Monitoring calcium handling by the plant endoplasmic reticulum with a low-Ca2+-affinity targeted aequorin reporter
| Autor: | Lorenzo Frigerio, Barbara Baldan, Ute C. Vothknecht, Roberto Moscatiello, Francesca Pettiti, Diego De Stefani, Luca Carraretto, Ildikò Szabò, Marisa Brini, Enrico Cortese, Lorella Navazio |
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| Jazyk: | angličtina |
| Rok vydání: | 2022 |
| Předmět: |
Signal peptide
biology Arabidopsis thaliana calcium homeostasis environmental stresses Endoplasmic reticulum Aequorin Photoprotein aequorin Cell Biology Plant Science biology.organism_classification QP Cell biology Cytosol QH301 endoplasmic reticulum chloroplasts signal transduction Genetics biology.protein Endomembrane system Signal transduction |
| ISSN: | 0960-7412 |
| Popis: | 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.\ud \ud |
| Databáze: | OpenAIRE |
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