Cristina Sgherri

Antioxidative responses of wheat treated with realistic concentration of cadmium

Abstract Two cvs. of wheat differently sensitive to many stress factors (cv. Ofanto less sensitive than cv. Adamello) were grown in a controlled environment with cadmium near threshold concentrations supplying the metal at equal-effect concentrations. Cd excess determined in both cvs. a reduction in water and turgor potential but a maintenance of relative water content. Cv Ofanto showed a higher capacity of Cd exclusion from roots but a higher translocation to shoots in comparison with cv. Adamello. Notwithstanding the higher metal concentration in leaves of cv. Ofanto, K + leakage was more pronounced in Adamello suggesting that mechanisms of Cd detoxification and tolerance such as vacuolar compartmentalisation were activated in the first one. In Adamello plants, ethylene rose at the lowest metal concentration and the activation in roots of the antioxidative enzymes catalase, ascorbate peroxidase and guaiacol peroxidase came into play whereas in Ofanto ethylene and catalase did not change. Following cadmium treatment, superoxide dismutase activity was reduced or remained at the control value in roots and in leaves. For both cultivars ascorbate peroxidase, syringaldazine peroxidase and guaiacol peroxidase activities were always higher in roots than in leaves. These activities were induced by Cd in Ofanto leaves, whereas in Adamello leaves they remained at control levels or increased somewhat at the highest metal concentration. Cadmium changed the peroxidase isozyme pattern in both cultivars. Cv. Ofanto showed, as for other stress such as drought, salinity, nickel and copper, a co-tolerance towards Cd. Analogies in the response to other metals such as copper could be found in activation of catalase at the lower metal concentration in cv. Adamello and in the induction of ascorbate peroxidase in leaves of cv. Ofanto.

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 mM NaCl) on protective enzyme activities under ambient (350 micromol mol(-1)) and elevated (700 micromol mol(-1)) CO(2) 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 CO(2), 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 CO(2) 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 CO(2) 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 CO(2) 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.

Lipoic acid: a unique antioxidant in the detoxification of activated oxygen species

Lipoic acid (1,2-dithiolane-pentanoic acid) is a dithiol which is effective in affording protection against oxidative stress by virtue of its two sulphydryl moieties. It is present in all kinds of eukaryotic and prokaryotic cells. As lipoamide, it functions as a cofactor in the multienzyme complexes that catalyse the oxidative decarboxylation of α-keto acids such as pyruvate, α-ketoglutarate, and branched-chain α-keto acids. The complete enzyme pathway responsible for the de novo synthesis of lipoic acid has not yet been elucidated. Octanoic acid appears to be the precursor for the eight-carbon fatty acid chain, and cysteine the source of sulfur. Lipoic acid is unique, among antioxidants, because it retains powerful antioxidant properties in both its reduced (dihydrolipoic acid) and oxidised (lipoic acid) forms. Both lipoic and dihydrolipoic acids have metal-chelating ability and quench activated oxygen species either in the cytosol or in the hydrophobic domains. Dihydrolipoic acid has more antioxidant properties than lipoic acid, and it plays an important role in the recycling of other oxidised radical scavengers such as glutathione, ascorbate and tocopherol. However, dihydrolipoic acid can also exert pro-oxidant properties both by its iron-reducing ability and by its ability to generate sulfur-containing radicals that can damage proteins. There are few quantitative data on lipoic acid contents in vegetables. It has been found in asparagus, wheat and potatoes, and recently, the presence of both lipoic and dihydrolipoic acids in roots, leaves and in the stroma of wheat has been demonstrated.

Antioxidant system in Sporobolus stapfianus: Changes in response to desiccation and rehydration

Abstract Changes in the amounts of ascorbic acid, dehydroascorbic acid and glutathione and in the levels of related enzyme activities were studied in Sporobolus stapfianus leaves, a desiccation-tolerant or resurrection plant, in response to desiccation and rehydration. Detached leaves of S. stapfianus were rapidly dried for 120 hr at 0% rel. hum. A 24 hr desiccation period was sufficient to achieve extremely low water contents (0.2 %). Dried leaves were then rehydrated for 24 hr. The soluble-protein content decreased after drying but did not increase after rehydration without death ensuing. The enzymes related to the glutathione-ascorbate cycle reacted differently to the rapid water loss: dehydroascorbate reductase (EC 1.8.5.1) and glutathione reductase (EC 1.6.4.2) activities increased while ascorbate peroxidase (EC 1.11.1.11) activity decreased. All these enzymes resumed their activities after rehydration. Oxidative processes were intensified following desiccation and even more after rehydration: i.e. the H 2 O 2 level and the dehydroascorbate/ ascorbate molar ratio increased in desiccated leaves and even more in rehydrated leaves, while the oxidized glutathione/reduced glutathione molar ratio, which decreased in desiccated leaves, reached the control value with rehydration.

