Antioxidant defences of the apoplast

Summary The apoplast of barley and oat leaves contained superoxide dismutase (SOD), catalase, ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase, and glutathione reductase activities. The activities of these enzymes in the apoplastic extracts were greatly modified 24 h after inoculation with the biotrophic fungal pathogenBlumeria graminis. The quantum efficiency of photosystem II, which is related to photosynthetic electron transport flux, was comparable in inoculated and healthy leaves during this period. Apoplastic soluble acid invertase activity was also modified in inoculated leaves. Inoculation-dependent increases in apoplastic SOD activity were observed in all lines. Major bands of SOD activity, observed in apoplastic protein extracts by activity staining of gels following isoelectric focusing, were similar to those observed in whole leaves but two additional minor bands were found in the apoplastic fraction. The apoplastic extracts contained substantial amounts of dehydroascorbate (DHA) but little or no glutathione (GSH). Biotic stress decreased apoplastic ascorbate and DHA but increased apoplastic GSH in resistant lines. The antioxidant cycle enzymes may function to remove apoplastic H₂O₂ with ascorbate and GSH derived from the cytoplasm. DHA and oxidized glutathione may be reduced in the apoplast or returned to the cytosol for rereduction.Abbreviations AA reduced ascorbate - APX ascorbate peroxidase - DHA dehydroascorbate (oxidised ascorbate) - DHAR dehydroascorbate reductase - G6PDH glucose-6-phosphate dehydrogenase - GSH reduced glutathione - GSSG glutathione disulphide - GR glutathione reductase - MDHA monodehydroascorbate - MDHAR monodehydroascorbate reductase - SOD superoxide dismutase     

转GST和CAT1基因水稻根系抗氧化系统对干旱高温胁迫的响应(英文)

以携带谷胱甘肽转移酶(GST)和过氧化氢酶(CAT1)的转基因水稻和非转基因水稻(Oryza sativa L.) 品种'中花11'的根系为材料, 比较分析了二者在PEG 6000、38℃及PEG 6000和38℃复合胁迫下抗氧化系统特别是抗坏血酸-谷胱甘肽循环系统的变化.结果显示, 6% PEG处理时,转基因水稻的CAT、GST、超氧化物歧化酶(SOD)、谷胱甘肽还原酶(GR)和脱氢抗坏血酸还原酶(DHAR)的活性都显著高于非转基因水稻;38℃处理时,前者的CAT、GST、SOD和GR的活性则显著低于后者;6% PEG和38℃复合处理时,前者的CAT、GST、抗坏血酸过氧化物酶(APX)和DHAR的活性也都显著高于后者,但前者的SOD和GR活性则显著低于后者.6%PEG 诱导的转基因水稻根系的抗坏血酸氧还状态显著低于非转基因水稻,但二者的谷胱甘肽氧还状态无显著差异; 而6% PEG和38℃同时处理时,转基因水稻的谷胱甘肽氧还状态则显著高于非转基因水稻,但二者的抗坏血酸氧还状态差异不显著.研究发现,干旱和高温复合胁迫时,转基因水稻和非转基因水稻的抗氧化组分的变化均不等于这2种单一胁迫的叠加;GST和CAT1基因的转入对水稻抗氧化系统内源功能相关组分尤其是抗坏血酸-谷胱甘肽循环系统产生了一定的影响,两种水稻的根系可能利用不同的抗氧化组分调节机制对这些胁迫做出应答.     

Constitutively Elevated Levels of Putrescine and Putrescine-Generating Enzymes Correlated with Oxidant Stress Resistance in Conyza bonariensis and Wheat

Changes in ascorbate and glutathione levels and in activities of ascorbate peroxidase, catalase, dehydroascorbate reductase (DHAR), glutathione reductase (GR), glutathione S-transferase (GST), and superoxide dismutase (SOD) were investigated in tobacco mosaic virus (TMV)-inoculated lower leaves and in non-inoculated upper leaves of Nicotiana tabacum L. cv Xanthi-nc. In separate experiments the effects of exogenous salicylic acid (SA) were also studied. Symptom appearance after TMV inoculation was preceded by a slight, transient decline of ascorbate peroxidase, GR, GST, and SOD activities in the inoculated lower leaves, but after the onset of necrosis these activities and the glutathione level substantially increased. Ascorbic acid level and DHAR activity declined and dehydroascorbate accumulated in the inoculated leaves. In upper leaves, the glutathione level and the activities of GR, GST, and SOD increased 10 to 14 d after TMV inoculation of the lower leaves, concomitantly with the development of systemic acquired resistance. From the six distinct SOD isoenzymes found in tobacco leaves, only the activities of Cu,Zn-SOD isoenzymes were affected by TMV. SA injection induced DHAR, GR, GST, and SOD activities. Catalase activities were not modified by TMV infection or SA treatment. It is supposed that stimulated antioxidative processes contribute to the suppression of necrotic symptom development in leaves with systemic acquired resistance.     

