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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 ISSN 0960-7412.

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Official URL: http://dx.doi.org/10.1111/tpj.15610

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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
Subjects: Q Science > QH Natural history > QH301 Biology
Q Science > QP Physiology
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:
DateEvent
February 2022Published
26 November 2021Available
22 November 2021Accepted
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 (Creative Commons)
Date of first compliant deposit: 26 January 2022
Date of first compliant Open Access: 26 January 2022
RIOXX Funder/Project Grant:
Project/Grant IDRIOXX Funder NameFunder ID
PRID 2018 prot. BIRD180317Universitá di Padovahttp://viaf.org/viaf/236210361
DOR 2019-2021Universitá di Padovahttp://viaf.org/viaf/236210361

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