activation of the plant mitochondrial potassium channel by free fatty

Journal of Experimental Botany, Vol. 62, No. 1, pp. 141–154, 2011doi:10.1093/jxb/erq256 Advance Access publication 27 August, 2010

RESEARCH PAPER

Activation of the plant mitochondrial potassium channel byfree fatty acids and acyl-CoA esters: a possible defencemechanism in the response to hyperosmotic stress

Maura N. Laus1,2, Mario Soccio1,2, Daniela Trono3, Maria T. Liberatore2 and Donato Pastore1,2,*

1 Dipartimento di Scienze Agro-ambientali, Chimica e Difesa Vegetale, Facolta di Agraria, Universita degli Studi di Foggia, Via Napoli,25-71122 Foggia, Italy2 Centro di Ricerca Interdipartimentale BIOAGROMED, Universita degli Studi di Foggia, Via Napoli, 52-71122 Foggia, Italy3 C.R.A.—Centro di Ricerca per la Cerealicoltura, S.S. 16, Km 675-71122 Foggia, Italy

* To whom correspondence should be addressed: E-mail: [email protected]

Received 7 May 2010; Revised 16 July 2010; Accepted 26 July 2010

Abstract

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 mM) 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 differentexperimental 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 mg21 protein, which was able to activate PmitoKATP strongly, was observed under

hyperosmotic stress conditions. Since PmitoKATP may act against environmental/oxidative stress, its activation byFFAs/acyl-CoAs is proposed to represent a physiological defence mechanism.

Key words: Acyl-CoA esters, durum wheat mitochondria, electrical membrane potential, free fatty acids, mitochondrial swelling,

osmotic stress, plant mitochondria, potassium channel, salt stress.

Introduction

Plant mitochondria possess protein-mediated K+ uptake

systems, the first of which, an ATP-sensitive K+ channel,

was discovered about ten years ago in durum wheat

mitochondria (DWM) (Pastore et al., 1999a). This channel

has been named PmitoKATP in analogy with the animal

counterpart, mitoKATP (Garlid, 1996). Other ATP-sensitive

Abbreviations: Dp, proton motive force; DW, electrical membrane potential; AOX, Alternative Oxidase; CsA, cyclosporin A; DWM, durum wheat mitochondria; EU,enzymatic units (lmol of substrate transformed min�1); FCCP, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone; FFAs, free fatty acids; MPT, mitochondrialpermeability transition; NCPT, non-classical CsA-insensitive permeability transition; PCD, programmed cell death; (P)IMAC, (Plant) Inner Membrane Anion Channel;PLA2, phospholipase A2; (P)mitoKATP, (plant) mitochondrial ATP-sensitive potassium channel; (P)UCP, (plant) uncoupling protein; PVP, polyvinylpyrrolidone; ROS,reactive oxygen species; SPE, solid-phase extraction; TEA+, tetraethylammonium; TRIS, tris-(hydroxymethyl)-aminomethane.ª The Author [2010]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.For Permissions, please e-mail: [email protected]

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K+ import pathways have been described in mitochondria

from etiolated pea stems (Petrussa et al., 2001, 2004;

Chiandussi et al., 2002; Casolo et al., 2003), from soybean

cell cultures (Casolo et al., 2005), from embryogenic

cultures of Picea abies (L.) Karst., Abies cephalonica Loud

(Petrussa et al., 2008a), Abies alba Mill. (Petrussa et al.,

2009), and from Arum spadix and tubers (Petrussa et al.,

2008b). The existence of an ATP-insensitive quinine-inhibited K+ transporter has been reported in mitochondria

from potato tubers, etiolated seedlings of maize, and

tomato fruits (Ruy et al., 2004). A large-conductance

calcium-activated K+ channel has also been described

recently in potato tuber mitochondria (Koszela-Piotrowska

et al., 2009). K+ uptake has also been shown in mitochon-

dria from etiolated seedlings of bread wheat, barley, spelt,

rye, from green leaves of spinach, from potato tubers(Pastore et al., 1999a) and from potato cell cultures

(Fratianni et al., 2001).

Among mitochondrial K+ channels, PmitoKATP shows

intriguing effects on the bioenergetics of isolated mitochon-

dria suspended in media containing K+. This channel

catalyses the electrophoretic uniport of K+ across the inner

mitochondrial membrane towards the matrix. The co-

operation between PmitoKATP and the K+/H+ exchanger,very active in plant mitochondria (Diolez and Moreau,

1985), allows the operation of a K+ cycle that may induce

H+ re-entry in the mitochondrial matrix. Whereas, in

mammals, the K+ cycle cannot uncouple mitochondria, the

maximal rate of this cycle being only about 20% of that of

the proton-ejection of the respiratory chain (Pastore et al.,

1999a), in plant mitochondria the activity of this K+

pathway is expected to be approximately equal to theproton-ejecting capacity. In fact, the K+ cycle flux is

sufficient to collapse the proton motive force (Dp) of

isolated mitochondria completely (Pastore et al., 1999a,

2007), by dissipating, in particular, the electrical membrane

potential (DW), the main component of Dp in plant

mitochondria (Douce, 1985, and references therein). By

lowering DW (and Dp), PmitoKATP may dampen the

generation of reactive oxygen species (ROS) in mitochon-dria purified in vitro (Pastore et al., 1999a, 2007). This is

explained according to the ‘mild uncoupling’ theory pro-

posed by Skulachev (Skulachev, 1998). Briefly, the occur-

rence of a deep positive relationship between DW and ROS

production by the respiratory chain has been well estab-

lished (in particular, the semiquinone CoQH. is involved in

the non-enzymatic one-electron reduction of oxygen to the

superoxide anion, that may generate other ROS): high DWvalues (over 140 mV) may induce a dramatic stimulation of

superoxide production; accordingly a small DW decrease,

below the critical level, may also markedly decrease

superoxide generation. This means that some increase in

the proton leak may prevent a burst of superoxide pro-

duction. In light of this, regulatory mechanisms able to

control DW levels, such as H+ re-entry generated by the K+

cycle due to the PmitoKATP-K+/H+ antiporter combined

function, as well as the free fatty acid (FFA)-induced H+

transport by the Uncoupling Proteins (UCPs), may decrease

ROS formation in mitochondria isolated in vitro. The

capability of PmitoKATP to control mitochondrial ROS

generation has been demonstrated in DWM from both

control (Pastore et al., 1999a) and salt- and osmotically

stressed seedlings (Trono et al., 2004). Therefore, Pmito-

KATP has been suggested to co-operate with the cellular

antioxidant systems and with the other plant mitochondrial

energy-dissipating systems, i.e. the PUCP (Pastore et al.,2000) and the Alternative Oxidase (AOX) (Pastore et al.,

2001), to defend the cell from oxidative stress occurring

when plants suffer environmental stresses (Pastore et al.,

2007).

