Plant Inner Membrane Anion Channel (PIMAC ... - Oxford Journals

Plant Cell Physiol. 49(7): 1039–1055 (2008) doi:10.1093/pcp/pcn082, available online at www.pcp.oxfordjournals.org ß The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected]

Plant Inner Membrane Anion Channel (PIMAC) Function in Plant Mitochondria Maura Nicoletta Laus

1, 2

, Mario Soccio

1, 2

, Daniela Trono 3, Luigi Cattivelli

3

and Donato Pastore

1, 2,

*

1

Dipartimento di Scienze Agro-ambientali, Chimica e Difesa Vegetale, Facolta` di Agraria, Universita` degli Studi di Foggia, Via Napoli, 25, 71100 Foggia, Italy 2 Centro di Ricerca Interdipartimentale BIOAGROMED, Universita` degli Studi di Foggia, Via Napoli, 52, 71100 Foggia, Italy 3 CRA-Centro di Ricerca per la Cerealicoltura, S.S. 16, Km 675, 71100 Foggia, Italy

To date, the existence of the plant inner membrane anion channel (PIMAC) has been shown only in potato mitochondria, but its physiological role remains unclear. In this study, by means of swelling experiments in Kþ and ammonium salts, we characterize a PIMAC-like anion-conducting pathway in mitochondria from durum wheat (DWM), a monocotyledonous species phylogenetically far from potato. DWM were investigated since they possess a very active potassium channel (PmitoKATP), so implying a very active matching anion uniport pathway and, possibly, a coordinated function. As in potato mitochondria, the electrophoretic uptake of chloride and succinate was inhibited by matrix [Hþ], propranolol, and tributyltin, and was insensitive to Mg2þ, N,N0 -dicyclohexylcarbodiimide (DCCD) and mercurials, thus showing PIMAC’s existence in DWM. PIMAC actively transports dicarboxylates, oxodicarboxylates, tricarboxylates and Pi. Interestingly, a novel mechanism of swelling in ammonium salts of isolated plant mitochondria is reported, based on electrophoretic anion uptake via PIMAC and ammonium uniport via PmitoKATP. PIMAC is inhibited by physiological compounds, such as ATP and free fatty acids, by high electrical membrane potential ( ), but not by acylCoAs or reactive oxygen species. PIMAC was found to cooperate with dicarboxylate carrier by allowing succinate uptake that triggers succinate/malate exchange in isolated DWM. Similar results were obtained using mitochondria from the dicotyledonous species topinambur, so suggesting generalization of results. We propose that PIMAC is normally inactive in vivo due to ATP and inhibition, but activation may occur in mitochondria de-energized by PmitoKATP (or other dissipative systems) to replace or integrate the operation of classical anion carriers. Keywords: Anion transport — Durum wheat mitochondria — Inner membrane anion channel — Jerusalem artichoke mitochondria — Potassium channel. Abbreviations: , electrical membrane potential; BSA, bovine serum albumin; DCCD, N,N0 -dicyclohexylcarbodiimide; DWM, durum wheat mitochondria; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; FFAs, free fatty acids; IMAC, inner membrane anion channel; JAM, Jerusalem artichoke

mitochondria; LA, linoleic acid; MDS, malate detecting system; NEM, N-ethylmaleimide; O2–, superoxide anion; PIMAC, plant inner membrane anion channel; PmitoKATP, ATP-sensitive plant mitochondrial potassium channel; PUCP, plant uncoupling protein; PVP, polyvinylpyrrolidone; ROS, reactive oxygen species; SHAM, salicylhydroxamate; TBT, tributyltin.

Introduction The mitochondrial inner membrane was originally assumed to be generally impermeable to ions as a prerequisite for efficient chemiosmotic coupling, but it is now clear that a number of ion channels exist in animal and plant mitochondria showing specific ion selectivities and sensitivities to modulators (O’Rourke 2000). As for the anion channels present in the inner membrane of mammalian mitochondria, it has been established by means of classical swelling experiments that heart and liver mitochondria contain a non-selective anion-conducting channel, known as the inner membrane anion channel (IMAC) (Garlid and Beavis 1986). IMAC catalyzes the electrophoretic uniport of a wide variety of physiological and nonphysiological, singly and multicharged, anions including chloride, sulfate, ferricyanide, bicarbonate, Pi, succinate, malate, citrate, etc. This pathway is inhibited by both matrix Hþ and divalent cations, such as Mg2þ and Ca2þ, the inhibition by Mg2þ being also pH-dependent (Beavis and Powers 1989). IMAC is normally closed or inactive due to regulation by matrix Mg2þ and Hþ, and it can be activated either by alkaline pH or by depletion of matrix divalent cations. IMAC is also reversibly inhibited by many cationic amphiphile drugs, such as propranolol (Beavis 1989a); although it is not absolutely specific (e.g. it is a b-blocker and also inhibits the Kþ/Hþ antiporter), this drug, that possess a hydrophobic moiety composed of two rings, strongly inhibits IMAC (IC50 ¼ 25 mM in rat liver mitochondria) by an interaction at a site located in the lipid bilayer (Beavis 1989a). IMAC is irreversibly inhibited by the alkylating agent N,N0 -dicyclohexylcarbodiimide (DCCD) (Warhurst et al. 1982). IMAC activity is completely

*Corresponding author: E-mail, [email protected]; Fax, þ39-0881589342. 1039

1040

PIMAC function in plant mitochondria

inhibited by triorganotins, one of the most potent being tributyltin (TBT) (Powers and Beavis 1991), and sensitive to mercurials, which both directly inhibit the transport and modulate the inhibition by Hþ, Mg2þ and propranolol (Beavis 1989b). IMAC is inhibited by palmitoyl-CoA (Halle-Smith et al. 1988), but CoA and the physiological nucletotides NADþ, NADH, NADPþ, NADPH and ATP have no effect on its activity (IMAC is able to transport ATP and AMP) (Powers et al. 1994). Recently, it has been reported that non-esterified long-chain fatty acids increase chloride permeation of isolated rat liver mitochondria suspended in slightly alkaline medium (Scho¨nfeld et al. 2000, Scho¨nfeld et al. 2001, Scho¨nfeld et al. 2002, Scho¨nfeld et al. 2003); it has been proposed that this increased anion permeability is due to the capability of fatty acids to activate IMAC by withdrawal of Mg2þ from intrinsic binding sites (Scho¨nfeld et al. 2004). IMAC activity is highly temperature dependent; this sensitivity is dependent on an effect of temperature on the inhibition by both Hþ (Liu et al. 1996) and Mg2þ (Beavis and Powers 2004). The physiological role of IMAC is still unclear. It has been suggested that IMAC, in conjunction with the Kþ/Hþ antiporter, is involved in mitochondrial volume homeostasis, thus allowing the respiratory chain proton pumps to drive salt efflux (Beavis 1992). In recent years, other roles have been proposed: it may also be involved in the efflux of the superoxide anion (O2–), from mitochondria during ischemic pre-conditioning (Vanden Hoek et al. 1998), and it may contribute to spontaneous synchronized oscillations of mitochondrial membrane potential in isolated cardiac myocytes (O’Rourke 2000). With respect to the IMAC counterpart in plant mitochondria, the information available is rather poor. This is despite the fact that the study of mitochondrial metabolite transport plays a crucial role in plant energy metabolism; in fact a rapid metabolite flux in and out of plant mitochondria is necessary for the maintenance of characteristic metabolic functions (photorespiration, nitrate assimilation, lipid–sugar transformation in germinating lipid-storing seeds) (Laloi 1999). To date, the occurrence of an IMAC-like anion channel has been demonstrated only in potato tuber mitochondria by swelling assays (Beavis and Vercesi 1992): such a channel, named PIMAC (plant inner membrane anion channel), has many of the characteristics of IMAC, such as the inhibition by propranolol (with an IC50 equal to 14 mM, even higher than that of IMAC), TBT and matrix Hþ, but differs in three important properties. Most significantly, the potato PIMAC does not appear to be blocked by matrix Mg2þ; it is also insensitive to inhibition by DCCD and mercurials. In tobacco, PIMAC was suggested to correspond to CLC-NT1, a putative chloride channel protein (Lurin et al. 2000). The physiological role of PIMAC has not been well characterized.

Anion transport via PIMAC is believed to play an important role in volume homeostasis maintenance (Beavis and Vercesi 1992). It was also suggested that in energized mitochondria, PIMAC may be involved in anion efflux out of the matrix, including the malate efflux in the operation of the malate/oxaloacetate shuttle (Beavis and Vercesi 1992). This proposal is in accordance with that of Zoglowek et al. (1988), who have suggested that both malate and oxaloacetate may be transported electrophoretically. Anyway, whether electrically coupled exchange may take place efficiently in the face of a large membrane potential remains to be demonstrated. On the other hand, the operation of a malate/oxaloacetate antiporter was shown in energized plant mitochondria (Pastore et al. 2003). It should be noted that plant mitochondria also possess a potassium channel that catalyzes the electrophoretic uniport of Kþ into the matrix compartment. The existence of a potassium channel inhibited by ATP in plant mitochondria was first shown in durum wheat mitochondria (DWM) (PmitoKATP; Pastore et al. 1999a); subsequently, an ATP-sensitive Kþ channel has also been described in pea mitochondria (Petrussa et al. 2001, Chiandussi et al. 2002, Casolo et al. 2003, Petrussa et al. 2004), and very recently in mitochondria from embryogenic cultures of Picea abies (L.) Karst. and Abies cephalonica Loud (Petrussa et al. 2008a) and from Arum spadix (Petrussa et al. 2008b). However, a protein-mediated mitochondrial Kþ uniport has been reported in many other plant species, including bread wheat, barley, spelt, rye, spinach (Pastore et al. 1999a), potato (Pastore et al. 1999a, Ruy et al. 2004), soybean (Casolo et al. 2005), maize and tomato (Ruy et al. 2004). In the light of the simple observation that PIMAC and PmitoKATP allow a matching ion transport, in this study, we have checked the idea that possible physiological roles of PIMAC should be sought by looking into the physiological roles of the PmitoKATP. Similarly, plant mitochondria possess another powerful dissipative system, the plant uncoupling protein (PUCP) (Vercesi et al. 1995), that may somehow affect PIMAC activity. To test these hypotheses, here we have investigated whether DWM, chosen in the light of the high PmitoKATP and PUCP activities (Pastore et al. 1999a, Pastore et al. 2000), also possess a channel similar to potato PIMAC. This preliminary investigation is worthwhile since durum wheat is a monocotyledonous plant species far from potato on a phylogenetic basis. Then, the specificity of the transport has been investigated by means of classical mitochondrial swelling assays in both Kþ and ammonium salts of metabolically relevant anions. Finally, possible physiological modulators of the PIMAC, as well as possible PIMAC cooperation with classical energy-dependent carriers, have been tested. In order to generalize results obtained with DWM to other plant mitochondria, we


