Quantitative Analysis of Microbe-Associated Molecular Pattern (MAMP)-Induced Ca(2+) Transients in Plants.

Abstract

Ca(2+) is a secondary messenger involved in early signaling events triggered in response to a plethora of biotic and abiotic stimuli. In plants, environmental cues that induce cytosolic Ca(2+) elevation include touch, reactive oxygen species, cold shock, and salt or osmotic stress. Furthermore, Ca(2+) signaling has been implicated in early stages of plant-microbe interactions of both symbiotic and antagonistic nature. A long-standing hypothesis is that there is information encoded in the Ca(2+) signals (so-called Ca(2+) signatures) to enable plants to differentiate between these stimuli and to trigger the appropriate cellular response. Qualitative and quantitative measurements of Ca(2+) signals are therefore needed to dissect the responses of plants to their environment. Luminescence produced by the Ca(2+) probe aequorin upon Ca(2+) binding is a widely used method for the detection of Ca(2+) transients and other changes in Ca(2+) concentrations in cells or organelles of plant cells. In this chapter, using microbe-associated molecular patterns (MAMPs), such as the bacterial-derived flg22 or elf18 peptides as stimuli, a protocol for the quantitative measurements of Ca(2+) fluxes in apoaequorin-expressing seedlings of Arabidopsis thaliana in 96-well format is described.