Tissue printing for myrosinase activity in roots of turnip and Japanese radish and horseradish: a technique for localizing myrosinases.

A method for analyzing the tissue distribution of myrosinase activity in Brassicaceous plants was developed. This technique is based on 'tissue printing' to visualize enzyme activity. The freshly-cut surface (transverse direction) of the root of three species, Japanese radish (Raphanus sativus), turnip (Brassica campestris) and Japanese horseradish (wasabi, Wasabia japonica), was pressed onto a polyvinylidene difluoride (PVDF) filter to immobilize the proteins onto the membrane. The sites of myrosinase activity on the membranes were visualized by the sinigrin-glucose oxidase-peroxidase system. Signals for myrosinase activity were observed in both the epidermis and vascular cambium of the root of the Japanese radish, turnip and wasabi. Measurement of myrosinase activity in protein extracts indicated that the level of myrosinase activity in the peeling, which consisted of the epidermis, cortex and vascular cambium, was much higher than that in the peeled root of the three species. These results support the image that myrosinase activity, obtained in tissue printing, corresponded well with the tissue distribution of myrosinase activity.