Publications - Stress and Develop Biology

Upon pathogen recognition, a transient rise in cytoplasmic calcium levels is one of the earliest events in plants and a prerequisite for defense initiation and signal propagation from a local site to systemic plant tissues. However, it is unclear if calcium signaling differs in the context of priming: Do plants exposed to a first pathogen stimulus and have consequently established systemic acquired resistance (SAR) display altered calcium responses to a second pathogen stimulus? Several calcium indicator systems including aequorin, YC3.6 or R-GECO1 have been used to document local calcium responses to the bacterial flg22 peptide but systemic calcium imaging within a single plant remains a technical challenge. Here, we report on an experimental approach to monitor flg22-induced calcium responses in systemic leaves of primed plants. The calcium-dependent protein kinase CPK5 is a key calcium sensor and regulator of the NADPH oxidase RBOHD and plays a role in the systemic calcium-ROS signal propagation. We therefore compared flg22-induced cytoplasmic calcium changes in Arabidopsis wild-type, cpk5 mutant and CPK5-overexpressing plants (exhibiting constitutive priming) by introgressing the calcium indicator R-GECO1-mTurquoise that allows internal normalization through mTurquoise fluorescence. Aequorin-based analyses were included for comparison. Based on the R-GECO1-mTurquoise data, CPK5-OE appears to reinforce an “oscillatory-like” Ca2+ signature in flg22-treated local tissues. However, no change was observed in the flg22-induced calcium response in the systemic tissues of plants that had been pre-challenged by a priming stimulus – neither in wild-type nor in cpk5 or CPK5-OE-lines. These data indicate that the mechanistic manifestation of a plant immune memory in distal plant parts required for enhanced pathogen resistance does not include changes in rapid calcium signaling upstream of CPK5 but rather relies on downstream defense responses.

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.