Monitoring calcium handling by the plant endoplasmic reticulum with a low‐Ca(2+)‐affinity targeted aequorin reporter

Precise measurements of dynamic changes in free Ca(2+) concentration in the lumen of the plant endoplasmic reticulum (ER) have been lacking so far, despite increasing evidence for the contribution of this intracellular compartment to Ca(2+) homeostasis and signalling in the plant cell. In the present study, we targeted an aequorin chimera with reduced Ca(2+) affinity to the ER membrane and facing the ER lumen. To this aim, the cDNA for a low‐Ca(2+)‐affinity aequorin variant (AEQmut) was fused to the nucleotide sequence encoding a non‐cleavable N‐terminal ER signal peptide (fl2). The correct targeting of fl2‐AEQmut was confirmed by immunocytochemical analyses in transgenic Arabidopsis thaliana (Arabidopsis) seedlings. An experimental protocol well‐established in animal cells – consisting of ER Ca(2+) depletion during photoprotein reconstitution followed by ER Ca(2+) refilling – was applied to carry out ER Ca(2+) measurements in planta. Rapid and transient increases of the ER luminal Ca(2+) concentration ([Ca(2+)](ER)) were recorded in response to different environmental stresses, displaying stimulus‐specific Ca(2+) signatures. The comparative analysis of ER and chloroplast Ca(2+) dynamics indicates a complex interplay of these organelles in shaping cytosolic Ca(2+) signals during signal transduction events. Our data highlight significant differences in basal [Ca(2+)](ER) and Ca(2+) handling by plant ER compared to the animal counterpart. The set‐up of an ER‐targeted aequorin chimera extends and complements the currently available toolkit of organelle‐targeted Ca(2+) indicators by adding a reporter that improves our quantitative understanding of Ca(2+) homeostasis in the plant endomembrane system.