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Monitoring calcium handling by the plant endoplasmic reticulum with a low‐Ca 2+ ‐affinity targeted aequorin reporter
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Cortese, Enrico, Moscatiello, Roberto, Pettiti, Francesca, Carraretto, Luca, Baldan, Barbara, Frigerio, Lorenzo, Vothknecht, Ute C., Szabo, Ildiko, De Stefani, Diego, Brini, Marisa and Navazio, Lorella (2022) Monitoring calcium handling by the plant endoplasmic reticulum with a low‐Ca 2+ ‐affinity targeted aequorin reporter. The Plant Journal, 109 (4). pp. 1014-1027. doi:10.1111/tpj.15610
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WRAP-Monitoring-calcium-handling-plant-endoplasmicreticulum-low-Ca2+-affinity-aequorin-2022.pdf - Published Version - Requires a PDF viewer. Available under License Creative Commons Attribution 4.0. Download (6Mb) | Preview |
Official URL: http://dx.doi.org/10.1111/tpj.15610
Abstract
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.
| Item Type: | Journal Article | |||||||||
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| Subjects: | Q Science > QH Natural history > QH301 Biology Q Science > QP Physiology |
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| Divisions: | Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) | |||||||||
| Library of Congress Subject Headings (LCSH): | Calcium -- Metabolism, Homeostasis, Endoplastic reticulum, Arabidopsis thaliana, Plant genetics, Plant cell membranes | |||||||||
| Journal or Publication Title: | The Plant Journal | |||||||||
| Publisher: | Wiley-Blackwell Publishing Ltd. | |||||||||
| ISSN: | 0960-7412 | |||||||||
| Official Date: | February 2022 | |||||||||
| Dates: |
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| Volume: | 109 | |||||||||
| Number: | 4 | |||||||||
| Page Range: | pp. 1014-1027 | |||||||||
| DOI: | 10.1111/tpj.15610 | |||||||||
| Status: | Peer Reviewed | |||||||||
| Publication Status: | Published | |||||||||
| Access rights to Published version: | Open Access | |||||||||
| RIOXX Funder/Project Grant: |
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