Antioxidative enzymes in wheat subjected to increasing water deficit and rewatering

Summary Two cultivars of wheat seedlings differently sensitive to drought (cv. Adamello more sensitive than cv. Ofanto) were subjected to water depletion and rehydration. Cv. Ofanto showed a higher relative water content in comparison with cv. Adamello during the whole stress period. Soluble and thylakoid- bound activities of ascorbate peroxidase (APX) and superoxide dismutase (SOD) showed different behaviours in the two cultivars. Differently from the sensitive cultivar, in which thylakoid-bound APX was induced as soon as water deficit was imposed, in the tolerant wheat the activation of the thylakoid-bound APX occurred only at high stress intensity. Controls of the more sensitive cultivar contained the double amount of soluble APX in comparison with the tolerant one and the activity of the enzyme was induced when a moderate water stress intensity was reached. As for APX, SOD and dehydroascorbate reductase (DHAR) activities were also higher in the sensitive cultivar, while thylakoid- bound SOD decreased all over the stress period. The electrophoretic pattern of the soluble extract showed at least 3 isozymes of SOD, 4 isozymes of APX and 2 isozymes of DHAR.

Lipid Composition and Protein Dynamics in Thylakoids of Two Wheat Cultivars Differently Sensitive to Drought

Two wheat (Triticum durum Desf.) cultivars with different sensitivities to drought were either grown under regular irrigation or subjected to water deficit by withholding water for 14 d. Water-stressed plants of both cultivars underwent similar decreases in leaf water potential, but the drought-tolerant cultivar showed higher relative water content and turgor. Neither osmotic nor elastic adjustment mechanisms appeared to be active under the conditions described here. Thylakoids isolated from the stressed, drought-tolerant wheat showed an increase in lipid-to-protein ratio, in comparison with the control, whereas this ratio remained unchanged in the sensitive wheat. In both cultivars, water deficit determined different rearrangements in the composition of the thylakoid individual polar lipids, but their unsaturation level remained unaffected with the exception of monogalactosyldiacylglycerol. In the drought-sensitive cultivar, an accumulation of free fatty acids together with a reduction in polar lipid amount was observed. Electron paramagnetic resonance measurements of spin-labeled proteins of stressed plants from the sensitive cv Adamello showed a higher spin label rotational correlation time together with lower sulphydryl group and mobile proteic portion levels, in comparison with the control. In the tolerant cv Ofanto, the first two parameters changed to a lesser extent following water depletion, and the mobile proteic portion was not altered.

Relation between lipoic acid and cell redox status in wheat grown in excess copper

Cell redox status and lipoic acid contents were analysed in wheat (Triticum durum Desf. cv. Creso) plants treated with 150 μM Cu to elucidate the role of the antioxidant lipoic acid against oxidative stress. In comparison with shoots, roots suffered a higher oxidative stress showing a decrease in NADPH contents and an oxidation of glutathione and ascorbate. Shoots did not evidence a clear oxidative damage since Cu was translocated in small amounts. Lipoic acid as reduced (DHLA) or oxidised (LA) form was present in both leaves and roots of wheat. Analysis of the cell sap showed that this antioxidant was present also as free form. The analyses showed that stroma contained significant amounts of free LA and that, after acidic hydrolysis, higher amounts of LA and DHLA were released. However, lipoic acid was undetectable in both thylakoids and microsomal membranes. Cu treatment did not determine changes in the contents of total LA and DHLA in roots, they being likely involved in Cu chelation. In contrast, in leaves after 48 h of treatment the metal induced an increase in DHLA, which could in part explain the reduction in the oxidised glutathione levels. In leaves free lipoic acid was more prone to be oxidised compared to the bound form, and the reduced form disappeared in both leaves and roots after Cu treatment.

Antioxidative responses of Ocimum basilicum to sodium chloride or sodium sulphate salinization.

