Jerusalem artichoke (Helianthus tuberosus L.) tubers were reported to be tolerant to cold and freezing. The aim of this study was to perform a kinetic characterization of the mitochondrial dicarboxylate-tricarboxylate carrier (HtDTC) and to assess a possible involvement of this carrier in the cold tolerance of tubers. The HtDTC was purified from isolated mitochondria by sequential chromatography on hydroxylapatite/celite and Matrex Gel Orange A. SDS gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent molecular mass of 31.6 kDa. A polyclonal antibody raised against the tobacco DTC cross-reacted with the purified protein on Western blot analysis. In gel trypsin, digestion of the purified HtDTC yielded peptides that exhibited strong amino acid sequence similarity to previously identified plant DTCs. Furthermore, using degenerate primers, a portion of the Htdtc cDNA was amplified and sequenced; this cDNA encoded for a protein with high sequence similarity to known plant homolog DTCs. When reconstituted in liposomes loaded with dicarboxylate (2-oxoglutarate, malate, malonate, succinate, and maleate) or tricarboxylate anions (citrate, trans-aconitate, and isocitrate), the purified HtDTC transported all these anions in exchange with external [14C]2-oxoglutarate. A kinetic characterization of HtDTC was performed: (a) the half-saturation constant Km and the Vmax at 25 degrees C of the 2-oxoglutarate/2-oxoglutarate exchange by reconstituted HtDTC were found to be 360 microM and 10.9 micromol/(min mg protein), respectively; (b) the activation energy Ea of the succinate/2-oxoglutarate exchange by the reconstituted HtDTC was found to be 50.7 kJ/mol constant between -5 and 35 degrees C. Similarly, the activation energy Ea of succinate respiration of isolated Jerusalem artichoke mitochondria, measured between -2 and 35 degrees C, was shown to be constant (65.3 kJ/mol). The physiological relevance of kinetic properties and temperature dependence of transport activities of HtDTC is discussed with respect to the cold tolerance ability of Jerusalem artichoke tubers.

The proteins performing the activity of the inner membrane anion channel (IMAC) and its plant counterpart (PIMAC) are still unknown. Lurin et al. (Biochem J 348: 291-295, 2000) indicated that a chloride channel (CLC) protein corresponds to PIMAC activity in tobacco seedling mitochondria. In this study, we investigated: (i) the presence of a CLC protein in maize seedling mitochondria; (ii) the involvement of this protein in plant cold tolerance; and (iii) its possible role in PIMAC activity. We validated the presence of a CLC protein (ZmCLCc) in maize mitochondria by immunoassay using a polyclonal antibody against its C-terminus. The differential expression of the ZmCLCc protein in mitochondria was measured in seedlings of maize populations divergently selected for cold tolerance and grown at different temperatures. The ZmCLCc protein level was higher in cold stressed than in non-stressed growing conditions. Moreover, the ZmCLCc level showed a direct relationship with the cold sensitivity level of the populations under both growing conditions, suggesting that selection for cold tolerance induced a constitutive change of the ZmCLCc protein amount in mitochondria. The anti-ZmCLCc antibody inhibited (about 60%) the channel-mediated anion translocations by PIMAC, whereas the same antibody did not affect the free diffusion of potassium thiocyanide through the inner mitochondrial membrane. For this reason, we conclude that the mitochondrial ZmCLCc protein can perform the PIMAC activity in maize seedlings.