Re-aeration-induced oxidative stress and antioxidative defenses in hypoxically pretreated lupine roots

The level of free radicals and activities of antioxidative enzymes were examined in roots of lupine seedlings (Lupinus luteus L.) that were deprived of oxygen by subjecting them to root hypoxia for 48 and 72 h and then re-aerated for up to 24 h. Using electron paramagnetic resonance (EPR), we found that the exposure of previously hypoxically grown roots to air caused the increase in free radicals level, irrespective of duration of hypoxic pretreatment. Immediately after re-aeration the level of free radicals was two times higher than in aerated control. The EPR signal with the g-values at the maximum absorption of 2.0057 and 2.0040 implied that the paramagnetic radicals are derived from a quinone. Directly after re-aeration of hypoxically pretreated roots, the activity of superoxide dismutase (SOD, EC 1.15.1.1) increased to its highest value, followed by a decline below the initial level, whereas activities of catalase (CAT, EC 1.11.1.6) and peroxidase (POX, EC 1.11.1.7) were diminished or only slightly influenced during re-aeration. The electrophoretic patterns of the soluble extracts show 4 isozymes of SOD, 4 isozymes of POX and 1 isozyme of CAT. The level of H2O2 was enhanced or lowered by re-aeration, depending on the previous duration of hypoxia. At the onset of re-aeration products of lipid peroxidation were present at a three-fourth of the levels found in aerobic control. Their levels increased after prolonged exposure to air but remained lower than those in aerobic control even after 24 h of re-aeration. Re-admission of oxygen resulted in about 20% rise in oxygen uptake by root axes segments immediately after transfer of roots from hypoxia and the high uptake rates were observed over whole re-aeration period. Oxygen consumption by root tips was significantly reduced just after transfer from hypoxic conditions as compared to aerated control but after 24 h of re-aeration even approached the control level. The results are discussed in relation to the ability of lupine roots to cope with oxidative stress caused by re-aeration following hypoxic pretreatment.     

Effect of a short-term hypoxic treatment followed by re-aeration on free radicals level and antioxidative enzymes in lupine roots.

To investigate whether re-aeration after a short-term hypoxic pre-treatment (for 2, 12 or 24 h) induces oxidative stress, the temporal sequence of physiological reactions, including the level of free radicals, hydrogen peroxide production, and changes in antioxidative enzymes, was characterized in roots of hydroponically grown lupine (Lupinus luteus L., cv. Juno) seedlings. By using electron paramagnetic resonance (EPR), we found that the exposure of hypoxically grown roots (hypoxic pre-treatment for 12 and 24 h) to air caused an increase in the level of free radicals. The amount of hydrogen peroxide also tended to increase when hypoxically pre-treated roots were re-aerated, which attests to a higher production of reactive oxygen species. Re-aeration caused a higher activity of superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6), whereas the activity of peroxidase (POX, EC 1.11.1.7) was only slightly influenced. The roots were less tolerant to longer hypoxic pre-treatments, with a significant decrease in viability, associated with death of root tips immediately after hypoxic stress. Roots exposed to hypoxia for 2 h showed less pronounced responses and their viability was not affected by hypoxic stress and re-aeration. These results indicate that re-aeration following short-term hypoxia imposes a mild oxidative stress. This led us to conclude that re-oxygenation stress per se was not the key factor for cell death in root tips.     

Ascorbate and glutathione metabolism in embryo axes and cotyledons of germinating lupine seeds

Changes in ascorbate and glutathione contents and the activities and isoenzyme patterns of enzymes of the ascorbate-glutathione cycle were investigated in embryo axes and cotyledons of germinating lupine (Lupinus luteus L.) seeds. Ascorbate content was not significantly affected over the initial 12 h of imbibition in embryo axes, but afterwards increased, with the most rapid accumulation coinciding with radicle emergence. A somewhat similar trend was observed for glutathione with significant increase in embryo axes shortly before radicle protrusion followed by decline in the next hours. In cotyledons the ascorbate pool rose gradually during germination but the amount of glutathione showed fluctuations during a whole germination period. The activity of ascorbate peroxidase (APX) rose progressively in embryo axes, while activities of dehydroascorbate reductase (DHAR) and glutathione reductase (GR) showed transient increase during germination. New isoforms of APX and GR were synthesized, suggesting that they play a relevant role during germination. All analyzed enzymes were already present in dry seeds which allowed them to be active immediately after imbibition.     