In plant cells, the K+ cytosol concentration is homeostati-

cally maintained at very high levels (80–100 mM) (Leigh

and Wyn Jones, 1984) by the activity of K+ influx and efflux

channels (for the regulation mechanisms see Lebaudy et al.,2007; Szczerba et al., 2009). Since, in DWM, a K+ content

ranging from 50–100 mM, similar to that of cytosol, was

measured by means of atomic absorption technique (D

Pastore et al., unpublished data), the electrophoretic K+

uptake via a fully opened PmitoKATP is mainly driven by

high DW (ranging from 170–200 mV in different DWM

preparations; Trono et al., 2004). This transport is active

enough to dissipate Dp completely, with the possible im-pairment of mitochondrial functions. Therefore, a careful

and fine modulation of PmitoKATP activity is expected to

occur in vivo. In light of this, studies of PmitoKATP

regulation to characterize physiological modulators appear

to be necessary to understand the in vivo function of this

channel fully, in particular, under environmental/oxidative

stress conditions. PmitoKATP is known to be modulated by

physiological compounds, including the inhibitors ATP andNADH and the activators GTP, palmitoyl-CoA, and CoA;

PmitoKATP is also activated as a consequence of mitochon-

dria treatment with the superoxide anion (Pastore et al.,

1999a; 2007). In particular, with respect to ROS and ATP

modulation, a feedback mechanism has been depicted under

hyperosmotic/oxidative stress conditions: the increased ROS

generation (Trono et al., 2004) and the inhibited ATP

synthesis (Flagella et al., 2006) may elicit PmitoKATP

activity, which, in turn, may control large-scale ROS

production (Trono et al., 2004; Pastore et al., 2007). For

a recent review about ROS homeostasis and signalling

during drought and salinity stresses see Miller et al. (2010).

In accordance with the involvement of the ROS-activated

PmitoKATP in the environmental stress response, it should

also be underlined that ROS can activate plasma membrane

non-selective cation influx channels in plant cells, as well asK+ efflux channels, both playing a critical role in ROS-

mediated plant regulatory reactions, including the plant cell

response to stress (Demidchik and Maathuis, 2007; Demid-

chik et al., 2010).

In this study, the possibility of a PmitoKATP modulation

by FFAs and their acyl-CoA esters derivatives has been

evaluated. This is in light of the ability of palmitoyl-CoA to

activate PmitoKATP strongly (Pastore et al., 1999a) and ofFFAs to activate or inhibit other mitochondrial dissipative

systems, such as (P)UCP (Vercesi et al., 2006; Echtay, 2007)

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and AOX (Sluse et al., 1998), respectively, as well as to

modulate the Plant Inner Membrane Anion Channel

(PIMAC) (Laus et al., 2008) and the mammalian mitochon-

drial K+ channel (Schonfeld et al., 2003).

Our results show that, in DWM, FFAs and acyl-CoAs

activate K+ transport; by enlarging the study to mitochon-

dria from different species and organs, this effect has been

found to be widespread in plants. Under hyperosmotic(NaCl or mannitol) stress conditions, endogenous FFAs

have been found to increase up to a content able to activate

PmitoKATP strongly. This suggests that activation may have

a role to play in environmental/oxidative stress because of

the crucial role of plant mitochondria in orchestrating

drought tolerance (Atkin and Macherel, 2009).

Materials and methods

Chemicals and plant material

All chemicals (at the highest commercially available purity) werepurchased from Sigma Chemical Co. (St Louis, MO, USA).Substrates were used as Tris-(hydroxymethyl)-aminomethane(TRIS) salts at pH 7.20. Carbonylcyanide p-(trifluoromethoxy)-phenylhydrazone (FCCP), valinomycin, and FFAs were dissolvedin ethanol.Certified seeds of durum wheat (Triticum durum Desf., cv.

Ofanto), bread wheat (Triticum aestivum L.), barley (Hordeumvulgare L.), triticale (Triticum3secale), maize (Zea mays L.),and tubers of topinambur (Jerusalem artichoke: Helianthustuberosus L.) were kindly supplied from the Cereal ResearchCentre (Foggia, Italy). Certified seeds of pea (Pisum sativum L.)and lentil (Lens esculenta M.) were purchased from an agricul-tural pharmacy.

Seedling growth

Durum wheat seeds were germinated in deionized water for 3 d indarkness, at 25 �C and 80–85% RH, as reported in Pastore et al.(1999b). Severe salt- and osmotic-stressed seedlings of durumwheat were obtained as described in Trono et al. (2004) and Soccioet al. (2010), by substituting water with 0.21 M NaCl solution(conductivity 20 dS m�1; Wp¼ –1.04 MPa) and 0.42 M mannitolsolution (having the same osmolarity as the NaCl solution),respectively.Bread wheat, barley, triticale, pea, and lentil seeds were grown

essentially as described for durum wheat seedlings in darkness, at25 �C and 80–85% RH, for times ranging from 2–3 d, as needed toreach the same shoot length as durum wheat. To obtain greenleaves, durum wheat and maize seeds were grown at 25 �C and 80–85% RH, for 4 d and 6 d, respectively (comprising 24 h and 60 h oflight exposure, respectively).

Mitochondria isolation

DWM were purified from 3-d-old etiolated shoots, as previouslyreported in Pastore et al. (1999b) with minor modifications. Thegrinding and washing buffers were: (i) 0.5 M sucrose, 4 mMcysteine, 1 mM EDTA, 30 mM TRIS-HCl (pH 7.5), 0.1% (w/v)defatted BSA, 0.6% (w/v) polyvinylpyrrolidone (PVP)-360; and (ii)0.5 M sucrose, 1 mM EDTA, 10 mM TRIS-HCl (pH 7.4), 0.1%(w/v) defatted BSA, respectively. Washed mitochondria werepurified by isopycnic centrifugation in a self-generating densitygradient containing 0.5 M sucrose, 10 mM TRIS-HCl (pH 7.2) and28% (v/v) Percoll (colloidal PVP-coated silica) in combination witha linear gradient of 0% (top) to 10% (bottom) PVP-40 (Moore andProudlove, 1987). The final mitochondrial suspension was diluted

with an appropriate volume of a sucrose-free washing buffer inorder to obtain a 0.3 M sucrose concentration. This protocol givesmitochondria with high intactness of inner and outer membranesand well-coupled (Pastore et al., 1999b).Mitochondria from salt- and osmotic-stressed seedlings of

