Reactive oxygen species inhibit the succinate oxidation-supported generation of membrane potential in wheat mitochondria

In order to gain a first insight into the effects of reactive oxygen species (ROS) on plant mitochondria, we studied the effect of the ROS producing system consisting of xanthine plus xanthine oxidase on the rate of membrane potential (ΔΨ) generation due to either succinate or NADH addition to durum wheat mitochondria as monitored by safranin fluorescence. We show that the early ROS production inhibits the succinate-dependent, but not the NADH-dependent, ΔΨ generation and oxygen uptake. This inhibition appears to depend on the impairment of mitochondrial permeability to succinate. It does not involve mitochondrial thiol groups sensitive to either mersalyl or N-ethylmaleimide and might involve both protein residues and/or membrane lipids, as suggested by the mixed nature. We propose that, during oxidative stress, early generation of ROS can affect plant mitochondria by impairing metabolite transport, thus preventing further substrate oxidation, ΔΨ generation and consequent large-scale ROS production. © 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved

Antioxidant capacity, phenolic and vitamin C contents of quinoa (Chenopodium quinoa Willd.) as affected by sprouting and storage conditions

Antioxidant capacity (AC) of quinoa (Chenopodium quinoa Willd. cv. Real) seeds and sprouts obtained after 4 days of seed germination at 20°C and 70% humidity was evaluated using trolox equivalent antioxidant capacity (TEAC) and oxygen radical absorbance capacity (ORAC) assays, able to highlight reducing activity and peroxyl radical scavenging capacity, respectively; phenolic content (PC) was also measured. Both TEAC and ORAC assays revealed a significantly higher (about 2- and 2.8-fold, respectively) AC of 4-day-old sprouts compared to seeds; consistently, also PC values of sprouts resulted about 2.6 times higher than seeds. In order to investigate the influence of storage on AC and PC, as well as on vitamin C content (VCC), 4-day-old sprouts were subjected for 7 days at 5°C to three different conditions of controlled atmosphere storage (CAS) compared with air. Interestingly, whatever the CAS conditions, storage of quinoa sprouts up to 7 days induced an increase of AC evaluated in terms of reducing activity by TEAC assay. Consistently, an increase of PC and VCC was measured during storage, positively correlated to TEAC values. Moreover, a decrease of peroxyl radical scavenging activity, measured by ORAC, was observed after 7 days of storage, in accordance with a shift of AC towards the reducing activity component. Overall, these findings indicate that sprouting approach using quinoa may provide highly antioxidant-enriched seedlings that may improve nutritional quality of diet or of functional foods. Interestingly, antioxidant properties of quinoa sprouts may be deeply influenced by storage, able to increase reducing activity by increasing phenols and vitamin C

First Evidence of a Protective Effect of Plant Bioactive Compounds against H2O2-Induced Aconitase Damage in Durum Wheat Mitochondria

In order to contribute to the understanding of the antioxidant behavior of plant bioactive compounds with respect to specific subcellular targets, in this study, their capability to protect aconitase activity from oxidative-mediated dysfunction was evaluated for the first time in plant mitochondria. Interest was focused on the Krebs cycle enzyme catalyzing the citrate/isocitrate interconversion via cis-aconitate, as it possesses a [4Fe-4S]2+ cluster at the active site, making it an early and highly sensitive target of reactive oxygen species (ROS)-induced oxidative damage. In particular, the effect on the aconitase reaction of five natural phenols, including ferulic acid, apigenin, quercetin, resveratrol, and curcumin, as well as of the isothiocyanate sulforaphane, was investigated in highly purified mitochondria obtained from durum wheat (DWM). Interestingly, a short-term (10 min) DWM pre-treatment with all investigated compounds, applied at 150 µM (75 µM in the case of resveratrol), completely prevented aconitase damage induced by a 15 min exposure of mitochondria to 500 µM H2O2. Curcumin and quercetin were also found to completely recover DWM-aconitase activity when phytochemical treatment was performed after H2O2 damage. In addition, all tested phytochemicals (except ferulic) induced a significant increase of aconitase activity in undamaged mitochondria. On the contrary, a relevant protective and recovery effect of only quercetin treatment was observed in terms of the aconitase activity of a commercial purified mammalian isoform, which was used for comparison. Overall, the results obtained in this study may suggest a possible role of phytochemicals in preserving plant mitochondrial aconitase activity, as well as energy metabolism, against oxidative damage that may occur under environmental stress conditions. Further investigations are needed to elucidate the physiological role and the mechanism responsible for this short-term protective effect

