Histochemical detection and image analysis of non-specific esterase activity and the amount of polyphenols during annual bud development in Norway spruce

This study focused on Norway spruce trees during their annual development and, specifically, on the histochemical analysis of the activity of the non-specific esterase and the amount of polyphenols in the vegetative buds. Ate-coupling reaction with naphthol-AS-acetate as a substrate and Fast Blue B Salt as a coupling reagent was applied to the longitudinal median cryo-sections of the formalin-fixed buds. A method of image analysis was developed to evaluate the intensity of the histochemical reaction quantitatively. High activity of non-specific esterase was detected in the cells of the collenchymatic plate, the youngest bud scales, the leaf primordia, in the zone of differentiation of vascular tissues and procambium of the dormant bud. The activity of non-specific, esterase decreased in all tissues during bud break. During the growth of a new shoot enzyme activity was detected only in the vascular bundles of the shoot, Enzyme activity re-occurred at the beginning of the autumn in the cells of the youngest bud scales and in the cells of the collenchymatic plate of the newly establishing buds. Three different types of polyphenols at different intracellular localizations (as verified by scanning electron microscopy) were observed: granular, vacuolar and drop-like polyphenols. The amount and localization of those polyphenols changed during the bud growing cycle. The positive reaction of all three types of polyphenols with vanillin-HCl proved that these polyphenols are tannin in nature. These findings are discussed from the viewpoint of frost tolerance and tissue differentiation.