durum wheat were purified as reported in Trono et al. (2004) andSoccio et al. (2010); they also showed high membrane intactnessand a sufficient functionality to study mitochondrial bioenergetics(Trono et al., 2004; Soccio et al., 2010).Mitochondria from etiolated shoots of bread wheat, barley,

triticale, and lentil and from pea roots were obtained essentially asabove reported for durum wheat. With respect to mitochondriafrom green leaves of durum wheat and maize, homogenization wascarried out by using a Waring blender homogenizer (speed setting333 s, three times); purification was performed as described forDWM, with the only exception that, in order to preventchlorophyll contamination, washed mitochondria were passed twoconsecutive times through the Percoll gradient. Mitochondria fromJerusalem artichoke (topinambur) tubers were isolated essentiallyas in Liden and Moller (1988) from fresh cut slices of fully maturetubers in the quiescent state, and stored at 4 �C for no more thanthree months. The grinding and washing buffers were: (i) 0.4 Msucrose, 1 mM EDTA, 30 mM TRIS-HCl (pH 8.2), 0.1% (w/v)defatted BSA, 0.05% (w/v) cysteine; and (ii) 0.3 M sucrose, 1 mMEDTA, 10 mM Tris-HCl (pH 7.4), 0.1% (w/v) defatted BSA,0.05% (w/v) cysteine, respectively. Mitochondria obtained fromdifferent plant species and organs showed good membrane in-tactness and functionality, as indicated by the capability togenerate high DW due to succinate oxidation and by the failure ofswelling in sucrose medium. Mitochondrial preparations lackingthese requisites were discarded.Mitochondrial protein content was determined by the method of

Lowry modified according to Harris (1987), using BSA asa standard.

Swelling experiments

Swelling experiments were performed as described by Pastore et al.(1999a). Absorbance changes at 546 nm of a mitochondrialsuspension (0.05 mg ml�1 protein) in 0.36 M sucrose, 0.18 M KClor 0.18 M tetraethylammonium chloride (TEACl) iso-osmoticsolutions (2 ml), buffered with 20 mM TRIS-HCl (pH 7.2), weremonitored at 25 �C as a function of time, by a PerkinElmerLambda 18 UV/Vis spectrophotometer (PerkinElmer, Wellesley,MA, USA). Mitochondrial swelling in iso-osmotic KCl solutiondepends on both K+ and Cl– influxes, that occur at different rates;in the light of this, the rate of the global process (swelling) reflectsthe rate of the slowest one. The rate-limiting step of the swelling inKCl is K+ rather Cl– uptake, as demonstrated by the increase inthe swelling rate induced by the addition of the K+ ionophorevalinomycin, which strongly increases K+ permeability across theinner mitochondrial membrane (see, for example, Fig. 1A). In thelight of this, swelling in KCl solution represents a useful tool tostudy K+ uptake, while swelling in KCl medium in the presence ofvalinomycin may allow Cl– transport to be measured.Swelling rate was expressed as DA546nm min-1 mg�1 protein and

calculated as the slope relative to the initial part of theexperimental trace. Swelling rate in KCl medium in the presenceof acyl-CoA esters was calculated as the highest slope of theexperimental curve, occurring about 15 s after the beginning of theswelling.

Fluorimetric measurements of DW changes

The fluorescent probe safranin O was used to estimate DWchanges, as reported by Moore and Bonner (1982). The fluores-cence intensity changes of safranin O were recorded at 25 �C, usingexcitation and emission wavelengths of 520 nm and 570 nm,respectively, by means of a PerkinElmer LS-50B spectrofluorime-ter. The incubation medium (2 ml) contained 0.3 M mannitol,

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5 mM MgCl2, 20 mM TRIS-HCl (pH 7.2), 2.5 lM safranin O, and0.1 mg ml�1 DWM protein [(safranin O)/(DWM protein) ratiovalue of 25]. The reaction was started by the addition of 5 mMsuccinate, as a respiratory substrate.Calibration of safranin O fluorescence changes as a function

of K+ diffusion potentials was performed using rat livermitochondria, as reported by Zottini et al. (1993). Rat livermitochondria were isolated according to Pastore et al. (1994);the K+ diffusion potential in rat liver mitochondria was inducedby the addition of 0.05 lg ml�1 valinomycin (Akerman andWikstrom, 1976).

Determination of FFA content in DWM from control and stressed

seedlings

Extraction of lipids and separation of FFAs: Total lipids wereextracted from mitochondrial preparations obtained from bothcontrol and severe salt- and osmotic-stressed seedlings. In brief,0.8 ml of mitochondrial suspension (1 mg ml�1 protein) wereadded together with 0.6 ml of diethyl ether-hexane 1:1 (v/v), towhich a known amount of heptadecanoic acid (17:0) had pre-viously been added as the internal standard; the resulting mixturewas vigorously shaken and subsequently centrifuged at 1700 g for10 min. The organic phase was collected and the extraction wasrepeated another three times. The combined organic phases werediluted with hexane to a final volume of 5 ml. The separation ofFFAs from bound FAs present in lipidic mitochondrial extractwas achieved by the solid-phase extraction (SPE) procedure. Theextraction was performed according to Gambacorta et al. (2009),with minor modifications. The sample obtained was evaporated todryness under a nitrogen stream.

Quantification of FFAs: Quantitative analysis of palmitate, linole-ate, and linolenate in the fraction obtained from SPE was carriedout by means of gas chromatography coupled with flame ionizationdetection. The FFA dry residue was subjected to acid-catalysedmethylation; the methylated FAs were extracted with 100 ll ofhexane, centrifuged at 1000 g for 1 min, and the supernatant wascollected. The sample (2 ll) was analysed by Carlo Erba HRGC5300 mega series chromatograph equipped with a SUPELCO-WAX10 capillary column (30 m30.25 mm i.d. 30.25 lm filmthickness, Supelco Inc, Bellerofonte, PA). Methyl esters wereidentified and quantified by a comparison of their retention timesand areas with that of known standards.Linoleate content in the SPE fraction was also confirmed by

means of gas chromatography coupled to mass spectrometry.In this case, the FFA dry residues were reconstituted in 10 ll ofdiethyl ether and 2 ll of the sample was injected into the gaschromatographic system. A 6890N series gas chromatograph(Agilent Technologies) with an Agilent 5973 mass selectivedetector and equipped with a ZB-FFAP capillary column(30 m30.25 mm i.d.30.25 lm film thikness, Restek, Bellerofonte,PA) was used.

Results

In order to gain a first insight into the effect of FFAs on K+

permeability in purified DWM, K+ uptake was studied by

means of swelling experiments in iso-osmotic KCl solutions.

