Protoplast isolation and transient gene expression in the single-cell C4 species, Bienertia sinuspersici

Although transient gene expression using reporters such as green fluorescent protein is a versatile tool for examining gene functions and intracellular protein trafficking, the establishment of a highly efficient gene manipulation method remains a challenge in many plant species. A reliable transformation protocol has not yet been established for the three single-cell C-4 species, despite their potential of serving as model systems for their extraordinary C-4 photosynthetic metabolism. We report the first protocol optimized for isolating a large-scale and homogenous population of protoplasts from chlorenchyma cells of the single-cell C-4 species Bienertia sinuspersici. Cytochemical staining confirmed the preservation of the unusual subcellular compartmentation of organelles in chlorenchyma cells after cell wall digestion. Approximately 84% of isolated protoplasts expressed the reporter fluorescent protein following our optimized polyethylene glycol-mediated transfection procedures. Fluorescent fusion protein tagged with various intracellular sorting signals demonstrated potential use of the transient gene expression system in subcellular protein localization and organelle dynamics studies. Further applications of the current protoplast isolation and transfection techniques in understanding the novel single-cell C-4 photosynthetic mechanism are discussed.