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.

CDNA cloning of radish (Raphanus sativus) myrosinase and tissue-specific expression in root.

Two cDNA clones of myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) were isolated from radish (Raphanus sativus) seedlings. Both clones were identified as MB (B type myrosinase) from their sequence homology at the amino acid level to MBs cloned from other Brassicaceae species. The tissue distribution of gene expression and enzyme activity of myrosinase corresponded well to the site of glucosinolate accumulation in different tissues of radish. The myrosinase-glucosinolate system was localized in the cotyledons in the seedlings and in the peel of the root in the mature plant. Tissue printing analysis showed that myrosinase mRNA and activity were localized in the epidermis and vascular cambium that were present in the peripheral part of the root but few signals were detected in the parenchyma inside of the vascular cambium. Since the myrosinase-glucosinolate system is known to be a defense system in higher plants, the localization of the myrosinase-glucosinolate system in the peel of the root may act to protect the sink organ from the attack of herbivores or pathogens in soil.

Preparation of High Yields of Algal Protoplasts Using Buccal Juice of Sea Hare and Commercial Cellulase.

; Buccal juice of the sea hare Aplysia juliana was found to degrade algal polysaccharides. The optimal enzyme composition for protoplast preparation from Undaria pinnatifida was protein at 48 µg/ml buccal juice from sea hare, 10 mg/ml cellulase Onozuka-RS, 0.4 M NaCl, 0.8 M sorbitol, 2 mg/ml dextran sulfate sodium salt, and 1 µl/ml 2-mercaptoethanol in 10 mM MES buffer (pH 6.0). Protoplasts of Eisenia bicyclis, Endarachne binghamiae (Phaeophyta), and Ulva pertusa (Chlorophyta) could also be prepared in a similar manner. Yields of these protoplasts were about 10(7) cells per gram of fresh weight alga.

cDNA sequence and expression of a cold-responsive gene in Citrus unshiu.

A cDNA clone encoding a protein (CuCOR19), the sequence of which is similar to Poncirus COR19, of the dehydrin family was isolated from the epicarp of Citrus unshiu. The molecular mass of the predicted protein was 18,980 daltons. CuCOR19 was highly hydrophilic and contained three repeating elements including Lys-rich motifs. The gene expression in leaves increased by cold stress.