Maura N. Laus

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.

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.

The existence of phospholipase A2 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.

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.

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.

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.

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.

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 +/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. [BMB Reports 2013; 46(8): 391-397]

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.

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.