Results In order to gain a first insight into the possible existence of a PIMAC-like anion channel in purified DWM, we studied chloride and succinate transport, the first being one of the anions most rapidly transported via PIMAC in potato tuber mitochondria (Beavis and Vercesi 1992) and the second a metabolically relevant anion. Chloride and succinate transport was studied by means of swelling experiments in iso-osmotic solutions of KCl and K2succinate, respectively. PIMAC in DWM DWM absorbance in 0.36 M sucrose solution was found to remain constant during the time of the experiment (Fig. 1, traces a and a0 ), thus demonstrating that DWM are intact; no swelling in sucrose solution was observed in the presence of the Kþ ionophore valinomycin, so indicating that this compound does not affect DWM intactness. Conversely, a clearly evident swelling was observed when DWM were suspended in iso-osmotic solutions of KCl (trace b) and K2succinate (trace b0 ), thus indicating that DWM are specifically permeable to Kþ and to chloride

A

0.36 M Sucrose ± Val.

a 0.18 M KC1 0.5 µg Val.

0.02 A546 nm

b

c 0.18 M KC1 + 0.5 µg Val. 1 min

B

a′ 0.12 M K2Succinate 0.5 µg Val. b′

0.36 M Sucrose ± Val.

0.02 A546 nm

have also verified the main results in Jerusalem artichoke (topinambur) mitochondria (JAM), topinambur being a dicotyledonous species. Our results show that DWM and JAM possess a PIMAC very similar to that of potato mitochondria, able to transport Pi, dicarboxylates, oxodicarboxylates and tricarboxylates. Interestingly, the study of swelling in ammonium salts allows definition of a new mechanism of swelling in isolated plant mitochondria, based on electrophoretic ammonium uniport via PmitoKATP compensated by anion uniport via PIMAC; therefore, classical interpretation based on ammonia diffusion accompanied by protoncompensated transport of Pi and its recycling around dicarboxylate carrier should be revised in these mitochondria. ATP and free fatty acids (FFAs) (but not acyl-CoAs) inhibit PIMAC as well as high electrical membrane potential ( ), while PIMAC is insensitive to reactive oxygen species (ROS). Interestingly, PIMAC was found to cooperate with dicarboxylate carrier by allowing succinate uptake that triggers the subsequent succinate/ malate exchange in isolated mitochondria. On the whole, the results are consistent with our working hypothesis, so PIMAC may enable metabolite movement when mitochondria generate low and low ATP. This may occur in nonenergized mitochondria isolated in vitro, as well as in the cell under either stress or spontaneous depolarization already described in vivo. Under these conditions, PIMAC may replace or integrate in the operation of classical anion carriers.

1041

c′

0.12 M K2Succinate + 0.5 µg Val. 1 min

Fig. 1 DWM swelling in iso-osmotic solutions of KCl (A) and K2succinate (B) in the presence and absence of valinomycin. DWM (0.1 mg prot.) were suspended in 2 ml of a medium consisting of 20 mM Tris–HCl (pH 7.20) and 0.36 M sucrose (A and B, traces a and a0 ), 0.18 M KCl (A, trace b) or 0.12 M K2succinate (B, trace b0 ). Where indicated, the medium contained 0.5 mg of valinomycin (A and B, traces c and c0 ). At the time indicated by the arrows, 0.5 mg of valinomycin was added. Swellings were continuously monitored by measuring the absorbance decrease at 546 nm and 258C, as reported in Materials and Methods. DWM absorbance in sucrose solution in the presence of valinomycin is also shown, demonstrating that DWM intactness is unaffected by valinomycin.

and succinate. The swelling rate was found to increase greatly when 0.5 mg of valinomycin was either added in the course of the swelling or present in the reaction medium (traces b and c, b0 and c0 ). This indicates that the swelling rate in both KCl and K2succinate solutions in the absence of valinomycin depends on the rate of Kþ uptake rather than chloride and succinate uptake. In this regard, we have

1042


B

100

V (% of the control)


A

80 60 40 20 0

0

200

400 600 800 [Propranolol] (µM)

100 80 60 40 20 0 0.0

1000

0.5

1.0

1.5 2.0 [TBT] (µM)

2.5

3.0

K2Succinate + Val. KC1 + Val. + nigericin

D

250 V (% of the control)


C

200 150 100 50 6.8

7.0

7.2

7.4 7.6 pH

7.8

8.0

− nigericin 250 200 150 100 50 6.8

7.0

7.2

7.4 7.6 pH

7.8

8.0

Fig. 2 Effect of propranolol, tributyltin (TBT) and pH on the swelling rate in iso-osmotic solutions of KCl and K2succinate. Swelling experiments were carried out in KCl and K2succinate solutions in the presence of valinomycin, as reported in Fig. 1 (traces c and c0 ). (A and B) Measurements were carried out in both the absence and presence of propranolol (A) or TBT (B) at the reported concentrations; the swelling rates (V) in KCl (filled squares) or K2succinate (open circles), expressed as a percentage of the rate measured in the absence of inhibitor (control), are reported as a function of inhibitor concentration. (C and D) Measurements were carried out at different pH values in both the presence (C) and absence (D) of 10 nM nigericin (an ionophore that promotes Kþ/Hþ exchange, so allowing the rapid equilibrium among internal and external pH); the swelling rates in KCl (filled squares) or K2succinate (open circles), expressed as a percentage of the rate measured at pH 7.20 (control), are plotted as a function of pH. The data are reported as mean value SD (n ¼ 3).

recently demonstrated that Kþ uniport in DWM is catalyzed by an ATP-sensitive Kþ channel, named PmitoKATP (Pastore et al. 1999a). In contrast, when DWM were suspended in KCl and K2succinate solutions in the presence of valinomycin (traces c and c0 ), a very high swelling rate was observed; it should be noted that a further increase of valinomycin concentration did not cause a further increase of swelling rate (not shown). This demonstrates that in the presence of 0.5 mg valinomycin, the rate-limiting step was chloride or succinate uptake and that the swelling rate is a measure of anion uptake. As shown in Fig. 2A and B, propranolol and TBT were found to inhibit the swelling rate in both KCl and K2succinate solutions (plus valinomycin); the inhibition resulted in an increase with increasing concentrations, with strong inhibition at 1 mM propranolol and 3 mM TBT. When DWM were suspended in KCl and K2succinate solutions (plus valinomycin) at different pHs and in the presence of nigericin, an ionophore that promotes Kþ/Hþ

exchange, so allowing the rapid equilibrium among external and internal pH, an evident increase in swelling rate was observed with increasing pH (Fig. 2C): the rate measured in KCl and K2succinate solutions at pH 8 was about 1.9 and 2.5 times, respectively, that measured at pH 7.2. A less evident increase of swelling rate with increasing pH was measured in the absence of nigericin (Fig. 2D), thus suggesting that chloride and succinate transport is activated by increasing both matrix and external pH, the matrix pH being more effective. Moreover, both chloride and succinate uptake by DWM was found to be insensitive to Mg2þ, DCCD and the mercurials mersalyl and N-ethylmaleimide (NEM), at 20 mM, 1 mM, 1 mM and 500 mM, respectively, i.e. at concentrations which inhibit IMAC (Beavis 1992). Preliminarily, a control was carried out to ensure that all the tested compounds at the reported concentrations had no effect on DWM intactness evaluated as maintenance of absorbance in sucrose solution.


Table 1 Swelling rate in iso-osmotic solutions of KCl or Kþ salts of dicarboxylates, oxodicarboxylates, tricarboxylates and Pi in the absence and presence of propranolol Anion

Chloride Succinate Malate Oxaloacetate 2-Oxoglutarate Citrate cis-Aconitate Pi

Swelling in Kþ Inhibition (%) salt þ valinomycin in the presence of (A min–1 mg–1) 500 mM propranolol 4.21 0.35a 4.27 0.46 4.69 0.14 3.75 0.70 4.05 0.14 3.89 0.39 3.50 0.88 1.25 0.26

75 6%a 95 8% 80 3% 75 8% 60 2% 90 9% 90 11% 85 9%

Swelling of DWM (0.1 mg prot.) was carried out in the presence of valinomycin as reported in Fig. 1, in 2.0 ml of 0.18 M KCl, 0.12 M K2-dicarboxylate(-oxodicarboxylate), 0.10 M K3-tricarboxylate or 0.15 M KPi. Measurements were carried out both in the absence and in the presence of 500 mM propranolol. Inhibition is expressed as a percentage of the control rate. a Mean value SD (n ¼ 3).

