased catalase (CAT) activity. Moreover, SA-induced protection against subsequent Cd toxicity could also be prevented by DPI and IMD. The inhibitory effect of DPI and IMD on SA-induced protection against subsequent Cd toxicity could be reversed by exogenous H₂O₂. All these results suggested that SA-induced protection against subsequent Cd toxicity is mediated through H₂O₂. This conclusion is supported further by the observations that exogenous H₂O₂ application resulted in an increase in SOD, APX, and GR activities, but not CAT activity and a protection against subsequent Cd toxicity of rice leaves.     

Nickel-induced Inhibition of Wheat Root Growth is Related to H2O2 Production, but not to Lipid Peroxidation

Effects of exogenous nickel (Ni: 10 and 200 μM) on growth, mitotic activity, Ni accumulation, H₂O₂ content and lipid peroxidation as well as the activities of various antioxidative enzymes, such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione peroxidase (GSH-Px) were investigated in wheat roots. A considerable Ni accumulation in the roots occurred at both the concentrations. Although Ni at 10 μM did not have any significant effect on root growth, it strongly inhibited the root growth at 200 μM. Mitotic activity in the root tips was not significantly affected by exposure of the seedlings to 10 μM Ni; however, it was almost completely inhibited at 200 μM treatment. Ni stress did not result in any significant changes in CAT and APX activities as well as lipid peroxidation. However, H₂O₂ concentration increased up to 82% over the control in the roots of seedlings exposed to 200 μM Ni. There was a significant decline in both SOD (50%) and GSH-Px (20–30%) activities in the roots when the seedlings were treated with 200 μM Ni. The results indicated that a strong inhibition of wheat root growth caused by Ni stress was not due to enhanced lipid peroxidation, but might be related to the accumulation of H₂O₂ in root tissue.     

Selenium Pretreatment Upregulates the Antioxidant Defense and Methylglyoxal Detoxification System and Confers Enhanced Tolerance to Drought Stress in Rapeseed Seedlings

In order to observe the possible regulatory role of selenium (Se) in relation to the changes in ascorbate (AsA) glutathione (GSH) levels and to the activities of antioxidant and glyoxalase pathway enzymes, rapeseed (Brassica napus) seedlings were grown in Petri dishes. A set of 10-day-old seedlings was pretreated with 25 μM Se (Sodium selenate) for 48 h. Two levels of drought stress (10% and 20% PEG) were imposed separately as well as on Se-pretreated seedlings, which were grown for another 48 h. Drought stress, at any level, caused a significant increase in GSH and glutathione disulfide (GSSG) content; however, the AsA content increased only under mild stress. The activity of ascorbate peroxidase (APX) was not affected by drought stress. The monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR) activity increased only under mild stress (10% PEG). The activity of dehydroascorbate reductase (DHAR), glutathione S-transferase (GST), glutathione peroxidase (GPX), and glyoxalase I (Gly I) activity significantly increased under any level of drought stress, while catalase (CAT) and glyoxalase II (Gly II) activity decreased. A sharp increase in hydrogen peroxide (H₂O₂) and lipid peroxidation (MDA content) was induced by drought stress. On the other hand, Se-pretreated seedlings exposed to drought stress showed a rise in AsA and GSH content, maintained a high GSH/GSSG ratio, and evidenced increased activities of APX, DHAR, MDHAR, GR, GST, GPX, CAT, Gly I, and Gly II as compared with the drought-stressed plants without Se. These seedlings showed a concomitant decrease in GSSG content, H₂O₂, and the level of lipid peroxidation. The results indicate that the exogenous application of Se increased the tolerance of the plants to drought-induced oxidative damage by enhancing their antioxidant defense and methylglyoxal detoxification systems.     

Regulation of exogenous spermidine on the reactive oxygen species level and polyamine metabolism in <Emphasis Type="Italic">Alternanthera philoxeroides</Emphasis> (Mart.) Griseb under copper stress

Effects of exogenous spermidine (Spd) on the reactive oxygen species level and polyamine metabolism against copper (Cu) stress in Alternanthera philoxeroides (Mart.) Griseb leaves were investigated. Cu treatment induced a marked accumulation of Cu and enhanced contents of malondialdehyde (MDA), hydrogen peroxide (H₂O₂) and the generation rate of O₂ ^·−. It also significantly increased putrescine (Put) levels but lowered spermidine (Spd) and spermine (Spm) levels. The activities of arginine decarboxylase (ADC), ornithine decarboxylase (ODC) and polyamine oxidase (PAO) were all elevated with the increase of Cu concentration. However, application of exogenous Spd effectively decreased H₂O₂ content and the generation rate of O₂ ^·−, prevented Cu-induced lipid peroxidation and reduced Cu accumulation. Moreover, it declined level of endogenous Put and increased levels of Spd and Spm. Activities of ADC, ODC and PAO were all inhibited by exogenous Spd. The results indicated that application of exogenous Spd could enhance the tolerance of A. philoxeroides to Cu stress by reducing the reactive oxygen level and balancing polyamine metabolism.     