Fungal pathogen-induced changes in the antioxidant systems of leaf peroxisomes from infected tomato plants

Peroxisomes, being one of the main organelles where reactive oxygen species (ROS) are both generated and detoxified, have been suggested to be instrumental in redox-mediated plant cell defence against oxidative stress. We studied the involvement of tomato (Lycopersicon esculentum Mill.) leaf peroxisomes in defence response to oxidative stress generated upon Botrytis cinerea Pers. infection. The peroxisomal antioxidant potential expressed as superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6) and glutathione peroxidase (GSH-Px, EC 1.11.1.19) as well as the ascorbate-glutathione (AA-GSH) cycle activities was monitored. The initial infection-induced increase in SOD, CAT and GSH-Px indicating antioxidant defence activation was followed by a progressive inhibition concomitant with disease symptom development. Likewise, the activities of AA-GSH cycle enzymes: ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and glutathione reductase (GR, EC 1.6.4.2) as well as ascorbate and glutathione concentrations and redox ratios were significantly decreased. However, the rate and timing of these events differed. Our results indicate that B. cinerea triggers significant changes in the peroxisomal antioxidant system leading to a collapse of the protective mechanism at advanced stage of infection. These changes appear to be partly the effect of pathogen-promoted leaf senescence.     

Time course of antioxidant responses of Capsicum annuum subjected to a progressive magnesium deficiency

The effect of magnesium (Mg²⁺)‐deficiency on the antioxidant responses of Capsicum annuum was investigated over a 60‐day period under controlled conditions. This Mg²⁺‐deficiency aimed to mimic the physiological conditions that plants may experience in the field. At each harvest time, five different leaf‐levels (L2 to L6) were distinguished. L2 and L6 correspond to the second and sixth youngest leaves, respectively. The following parameters were determined: Mg²⁺, chlorophyll and protein contents, total and redox pools of ascorbate and glutathione, and the activities of superoxide dismutase, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. Under Mg²⁺‐deficiency, leaf Mg²⁺ contents decreased over time in all leaf‐levels except in the second youngest leaves (L2), where they remained constant at about 0.25% (dry weight basis). Mg²⁺‐deficiency led to an increase in the antioxidant enzyme activities concomitant with an increase in the ascorbate and glutathione pools, whereas total chlorophyll and soluble protein contents decreased. The L2 leaves showed an increase in glutathione reductase activity and in the ascorbate redox state whereas no difference was observed for the other parameters. Superoxide dismutase activities increased in L5 leaves from day 15 and, afterwards, in L3 to L5 leaves, irrespective of Mg²⁺ content. At day 30, glutathione reductase activities increased in L2 to L4 leaves and dehydroascorbate reductase activities in L4 leaves. At day 45, we observed an increase in the ascorbate peroxidase activities in L3 to L5 leaves. At the same time, ascorbate and glutathione pools increased in intermediate leaves, whereas chlorophyll content decreased in L3 and L4 leaves, and protein content decreased in L4 leaves. Results suggest that pepper leaves enhance their defence capacities against oxidative stress by increasing ascorbate more than glutathione synthesis. However, cells showed higher regeneration rates for the glutathione redox state than for the ascorbate redox state.     

Changes in the antioxidative systems in mitochondria during ripening of pepper fruits

The presence of enzymes of the ascorbate–glutathione cycle was studied in mitochondria purified from green and red pepper (Capsicum annuum L.) fruits. All four enzymes, ascorbate peroxidase (APX; EC 1.11.1.11), monodehydroascorbate reductase (MDHAR; EC 1.6.5.4), dehydroascorbate reductase (DHAR; EC 1.8.5.1) and glutathione reductase (GR; EC 1.6.4.2) were present in the isolated mitochondria of both fruit ripening stages. The activity of the reductive ascorbate–glutathione cycle enzymes (MDHAR, GR and DHAR) was higher in mitochondria isolated from green than from red fruits, while APX and the antioxidative enzyme superoxide dismutase (SOD; EC 1.15.1.1) were higher in the red fruits. The levels of ascorbate and L-galactono-γ-lactone dehydrogenase (GLDH; EC 1.3.2.3) activity were found to be similar in the mitochondria of both fruits. The higher APX and Mn-SOD specific activities in mitochondria from red fruits might play a role in avoiding the accumulation of any activated oxygen species generated in these mitochondria, and suggests an active role for these enzymes during ripening.     