As a whole, the data show that in DWM both chloride and succinate uniport is mediated by a PIMAC functionally similar to that of potato. In order to investigate DWM–PIMAC specificity, we compared the swelling rate in Kþ (plus valinomycin) salts of chloride, of the dicarboxylates succinate and malate, of the oxodicarboxylates oxaloacetate and 2-oxoglutarate, of the tricarboxylates citrate and cis-aconitate, as well as of Pi. All the anions were found to be transported at a high rate and all the transport was strongly inhibited by propranolol (Table 1), thus showing that DWM–PIMAC can mediate the electrophoretic transport of metabolically relevant anions. Pi can also be transported via PIMAC, but at a rate 3- to 3.5-fold lower with respect to the other anions. This finding prompted us to reinvestigate the mechanism of classical swelling in NH4þ salt solutions to ascertain whether PIMAC may be somehow involved. Interestingly, DWM show a fast and large amplitude swelling in isoosmotic solution of NH4Cl (Fig. 3A, B). The swelling rate is further increased by the protonophore (Hþ ionophore) carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), either added in the course of the swelling or present in the reaction medium. This clearly excludes a NH4Cl influx due to a proton-compensated Cl– transport accompanying NH3 diffusion across the inner membrane; on the contrary, it suggests the occurrence in these mitochondria of an electrophoretic uniport of both NH4þ and Cl–. Activation of the swelling rate due to 1 mM FCCP addition is the consequence of FCCP-stimulated NH4þ

1043

influx: in fact, FCCP, allowing Hþ entry into the matrix, promotes NH4þ dissociation in Hþ and NH3, that can freely and rapidly permeate the inner membrane (see Fig. 4 and Beavis 1992). Therefore, FCCP activation of DWM swelling in NH4Cl solution indicates NH4þ uptake as the rate-limiting step, so this swelling may be suitable to study the mechanism of NH4þ uptake. In this regard, the hypothesis of an involvement of PmitoKATP in NH4þ uptake was tested, by evaluating the effect on DWM swelling in NH4Cl solution of inhibitors (ATP and NADH) and activators (mersalyl and palmitoyl-CoA) of PmitoKATP (Pastore et al. 1999a), using concentrations known to affect PmitoKATP activity. The swelling was clearly inhibited by 1 mM ATP and even more so by 1 mM NADH (Fig. 3A); consistently, 5 mM palmitoyl-CoA and 500 mM mersalyl strongly activated the swelling (Fig. 3B). In particular, ATP inhibition was observed in the presence of atractyloside, that inhibits ATP transport via ATP/ADP carrier, and of oligomycin, an inhibitor of ATP hydrolysis via ATP synthase, thus showing that ATP per se inhibits swelling at the outer side of the inner membrane. These results are consistent with the electrophoretic uptake of NH4þ via PmitoKATP (Pastore et al. 1999a). At the same time, a PIMAC-mediated electrophoretic Cl– uptake, accompanying NH4þ via PmitoKATP, was evaluated. To do this, we studied the effect on swelling in NH4Cl solution of propranolol (Fig. 3C, D), used as a typical PIMAC inhibitor, and DCCD (Fig. 3D), that affects the transport of many compounds, but not Cl– transport via PIMAC as reported above. Propranolol (1 mM) induced a 45% inhibition of the swelling rate (Fig. 3C), that resulted in no further increase by 1 mM FCCP addition, so showing a PIMAC involvement in NH4Cl swelling. As expected, the inhibition by propranolol is much smaller compared with that shown in Fig. 2A: in fact, the rate of NH4Cl swelling is limited by NH4þ uptake (note the increase induced by FCCP) rather than Cl– transport (Fig. 3C, control). In order to quantify accurately the inhibition exerted by propranolol and to verify the lack of inhibition by DCCD, in a parallel set of experiments we adopted experimental conditions making the rate of Cl– uptake limiting with respect to NH4þ uptake. This was accomplished by carrying out swelling in NH4Cl solution according to Selwyn assay (as reported in Beavis 1992), by including in the reaction medium the uncoupler FCCP (10 mM) and by adopting alkaline conditions (pH 8.00) (Fig. 3D). This swelling was strongly inhibited by propranolol [78 8% (SD), in three different experiments] and insensitive to DCCD, thus demonstrating PIMAC as the Cl– transport pathway in NH4Cl swelling. Swelling of DWM was also observed in iso-osmotic (NH4)2succinate solution, although slower than in NH4Cl (see the explanation in the text of Fig. 7). Strong activation

1044


A

0.36 M Sucrose

B

0.36 M Sucrose

+ 1 mM NADH 0.02 A546 nm

0.02 A546 nm

1 µM FCCP

+ 1 mM ATP + Oligo/Atr + 1 µM FCCP

0.18 M NH4Cl

0.18 M NH4Cl + 500 µM Mersalyl 1 µM FCCP

+ 5 µM PalmitoylCoA

1 min

1 min

C

0.36 M Sucrose

D

0.36 M Sucrose

1 µM FCCP

+1 mM Propranolol

0.18 M NH4Cl

1 min

0.02 A546 nm

0.02 A546 nm

+ 1 mM Propranolol

+1 µM DCCD

0.18 M NH4Cl + 10 µM FCCP (pH 8.00) 1 min

Fig. 3 Effect of ATP, NADH, mersalyl, palmitoyl-CoA, FCCP, propranolol and DCCD on DWM swelling in iso-osmotic solution of NH4Cl. Swelling experiments were carried out as reported in Fig. 1. (A–C) DWM (0.1 mg prot.) were suspended in 2 ml of a medium consisting of Tris–HCl 20 mM (pH 7.20) and 0.18 M NH4Cl, and in the same medium supplemented with: 1 mM NADH or 1 mM ATP [in the presence of 2 mg of oligomycin (Oligo) and 10 mM atractyloside (Atr), inhibitors of ATP synthase and of ADP/ATP translocator, respectively] or 1 mM FCCP (A); 500 mM mersalyl or 5 mM palmitoyl-CoA (B); 1 mM propranolol (C). At the time indicated by the arrows, 1 mM FCCP was added. (D) DWM (0.1 mg prot.) were suspended in 2 ml of a medium consisting of 20 mM Tris–HCl (pH 8.00), 0.18 M NH4Cl and 10 mM FCCP and in the same medium supplemented with 1 mM propranolol or 1 mM DCCD. The absorbance in sucrose solution is also shown, demonstrating DWM intactness.

by FCCP either added in the course of the swelling or present in the reaction medium (Fig. 5A, B), as well as inhibition by ATP and NADH (Fig. 5A) and activation by mersalyl and palmitoyl-CoA (Fig. 5B) was observed, so demonstrating, also in this case, the occurrence of an electrophoretic NH4þ uptake via PmitoKATP. To conserve parallelism between Cl– and succinate experiments, we also studied the effect of propranolol and DCCD on swelling in (NH4)2succinate solution (Fig. 5C, D). The experiments were carried out as in Fig. 3C and D, and gave the same results. In Fig. 5D, propranolol (1 mM) inhibition was

(82 9% SD in three different experiments). These results indicate PIMAC involvement also in (NH4)2succinate swelling. Surprisingly, DWM swelling in (NH4)2succinate solution was insensitive to externally added Pi (strictly necessary to trigger the swelling in animal mitochondria) and to NEM, a known inhibitor of Pi carrier (Laloi 1999, and references therein) (Table 2). DWM swelling in iso-osmotic solutions of NH4þ salt of malate, oxaloacetate, 2-oxoglutarate, citrate, cis-aconitate or Pi was also studied and compared with that of succinate (Table 2). No swelling was

PIMAC function in plant mitochondria NH4A H+

+

NH4

A−

PmitoKATP

PIMAC

NH3

FCCP

H+

NH3 ATP NADH

matrix

Mersalyl + Palmitoyl-CoA

NH4

A−

i.m.m.

intermembrane space

NH4A

Fig. 4 Possible mechanism of DWM swelling in iso-osmotic solution of NH4þ salt of an anion. Anion (A–) uptake may be mediated by PIMAC. NH4þ is transported via PmitoKATP. In the presence of FCCP, that allows Hþ entry into the matrix, NH4þ dissociation into Hþ and NH3 is promoted, with a consequent NH3 diffusion across the inner membrane and increase of swelling rate. PIMAC, plant inner membrane anion channel; PmitoKATP, plant mitochondrial ATP-dependent Kþ channel; i.m.m., inner mitochondrial membrane. Activators and inhibitors of PmitoKATP used in Fig. 3 and 4 are also reported.

enhanced by Pi or Pi/malate (generally supposed to be essential to trigger the swelling in the NH4þ salt of oxodicarboxylates and tricarboxylates). Swellings were activated by mersalyl in all the cases except oxaloacetate and Pi; this despite the fact that mersalyl is a well known inhibitor of dicarboxylate, tricarboxylate and Pi carriers (Douce 1985, and references therein); FCCP also strongly increased swellings, with the exception of slow inhibition observed in the case of Pi. So, DWM swelling in NH4þ salt of dicarboxylates, oxodicarboxylates and tricarboxylates should occur via cooperation between PmitoKATP and PIMAC, as reported in Fig. 4. Different behavior was observed in the case of Pi: in fact, swelling in NH4Pi was found to be insensitive to mersalyl and NEM, but slight inhibition was observed in the presence of FCCP and when DWM were incubated for 5 min in the presence of NEM before swelling (see Table 2 footnote). In order to identify physiological modulators of PIMAC activity and its possible physiological role, we evaluated the effect of FFAs and ATP, modulators of other ion-conducting pathway, such as PUCP (Pastore et al. 2000) and PmitoKATP (Pastore et al. 1999a). Several FFAs were found to inhibit chloride and succinate transport via PIMAC (Table 3). At 10 mM concentration, higher inhibition was exerted by unsaturated FFAs (mainly oleic and linoleic acids), while the saturated FFAs were not active on succinate transport. Moreover, 10 mM linoleic acid (LA) inhibited oxaloacetate and citrate (55% in both cases), but not Pi transport (data not shown). A dose-dependent inhibition was observed when chloride and any more succinate uptake were inhibited by LA in

