The assays commonly used to determine ATP content in biological samples generally measure total cellular ATP content, but not the different subcellular pools. In this study a new simple method for measuring ATP content in a cytosol-enriched fraction (CEF) was developed, based on a rapid cytosolic ATP extraction (by an isotonic grinding medium that preserves organelle integrity) and its detection monitoring the NADPH fluorescence generated via hexokinase/glucose-6-phosphate dehydrogenase coupled reactions. Four protocols, differing for timing of NADPH generation and for either the presence or absence of some inhibitors of ATP and NADPH metabolism, were compared by determining CEF-ATP, as well as total ATP, in durum wheat (Triticum durum Desf.) etiolated seedlings. The best protocol was the one adopting both simultaneous NADPH generation and use of inhibitors during tissue homogenization. This protocol also showed higher performance than the classical trichloroacetic acid extraction. Using the new method, CEF-ATP content was assessed in control, salt- and osmotic-stressed seedlings, resulting 2. 68 ± 0. 04, 1. 69 ± 0. 12 (-40%) and 1. 35 ± 0. 16 (-50%) μmol/g dry weight, respectively. Finally, the effects of this stress-dependent decrease of cytosolic ATP were evaluated with respect to a possible modulation of two mitochondrial energy-dissipating systems, the uncoupling protein (PUCP) and the K+ channel (PmitoKATP), both inhibited by cytosolic ATP. Experiments carried out at different physiological ATP concentrations suggest that the decreased cytosolic ATP content occurring under hyperosmotic stress may contribute to attenuate inhibition of PmitoKATP, thus promoting its activity (up to about 90%), but not of PUCP, that appears to lose ATP sensitivity under stress condition.

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 (PmitoKATP), that may confer resistance to environmental stresses. This was made by swelling experiments in KCl solution under experimental conditions in which PmitoKATP 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 PmitoKATP 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 PmitoKATP. 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 PmitoKATP strongly, was observed under hyperosmotic stress conditions. Since PmitoKATP may act against environmental/oxidative stress, its activation by FFAs/acyl-CoAs is proposed to represent a physiological defence mechanism.

An improved spectrophotometric assay of phospholipase A2 (PLA2) activity based on the coupled PLA2/lipoxygenase (LOX) reactions using 1-palmitoyl-2-linoleoyl-sn-glycero-3- phosphatidylcholine (PCLIN) as substrate is reported. The PLA2- mediated release of free linoleate is continuously monitored by following the absorbance increase at 234 nm caused by its conversion into the conjugated diene hydroperoxide catalyzed by the coupled soybean LOX-1 reaction. The new protocol includes the use of Tween 20 (3 μL/mmol phospholipid) as surfactant and of ethanol (15 μL/mL reaction mixture), that ensure clearness of reaction mixture and linear increase of absorbance in the course of reaction. This method was tested on a purified secretory PLA2 from honey bee venom (HBV-PLA2). The enzyme did not discriminate among PCLIN, phosphatidylcholine, and phosphatidylethanolamine, but showed the highest rate using 1,2- dilinoleoyl-sn-glycero-3-phosphatidylcholine (PCDILIN). Nevertheless, the use of PCDILIN is not recommended, as it may induce an overestimation of enzyme activity, because not only the free linoleate, but also the reaction product 1-linoleoyl-lysophosphatidylcholine, are known to be oxidized by LOX. HBV-PLA2 showed maximal activity at pH 9.0, hyperbolic kinetics (Km, 74.22.9 μM; Vmax, 8277 μmol/min/mg protein) and competitive inhibition (Ki about 5 μM) by palmityl trifluoromethyl ketone, a classical PLA2 inhibitor. Interestingly, the HBV-PLA2/soybean LOX-1 coupled reactions also allow an accurate assay of PCLIN concentration. In the whole, these results demonstrate that this improved PLA2/LOX assay allows a very accurate, simple, and rapid measurement of enzyme activity and substrate concentration.

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 Sch¨ubler) 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 (AAH+L+FSP), containing freely solvent-soluble antioxidants, and AA values of IBP extracts (AAIBP), representing the phenolic fraction ester-linked to insoluble cell wall polymers. The LOX/RNO and ORAC methods measured in quinoa flour a remarkable AAH+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 AAH+L+FSP for quinoa. Interestingly, the ratio AAH+L+FSP/AAH+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.

Assessment of Antioxidant Capacity (AC) of foods is useful to consider cumulative/synergistic action of all dietary antioxidants, thus providing a more integrated information than the simple sum of measurable antioxidants. Among the different AC assays, the QUENCHERABTS (QUick, Easy, New, CHEap and Reproducible)procedure is based on the direct reaction of ABTS•+reagent with fine solid food particles without extraction of antioxidants. Thisassay is able to measure both soluble and insoluble antioxidants, that simultaneously come into contact with ABTS•+ molecules byeither liquid-liquid or solid-liquid interactions,respectively.These interactions may change depending on the particle diameter. Usually, particles having 0.1-0.3 mm size are used. Here, AC was evaluated on whole flour (WF),derived from a mix of grains of ten durum wheatvarieties, characterized by three different particle sizes:a smaller one, <0.2 mm (control, WF0.2), and two larger ones,<0.5 mm and <1 mm (WF0.5 and WF1, respectively). Moreover, a novel AC calculation procedure based on the slope value of the regression line of ABTS•+ response vs flour amount is presented in detail. The classical QUENCHERABTS procedure provided for WF0.2 an AC value of 42.0±2.7 mmol eq. Trolox/g d.w. A similar result was obtained for WF0.5 (38.3±0.9 mmol eq. Trolox/g d.w.), thus indicating that these large particles may be analyzed by the QUENCHERABTS assay provided that the “slope” calculation procedure is used.On the contrary, WF1 showed about half AC (20.3±0.2mmol eq. Trolox/g d.w.), thus showing that very large particles cannot be used even adopting the “slope” calculation.

