Precise measurements of dynamic changes in free Ca²⁺ 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 Ca²⁺ homeostasis and signalling in the plant cell. In the present study, we targeted an aequorin chimera with reduced Ca²⁺ affinity to the ER membrane and facing the ER lumen. To this aim, the cDNA for a low-Ca²⁺ -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 Ca²⁺ depletion during photoprotein reconstitution followed by ER Ca²⁺ refilling - was applied to carry out ER Ca²⁺ measurements in planta. Rapid and transient increases of the ER luminal Ca²⁺ concentration ([Ca²⁺ ]_ER ) were recorded in response to different environmental stresses, displaying stimulus-specific Ca²⁺ signatures. The comparative analysis of ER and chloroplast Ca²⁺ dynamics indicates a complex interplay of these organelles in shaping cytosolic Ca²⁺ signals during signal transduction events. Our data highlight significant differences in basal [Ca²⁺ ]_ER and Ca²⁺ 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 Ca²⁺ indicators by adding a reporter that improves our quantitative understanding of Ca²⁺ homeostasis in the plant endomembrane system.

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