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.     

Ability of lupine seeds to germinate and to tolerate desiccation as related to changes in free radical level and antioxidants in freshly harvested seeds

Seeds of yellow lupine (Lupinus luteus L. cv. Juno) were collected throughout their development on the mother plant to determine whether the ability to germinate and to tolerate desiccation is related to the level of free radicals and the changes in the redox state of ascorbate and glutathione as well as the activities of antioxidative enzymes. Electron paramagnetic resonance (EPR)-based analyses showed that development of lupine seed was accompanied by generation of free radicals with g₁ and g₂ values of 2.0049 ± 0.0004 and 2.0029 ± 0.0003, respectively. Free radical level increased significantly 25 DAF and decreased thereafter. The amount of hydrogen peroxide was high in fresh immature seeds and decreased during maturation drying. Ascorbate accumulated in lupine embryos during early seed filling stage whereas glutathione content increased during late seed filling phase. During maturation drying the redox state of both ascorbate and glutathione pools shifted towards the oxidized forms. While superoxide dismutase (SOD, EC 1.15.1.1), and ascorbate peroxidase (APX, EC 1.11.1.11) activities remained high at the early seed filling stage the activities of both dehydroascorbate reductase (DHAR, EC 1.8.5.1) and glutathione reductase (GR, EC 1.6.4.2) and that of catalase (CAT, EC 1.11.1.6) increased before seeds reached physiological maturity and decreased thereafter. The changes of isoform patterns of antioxidative enzymes were observed during seed maturation. Immature lupine seeds tested immediately after harvest acquired the ability to germinate when less than half-filled and reached high tolerance to desiccation just after physiological maturity. The physiological implications of the changes in antioxidative machinery for the acquisition of desiccation tolerance and seeds germinability are discussed.     

Antioxidative defense system in an upland rice cultivar subjected to increasing intensity of water stress followed by recovery

Rice ( Oryza sativa L.) cv. Tulsi is recommended for Eastern India, for upland ecological cultivation systems where a crop experiences natural cycles of water deficit and water sufficiency, depending upon the monsoon rains. In this experiment, this cultivar was subjected to three cycles of water stress of increasing stress intensity. Each stress cycle was terminated by rewatering the plants for a 48-h period. The level of stress was measured by quantification of H₂O₂. The response of antioxidant metabolites such as ascorbate and glutathione, and enzymes such as superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), glutathione reductase (GR, EC 1.6.4.2) and guaiacol peroxidase (POX, EC 1.11.1.7) was analysed in terms of activity and isozyme pattern for each cycle of stress and recovery. The differential response of the antioxidant enzymes with increasing stress intensity followed by recovery, highlight the different role of each in the drought acclimation process of upland rice. SOD and POX activity in stressed plants was higher than the controls in all the three cycles. The second level of stress saw an increase in all the enzymes with APX and GR showing its maximum activity and there was a better management of H₂O₂ levels. There was an induction of a new CAT isoform in stressed plants in the third cycle of water stress. The co-ordinated defense helped the plants to recover in terms of growth on rewatering after stress cycles.     

Changes in antioxidative enzymes in cucumber cotyledons during natural senescence: comparison with those during dark-induced senescence

Changes in 7 antioxidative enzymes in naturally senescent cotyledons of cucumber ( Cucumis sativus ) were investigated. The activities of superoxide dismutase (SOD; EC 1.15.1.1), catalase (EC 1.11.1.6), dehydroascorbate reductase (EC 1.8.5.1) and glutathione reductase (GR; EC 1.6.4.2) gradually decreased during the progression of senescence, while those of ascorbate peroxidase (APX; EC 1.11.1.11) and guaiacol peroxidase (GPX; EC 1.11.1.7) gradually increased. The activity of monodehydroascorbate reductase (MDAR; EC 1.6.5.4) was not significantly changed. Western blot analysis showed that the protein level of mitochondrial SOD gradually declined. The protein level of catalase transiently decreased and then increased in the later stages of senescence, despite the decrease in its activity. The overall behavior was markedly different from that found in cotyledons of artificially senescing seedlings transferred into darkness; the activities of SOD, catalase, APX, GPX and GR gradually increased.     