1045

the 5–20 mM range (Fig. 6A, B). Inhibition by FFAs appears to be a particularity of PIMAC being the animal counterpart activated by FFAs (Scho¨nfeld et al. 2004); moreover, while palmitoyl-CoA is known to inhibit IMAC (Halle-Smith et al. 1988), no inhibition of either chloride or succinate uptake by DWM–PIMAC was observed in the presence of 10 mM acyl-CoA esters of the FFAs reported in Table 3 and in the 5–30 mM range in the case of linoleoyl-CoA. Interestingly, as shown in Fig. 6A0 and B0 , DWM swelling in both KCl and K2succinate solutions (plus valinomycin) was found to be inhibited by externally added ATP. In particular, in the 1–5 mM range, ATP induced a progressive (from 60 to 85%) inhibition of the swelling rate in K2succinate (B0 ); swelling in KCl was also inhibited by 1–2 mM ATP (A0 ). Unexpectedly, in KCl plus 3 mM ATP, a little swelling followed by mitochondrial shrinkage was observed and a further increase of ATP concentration up to 5 mM induced a clearly evident mitochondrial shrinkage; these findings will merit further investigation. We also tested sensitivity to ATP of Pi, oxaloacetate and citrate transport and observed that 5 mM ATP induced 45, 80 and 63% inhibition, respectively (data not shown). All measurements were carried out in the presence of oligomycin and atractyloside (see above), so the observed inhibition is probably exerted by ATP on the outer side of the inner membrane; moreover, differently from mammalian mitochondria, we observed that ATP is not transported by PIMAC (data not shown). PmitoKATP and PUCP may strongly affect in DWM (Pastore et al. 1999a, Pastore et al. 2000); so, in order to evaluate how influences PIMAC activity, we monitored generation and mitochondrial swelling simultaneously in iso-osmotic (NH4)2succinate solution (Fig. 7). When DWM were suspended in iso-osmotic sucrose solution in the presence of 5 mM Na-succinate, a high (180 mV) was generated due to succinate uptake and oxidation by the respiratory chain (Fig. 7A, trace a). As expected, in (NH4)2succinate solution, DWM were found to generate at a lower rate and extent (110 mV after about 3 min) (Fig. 7A, trace b); this is due to NH4þ uptake via PmitoKATP which consumes . In both traces a and b of Fig. 7A, the addition of the uncoupler FCCP quickly collapses . No was generated by DWM in (NH4)2succinate solution in the presence of KCN plus salicylhydroxamate (SHAM), inhibitors of the cytochrome oxidase and alternative oxidase (AOX), respectively, and FCCP that prevented generation (Fig. 7A, traces c and d). Swellings recorded simultaneously with measurements are reported in Fig. 7B. Interestingly, electrophoretic uptake of ammonium and succinate ions carries on with time, although a 110 mV was reached after 3 min as seen in Fig. 7A trace b, and electrical charges (negative inside)

1046


B

0.36 M Sucrose 0.12 M (NH4)2Succinate 1 µM FCCP

0.02 A546 nm

0.12 M (NH4)2Succinate

0.36 M Sucrose

+ 1 mM ATP + Oligo/Atr + 1 mM NADH 1 µM FCCP

0.02 A546 nm

A

+500 µM Mersalyl

+ 5 µM PalmitoylCoA

+ 1 µM FCCP

1 min

1 min

C

D

0.36 M Sucrose

0.36 M Sucrose 1 µM FCCP

+ 1 mM Propranolol

0.02 A546 nm

0.02 A546 nm

+ 1 mM Propranolol

+ 1 µM DCCD 0.12 M (NH4)2Succinate

0.12 M(NH4)2Succinate + 10 µM FCCP (pH 8.00)

1 min 1 min

Fig. 5 Effect of ATP, NADH, mersalyl, palmitoyl-CoA, FCCP, propranolol and DCCD on DWM swelling in iso-osmotic solution of (NH4)2succinate. Swelling experiments were carried out as reported in Fig. 3, with the only exception that the reaction medium contained 0.12 M (NH4)2succinate instead of 0.18 M NH4Cl.

Table 2 Effect of Pi (or Pi/malate), mersalyl or FCCP on DWM swelling in isosmotic solutions of NH4þ salts of dicarboxylates, oxodicarbolxylates, tricarboxylates and Pi Swelling in NH4þ salt of anion (% of the control) Succinate

Malate

Oxaloacetate 2-Oxoglutarate

Citrate

Pi

cis-Aconitate

þ 1 mM Pi No effect No effect – þ 1 mM Pi þ 1 mM malate – – No effect

– No effect

– – No effect No effect

– _

þ 500 mM mersalyl þ 1 mM FCCP

þ50% þ450%

þ115% þ450%

No effecta 20%

þ20% þ360%

þ305% þ375%

No effect þ360%

þ50% þ135%

Swelling experiments were carried out as reported in Fig. 5B, in 0.12 M (NH4)2-dicarboxylate(-oxodicarboxylate), 0.10 M (NH4)3tricarboxylate or 0.15 M NH4Pi solutions, in the absence and presence of Pi (or Pi/malate), mersalyl or FCCP at the reported concentrations. Activation or inhibition was expressed as a percentage of the control rate. a No effect was observed in the presence of 500 mM NEM instead of mersalyl; about 20% inhibition was obtained in the presence of 500 mM NEM incubated 5 min .


Table 3

1047

Effect of several FFAs on the swelling rate in iso-osmotic solutions of KCl and K2succinate

FFA

12 : 0 16 : 0 18 : 0 16 : 19 18 : 19 18 : 29,12 20 : 45,8,11,14

Lauric Palmitic Stearic Palmitoleic Oleic Linoleic Arachidonic

Inhibition (%) of swelling rate in KCl þ valinomycin

Inhibition (%) of swelling rate in K2succinate þ valinomycin

40 4%a 40 3% No effect No effect 90 7% 40 3% 40 2%

No effect No effect No effect 25 2% 50 4% 65 5% 60 4%

Swelling experiments were carried out in the presence of valinomycin as reported in Fig. 1. Measurements were carried out both in the absence and in the presence of 10 mM FFA. Inhibition was expressed as a percentage of the control rate. a Mean value SD (n ¼ 3).

oppose succinate uptake. Prevention of generation by KCN/SHAM increases the swelling rate and extent, and a further increase was induced by FCCP that stimulates swelling both by preventing generation and by promoting ammonium uptake as reported in Fig. 4. On the whole, inhibits PIMAC, but a relevant activity may still be observed at 110 mV. In the experiment reported in Fig. 8, the possible cooperation between PIMAC and classical dicarboxylate carrier was investigated. Succinate transport was checked by monitoring the succinate/malate exchange in DWM, evaluated as malate appearance in the extramitochondrial phase. This was continuously measured by following the absorbance increase at 340 nm due to NADPH generated by the malate detecting system (MDS) (see Materials and Methods). Simultaneously, generation due to succinate addition to DWM was monitored (dotted line). Succinate (Naþ salt) addition to DWM generated in about 30 s a of 180 mV due to succinate uptake and oxidation; conversely, a malate efflux outside DWM was detected only after a lag phase of about 1 min (see the traces reported in the inset), i.e. when a high was already reached. No malate efflux occurred when 30 mM phenylsuccinate, an inhibitor of dicarboxylate carrier that cannot enter mitochondria (La Noue and Schoolwerth 1984, Fratianni et al. 2001), was either added in the course of the reaction or was present in the reaction medium (Fig. 8, traces b and d, respectively). In trace b, the absorbance decrease induced by phenylsuccinate addition is probably due to NADPH oxidation by external NADPH dehydrogenase. When the PIMAC inhibitor propranolol was present in the reaction medium, a clearly evident inhibition of the rate of malate appearance (45%) was found (Fig. 8, trace c). In contrast, if propranolol was added in the course of the reaction, only a slight inhibition of the malate efflux rate (515%) was observed (Fig. 8, trace a). These results may be explained by a cooperation between PIMAC and dicarboxylate carrier.

PIMAC should mediate the initial uptake of succinate, that is not accompanied by malate efflux and occurs in conditions of low ; in this regard, it should be noted that the reaction medium did not contain Pi in order to inhibit the possibility of a succinate/Pi exchange. Therefore, in this first phase, a PIMAC-dependent succinate accumulation inside mitochondria may occur, so triggering a second phase based on succinate/malate exchange due to dicarboxylate carrier (in trace c, lower succinate accumulation resulted in a lower exchange rate). In this second phase, PIMAC contribution to the transport is negligible, probably because of the high inhibition. PIMAC in JAM The main characteristics of DWM–PIMAC were also investigated in mitochondria isolated from tubers of topinambur (Jerusalem artichoke, Helianthus tuberosus L.), a dicotyledonous species very different from durum wheat. As shown in Fig. 9A and B, in JAM, chloride and succinate can be transported electrophoretically, as demonstrated by the large amplitude swellings measured in both KCl and K2succinate solutions (plus valinomycin). Propranolol (1 mM) strongly inhibited both chloride and succinate uptake (85 and 80%, respectively), so indicating the involvement of PIMAC in chloride and succinate transport. Both swellings were inhibited by ATP and LA. Once again, in the presence of high ATP concentration, a little swelling followed by mitochondrial shrinkage in KCl was observed (Fig. 9A). Malate, oxaloacetate, 2-oxoglutarate, citrate, cisaconitate and Pi transport were also tested: they were found to be transported electrophoretically and the transport was inhibited by 1 mM propranolol about 85, 60, 60, 70, 45 and 60%, respectively. The sensitivity of JAM-PIMAC to ATP and LA was also investigated on oxaloacetate, citrate and Pi transport: inhibition by 5 mM ATP was found to be 80, 70 and 50%, respectively, while 10 mM LA inhibited oxaloacetate and citrate uptake (32 and 45%, respectively),

1048


+ 5 mM ATP + Oligo/Atr

A

A′ 0.36 M Sucrose ± 5 mM ATP + Oligo/Atr

0.02 A546 nm

0.02 A546 nm

0.36 M Sucrose ± 20 µM LA

+ 20 µM LA



+ 10 µM LA + 5 µM LA


0.18 M KC1 + 0.5 µg Val.

0.18 M KC1 + 0.5 µg Val.

1 min

1 min

0.36 M Sucrose ± 20 µM LA

B

B′

+ 5 mM ATP + Oligo/Atr 0.02 A546 nm

0.02 A546 nm

+ 20 µM LA

0.36 M Sucrose ± 5 mM ATP + Oligo/Atr

+ 15 µM LA

+ 10 µM LA + 5 µM LA 0.12 M K2Succinate + 0.5 µg Val. 1 min

+ 3 mM ATP + Oligo/Atr + 2 mM ATP + Oligo/Atr

+ 1 mM ATP + Oligo/Atr 0.12 M K2Succinate + 0.5 µg Val. 1 min

Fig. 6 Effect of linoleic acid (LA) and ATP on DWM swelling in iso-osmotic solutions of KCl (A, A0 ) and K2succinate (B, B0 ). Swelling experiments were carried out in the presence of valinomycin as reported in Fig. 1, in KCl and K2succinate solutions in the absence and presence of LA (A, B) or ATP (A0 , B0 ) at the reported concentrations. Where indicated, the medium also contained 2 mg of oligomycin (Oligo) and 10 mM atractyloside (Atr). DWM absorbance in sucrose solution in the absence and presence of 5 mM ATP (plus Oligo/Atr) or 20 mM LA is also shown, demonstrating that DWM intactness is unaffected by ATP and LA.

but not Pi uptake. In JAM, PIMAC regulation by and cooperation between PIMAC and dicarboxylate carrier were found to be similar to those reported in Fig. 7 and 8 (data not shown). Taken together, these results show the existence of a JAM–PIMAC with almost the same properties as DWM–PIMAC. In the light of ROS activation of PmitoKATP (Pastore et al. 1999a) and PUCP (Pastore et al. 2000, Considine et al. 2003, Paventi et al. 2006), a possible modulation of PIMAC by ROS was investigated in JAM. The effect of H2O2 and

O2–, generated in situ by the xanthine/xanthine oxidase system, was studied on chloride uptake, but no change in swelling rate was observed (Fig. 9C), thus suggesting that ROS cannot modulate PIMAC.

