EFFECTS OF LIGHT EXPOSURE OR SHADING DURING GROWTH OF KIWIFRUIT ON THE RESPIRATION OF MITOCHONDRIA ISOLATED FROM THE PULP OF COLD-STORED FRUIT

We have established an experimental procedure for the isolation of intact mitochondria from fruit of kiwifruit vines. The effects of enhanced light and of shading during fruit growth on respiratory rate and on metabolite transport through purified inner membrane carrier proteins were evaluated in mitochondria isolated from kiwifruit pulp. Fruit were harvested from organically grown plants and stored for 7 months at 5°C. Rates of succinate respiration and of external NADH oxidation in the presence of Ca²⁺ or Mg²⁺ ions, appeared to be higher in mitochondria isolated from kiwifruit produced on shaded plants than in those isolated from fruit produced on light-exposed plants. Addition of salicylhydroxamic acid (SHAM), a specific inhibitor of alternative oxidase protein (AOX), caused a greater inhibition of these respiratory activities in mitochondria obtained from fruit of light-exposed plants, than in those from the shaded ones. In mitochondria isolated from fruit of shaded plants, Arrhenius plots of substrate fluxes by partially purified adenine nucleotide translocator protein (ANT) or by dicarboxylates-tricarboxylates carrier protein (DTC), reconstituted in proteoliposomes, were found to be linear, with an activation energy (Ea) of 56.8 kJ/mol for ANT and 61.4 kJ/mol for DTC. A partial aox cDNA from kiwifruit pulp was amplified using degenerate primers and sequenced. A partial amino acid sequence of the AdAOX protein is very similar to that reported from other plant sources. AdAOX expression level was higher in fruit pulp of light-exposed plants than in those from shaded-plants. Since high levels of expression and activities of external NAD(P)H dehydrogenases and AOX were associated with cold stress occurrence in various plant tissues and linear temperature dependence of mitochondrial carrier activities was reported to be a cold tolerance indicator, our conclusion is that kiwifruit, harvested from shaded plants and stored for 7 months at 5°C, have a greater chilling tolerance than fruit grown on light-exposed plants.