Transport pathways--proton motive force interrelationship in durum wheat mitochondria
These results are also in line with the frequently reported inefficiency of purine nucleotides in inhibiting the induced activity of UCPs [92]. In this regard, it is widely accepted that plants suffering from biotic or abiotic stress often show an over-reduction of electron carriers such as ubiquinone (Q), that causes electron leakage from the system with consequent ROS generation [93]; under these conditions, a decrease of PUCP sensitivity to purine nucleotides may help to better control ROS.
In the whole, modulation of PUCP is similar to the one of PmitoKATP, thus suggesting a possible reinforcement of the feedback control described for potassium channel (Figure 2). So, DWM appear to be ready to dissipate pmf in order to avoid ROS over-production. Anyway, the loss of ΔΨ and ΔpH may modulate transports by impairing carriers, but at the same time also unlock PIMAC activity (Figure 2 and Section 3.3).
The activity of inner membrane anion channels (IMAC) has been studied in liver and heart mitochondria [94]. 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, ATP etc., and it has been demonstrated to play crucial functions, such as the regulation of the mitochondrial volume and respiratory rates [95], and the control of the superoxide traffic among mitochondria [96].
In plant cells, the occurrence of the plant inner membrane anion channel (PIMAC) has been demonstrated in mitochondria from tubers of potato [40] and topinambur [39], and seedlings of durum wheat [39] and maize [38]. As previously observed for IMAC of mammalian mitochondria [97], evidence has been reported in seedlings of maize that a chloride channel protein, namely the ZmCLCc, can perform the PIMAC activity [42].
Similarly to mitochondria from tubers of potato and topinambur, DWM present a clearly evident swelling when suspended in iso-osmotic solutions of KCl or K+ salts of dicarboxylates, tricarboxylates, oxodicarboxylates and Pi (but not ATP), thus indicating that DWM are specifically permeable to both K+ and counteranions. As stated above, the K+ uniport in DWM is catalyzed by the PmitoKATP [44]; as far as the anion transport, it is strongly inhibited by propranolol, a specific IMAC and PIMAC inhibitor [40,98], thus showing that DWM–PIMAC can mediate the electrophoretic transport of these metabolically relevant anions. Chloride, succinate, malate, oxaloacetate, 2-oxoglutarate, citrate and cis-aconitate are transported at high rate by DWM-PIMAC, while Pi is also transported via PIMAC but at a rate 3- to 3.5-fold lower with respect to the other anions [39].
Regarding modulator sensitivity, DWM-PIMAC presents many of the characteristics of other animal and plant anion channels; in addition to the inhibition by propranolol, DWM-PIMAC is inhibited also by tributyltin. On the other hand, DWM-PIMAC, similarly to other plant anion channels, differs from IMAC in other important properties; notably, it is insensitive to mercurials and N,N′-dicyclohexylcarbodiimide at concentrations which inhibit IMAC [95]. As far as physiological modulators, DWM-PIMAC, similarly to PIMAC from potato and topinambur tubers, is not affected by Mg2+, which represents the most important inhibitor of IMAC [99]; on the contrary, it is inhibited, but not completely inactivated, by physiological concentrations of ATP [39,87], that acts at the outer side of the inner mitochondrial membrane; moreover, PIMAC is activated by alkaline pH, mainly at the matrix side [39]. To date, no information is available regarding the ATP sensitivity of the anion channels 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 [100,101]. As for FFAs, several unsaturated and, to a lesser extent, saturated FFAs inhibit DWM-PIMAC, while acyl-CoAs, that were found to exert an inhibitory action on IMAC of rat liver mitochondria [102], had no effect on DWM-PIMAC; moreover, no effect on DWM-PIMAC, as well as on PIMAC of topinambur tubers, has been observed by ROS [39].
As expected, the PIMAC-mediated electrophoretic anion flux inside DWM is inhibited by high ΔΨ values, while it is fully promoted by a complete ΔΨ depolarization; nevertheless, at a ΔΨ of about 110 mV, an anion uptake through PIMAC, although inhibited, has been observed. In this regard, in vivo determination under different physiological conditions has shown the existence of low ΔΨ values in mitochondria from both plant [103] and animal [104–106] sources, thus suggesting that in vivo a PIMAC activity occurs at ΔΨ values that resemble the physiological ones, and that also little ΔΨ oscillations may greatly affect PIMAC activity.
