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.

The Effect of Sulforaphane on Glyoxalase I Expression and Activity in Peripheral Blood Mononuclear Cells.

Studies demonstrate that the potential health-beneficial effect of sulforaphane (SR), a compound formed in broccoli, is the result of a number of mechanisms including upregulation of phase two detoxification enzymes. Recent studies suggest that SR increases expression/activity of glyoxalase 1 (Glo1), an enzyme involved in the degradation of methylglyoxal, is major precursor of advanced glycation end products. Those compounds are associated with diabetes complications and other age-related diseases. In this study, the effect of SR on the expression/activity of Glo1 in peripheral blood mononuclear cells (PBMCs) from 8 healthy volunteers was investigated. PBMCs were isolated and incubated with SR (2.5 µM-concentration achievable by consuming a broccoli portion) for 24 h and 48 h. Glo1 activity/expression, reduced glutathione (GSH), and glutathione-S-transferase gene expression were measured. Glo1 activity was not affected while after 48 h a slight but significant increase of its gene expression (1.03-fold) was observed. GSTP1 expression slightly increased after 24 h incubation (1.08-fold) while the expressions of isoform GSTT2 and GSTM2 were below the limit of detection. GSH sharply decreased, suggesting the formation of GSH-SR adducts that may have an impact SR availability. Those results suggest that a regular exposure to SR by broccoli consumption or SR supplements may enhance Glo1.

Measuring Activity of Native Plant Sirtuins - The Wheat Mitochondrial Model.

Sirtuins are NAD+-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 (HTRF®) 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 mU⋅mg-1 protein, as measured by HTRF® assay, and 166 ± 12 ng hSIRT1 eq.⋅mg-1 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 high-throughput 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.

Different effectiveness of two pastas supplemented with either lipophilic or hydrophilic/phenolic antioxidants in affecting serum as evaluated by the novel Antioxidant/Oxidant Balance approach.

Effectiveness in improving serum antioxidant status of two functional pastas was evaluated by the novel Antioxidant/Oxidant Balance (AOB) parameter, calculated as Antioxidant Capacity (AC)/Peroxide Level ratio, assessed here for the first time. In particular, Bran Oleoresin (BO) and Bran Water (BW) pastas, enriched respectively with either lipophilic (tocochromanols, carotenoids) or hydrophilic/phenolic antioxidants extracted from durum wheat bran, were studied. Notably, BO pasta was able to improve significantly (+65%) serum AOB during four hours after intake similarly to Lisosan G, a wheat antioxidant-rich dietary supplement. Contrarily, BW pasta had oxidative effect on serum so as conventional pasta and glucose, thus suggesting greater effectiveness of lipophilic than hydrophilic/phenolic antioxidants under our experimental conditions. Interestingly, no clear differences between the two pastas were observed, when AC measurements of either serum after pasta intake or pasta extracts by in vitro assays were considered, thus strengthening effectiveness and reliability of AOB approach.

Serum antioxidant capacity and peroxide level of seven healthy subjects after consumption of different foods.

This article reports experimental data related to the research article entitled "Different effectiveness of two pastas supplemented with either lipophilic or hydrophilic/phenolic antioxidants in affecting serum as evaluated by the novel Antioxidant/Oxidant Balance approach" (M.N. Laus, M. Soccio, M. Alfarano, A. Pasqualone, M.S. Lenucci, G. Di Miceli, D. Pastore, 2016) [1]. Antioxidant status of blood serum of seven healthy subjects was evaluated during four hours after consumption of two functional pastas, supplemented with either bran oleoresin or bran water extract obtained from durum wheat. For comparison, the effect of a non-supplemented reference pasta was also evaluated, as well as the effects of glucose, of the wheat grain dietary supplement Lisosan G, and of the reference pasta consumed together with Lisosan G. Serum antioxidant status was evaluated by measuring both the serum antioxidant capacity, using LOX-FL, ORAC and TEAC methods, and the serum oxidant status, assessed as peroxide level.

Modulation of Potassium Channel Activity in the Balance of ROS and ATP Production by Durum Wheat Mitochondria-An Amazing Defense Tool Against Hyperosmotic Stress.