As shown in Fig. 1A, DWM absorbance in 0.36 M

sucrose solution was found to remain constant during thetime, thus demonstrating the intactness of mitochondrial

membranes. No swelling in the sucrose solution was

observed after the addition of either 10 lM linoleate or

linoleoyl-CoA. The ineffectiveness of linoleate and linoleoyl-

CoA excludes damage to the inner membrane under the

adopted experimental conditions as a consequence of de-

tergent action. Moreover, a control was made that linoleate

did not promote aspecific permeabilization processes attrib-

utable to the cyclosporin A (CsA)-sensitive mitochondrial

permeability transition (MPT) (for a review see Halestrap,

2009) or to the non-classical CsA-insensitive permeability

transition (NCPT) (Sultan and Sokolove, 2001), evaluated

Fig. 1. Effect of linoleate and linoleoyl-CoA on DWM swelling in

iso-osmotic solution of KCl, of KCl plus valinomycin, and of TEACl.

(A) DWM (0.1 mg protein) were suspended in 2.0 ml of a medium

consisting of 20 mM TRIS-HCl pH 7.2 and 0.36 M sucrose or

0.18 M KCl or in KCl medium supplemented with: 10 lM linoleate

(Lin); 10 lM linoleoyl-CoA (Lin-CoA); 10 lM Lin plus 0.1% (w/v)

BSA; 10 lM Lin-CoA plus 0.1% (w/v) BSA; 0.5 lg valinomycin

(Val). At the time indicated by the arrows 10 lM Lin or Lin-CoA

and 0.5 lg Val were added. Swelling was continuously monitored

by measuring the absorbance decrease at 546 nm and 25 �C, as

reported in the Materials and methods. (B) Swelling rates in 0.36 M

sucrose, 0.18 M TEACl, 0.18 M KCl, and 0.18 M KCl plus 0.5 lg

Val either in the absence or presence of Lin or Lin-CoA are

reported as a function of Lin (full symbol) or Lin-CoA (empty

symbol) concentration. Data are expressed as mean

value 6standard deviation (n¼3).

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essentially as reported in Schonfeld and Bohnensack (1997)

and Sultan and Sokolove (2001), respectively (data not

shown).

On the other hand, DWM exhibited a fast and clearly

evident swelling in iso-osmotic KCl solution, depending on

K+ and Cl– uptake mediated by PmitoKATP (Pastore et al.,

1999a) and PIMAC (Laus et al., 2008), respectively.

Swelling rate in KCl was strongly increased by the K+

ionophore valinomycin, either added in the course of the

swelling or already present in the reaction medium, thus

indicating that the swelling rate in KCl solution is a measure

of the rate of K+ uptake, while the swelling rate in KCl plus

valinomycin reflects the rate of Cl– uptake (see also the

Materials and methods). When 10 lM linoleate was added

to KCl medium, a large increase in the rate of K+ uptake of

about 160% with respect to the control was observed,further increased by valinomycin, so indicating K+ uptake

as the rate-limiting step under these conditions as well.

About 55% inhibition of this linoleate-stimulated swelling

rate was induced by the addition of 0.1% BSA, that binds

FFAs. Complete recovery was not obtained in the presence

of higher BSA concentrations [up to 0.5% (w/v)] (data not

shown). Since acyl-CoAs are obligate intermediates of FFA

metabolism, the effect of linoleoyl-CoA on K+ transportwas also evaluated. In the presence of 10 lM linoleoyl-CoA,

a much more evident increase of about 550% of swelling

rate in KCl solution was found, slightly inhibited by BSA,

in accordance with the BSA ability to bind acyl-CoAs

(Richards et al., 1990).

The effect of increasing linoleate and linoleoyl-CoA

concentrations on DWM swelling in sucrose, KCl, and KCl

plus valinomycin was also evaluated (Fig. 1B). Both linoleateand linoleoyl-CoA, up to 30 lM, did not significantly affect

DWM permeability to sucrose. Consistently, under these

experimental conditions, no significant release into the

extramitochondrial phase of the matrix malate dehydroge-

nase was observed (data not shown). By contrast, an

increasing stimulation of K+ uptake (30–285%) was observed

with increasing linoleate concentrations from 2 lM to 20

lM. As expected, in the light of the recently demonstratedFFA-dependent inhibition of anion uptake via PIMAC

(Laus et al., 2008), linoleate also induced a progressive

decrease of the swelling rate in KCl plus valinomycin

(representing the rate of Cl– uptake). As a result of the

combined linoleate capability to activate K+ uptake and

inhibit anion transport, when FFA concentration was higher

than 20 lM, the swelling rates in KCl were the same in both

the absence and presence of valinomycin and representeda measure of Cl– uptake. Increasing concentrations of

linoleoyl-CoA from 1 lM to 20 lM caused an increasing

activation of K+ transport, which resulted at any concentra-

tion higher than the linoleate-dependent one. Since linoleoyl-

CoA does not affect PIMAC activity (Laus et al., 2008),

under these experimental conditions, at 20 lM linoleoyl-CoA

it is possible to observe maximal measurable activation of K+

transport.To unravel the role of PmitoKATP in the activation of

K+ transport by linoleate and linoleoyl-CoA, swelling in

the TEACl medium was studied. This depends on the

uptake of TEA+, a cation not transported by either

mitoKATP (Beavis et al., 1993) or PmitoKATP (Pastore

et al., 1999a) (Fig. 1B). Although no significant increase

in the swelling rate was observed up to about 15 lMlinoleate, an evident stimulation of TEA+ transport was

observed at higher linoleate concentrations. This stimula-

tion was prevented by 0.1% BSA (data not shown).A comparison between swellings in KCl and TEACl

suggests that, at linoleate concentrations higher than

15 lM, the involvement of a ‘valinomycin-like effect’ due

to the formation of cation–FFA complexes (Sharpe et al.,

1994; Zeng et al., 1998) should also be considered to

explain the activation fully. By contrast, the activation of

K+ transport observed up to about 15 lM linoleate

appears only to be PmitoKATP-dependent, the contribu-tion of the ‘valinomycin-like effect’ to K+ permeabiliza-

tion being negligible. Consistently, linoleoyl-CoA, which

is unable to form the cation complex, was found not to

affect TEA+ uptake in the 2.5–15 lM concentration range

(Fig. 1B).

On the whole, the results of Fig. 1 show that FFA

concentrations lower than 15 lM have to be used to

evaluate the effect of FFAs on K+ transport via Pmito-KATP. This is in order to avoid confusion consequent on

both a possible rate-limiting step due to Cl– uptake (see

above) and a ‘valinomycin-like effect’.

