Effect of High Temperature on Oxidation of NAD-Dependent Substrates and Alternative Oxidase Activity in Mitochondria of Lupine Cotyledons

Isolated mitochondria from lupine ( Lupinus angustifolius L.) cotyledons were used to study the effect of temperature in the range of 20–40°C on oxidation of malate and other NAD-dependent respiratory substrates as well as on the activity of alternative CN-resistant oxidase. The metabolic responses of mitochondria to heat treatment analyzed in vitro were compared with metabolic activities of organelles isolated from seedlings exposed to elevated temperature in vivo. Mild warming of the incubation medium (20–30°C) accelerated oxygen uptake by mitochondria during malate oxidation in the presence of glutamate in the active phosphorylating state (state 3) and, to a lesser extent, in the state 4 (in the absence of ADP). The enhancement of mitochondrial respiration at increasing temperature in this range was entirely due to the activation of the cytochrome pathway in electron-transport chain (ETC). At temperatures of 35–40°С, an appreciable inhibition of the alternative respiration pathway was observed. The successive additions of ADP during malate oxidation under hyperthermia (35–40°С) caused the improvement (phenomenon of conditioning) of oxidative phosphorylation parameters. After the second addition of 100–200 μM ADP, the rate of substrate oxidation in state 3 and the respiratory control ratio (RCR) increased significantly. Similar changes in mitochondrial respiration under hyperthermia were found upon oxidation of other NAD-dependent substrates, but they did not occur during succinate oxidation catalyzed by ETC complex II. In mitochondria isolated from cotyledons of lupine seedlings that were exposed in vivo to high temperature (35°C for 12 h), the malate oxidation at 25 or 35°C was also characterized by a sharp decrease in RCR and the state 3 rate after the first addition of ADP, but these parameters were restored during several cycles of phosphorylation. Oxidation of malate and other NAD-dependent substrates catalyzed by complex I after heating of mitochondria at 40°C in the presence of 10 mM MgCl 2 was substantially inhibited and was carried out by rotenone-insensitive NADH dehydrogenases. The results suggest that the suppressed oxidation of NAD-dependent substrates in lupine cotyledon mitochondria at high temperature is probably due to the reversible transformation of complex I from the active to the deactivated form (A/D transition). Possible physiological significance of such regulatory changes in plant respiration in response to adverse environmental conditions is discussed.