Preparation of Arabidopsis mesophyll protoplasts with high rates of photosynthesis

Abstract

A simple and quick method is described for rapid isolation of metabolically active mesophyll protoplasts from leaves of Arabidopsis thaliana. The optimal composition of the digestion medium, period of digestion and stability of protoplast preparation were examined. A large number of protoplasts could be prepared within an hour. The isolated protoplasts were intact, stable and metabolically very active, as indicated by their high rates of photosynthetic oxygen evolution. The important factors during the preparation of protoplasts are short time of digestion, composition of medium, use of nylon nets for filtration, centrifugation at low speed and use of pH 7.0 for storage. The highest rate of photosynthesis obtained in these experiments was 130±4 μmol O₂ evolved mg⁻¹ Chl h⁻¹, at 1 mM sodium bicarbonate and at a light intensity of 600 μE m⁻²s⁻¹. The present technique of isolation can be very useful for making Arabidopsis protoplasts for studies on not only metabolic processes, such as photosynthesis, but also metabolomics, proteomics and genomics.