Loading acetoxymethyl ester fluorescent dyes into the cytoplasm of Arabidopsis and Commelina guard cells

Cytosolic calcium and pH changes are integral components of guard cell signal transduction. Acetoxymethyl (AM) ester-linked Ca²⁺ sensitive dyes are usually degraded before loading by extracellular esterases or partitioned into plant vacuoles using standard techniques, thereby preventing cytoplasmic Ca²⁺ imaging. Here a method is described for improved loading of the calcium sensitive fluorescent dyes Calcium Green-1 and Fura-2 AM ester into the cytoplasm of Commelina and Arabidopsis guard cells in epidermal strips, allowing fluorescence from several guard cells in an epidermal strip to be imaged and measured simultaneously. Calcium Green-1 based imaging, external Ca²⁺ buffering and Mn²⁺ quenching in Commelina guard cells suggest that abscisic acid stimulates plasma membrane Ca²⁺ influx. The Ca²⁺ sensitive dye Fura-2 was loaded into the cytoplasm of Arabidopsis guard cells, allowing ratiometric analyses of these cells. Data indicated that intact Ca²⁺ homeostasis mechanisms were present in Fura-2 AM loaded cells. Loading of acetoxymethyl ester dyes provides a viable alternative method to cameleon imaging, which will be useful in loading Arabidposis mutants that are difficult to transform. This method may also be applicable to loading other ester linked dyes into the cytoplasm of plant cells.