Analyses of Ca2+ accumulation and dynamics in the Endoplasmic Reticulum of Arabidopsis thaliana root cells using genetically encoded Cameleon sensors

Calcium is a versatile second messenger in plants that triggers developmental programs and translates environmental stimuli into specific cellular responses. Cytoplasmic Ca2+ signals are the results of a precised orchestration of Ca2+ influxes and effluxes occurring at different cellular membranes, including plasma, endoplasmic reticulum (ER), vacuolar and other organellar membranes. So far, in planta, only very limited information is available about how the ER contributes to cellular Ca2+ dynamics and homeostasis. Here we report the generation of new ER-targeted Cameleon reporter proteins (CRT-D1ER and CRT-D4ER) for suitable analysis of Ca2+ accumulation and dynamic in the lumen of the ER in plant cells. Using stably transformed Arabidopsis plants expressing the new reporter proteins we observed a transiently enhanced accumulation of Ca2+ in the ER in response to stimuli inducing cytosolic Ca2+ rises in root tip cells. In all experimental conditions ER Ca2+ dynamics were substantially different from those monitored in the cytosol. A pharmacological approach allowed us to evaluate the contribution of the different ER resident Ca2+-ATPase classes in the regulation of the ER Ca2+ homeostasis. Together, our results do not provide evidence for a role of the ER as a major source for stimulus-induced increases in cytosolic Ca2+ concentration. Instead, our results identify a critical role of the ER as a Ca2+ buffering system. These findings suggest fundamental differences for the function of the ER in cellular Ca2+ homeostasis in plants and animals.