Induced resistance in cells exposed to repeated low doses of H2O2 involves enhanced activity of antioxidant enzymes

We have derived cells from the Chinese hamster V79 cell line by conditioning them with repeated low doses of hydrogen peroxide (H(2)O(2)). This mimics the physiological condition where cells are repeatedly exposed to low levels of oxidants. In an attempt to characterize such cells, we have exposed both conditioned cells (V79(C)) and the parental V79 cells (V79(P)) to different types of cytotoxic agents and compared their sensitivity to cell killing. The V79(C) cells were found to be stably resistant to killing by agents that produced toxicity through oxidative stress, e.g. H(2)O(2) and cisplatin. It was also found that the lipid peroxidation produced by these agents were considerably lower in the V79(C) cells. Thus, the difference in sensitivity could be due to lesser extent of damage to these cells. V79(C) cells had greater antioxidant defense through higher GSH content and greater activity of enzymes such as Cu-Zn superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), which provided protection from damage. Enzyme activities were also assayed at different times after treatment with various cytotoxic agents; there was a relatively large increase in SOD activity which perhaps plays a key role in determining the resistance of the V79(C) cells to killing.     

Differential accumulation of antioxidant mRNAs in Arabidopsis thaliana exposed to ozone.

Antioxidant isoenzymes function to eliminate free radicals and are localized to several different subcellular compartments within the plant cell. In Arabidopsis thaliana exposed to ozone (O3), we have monitored the accumulation of mRNAs encoding both cytosolic and chloroplastic antioxidant isoenzymes. Two different O3 exposure protocols yielded similar results. Upon O3 exposure, the steady-state levels of three mRNAs encoding cytosolic antioxidant isoenzymes (ascorbate peroxidase, copper/zinc superoxide dismutase, and glutathione S-transferase) increase. The glutathione S-transferase mRNA responds very quickly to the oxidative stress (2-fold increase in 30 min) and is elevated to very high levels, especially in plants grown with a 16-h photoperiod. In contrast, O3 exposure causes a decline in the levels of two chloroplastic antioxidant mRNAs (iron superoxide dismutase and glutathione reductase) and two photosynthetic protein mRNAs (chlorophyll a/b-binding protein and ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit). We show that this decline does not include all mRNAs encoding chloroplast-targeted proteins, since O3 causes an elevation of mRNA encoding the chloroplast-localized tryptophan biosynthetic enzyme phosphoribosylanthranilate transferase. Two alternative hypotheses that could explain this differential mRNA accumulation in response to O3 are discussed.     

拟南芥H_2O_2突变体的筛选及特性分析

通过化学诱变剂甲基磺酸乙酯(EMS)诱变模式植物拟南芥(Arabidopsis thaliana)获得突变体筛选群体.在5 mmol/L H2O2胁迫下,以叶片温度差异为筛选指标,利用远红外成像技术进行突变体的筛选,获得了对H2O2不敏感突变体hpi1(hydrogen peroxide-insensitive1)和敏感突变体hps1(hydrogen peroxide-sensitive1).进一步研究发现,两种突变均为单基因隐性突变,气孔密度同野生型一样,而叶片温度、气孔开度和叶片失水率则有明显的差异.种子萌发实验表明,hpi1对甘露醇(Man)和NaCl不敏感而对ABA敏感,hps1则对3种胁迫都表现出敏感特性.     

Ultraviolet-B- and ozone-induced biochemical changes in antioxidant enzymes of Arabidopsis thaliana.