Relationship between Ethylene and Spermidine in the Leaves of Glycyrrhiza uralensis Seedlings under Root Osmotic Stress1

The correlation between ethylene (ETH) production and spermidine (Spd) content was studied in the leaves of Glycyrrhiza uralensis seedlings under root osmotic stress. After 4-h root osmotic stress, Spd inhibited ETH production significantly while ETH had no significant influence on Spd content in leaves. After 24-h root osmotic stress, ETH production and Spd content showed significant negative correlations in leaves, and the negative correlations were still significant even if the common precursor S-adenosylmethionine (SAM) was abundant. This result suggested that the significant negative correlations were not mainly caused by the competition for the common precursor SAM. The results also showed that, after 24-h root osmotic stress, ETH enhanced while Spd reduced the production rate of H₂O₂ and markedly in leaves; furthermore, exogenous H₂O₂ accelerated the increase in ETH production and the decrease in Spd content caused by deep stress. So it could be concluded that reactive oxygen species played important roles in the significant negative correlations in the deeply stressed leaves of G. uralensis seedlings.     

Abscisic acid-induced apoplastic H<Subscript>2</Subscript>O<Subscript>2</Subscript> accumulation up-regulates the activities of chloroplastic and cytosolic antioxidant enzymes in maize leaves

The histochemical and cytochemical localization of abscisic acid (ABA)-induced H₂O₂ production in leaves of maize (Zea mays L.) plants were examined, using 3,3-diaminobenzidine (DAB) and CeCl₃ staining, respectively, and the relationship between ABA-induced H₂O₂ production and ABA-induced subcellular activities of antioxidant enzymes was studied. H₂O₂ generated in response to ABA treatment was detected within 0.5 h in major veins of the leaves and maximized at about 2–4 h. In mesophyll and bundle sheath cells, ABA-induced H₂O₂ accumulation was observed only in apoplast, and the greatest accumulation occurred in the walls of mesophyll cells facing large intercellular spaces. Meanwhile, ABA treatment led to a significant increase in the activities of the leaf chloroplastic and cytosolic antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), and pretreatment with the NADPH oxidase inhibitor diphenyleneiodonium (DPI), the O ₂ ⁻ scavenger Tiron and the H₂O₂ scavenger dimethylthiourea (DMTU) almost completely arrested the increase in the activities of these antioxidant enzymes. Our results indicate that the accumulation of apoplastic H₂O₂ is involved in the induction of the chloroplastic and cytosolic antioxidant enzymes. Moreover, an oxidative stress induced by paraquat (PQ), which generates O ₂ ⁻ and then H₂O₂ in chloroplasts, also up-regulated the activities of the chloroplastic and cytosolic antioxidant enzymes, and the up-regulation was blocked by the pretreatment with Tiron and DMTU. These data suggest that H₂O₂ produced at a specific cellular site could coordinate the activities of antioxidant enzymes in different subcellular compartments.     

Role of hydrogen peroxide in regulating glucose-6-phosphate dehydrogenase activity under salt stress

The role of hydrogen peroxide in the regulation of glucose-6-phosphate dehydrogenase (G6PDH) activity in the red kidney bean (Phaseolus vulgaris L.) roots under salt stress (100 mM NaCl) was investigated. Salt stress caused the increase of the activities of G6PDH and antioxidative enzymes including ascorbate peroxidase (APX), catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), as well as H₂O₂ production. The application of H₂O₂ (1 mM) also enhanced the activities of G6PDH as well as antioxidative enzymes. In the presence of exogenous CAT, H₂O₂ content was decreased, and the enhanced activities of G6PDH and antioxidative enzymes induced by NaCl or by exogenous H₂O₂ were also abolished, suggesting that the enhancement of the above enzyme activities under salt stress was a result of the increased endogenous H₂O₂ levels. Further results showed that the effects of NaCl and H₂O₂ on the activities of antioxidative enzymes were diminished by Na₃PO₄ (a G6PDH inhibitor), suggesting G6PDH activity is required in enhancing the activities of antioxidative enzymes. The enhanced membrane leakage, lipid peroxidation, H₂O₂ and O₂ — contents, G6PDH and antioxidative enzyme activities under salt stress were all recovered to control level when the red kidney bean seedlings treated with 100 mM NaCl for 6 d were transferred to the control conditions for 8 d.     

