CHALCONE SYNTHASE LOCALIZATION IN EARLY STAGES OF PLANT DEVELOPMENT .1. IMMUNOHISTOCHEMICAL USE OF PLASMOLYSIS FOR LOCALIZING THE ENZYME INEPIDERMAL-CELL CYTOPLASM OF ILLUMINATED BUCKWHEAT HYPOCOTYLS

Immunohistochemical methods combined with progressive plasmolysis were used to localize chalcone synthase (CHS), an important enzyme for pla nt metabolism of aromatics in hypocotyls of illuminated buckwheat (Fag opyrum esculentum M.) seedlings, Illumination of etiolated seedlings w ith white light results in anthocyanin synthesis in the epidermal laye r of the hypocotyl. Anthocyanin-containing epidermal peels, after fixa tion for 30 min in 4% paraformaldehyde, 2.5% glutaraldehyde, 0.1% caff eine, were treated with a specific rabbit anti-buckwheat CHS antibody and a 20 nm goat anti-rabbit IgG gold conjugate. CHS is specifically s hown in epidermal cells as pink to dark red deposits, Progressive plas molysis combined with our immunohistochemical method showed that CHS w as located exclusively in the cytoplasm of the epidermal cells of buck wheat hypocotyls except for the guard cells, which contained no detect able CHS,