Protoplasts as a tool for isolating functional chloroplasts from leaves

Abstract

Leaf protoplasts from various grasses can be used for isolating chloroplasts with high photosyndietic activity. The protoplasts were stable for more than 20 hr during which time chloroplasts could be isolated from protoplasts without any loss of original CO₂ fixation capacity (100–157 μmoles/mg chl-hr). Using Triticum aestiuum to optimize assay conditions, the pH optimum for CO₂ fixation by the chloroplasts isolated from protoplasts was between 8.2 and 8.6. Magnesium (0.75 mM) was required for maximum CO₂ fixation by the isolated chloroplasts and sodium ascorbate in the medium allowed a more linear increase in CO₂ fixation with time. Based upon ¹⁴CO₂ fixation and ferricyanide-dependent oxygen evolution as criteria of intactness, chloroplasts from protoplasts exhibited a high degree of intactness compared to those obtained by mechanical grinding. Chloroplasts isolated from grass leaves by mechanical grinding had a relatively low capacity for endogenous CO₂ fixation and required addition of ribose-5-phosphate and ADP for maximum activity.