Cytosolic calcium signals elicited by the pathogen-associated molecular pattern flg22 in stomatal guard cells are of an oscillatory nature

Abstract

Changes in cytosolic free calcium ([Ca2+]cyt) are an early and essential element of signalling networks activated by the perception of pathogen-associated molecular patterns (PAMPs), such as flg22. The flg22-induced calcium signal has been described on whole-plant, but not on single-cell scale so far. Also, the Ca2+ sources and channels contributing to its generation are still obscure. Ratiometric fluorescence imaging employing the calcium reporter Yellow Cameleon 3.6 was performed to analyse the flg22-induced calcium signature in single guard cells of Arabidopsis thaliana. Calcium stores and channel types involved in its generation were determined by a pharmacological approach. In contrast to the calcium signal determined on whole-plant level, the signature on single-cell level is not characterized by one sustained response, but by oscillations in [Ca2+]cyt. These oscillations were abolished by EGTA and lanthanum, as well as by U73122, neomycin and TMB-8, but only partially or not at all affected by inhibitors of glutamate receptor-like channels and cyclic nucleotide-gated channels. Our analyses suggest that the response observed on whole-plant level is the summary of oscillations occurring in single cells. Parallel to external calcium, influx via channels located at internal stores contributes to the signal.