Immunocytochemical studies of the mitochondrial alternative oxidase in the thermogenic appendix of five members of the Araceae family

Abstract

Summary The distribution of mitochondria in fixed sections of thermogenic appendices of five members of the Araceae family was visualized by using indirect immunofluorescence of the monoclonal antibody against the mitochondrial alternative oxidase. It revealed tissue specific distribution of mitochondria in the different appendices. Clustering of mitochondria at the perinuclear region was observed in the appendix of Amorphophallus konjac, Dracunculus vulgaris , and Arum italicum. In the Sauromatum guttatum appendix, clustering of mitochondria at the perinuclear region was observed only in cells near the vascular elements, and in A. dioscoridis , the distribution was homogenous throughout the cell. A comparative staining with anti-Cyt-actin and anti-β-tubulin subunit revealed a similar but not absolutely identical distribution of these two cytoskeletal proteins. On Western blots of acetone extracts of total appendix proteins of the Sauromatum appendix, anti-AOX recognized three major protein bands. A major protein band with an apparent molecular weight of 37 kDa, and a lower band that consisted of a closely spaced double protein band at an apparent molecular weight of 36 kDa. There was also a new additional band with an apparent molecular weight of 32 kDa. The level of AOX forms increased two-fold from D − 3 to D-day, the day of heat production, and it declined on D + 1. The protein band were also detected in the appendix of A. konjac, D. vulgaris, A. italicum , and A. dioscoridis during their thermogenic activity. When parts of an immature Sauromatum appendix were supplied with one of the three thermogenic inducers: salicylic acid, aspirin, and 2,6-dihydroxybenzoic acid at a 10 μM concentration that is sufficient to trigger heat production, the level of AOX increased twofold in the presence of salicylic acid, 1.5-fold in the presence of aspirin, and only 10% with 2,6-dihydroxybenzoic acid.