Rapid isolation and study of protoplasts obtained throughout the day-night cycle from leaves of Kalanchoe blossfeldiana showing different levels of crassulacean acid metabolism

Abstract

A method is described for rapid enzymatic isolation of protoplasts from the Crassulacean acid metabolism (CAM) plant Kalanchoe blossfeldiana cv. Tom Thumb. Young leaves were sampled at low, middle or full CAM levels induced by increasing number of short-days (14, 31 and 49 SD). Maximum O 2 exchange in light or dark and maximum CO 2 fixation in light occur with protoplasts obtained at 1730 (end of the day) for all CAM levels. Dark CO 2 fixation, typical of CAM, is performed by protoplasts isolated in the middle of the night from plants having received at least 31 SD. Rates of dark CO 2 fixation by these protoplasts are of the same order as those of intact leaves. The capacity for O 2 exchange and CO 2 fixation increases with the level of CAM. These protoplasts retain characteristics typical of CAM, such as diurnal oscillations in phospho enolpyruvate carboxylase (EC 4.1.1.31; PEPC) capacity and malate content.