Hydrogen-peroxide-scavenging systems within pea chloroplasts

The subcellular distribution of ascorbate peroxidase and glutathione reductase (EC 1.6.4.2) in pea leaves was compared with that of organelle markers. Enzyme distribution was found to be similar to that of the chloroplast enzyme NADPH-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13). Isolated chloroplasts showed a close correlation between intactness and the percentage of enzyme activity recovered. Chloroplasts of 85% intactness were found to contain a high proportion of leaf dehydroascorbate reductase activity (EC 1.8.5.1), 10% of leaf glutathione and 30% of leaf ascorbate. These results are discussed in relation to the potential role of chloroplast antioxidant systems in plant resistance to environmental and other stress conditions.Abbreviations GSH reduced glutathione - GSSG oxidized glutathione - NADPH-GPD glyceraldehyde-3-phosphate dehydrogenase - SOD superoxide dismutase     

Antioxidant enzymes in the liver of Chelidonichthys obscurus from the Montenegrin coastline

The activities of antioxidant defence enzymes — total, manganese and copper zinc containing superoxide dismutase (Tot SOD, Mn SOD, CuZn SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR) and biotransformation phase II enzyme glutathione-S-transferase (GST) — in the liver of longfin gurnard (Chelidonichthys obscurus) from the Montenegrin coastline (Adriatic sea) were investigated. The specimens were collected in winter (February) and late spring (May) at two localities: Platamuni (PL, potentially unpolluted) and the Estuary of the River Bojana (EB, potentially polluted). The obtained results show that the activities of Mn SOD, CAT, GSH-Px and GST in winter were significantly lower at EB than at PL. In spring, the activities of CAT and GST were decreased, while GR activity was increased at EB in comparison to PL. The activities of Mn SOD and GST at PL were decreased and GSH-Px, GR and GST activities at EB were increased in spring compared to winter. Our work represents the first study of liver antioxidant enzymes of longfin gurnard from the Montenegrin coastline and reveals that locality, as a variable, has a greater influence on antioxidant enzymes and biotransformation phase II enzyme GST activities compared to season.     

Modified responses of root growth and reactive oxygen species-scavenging system to combined salt and heat stress in transgenic rice

Root growth and modifications of ROS-scavenging systems were investigated in transgenic rice (Oryza sativa L., cv. Zhonghua no. 11) co-expressing glutathione-S-transferase (GST, EC. 2.5.1.18) and catalase 1 (CAT1, EC 1.11.1.6) and nontransgenic rice exposed to just salt or heat and their combination. The higher number of adventitious roots but the lower root to shoot ratio were observed in the stressed transgenics as compared with nontransgenics. Most antioxidant enzymes, such as CAT, GST, ascorbate peroxidase (APX, EC 1.11.1.11), glutathione reductase (GR, EC.1.6.4.2), dehydroascorbate reductase (DHAR, EC.1.8.5.1), and the redox state of glutathione and ascorbate, measured in the transformant roots, were significantly different from those in nontransformant roots following the three types of stress. The variations of root growth and antioxidant systems in the stressed transgenic rice may be attributed to not only the GST and CAT1 transgenes but also the coordination of the ascorbate-glutathione cycle.     

Methyl jasmonate and salicylic acid elicitation induces ginsenosides accumulation, enzymatic and non-enzymatic antioxidant in suspension culture Panax ginseng roots in bioreactors

The effects of methyl jasmonate (MJ) and salicylic acid (SA) on changes of the activities of major antioxidant enzymes, superoxide anion accumulation (O₂ ⁻), ascorbate, total glutathione (TG), malondialdehyde (MDA) content and ginsenoside accumulation were investigated in ginseng roots (Panax ginseng L.) in 4 l (working volume) air lift bioreactors. Single treatment of 200 μM MJ and SA to P. ginseng roots enhanced ginsenoside accumulation compared to the control and harvested 3, 5, 7 and 9 days after treatment. MJ and SA treatment induced an oxidative stress in P. ginseng roots, as shown by an increase in lipid peroxidation due to rise in O₂ ⁻ accumulation. Activity of superoxide dismutase (SOD) was inhibited in MJ-treated roots, while the activities of monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), SOD, guaiacol peroxidase (G-POD), glutathione peroxidase (GPx) and glutathione reductase (GR) were induced in SA-treated roots. A strong decrease in the activity of catalase (CAT) was obtained in both MJ- and SA-treated roots. Activities of ascorbate peroxidase (APX) and glutathione S transferase (GST) were higher in MJ than SA while the contents of reduced ascorbate (ASC), redox state (ASC/(ASC+DHA)) and TG were higher in SA- than MJ-treated roots while oxidized ascorbate (DHA) decreased in both cases. The result of these analyses suggests that roots are better protected against the O₂ ⁻ stress, thus mitigating MJ and SA stress. The information obtained in this work is useful for efficient large-scale production of ginsenoside by plant-root cultures.     

