Abstract
SUMMARYPrecise 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.
Highlights
Calcium is a fundamental intracellular messenger that plays a key role in the transduction of a wide range of stimuli in all living organisms (Berridge et al, 2000; Dodd et al, 2010; Domınguez et al, 2015)
We developed a novel Ca2+ reporter for the plant endoplasmic reticulum (ER) that is useful for quantitative analyses of Ca2+ signatures in this compartment
AEQmut had previously been used as a suitable Ca2+ probe to quantitatively measure ER Ca2+ levels ([Ca2+]ER) in mammalian cells (Brini, 2008; Montero et al, 1995; Ottolini et al, 2014)
Summary
Calcium is a fundamental intracellular messenger that plays a key role in the transduction of a wide range of stimuli in all living organisms (Berridge et al, 2000; Dodd et al, 2010; Domınguez et al, 2015). Implementation of Ca2+ reporter-based technologies (Alonso et al, 2017; Perez Koldenkova and Nagai, 2013) into cellular and molecular studies has helped to provide a more detailed picture of the complexity of intracellular Ca2+ signalling networks, paving the way to investigate the fine-tuned integration of internal mobilizable Ca2+ stores in achieving an efficient Ca2+ homeostasis and signal transduction (Brini et al, 2013; Costa et al., 2018). Information on the precise role of different intracellular compartments in Ca2+ handling in the plant cell is still incomplete, especially regarding compartments such as the endoplasmic reticulum (ER), for which only putative estimates of basal [Ca2+] and its changes are available (Costa et al, 2018; Stael et al, 2012).
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