Interestingly, DWM also swell in iso-osmotic solutions of NH4 + salts of chloride, malate, oxaloacetate, 2-oxoglutarate, citrate, cis-aconitate and Pi in a manner involving PmitoKATP and PIMAC. This is suggested by some observations. Firstly, none of the dicarboxylate, tricarboxylate and oxodicarboxylate swellings were found to be enhanced by Pi (or Pi plus malate), which is essential to trigger the swelling in intact mammalian/plant mitochondria according to the classical scheme reported in Figure 3A(I–III). NH3 diffusion across the membrane, Pi symport with proton via Pi carrier (I), dicarboxylate entry in exchange with Pi via the DIC (I and II) and tricarboxylate or oxodicarboxylate entry in exchange with the dicarboxylate via DTC in plants (I, II and III). Secondly, all these swellings are strongly inhibited by the PIMAC inhibitor propranolol, but not by the carrier inhibitor mersalyl. Finally, an increase in the rate of swelling in NH4 + salt is generally observed in the presence of the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) [39]; FCCP allows H+ entry into the matrix in competition with PiC; this activation is inconsistent with mechanisms in Figure 3A(I–III).
These findings allow explanation of some previously incomprehensible behaviors of plant mitochondria swelling observed in the past ([1], and references therein, [107]), thus also assessing that the classical interpretation of swelling experiments should be revised in plant mitochondria possessing active PmitoKATP and PIMAC. Swelling experiments of DWM in NH4 + salts show that transport mode of Figure 3B is much more active than that involving DIC and DTC [39]; in any case, Pi transport merits specification. It is not activated by FCCP, but even slightly inhibited; moreover, DWM incubation in the presence of NEM, a known inhibitor of Pi carrier ([108] and references therein) allows some (20%) inhibition. These observations suggest that both PIMAC and Pi carrier contribute significantly to the overall rate of swelling in NH4Pi in DWM and presumably in other plant mitochondria.
A central role of mitochondria is to supply the cell with the ATP produced by oxidative phosphorylation. To do this, mitochondria have an ADP/ATP carrier (AAC), which exchanges cytosolic ADP with matrix ATP [109]; due to its central role in cell energetics, the AAC represents the most abundant carrier in the inner mitochondrial membrane [110]. The exchange involves ingoing ADP3− and outgoing ATP4−, thus exploiting mitochondrial ΔΨ as driving force.
In plants, the existence of genes encoding AAC putative proteins has been reported for different species; in particular, three genes (AAC1, AAC2 and AAC3) encoding different ACC isoforms and up to seven AAC-like genes have been identified in Arabidopsis thaliana, while two ACC genes and two AAC-like genes have been identified in the monocot species Brachypodium distachyon [7]. Investigations carried out on purified ACC from maize [111] and topinambur [112], as well as on recombinant AAC proteins and intact mitochondria from both potato and Arabidopsis thaliana [6] have revealed that, in analogy with the animal counterpart, the activity of the plant mitochondrial AAC is strongly reduced in the presence of bongkrekic acid and/or carboxyatractyloside (CATR), two specific AAC inhibitors [6]; moreover, compared to the nucleotide affinity of mammalian AACs (Km ranging between 40 and 140 μM), a significantly higher external affinity for ADP and ATP has been measured both in purified (Km ranging between 17 and 26 μM) and in recombinant AACs (Km ranging between 10 and 22 μM), as well as for adenine nucleotide transport in intact mitochondria (Km ranging between 1 and 2 μM).
