Preparation and applications of Arabidopsis thaliana guard cell protoplasts

Guard cells play an important role in the physiology and development of plants. The genetic resources available for Arabidopsis thaliana make it the most favorable plant species for the study of guard cell processes, but it is not easy to isolate highly purified preparations of large numbers of guard cells from this species. Here, we describe methods for isolation of both guard cell and mesophyll cell protoplasts from A. thaliana and their use in the study of unique biochemical and cellular properties of these cell types. Protocols developed for large- and small-scale preparation of guard cell protoplasts and mesophyll cell protoplasts are described, followed by specific examples of their use in electrophysiological, biochemical and molecular approaches such as patch clamping, enzyme assays, and reverse-transcription polymerase chain reaction. The protocols described yield millions of highly purified, viable guard cell protoplasts and mesophyll cell protoplasts from A. thaliana. These protoplasts have been used successfully in the study of ion channel properties, assay of ABA activation in phospholipase D activity and comparisons of gene and protein expression levels. These techniques make it possible to elucidate electrophysiological, biochemical and molecular genetic pathways of guard cell function.