Evaluation of synergistic interactions of antioxidants from plant foods by a new method using soybean lipoxygenase

A proper evaluation of synergism among antioxidants remains so far rather difficult to obtain. We recently reported that a new method for antioxidant activity measurement, based on the secondary reaction of soybean lipoxygenase (LOX)-1 isoenzyme with 4-nitroso-N,N-dimethylaniline (RNO), so-called LOX/RNO method, may assess very well the synergism among antioxidants from durum wheat whole semolina. To evaluate whether this behaviour is generalizable to different food matrices, herein, antioxidants from other very different sources were analysed. For this purpose, antioxidant activity of food-grade preparations enriched in catechins, quercetin, resveratrol, tyrosol/hydroxytyrosol and lycopene was evaluated by the LOX/RNO method in comparison with the Trolox equivalent antioxidant ca pacity (TEAC) assay. The antioxidant activity values obtained by LOX/RNO method were 2–90-times higher than those obtained by the TEAC protocol, depending on the tested antioxidant. Synergism was evaluated by comparing antioxidant activity of the mixture of compounds (AAmix) with the sum of antioxidant activity values of individual compounds (AAsum). The LOX/RNO method revealed a strong synergism, being AAmix 5.69 ± 0.31 times higher than AAsum (statistically significant, p < 0.001), while the TEAC method showed a synergistic increase of only 0.31 ± 0.04 (statistically significant, p < 0.01). These findings suggest that the LOX/RNO method is able to assess very well the synergism in various food samples

Assessment of Antioxidant Capacity and Putative Healthy Effects of Natural Plant Products Using Soybean Lipoxygenase-Based Methods. An Overview

In the last decades, increasing demand of antioxidant-rich foods and growing interest in their putative role in prevention of degenerative diseases have promoted development of methods for measuring Antioxidant Capacity (AC). Nevertheless, most of these assays use radicals and experimental conditions far from the physiological ones, and are able to estimate only one or a few antioxidant mechanisms. On the other hand, the novel LOX/RNO and LOX–FL methods, based on secondary reactions between the soybean lipoxygenase (LOX)-1 isoenzyme and either 4-nitroso-N,N-dimethylaniline (RNO) or fluorescein (FL), may provide a more comprehensive AC evaluation. In fact, they are able to detect simultaneously many antioxidant functions (scavenging of some physiological radical species, iron ion reducing and chelating activities, inhibition of the pro-oxidant apoenzyme) and to highlight synergism among phytochemicals. They are applied to dissect antioxidant properties of several natural plant products: food-grade antioxidants, cereal and pseudocereal grains, grain-derived products, fruits. Recently, LOX–FL has been used for ex vivo AC measurements of human blood samples after short- and long-term intakes of some of these foods, and the effectiveness in improving serum antioxidant status was evaluated using the novel Antioxidant/Oxidant Balance (AOB) parameter, calculated as an AC/Peroxide Level ratio. An overview of data is presented

Transport Pathways—Proton Motive Force Interrelationship in Durum Wheat Mitochondria

In durum wheat mitochondria (DWM) the ATP-inhibited plant mitochondrial potassium channel (PmitoKATP) and the plant uncoupling protein (PUCP) are able to strongly reduce the proton motive force (pmf) to control mitochondrial production of reactive oxygen species; under these conditions, mitochondrial carriers lack the driving force for transport and should be inactive. However, unexpectedly, DWM uncoupling by PmitoKATP neither impairs the exchange of ADP for ATP nor blocks the inward transport of Pi and succinate. This uptake may occur via the plant inner membrane anion channel (PIMAC), which is physiologically inhibited by membrane potential, but unlocks its activity in de-energized mitochondria. Probably, cooperation between PIMAC and carriers may accomplish metabolite movement across the inner membrane under both energized and de-energized conditions. PIMAC may also cooperate with PmitoKATP to transport ammonium salts in DWM. Interestingly, this finding may trouble classical interpretation of in vitro mitochondrial swelling; instead of free passage of ammonia through the inner membrane and proton symport with Pi, that trigger metabolite movements via carriers, transport of ammonium via PmitoKATP and that of the counteranion via PIMAC may occur. Here, we review properties, modulation and function of the above reported DWM channels and carriers to shed new light on the control that they exert on pmf and vice-versa

