Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells

Cytoplasmic free calcium ([Ca2+](cyt)) acts as a stimulus-induced second me ssenger in plant cells and multiple signal transduction pathways regulate [ Ca2+](cyt) in stomatal guard cells. Measuring [Ca2+](cyt) in guard cells ha s previously required loading of calcium-sensitive dyes using invasive and technically difficult micro-injection techniques. To circumvent these probl ems, we have constitutively expressed the pH-independent, green florescent protein-based calcium indicator yellow cameleon 2.1 in Arabidopsis thaliana (Miyawaki et al., 1999; Proc. Natl. Acad. Sci. USA 96, 2135-2140). This ye llow cameleon calcium indicator was expressed in guard cells and accumulate d predominantly in the cytoplasm. Fluorescence ratio imaging of yellow came leon 2.1 allowed time-dependent measurements of [Ca2+](cyt) in Arabidopsis guard cells. Application of extracellular calcium or the hormone abscisic a cid (ABA) induced repetitive [Ca2+](cyt) transients in guard cells. [Ca2+]( cyt) changes could be semi-quantitatively determined following correction o f the calibration procedure for chloroplast autofluorescence. Extracellular calcium induced repetitive [Ca2+](cyt) transients with peak values of up t o approximately 1.5 mu M, whereas ABA-induced [Ca2+](cyt) transients had pe ak values up to approximately 0.6 mu M. These values are similar to stimulu s-induced [Ca2+](cyt) changes previously reported in plant cells using rati ometric dyes or aequorin. In some guard cells perfused with low extracellul ar KCI concentrations, spontaneous calcium transients were observed. As yel low cameleon 2.1 was expressed in all guard cells, [Ca2+](cyt) was measured independently in the two guard cells of single stomates for the first time . ABA-induced, calcium-induced or spontaneous [Ca2+](cyt) increases were no t necessarily synchronized in the two guard cells. Overall, these data demo nstrate that that GFP-based cameleon calcium indicators are suitable to mea sure [Ca2+](cyt) changes in guard cells and enable the pattern of [Ca2+](cy t) dynamics to be measured with a high level of reproducibility in Arabidop sis cells. This technical advance in combination with cell biological and m olecular genetic approaches will become an invaluable tool in the dissectio n of plant cell signal transduction pathways.