Discussion Although in the past the existence of an aniontranslocating pathway has been suggested in mitochondria from many different plant sources (Hanson and Koeppe


A 50

0.2 mg DWM prot.

+ 1 µM FCCP

1049

B d

0.36 M Sucrose ± KCN/SHAM or FCCP

c + 1 mM KCN/ 1 mM SHAM

0.12 M (NH4)2Succinate 0.01 A546 nm

∆ψ (mV)

100 b 0.12 M (NH4)2Succinate

+ 1 mM KCN/ 1 mM SHAM + 1 µM FCCP

150 a

1 µM FCCP

1 min

0.36 M Sucrose 200

2 min

Fig. 7 Effect of KCN/SHAM and FCCP on membrane potential ( ) generation (A) and on DWM swelling (B) in an iso-osmotic solution of (NH4)2succinate. generation (A) and mitochondrial swelling (B) in (NH4)2succinate solution were monitored simultaneously. (A) Fluorimetric measurements of changes were carried out as reported in Materials and Methods. DWM (0.2 mg prot.) were suspended in 2 ml of a medium containing 0.12 M (NH4)2succinate, 20 mM Tris–HCl (pH 7.20), 2.5 mM safranin O (trace b) and in the same medium with 1 mM KCN and 1 mM SHAM (trace c) or 1 mM FCCP (trace d) added. measurement in 0.36 M sucrose solution, containing 20 mM Tris–HCl (pH 7.20), 2.5 mM safranin and 5 mM Na-succinate, is also reported for comparison (trace a). The additions of 0.2 mg of DWM prot. and 1 mM FCCP were carried out at the time indicated by the arrows. (B) Swelling experiments were carried out as reported in Fig. 5, in (NH4)2succinate solution in the absence and presence of either 1 mM KCN plus 1 mM SHAM or 1 mM FCCP. The absorbance in sucrose solution containing 5 mM Na-succinate in the absence and presence of KCN/SHAM or FCCP is also shown.

1975, Hensley and Henson 1975, Zoglowek et al. 1988), to the best of our knowledge, PIMAC activity has been detected, to date, by means of classical swelling assays, only in potato tuber mitochondria (Beavis and Vercesi 1992). In this study, for the first time, PIMAC is shown to occur in a monocotyledonous species, such as durum wheat. The existence of PIMAC in DWM is demonstrated on the basis of the similarities of the electrophoretic chloride transport in durum wheat and potato tuber mitochondria with respect to the inhibition by propranolol, TBT and matrix Hþ, and the insensitivity to Mg2þ, mercurials and DCCD; PIMAC inhibition by propranolol, TBT and Hþ are properties common to IMAC of mammalian mitochondria (Beavis 1992), while the absence of inhibition by Mg2þ, mercurials and DCCD are the most important differences from the animal counterpart. An important novelty of this study regards anion selectivity of PIMAC. In DWM and JAM, PIMAC can mediate the transport of metabolically relevant anions, including dicarboxylates, oxodicarboxylates, tricarboxylates and Pi, as demonstrated by the electrophoretic nature of the transport and the inhibition by propranolol. In animal mitochondria, the most important physiological regulators of IMAC are matrix Mg2þ and Hþ (Beavis 1992); moreover, in the past, an inhibitory

action on IMAC of palmitoyl-CoA was demonstrated in rat liver mitochondria (Halle-Smith et al. 1988) and, only recently, in the same mitochondria, the activation by non-esterified long-chain fatty acids has been shown (Scho¨nfeld et al. 2004). In plant mitochondria, lacking the inhibition by Mg2þ, the only known physiological mechanism of PIMAC modulation is the endogenous Hþ inhibition (Beavis and Vercesi 1992). Here, by using both DWM and JAM, the first physiological inhibitors of PIMAC were identified: ATP and FFAs. PIMAC was found to be strongly inhibited by ATP in a physiological concentration range, acting at the outer side of the inner membrane. To date, no information is available regarding the ATP sensitivity of the anion-conducting pathway in mammalian mitochondria (it is known that ATP is a substrate of IMAC), while, interestingly, in yeast mitochondria matrix ATP was found to regulate, with a different mechanism, two anion channels with different conductance (Ballarin and Sorgato 1995, Ballarin and Sorgato 1996). As for FFAs, several unsaturated and, to a lesser extent, saturated FFAs inhibit PIMAC, while acylCoAs had no effect. Therefore, different regulation is observed with respect to rat liver mitochondria–IMAC (Halle-Smith et al. 1988, Scho¨nfeld et al. 2004), thus also

1050


1 mM Propranolol a 0.2 A340 nm

30 mM Phenylsuccinate

+ 1 mM Propranolol b c

5 mM Succinate 50 d e

+ 30 mM Phenylsuccinate

∆ψ (mV)

0.05 A340 nm

100

50 ∆ψ (mV)

150

75

200

5 mM Succinate

a

e 1 min

2 min

Fig. 8 Succinate/malate exchange and generation due to succinate addition to DWM. Measurement were monitored simultaneously; succinate/malate exchange was studied as malate appearance in the extramitochondrial phase by measuring spectrophotometrically the NADPH absorbance increase at 340 nm, as reported in Materials and Methods. The MDS was added to a reaction medium containing 0.3 M mannitol, 5 mM MgCl2, 20 mM Tris–HCl (pH 7.20), and then the reaction was started by adding 5 mM Na-succinate. Traces a and b: 1 mM propranolol or 30 mM phenylsuccinate was added in the course of the reaction at the time indicated by the arrows, respectively. Traces c and d: 1 mM propranolol or 30 mM phenylsuccinate was already present in the reaction medium, respectively. Trace e (dotted line): measurements were carried out in 2 ml of the medium reported above, supplemented with 2.5 mM safranin O and 0.2 mg of DWM prot.; the reaction was started by the addition of 5 mM Na-succinate. Naþ was used to avoid depolarization induced by Kþ via PmitoKATP. In the inset, a detail of the traces a and e is reported.

suggesting possible different physiological roles. ROS had no effect on JAM–PIMAC, while JAM–PUCP was found to be strongly activated by ROS (Paventi et al. 2006), as are PUCP and PmitoKATP in other plant mitochondria (Pastore et al. 1999a, Pastore et al. 2000, Considine et al. 2003). Since high mitochondrial (negative inside) opposes electrophoretic anion uptake, the effect of on PIMAC activity was evaluated. As expected, activity was promoted by complete collapse of , but, at a around 110 mV, anion uptake via PIMAC, although inhibited, was still clearly observed under our experimental conditions. Quantification of actual mitochondrial in living cells is very difficult, but it is probably not so high: in perfused rat heart, values ranging from 101 to 145 mV

were determined under different experimental conditions (Wan et al. 1993); in fibroblasts and neuroblastoma cells values of 105 and 81 mV were measured (Kadenbach 2003). Therefore, in in vitro experiments we showed PIMAC activity at a value that might resemble the physiological value, thus suggesting that in vivo also little oscillation may greatly affect PIMAC activity. PIMAC function in plant mitochondria isolated in vitro An important target of this study concerns the mechanism of mitochondrial swelling in iso-osmotic ammonium salt solutions of Pi, dicarboxylates, tricarboxylates and oxodicarboxylates. In DWM, activation by FCCP, sensitivity to modulators of PmitoKATP and insensitivity to


A 0.36 M Sucrose

0.02 A546 nm

+ 5 mM ATP + Oligo/Atr + 1 mM Propranolol

+ 20 µM LA

0.18 M KC1 + 0.5 µg Val. 30 s

B

0.36 M Sucrose + 5 mM ATP + Oligo/Atr

0.02 A546 nm

+ 1 mM Propranolol

+ 20 µM LA 0.12 M K2Succinate + 0.5 µg Val. 30 s 0.36 M Sucrose

C

0.02 A546 nm

+ Xan/XOX + 0.5 mM H2O2

0.18 M KC1 + 0.5 µg Val. + 0.5 mM H2O2

+ Xan/XOX

30 s

Fig. 9 Effect of propranolol, ATP, linoleic acid (LA), hydrogen peroxide and superoxide anion on JAM swelling in iso-osmotic solutions of Kþ anions. Swelling of JAM (0.1 mg prot.) was carried out in the presence of valinomycin as reported in Fig. 1, in KCl (A) and K2succinate (B) solutions in the absence and presence of 5 mM ATP, 20 mM LA or 1 mM propranolol, and in KCl solution (C) in the absence and presence of 0.5 mM H2O2 or the superoxide aniongenerating system (Xan/XOX). Where indicated, the medium also contained 2 mg of oligomycin (Oligo) and 10 mM atractyloside (Atr). In (C), the absorbance in sucrose solution in the absence and presence of H2O2 and Xan/XOX is also shown. No effect on sucrose absorbance was due to all the tested compounds.