Earlier studies with Arabidopsis thaliana exposed to ultraviolet B (UV-B) and ozone (O3) have indicated the differential responses of superoxide dismutase and glutathione reductase. In this study, we have investigated whether A. thaliana genotype Landsberg erecta and its flavonoid-deficient mutant transparent testa (tt5) is capable of metabolizing UV-B- and O3-induced activated oxygen species by invoking similar antioxidant enzymes. UV-B exposure preferentially enhanced guaiacol-peroxidases, ascorbate peroxidase, and peroxidases specific to coniferyl alcohol and modified the substrate affinity of ascorbate peroxidase. O3 exposure enhanced superoxide dismutase, peroxidases, glutathione reductase, and ascorbate peroxidase to a similar degree and modified the substrate affinity of both glutathione reductase and ascorbate peroxidase. Both UV-B and O3 exposure enhanced similar Cu,Zn-superoxide dismutase isoforms. New isoforms of peroxidases and ascorbate peroxidase were synthesized in tt5 plants irradiated with UV-B. UV-B radiation, in contrast to O3, enhanced the activated oxygen species by increasing membrane-localized NADPH-oxidase activity and decreasing catalase activities. These results collectively suggest that (a) UV-B exposure preferentially induces peroxidase-related enzymes, whereas O3 exposure invokes the enzymes of superoxide dismutase/ascorbate-glutathione cycle, and (b) in contrast to O3, UV-B exposure generated activated oxygen species by increasing NADPH-oxidase activity.     

H2O2介导的H2S产生参与干旱诱导的拟南芥气孔关闭

以野生型拟南芥(Arabidopsis thaliana)及其突变体(atrbohD、atrbohF、atrbohD/F、atl-cdes、atd-cdes)和过表达株系(OEL-CDes、OED-CDes)为材料,利用药理学实验,结合分光光度法和激光共聚焦显微技术,探讨硫化氢(hydrogen sulfide,H2S)在干旱诱导的拟南芥气孔关闭中的作用及其与过氧化氢(hydrogen peroxide,H2O2)的关系.结果表明,H2S清除剂次牛磺酸(hypotaurine,HT)及合成抑制剂氨氧基乙酸(aminooxy acetic acid,AOA)、羟胺(hydroxylamine,NH2OH)和丙酮酸钾(potasium pyruvate,C3H3KO3)+氨水(ammonia,NH3)均可不同程度抑制干旱诱导的气孔关闭;干旱对OEL-CDes和OED-CDes植株气孔关闭的诱导作用明显,而atl-cdes和atd-cdes叶片气孔对干旱胁迫反应的敏感性下降;干旱胁迫能明显增加拟南芥保卫细胞中H2O2水平及叶片中H2S含量,提高D-/L-半胱氨酸脱巯基酶活性及基因表达量,而对突变体atrbohD、atrbohF和atrbohD/F没有显著影响.清除H2O2可减弱干旱胁迫对H2S含量和D-/L-半胱氨酸脱巯基酶活性的诱导效应.研究结果表明H2S位于H2O2下游参与干旱诱导拟南芥气孔关闭的信号转导过程.     

Effect of short-term salt stress on oxidative stress markers and antioxidant enzymes activity in tocopherol-deficient Arabidopsis thaliana plants

Changes of carotenoids and anthocyanins content, lipid peroxidation, and activity of antioxidant enzymes were studied in wild type and tocopherol-deficient lines vte1 and vte4 of Arabidopsis thaliana subjected to 200 mM NaCI during 24 h. The salt stress enhanced the intensity of lipid peroxidation to different extent in all three plant lines. Salt stress resulted in an increase of carotenoid content and activity of catalase, ascorbate peroxidase, guaiacol peroxidase and glutathione reductase in wild type and tocopherol-deficient vte1 mutant. However, the increase in anthocyanins concentration was observed in vte1 mutants only. In vte4 mutant, which contain gamma-tocopherol instead of alpha-tocopherol, the response to salt stress occurred via coordinative action of superoxide dismutase and enzymes of ascorbate-glutathione cycle, in particular, ascorbate peroxidase, glutathione reductase, dehydroascorbate reductase, and glutathione-S-transferase. It can be concluded, that salt stress was accompanied by oxidative stress in three studied lines, however different mechanisms involved in adaptation of wild type and tocopherol-deficient lines to salt stress.     

