Chalcone Synthase Localization in Early Stages of Plant Development. I. Immunohistochemical Use of Plasmolysis for Localizing the Enzyme in Epidermal Cell Cytoplasm of Illuminated Buckwheat Hypocotyls

Abstract

Immunohistochemical methods combined with progressive plasmolysis were used to localize chalcone synthase (CHS), an important enzyme for plant metabolism of aromatics in hypocotyls of illuminated buckwheat (Fagopyrum esculentum M) seedlings. Illumination of etiolated seedlings with white light results in anthocyanin synthesis in the epidermal layer of the hypocotyl. Anthocyanin-containing epidermal peels, after fixation for 30 min in 4% paraformaldehyde, 2.5% glutaraldehyde, 0.1% caffeine, were treated with a specific rabbit anti-buckwheat CHS antibody and a 20 nm goat anti-rabbit IgG gold conjugate. CHS is specifically shown in epidermal cells as pink to dark red deposits. Progressive plasmolysis combined with our immunohistochemical method showed that CHS was located exclusively in the cytoplasm of the epidermal cells of buckwheat hypocotyls except for the guard cells, which contained no detectable CHS.

Key Words:

• chalcone synthase
• epidermal anthocyanins
• epidermis
• histochemical localization
• immunogold labeling
• plasmolysis