1051

Pi (or Pi plus malate) and NEM are inconsistent with the widely accepted mechanism that explains these swellings in intact mammalian/plant mitochondria by (i) NH3 diffusion across the membrane; (ii) Pi influx in symport with Hþ via Pi carrier; (iii) dicarboxylate entry in exchange with Pi by means of dicarboxylate transporter; and (iv) tricarboxylate or oxodicarboxylate entry in exchange with a dicarboxylate (Douce 1985, and references therein, Laloi 1999). In contrast we show that these swellings should occur by electrophoretic anion uptake via PIMAC compensated by NH4þ uptake via PmitoKATP (Fig. 4). This finding is able to clarify some evidence obtained in the past by swelling experiments in ammonium salt solutions of other plant mitochondria (Wiskich 1974, Zogloweck et al. 1988); in these studies the occurrence in plant mitochondria of an electrophoretic Pi-independent uptake of dicarboxylates and tricarboxylates was already suggested, but not explained, the existence of PIMAC and PmitoKATP still being unknown. On the other hand, in DWM, both PIMAC and classical Pi carrier appear to contribute significantly to the overall rate of swelling in NH4Pi; therefore, it should be supposed that in different plant mitochondria, the contribution of the old and new mechanism should be dependent on the relative activity of PmitoKATP, PIMAC and classical carriers. Interestingly, PIMAC and classical energy-dependent carriers may cooperate with each other. In actively succinate respiring mitochondria, the succinate/malate exchange occurs by means of the specific dicarboxylate carrier. On the other hand, malate appearance outside mitochondria is sensitive to propranolol and shows a lag phase. This is in agreement with the proposal of an initial electrophoretic uptake of succinate via PIMAC at low . PIMAC functioning should allow succinate to accumulate inside mitochondria, with consequent oxidation and malate as well as generation; when the malate concentration becomes sufficiently high, the succinate/malate exchange may be activated via the dicarboxylate carrier. The existence of a first phase of substrate uptake followed by a second phase of exchange was already proposed as a general mechanism operating in several transport systems in mitochondria isolated in vitro (Passarella et al. 2003, Di Martino et al. 2006). Hypothesis about PIMAC function in plant mitochondria in vivo PIMAC is believed to contribute to volume homeostasis maintenance (Beavis and Vercesi 1992) and to be involved in anion efflux out of the matrix in energized mitochondria, including the malate efflux in the operation of the malate/oxaloacetate shuttle (Beavis and Vercesi 1992). Anyway, our results suggest that in vivo,

1052


in energized mitochondria, the anion efflux via PIMAC will be inhibited by ATP and anion influx by both ATP and high (although not so high as generally believed). In the light of these observations, PIMAC should play a role under conditions in which mitochondria are depolarized. Mitochondrial depolarization should not be so unusual in vivo; for example, in animal mitochondria, it has been demonstrated that, due to spontaneous opening and closure of permeability transition pore, mitochondria can sometimes be exposed to complete depolarization followed by recovery (Huser and Blatter 1999). In plant mitochondria, depolarization is even more likely to occur in the light of the presence of powerful energy-dissipating systems such as AOX, PmitoKATP and PUCP. In particular, cooperation of AOX with external NAD(P)H dehydrogenases may promote an active non-phosphorylating pathway that may prevent protonmotive force generation and ATP synthesis in plant mitochondria (Møller 2001, and references therein, Pastore et al. 2001). Moreover, it has been recently reported that PmitoKATP and PUCP may be involved in cell adaptation to some abiotic stresses (e.g. drought and salt stress) that may induce oxidative stress at the mitochondrial level (Pastore et al. 2007). In osmotic- and salt stressedDWM, ROS activate PmitoKATP and PUCP activity. This causes an increase of electrophoretic Kþ influx into the mitochondrial matrix as well as uncoupling by PUCP; as a consequence, a decrease of to control large-scale harmful ROS generation is observed according to a feedback mechanism (Trono et al. 2004, Pastore et al. 2007). Furthermore, under the same osmotic and salt stress conditions, a stress intensity-dependent decrease of ATP synthesis was reported in DWM (Flagella et al. 2006), as well as an increase of FFAs that activate PUCP (Trono et al. 2006) and some increase of ROS (Trono et al. 2004) that may impair classical anion carriers (Pastore et al. 2002). On the whole, old and new data suggest that under stress the lowering of and ATP may activate PIMAC, while an increase of FFAs may put a brake on excess activation; simultaneously, a decrease of the protonmotive force and an increase of ROS may contribute to shift anion transport from energy-dependent (and ROSsensitive) anion carriers toward electrophoretic anion transport via PIMAC. In general, we propose that when mitochondria are somehow de-energized displaying low and ATP synthesis, PIMAC may allow, alone or in cooperation with the anion carriers, the rapid exchange of metabolites between mitochondria and other organelles that is fundamental for the maintenance of efficient plant mitochondria function (Fig. 10).

Materials and Methods Chemicals and plant materials All reagents at the highest purity commercially available were purchased from Sigma Chemical Co. (St Louis, MO, USA). All solutions were adjusted to the desired pH with Tris or HCl. Substrates were used as Tris salts at pH 7.20. FCCP, valinomycin, nigericin, oligomycin, linoleate and other FFAs were dissolved in ethanol; DCCD was dissolved in dimethylsulfoxide. Certified seeds of durum wheat (Triticum durum Desf., cv Ofanto) and tubers of Jerusalem artichoke (Helianthus tuberosus L.) were kindly supplied from the Cereal Research Centre (Foggia, Italy). DWM and JAM isolation DWM were purified from 72-h-old etiolated seedlings, as reported in Pastore et al. (1999b) with minor modifications. The grinding and washing buffers were: (i) 0.3 M mannitol, 4 mM cysteine, 1 mM EDTA, 30 mM Tris–HCl (pH 7.50), 0.1% (w/v) defatted bovine serum albumin (BSA), 0.6% (w/v) polyvinylpyrrolidone (PVP)-360; and (ii) 0.3 M mannitol, 1 mM EDTA, 10 mM Tris–HCl (pH 7.40), 0.1% (w/v) defatted BSA, respectively. Washed mitochondria were purified by isopycnic centrifugation in a self-generating density gradient containing 0.3 M sucrose, 10 mM Tris–HCl (pH 7.20) and 28% (v/v) Percoll (colloidal PVPcoated silica, Amersham Pharmacia Biotech) in combination with a linear gradient of 0% (top) to 10% (bottom) PVP-40 (Moore and Proudlove 1987). The final mitochondrial suspension was diluted with an appropriate volume of a sucrose-free washing buffer in order to obtain a 0.3 M sucrose concentration. JAM were isolated essentially as in Liden and Moller (1988) from fresh-cut slices of fully mature tubers in quiescence status, stored at 48C for no more than 3 months. The grinding and washing buffers were: (i) 0.4 M sucrose, 1 mM EDTA, 30 mM Tris–HCl (pH 8.20), 0.1% (w/v) defatted BSA, 0.05% (w/v) cysteine; and (ii) 0.3 M sucrose, 1 mM EDTA, 10 mM Tris–HCl (pH 7.40), 0.1% (w/ v) defatted BSA, 0.05% (w/v) cysteine, respectively. Purified DWM and isolated JAM showed 95% intactness of the inner membrane, and 90 and 93% intactness of the outer membrane, respectively (Pastore et al. 1999b, Paventi et al. 2006). The intactness of inner and outer mitochondrial membranes was determined as in Douce et al. (1987). Both DWM and JAM proved to be tightly coupled: using 10 mM 2-oxoglutarate as a substrate, DWM show respiratory control and ADP/O ratios equal to 6.7 1.4 and 3.6 0.15 (n ¼ 4), respectively (Pastore et al. 1999b); using 5 mM succinate as a substrate, JAM show respiratory control and ADP/O ratios equal to 2.6 0.24 and 2.0 0.1 (n ¼ 5), respectively (Paventi et al. 2006). Oxygen uptake was measured at 258C by means of a GILSON Oxygraph model 5/6-servo Channel pH 5, equipped with a Clark-type electrode (5331 YSI, Yellow Spring, OH, USA), in a medium consisting of 0.3 M mannitol, 5 mM MgCl2, 10 mM KCl, 0.1% (w/v) defatted BSA and 10 mM potassium Pi buffer (pH 7.20). DWM and JAM protein content was determined by the method of Lowry modified according to Harris (1987), using BSA as a standard. Swelling experiments Swelling experiments were performed as described by Pastore et al. (1999a). Absorbance changes at 546 nm of a DWM or JAM suspension (0.05 mg ml–1) in different iso-osmotic media were monitored at 258C as a function of time by means of a Perkin


∆ψ

A H+

Pi

Dic

DpH ATP Pi ADP

Tric

1053

H+ ATPase

matrix SH2

S

O2 H2O

F1 PiC

H+

DTP

Pi

CTP

Dic

PIMAC

Tric

ANT F0

PUCP respiratory AOX i.m.m. PmitoKATP

chain H+

H+

ADP

ATP

H+ H+

∆ψ

B

H+

intermembrane space

DpH matrix

H+

H+

Pi

Dic

Tric

ATPase F1

PiC

H+

H+

DTP

CTP

PIMAC

ANT F0

SH2 PUCP/FFAs PmitoKATP

S

respiratory AOX i.m.m. chain H+

Pi

Dic

Tric

O2 H2O

H+

intermembrane space

Fig. 10 Mitochondrial uptake of Pi, dicarboxylates (Dic) and tricarboxylates (Tric) in conditions of high or low and ATP. (A) Under conditions in which high is generated by the respiratory chain and ATP is synthesized at a high rate, PIMAC is inactive due to ATP inhibition and regulation; metabolically relevant anions are transported by the specific energy-dependent carriers [in the figure, Pi, Dic and Tric carriers are reported, but it should be noted that the existence of a Dic–Tric carrier protein has also been suggested in plant mitochondria (Picault et al. 2002)]. (B) When is lowered by the functioning of AOX, PmitoKATP or PUCP and ATP synthesis is limited, anion transport can be mediated by PIMAC. This may occur under some stress conditions. PiC, Pi carrier; DTP, dicarboxylate transport protein; CTP, citrate transport protein or tricarboxylate transporter (Laloi 1999); PIMAC, plant inner membrane anion channel; ANT, adenine nucleotide translocator; PUCP, plant uncoupling protein; FFAs, free fatty acids; PmitoKATP, plant mitochondrial ATP-dependent Kþ channel; AOX, alternative oxidase; SH2, reduced substrates; S, oxidized substrates; i.m.m., inner mitochondrial membrane.