NaCl treatment markedly enhances H2O2-scavenging system in leaves of halophyte Suaeda salsa

The C₃ halophyte Suaeda salsa L. grown under the high concentration of NaCl (200 m M ) was used to investigate the role of the hydrogen peroxide (H₂O₂)-scavenging system [catalase, ascorbate peroxidase, glutathione reductase (GR), ascorbic acid, and glutathione (GSH)] in removal of reactive oxygen species. The activity of catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), and GR (EC 1.6.4.2) increased significantly after 7 days of NaCl treatment. The isoform patterns of CAT and GR were not affected, but the staining intensities were significantly increased by NaCl treatment. Activities of both the thylakoid-bound APX or GR and stromal APX (S-APX) or GR in the chloroplasts were markedly enhanced under high salinity. Fifty percent of APX in the chloroplasts is thylakoid-bound APX. S-APX and GR activity represented about 74–78 and 64–71% of the total soluble leaf APX and GR activity, respectively. Salt treatment increased the contents of ascorbic acid and GSH. By contrast, a decreased content of H₂O₂ was found in the leaves of NaCl-treated S . salsa . The level of membrane lipid peroxidation decreased slightly after NaCl treatment. The plants grew well with high rate of net photosynthesis under high salinity. These data suggest that upregulation of the H₂O₂-scavenging system in plant cells, especially in the chloroplasts, is at least one component of the tolerance adaptations of halophytes to high salinity.     

Antioxidative responses of Calendula officinalis under salinity conditions.

To gain a better insight into long-term salt-induced oxidative stress, some physiological parameters in marigold (Calendula officinalis L.) under 0, 50 and 100 mM NaCl were investigated. Salinity affected most of the considered parameters. High salinity caused reduction in growth parameters, lipid peroxidation and hydrogen peroxide accumulation. Under high salinity stress, a decrease in total glutathione and an increase in total ascorbate (AsA + DHA), accompanied with enhanced glutathione reductase (GR, EC 1.6.4.2) and ascorbate peroxidase (APX, EC 1.11.1.11) activities, were observed in leaves. In addition, salinity induced a decrease in superoxide dismutase (SOD, EC 1.15.1.1) and peroxidase (POX, EC 1.11.1.7) activities. The decrease in dehydroascorbate reductase (DHAR, EC 1.8.5.1) and monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) activities suggests that other mechanisms play a major role in the regeneration of reduced ascorbate. The changes in catalase (CAT, EC 1.11.1.6) activities, both in roots and in leaves, may be important in H2O2 homeostasis.     

Age-dependence of the antioxidative system in tobacco with enhanced glutathione reductase activity or senescence-induced production of cytokinins

Tobacco leaves of plants with enhanced glutathione reductase activity (GR46-27, Nicotiana tabacum L. cv. Samsun) or with autoregulated senescence-induced production of cytokinins (P_SAG12-IPT, N. tabacum L. cv. Wisconsin) were studied during the course of leaf development and senescence by measuring photosynthesis, chlorophyll and protein content, the antioxidants ascorbate, glutathione and α -tocopherol as well as the antioxidative enzymes ascorbate peroxidase (APX, EC 1.11.1.11), glutathione reductase (GR, EC 1.6.4.2) and superoxide dismutase (SOD, EC 1.15.1.1). The photosynthetic rate, as well as the chlorophyll and protein content, dropped with increasing leaf age after having reached a maximum at the end of the exponential growth phase. The concentrations of the water-soluble antioxidants ascorbate and glutathione fell continuously with age, whereas the concentration of the lipophilic α -tocopherol increased. The activities of the antioxidative enzymes APX, GR and SOD reached their maximum at the beginning of leaf development, but were reduced in senescing leaves. The age-dependent course of the measured leaf parameters in GR46-27 leaves was similar to the one in wild-type leaves, with the exception of an overall enhanced GR activity. In contrast, in old leaves of P_SAG12-IPT plants, which possess a much higher life span, the chlorophyll and protein content, the photosynthetic rate, the antioxidant concentrations of ascorbate and glutathione as well as the activities of the antioxidative enzymes were higher than in wild-type leaves. The results show that the capacity of the antioxidative system to scavenge radicals is sufficiently balanced with the plant metabolism, and its decline with increasing age is not the cause, but a consequence of senescence and ageing in plants.     