Soils and ground water in nature are dominated by chloride and sulphate salts. There have been several studies concerning NaCl salinity, however, little is known about the Na(2)SO(4) one. The effects on antioxidative activities of chloride or sodium sulphate in terms of the same Na(+) equivalents (25 mM Na(2)SO(4) and 50 mM NaCl) were studied on 30 day-old plants of Ocimum basilicum L., variety Genovese subjected to 15 and 30 days of treatment. Growth, thiobarbituric acid reactive substances (TBARS), relative ion leakage ratio (RLR), hydrogen peroxide (H(2)O(2)), ascorbate and glutathione contents as well as the activities of ascorbate peroxidase (APX, EC 1.11.1.11); glutathione reductase (GR, EC 1.6.4.2) and peroxidases (POD, EC 1.11.1.7) were determined. In leaves, growth was more depressed by 25 mM Na(2)SO(4) than 50 mM NaCl. The higher sensitivity of basil to Na(2)SO(4) was associated with an enhanced accumulation of H(2)O(2), an inhibition of APX, GR and POD activities (with the exception of POD under the 30-day-treatment) and a lower regeneration of reduced ascorbate (AsA) and reduced glutathione (GSH). However, the changes in the antioxidant metabolism were enough to limit oxidative damage, explaining the fact that RLR and TBARS levels were unchanged under both Na(2)SO(4) and NaCl treatment. Moreover, for both salts the 30-day-treatment reduced H(2)O(2) accumulation, unchanged RLR and TBARS levels, and enhanced the levels of antioxidants and antioxidative enzymes, thus achieving an adaptation mechanism against reactive oxygen species.

Superoxide and hydroxyl radical generation, and superoxide dismutase in PSII membrane fragments from wheat

Illumination of photosystem II (PSII) membrane fragments of wheat (Triticum durum Desf. cv. Adamello) gave rise to both O2*- and *OH radicals adducts of the novel spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO). With time, *OH became predominant displaying the conversion of O2*- into *OH. An intrinsic activity of superoxide dismutase (SOD) was found in PSII. Photoreduction of nitroblue tetrazolium (NBT) by PSII membrane fragments was induced by the addition of sodium azide and hydrogen peroxide. Western blotting of PSII proteins showed that a 29 kDa protein was recognised by an antibody against chloroplastic Fe-SOD from water lily. An increased formation rate of O2*- was observed in damaged PSII where the SOD activity decreased following a treatment with a free radical-generating system. Damage in PSII consisted also in a decrease in chlorophyll and in carotenoids as well as in a change in the lipid : chlorophyll : protein ratio. Upon treatment a decrease in the unsaturation of PSII membrane fragments was also monitored together with a degradation towards more saturated molecular species of monogalactosyldiacylglycerol.

Antioxidant system in Boea hygroscopica: Changes in response to desiccation and rehydration

Abstract In leaves of Boea hygroscopica subjected to either rapid or slow dehydration and rehydration, the response to H 2 O 2 production was studied by monitoring the changes in the amounts of ascorbic and dehydroascorbic acids as well as the amounts of reduced and oxidized glutathione and related enzyme activities. Boea hygroscopica is a resurrection plant in which drought tolerance depends on the rate of desiccation. For this reason, excised leaves of B. hygroscopica were subjected either to rapid or slow dehydration, by a 144-hr exposure to 0% or 80% relative humidity, respectively. Slowly dried leaves, rehydrated for 36 hr, were able to resume their activities completely, while rapidly dried leaves were not able to revive. After dehydration, H 2 O 2 production decreased both in slowly and in rapidly dried leaves, whereas the levels of total ascorbate and glutathione became two and 50 times as high, respectively, as in the control. During slow drying, reduced glutathione was oxidized, since its content in slowly dried leaves was 50% lower than in rapidly dried leaves. The levels of soluble proteins were found to be, respectively, five and six times as high in rapidly and slowly dried leaves as in the control and rehydrated leaves. The electrophoretic patterns of soluble proteins were similar in both dehydrated leaves, but different from control and rehydrated leaves, which also presented similar patterns. The specific activities of dehydroascorbate reductase (EC 1.8.5.1), glutathione reductase (EC 1.6.4.2) and ascorbate peroxidase (EC 1.11.1.11) also had the same values in both slowly and rapidly dried leaves. Following rehydration, oxidative processes were intensified: H 2 O 2 levels and the oxidation of ascorbate and glutathione mostly increased during this period in which the functioning of the ascorbate/glutathione cycle was induced.