Peach (Prunus persica L.) fruits contain several health-promoting phytochemicals. Among these, carotenoids, in addition to being involved in determining flesh color, play a relevant role in cell protection against oxidative stress. Nevertheless, antioxidant activity (AA) of peach carotenoids so far has not been investigated in as much detail as phenols. In the present study, for the first time, AA of peach carotenoid extracts was evaluated using the innovative lipoxygenase/4- nitroso-N,N-dimethylaniline (LOX/RNO) method, able to simultaneously detect different antioxidant mechanisms and synergistic antioxidant interactions, as well as using the well-known ORAC and TEAC assays. In particular, extracts were obtained from fruits collected in S4 development stage from two yellow-fleshed (Armking and Redhaven) and three white-fleshed (Silverking, Caldesi 2000, IFF331) varieties. The LOX/RNO method gave high AA values (10-150 μmol eq. Trolox/g f.w.), about 85-1900-fold higher than ORAC and TEAC methods. Moreover, the ratio between AA values, measured by the LOX/RNO method, of yellow- and white-fleshed peaches resulted equal to 14, but only 2.6 and 3.6 for ORAC and TEAC, respectively. Results of this study indicate that the LOX/RNO method, measuring high AA values and easily discriminating among samples, is an advisable tool to assess the AA of the carotenoid component in peach.

Antioxidant activity (AA) of durum wheat (Triticum durum Desf.) grains was studied using the innovative LOX/RNO method, able to simultaneously detect different antioxidant mechanisms, and the TEAC assay, one of the most widely used assays. Insoluble-bound and free-soluble phenols, hydrophilic and lipophilic compounds were extracted from eight different whole flour samples; extracts were analyzed for AA and their content in several antioxidants. The LOX/RNO method measured very high AA values, with the highest ones [850-1500 μmol Trolox eq./g whole flour (dry weight)] for insoluble-bound phenolic extracts, highly correlated to total phenolic (r = 0.761, P < 0.001) and ferulic acid (r = 0.816, P < 0.001) contents. Hydrophilic and lipophilic extracts showed lower AA [70-140 and 40-60 μmol Trolox eq./g (dry weight), respectively], highly correlated to flavonoid (r = 0.583, P < 0.01) and protein (r = 0.602, P < 0.01), as well as β-tocotrienol (r = 0.684, P < 0.05) contents, respectively. Interestingly, the LOX/RNO method suggests that insoluble-bound phenolic compounds may exert very strong synergistic interactions within the extract. Contrarily, the TEAC assay did not correlate to any antioxidant content, resulted unable to highlight differences among samples, measured much lower AA values and did not suggest synergism. The use of the LOX/RNO method is useful to unearth new properties of phytochemicals from durum wheat grains, potentially giving health benefits. © 2012 Elsevier Ltd.

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.

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 (PmitoK(ATP)). Since then, both properties of the original PmitoK(ATP) 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 PmitoK(ATP) 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. PmitoK(ATP) 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 (Delta Psi), the main component of the protonmotive force (Delta p) in plant mitochondria; moreover, cooperation between PmitoK(ATP) and the K+/H+ antiporter allows a potassium cycle able to dissipate also Delta pH. Interestingly, Delta Psi 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, Delta Psi collapse by PmitoK(ATP) 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 PmitoK(ATP); this brake to the channel activity may allow a loss of the bulk phase Delta p, 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), PmitoK(ATP) was found to be activated by ROS, so inhibiting further large-scale ROS production according to a feedback mechanism; moreover, a stress-activated phospholipase A(2) may generate FFAs, further activating the channel. In conclusion, a main property of PmitoK(ATP) 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.

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. The manuscript deals with the effect of the mitochondrial ATP-sensitive potassium channel (PmitoKATP) activity on the oxidative phosphorylation (OXPHOS) in durum wheat. The PmitoKATP may collapse membrane potential (ΔY) in such a manner to dampen mitochondrial ROS production. So, the channel may act as a mitochondrial and cell defence mechanism under environmental/oxidative stress condition. Interestingly, ΔY collapse may occur without affecting mitochondrial ATP synthesis via OXPHOS in mitochondria from seedlings grown under both control and moderate hyperosmotic stress condition, while under severe hyperosmotic stress OXPHOS is impaired. Results suggest a major role of the channel at the early phase of the stress when it may control ROS generation without affecting ATP synthesis. © 2011 Blackwell Publishing Ltd.

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 activity of mitochondrial phospholipase A2 (PLA2) 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 PLA2/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 PLA2 (DWM-PLA2) showed maximal activity at pH 9.0 and 1mM Ca2+, hyperbolic kinetics (Km=90±6μM, Vmax=29±1nmolmin-1mg-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-PLA2, 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 Ca2+-independent activity, having optimum at pH 7.0, was stress-insensitive. We propose that the activation of DWM-PLA2 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.

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.

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.