Monitoring calcium handling by the plant endoplasmic reticulum with a low-Ca -affinity targeted aequorin reporter.

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