As for DWM, evidence has been reported that these mitochondria are able to synthesise ATP from externally added ADP, and to export the newly synthesised ATP outside mitochondria [113]. In DWM translocation is the rate-limiting step of ATP appearance outside mitochondria; so, the ADP/ATP translocator can regulate the rate of ATP synthesis. Similarly to the adenine nucleotide transport from other plant mitochondrial systems, the ADP/ATP translocator in DWM displays a Km for the ADP significantly lower (21.7 ± 3.06 μM) than that reported for mammalian AACs. As far as sensitivity to inhibitors, the ADP/ATP exchange in DWM is inhibited by both atractyloside (ATR) and CATR, up to a complete stop at 3 μM CATR and 10 μM ATR [68]. In particular, incubation of DWM with 3 μM CATR causes a strong inhibition of the initial ATP efflux from mitochondria followed by a complete stop after few minutes, while incubation with 10 μM ATR completely prevents the ATP efflux. In addition to its main function of exchanging intra-mitochondrial ATP with the cytosolic ADP, evidence has been reported that the ADP/ATP carrier is also involved in the uncoupling effect of low concentrations of FFAs, raising the hypothesis that it is able to translocate the FFA anion by virtue of its role as a transporter of anionic substrates [114]. The effect is weak in liver and stronger in muscle mitochondria, which have a higher AAC content, but, in any case, it is weak as compared to uncouplers or to UCP [8]. Similar evidence has been reported also for mitochondria of different plant species. In pea stem and sunflower hypocotyls, CATR addition to mitochondria causes a weak (few mV) restoration of ΔΨ in organelles partially uncoupled by palmitate addition [115], but no ΔΨ recovery is observed at high FFA concentrations [116].
A more evident effect has been observed in durum wheat, in which the addition of CATR or ATR to mitochondria completely depolarised by a saturating linoleate concentration induces a ΔΨ recovery of about 25 mV [68]. So, although to a limited extent, the AAC carrier may cooperate with PUCP and PmitoKATP in the control of ΔΨ.
Uptake of Pi into mitochondria through a Pi/OH− antiport [117] or a Pi/H+ symport [118] is essential for the oxidative phosphorylation of ADP to ATP [119]. Inside the matrix, Pi plays also additional roles, since it allows the uptake of other metabolites via exchange mechanisms catalysed by other mitochondrial transporters [2,5]. Recently, involvement of mitochondrial Pi carrier (MPC/PiC) in the Pi activation of the permeability transition pore opening has been also postulated [120]. The electroneutral Pi transport is driven by the pH gradient across the membrane, which is generated by the mitochondrial electron transport chain, to accumulate high levels of Pi in the matrix.
In plants, the first gene encoding a putative PiC has been cloned from birch (Betula pendula Roth) [121]. Afterwards, cDNAs encoding the mitochondrial PiC have been isolated from various species including Arabidopsis thaliana, in which three PiC genes have been identified, AtMPT1, AtMPT2 and AtMPT3, also named AT2, AT3, AT5 and PHT3;3, PHT3;2, PHT3;1 ([122], and references therein). The existence of PiC activity in plant mitochondria has been demonstrated by means of biochemical approaches; indeed, evidence has been reported that soybean recombinant PiC protein reconstituted in liposomes presents high Pi transport activity inhibited by NEM, like those of mammalian Pi transporters [123]. As far as Arabidopsis thaliana, the functional complementation strategy has demonstrated that two putative PiC proteins can restore Pi transport activity in a yeast mutant lacking the endogenous PiC [28].
As far as DWM, these mitochondria possess PiC since they swell in an iso-osmotic solution of NH4Pi in a NEM-sensitive way [39,44]. Consistently, Pi addition to succinate-energized mitochondria causes a slight increase of ΔΨ [44], as expected if Pi enters via proton-compensated symport [124,125].
Evidence has been reported in the literature supporting the idea that, among the members of the MCF, there are two types of carrier proteins potentially involved in succinate transport in plant mitochondria: the SFC and the DICs.
In the Arabidopsis thaliana genome, a gene encoding a SFC, named SFC1 has been identified by the functional complementation strategy in the yeast mutant, characterized by a deletion of the gene acr1 that encodes this transporter [16]. ACR1 has the highest affinities for succinate and fumarate, but also recognizes a broad spectrum of di- and tricarboxylates, such as oxoglutarate, oxalacetate, malate, phosphoenolpyruvate, citrate or isocitrate [15]. The substrate specificity of the recombinant plant SFC1 has not been analyzed in detail to date, but it has been suggested that the plant carrier SFC1 may act in a similar way to the yeast ACR1.