Other natural unsaturated and saturated FFAs with

different chain length and their CoA ester derivatives were

evaluated for their capability to activate K+ uptake in

DWM (Fig. 2A, B). To begin with it was determined that

all the FFAs/acyl-CoAs tested did not affect DWM perme-ability to sucrose in a 1–20 lM concentration range. All the

FFAs and acyl-CoAs (10 lM) tested were found to promote

K+ uptake; in all cases the activation due to the acyl-CoA

was much higher, from 3–6 times, than that of the respective

FFA. Two non-natural FFAs, 1-undecanesulphonate and

5-phenylvalerate, were also found to activate the swelling

rate in KCl strongly (Fig. 2B). Since these latter two FFAs

are not transported by PUCP in DWM (Pastore et al.,2000), as well as in other plant mitochondria (Jezek et al.,

1997), our results rule out a possible mechanism of K+

transport activation involving a combined action of PUCP

with a K+/H+ antiporter (see Ruy et al., 2004, and the

Discussion).

In order to confirm the involvement of PmitoKATP in the

FFA-induced stimulation of K+ transport, another method

based on DW measurement using the fluorimetric probesafranin O was used. This method allows PmitoKATP

activity to be evaluated by measuring the decrease in DWdue to the addition of KCl to respiring mitochondria. This

can be done at much lower KCl concentrations with respect

to the swelling experiments (Pastore et al., 1999a), so

allowing the effect of FFAs and acyl-CoAs to be checked

with a completely different experimental condition. To

exclude PUCP activity (Fig. 3B, C), use was made of thenon-natural FFA phenylvalerate in DW experiments. More-

over, the use of this FFA has another advantage: the


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activation of K+ transport (Fig. 3A), but the inability toexhibit a flip-flop movement across membranes (Jezek et al.,

1997) and, as a consequence, the inability to exhibit

a ‘valinomycin-like effect’, as confirmed by the lack of

permeability to TEA+ (Fig. 3A).

The effect of phenylvalerate on DW changes is reported in

Fig. 3B. The reaction was started by adding succinate to

DWM in order to induce a rapid DW increase until about

180 mV. In the absence of phenylvalerate, sequentialadditions of KCl were found progressively to depolarize

mitochondria; ATP restored DW up to about 165 mV. This

confirms that the K+-induced DW decrease depends on K+

entry via PmitoKATP (Pastore et al., 1999a, 2007). It should

be noted that ATP was added together with oligomycin and

atractyloside, inhibitors of ATP synthase and ADP/ATP

translocator, respectively, in order to avoid ATP entry and

DW generation via hydrolysis by ATP synthase. Then, DWwas completely abolished by the addition of valinomycin

and by the uncoupler FCCP. In the other trace in Fig. 3B,

the addition of 10 lM phenylvalerate induced only a very

small depolarization, as expected for a FFA not efficiently

transported by PUCP, while the addition of KCl caused

much faster and larger-amplitude DW decreases compared

with the control experiment, thus confirming the activation

of K+ transport by a FFA, by means of a DW experiment.

Once again, the addition of ATP restored DW, thusconfirming the involvement of PmitoKATP in this phenom-

enon, but, in this case, only a value of about 130 mV DWwas reached. Consistently, when ATP was already present

in the reaction medium (Fig. 3C), K+-induced depolariza-

tion was inhibited less efficiently when the FFA was

present, thus suggesting that FFAs may cause some

activation on the ATP-inhibited channel.

DW experiments were also carried out in order to checkPmitoKATP activation by acyl-CoAs. In Fig. 4 the effect of

palmitoyl-CoA on DWM DW changes is shown; when

palmitoyl-CoA was added to succinate-respiring DWM,

only a negligible DW decrease was observed, thus showing

that it cannot activate PUCP (Fig. 4A); by contrast,

addition of KCl was found to decrease DW to a much

higher rate and extent compared with the control. DW was

restored by externally added ATP up to values of about 155mV and 125 mV in the absence and presence of palmitoyl-

CoA, respectively. Consistently, ATP was able to prevent

a K+-dependent DW decrease more efficiently in control

mitochondria than in DWM treated with palmitoyl-CoA

(Fig. 4B). Similar results were obtained in the presence of

linoleoyl-CoA and arachidonoyl-CoA (data not shown).

Therefore, acyl-CoAs may also cause some activation of the

ATP-inhibited channel.Some experiments were also performed to evaluate the

K+/Na+ selectivity with respect to the FFA/acyl-CoA

effect. To do this, DW experiments were carried out, since

they allow ion concentrations resembling the physiological

ones to be adopted. In particular, the effect on Na+ uptake

of a FFA (phenylvalerate) and an acyl-CoA (palmitoyl-

CoA) was tested by measuring the Na+-induced DWdecrease in succinate-respiring DWM (Fig. 5). As shownin Fig. 5A, sequential additions of 5 mM NaCl did not

affect the DW of succinate-respiring DWM, while, as

expected, DWM were depolarized at a significant rate by

the addition of 15 mM KCl; no Na+-dependent DWdecrease was observed in the presence of phenylvalerate,

that induced, on the contrary, a K+-dependent depolariza-

tion at a higher rate and extent with respect to the control.

Similarly, in the presence of palmitoyl-CoA (Fig. 5B),a slight DW decrease was found due to NaCl additions,

while a very strong K+-dependent DW decrease was

observed with respect to the control. These observations

confirm the selectivity of the transport of K+ with respect

to Na+ by FFAs/acyl-CoAs.

In order to ascertain whether activation of K+ uptake by

FFAs and acyl-CoAs is a property common to other plant

mitochondria, K+ transport was studied by means ofswelling experiments in mitochondria isolated from different

mono and dicotyledonous, C3 and C4 plant species,

Fig. 2. Effect of several FFAs and acyl-CoA esters on the swelling

rate in KCl solution. Swelling experiments were carried out as

reported in Fig. 1A, in 2.0 ml of 0.18 M KCl solution in the absence

and presence of either 10 lM FFAs or 10 lM acyl-CoAs. Swelling

rates are expressed as a percentage of the control. In (A) the effect

of unsaturated FFAs and their acyl-CoA ester derivatives and in (B)

the effect of saturated FFAs and their acyl-CoAs as well as of

5-phenylvalerate and 1-undecanesulphonate are shown.

The statistical analysis was carried out according to the Tukey’s

test (a¼0.01).