In plants, the existence of a mitochondrial potassium channel was firstly demonstrated about 15 years ago in durum wheat as an ATP-dependent potassium channel (PmitoKATP). Since then, both properties of the original PmitoKATP and occurrence of different mitochondrial potassium channels in a number of plant species (monocotyledonous and dicotyledonous) and tissues/organs (etiolated and green) have been shown. Here, an overview of the current knowledge is reported; in particular, the issue of PmitoKATP physiological modulation is addressed. Similarities and differences with other potassium channels, as well as possible cross-regulation with other mitochondrial proteins (Plant Uncoupling Protein, Alternative Oxidase, Plant Inner Membrane Anion Channel) are also described. PmitoKATP is inhibited by ATP and activated by superoxide anion, as well as by free fatty acids (FFAs) and acyl-CoAs. Interestingly, channel activation increases electrophoretic potassium uptake across the inner membrane toward the matrix, so collapsing membrane potential (ΔΨ), the main component of the protonmotive force (Δp) in plant mitochondria; moreover, cooperation between PmitoKATP and the K(+)/H(+) antiporter allows a potassium cycle able to dissipate also ΔpH. Interestingly, ΔΨ collapse matches with an active control of mitochondrial reactive oxygen species (ROS) production. Fully open channel is able to lower superoxide anion up to 35-fold compared to a condition of ATP-inhibited channel. On the other hand, ΔΨ collapse by PmitoKATP was unexpectedly found to not affect ATP synthesis via oxidative phosphorylation. This may probably occur by means of a controlled collapse due to ATP inhibition of PmitoKATP; this brake to the channel activity may allow a loss of the bulk phase Δp, but may preserve a non-classically detectable localized driving force for ATP synthesis. This ability may become crucial under environmental/oxidative stress. In particular, under moderate hyperosmotic stress (mannitol or NaCl), PmitoKATP was found to be activated by ROS, so inhibiting further large-scale ROS production according to a feedback mechanism; moreover, a stress-activated phospholipase A2 may generate FFAs, further activating the channel. In conclusion, a main property of PmitoKATP is the ability to keep in balance the control of harmful ROS with the mitochondrial/cellular bioenergetics, thus preserving ATP for energetic needs of cell defense under stress.

Evaluation of Phenolic Antioxidant Capacity in Grains of Modern and Old Durum Wheat Genotypes by the Novel QUENCHERABTS Approach.

The QUENCHERABTS (QUick, Easy, New, CHEap and Reproducible) approach for antioxidant capacity (AC) determination is based on the direct reaction of 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation with fine solid food particles. So, it may resemble the antioxidant action in foods or in human gastrointestinal trait. Here, the QUENCHER approach was used to study AC of durum wheat (Triticum durum Desf.) grains. Firstly, it was assessed which kind of antioxidants determines QUENCHER response. This has been performed by comparing AC measured by QUENCHERABTS and that measured by classical TEACABTS (Trolox equivalent antioxidant capacity) in four different extracts from whole flour of 10 durum wheat varieties containing: lipophilic, hydrophilic, insoluble-bound phenolic (IBP) and free-soluble phenolic (FSP) compounds. QUENCHERABTS data were unrelated to AC of water-extractable antioxidants and weakly correlated (r = 0.405, P < 0.05) to AC of the lipophilic ones; on the contrary, QUENCHERABTS response was mainly related to AC of IBP (r = 0.907, P < 0.001) and to a lesser extent of FSP extracts (r = 0.747, P < 0.001). Consistently, correlation was also found with the phenolic content of IBP and FSP (r = 0.760, P < 0.001 and r = 0.522, P < 0.01, respectively), thus confirming that QUENCHERABTS assay mainly assesses AC due to IBP. So, this assay was used in a first screening study to compare AC of bioactive IBP of thirty-six genotypes/landraces covering a century of cultivation in Italy. Interestingly, no relevant AC difference between modern and old genotypes was found, thus suggesting that a century of plant breeding did not decrease phenol-dependent health potential in durum wheat.

Transport pathways--proton motive force interrelationship in durum wheat mitochondria.

In durum wheat mitochondria (DWM) the ATP-inhibited plant mitochondrial potassium channel (PmitoK(ATP)) 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 PmitoK(ATP) 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 PmitoK(ATP) 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 PmitoK(ATP) 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.

Stay-green trait-antioxidant status interrelationship in durum wheat (Triticum durum) flag leaf during post-flowering.