As far as DICs, three potential homologues of mammalian and yeast mitochondrial DICs, designated as DIC1, DIC2 and DIC3, have been identified in Arabidopsis thaliana [13]. Characterization of the recombinant and reconstituted DIC isoforms has revealed that they present characteristics that resemble those of the non-plant organisms. Notably, similarly to animal and yeast, plant DICs transport a wide range of dicarboxylic acids including succinate and malate, and their activity is affected by phenylsuccinate, an impermeable dicarboxylate analogue, which has been found to markedly inhibit the activity of DIC1 and DIC2 and, to a lesser extent, that of DIC3 [13]. It is likely that DICs have an anaplerotic function, that allows transport of dicarboxylic acids into mitochondria, which are then used as respiratory substrates; this function is supported by evidence that succinate and malate oxidation by plant mitochondria is inhibited by DIC inhibitors ([13], and references therein).
Consistent with these findings, we have reported evidence that succinate is oxidized by DWM and that in these mitochondria transport across the inner mitochondrial membrane represents the limiting step of succinate oxidation; also in this case, competitive inhibition of the transport by phenylsuccinate (Ki ~0.63 mM, [126]) suggests an involvement of DICs. A similar result has been obtained in mitochondria from potato cell culture, in which succinate has been found to enter mitochondria in exchange with malate [53], thus further supporting the idea that, in plant mitochondria, succinate transport may occur via DICs.
Our findings show that in DWM succinate uptake is inhibited by ROS after a few minutes of exposure; indeed, in the presence of the superoxide anion producing system, xanthine/xanthine oxidase system, a mixed inhibition has been observed in the succinate-induced ΔΨ generation [126]. The inhibition of succinate transport in DWM is not dependent on the interaction between ROS and thiol groups; on the basis of the mixed nature of the inhibition, it is feasible that the inhibition could depend on the interaction of ROS with a variety of mitochondrial components, including protein carrier domains and membrane lipids close to the carrier. As far as the physiological implications of such an inhibition, it is likely that the impairment of succinate translocator by ROS may act as a defence mechanism by reducing the energy metabolism and the consequent large-scale ROS generation by mitochondria during environmental/oxidative stress in plants.
Interestingly, in DWM, succinate/malate exchange consists of two phases: the first phase mediated exclusively by PIMAC, that, at low ΔΨ, favours the accumulation of succinate inside mitochondria with consequent oxidation to malate as well as ΔΨ generation; this triggers the second phase in which succinate is exchanged with malate through the DIC; during this second phase the contribution of PIMAC should be negligible due to the high ΔΨ. So, PIMAC and energy-dependent carriers may cooperate with each other. This result is not unique; the existence of a first phase of substrate uptake followed by a second phase of exchange has been already proposed as a general mechanism that operates in several transport systems both in animal [127] and plant mitochondria [43].
In addition to their function in the exchange of organic acid intermediates that connects the Krebs cycle with diverse metabolic processes (i.e., gluconeogenesis, glyoxylate cycle, synthesis of amino acids and nucleic acids), DICs may function as malate/oxaloacetate shuttle providing other cell compartments with reducing equivalents [13]. Because of the redox gradient between the NADH/NAD+ systems in the mitochondria and cytosol, the malate/oxaloacetate shuttle is thought to be more aligned energetically to export NADH from mitochondria than to import it. This could be important in photosynthetic tissues, in which the mitochondrial malate/oxaloacetate shuttle is involved, together with the chloroplastic malate valve, in the shift of malate to peroxisomes for the production of the NADH needed for the synthesis of glycerate from hydroxypyruvate in the photorespiratory cycle [128].
We have reported evidence that in DWM, as well as in potato cell mitochondria, a malate/oxaloacetate exchange occurs [129] and that this exchange is inhibited in a competitive manner by phenylsuccinate and butylmalonate, thus suggesting that it is due to a malate/oxaloacetate antiporter rather than to the combined activities of possible electrogenic uniporters of malate and oxaloacetate linked to each other for the charge compensation as reported by Zoglowek et al. [107]. Interestingly, our findings demonstrate that, both in durum wheat and potato cells, the malate/oxaloacetate antiporter, together with the cytosolic and mitochondrial malate dehydrogenase (cMDH and mMDH, respectively), allows the operation of a malate/oxaloacetate shuttle, which is responsible for the transfer of reducing equivalents inside mitochondria: malate enters mitochondria in exchange with endogenous oxaloacetate; once inside the matrix, malate is oxidized via the very active mMDH to oxaloacetate [113,130], which, in turn, can exit mitochondria, where it is reduced by the cMDH, with NADH consumption. Once inside mitochondria, the NADH can be oxidized by the respiratory chain-linked NADH dehydrogenase. Our results indicate that the antiporter regulates the rate of the overall operation of the malate/oxaloacetate shuttle.