拟南芥保卫细胞中茉莉酸甲酯诱导的H2O2产生与MAPK信号转导体系的可能关系

蛋白激酶MEK1/2的专一抑制剂PD98059可抑制茉莉酸甲酯(MeJA)诱导的拟南芥保卫细胞中H2O2的产生和气孔的关闭.MeJA和H2O2诱导气孔关闭后,再用PD98059处理,可使关闭的气孔重新开放,同样,外源PD98059处理,能使MeJA诱导增强的H2O2探针的荧光强度降低.此结果表明,类属于MAPKK的蛋白激酶MEK1/2参与了MeJA诱导的拟南芥气孔关闭的信号转导过程,其作用机制可能是通过调节MeJA诱导保卫细胞产生和积累H2O2而起作用.     

Increased activity of H2O2 in aorta isolated from chronically streptozotocin-diabetic rats: effects of antioxidant enzymes and enzymes inhibitors.

The effects of hydrogen peroxide (H2O2, 1 nM-5 mM) on the tone of the rings of aorta precontracted with phenylephrine (PE) were studied in 4-5 months streptozotocin (STZ)-diabetic rats and their age-matched controls. H2O2 induced brief contraction before relaxation in endothelium-containing rings that was more pronounced in diabetic rats. Removal of the endothelium or pretreatment of rings with N(G)-nitro-L-arginine methyl ester (L-NAME, 100 microM) abolished H2O2-induced immediate and transient increase in tone, but preincubation with indomethacin (10 microM) had no effect on contractions induced by H2O2 in both group of animals. Pretreatment with L-NAME or indomethacin as well as absence of endothelium produced an inhibition of H2O2-induced relaxation that was more pronounced in diabetic rings. Chronically STZ-diabetes resulted in a significant increase in H2O2-induced maximum relaxation that was largely endothelium-dependent. Decreased sensitivity (pD2) of diabetic vessels to vasorelaxant action of H2O2 was normalized by superoxide dismutase (SOD, 80 U/ml). Pretreatment with SOD had no effect on H2O2-induced maximum relaxations in both group of animals but led to an increase in H2O2-induced contractions in control rats. When the rings pretreated with diethyldithiocarbamate (DETCA, 5 mM), H2O2 produced only contraction in control rats, and H2O2-induced relaxations were markedly depressed in diabetic rats. H2O2 did not affect the tone of intact or endothelium-denuded rings in the presence of catalase (2000 U/ml). Aminotriazole (AT, 10 mM) failed to affect H2O2-induced contractions or relaxations in all rings. Our observations suggest that increased production of oxygen-derived free radicals (OFRs) in diabetic state leads to a decrease in SOD activity resulting an increase in endogenous superoxide anions (O2*-), that is limited cytotoxic actions, and an increase in catalase activity resulting a decrease in both H2O2 concentrations and the production of harmful hydroxyl radical (*OH) in diabetic aorta in long-term. Present results indicate that increased vascular activity of H2O2 may be an important factor in the development of vascular disorders associated with chronically diabetes mellitus. Enhanced formation of *OH, that is a product of exogenous H2O2 and excess O2*, seems to be contribute to increased relaxations to exogenously added H2O2 in chronically diabetic vessels.     

Ca2+位于H2O2上游参与H2S诱导的拟南芥气孔关闭过程

以拟南芥为材料,利用药理学实验,结合分光光度法和激光共聚焦显微技术,研究了Ca2+在硫化氢(H2S)诱导拟南芥气孔关闭过程中的作用及其与过氧化氢(H2O2)的关系。结果表明： H2S诱导气孔关闭, Ca2+螯合剂EGTA和质膜Ca2+通道阻断剂硝苯地平(Nif)能不同程度抑制H2S诱导的气孔关闭,而内质网钙泵阻断剂毒胡萝卜素(Thaps)对H2S的作用无显著影响。由此推测, Ca2+参与调节H2S诱导的拟南芥气孔关闭过程,且胞质中Ca2+来源于胞外Ca2+的内流。另外, H2S诱导拟南芥叶片NADPH氧化酶基因AtRBOHD和AtRBOHF以及细胞壁过氧化物酶基因AtPRX34表达增强,促进叶片和保卫细胞中H2O2积累, EGTA对此起抑制作用,而外源CaCl2处理上调AtRBOHD、AtRBOHF和AtPRX34的表达。表明Ca2+可能位于H2O2上游参与H2S诱导的拟南芥气孔关闭过程。     