Exogenous Nitric Oxide (as Sodium Nitroprusside) Ameliorates Polyethylene Glycol-Induced Osmotic Stress in Hydroponically Grown Maize Roots

The present study was designed to examine whether exogenous sodium nitroprusside (SNP) supplementation has any ameliorating action against PEG-induced osmotic stress in Zea mays cv. FRB-73 roots. Twenty percent or 40 % polyethylene glycol (PEG6000; ?0.5 MPa and ?1.76 MPa, respectively) treatment alone or in combination with 150 and 300 μM SNP was applied to hydroponically grown maize roots for 72 h. Although only catalase (CAT) activity increased when maize roots were exposed to PEG-induced osmotic stress, induction of this antioxidant enzyme was inadequate to detoxify the extreme levels of reactive oxygen species, as evidenced by growth, water content, superoxide anion radical (O ₂ ^?? ), hydroxyl radical (OH^?) scavenging activity, and TBARS content. However, supplementation of PEG-exposed specimens with SNP significantly alleviated stress-induced damage through effective water management and enhancement of antioxidant defense markers including the enzymatic/non-enzymatic systems. Exogenously applied SNP under stress resulted in the up-regulation of glutathione peroxidase (GPX), glutathione S-transferase (GST), ascorbate peroxidase (APX), glutathione reductase (GR), total ascorbate, and glutathione contents involved in ascorbate–glutathione cycle. On the other hand, growth rate, osmotic potential, CAT, APX, GR, and GPX increased in maize roots exposed to both concentrations of SNP alone, but activities of monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase decreased. Based on the above results, an exogenous supply of both 150 and 300 μM SNP to maize roots was protective for PEG-induced toxicity. The present study provides new insights into the mechanisms of SNP (NO donor) amelioration of PEG-induced osmotic stress damages in hydroponically grown maize roots.     

The inductive responses of the antioxidant enzymes by salt stress in the rice (Oryza sativa L.)

To access contributions of inductive responses of the antioxidant enzymes in the resistance to salt stress, activities of the enzymes were determined in the rice (Oryza sativaL. cv. Dongjin) plant. In the leaves of the rice plant, salt stress preferentially enhanced the content of H₂O₂ as well as the activities of the superoxide dismutase (SOD), ascorbate peroxidase (APX), and peroxidase specific to guaiacol, whereas it induced the decrease of catalase activity. On the other hand, salt stress had little effect on the activity levels of glutathione reductase (GR). In order to analyze the changes of antioxidant enzyme isoforms against salt stress, plant extracts were subjected to native PAGE. Leaves of the rice plant had two isoforms of Mn-SOD and five isoforms of Cu/Zn-SOD. Fe-SOD isoform was not observed in the activity gels. Expression of Cu/Zn-1, -2, and Mn-SOD-2 isoforms was preferentially enhanced by salt stress. Seven APX isoforms were presented in the leaves of the rice plants. The intensities of APX-4 to -7 were enhanced by salt stress, whereas those of APX-1 to -3 were minimally in changed response to salt stress. There were seven GR isoforms in the leaves of rice plants. Levels of activity for most GR isoforms did not change in the stressed plants compared to the control plants. On the other hand, the levels of activity for most antioxidant enzymes changed little in the roots of stressed plants compared to the control plants. These results collectively suggest that SOD leads to the overproduction of hydrogen peroxide in the leaves of rice plants subjected to salt stress: The overproduction of hydrogen peroxide functions as the signal of salt stress, which induces the induction of specific APX isoforms but not specific GR isoforms under catalase deactivation.     

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.     

