Quantitative Analysis of Microbe-Associated Molecular Pattern (MAMP)-Induced Ca2+ Transients in Plants
Ca2+ 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 Ca2+ elevation include touch, reactive oxygen species, cold shock, and salt or osmotic stress. Furthermore, Ca2+ 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 Ca2+ signals (so-called Ca2+ signatures) to enable plants to differentiate between these stimuli and to trigger the appropriate cellular response. Qualitative and quantitative measurements of Ca2+ signals are therefore needed to dissect the responses of plants to their environment. Luminescence produced by the Ca2+ probe aequorin upon Ca2+ binding is a widely used method for the detection of Ca2+ transients and other changes in Ca2+ 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 Ca2+ fluxes in apoaequorin-expressing seedlings of Arabidopsis thaliana in 96-well format is described.
Key wordsCa2+ measurements Aequorin Microbe-associated molecular pattern (MAMP)