Three independent durum wheat mutant lines that show delayed leaf senescence or stay-green (SG) phenotype, SG196, SG310 and SG504, were compared to the parental genotype, cv. Trinakria, with respect to the photosynthetic parameters and the cellular redox state of the flag leaf in the period from flowering to senescence. The SG mutants maintained their chlorophyll content and net photosynthetic rate for longer than Trinakria, thus revealing a functional SG phenotype. They also showed a better redox state as demonstrated by: (1) a lower rate of superoxide anion production due to generally higher activity of the antioxidant enzymes superoxide dismutase and catalase in all of the SG mutants and also of the total peroxidase in SG196; (2) a higher thiol content that can be ascribed to a higher activity of the NADPH-providing enzyme glucose-6-phosphate dehydrogenase in all of the SG mutants and also of the NADP(+)-dependent malic enzyme in SG196; (3) a lower pro-oxidant activity of lipoxygenase that characterises SG196 and SG504 mutants close to leaf senescence. Overall, these results show a general relationship in durum wheat between the SG phenotype and a better redox state. This relationship differs across the different SG mutants, probably as a consequence of the different set of altered genes underlying the SG trait in these independent mutant lines.

The uniqueness of the plant mitochondrial potassium channel.

The ATP-inhibited Plant Mitochondrial K(+) Channel (PmitoKATP) was discovered about fifteen years ago in Durum Wheat Mitochondria (DWM). PmitoKATP catalyses the electrophoretic K(+) uniport through the inner mitochondrial membrane; moreover, the co-operation between PmitoKATP and K(+)/H(+) antiporter allows such a great operation of a K(+) cycle to collapse mitochondrial membrane potential (ΔΨ) and ΔpH, thus impairing protonmotive force (Δp). A possible physiological role of such ΔΨ control is the restriction of harmful reactive oxygen species (ROS) production under environmental/oxidative stress conditions. Interestingly, DWM lacking Δp were found to be nevertheless fully coupled and able to regularly accomplish ATP synthesis; this unexpected behaviour makes necessary to recast in some way the classical chemiosmotic model. In the whole, PmitoKATP may oppose to large scale ROS production by lowering ΔΨ under environmental/oxidative stress, but, when stress is moderate, this occurs without impairing ATP synthesis in a crucial moment for cell and mitochondrial bioenergetics.

The existence of phospholipase A(2) activity in plant mitochondria and its activation by hyperosmotic stress in durum wheat (Triticum durum Desf.).

The activity of mitochondrial phospholipase A(2) (PLA(2)) was shown for the first time in plants. It was observed in etiolated seedlings from durum wheat, barley, tomato, spelt and green seedlings of maize, but not in potato and topinambur tubers and lentil etiolated seedlings. This result was achieved by a novel spectrophotometric assay based on the coupled PLA(2)/lipoxygenase reactions using 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine as substrate; the mitochondrial localisation was assessed by checking recovery of marker enzymes. Durum wheat mitochondrial PLA(2) (DWM-PLA(2)) showed maximal activity at pH 9.0 and 1mM Ca(2+), hyperbolic kinetics (K(m)=90±6µM, V(max)=29±1nmolmin(-1)mg(-1) of protein) and inhibition by methyl arachidonyl fluorophosphonate, 5-(4-benzyloxyphenyl)-4S-(7-phenylheptanoylamino)pentanoic acid and palmityl trifluoromethyl ketone. Reactive oxygen species had no effect on DWM-PLA(2), that instead was activated by about 50% and 95%, respectively, under salt (0.21M NaCl) and osmotic (0.42M mannitol) stress imposed during germination. Contrarily, a secondary Ca(2+)-independent activity, having optimum at pH 7.0, was stress-insensitive. We propose that the activation of DWM-PLA(2) is responsible for the strong increase of free fatty acids recently measured in mitochondria under the same stress conditions [Laus, et al., J. Exp. Bot. 62 (2011) 141-154] that, in turn, activate potassium channel and uncoupling protein, able to counteract hyperosmotic stress.

Antioxidant activity of free and bound compounds in quinoa (Chenopodium quinoa Willd.) seeds in comparison with durum wheat and emmer.