Changes in the ascorbate system in the response of pumpkin (Cucurbita pepo L.) roots to aluminium stress

The involvement of the ascorbate (AsA) system in the response of pumpkin (Cucurbita pepo L.) roots to aluminium stress was studied. The treatment of 5-day-old pumpkin seedlings with 50 microM aluminium sulphate resulted in approximately 60% inhibition of root growth within 48-60 h of treatment, while aluminium accumulated in the roots reaching a maximum within 48h. During the same period, the hydrogen peroxide content of the roots was strongly enhanced. The increased level of hydrogen peroxide was matched by both increased ascorbate peroxidase (APX) (EC 1.11.1.11) activity and ascorbate free radical reductase (AFRR) (EC 1.1.5.4) activity, while dehydroascorbate reductase (DHAR) (EC 1.8.5.1) and glutathione reductase (GR) (EC 1.6.4.2) did not change. The levels of AsA in the roots were also increased by the Al treatment. It was concluded that an oxidative burst is probably involved in the toxicity of Al in pumpkin roots and that plants react to the enhanced production of reactive oxygen species by expressing higher levels of scavenging systems such as the AsA-APX system.     

Defense potentials to NaCl in a mangrove, Bruguiera parviflora: differential changes of isoforms of some antioxidative enzymes

In order to assess the role of the antioxidative defense system against salt treatment, the activities of some antioxidative enzymes and levels of antioxidants were monitored in a true mangrove, Bruguiera parviflora, subjected to varying levels of NaCl under hydroponic culture. In the leaves of B. parviflora, salt treatment preferentially enhanced the content of H2O2 as well as the activity of ascorbate peroxidase (APX), guaiacol peroxidase (GPX), glutathione reductase (GR), and superoxide dismutase (SOD), whereas it induced the decrease of total ascorbate and glutathione (GSH+GSSG) content as well as catalase (CAT) activity. Analysis of isoforms of antioxidative enzymes by native PAGE and activity staining revealed that leaves of B. parviflora had one isoform each of Mn-SOD and Cu/Zn-SOD and three isoforms of Fe-SOD. Expression of Mn-SOD and Fe-SOD-2 was preferentially elevated by NaCl. Similarly, out of the six isoforms of GPX, the GPX-1, 2, 3 and 6 were enhanced by salt treatment but the levels of GPX-4 and -5 changed minimally as compared to those of a control. Activity staining gel revealed only one prominent isoform of APX and two isoforms of GR (GR-1 and GR-2), all of these isoforms increased upon salt exposure. Four CAT-isoforms were identified, among which the prominent CAT-2 isoform level was maximally reduced, suggesting differential down regulation of CAT isoforms by NaCl. The concentrations of malondialdehyde (MDA), a product of lipid peroxidation, remained unchanged in leaves of the plant treated with different concentrations of NaCl. This suggests that plants are protected against activated oxygen species by the elevated levels of certain antioxidative enzymes, thus avoiding lipid peroxidation during salt exposure. The differential changes in the levels of the isoforms due to NaCl treatment may be useful as markers for recognizing salt tolerance in mangroves.     

Nickel-induced oxidative stress and the role of antioxidant defence in rice seedlings

Seedlings of rice (Oryza sativa L.) cv. Pant-12 grown in sand cultures containing 200 and 400 μM NiSO₄, showed a decrease in length and fresh weight of roots and shoots. Nickel was readily taken up by rice seedlings and the concentration was higher in roots than shoots. Nickel-treated seedlings showed increased rates of superoxide anion (O₂ ^•− ) production, elevated levels of H₂O₂ and thiobarbituric acid reactive substances (TBARS) demonstrating enhanced lipid peroxidation, and a decline in protein thiol levels indicative of increased protein oxidation compared to controls. With progressively higher Ni concentrations, non-protein thiol and ascorbate (AsA) increased, whereas the level of low-molecular-weight thiols (such as glutathione and hydroxyl-methyl glutathione), the ratio of these thiols to their corresponding disulphides, and the ratio of AsA to dehydroascorbic acid declined in the seedlings. Among the antioxidant enzymes studied, the activities of all isoforms of superoxide dismutase (Cu-Zn SOD, Mn SOD and Fe SOD), guaiacol peroxidases (GPX) and ascorbate peroxidase (APX) increased in Ni-treated seedlings, while no clear alteration in catalase activity was evident. Activity of the ascorbate-glutathione cycle enzymes monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR)—significantly increased in Ni-treated seedlings. However such increase was apparently insufficient to maintain the intracellular redox balance. Results suggest that Ni induces oxidative stress in rice plants, resulting in enhanced lipid peroxidation and decline in protein thiol levels, and that (hydroxyl-methyl) glutathione and AsA in conjunction with Cu-Zn SOD, GPX and APX are involved in stress response.     

