Activities of SOD and the ascorbate-glutathione cycle enzymes in subcellular compartments in leaves and roots of the cultivated tomato and its wild salt-tolerant relative Lycopersicon pennellii

The activities of the ascorbate-glutathione cycle enzymes ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) and SOD were studied in cell organelles of the cultivated tomato Lycopersicon esculentum (M82) and its wild salt-tolerant related species Lycopersicon pennellii (Lpa). All four enzymes of the ascorbate-glutathione cycle were present in chloroplasts/plastids, mitochondria and peroxisomes of leaf and root cells of both tomato species. In all leaf and root organelles of both species, the activity of MDHAR was similar to, or higher than, that of APX, while the activity of DHAR was one order of magnitude lower than that of MDHAR. Based on these results, it is suggested that in the organelles of both tomato species, ascorbate is regenerated mainly by MDHAR. In both tomato species, GR activity, and to a lesser extent DHAR activity, was found to reside in the soluble fraction of all leaf and root cell organelles, while APX and MDHAR activities were distributed between the membrane and soluble fractions. A higher SOD to APX activity ratio in all Lpa organelles was the major difference between the two tomato species. It is possible that this higher ratio contributes to the inherently better protection of Lpa from salt stress, as was previously reported.     

Role of the Ascorbate-Glutathione Cycle of Mitochondria and Peroxisomes in the Senescence of Pea Leaves

We investigated the relationship between H₂O₂ metabolism and the senescence process using soluble fractions, mitochondria, and peroxisomes from senescent pea (Pisum sativum L.) leaves. After 11 d of senescence the activities of Mn-superoxide dismutase, dehydroascorbate reductase (DHAR), and glutathione reductase (GR) present in the matrix, and ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR) activities localized in the mitochondrial membrane, were all substantially decreased in mitochondria. The mitochondrial ascorbate and dehydroascorbate pools were reduced, whereas the oxidized glutathione levels were maintained. In senescent leaves the H₂O₂ content in isolated mitochondria and the NADH- and succinate-dependent production of superoxide (O₂^·−) radicals by submitochondrial particles increased significantly. However, in peroxisomes from senescent leaves both membrane-bound APX and MDHAR activities were reduced. In the matrix the DHAR activity was enhanced and the GR activity remained unchanged. As a result of senescence, the reduced and the oxidized glutathione pools were considerably increased in peroxisomes. A large increase in the glutathione pool and DHAR activity were also found in soluble fractions of senescent pea leaves, together with a decrease in GR, APX, and MDHAR activities. The differential response to senescence of the mitochondrial and peroxisomal ascorbate-glutathione cycle suggests that mitochondria could be affected by oxidative damage earlier than peroxisomes, which may participate in the cellular oxidative mechanism of leaf senescence longer than mitochondria.     

Postharvest chilling induces oxidative stress response in the dwarf tomato cultivar Micro-Tom

Oxidative stress is involved in the response of Lycopersicon esculentum fruits (cultivar Micro-Tom) to chilling. Changes in activated oxygen scavenging enzymes, superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), and glutathione reductase (GR, EC 1.6.4.2) were examined during ripening after postharvest chilling. Also, lipid peroxidation, respiration, and pigment contents were determined. These parameters were affected by chilling, especially the lycopene content and the respiration rate that showed a high value when the fruits were transferred to higher temperatures. CAT activity increased the day after the fruits were re-warmed, while the activity of GR was higher in the chilled than in the non-chilled green fruits. Lipid peroxidation was more evident at the 'pre-chilled' yellow and red fruits. APX and SOD were not affected by previous chilling in ripening fruits. These results indicate that oxidative stress is generated by conservation at 4°C. The antioxidant response of tomato fruit could be mediated by CAT and GR but not by SOD or APX. Moreover, CAT seemed to respond to the increase in the respiration rate.     