Free radical formation and activity of antioxidant enzymes in lupin roots exposed to lead

Inhibition of root growth and modification of root morphology are the most sensitive responses of Lupinus luteus cv. Ventus L. to lead ions - Pb(NO₃)₂. Using electron paramagnetic resonance (EPR), we found that at the lead concentration of 150 mg.L^–1, the level of free radicals remained at control level, whereas at the higher, sublethal concentration of 350 mg.L^–1, they markedly increased. The EPR signal with the g-value at the maximum absorption of 2.0053 implied that the paramagnetic radical is derived from a quinone. The response of antioxidant enzymes, such as superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), peroxidase (POX, EC 1.11.1.7) and ascorbate peroxidase (APOX, EC 1.11.1.11), to exogenously applied lead ions was also examined. Enzyme activity was estimated as a function of time and concentration. Native polyacrylamide gel electrophoresis followed by specific staining revealed an increase in the activity of SOD, CAT, POX and APOX coinciding with the time of cultivation. A lead-dependent increase in activities of SOD and POX from root tip extracts was observed, whereas CAT and APOX activities decreased at the higher lead concentrations. These results suggest that at higher lead concentrations, the formation of both free radicals and reactive oxygen species is beyond the capacity of the antioxidant system, which in turn may contribute to the reduced root growth.     

The enzymatic activity of the antioxidant system in the ontogeny of mutant norflurazone-tolerant Arabidopsis thaliana

The activities of superoxide dismutase and guaiacol-dependent peroxidase were studied in the ontogenesis of recessive homozygous mutants of Arabidopsis thaliana Heynh. le-2 and nfz24, which are characterized by two- to threefold increases in tolerance to the herbicide norflurazone. The mutants le-2 and nfz24 differed from the initial race Dijon in some phenotypic features, duration of ontogenetic stages, and dynamics of the superoxide dismutase and peroxidase activities in ontogenesis. A single treatment of plants with norflurazone induced an accelerated increase in the level of both enzymes in the mutants as compared to the wild type plants. Under the conditions of multiple treatment with norflurazone, the mutants le-2 and nfz24 displayed a higher tolerance to the bleaching effect of the herbicide and were characterized by a higher level of superoxide dismutase. The data obtained suggest that the superoxide dismutase and peroxidase activities are controlled by both ontogenetic factors and stress signals. Mutations in the lines le-2 and nfz24 increase sensitivity to a stress signal or increase efficiency of an adaptive response due to long-term maintenance of a high level of the antioxidant enzymes under the conditions of stress.     

Enhanced chloroplastic generation of H2O2 in stress‐resistant Thellungiella salsuginea in comparison to Arabidopsis thaliana

In order to find some basis of salinity resistance in the chloroplastic metabolism, a halophytic Thellungiella salsuginea was compared with glycophytic Arabidopsis thaliana. In control T.s. plants the increased ratios of chlorophyll a/b and of fluorescence emission at 77 K (F₇₃₀/F₆₈₅) were documented, in comparison to A.t.. This was accompanied by a higher Y_II and lower NPQ (non‐photochemical quenching) values, and by a more active PSI (photosystem I). Another prominent feature of the photosynthetic electron transport (PET) in T.s. was the intensive production of H₂O₂ from PQ (plastoquinone) pool. Salinity treatment (0.15 and 0.30 M NaCl for A.t. and T.s., respectively) led to a decrease in ratios of chl a/b and F₇₃₀/F₆₈₅. In A.t., a salinity‐driven enhancement of Y_II and NPQ was found, in association with the stimulation of H₂O₂ production from PQ pool. In contrast, in salinity‐treated T.s., these variables were similar as in controls. The intensive H₂O₂ generation was accompanied by a high activity of PTOX (plastid terminal oxidase), whilst inhibition of this enzyme led to an increased H₂O₂ formation. It is hypothesized, that the intensive H₂O₂ generation from PQ pool might be an important element of stress preparedness in Thellungiella plants. In control T.s. plants, a higher activation state of carboxylase ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) was also documented in concert with the attachment of Rubisco activase (RCA) to the thylakoid membranes. It is supposed, that a closer contact of RCA with PSI in T.s. enables a more efficient Rubisco activation than in A.t.     