Elmer 18 UV/VIS spectrometer (Perkin Elmer, Wellesley, MA, USA). The iso-osmotic solutions (2 ml), buffered with 20 mM Tris– HCl (pH 7.20), contained: (i) sucrose; or (ii) Kþ or NH4þ salts of the following anions: chloride, succinate, malate, oxaloacetate, 2-oxoglutarate, citrate, cis-aconitate and Pi. Swelling experiments in Kþ salts were performed in the presence of valinomycin, a specific ionophore that allows Kþ permeability across the inner mitochondrial membrane. In preliminary experiments in Kþ salts we verified that anion uptake became the limiting step of the swelling rate, when 5 mg mg–1 prot. valinomycin or higher concentrations were used. Thus, the swelling rate in Kþ salt solutions in the presence of 5 mg mg–1 prot. valinomycin depends on the rate of anion uptake. Fluorimetric measurements of electrical membrane potential ( ) changes The fluorescent probe safranin O was used to estimate changes, as reported by Moore and Bonner (1982). Safranin fluorescence changes (ex ¼ 520 nm, em ¼ 570 nm) were monitored at 258C by means of a Perkin-Elmer LS-50B spectrofluorimeter. Measurements were carried out in 2 ml of an iso-osmotic solution

consisting of 20 mM Tris–HCl (pH 7.20), 2.5 mM safranin O and either (NH4)2succinate or sucrose plus 5 mM Na-succinate; the reaction was started by the addition of 0.1 mg ml–1 DWM prot. ([safranin O]/[DWM prot.] ratio value of 25). In another set of experiments, measurements were carried out in 2 ml of a medium consisting of 0.3 M mannitol, 5 mM MgCl2, 20 mM Tris–HCl (pH 7.20), 2.5 mM safranin O and 0.1 mg ml–1 DWM prot.; the reaction was started by the addition of 5 mM Na-succinate. Calibration of as a function of safranin O fluorescence decrease in DWM was carried out by using safranin O response as a function of Kþ diffusion potential in rat liver mitochondria as reported by Zottini et al. (1993). Rat liver mitochondria were isolated according to Pastore et al. (1994); the Kþ diffusion potential in rat liver mitochondria was induced by the addition of 0.05 mg ml–1 valinomycin (A˚kerman and Wikstro¨m, 1976). Spectrophotometric measurements of succinate/malate exchange The succinate/malate exchange was monitored essentially as in Atlante et al. (1998) and Fratianni et al. (2001), by measuring the rate of malate appearance in the extramitochondrial phase due to succinate addition to DWM. This was done by monitoring

1054


spectrophotometrically the absorbance increase at 340 nm due to NADPH ("340 ¼ 6.22 mM–1 cm–1) generated by the MDS consisting of 0.4 mM NADPþ and 0.2 EU malic enzyme (EC 1.1.1.40) from chicken liver (Sigma M-5257), previously dialyzed against 100 mM Tris–HCl (pH 7.20). DWM (0.2 mg prot.) were incubated at 258C in 2 ml of a medium containing 0.3 M mannitol, 5 mM MgCl2, 20 mM Tris–HCl (pH 7.20). Then MDS was added to the reaction medium and the reaction was started by adding 5 mM Nasuccinate. O2– production O2– was generated as described in Pastore et al. (1999a, 2000), by a system consisting of 0.1 mM xanthine (Xan) plus the amount of xanthine oxidase (XOX) (xanthine: oxygen oxidoreductase, EC 1.1.3.22) from buttermilk (Sigma X-4376) required to generate O2– at an initial rate of 20 nmol min–1 (usually about 100–200 mg). The rate of O2– generation was determined oxygraphically by measuring the rate of oxygen consumption due to xanthine oxidase reaction at 258C in 1 ml of a medium consisting of 0.3 M mannitol, 5 mM MgCl2, 20 mM Tris–HCl (pH 7.20).

Funding Ministero dell’Istruzione, dell’Universita´ e della Ricerca (MIUR) ‘AGROGEN’ and Ministero delle Politiche Agricole e Forestali (MiPAF) ‘SICERME’.

Acknowledgments We gratefully acknowledge the skilful cooperation of Dr. Marianna Pompa, who participated as a student in this work, and of Professor Salvatore Passarella for stimulating discussions at the early stage of this work.

References A˚kerman, E.O.K. and Wikstro¨m, K.F.M. (1976) Safranine as a probe of the mitochondrial membrane potential. FEBS Lett. 68: 191–197. Atlante, A., Gagliardi, S. and Passarella, S. (1998) Fumarate permeation in normal and acidotic rat kidney mitochondria: fumarate/malate and fumarate/aspartate translocators. Biochem. Biophys. Res. Commun. 243: 711–718. Ballarin, C. and Sorgato, M.C. (1995) An electrophysiological study of yeast mitochondria. Evidence for two inner membrane anion channels sensitive to ATP. J. Biol. Chem. 270: 19262–19268. Ballarin, C. and Sorgato, M.C. (1996) Anion channel of the inner membrane of mammalian and yeast mitochondria. J. Bioenerg. Biomembr. 28: 125–130. Beavis, A.D. (1989a) On the inhibition of the mitochondrial inner membrane anion uniporter by cationic amphiphiles and other drugs. J. Biol. Chem. 264: 1508–1515. Beavis, A.D. (1989b) The mitochondrial inner-membrane anion channel possesses two mercurial-reactive regulatory sites. Eur. J. Biochem. 185: 511–519. Beavis, A.D. (1992) Properties of the inner membrane anion channel in intact mitochondria. J. Bioenerg. Biomembr. 24: 77–90. Beavis, A.D. and Powers, M.F. (1989) On the regulation of the mitochondrial inner membrane anion channel by magnesium and protons. J. Biol. Chem. 264: 17148–17155. Beavis, A.D. and Powers, M. (2004) Temperature dependence of the mitochondrial inner membrane anion channel. J. Biol. Chem. 279: 4045–4050.

Beavis, A.D. and Vercesi, A.E. (1992) Anion uniport in plant mitochondria is mediated by a Mg2þ-insensitive inner membrane anion channel. J. Biol. Chem. 267: 3079–3087. Casolo, V., Braidot, E., Chiandussi, E., Vianello, A. and Macrı` , F. (2003) KþATP channel opening prevents succinate-dependent H2O2 generation by plant mitochondria. Physiol. Plant. 118: 313–318. Casolo, V., Petrussa, E., Krajnˇa´kova´, J., Macrı` , F. and Vianello, A. (2005) Involvement of the mitochondrial KþATP channel in H2O2- or NO-induced programmed death of soybean suspension cell cultures. J. Exp. Bot. 56: 997–1006. Chiandussi, E., Petrussa, E., Macrı` , F. and Vianello, A. (2002) Modulation of a plant mitochondrial KþATP channel and its involvement in cytochrome c release. J. Bioenerg. Biomembr. 34: 177–184. Considine, M.J., Goodman, M., Echtay, K.S., Laloi, M., Whelan, J., Brand, M.D. and Sweetlove, L.J. (2003) Superoxide stimulates a proton leak in potato mitochondria that is related to the activity of uncoupling protein. J. Biol. Chem. 278: 22298–22302. Di Martino, C., Pizzuto, R., Pallotta, M.L., De Santis, A. and Passarella, S. (2006) Mitochondrial transport in proline catabolism in plants: the existence of two separate translocators in mitochondria isolated from durum wheat seedlings. Planta 223: 1123–1133. Douce, R., Bourguignon, J., Brouquisse, R. and Neuburger, M. (1987) Isolation of plant mitochondria: general principles and criteria of integrity. Methods Enzymol. 148: 403–415. Douce, R. (1985) Composition and function of plant mitochondrial membranes. In Mitochondria in Higher Plants. Structure, Function, and Biogenesis. pp. 125–146. Academic Press, Orlando. Flagella, Z., Trono, D., Pompa, M., Di Fonzo, N. and Pastore, D. (2006) Seawater stress applied at germination affects mitochondrial function in durum wheat (Triticum durum) early seedlings. Funct. Plant Biol. 33: 357–366. Fratianni, A., Pastore, D., Pallotta, M.L., Chiatante, D. and Passarella, S. (2001) Increase of membrane permeability of mitochondria isolated from water stress adapted potato cells. Biosci. Rep. 21: 81–91. Garlid, K.D. and Beavis, A.D. (1986) Evidence for the existence of an inner membrane anion channel in mitochondria. Biochim. Biophys. Acta 853: 187–204. Halle-Smith, S.C., Murray, A.G. and Selwyn, M.J. (1988) Palmitoyl-CoA inhibits the mitochondrial inner membrane anion-conducting channel. FEBS Lett. 236: 155–158. Hanson, J.B. and Koeppe, D.E. (1975) Mitochondria. In Ion Transport in Plant Cells and Tissues. Edited by Baker, D.A. and Hall, J.L. pp. 79–99. North Holland Publishing Co., Amsterdam. Harris, D.A. (1987) Spectrophotometric assays. In Spectrophotometry and Spectrofluorimetry: A Practical Approach. Edited by Bashford, C.L. and Harris, D.A. pp. 59–61. IRL Press, Oxford. Hensley, J.R. and Henson, J.B. (1975) The action of valinomycin in uncoupling corn mitochondria. Plant Physiol. 56: 13–18. Hu¨ser, J. and Blatter, L.A. (1999) Fluctuations in mitochondrial membrane potential caused by repetitive gating of the permeability transition pore. Biochem. J. 343: 311–317. Kadenbach, B. (2003) Intrinsic and extrinsic uncoupling of oxidative phosphorylation. Biochim. Biophys. Acta 1604: 77–94. La Noue, K.F. and Schoolwerth, A.C. (1984) Metabolite transport in mammalian mitochondria. In Bioenergetics. Edited by Ernster, L. pp. 221–226. Elsevier, Amsterdam. Laloi, M. (1999) Plant mitochondrial carriers: an overview. Cell Mol. Life Sci. 56: 918–944. Liden, A.C. and Møller, I.M. (1988) Purification, characterization and storage of mitochondria from Jerusalem artichoke tubers. Physiol. Plant. 72: 265–270. Liu, G., Hinch, B., Davatol-Hag, H., Lu, Y., Powers, M. and Beavis, A.D. (1996) Temperature dependence of the mitochondrial inner membrane anion channel. The relationship between temperature and the inhibition by protons. J. Biol. Chem. 271: 19717–19723. Lurin, C., Gu¨clu¨, J., Cheniclet, C., Carde, J.P., Barbier-Brygoo, H. and Maurel, C. (2000) CLC-Nt1, a putative chloride channel protein of tobacco, co-localizes with mitochondrial membrane markers. Biochem. J. 348: 291–295.