Copper affects the enzymes of the ascorbate-glutathione cycle and its related metabolites in the roots of Phaseolus vulgaris

The involvement of the ascorbate-glutathione cycle in the defence against Cu-induced oxidative stress was studied in the roots of Phaseolus vulgaris L. cv. Limburgse vroege. All the enzymes of this cycle [ascorbate peroxidase (APOD), EC 1.11.1.11; monodehydroascorbate reductase (MDHAR), EC 1.6.5.4; dehydroascorbate reductase (DHAR), EC 1.8.5.1; glutathione reductase (GR), EC 1.6.4.2] were increased, and the total ascorbate and glutathione pools rose after a 15 μ M root Cu treatment. In the first hours after the start of the experiment, the accumulation of dehydroascorbate (DHA), formed as a result of a Cu-mediated direct oxidation of ascorbate (AA), was limited by a non-enzymatic reduction using glutathione (GSH) as the reductant. At 24 h, the enzyme capacities of both DHAR and GR were increased to maintain the redox status of the AA and GSH pools. After 72 h of Cu application, the DHAR capacity was inhibited and MDHAR was responsible for maintaining the AA pool in its reduced form. Although the GR capacity was enhanced after 72 h in the treated plants, the GSSG/GSH ratio was increased. This could be due to direct participation of GSH in the detoxification of Cu through reduction and complexation.     

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。     

Effect of Selenium on Ascorbate�CGlutathione Metabolism During PEG-induced Water Deficit in Trifolium repens L.

To elucidate the effect of selenium (Se) on the ascorbate?Cglutathione (ASC?CGSH) cycle under drought stress, the activities of antioxidant enzymes and the levels of molecules involved in ASC?CGSH metabolism were studied in Trifolium repens seedlings subjected to polyethylene glycol (PEG)-induced water deficit alone or combined with 5???M Na₂SeO₄. Compared to the control, H₂O₂, thiobarbituric acid reactive substances (TBARS), ascorbate (ASC), dehydroascorbate (DHA), and glutathione disulfide (GSSG) contents increased, whereas a constant content of glutathione (GSH) and decreases in ASC/DHA and GSH/GSSG ratios were observed in the presence of PEG. The activities of ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) were upregulated, except for monodehydroascorbate reductase (MDHAR) activity during PEG-induced water deficit. Se application decreased the contents of H₂O₂, TBARS, DHA, and GSSG, increased the levels of GSH and ASC, and inhibited the decreases of ASC/DHA and GSH/GSSG ratios. Although it did not affect APX activity significantly, Se addition improved the activities of MDHAR, DHAR, and GR. Furthermore, GR activity showed the highest increase followed by that of DHAR and MDHAR in decreasing order. These data indicated that fluctuations in ASC?CGSH metabolism resulting from Se may have a positive effect on drought stress mitigation, and the regulation in the ASC?CGSH cycle can be attributed mainly to GR and DHAR in PEG?+?Se-treated T. repens seedlings.     

Ascorbate-glutathione metabolism during PEG-induced water deficit in <Emphasis Type="Italic">Trifolium repens</Emphasis>

In order to elucidate the response of the ascorbate-glutathione (ASC-GSH) cycle to drought stress, the activities of antioxidant enzymes and the levels of molecules involved in the ASC-GSH metabolism were studied in Trifolium repens L. seedlings subjected to PEG-induced water deficit. Compared to the control, the contents of H₂O₂, thiobarbituric acid reactive substances (TBARS), ascorbate (ASC), dehydroascorbate (DHA), and glutathione disulfide (GSSG) increased in PEG-treated seedlings, whereas the glutathione (GSH) content kept constant during the drought period. Further more, the ASC/DHA and GSH/GSSG ratios decreased in the presence of PEG. Except for that of monodehydroascorbate reductase (MDHAR), the activities of ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) were up-regulated during water deficit, and the increases in APX and DHAR activities were much higher than those in GR activity. These data indicate that fluctuations in the ASC-GSH metabolism resulted from PEG treatment may have a positive effect on drought stress mitigation in T. repens.     

Antioxidative enzymes from chloroplasts, mitochondria, and peroxisomes during leaf senescence of nodulated pea plants

In this work the influence of the nodulation of pea (Pisum sativum L.) plants on the oxidative metabolism of different leaf organelles from young and senescent plants was studied. Chloroplasts, mitochondria, and peroxisomes were purified from leaves of nitrate-fed and Rhizobium leguminosarum-nodulated pea plants at two developmental stages (young and senescent plants). In these cell organelles, the activity of the ascorbate-glutathione cycle enzymes ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR), and the ascorbate and glutathione contents were determined. In addition, the total superoxide dismutase (SOD) activity, the pattern of mitochondrial and peroxisomal NADPH-generating dehydrogenases, some of the peroxisomal photorespiratory enzymes, the glyoxylate cycle and oxidative metabolism enzymes were also analysed in these organelles. Results obtained on the metabolism of cell organelles indicate that nodulation with Rhizobium accelerates senescence in pea leaves. A considerable decrease of the ascorbate content of chloroplasts, mitochondria, and peroxisomes was found, and in these conditions a metabolic conversion of leaf peroxisomes into glyoxysomes, characteristic of leaf senescence, took place.     

