Intracellular Localization of Individual Dehydro-Genases in Frozen Plant Tissues by Means of Specific Substrates and Coenzymes

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Abstract
In fresh sections of Pinus bark infected with a Cronartium parasite, reduction of Nitro-blue tetrazolium by several dehydrogenases concomitantly obscured the activity of the enzymes individually. This nonspecific enzymatic reduction was avoided by using frozen sections. Freezing resulted in complete loss of dye reduction but this inactivation was overcome by supplying both the individual substrate and the coenzyme for each dehydrogeuase. Centers of intense activity of malic, isocitric, α-glycerophosphate, glucose-6-phosphate and 6-phosphogluconate dehydrogenases and of NAD and NADP diaphorases were observed within spores and sporogenous cells of the fungus and in underlying hyphae and haustoria. Activity of the dehydrogenases was less in host cells than in fungal cells. Succinic dehydrogenase caused weak staining in fungal and host cells. Ethanol dehydrogenase activity was variable among samples and it and lactic dehydrogenase activity were variable within sections. Pyruvic and β-hydroxy-butyric dehydrogenases were not found in either organism. Differences were observed in the distribution of glutamic and isocitric dehydrogenases and NAD diaphorase between fungal and host cell types in different positions within the bark.