Response of mitochondrial antioxidant system and respiratory pathways to reactive nitrogen species in pea leaves

Nitric oxide (NO) has emerged as an important signaling molecule in plants, but little is known about the effects of reactive nitrogen species in plant mitochondria. In this study, the effects of DETA‐NONOate, a pure NO slow generator, and of SIN‐1 (3‐morpholinosydnonimine), a peroxynitrite producer, on the activities of respiratory pathways, enzymatic and non‐enzymatic antioxidants have been investigated in isolated mitochondria from pea leaves. No significant changes in lipid peroxidation, protein oxidation or in ascorbate and glutathione redox state were observed after DETA‐NONOate treatments whereas cytochrome pathway (CP) respiration was reversibly inhibited and alternative pathway (AP) respiration showed little inhibition. On the other hand, NO did not affect neither activities of Mn superoxide dismutase (Mn‐SOD) nor enzymes involved in the ascorbate and glutathione regeneration in mitochondria except for ascorbate peroxidase (APX), which was reversely inhibited depending on ascorbate concentration. Finally, SIN‐1 treatment of mitochondria produced a decrease in CP respiration, an increase in protein oxidation and strongly inhibited APX activity (90%), with glutathione reductase and dehydroascorbate reductase (DHAR) being moderately inhibited (30 and 20%, respectively). This treatment did not affect monodehydroascorbate reductase (MDHAR) and Mn‐SOD activities. Results showed that mitochondrial nitrosative stress was not necessarily accompanied by oxidative stress. We suggest that NO‐resistant AP and mitochondrial APX may be important components of the H₂O₂‐signaling pathways under nitrosative stress induced by NO in this organelle. Also, MDHAR and DHAR, via ascorbate regeneration, could constitute an essential antioxidant defense together with Mn‐SOD, against NO and ONOO^? stress in plant mitochondria.     

Antioxidants and Manganese Deficiency in Needles of Norway Spruce (Picea abies L.) Trees

Chlorotic and green needles from Norway spruce (Picea abies L.) trees were sampled in the Calcareous Bavarian Alps in winter. The needles were used for analysis of the mineral and pigment contents, the levels of antioxidants (ascorbate, glutathione), and the activities of protective enzymes (superoxide dismutase, catalase, ascorbate peroxidase, monodehydroascorbate radical reductase, dehydroascorbate reductase, glutathione reductase). In addition, the activities of two respiratory enzymes (glucose-6-phosphate dehydrogenase, NAD-malate dehydrogenase), which might provide the NADPH necessary for functioning of the antioxidative system, were determined. We found that chlorotic needles were severely manganese deficient (3 to 6 micrograms Mn per gram dry weight as compared with up to 190 micrograms Mn per gram dry weight in green needles) but had a similar dry weight to fresh weight ratio, had a similar protein content, and showed no evidence for enhanced lipid peroxidation as compared with green needles. In chlorotic needles, the level of total ascorbate and the activities of superoxide dismutase, monodehydroascorbate radical reductase, NAD-malate dehydrogenase, and glucose-6-phosphate dehydrogenase were significantly increased, whereas the levels of ascorbate peroxidase, dehydroascorbate reductase, glutathione reductase, and glutathione were not affected. The ratio of ascorbate to dehydroascorbate was similar in both green and chlorotic needles. These results suggest that in spruce needles monodehydroascorbate radical reductase is the key enzyme involved in maintaining ascorbate in its reduced state. The reductant necessary for this process may have been supplied at the expense of photosynthate.     