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.     

Enzymes of the ascorbate biosynthesis and ascorbate-glutathione cycle in cultured cells of tobacco Bright Yellow 2

The ascorbate (ASC) and glutathione (GSH) metabolisms were studied in cultured Nicotiana tabacum cv. Bright Yellow 2 (TBY-2) cells. TBY-2 cells were found to be endowed with L-galactono-γ-lactone dehydrogenase (GLDH) (EC 1.3.2.3), an enzyme that converts L-galactono-γ-lactone into ASC. Cellular fractionation of TBY-2 protoplasts indicated that this enzyme is exclusively localised in mitochondria and associated to the membrane fractions. During the growth cycle of TBY-2 cell culture, GLDH transiently increased, reaching the maximum value on the third day of culture, at the beginning of the exponential phase, when the cell proliferative activity was also higher. Similar behaviour has been observed for ASC and GSH contents. The activities of ascorbate peroxidase (APX) (EC 1.11.1.11), ascorbate-free radical reductase (AFRR) (EC 1.6.5.4), dehydroascorbic acid reductase (DHAR) (EC 1.8.5.1) and glutathione reductase (GR) (EC 1.6.4.2) also transiently raised. However, the scale of the increases varied being about 4-fold for APX and AFRR, 2-fold for DHAR and more than 11-fold for GR. The behaviour of the ASC and GSH recycling enzymes allowed TBY-2 cells to maintain both dehydroascorbic acid and glutathione disulphide at low levels, even under conditions of high ASC and GSH utilisation. The relationship between the ASC and GSH metabolisms during the growth cycle of TBY-2 cell suspension cultures is also discussed.     

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.     

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.     

Red 'Anjou' pear has a higher photoprotective capacity than green 'Anjou'

Photoprotective function of anthocyanins along with xanthophyll cycle and antioxidant system in fruit peel was investigated in red 'Anjou' vs green 'Anjou' pear (Pyrus communis) during fruit development and in response to short-term exposure to high light. The sun-exposed peel of red 'Anjou' had higher maximum quantum yield of photosystem II (F(V)/F(M)) than that of green 'Anjou' and both the sun-exposed peel and the shaded peel of red 'Anjou' had smaller decreases in F(V)/F(M) after 2-h high light (photon flux density of 1500 mumol m(-2) s(-1)) treatment than those of green 'Anjou'. At the middle and late developmental stages, the xanthophyll cycle pool size on a chlorophyll basis, the activity of superoxide dismutase, ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) and the level of reduced ascorbate and total ascorbate pool in the sun-exposed peel were either the same or lower in red 'Anjou' than in green 'Anjou', whereas the xanthophyll cycle pool size on a chlorophyll basis and the activity of APX, catalase, MDAR, DHAR and GR in the shaded peel were higher in red 'Anjou' than in green 'Anjou'. It is concluded that red 'Anjou' has a higher photoprotective capacity in both the sun-exposed peel and the shaded peel than green 'Anjou'. While the higher anthocyanin concentration along with the larger xanthophyll cycle pool size and the higher activity of some antioxidant enzymes may collectively contribute to the higher photoprotective capacity in the shaded peel of red 'Anjou', the higher photoprotective capacity in the sun-exposed peel of red 'Anjou' is mainly attributed to its higher anthocyanin concentration.     