UNLABELLED: Antioxidant activity (AA) of quinoa (Chenopodium quinoa Willd.) seeds, as well as of durum wheat (Triticum turgidum L. ssp. durum Desf.) and of emmer (T. turgidum L. ssp. dicoccum Schübler) grains, was evaluated by studying hydrophilic (H), lipophilic (L), free-soluble (FSP) and insoluble-bound (IBP) phenolic extracts using the new lipoxygenase/4-nitroso-N,N-dimethylaniline (LOX/RNO) method, able to simultaneously detect different antioxidant mechanisms, as well as using the Oxygen Radical Absorbance Capacity (ORAC) and the Trolox Equivalent Antioxidant Capacity (TEAC) assays, which measure the scavenging activity against peroxyl and ABTS [2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonate)] radicals, respectively. The species under study were compared with respect to the sum of AA values of H, L and FSP extracts (AA(H+L+FSP)), containing freely solvent-soluble antioxidants, and AA values of IBP extracts (AA(IBP)), representing the phenolic fraction ester-linked to insoluble cell wall polymers. The LOX/RNO and ORAC methods measured in quinoa flour a remarkable AA(H+L+FSP) higher than durum wheat, although lower than emmer; according to the same assays, the IBP component of quinoa resulted less active than the durum wheat and emmer ones. The TEAC protocol also revealed a high AA(H+L+FSP) for quinoa. Interestingly, the ratio AA(H+L+FSP)/AA(H+L+FSP+IBP), as evaluated by the LOX/RNO and ORAC assays, resulted in quinoa higher than that of both durum wheat and emmer, and much higher than durum wheat, according to the TEAC protocol. This may suggest that antioxidants from quinoa seeds may be more readily accessible with respect to that of both the examined wheat species. PRACTICAL APPLICATIONS: Quinoa seeds may represent an excellent source of natural antioxidant compounds and, in particular, of the free-soluble antioxidant fraction. These compounds may improve nutritive and health-beneficial properties of quinoa-based gluten-free products, thus expanding interest for quinoa utilization from celiac patients to the general population.

Potassium channel-oxidative phosphorylation relationship in durum wheat mitochondria from control and hyperosmotic-stressed seedlings.

Durum wheat mitochondria (DWM) possess an ATP-inhibited K(+) channel, the plant mitoK(ATP) (PmitoK(ATP) ), which is activated under environmental stress to control mitochondrial ROS production. To do this, PmitoK(ATP) collapses membrane potential (ΔΨ), thus suggesting mitochondrial uncoupling. We tested this point by studying oxidative phosphorylation (OXPHOS) in DWM purified from control seedlings and from seedlings subjected both to severe mannitol and NaCl stress. In severely-stressed DWM, the ATP synthesis via OXPHOS, continuously monitored by a spectrophotometric assay, was about 90% inhibited when the PmitoK(ATP) was activated by KCl. Contrarily, in control DWM, although PmitoK(ATP) collapsed ΔΨ, ATP synthesis, as well as coupling [respiratory control (RC) ratio and ratio between phosphorylated ADP and reduced oxygen (ADP/O)] checked by oxygen uptake experiments, were unaffected. We suggest that PmitoK(ATP) may play an important defensive role at the onset of the environmental/oxidative stress by preserving energy in a crucial moment for cell and mitochondrial bioenergetics. Consistently, under moderate mannitol stress, miming an early stress condition, the channel may efficiently control reactive oxygen species (ROS) generation (about 35-fold from fully open to closed state) without impairing ATP synthesis. Anyway, if the stress significantly proceeds, the PmitoK(ATP) becomes fully activated by decrease of ATP concentration (25-40%) and increase of activators [free fatty acids (FFAs) and superoxide anion], thus impairing ATP synthesis.

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

The effect of free fatty acids (FFAs) and acyl-CoA esters on K(+) uptake was studied in mitochondria isolated from durum wheat (Triticum durum Desf.), a species that has adapted well to the semi-arid Mediterranean area and possessing a highly active mitochondrial ATP-sensitive K(+) channel (PmitoK(ATP)), that may confer resistance to environmental stresses. This was made by swelling experiments in KCl solution under experimental conditions in which PmitoK(ATP) activity was monitored. Linoleate and other FFAs (laurate, palmitate, stearate, palmitoleate, oleate, arachidonate, and the non-physiological 1-undecanesulphonate and 5-phenylvalerate), used at a concentration (10 µM) unable to damage membranes of isolated mitochondria, stimulated K(+) uptake by about 2-4-fold. Acyl-CoAs also promoted K(+) transport to a much larger extent with respect to FFAs (about 5-12-fold). In a different experimental system based on safranin O fluorescence measurements, the dissipation of electrical membrane potential induced by K(+) uptake via PmitoK(ATP) was found to increase in the presence of 5-phenylvalerate and palmitoyl-CoA, both unable to elicit the activity of the Plant Uncoupling Protein. This result suggests a direct activation of PmitoK(ATP). Stimulation of K(+) transport by FFAs/acyl-CoAs resulted in a widespread phenomenon in plant mitochondria from different mono/dicotyledonous species (bread wheat, barley, triticale, maize, lentil, pea, and topinambur) and from different organs (root, tuber, leaf, and shoot). Finally, an increase in mitochondrial FFAs up to a content of 50 nmol mg(-1) protein, which was able to activate PmitoK(ATP) strongly, was observed under hyperosmotic stress conditions. Since PmitoK(ATP) may act against environmental/oxidative stress, its activation by FFAs/acyl-CoAs is proposed to represent a physiological defence mechanism.

