Isolation of high purity guard cell protoplasts of Arabidopsis thaliana for omics research

Abstract

Stomata are unique plant structures responsible for photosynthesis, transpiration and innate immunity to pathogens, and guard cells are one of the most studied cell types with respect to plant cell functioning, signalling, and stress responses. The ability to easily purify large quantities of high purity guard cell protoplasts (GCPs) would facilitate further studies, as current methods for GCP isolation are barely sufficient for omics research. Here, we report a new procedure for isolating high purity GCPs. For the isolation of GCPs, detached epidermal peels were used to extract GCPs instead of whole leaves. GCPs and mesophyll cell protoplasts (MCPs) were found to have diameters of 2.5–9.1 µm and 6.5–43.5 µm, respectively. The overlap in sizes of GCPs and MCPs suggests that blending and filtering of whole leaves used in previous methods may not necessarily avoid contamination with MCPs during GCP extraction. There were, on average, 8.4 ± 0.18 chloroplasts in GCPs and 34.6 ± 1.5 in MCPs. For MCPs and GCPs with similar sizes, there were fourfold more chloroplasts in MCPs, which made MCPs readily distinguishable from GCPs by microscopic inspection. The protocol enabled the isolation of over 1.44 × 106 GCPs from about 150 cm2Arabidopsis abaxial epidermis with over 97% purity. These protocols provide an advance in isolating sufficient, high purity, and viable protoplasts for RNA extraction and transcriptomic analysis. The application of this protocol to other plant species may accelerate the research and development of plant cell-type specific omics.