The role of lipoic acid in the regulation of the redox status of wheat irrigated with 20% sea water.

The effect of irrigation with 20% sea water was studied in 14 and 21-day-old seedlings of durum wheat (Triticum durum, cv. Ofanto). Comparisons between control (Hoagland's 2 solution) and treated (20% sea water in Hoagland's solution) plants included, besides HPLC determination of reduced (DHLA) and oxidised (LA) forms of lipoic acid, ascorbate and glutathione contents, their redox status, the activity of ascorbate peroxidase (APX, EC 1.11.1.11.) and glutathione reductase (GR, EC 1.6.4.2.). The results indicated a more relevant presence of lipoic acid in the roots in comparison to the shoots. An involvement of its reduced form in the regeneration of the reduced glutathione, at least at 14 days of treatment, suggested, besides its role as dehydrogenase enzyme cofactor, a role in the recycling of the other antioxidants. The amount of LA always increased with growth in shoots and decreased in roots, while DHLA remained constant in control and increased in treated plants. Besides, the oxidised form always decreased with sea water while the reduced form decreased in shoots and increased in roots. The ascorbate pool exerted its positive influence especially in the shoots, while APX and GR activities resulted differently modulated by the salinity level.     

Glutathione reductase activity and isoforms in leaves and roots of wheat plants subjected to cadmium stress

The behavior of glutathione reductase (GR, EC 1.6.4.2) activity and isoforms were analyzed in wheat (Triticum aestivum L.) leaves and roots exposed to a chronic treatment with a toxic cadmium (Cd) concentration. A significant growth inhibition (up to 55%) was found in leaves at 7, 14 and 21 days, whereas roots were affected (51%) only after three weeks. Wheat plants grown in the presence of 100microM Cd showed a time-dependent accumulation of this metal, with Cd concentration being 10-fold higher in roots than in leaves. Nevertheless, lipid peroxidation was augmented in leaves in all experiments, but not in roots until 21 days. Cadmium treatment altered neither the GR activity nor the isoform pattern in the leaves. However, GR activity increased 111% and 200% in roots at 7 and 14 days, respectively, returning to control levels after 21 days. Three GR isoforms were found in roots of control and treated plants, two of which were enhanced by Cd treatment at 7 and 14 days, as assessed by activity staining on native gels. The changes in the isoform pattern modified the global kinetic properties of GR, thereby decreasing significantly (2.5-fold) the Michaelis constant (K(m)) value for oxidized glutathione. Isozyme induction was not associated with an enhancement of GR mRNA and protein expression, indicating that post-translational modification could occur. Our data demonstrated that up-regulation of GR activity by the induction of distinctive isoforms occurs as a defense mechanism against Cd-generated oxidative stress in roots.     

Antioxidative enzymes in seedlings of Nelumbo nucifera germinated under water

Dry seeds of anoxia-tolerant lotus ( Nelumbo nucifera Gaertn= Nelumbium speciosum Willd.) have green shoots with plastids containing chlorophyll, so photosynthesis starts even in seedlings germinated under water, namely hypoxia. Here we investigated antioxidative enzyme changes in N. nucifera seedlings responding to oxygen deficiency. The activity of superoxide dismutase (SOD; EC 1.15.1.1), dehydroascorbate reductase (DHAR; EC 1.8.5.1) and glutathione reductase (GR; EC 1.6.4.2) were lower in seedlings germinated under water (submerged condition) in darkness (SD seedlings) than those found in seedlings germinated in air and darkness (AD seedlings). In contrast, ascorbate peroxidase (APX; EC 1.11.1.11) activity was higher in SD seedlings and the activity of catalase (EC 1.11.1.6) and monodehydroascorbate reductase (MDAR; EC 1.6.5.4) in SD seedlings was nearly the same as in AD seedlings. When SD seedlings were exposed to air, the activity of SOD, DHAR and GR increased, while the activity of catalase and MDAR decreased. Seven electrophoretically distinct SOD isozymes were detectable in N. nucifera . The levels of plastidic Cu,Zn-SODs and Fe-SOD in SD seedlings were comparable with those found in AD seedlings, which may reflect the maintenance of green plastids in SD seedlings as well as in AD seedlings. These results were substantially different from those previously found in rice seedlings germinated under water.