ECS1参与调节CO2诱导的拟南芥气孔关闭和H2O2积累

CO2浓度升高可以诱导植物叶片气孔关闭,提高植物对高浓度CO2的适应性.但植物如何感知CO2浓度变化并启动气孔关闭反应的分子机制至今仍不十分清楚.利用高通量、非侵入的远红外成像技术,建立了拟南芥(Arabidopsis thaliana)气孔对CO2浓度变化反应相关的突变体筛选技术,筛选出对环境CO2浓度敏感的拟南芥突变体ecs1.遗传学分析表明,ecs1 为单基因隐性突变体,突变基因ECS1编码一个跨膜钙离子转运蛋白.与野生型拟南芥相比,360 μL·L-1CO2可引起ecs1突变体叶片温度上升和气孔关闭,ecs1突变体对900 μL·L-1CO2长时间处理具有较强的适应性.进一步的实验表明,360 μL·L-1CO2即可诱导ecs1突变体叶片积累较高浓度的H2O2,而900 μL·L-1CO2才能够诱导野生型拟南芥叶片积累H2O2.因此,ECS1可能参与调节高浓度CO2诱导的拟南芥气孔关闭和H2O2产生,H2O2可能作为第二信号分子介导CO2诱导拟南芥气孔关闭的反应.     

Genomic mutation in lines of Arabidopsis thaliana exposed to ultraviolet-B radiation

Studies that have attempted to estimate the rate of deleterious mutation have typically been conducted under low levels of ultraviolet-B (UV-B) radiation, a naturally occurring mutagen. We conducted experiments to test whether the inclusion of natural levels of UV-B radiation in mutation-accumulation (MA) experiments influences the rate and effects of mildly deleterious mutation in the plant Arabidopsis thaliana. Ten generations of MA proved insufficient to observe significant changes in means or among-line variances in experimental lines maintained either with or without supplemental UV-B radiation. Maximum-likelihood estimates of mutation rate for total flower number revealed a small but significant rate of mutation for MA lines propagated under supplemental UV-B exposure, but not for those in which supplemental UV-B was omitted. A fraction of the flower number mutations under UV-B (approximately 25-30%) are estimated to increase flower number. Results from the application of transposon display to plant materials obtained after MA, in both the presence and absence of supplemental UV-B, suggest that the average rate of transposition for the class I and II transposable elements (TEs) surveyed was no more than 10(-4). Overall, the estimates of mutation parameters are qualitatively similar to what has been observed in other MA experiments with this species in which supplemental UV-B levels have not been used. As well, it appears that naturally occurring levels of UV-B do not lead to detectable increases in levels of transposable element activity.     

Responses of antioxidant enzymes to cold and high light are not correlated to freezing tolerance in natural accessions of Arabidopsis thaliana

Low temperatures and high light cause imbalances in primary and secondary reactions of photosynthesis, and thus can result in oxidative stress. Plants employ a range of low‐molecular weight antioxidants and antioxidant enzymes to prevent oxidative damage, and antioxidant defence is considered an important component of stress tolerance. To figure out whether oxidative stress and antioxidant defence are key factors defining the different cold acclimation capacities of natural accessions of the model plant Arabidopsis thaliana, we investigated hydrogen peroxide (H₂O₂) production, antioxidant enzyme activity and lipid peroxidation during a time course of cold treatment and exposure to high light in four differentially cold‐tolerant natural accessions of Arabidopsis (C24, Nd, Rsch, Te) that span the European distribution range of the species. All accessions except Rsch (from Russia) had elevated H₂O₂ in the cold, indicating that production of reactive oxygen species is part of the cold response in Arabidopsis. Glutathione reductase activity increased in all but Rsch, while ascorbate peroxidase and superoxide dismutase were unchanged and catalase decreased in all but Rsch. Under high light, the Scandinavian accession Te had elevated levels of H₂O₂. Te appeared most sensitive to oxidative stress, having higher malondialdehyde (MDA) levels in the cold and under high light, while only high light caused elevated MDA in the other accessions. Although the most freezing‐tolerant, Te had the highest sensitivity to oxidative stress. No correlation was found between freezing tolerance and activity of antioxidant enzymes in the four accessions investigated, arguing against a key role for antioxidant defence in the differential cold acclimation capacities of Arabidopsis accessions.     