Activation of ascorbate-glutathione cycle inArabidopsis leaves in response to aminotriazole

Aminotriazole(AT)-induced changes in growth, hydrogen peroxide content and activities of H₂O₂-scavenging antioxidant enzymes were investigated in the growing leaves ofArabidopsis plants (Arabidopsis thaliana cv Columbia). Catalase activity of rosette leaves was reduced by 65% with an application of 0.1 mM AT (a herbicide known as a catalase inhibitor), whereas the leaf growth and H₂O₂ content were almost unaffected. However, an approximate 1.6 to 2-fold increase in cytosolic ascorbate peroxidase (APX) activity concomitant with a substantial activation of glutathione reductase (GR) (approx. 22% increase) was observed during leaf growth in the presence of 0.1 mM AT. The activity of cytosolic APX in leaves was also increased by 1.8-fold with an application of exogenous 2 mM paraquat (an inducer of H₂O₂ production in plant cells) in the absence of AT. These results collectively suggest that (a) cytosolic APX and GR operate to activate an ascorbate-glutathione cycle for the removal of H₂O₂ under severe catalase deactivation, and (b) the expression of APX seems to be regulated by a change of the endogenous H₂O₂ level in leaf cells.     

Exogenous hydrogen peroxide can enhance tolerance of wheat seedlings to salt stress

Hydrogen peroxide (H₂O₂), an active oxygen species, is widely generated in many biological systems and mediates various physiological and biochemical processes in plants. In this study, we demonstrated that exogenous H₂O₂ was able to improve the tolerance of wheat seedlings to salt stress. Treatments with exogenous H₂O₂ for 2 days significantly enhanced salt stress tolerance in wheat seedlings by decreasing the concentration of malondialdehyde (MDA), the production rate of superoxide radical (O₂ ⁻), and increasing the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX), and the concentration of glutathione (GSH) and carotenoids (CAR). To further clarify the role of H₂O₂ in preventing salt stress damage, CAT and ascorbate (AsA), the specific H₂O₂ scavengers, were used. The promoting effect of exogenous H₂O₂ on salt stress could be reversed by the addition of CAT and AsA. It was suggested that exogenous H₂O₂ induced changes in MDA, O₂ ⁻, antioxidant enzymes and antioxidant compounds were responsible for the increase in salt stress tolerance observed in the experiments. Therefore, H₂O₂ may participate in antioxidant enzymes and antioxidant compounds induced tolerance of wheat seedlings to salt stress. The results also showed that exogenous H₂O₂ had a positive physiological effect on the growth and development of salt-stressed seedlings.     

Effects of superoxide radicals on ACC synthase activity in chilling-stressed etiolated mungbean seedlings

The activity of 1-aminocyclopropane-1-carboxylic acid synthase (ACC synthase, ACS) and the concentrations of superoxide radical (O₂^−.) and hydrogen peroxide (H₂O₂) were measured in etiolated mungbean seedlings following their transfer to a growth chamber at 25°C after a 5-h-chilling treatment at 5°C. All of these variables increased dramatically after the transfer, and strong correlations were found between ACS activity and the concentrations of superoxide and H₂O₂. Exogenous applications of two generators of superoxide radicals, methylviologen (MV) and xanthine–xanthine oxidase (X–XOD), enhanced ACS activity in seedlings, but their effects were inhibited by exogenous applications of specific scavengers of O₂^−.. However, applications of H₂O₂ or specific H₂O₂-scavengers had no significant effects on seedlings ACS activity. The results indicate that O₂^−. was involved in the chilling-induced increases in ACS activity, but not H₂O₂. ACS activity peaked ca. 8 h after the transfer, and then declined, but the decline could be counteracted by exogenous applications of specific O₂^−. scavengers, this suggests that damage was caused by superoxide radicals influencing ACS activity in etiolated mungbean seedlings. Further analysis of changes in two key kinetic parameters of ACS activity—V _max (maximum velocity) and K _m (the Michaelis constant)—in the seedlings indicated that the presence of O₂^−. may reduce K _m, i.e. increase substrate (S-adenosyl methionine, SAM) affinity. That would be the main mechanism responsible for the observed chilling-induced increases in ACS activity in etiolated mungbean seedlings.     

Triple mutation of Cys26, Trp35, and Cys126 in stromal ascorbate peroxidase confers H2O2 tolerance comparable to that of the cytosolic isoform

Ascorbate peroxidase (APX) isoforms localized in the stroma and thylakoid of the chloroplast play a principle role in detoxifying hydrogen peroxide (H₂O₂) generated in photosystem I; however, once the ascorbate is depleted, the enzyme is attacked by H₂O₂ and rapidly loses its activity. Here, we report that radical transfer across the porphyrin moiety and amino acid residues in the reaction intermediate and H₂O₂-mediated enzyme inactivation involve cooperative interactions of the Cys26, Trp35, and Cys126 residues of stromal APX. The wild-type enzyme had a half-time of inactivation of <10 s, while the triple mutant of the three residues retained 50% of the initial activity after H₂O₂ treatment for 3 min. The H₂O₂ tolerance of this mutant was comparable to that of the H₂O₂-tolerant APX isoform localized in the cytosol.