黄土高原冰草叶片抗坏血酸和谷胱甘肽合成及循环代谢对干旱胁迫的生理响应

通过盆栽实验, 对干旱胁迫下黄土高原地区冰草(Agropyron cristatum)叶片的抗坏血酸和谷胱甘肽合成及循环代谢相关酶及物质含量进行了研究。结果表明: 冰草可以通过增强叶片的抗坏血酸和谷胱甘肽合成及循环代谢酶: 抗坏血酸过氧化物酶、谷胱甘肽还原酶、脱氢抗坏血酸还原酶、单脱氢抗坏血酸还原酶、L-半乳糖酸-1, 4-内酯脱氢酶和γ-谷氨酰半胱氨酸合成酶活性, 维持植物体内抗坏血酸和谷胱甘肽水平及氧化还原状态, 从而抵御干旱造成的氧化胁迫。但叶片抗坏血酸和谷胱甘肽合成及循环代谢对不同水平干旱胁迫的响应, 随胁迫时间的延长而不同。在胁迫24天以前, 严重干旱下叶片的抗坏血酸和谷胱甘肽合成及循环代谢增强较显著; 在胁迫24天后, 由于该胁迫下植物所遭受的氧化胁迫较为严重, 叶片中上述6种酶的活性均呈降低趋势。而在中度干旱下叶片抗坏血酸和谷胱甘肽合成及循环代谢相关的6种酶在整个胁迫过程中均保持较高的活性。这说明, 冰草能够长时间有效地抵御中度干旱所造成的氧化胁迫, 但只能在一定时间范围内有效地抵御严重干旱所造成的氧化胁迫, 胁迫时间延长则会降低其抵御严重干旱的能力。     

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.     

Role of glutathione in adaptation and signalling during chilling and cold acclimation in plants

To test the hypothesis that antioxidant systems flexibly adjust to short-term, diurnal fluctuations of ambient environmental conditions, ascorbate-related systems were studied over several day/night cycles in mature sun-acclimated leaves of field-grown beech trees ( Fagus sylvatica ). Light-dependent increases in the activities of ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate radical reductase (MDAR, EC 1.1.5.4) and glutathione reductase (GR, EC 1.6.4.2) were not observed. Lowest activities of APX and MDAR were found on hot, sunny days. A strong negative correlation occurred between APX activities and ambient temperatures suggesting that this enzyme was temperature- rather than light-regulated. In contrast to the enzymatic defences, ascorbate levels increased by about 30% under bright sunlight suggesting that protection from excess light is mediated via the adjustment of metabolites. Under these conditions the apparent electron transport rate exceeded the capacity for assimilation and the dehydroascorbate pool increased twofold. Since dehydroascorbate reductase activities were hardly detected, MDAR activities seemed to be the major enzyme to keep ascorbate in its reduced state. However, MDAR appeared to be insufficient to maintain the redox balance of the ascorbate pool under high light intensities in the field.     

外源一氧化氮对镉胁迫下绿豆幼苗根尖抗氧化酶的影响

采用水培法研究外源一氧化氮对镉(Cd)胁迫下绿豆幼苗根尖抗氧化酶活性的影响。结果表明:0.01mmol/L和0.1mmol/L一氧化氮供体硝普钠(sodium nitroprusside,SNP)显著促进上胚轴生长,1mmol/LSNP则抑制绿豆幼苗生长。Cd单独处理抑制根尖抗坏血酸过氧化物酶(ascorbate peroxidase,APX)和超氧化物歧化酶(superoxide dismutase,SOD)活性而刺激脂氧合酶(lipoxygenase,LOX)、谷胱甘肽转硫酶(glutathione S-transferase,GST)、谷胱甘肽还原酶(glutathione reductase,GR)和过氧化物酶(guaiacol peroxidase,POD)活性上升。0.1mmol/LSNP预处理能够明显缓解Cd对根生长的抑制,降低根尖中MDA含量,提高根尖APX和SOD活性,降低LOX和POD活性,但不影响GST和GR活性。     

Antioxidant defense system in <Emphasis Type="Italic">Phragmites communis</Emphasis> Trin. ecotypes

The antioxidant defense system in three ecotypes of reed (Phragmites communis Trin.), swamp reed (SR), dune reed (DR), and heavy salt meadow reed (HSMR), from northwest China were investigated. The HSMR possessed the highest ratio of ascorbate (ASC)/dehydroascorbate (DHA) and activities of superoxide dismutase (SOD) and catalase among the three reed ecotypes, whereas, the DR exhibited the highest ratio of glutathione/glutathione disulfide and activities of ASC peroxidase (APX) and DHA reductase. Malondialdehyde and hydrogen peroxide contents were highest in HSMR, intermediate in SR, and lowest in DR. In addition, different isoenzymes of glutathion reductase, APX, SOD and DHA were also observed in three reed ecotypes.