Short communication. H2O2 activates a MAP kinase-like enzyme in Arabidopsis thaliana suspension cultures

Treatment of Arabidopsis thaliana suspension cultures with H2O2 results in the activation of a 44 kDa protein kinase with the characteristics of a mitogen activated protein (MAP) kinase. It preferentially phosphorylates myelin basic protein as an artificial substrate, it requires tyrosine phosphorylation for its activity, is tyrosine and threonine phosphorylated upon activation, and its activation is rapid, transient, and occurs in a dose-dependent manner. This is the first demonstration of the activation of a MAP kinase-like enzyme by H2O2 in plants.     

Copper-induced oxidative stress and antioxidant defence in Arabidopsis thaliana

Content of reactive oxygen species (ROS): O2*-, H2O2 and OH* as well as activities of antioxidant enzymes: superoxide dismutase (SOD), guaiacol peroxidase (POX) and catalase (CAT) were studied in leaves of Arabidopsis thaliana ecotype Columbia, treated with Cu excess (0, 5, 25, 30, 50, 75, 100, 150 and 300 microM). After 7 days of Cu action ROS content and the activity of SOD and POX increased, while CAT activity decreased in comparison with control. Activities of SOD, POX and CAT were correlated both with Cu concentration (0-75 microM) in the growth medium and with OH* content in leaves. Close correlation was also found between OH* content and Cu concentration. Oxidative stress in A. thaliana under Cu treatment expressed in elevated content of O2*-, H2O2 and OH* in leaves. To overcome it very active the dismutase- and peroxidase-related (and not catalase-related, as in other plants) ROS scavenging system operated in A. thaliana. Visual symptoms of phytotoxicity: chlorosis, necrosis and violet colouring of leaves as well as a reduction of shoot biomass occurred in plants.     

PLC1 and H2O2 Mediated the Allelopathic Effect of Oridonin on Root Growth in Arabidopsis thaliana

Oridonin is a diterpenoid isolated from medicinal herb Rabdosia rubescens (Hemsl.) Hara (Lamiaceae), which has an allelopathic effect on plants. Phospholipase C (PLC1) and hydrogen peroxide (H₂O₂) are involved in many biotic or abiotic stress responses. Using the 16-day-old seedlings of Arabidopsis thaliana ecotype (WT) and PLC1-deficient mutant (plc1) as materials (treated with 10 μM or 60 μM oridonin for 72 h), the effect of oridonin on root growth regulating by PLC1 and H₂O₂ was investigated. The results showed that the promoting of root growth was about 6.9% at 10 μmol L^?1 oridonin and the inhibiting of root growth was about 19.73% at 60 μmol L^?1 oridonin in WT, the inhibiting of root growth was about 10.5% and 41.2% at 10 mol L^?1 and 60 mol L^?1 oridonin, respectively, in plc1. The expression of ARR1, ARR12, and AHK3 was promoted at low concentrations of oridonin and inhibited at high concentrations in WT, whereas the expression of ARR1 and ARR12 was inhibited with the increase of oridonin concentration in plc1. This suggested that PLC1 was involved in the root growth regulation of oridonin. H₂O₂ was promoted by oridonin with concentration dependence pattern in root cells. Oridonin increased the activity of antioxidant enzymes in both WT and plc1, but the activity of antioxidant enzymes in plc1 was lower than WT. This indicated that PLC1 involved in the activation of antioxidant enzymes promoted by the oridonin. Exogenous CaCl₂ facilitated the accumulation of H₂O₂ in both WT and plc1. And the H₂O₂ of WT was obviously higher than that of plc1. The root growth of WT was inhibited by CaCl₂ with the increase of oridonin. However, there is no effect of CaCl₂ on the root growth in plc1. This reflected that PLC1 positively involved in the regulation of Ca²⁺ on the H₂O₂ and the inhibition effect of Ca²⁺ on the root growth under oridonin treatment. PA promoted the H₂O₂ and suppressed the root growth under oridonin treatment in both WT and plc1. In plc1, PA facilitated the root growth with no oridonin and inhibited the root growth with the increase of oridonin. This reflected that PLC1 positively regulated the promotion effect of PA on the root growth under high oridonin treatment. PLC1 mediated oridonin (10 and 60 mol L^?1) to regulate H₂O₂ levels in A. thaliana seedlings, thereby regulating root tip cell morphology and mitosis. These results demonstrated that PLC1 mediated the low-promotion and high-inhibition effect of oridonin on the root growth in A. thaliana by regulating the concentrations of Ca²⁺ and PA, and further affecting the intracellular H₂O₂ level.

