Protoplast Isolation from Leaves of C3, C4 and CAM Plants and Biochemical Activities
Leaf protoplasts have been isolated from a number of plants by enzymatic digestion with 2% cellulase (Onozuka R-10, All Japan Biochem. Co.) and 0.1% pectinase (1). Leaf segments less than lmm wide were incubated in the isolation medium for 3 hours at 30°C. The protoplasts released were purified in a liquid-liquid two-phase system using dextran T20 and polyethylene glycol 6000. Intact mesophyll protoplasts were collected at the interphase. Bundle sheath strands from C4 plants were collected by filtration techniques through nylon sieves of different porosity. For microscopy observations isolated protoplasts and bundle sheath cells were suspended in 0.5% Evans Blue. The exclusion of the dye was used as an indication of the intactness of the preparation. Comparative studies on the photosynthetic activities by mesophyll protoplasts from C3 and Crassulcean Acie Metabolism plants further showed the viability of the isolated cells. C4 plants, such as maize, sugarcane and Panicum miliaceum, are characterized as having two types of chlorophyllous cells, the mesophyll and the bundle sheath cells, and two carboxylating enzymes, PEP carboxylase of the C4-dicarboxylic acid pathway and RuDP carboxylase of the ribulose pentose phosphate pathway. PEP carboxylase and other enzymes of the C4 pathway were almost exclusively localized in mesophyll protoplasts while RuDP carboxylase and phosphoribulose kinase were localized in bundle sheath cells. Enzyme distribution, photochemical activities and CO2 fixation have been studies in relation to the compartmentation of photosynthetic carbon metabolism (2,3).