The reaction of mitochondria in the coleoptiles of rice (Oryza sativa L.) with diaminobenzidine

Diaminobenzidine, DAB, was applied to segments of aerobically and anaerobically grown coleoptiles of rice, Oryza sativa L., with the object of studying the location of cytochrome oxidase at the electron-microscope level. A specific staining of mitochondrial cristae and inner membrane was obtained, with no reaction in other organelles; with extended periods of incubation, the reaction product filled the mitochondria completely. In anaerobically grown coleoptiles, the reaction was much slower and the difference was particularly marked in vascular bundle companion cells and parenchyma, which gave the strongest reaction in aerobic tissue, but in the anaerobic stained even less than the cortical parenchyma. The reaction was inhibited by boiling and slowed very much by lowering of the incubation temperature from 27 to 4 degrees C. This indicated the involvement of an enzymic reaction and cyanide inhibition indicated that a haem enzyme was involved. The catalase inhibitor aminotriazole did not inhibit DAB oxidation. Nevertheless the specificity of the reaction for cytochrome oxidase must be questioned, because preheating of the tissue to 60 degrees C before incubation, which would be expected to destroy cytochrome oxidase activity, failed to decrease the oxidation, at least in aerobically grown coleoptiles. It is concluded that DAB is oxidized in the rice coleoptile tissue by a cytochrome system, and the development of this system is inhibited by anaerobiosis, but the oxidation cannot be claimed to represent cytochrome oxidase activity exclusively. Perhaps other autoxidizable, more heat-stable cytochromes participate in the reaction.