Imaging long distance propagating calcium signals in intact plant leaves with the BRET-based GFP-aequorin reporter

Calcium (Ca(2+)) is a second messenger involved in many plant signaling processes. Biotic and abiotic stimuli induce Ca(2+) signals within plant cells, which, when decoded, enable these cells to adapt in response to environmental stresses. Multiple examples of Ca(2+) signals from plants containing the fluorescent yellow cameleon sensor (YC) have contributed to the definition of the Ca(2+) signature in some cell types such as root hairs, pollen tubes and guard cells. YC is, however, of limited use in highly autofluorescent plant tissues, in particular mesophyll cells. Alternatively, the bioluminescent reporter aequorin enables Ca(2+) imaging in the whole plant, including mesophyll cells, but this requires specific devices capable of detecting the low amounts of emitted light. Another type of Ca(2+) sensor, referred to as GFP-aequorin (G5A), has been engineered as a chimeric protein, which combines the two photoactive proteins from the jellyfish Aequorea victoria, the green fluorescent protein (GFP) and the bioluminescent protein aequorin. The Ca(2+)-dependent light-emitting property of G5A is based on a bioluminescence resonance energy transfer (BRET) between aequorin and GFP. G5A has been used for over 10 years for enhanced in vivo detection of Ca(2+) signals in animal tissues. Here, we apply G5A in Arabidopsis and show that G5A greatly improves the imaging of Ca(2+) dynamics in intact plants. We describe a simple method to image Ca(2+) signals in autofluorescent leaves of plants with a cooled charge-coupled device (cooled CCD) camera. We present data demonstrating how plants expressing the G5A probe can be powerful tools for imaging of Ca(2+) signals. It is shown that Ca(2+) signals propagating over long distances can be visualized in intact plant leaves and are visible mainly in the veins.