PIMAC function in plant mitochondria Møller, I.M. (2001) Plant mitochondria and oxidative stress: electron transport, NADPH turnover, and metabolism of reactive oxygen species. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52: 561–591. Moore, A.L. and Bonner, W.D., Jr (1982) Measurement of membrane potential in plant mitochondria with the safranine method. Plant Physiol. 70: 1271–1276. Moore, A.L. and Proudlove, M.O. (1987) Purification of plant mitochondria on silica sol gradients. Methods Enzymol. 148: 415–420. O’Rourke, B. (2000) Pathophysiological and protective roles of mitochondrial ion channels. J. Physiol. 529: 23–36. Passarella, S., Atlante, A., Valenti, D. and de Bari, L. (2003) The role of mitochondrial transport in energy metabolism. Mitochondrion 2: 319–343. Pastore, D., Di Pede, S. and Passarella, S. (2003) Isolated durum wheat mitochondria and potato cell mitochondria oxidize externally added NADH mostly via the malate/oxaloacetate shuttle with a rate that depends on the carrier-mediated transport. Plant Physiol. 133: 2029–2039. Pastore, D., Fratianni, A., Di Pede, S. and Passarella, S. (2000) Effect of fatty acids, nucleotides and reactive oxygen species on durum wheat mitochondria. FEBS Lett. 470: 88–92. Pastore, D., Greco, M., Petragallo, V.A. and Passarella, S. (1994) Increase in Hþ/e– ratio of the cytochrome c oxidase reaction in mitochondria irradiated with helium–neon laser. Biochem. Mol. Biol. Int. 34: 817–826. Pastore, D., Laus, M.N., Di Fonzo, N. and Passarella, S. (2002) Reactive oxygen species inhibit the succinate oxidation-supported generation of membrane potential in wheat mitochondria. FEBS Lett. 516: 15–19. Pastore, D., Stoppelli, M.C., Di Fonzo, N. and Passarella, S. (1999a) The existence of the Kþ channel in plant mitochondria. J. Biol. Chem. 274: 26683–26690. Pastore, D., Trono, D., Laus, M.N., Di Fonzo, N. and Flagella, Z. (2007) Possible plant mitochondria involvement in cell adaptation to drought stress. A case study: durum wheat mitochondria. J. Exp. Bot. 58: 195–210. Pastore, D., Trono, D., Laus, M.N., Di Fonzo, N. and Passarella, S. (2001) Alternative oxidase in durum wheat mitochondria. Activation by pyruvate, hydroxypyruvate and glyoxylate and physiological role. Plant Cell Physiol. 42: 1373–1382. Pastore, D., Trono, D. and Passarella, S. (1999b) Substrate oxidation and ADP/ATP exchange in coupled durum wheat (Triticum durum Desf.) mitochondria. Plant Biosyst. 133: 219–228. Paventi, G., Pastore, D., Bobba, A., Pizzuto, R., Di Pede, S. and Passarella, S. (2006) Plant uncoupling protein in mitochondria from aged-dehydrated slices of Jerusalem artichoke tubers becomes sensitive to superoxide and to hydrogen peroxide without increase in protein level. Biochimie 88: 179–188. Petrussa, E., Bertolini, A., Krajnˇa´kova´, J., Casolo, V., Macrı` , F. and Vianello, A. (2008a) Isolation of mitochondria from embryogenic cultures of Picea abies (L.) Karst. and Abies cephalonica Loud.: characterization of K(ATP) (þ) channel. Plant Cell Rep. 27: 137–146. Petrussa, E., Casolo, V., Braidot, E., Chiandussi, E., Macrı` , F. and Vianello, A. (2001) Cyclosporin A induces the opening of a potassiumselective channel in higher plant mitochondria. J. Bioenerg. Biomembr. 33: 107–117. Petrussa, E., Casolo, V., Peresson, C., Braidot, E., Vianello, A. and Macrı` , F. (2004) The KþATP channel is involved in a low-amplitude permeability transition in plant mitochondria. Mitochondrion 3: 297–307. Petrussa, E., Casolo, V., Peresson, C., Krajnˇa´kova´, J., Macrı` , F. and Vianello, A. (2008b) Activity of a K(ATP)(þ) channel in Arum spadix mitochondria during thermogenesis. J. Plant Physiol. doi:10.1016/ j.jplph.2007.10.012.

1055

Picault, N., Palmieri, L., Pisano, I., Hodges, M. and Palmieri, F. (2002) Identification of a novel transporter for dicarboxylates and tricarboxylates in plant mitochondria. Bacterial expression, reconstitution, functional characterization, and tissue distribution. J. Biol. Chem. 277: 24204–24211. Powers, M.F. and Beavis, A.D. (1991) Triorganotins inhibit the mitochondrial inner membrane anion channel. J. Biol. Chem. 266: 17250–17256. Powers, M.F., Smith, L.L. and Beavis, A.D. (1994) On the relationship between the mitochondrial inner membrane anion channel and the adenine nucleotide translocase. J. Biol. Chem. 269: 10614–10620. Ruy, F., Vercesi, A.E., Andrade, P.B.M., Bianconi, M.L., Chaimovich, H. and Kowaltowski, A.J. (2004) A highly active ATP-insensitive Kþ import pathway in plant mitochondria. J. Bioenerg. Biomembr. 36: 195–202. Scho¨nfeld, P., Gerke, S., Bohnensack, R. and Wojtczak, L. (2003) Stimulation of potassium cycling in mitochondria by long-chain fatty acids. Biochim. Biophys. Acta 1604: 125–133. Scho¨nfeld, P., Sayeed, I., Bohnensack, R. and Siemen, D. (2004) Fatty acids induce chloride permeation in rat liver mitochondria by activation of the inner membrane anion channel (IMAC). J. Bioenerg. Biomembr. 36: 241–248. Scho¨nfeld, P., Schlu¨ter, T., Schu¨ttig, R. and Bohnensack, R. (2001) Activation of ion-conducting pathways in the inner mitochondrial membrane—an unrecognized activity of fatty acid? FEBS Lett. 491: 45–49. Scho¨nfeld, P., Schu¨ttig, R. and Wojtczak, L. (2002) Rapid release of Mg(2þ) from liver mitochondria by nonesterified long-chain fatty acids in alkaline media. Arch. Biochem. Biophys. 403: 16–24. Scho¨nfeld, P., Wieckowski, M.R. and Wojtczak, L. (2000) Long-chain fatty acid-promoted swelling of mitochondria: further evidence for the protonophoric effect of fatty acids in the inner mitochondrial membrane. FEBS Lett. 471: 108–112. Trono, D., Flagella, Z., Laus, M.N., Di Fonzo, N. and Pastore, D. (2004) The uncoupling protein and the potassium channel are activated by hyperosmotic stress in mitochondria from durum wheat seedlings. Plant Cell Environ. 27: 437–448. Trono, D., Soccio, M., Mastrangelo, A.M., De Simone, V., Di Fonzo, N. and Pastore, D. (2006) The transcript levels of two plant mitochondrial uncoupling protein (pUCP)-related genes are not affected by hyperosmotic stress in durum wheat seedlings showing an increased level of pUCP activity. Biosci. Rep. 26: 251–261. Vanden Hoek, T.L., Becker, L.B., Shao, Z., Li, C. and Schumacker, P.T. (1998) Reactive oxygen species released from mitochondria during brief hypoxia induce preconditioning in cardiomyocytes. J. Biol. Chem. 273: 18092–18098. Vercesi, A.E., Martins, I.S., Silva, M.A.P., Leite, H.M.F., Cuccovia, I.M. and Chaimovich, H. (1995) PUMPing plants. Nature 375: 24. Wan, B., Doumen, C., Duszynski, J., Salama, G., Vary, T.C. and La Noue, K.F. (1993) Effects of cardiac work on electrical potential gradient across mitochondrial membrane in perfused rat hearts. Amer. J. Physiol. 265: H453–H460. Warhurst, I.W., Dawson, A.P. and Selwyn, M.J. (1982) Inhibition of electrogenic anion entry into rat liver mitochondria by N,N0 -dicyclohexylcarbodiimide. FEBS Lett. 149: 249–252. Wiskich, J.T. (1974) Substrate transport into plant mitochondria: swelling studies. Aust. J. Plant Physiol. 1: 177–181. Zoglowek, C., Kro¨mer, S. and Heldt, H.W. (1988) Oxaloacetate and malate transport by plant mitochondria. Plant Physiol. 87: 109–115. Zottini, N., Mandolino, G. and Zannoni, D. (1993) Oxidation of external NAD(P)H by mitochondria from taproots and tissue cultures of sugar beet (Beta vulgaris). Plant Physiol. 102: 579–585.

(Received April 30, 2008; Accepted May 23, 2008)