Transport pathways--proton motive force interrelationship in durum wheat mitochondria

In energized mitochondria, PIMAC functioning should be inhibited by ATP and high ΔΨ values; under these conditions ΔΨ allows for functioning of the AAC and ΔpH represents the driving force for the PiC, that triggers DIC and DTC activity (Figure 4A). The set-up is different in de-energized mitochondria. PIMAC should play a role that gradually increases under conditions in which mitochondria are progressively de-energized by replacing or integrating the operation of anion carriers to ensure the exchange of metabolites between mitochondria and other organelles. This is likely to occur in DWM in the light of the presence of powerful energy-dissipating systems such as PmitoKATP and PUCP (see above) as well as alternative oxidase (AOX) [130], whose functioning under abiotic stress conditions allows the control of mitochondrial ROS generation.

As already shown in Figure 2, under stress conditions the decrease in both ΔΨ and ATP activates PIMAC, while the increase in FFAs may put a brake on excess activation, which might cause swelling and rupture of outer mitochondrial membrane. At the same time, the decreased ΔΨ and ΔpH, as well as the increased ROS generation, may impair anion carriers; as a consequence, the anion transport across the inner mitochondrial membrane may shift from the energy-dependent anion carriers toward the electrophoretic flux through the PIMAC (Figure 4B).

Consistently, evidence for a role of PIMAC in plant response to abiotic stress has been also reported in maize seedlings; the analysis of three maize populations differing in terms of cold tolerance highlighted a relationship between the PIMAC activity kinetics and the level of cold tolerance [38]; in addition, in the same plant material, the level of the ZmCLCc protein, responsible for the PIMAC activity in maize seedling, was found to be higher in cold stressed than in non-stressed growing conditions [42].

The picture reported in Figure 4B raises two main questions: how ATP may be synthesized at low driving force and how it may be exported outside mitochondria, as PIMAC cannot transport ATP (see Section 3.1).

In this regard, we have reported evidence in a recent paper that ATP synthesis and efflux may be maintained when de-energization of succinate oxidising DWM is obtained via PmitoKATP in a high KCl medium [55]. In particular, under these conditions DWM are unexpectedly fully coupled as they preserve ADP/O ratio and do not change their rate of ATP synthesis and efflux despite complete pmf decrease. A possible explanation for this apparently inconsistent behaviour resides in the inhibition of the PmitoKATP by ATP; in practice, the channel may strongly lower measurable bulk phase pmf, but ATP inhibition represents a brake, so it appears unable to fully compete with ATP synthase for protons, thus preserving a latent proton movement non-classically detectable [55,138]. As for transports, it is likely that succinate and Pi transport may mainly occur via PIMAC (Figure 5). On the other hand, ADP/ATP exchange resulted in full inhibition by ATR [55] (see also Figure 5); this surprising finding strongly suggests that adenine nucleotide movement is dependent on the ADP/ATP carrier despite the absence of ΔΨ. To date, how this transport may actively occur in fully de-energized DWM remains to be established. Anyway, these in vitro results show that mitochondria are able to maintain transport activity even in the absence of any measurable driving force.

It should be also underlined that in potato tuber mitochondria, under phosphorylating conditions, the coupling parameters, i.e., the ADP/O ratio and the respiratory control ratio, were unchanged in mitochondria respiring either in the absence or in the presence of 100 mM KCl (at constant osmolarity). These measurements provide direct evidence against significant uncoupling as a consequence of K+ channel opening in mitochondria of potato tubers [41].

Studies about DWM have shed some light about the interrelationship between transport systems and pmf in plant mitochondria. In particular, PmitoKATP and PUCP may actively dissipate pmf, but, unexpectedly, de-energization of mitochondria in vitro does not impair metabolite transport. Indeed, PIMAC may cooperate with carriers, and the ADP/ATP carrier may show activity also in the absence of measurable ΔΨ. These findings may have a relevant physiological importance. In this regard, evidence has been reported that mitochondria depolarization is not unusual in vivo. As described above, dissipative systems may lower ΔΨ to avoid excess ROS production under environmental stresses. Moreover, determination of the in vivo dynamics of individual mitochondrial membrane potentials in Arabidopsis thaliana roots has demonstrated that plant mitochondria undergo sporadic and rapid cycles of partial dissipation and restoration of ΔΨ [139]. Probably, PmitoKATP may also induce partial ΔΨ dissipation in vivo, as a result of a balance between positive and negative modulators. Under these ΔΨ dissipation cycles cooperation between PIMAC and carriers might ensure mitochondrial metabolism in vivo. Similarly, animal mitochondria are reported to be exposed to complete depolarization as a consequence of spontaneous opening and closure of the permeability transition pore [140]. Finally, it should be considered that ΔΨ values measured in vivo are generally low. In animals, Zhang et al. [106] measured a mitochondrial ΔΨ of about 105 mV in fibroblasts and 81 mV in neuroblastoma cells; in perfused hearts [104] and single hepatocytes [105] values of 100–140 mV were measured under different metabolic conditions. This is true also in plant mitochondria, as Igamberdiev and Kleczkowski [103] estimated a mitochondrial ΔΨ of 70–95 mV in barley leaf protoplasts under different physiological conditions.

So, the transport systems in plant mitochondria must be active under low driving force also in vivo. Further studies are required to fully understand this behaviour.