ATP-sensitive cation-channel in wheat (Triticum durum Desf.): identification and characterization of a plant mitochondrial channel by patch-clamp.

Indirect evidence points to the presence of K(+) channels in plant mitochondria. In the present study, we report the results of the first patch clamp experiments on plant mitochondria. Single-channel recordings in 150 mM potassium gluconate have allowed the biophysical characterization of a channel with a conductance of 150 pS in the inner mitochondrial membrane of mitoplasts obtained from wheat (Triticum durum Desf.). The channel displayed sharp voltage sensitivity, permeability to potassium and cation selectivity. ATP in the mM concentration range completely abolished the activity. We discuss the possible molecular identity of the channel and its possible role in the defence mechanisms against oxidative stress in plants.

New tool to evaluate a comprehensive antioxidant activity in food extracts: bleaching of 4-nitroso-N,N-dimethylaniline catalyzed by soybean lipoxygenase-1.

In this study the 4-nitroso-N,N-dimethylaniline (RNO) bleaching associated with linoleic acid hydroperoxidation catalyzed by the soybean lipoxygenase (LOX)-1 isoenzyme (LOX/RNO reaction) was used to determine the antioxidant activity (AA) of hydrophilic and lipophilic pure antioxidant compounds and of mixtures of antioxidants extracted from durum wheat whole flour (DWWF). By means of a simple and rapid experimental protocol (about 3 min/assay), the LOX/RNO reaction may simultaneously detect many antioxidant functions (scavenging of some physiological radical species, iron ion reducing and chelating activities, inhibition of the pro-oxidant apoenzyme), thus providing a comprehensive AA evaluation. Consistently, the LOX/RNO assay was very sensitive to hydrophilic, lipophilic, and phenolic antioxidant extracts from DWWF, providing AA values at least 35 and 30 times higher than those by TEAC and ORAC methods, respectively. Moreover, the new method was able to highlight synergism (among extracts) 3 times more than the ORAC method, whereas TEAC did not measure synergism under our experimental conditions.

Plant inner membrane anion channel (PIMAC) function in plant mitochondria.

To date, the existence of the plant inner membrane anion channel (PIMAC) has been shown only in potato mitochondria, but its physiological role remains unclear. In this study, by means of swelling experiments in K(+) and ammonium salts, we characterize a PIMAC-like anion-conducting pathway in mitochondria from durum wheat (DWM), a monocotyledonous species phylogenetically far from potato. DWM were investigated since they possess a very active potassium channel (PmitoK(ATP)), so implying a very active matching anion uniport pathway and, possibly, a coordinated function. As in potato mitochondria, the electrophoretic uptake of chloride and succinate was inhibited by matrix [H(+)], propranolol, and tributyltin, and was insensitive to Mg(2+), N,N'-dicyclohexylcarbodiimide (DCCD) and mercurials, thus showing PIMAC's existence in DWM. PIMAC actively transports dicarboxylates, oxodicarboxylates, tricarboxylates and Pi. Interestingly, a novel mechanism of swelling in ammonium salts of isolated plant mitochondria is reported, based on electrophoretic anion uptake via PIMAC and ammonium uniport via PmitoK(ATP). PIMAC is inhibited by physiological compounds, such as ATP and free fatty acids, by high electrical membrane potential (Delta Psi), but not by acyl-CoAs or reactive oxygen species. PIMAC was found to cooperate with dicarboxylate carrier by allowing succinate uptake that triggers succinate/malate exchange in isolated DWM. Similar results were obtained using mitochondria from the dicotyledonous species topinambur, so suggesting generalization of results. We propose that PIMAC is normally inactive in vivo due to ATP and Delta Psi inhibition, but activation may occur in mitochondria de-energized by PmitoK(ATP) (or other dissipative systems) to replace or integrate the operation of classical anion carriers.

