Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells

Cytoplasmic free calcium (Ca2+]cyt) acts as a stimulus-induced second messenger in plant cells and multiple signal transduction pathways regulate Ca2+]cyt in stomatal guard cells. Measuring Ca2+]cyt in guard cells has previously required loading of calcium-sensitive dyes using invasive and technically difficult micro-injection techniques. To circumvent these problems, we have constitutively expressed the pH-independent, green fluorescent protein-based calcium indicator yellow cameleon 2.1 in Arabidopsis thaliana (Miyawaki et al. 1999; Proc. Natl. Acad. Sci. USA 96, 2135-2140). This yellow cameleon calcium indicator was expressed in guard cells and accumulated predominantly in the cytoplasm. Fluorescence ratio imaging of yellow cameleon 2.1 allowed time-dependent measurements of Ca2+]cyt in Arabidopsis guard cells. Application of extracellular calcium or the hormone abscisic acid (ABA) induced repetitive Ca2+]cyt transients in guard cells. Ca2+]cyt changes could be semi-quantitatively determined following correction of the calibration procedure for chloroplast autofluorescence. Extracellular calcium induced repetitive Ca2+]cyt transients with peak values of up to approximately 1.5 microM, whereas ABA-induced Ca2+]cyt transients had peak values up to approximately 0.6 microM. These values are similar to stimulus-induced Ca2+]cyt changes previously reported in plant cells using ratiometric dyes or aequorin. In some guard cells perfused with low extracellular KCl concentrations, spontaneous calcium transients were observed. As yellow 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 not necessarily synchronized in the two guard cells. Overall, these data demonstrate that that GFP-based cameleon calcium indicators are suitable to measure Ca2+]cyt changes in guard cells and enable the pattern of Ca2+]cyt dynamics to be measured with a high level of reproducibility in Arabidopsis cells. This technical advance in combination with cell biological and molecular genetic approaches will become an invaluable tool in the dissection of plant cell signal transduction pathways.