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including durum wheat, bread wheat, barley, triticale,

maize, pea, topinambur, and lentil and from different

organs, such as the shoot, leaf, root, and tuber (see the

Materials and methods). All the species/organs investigated

exhibited no swelling in the sucrose solution (data not

shown) and evident swelling in the KCl medium occurring

at different rates (Table 1), but it was always increased by

valinomycin, as well as being promoted by linoleate (datanot shown). In this case, analysis of the effect of FFAs/acyl-

CoAs on plant mitochondrial K+ uptake was extended to

the palmitate/palmitoilCoA and oleate/oleoyl-CoA pairs

(Table 1). In all the species/organs tested, the swelling rate

in KCl was activated by FFAs up to about 4 times with

respect to the control. In all cases, acyl-CoAs induced an

activation of K+ uptake much higher than FFAs, ranging

from 3–11 times compared with the control. In the light ofthese results, activation of K+ transport by FFAs/acyl-CoAs

may be considered a widespread phenomenon in the plant

kingdom, thus suggesting some basic physiological signifi-

cance.

In the light of the role of PmitoKATP against environ-

mental/oxidative stress, the hypothesis of the occurrence

under stress conditions of a PmitoKATP regulation pathway

involving FFAs and their CoA ester derivatives waschecked. As shown in Fig. 6A, mitochondria purified from

severely salt-stressed seedlings, that showed a good in-

tactness as demonstrated by the constancy of the absor-

bance in sucrose solution, exhibited a large amplitude

swelling in KCl solution, which was found to be much

higher than that obtained with DWM from control

seedlings (dotted line), and to be largely prevented by BSA.

This suggests an activation by FFAs. Consistently, a smallamount of commercial phospholipase A2 (PLA2) purified

from bee venom, an enzyme able specifically to hydrolyse

membrane glycerophospholipids at the sn-2 position to

yield FFAs and lysophospholipids, caused an evident BSA-

sensitive increase of the swelling rate in KCl solution of

DWM from control seedlings; under our experimental

conditions, PLA2 did not affect mitochondrial integrity, as

shown by the lack of any effect on sucrose trace (Fig. 6B).To confirm whether and to what extent the mitochondrial

content of FFAs may increase under hyperosmotic stress

conditions, it was studied in NaCl- and mannitol-stressed

DWM (Table 2). The content of all the tested FFAs was

found to rise remarkably, with higher increases under salt

stress with respect to the osmotic one. In particular,

Fig. 3. Effect of 5-phenylvalerate on the swelling in KCl and TEACl

(A) and on the K+-induced DW decrease in succinate-respiring

DWM either in the absence (B) or presence (C) of ATP. (A) Swelling

experiments were carried out as reported in Fig. 1A, in 2.0 ml of

0.18 M KCl or 0.18 M TEACl in the presence and absence of

10 lM phenylvalerate. DWM absorbance in sucrose and the

swelling in KCl in the presence of valinomycin (Val) are also

reported. (B) Fluorimetric measurements of DW changes were

carried out as reported in the Materials and methods. DWM

(0.2 mg protein) were suspended in 2.0 ml of a medium consisting

of 20 mM TRIS-HCl pH 7.2, 0.3 M mannitol, 5 mM MgCl2, 2.5 lM

safranin O, with the safranin O fluorescence continuously moni-

tored as a function of time. At the time indicated by the arrows

succinate, phenylvalerate, KCl, ATP (together with 2 lg oligomycin

and 10 lM atractyloside), Val and FCCP were added at the

reported concentrations. In (C) 0.5 mM ATP plus 10 lM atractylo-

side and 2 lg oligomycin were already present in the reaction

medium.


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linoleate and palmitate, the most abundant and the most

active FFA, respectively, were found to undergo about 30-

fold and 26-fold increases with respect to the control undersalt stress and about 27-fold and 18-fold increases under

osmotic stress, respectively. The total content of FFAs was

found to rise, as a result of stress imposition, from about

2 nmol mg�1 protein to about 50 nmol mg�1 protein. The

possible occurrence of a significant increase of PmitoKATP

activity within this concentration range was evaluated. This

was done by using palmitate and palmitoyl-CoA as the

FFA/acyl-CoA pair. As expected, an increase in swellingrate with increasing either palmitate or palmitoyl-CoA

concentrations was observed, with a much higher activation

in the case of the acyl-CoA (Fig. 7). Interestingly, when lowincreasing concentrations (20–50 nmol mg�1 protein or 1–

2.5 lM) of palmitate were added to KCl medium already

containing palmitoyl-CoA at a concentration (2.5 lM)

resembling the physiological one (Larson and Graham,

2001), a synergistic action was observed with a very strong

activation of K+ transport.

In the whole, these results are in accordance with the

hypothesis of PmitoKATP activation as a result of FFA releasefrom mitochondrial membrane phospholipids under stress.

Fig. 5. Enhancement of DW decrease in succinate-respiring DWM

due to phenylvalerate (A) and palmitoyl-CoA (B): effect of Na+

versus K+. DW changes were monitored as reported in Fig. 3B.

At the time indicated by the arrows succinate, phenylvalerate (A) or

palmitoyl-CoA (Palm-CoA) (B), NaCl, KCl, valinomycin (Val), and

FCCP were added at the reported concentrations.

Fig. 4. Effect of palmitoyl-CoA on the K+-induced DW decrease in

succinate-respiring DWM either in the absence (A) or presence (B) of

ATP. (A) DW changes were monitored as reported in Fig. 3B and C.

At the time indicated by the arrows succinate, palmitoyl-CoA

(Palm-CoA), KCl, ATP (together with 2 lg oligomycin and 10 lM

atractyloside), valinomycin (Val), and FCCP were added at the

reported concentrations. In (B) 0.5 mM ATP plus 10 lM atractyloside

and 2 lg oligomycin were already present in the reaction medium.

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Discussion

In this study, the property of FFAs/acyl-CoAs to enhance

K+ permeability of DWM and plant mitochondria from

different species/organs has been shown to involve the

PmitoKATP activation. Under our experimental conditions

(1–10 lM FFAs/acyl-CoAs), the occurrence of an impair-

ment to the inner membrane due to FFA/acyl-CoA de-tergent action was ruled out, and the contribution of FFA

ionophoric activity was shown to be negligible. The in-

volvement of MPT- or NCPT-like processes was also

excluded in the light of (i) the absence of DWM swelling in

sucrose medium in the presence of linoleate and the classical

MPT/NCPT inducers, Pi and Ca2+ (data not shown) and

(ii) the FFA-stimulated K+ uptake in the absence of Pi and

Ca2+, as well as in the presence of CsA and the chelatingagent EGTA (data not shown). Moreover, the hypothesis of

a PUCP involvement was rejected considering the effective-

ness in promoting K+ transport of phenylvalerate and

undecanesulphonate, FFAs not transported by PUCP in

DWM (Pastore et al., 2000). These observations also

exclude the operation in DWM of the mechanism proposed

by Ruy et al. (2004) to explain the sensitivity to BSA

and ATP of the swelling of potato mitochondria in KCl.This is based on the occurrence of an ATP-insensitive K+

transporter and an ATP-sensitive and FFA-promoted

K+ pathway involving (i) K+ uptake through the K+/H+

antiporter in co-operation with (ii) PUCP-mediated H+

cycling.

