A simple method for isolating import-competent Arabidopsis chloroplasts

Abstract

We present a simple, rapid and low-cost method for isolating a high yield of Arabidopsis chloroplasts that can be used to study chloroplast protein import. Efficiency of chloroplast isolation was dependent upon the ratio between amount of plant tissue and the buffer volume, the size and speed of the homogenisation equipment, and the size of the homogenisation beaker. The import method proved useful when characterising different precursor proteins, developmental stages and import-defective mutants. Time-course experiments enabled the measurement of import rates in the linear range. Compared to protoplastation, this isolation method has significant time and cost savings (∼80% and ∼95%, respectively), and yields chloroplasts with a higher capacity to import proteins.