At the physiological level, the malate/oxaloacetate shuttle plays a major role in the oxidation of cytosolic NADH both in durum wheat and potato cells. Indeed, on the basis of the kinetic parameters of the antiporter, of the external NADH dehydrogenase (NADH DHext) and of the cMDH, and assuming that the cytosolic NADH concentration is about 1 μM [131], we conclude that the shuttle-dependent oxidation rate is about 100- and 10-fold higher than that due to the NADH DHext in durum wheat and potato cells, respectively. Both durum wheat and potato cells present a very high cMDH reaction rate, which does not limit the maximal rates of malate/oxaloacetate exchange even in the presence of physiological concentration of the product pair (1 mM malate and 0.5 mM NAD+). It is feasible that the contribution of the NADH DHext to NADH oxidation could increase under conditions in which a decrease in the cytosolic reduction potential occurs; for example, as observed under environmental stress, when plants suffer over-reduction of the carriers of the photosynthetic electron transport [132,133].
Due to the specificity of the cytosolic MDH for NADH, the malate/oxaloacetate shuttle cannot oxidize NADPH, whose oxidation occurs via the external NADPH dehydrogenase(s).
Proline is an amino acid that serves different functions in the plant cell. Besides its structural role as component of proteins, proline also acts as a compatible solute and its accumulation to high levels in response to the imposition of a wide range of biotic and abiotic stresses represents one of the major strategies to support the plant cell functionality under adverse conditions [134]. In addition, proline plays other roles during stress, i.e., as antioxidant, metal chelator and signaling molecule. Evidence exists that the cellular levels of proline are regulated by the rate of both biosynthesis and degradation ([135], and references therein). Moreover, due to the compartmentation of the anabolic and catabolic processes in chloroplast and mitochondria, respectively, regulation of the intracellular proline transports cannot also be excluded; unfortunately, to date, little information is available in plants about the identity and the regulation of transporters responsible for the flux of proline into and out of specific intracellular compartments.
Mitochondria are devoted to proline metabolization to glutamate via proline dehydrogenase and Δ1 pyrroline-5-carboxylate dehydrogenase, which implies a proline flux from the cytosol to the matrix. In this regard, evidence has been reported, in DWM, about the existence of two different carriers for proline: a proline uniporter, which facilitates proline transport into the mitochondrial matrix; and a proline/glutamate antiporter, which appears to have an important role in the proline/glutamate exchange between the mitochondrial matrix and the cytosol [43]. However, the genes encoding proline transporters have not been identified in any plant [2].
Evidence that the two carriers differ from each other derives from their kinetic properties. In particular, the two transporters show a different affinity for proline, with a Km value of 2 and 8 mM for the uniporter and the antiporter, respectively. Moreover, the uniporter is inhibited by the non-penetrant thiol reagent mersalyl, but it is insensitive to the penetrant thiol reagent NEM, which, in contrast inhibits the antiporter; this suggests that the uniporter contains thiol/s in a hydrophilic environment whereas thiol/s belonging to the antiporter resides in the hydrophobic protein domain. The occurrence of two separate carriers for proline in DWM is further confirmed by the time course of ΔΨ generation, which starts immediately after the addition of proline to DWM, and that of the glutamate efflux, which starts about 1 min after the addition of proline, that is after reaching the maximum ΔΨ. On the basis of these findings the following mechanism can be proposed: the net uptake of proline occurs, and then, as a result of proline catabolism inside DWM [136], glutamate is generated and is then exported outside mitochondria in exchange with further proline via the proline/glutamate antiporter. Notice that these findings are consistent with the occurrence of the proline/glutamate shuttle of cytosolic NADH. This proposed shuttle might be considered to join the malate/oxaloacetate shuttle [129] in transferring the reducing equivalents from cytosol to mitochondria and vice-versa.
In addition to protein-mediated transports, an additional diffusion process has been observed in spinach leaf mitochondria [137]; this latter could contribute to proline uptake inside mitochondria under stress conditions, when high proline concentrations in the cytosol may be reached.