Cameleon calcium indicator in Arabidopsis stomata

Photo credit: Wiley Online Library

Yellow cameleon 2.1 (YC 2.1) expression in Arabidopsisguard cells.

(a,b) Yellow cameleon 2.1 was expressed in guard cells as visualized by scanning confocal microscopy. YC2.1 expression was observed in guard cells in epidermal fragments. Bar = 12 μm. (c,d) Cytoplasmic localization of YC2.1. At higher magnification, fluorescence was observed in the area surrounding the nucleus, in the thin layer of cytoplasm at the edge and ends of the cells, and in cytoplasmic strands traversing the vacuoles and surrounding chloroplasts. Bar = 5 μm. In both (b) and (d), excitation was at 488 nm, emission at 522 nm (DF32). (a) and (c) are confocal, bright-field images collected at the same time. (e–h) One-image projection from a three-dimensional reconstruction of deconvoluted, 0.2 μm z-series images of one stomate expressing yellow cameleon 2.1. (e) Stomate imaged at the YC2.1 emission wavelength 535 nm. (f) Stomate imaged at the YC2.1 emission wavelength 480 nm. (g) Superimposed images at both 535 and 480 nm wavelengths indicating coincident fluorescence of the 535 and 480 nm signals except from the chloroplasts. (h) Ratiometric image of the 535/480 nm wavelength emissions to accurately demonstrate YC2.1 distribution (pseudo-coloured red for clarity). No ratiometric signal is observed from the chloroplasts. Bar = 10 μm. (i–k) One-image projection from a three-dimensional reconstruction of deconvoluted 0.2 μm z-series images of one non-transformed stomate. (i) Stomate imaged at 535 nm emission, enhanced relative to 480 nm by a three times longer excitation period. (j) Stomate imaged at 480 nm emission. (g) Overlaying images at 535 and 480 nm wavelength indicating coincident fluorescence of the weak 535 and strong 480 signals in the chloroplasts, and some signal at 535 nm from the lignified cell wall at the lip of the stomatal pore.

 

Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells

by Allen G. J., Kwak J. M., Chu S. P., Llopis J., Tsien R. Y., Harper J. F., Schroeder J. I. (1999)

in Plant Journal 19: 735–738. – DOI: 10.1046/j.1365-313x.1999.00574.x

Wiley Online Library |PubMed |CAS |

http://onlinelibrary.wiley.com/doi/10.1046/j.1365-313x.1999.00574.x/full

http://www.ncbi.nlm.nih.gov/pubmed/10571859

http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=wiley&version=1%2E0&coi=1%3aCAS%3a528%3aDyaK1MXntFOnsbY%253D&md5=f5dd8b58fe6aa001e5db2b4bd5ec5072

Summary

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 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 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 μM, whereas ABA-induced [Ca²⁺]_cyt transients had peak values up to approximately 0.6 μM. 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.