The direct involvement of PmitoKATP in the activation of

K+ transport by FFAs was suggested by swelling experi-

ments with phenylvalerate, unable to activate PUCP and toexhibit a flip-flop movement across membranes and, as

a consequence, an ionophoric ‘valinomycin-like’ activity.

Phenylvalerate, in fact, remarkably enhanced K+ transport,

but not the PmitoKATP-independent TEA+ uptake. The

activation of PmitoKATP by FFAs/acyl-CoAs was clearly

demonstrated in energized DWM by means of fluorimetric

measurements of DW changes using safranin O as the probe.

Phenylvalerate and palmitoyl-CoA were found to stimulate

depolarization, induced by the addition of low KCl (but not

NaCl) concentrations to succinate-respiring DWM. This

stimulation was partially recovered/prevented by the Pmito-

KATP inhibitor ATP, so indicating that FFAs/acyl-CoAs are

activators of PmitoKATP able to modulate ATP sensitivity.

The capability of FFAs/acyl-CoAs to promote K+ uptakeappears to be a widespread property in the plant kingdom,

but this is not surprising. In many different mammalian

tissues a role of FFAs in the modulation of different plasma

membrane K+ channels, including also the ATP-regulated

channels, has been reported (Kim and Pleumsamran, 2000;

Liu et al., 2001; Zhao et al., 2008). Moreover, it should also

be considered that the interactions of voltage-sensing

domains of voltage-activated plasma membrane K+ chan-

nels with the surrounding lipid membrane have recently

been demonstrated to play a critical role in pore gating

(Swartz, 2008; Krepkiy et al., 2009; Milescu et al., 2009).

With respect to plant cells, the FFA capability both to

activate inwardly rectifying K+ channels and to inhibit

outward K+ channels has been demonstrated in guard cell

plasma membranes (Lee et al., 1994). By contrast, very few

literature data are available about an FFA-dependent

modulation of mitochondrial K+ channels. Long-chain

FFAs have been demonstrated to permeabilize the inner

membrane of rat liver mitochondria to K+ (and Cl–), by

promoting a rapid depletion of mitochondrial Mg2+, that

activates latent Mg2+-sensitive ion-conducting pathways,

including the K+ transporter, the K+/H+ exchanger, and

the IMAC, probably by a direct Mg2+ withdrawal from

protein-binding sites (Schonfeld et al., 2003, 2004, and

references therein). To the best of our knowledge, this is

the first demonstration of an FFA-induced activation of

K+ uniport in plant mitochondria. Unlike rat liver

Table 1. Swelling rate in iso-osmotic KCl solution of mitochondria from different plant species and organs and effect of palmitate, oleate,

palmitoyl-CoA, and oleoyl-CoA

Swelling experiments were carried out as described in Fig. 1A. Mitochondrial proteins (0.1 mg) were suspended in 2 ml of 0.18 M KCl solutioneither in the absence or presence of the listed compounds at the reported concentrations. For each plant species the organs used to obtainmitochondria are indicated.

Monocotyledonous Dicotyledonous

Shoot Leaf Root Tuber Shoot

Durum wheat Bread wheat Barley Triticale Maizea Durum wheat Pea Topinambur Lentil

Swelling rate in 0.18 M KCl (DA546nm min�1 mg�1 protein)

0.3360.01b 0.2860.01 1.5460.09 0.6060.04 0.5460.02 0.6060.05 0.6360.04 0.6960.05 0.5260.01

Swelling rate (% of the control)

+10 lM Palmitate 277626 32664 175627 210614 276618 230621 295620 287626 20268

+10 lM Oleate 287625 430630 138617 19468 296623 185615 179617 21765 173616

+5 lM Palmitoyl-CoA 550654 964636 417611 873663 705627 538652 914619 402617 708611

+5 lM Oleoyl-CoA 667665 1160625 411645 916683 726617 685661 503633 326651 88567

a C4 species.b Mean value 6standard deviation (n¼3).


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mitochondria, in DWM this activation should not involve

an endogenous Mg2+ release, PmitoKATP activity being

Mg2+-insensitive (Pastore et al., 1999a).As for acyl-CoAs, they represent one of the most

important classes of activators of plasma membrane ATP-

regulated K+ channels in pancreatic b-cells (Schulze et al.,

2003; Branstrom et al., 2007, and references therein;

Webster et al., 2008, and references therein) and in cardiac

muscle cells (Liu et al., 2001; Schulze et al., 2003). On the

other hand, in rat liver mitochondria palmitoyl-CoA and

oleoyl-CoA are known to inhibit mitoKATP strongly(Paucek et al., 1996). Unlike rat liver mitochondria, acyl-

CoAs act as powerful activators of PmitoKATP in DWM, as

observed for the plasma membrane KATP channels of

cardiac and pancreatic cells.

Since PmitoKATP was reported to act against environ-

mental/oxidative stress, a possible physiological role of

Fig. 6. Sensitivity to BSA of the swelling in KCl solution of DWM

from salt-stressed seedlings (A) and the effect of phospholipase A2

(PLA2) on the swelling in KCl solution of DWM from control

seedlings (B). (A) Swelling experiments were carried out as

described in Fig. 1A, by using DWM purified from severe salt-

stressed seedlings (see the Materials and methods), in 0.18 M KCl

solution in the absence and presence of 0.1% (w/v) BSA. At the

arrows 0.5 lg valinomycin (Val) was added. DWM absorbance in

sucrose solution and the swelling in KCl solution in the presence of

Val are also reported. For comparison the swelling in KCl of DWM

from control seedlings is shown (dotted line). (B) Swelling experi-

ments were carried out with DWM purified from control seedlings,

in 0.18 M KCl solution and in the same medium supplemented

with: 0.01 enzymatic units (EU) of PLA2 from bee venom

(phosphatidylcholine 2-acylhydrolase, EC 3.1.1.4); 0.01 EU PLA2

plus 0.1% (w/v) BSA. At the arrows 0.5 lg Val was added. DWM

absorbance in sucrose solution either in the absence or presence

of 0.01 EU PLA2 and the swelling in KCl in the presence of Val are

also reported.

Table 2. FFA content in DWM from control and severe salt- and

osmotic-stressed seedlings

FFA content was measured as described in the Materials andmethods.