Measuring Activity of Native Plant Sirtuins - The Wheat Mitochondrial Model

Sirtuins are NADC-dependent deacetylase enzymes that have gained considerable interest in mammals for their recognized importance in gene silencing and expression and in cell metabolism. Conversely, knowledge about plant sirtuins remains limited, although a sirtuin-mediated regulation of mitochondrial energy metabolism has been recently reported in Arabidopsis. However, so far, no information is available about direct measurement of intracellular plant sirtuin activity, i.e., in cell extracts and/or subcellular organelles. In this study, a novel approach was proposed for reliable evaluation of native sirtuin activity in plant samples, based on (i) an adequate combinatory application of enzymatic assays very different for chemical basis and rationale and (ii) a comparative measurement of activity of a recombinant sirtuin isoform. In particular, two sirtuin assays were applied, based on bioluminescence emission and Homogeneous Time-Resolved Fluorescence (HTRFR ) technology, and the human SIRT1 isoform (hSIRT1) was used for comparison. For the first time in plants, this new approach allowed measuring directly a high and nicotinamide-sensitive sirtuin activity in highly purified mitochondrial fraction obtained from durum wheat (WM). WM-sirtuin activity was 268 10 mUmg1 protein, as measured by HTRFR assay, and 166 12 ng hSIRT1 eq.mg1 protein, as evaluated by the bioluminescent assay and calculated on the basis of the hSIRT1 calibration curve. Moreover, effects of resveratrol and quercetin, reported as potent hSIRT1 activators, but whose activation mechanism is still debated, were also studied. No effect of resveratrol was found on both WM-sirtuin and hSIRT1 activities, while only a slight increase, up to about 20%, of hSIRT1 activity by quercetin was observed. In the whole, results of this study indicate that WM may represent a good system for studying native plant sirtuins. In fact, the high yield of purified WM and their high sirtuin activity, together with use of microplate readers, allow performing a large number of measurements from the same preparation, so qualifying the approach for application to large-scale highthroughput screening. Moreover, WM may also represent an excellent tool to investigate physiological role and modulation of plant sirtuins under experimental conditions more physiologically relevant with respect to recombinant purified enzymes

Journal of Experimental Botany, Page 1 of 14

Activation of the plant mitochondrial potassium channel by free fatty acids and acyl-CoA esters: a possible defence mechanism in the response to hyperosmotic stres

Alternative oxidase in durum wheat mitochondria. Activation by pyruvate, hydroxypyruvate and glyoxylate and physiological role

In order to gain a first insight into the alternative oxidase (AO) function in durum wheat mitochondria (DWM), we investigated some activation pathways of this enzyme in DWM purified from both etiolated shoots and green leaves. AO was activated when DWM were added with either pyruvate, known as an AO activator in other plant mitochondria, or alanine plus 2-oxoglutarate, which can generate intramitochondrial pyruvate and glutamate via transamination. In contrast, no AO activity was observed during oxidation of malate plus glutamate or succinate (which can generate malate). In this regard DWM differ from other plant mitochondria. Moreover, DWM were found: (i) to have a very low malic enzyme (ME) activity, (ii) to release oxaloacetate rather than pyruvate during malate oxidation and (iii) to poorly oxidise malate in the absence of glutamate, which removes oxaloacetate via transamination. Therefore, we show that, unlike other plant mitochondria, no pyruvate is generated inside DWM from malate via ME, allowing no AO activity. Other AO activators, alternative to pyruvate, were checked by evaluating the capability of several compounds to induce oxygen uptake and/or electrical membrane potential (ΔΨ) in cyanide-treated DWM. Hydroxypyruvate and glyoxylate, photorespiratory cycle intermediates, were found to be powerful AO activators, capable of inducing a maximal rate of cyanide-insensitive oxygen uptake 1.7 times and 2.3 times higher than pyruvate, respectively. These results suggest that in durum wheat a link may exist between AO activity and photorespiratory metabolism rather than malate metabolism. Moreover, we observed that AO activation resulted in both a partially coupled respiration and a reduction by half of the rate of superoxide anion generation; therefore, AO is expected to work as an antioxidative defence system when the photorespiratory cycle is highly active, as under environmental stress