乙烯与NO互作对镉胁迫下荷花的抗坏血酸-谷胱甘肽循环的影响

以荷花‘微山湖红莲’实生苗为试验材料,研究镉(Cd,50 μmol·L^-1)胁迫下,外源乙烯前体1-氨基环丙烷羧酸(ACC,100 μmol·L^-1)、ACC与一氧化氮合酶(NOS)抑制剂N-硝基-L-精氨酸(L-NNA,200 μmol·L^-1)、ACC与硝酸还原酶(NR)抑制剂钨酸钠(Tu,1 mmol·L^-1),ACC与一氧化氮(NO)清除剂2-苯基-4,4,5,5-四甲基咪唑啉-3-氧代-1-氧(PTIO,200 μmol·L^-1),外源NO供体硝普钠(SNP,500 μmol·L^-1)、SNP与乙烯信号转导抑制剂硫代硫酸银(STS,100 μmol·L^-1)处理下荷花幼苗叶片的受害程度及抗坏血酸(AsA)-谷胱甘肽(GSH)循环的变化情况.结果表明: Cd胁迫下,荷花叶片受害症状明显,其相对电导率、丙二醛(MDA)、AsA和GSH含量显著上升,抗坏血酸过氧化物酶(APX)、谷胱甘肽还原酶(GR)、单脱氢抗坏血酸还原酶(MDHAR)和脱氢抗坏血酸还原酶(DHAR)活性明显降低;ACC的添加进一步增加了Cd对荷花叶片的毒害症状,并加剧了4种抗氧化酶活性的降低,但增加了抗氧化剂的含量;SNP的添加对荷花叶片的伤害起到加重作用,并导致GR和MDHAR活性降低以及AsA和GSH含量的升高;PTIO可显著提高Cd和ACC复合处理下荷花叶片APX、GR、MDHAR和DHAR的活性并降低AsA和GSH的含量,而L-NNA和Tu效果不如PTIO明显;STS可显著缓解Cd和SNP复合处理下荷花叶片的毒害症状,并提高4种抗氧化酶的活性、降低AsA和GSH的含量.由此说明,乙烯和NO在AsA-GSH循环中存在互作,二者相互促进,共同调控AsA-GSH循环,进而参与调控荷花对Cd胁迫的响应.     

The oxidative stress caused by salinity in two barley cultivars is mitigated by elevated CO2

Changes in antioxidant metabolism because of the effect of salinity stress (0, 80, 160 or 240 m M NaCl) on protective enzyme activities under ambient (350 μmol mol⁻¹) and elevated (700 μmol mol⁻¹) CO₂ concentrations were investigated in two barley cultivars ( Hordeum vulgare L., cvs Alpha and Iranis). Electrolyte leakage, peroxidation, antioxidant enzyme activities [superoxide dismutase (SOD), EC 1.15.1.1; ascorbate peroxidase (APX), EC 1.11.1.11; catalase (CAT), EC 1.11.1.6; dehydroascorbate reductase (DHAR), EC 1.8.5.1; monodehydroascorbate reductase (MDHAR), EC 1.6.5.4; glutathione reductase (GR), EC 1.6.4.2] and their isoenzymatic profiles were determined. Under salinity and ambient CO₂, upregulation of antioxidant enzymes such as SOD, APX, CAT, DHAR and GR occurred. However, this upregulation was not enough to counteract all ROS formation as both ion leakage and lipid peroxidation came into play. The higher constitutive SOD and CAT activities together with a higher contribution of Cu,Zn-SOD 1 detected in Iranis might possibly contribute and make this cultivar more salt-tolerant than Alpha. Elevated CO₂ alone had no effect on the constitutive levels of antioxidant enzymes in Iranis, whereas in Alpha it induced an increase in SOD, CAT and MDHAR together with a decrease of DHAR and GR. Under combined conditions of elevated CO₂ and salinity the oxidative damage recorded was lower, above all in Alpha, together with a lower upregulation of the antioxidant system. So it can be concluded that elevated CO₂ mitigates the oxidative stress caused by salinity, involving lower ROS generation and a better maintenance of redox homeostasis as a consequence of higher assimilation rates and lower photorespiration, being the response dependent on the cultivar analysed.     

Regulation of the ascorbate-glutathione cycle in wild almond during drought stress

In wild species of almond (Prunus spp.), the activities of ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR), as well as the levels of ascorbate/glutathione pools and H₂O₂ were subjected to water deficit and shade conditions. After 60 days of water shortage, the species were subjected to a rewatering treatment. During water recovery, leaves exposed to sunlight and leaves under shade conditions of about 20–35% of environmental irradiance were sampled. After 70 days without irrigation, mean predawn leaf water potential of all the species fell from −0.32 to −2.30 MPa and marked decreases in CO₂ uptake and transpiration occurred. The activities of APX, MDHAR, DHAR, and GR increased in relation to the severity of drought stress in all the wild species studied. Generally, APX, MDHAR, DHAR, and GR were down-regulated during the rewatering phase and their activities decreased faster in shaded leaves than in sun-exposed leaves. The levels in total ascorbate, glutathione, and H₂O₂ were directly related to the increase in drought stress and subsequently decreased during rewatering. The antioxidant response of wild almond species to drought stress limits cellular damage caused by reactive oxygen species during periods of water deficit and may be of key importance for the selection of drought-resistant rootstocks for cultivated almond.