Possible plant mitochondria involvement in cell adaptation to drought stress. A case study: durum wheat mitochondria.

Although plant cell bioenergetics is strongly affected by abiotic stresses, mitochondrial metabolism under stress is still largely unknown. Interestingly, plant mitochondria may control reactive oxygen species (ROS) generation by means of energy-dissipating systems. Therefore, mitochondria may play a central role in cell adaptation to abiotic stresses, which are known to induce oxidative stress at cellular level. With this in mind, in recent years, studies have been focused on mitochondria from durum wheat, a species well adapted to drought stress. Durum wheat mitochondria possess three energy-dissipating systems: the ATP-sensitive plant mitochondrial potassium channel (PmitoK(ATP)); the plant uncoupling protein (PUCP); and the alternative oxidase (AOX). It has been shown that these systems are able to dampen mitochondrial ROS production; surprisingly, PmitoK(ATP) and PUCP (but not AOX) are activated by ROS. This was found to occur in mitochondria from both control and hyperosmotic-stressed seedlings. Therefore, the hypothesis of a 'feed-back' mechanism operating under hyperosmotic/oxidative stress conditions was validated: stress conditions induce an increase in mitochondrial ROS production; ROS activate PmitoK(ATP) and PUCP that, in turn, dissipate the mitochondrial membrane potential, thus inhibiting further large-scale ROS production. Another important aspect is the chloroplast/cytosol/mitochondrion co-operation in green tissues under stress conditions aimed at modulating cell redox homeostasis. Durum wheat mitochondria may act against chloroplast/cytosol over-reduction: the malate/oxaloacetate antiporter and the rotenone-insensitive external NAD(P)H dehydrogenases allow cytosolic NAD(P)H oxidation; under stress this may occur without high ROS production due to co-operation with AOX, which is activated by intermediates of the photorespiratory cycle.

The transcript levels of two plant mitochondrial uncoupling protein (pUCP)-related genes are not affected by hyperosmotic stress in durum wheat seedlings showing an increased level of pUCP activity.

Etiolated early seedlings of durum wheat submitted to moderate and severe salt (NaCl) and osmotic (mannitol) stress showed no relevant increase of both transcript levels of two plant uncoupling protein (pUCP)-related genes and maximal pUCP activity in purified mitochondria (which estimates protein level); contrarily, pUCP functioning due to endogenous free fatty acids strongly increased. These results show that pUCP activation under hyperosmotic stress may be due to modulation of pUCP reaction rather than to an increased protein synthesis. Finally, a properly developed method, based on a single membrane potential measurement, to evaluate both pUCP maximal activity and functioning, is reported.

Seawater stress applied at germination affects mitochondrial function in durum wheat (Triticum durum) early seedlings.

Seawater stress effects on mitochondrial ATP synthesis and membrane potential (ΔΨ) were investigated in germinating durum wheat seedlings under moderate (22% seawater osmolarity, -0.62 MPa) and severe (37% seawater osmolarity, -1.04 MPa) stress. To estimate the osmotic component of salt stress, mannitol solutions (0.25 and 0.42 m) iso-osmotic with the saline ones were used. Moderate stress intensity only delayed mean germination time (MGT), whereas higher seawater osmolarity reduced germination percentage as well. In contrast, Na+ and Cl- accumulation showed a sharp increase under moderate stress and only a small further increase under severe stress, which was more pronounced for Cl-. Only severe stress significantly damaged succinate-dependent oxidative phosphorylation, which may be related to the stress-induced alteration in inner mitochondrial membrane permeability, as indicated by changes in ΔΨ profiles. Proline-dependent oxidative phosphorylation, however, was inhibited under moderate stress. This suggests the occurrence of an adaptation mechanism leading to proline accumulation as an osmoprotectant. Moreover, both the osmotic and the toxic components of seawater stress were detrimental to oxidative phosphorylation. Damage to germination and MGT, in contrast, were mainly caused by osmotic stress. Therefore, mitochondrial function appears to be a more sensitive target of toxic stress than growth. In conclusion, the effects of seawater stress on mitochondrial ATP synthesis vary in relation to the substrate oxidised and stress level, inducing both adaptive responses and damage.