     

Arabidopsis thaliana overexpressing glycolate oxidase in chloroplasts: H2O2-induced changes in primary metabolic pathways

Reactive oxygen species (ROS) represent both toxic by-products of aerobic metabolism as well as signaling molecules in processes like growth regulation and defense pathways. The study of signaling and oxidative-damage effects can be separated in plants expressing glycolate oxidase in the plastids (GO plants), where the production of H₂O₂ in the chloroplasts is inducible and sustained perturbations can reproducibly be provoked by exposing the plants to different ambient conditions. Thus, GO plants represent an ideal non-invasive model to study events related to the perception and responses to H₂O₂ accumulation. Metabolic profiling of GO plants indicated that under high light a sustained production of H₂O₂ imposes coordinate changes on central metabolic pathways. The overall metabolic scenario is consistent with decreased carbon assimilation, which results in lower abundance of glycolytic and tricarboxylic acid cycle intermediates, while simultaneously amino acid metabolism routes are specifically modulated. The GO plants, although retarded in growth and flowering, can complete their life cycle indicating that the reconfiguration of the central metabolic pathways is part of a response to survive and thus, to adapt to stress conditions imposed by the accumulation of H₂O₂ during the light period.Key words: Arabidopsis thaliana, H₂O₂, oxidative stress, reactive oxygen species, signalingReactive oxygen species (ROS) are key molecules in the regulation of plant development, stress responses and programmed cell death. Depending on the identity of ROS species or its subcellular production site, different cellular responses are provoked.¹ To assess the effects of metabolically generated H₂O₂ in chloroplasts, we have recently generated Arabidopsis plants in which the peroxisomal GO was targeted to chloroplasts.² The GO overexpressing plants (GO plants) show retardation in growth and flowering time, features also observed in catalase, ascorbate peroxidase and MnSOD deficient mutants.^3–5 The analysis of GO plants indicated that H₂O₂ is responsible for the observed phenotype. GO plants represent an ideal non-invasive model system to study the effects of H₂O₂ directly in the chloroplasts because H₂O₂ accumulation can be modulated by growing the plants under different ambient conditions. By this, growth under low light or high CO₂ concentrations minimizes the oxygenase activity of RubisCO and thus the flux through GO whereas the exposition to high light intensities enhances photorespiration and thus the flux through GO.Here, we explored the impact of H₂O₂ production on the primary metabolism of GO plants by assessing the relative levels of various metabolites by gas chromatography coupled to mass spectrometry (GC-MS)⁶ in rosettes of plants grown at low light (30 µmol quanta m⁻² s⁻¹) and after exposing the plants for 7 h to high light (600 µmol quanta m⁻² s⁻¹). The results obtained for the GO5 line are shown in