Abstract
Cytoplasmic calcium ([Ca2+]cyt) acts as a stimulus-induced second messenger in multiple signal transduction cascades (Allen et al., 1999). In plant cells, a dramatic and readily assayed response to stimulus is the change of stomatal aperture. Changes in [Ca2+]cyt of stomatal guard cells were involved in stomatal movement in response to various stimuli and cellular processes. In general, there are two available ways to measure [Ca2+]cyt in guard cells, i.e., loading of calcium-sensitive fluorescence dyes such as fluo-3 AM and fura-2 or expressing genetically encoded calcium indicators such as yellow cameleon (Krebs et al., 2012). In this protocol, we aim at describing the experimental procedure to record [Ca2+]cyt fluctuation in guard cells with loading of fluo-3 AM upon ABA or PA treatment combining with fluorescence imaging performed with confocal laser scanning microscope.
Materials and Reagents
Equipment
Software
Procedure
Representative data
Figure 1. Elevation of cytoplasmic calcium induced by ABA and PA in Arabidopsis guard cells. The confocal images of [Ca2+]cyt in guard cells were monitored by fluo-3 AM dye before (A) or after treatment with10 μM ABA (B) or 50 μM PA (C) for 12 min. The color bars showed the intensity as the arrow indicated. D. Changes in the relative levels of [Ca2+]cyt in ABA- or PA-treated guard cells. Regions of interests used to measure the intensities were indicated by white rectangle with background subtraction. Values are the mean ± SD (n = 50-60 from not less than 10 cotyledons) from three independent experiments.
Recipes
Acknowledgments
The methods were adapted from (Jiang et al., 2014). Techniques were also adapted from all of the references cited. This work was supported by grants from National Basic Research Program of China (31100194 and 31470364) and the Fundamental Research Funds for the Central Universities (KYZ201423) to Q Zhang.
References
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