FFA FFA content (nmol mg�1 protein)

Control Salt stress Osmotic stress

16:0 Palmitate 0.460.1a 10.761.9 7.561.8

18:2D9,12 Linoleate 1.160.2 34.668.7 30.766.6

18:3D9,12,15 or D6,9,12 Linolenate 0.460.1 10.062.2 8.861.7

Total 1.960.4 55.3612.8 47.0610.1

a Mean value 6standard deviation (n¼3).

Fig. 7. Dependence of the swelling rate in KCl solution on the

concentration of palmitate and palmitoyl-CoA present either

separately or together. Swelling experiments were carried out as

reported in Fig. 1A, in 2.0 ml of 0.18 M KCl solution in the absence

and presence of palmitate (black squares) or palmitoyl-CoA (black

circles) at the reported concentrations; swelling rates in KCl

medium in the presence of 2.5 lM palmitoyl-CoA (open squares)

are reported as a function of palmitate concentration. Swelling

rates are expressed as percentage of the control. Data are

expressed as mean value 6standard deviation (n¼3).

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PmitoKATP modulation by FFAs/acyl-CoAs under salt

stress was evaluated. Salinity, in fact, heavily affects in-

tracellular Na+/K+ homeostasis and K+ transporters are

known to play a crucial role in the plant response to salt

stress (Chen et al., 2007, and references therein). In

particular, it was evaluated whether and how PmitoKATP

activity may be related to FFA content in DWM from salt-and osmotically stressed seedlings. Under NaCl and manni-

tol stress applied at germination, an increase in FFA-

dependent PUCP activity was measured in DWM, that was

suggested to depend on an increased availability of endog-

enous FFAs (Trono et al., 2006). At the same time, an

increase of PmitoKATP activity was observed (Trono et al.,

2004; Pastore et al., 2007); here, it appears that this may be

dependent, in part, on an increased content of endogenousFFAs. Consistently, several studies have related changes in

the structural and functional properties of mitochondrial

membranes with the plant’s adaptation to stresses. Mod-

ifications of linolenic acid content of mitochondrial mem-

branes have been observed in soybean seedlings in response

to salicylic acid treatment (Matosa et al., 2009). A higher

ratio between unsaturated and saturated 18-carbon FAs of

mitochondrial membranes and a higher membrane fluiditywere suggested to be involved in chilling tolerance of maize

seedlings (De Santis et al., 1999). A loss of membrane

phospholipids was measured in mitochondria from castor

bean endosperm exposed to hydrogen peroxide treatment

(Kwon et al., 1998). Mitochondria isolated from mild

osmotically stressed wheat shoots showed altered FA and

phospholipid composition and transport processes (Klein

et al., 1986).

The increase of FFAs measured in DWM under severe

salt and osmotic stress conditions was remarkable (up to

about 50 nmol mg�1 protein). The same amount of an

externally added FFA (palmitate) caused a significant 2-fold

activation of PmitoKATP. Interestingly, when the FFA

was added in the presence of a basal concentration of an

acyl-CoA, the activation was very strong: the palmitate/palmitoyl-CoA pair generated a 12-fold activation of the

PmitoKATP when palmitate and palmitoyl-CoA were both

2.5 lM (i.e. 50 nmol mg�1 protein). This PmitoKATP

activation is comparable with that of PUCP (from 7–15-

fold) measured under severe salt and osmotic stress (Trono

et al., 2004). It should be noted that the acyl-CoA basal

concentration (2.5 lM) used in our experiment may be

considered physiological, since it was found to rangebetween about 2.5 lM and 6 lM in different species and

developmental stages (Larson and Graham, 2001).

Now the question arises about how the FFA content may

increase under stress. Interestingly, mammalian mitochon-

dria possess some PLA2 isoforms able to hydrolyse

membrane glycerophospholipids and to release FFAs

(Nakamura et al., 1991; Ghosh et al., 2006; Kinsey et al.,

2007, and references therein); these enzymes may beactivated by ROS (Madesh and Balasubramanian, 1997;

Goto et al., 1999; Guidarelli and Cantoni, 2002) or under

stress conditions, for example, under cerebral (Bonventre

and Koroshetz, 1993; Adibhatla and Hatcher, 2006) and

renal (Nakamura et al., 1991) ischemia-reperfusion, and are

implicated in the pathogenesis of some neurodegenerative

diseases (Sun et al., 2007). To date, no information is

available concerning the occurrence of a PLA2 activity in

Fig. 8. Hypothetical mechanism of PmitoKATP activation under hyperosmotic stress conditions based on the modulation of FFAs/

acylCoAs. Under salt and osmotic stress, in DWM an increase of mitochondrial FFA content occurs, that may activate PmitoKATP;

acylCoAs, derived from FFAs, also strongly activate this channel. PmitoKATP functioning is able to collapse DW, so damping excess ROS

production, according to a feedback mechanism (see text). ROS, per se, also activate the channel (Pastore et al., 2007). A possible

involvement of a mitochondrial PLA2 in the increase in FFA content is also hypothesized. The topology of ROS and FFA/acyl-CoA

interactions with PmitoKATP is not considered. OUT: intermembrane space; I.M.M.: inner mitochondrial membrane; IN: matrix space.


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plant mitochondria, but the ability of a commercial PLA2

to activate the DWM swelling in KCl suggests that a similar

mechanism might operate in durum wheat under hyper-

osmotic stress conditions to activate PmitoKATP. This point

will merit further investigation.

In conclusion, the PmitoKATP activation by FFAs/acyl-

CoAs demonstrated in this study allows a regulation

pathway operating under hyperosmotic/oxidative stress tobe suggested: under these conditions an increase in mito-

chondrial FFA content occurs that may activate Pmito-

KATP and also generate acyl-CoAs (Fig. 8). The combined

FFA/acyl-CoA activation of PmitoKATP may be about as

strong as that of PUCP under the same stress conditions.

This pathway is to be added to the other, recently described,

pathway involving ATP and ROS (see Introduction).

PmitoKATP activation under stress conditions due to thefunctioning of both the FFA/acyl-CoA and ROS/ATP

pathways may collapse DW, so controlling the harmful

large-scale ROS production according to a feedback mech-

anism. To date, it is not clear whether this FFA/acyl-CoA

modulation of PmitoKATP may play a role in the manifes-

tation of programmed cell death (PCD). However, the

involvement of plasma membrane ROS-activated K+ chan-

nels in stress-induced PCD (Demidchik et al., 2010, andreferences therein), as well as some mitochondrial Kþ

ATP

channels (Casolo et al., 2005; Petrussa et al., 2009) is well

established.

Acknowledgements

This work was supported by the projects MIUR

‘AGROGEN’. We gratefully acknowledge the skilful co-

operation of Drs Vanessa De Simone and Damiana

Tozzi, who participated as students in this work.

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