title: Chloroplast-Specific in Vivo Ca2+ Imaging Using Yellow Cameleon Fluorescent Protein Sensors Reveals Organelle-Autonomous Ca2+ Signatures in the Stroma.
abstract: In eukaryotes, subcellular compartments such as mitochondria, the endoplasmic reticulum, lysosomes, and vacuoles have the capacity for Ca(2+) transport across their membranes to modulate the activity of compartmentalized enzymes or to convey specific cellular signaling events. In plants, it has been suggested that chloroplasts also display Ca(2+) regulation. So far, monitoring of stromal Ca(2+) dynamics in vivo has exclusively relied on using the luminescent Ca(2+) probe aequorin. However, this technique is limited in resolution and can only provide a readout averaged over chloroplast populations from different cells and tissues. Here, we present a toolkit of Arabidopsis (Arabidopsis thaliana) Ca(2+) sensor lines expressing plastid-targeted FRET-based Yellow Cameleon (YC) sensors. We demonstrate that the probes reliably report in vivo Ca(2+) dynamics in the stroma of root plastids in response to extracellular ATP and of leaf mesophyll and guard cell chloroplasts during light-to-low-intensity blue light illumination transition. Applying YC sensing of stromal Ca(2+) dynamics to single chloroplasts, we confirm findings of gradual, sustained stromal Ca(2+) increases at the tissue level after light-to-low-intensity blue light illumination transitions, but monitor transient Ca(2+) spiking as a distinct and previously unknown component of stromal Ca(2+) signatures. Spiking was dependent on the availability of cytosolic Ca(2+) but not synchronized between the chloroplasts of a cell. In contrast, the gradual sustained Ca(2+) increase occurred independent of cytosolic Ca(2+), suggesting intraorganellar Ca(2+) release. We demonstrate the capacity of the YC sensor toolkit to identify novel, fundamental facets of chloroplast Ca(2+) dynamics and to refine the understanding of plastidial Ca(2+) regulation.
authors.affiliation: Department of Biosciences, University of Milan, 20133 Milan, Italy (G.L., F.G.D., S.B., A.C.);Department of Biology, University of Padua, Italy, 35131 Padua, Italy (G.L., M.Z.);Institut für Biologie und Biotechnologie der Pflanzen, Universität Münster, 48149 Münster, Germany (S.We., J.K.);Plant Energy Biology Lab, Institute of Crop Science and Resource Conservation (INRES), University of Bonn, 53113 Bonn, Germany (S.Wa., M.S.); andInstitute of Biophysics, Consiglio Nazionale delle Ricerche, 20133 Milan, Italy (A.C.).
authors.forename: Giovanna
authors.initials: G
authors.lastname: Loro
authors.affiliation: Department of Biosciences, University of Milan, 20133 Milan, Italy (G.L., F.G.D., S.B., A.C.);Department of Biology, University of Padua, Italy, 35131 Padua, Italy (G.L., M.Z.);Institut für Biologie und Biotechnologie der Pflanzen, Universität Münster, 48149 Münster, Germany (S.We., J.K.);Plant Energy Biology Lab, Institute of Crop Science and Resource Conservation (INRES), University of Bonn, 53113 Bonn, Germany (S.Wa., M.S.); andInstitute of Biophysics, Consiglio Nazionale delle Ricerche, 20133 Milan, Italy (A.C.).
authors.forename: Stephan
authors.identifier: 0000-0001-5369-7911
authors.initials: S
authors.lastname: Wagner
authors.affiliation: Department of Biosciences, University of Milan, 20133 Milan, Italy (G.L., F.G.D., S.B., A.C.);Department of Biology, University of Padua, Italy, 35131 Padua, Italy (G.L., M.Z.);Institut für Biologie und Biotechnologie der Pflanzen, Universität Münster, 48149 Münster, Germany (S.We., J.K.);Plant Energy Biology Lab, Institute of Crop Science and Resource Conservation (INRES), University of Bonn, 53113 Bonn, Germany (S.Wa., M.S.); andInstitute of Biophysics, Consiglio Nazionale delle Ricerche, 20133 Milan, Italy (A.C.).
authors.forename: Fabrizio Gandolfo
authors.identifier: 0000-0002-4499-2297
authors.initials: FG
authors.lastname: Doccula
authors.affiliation: Department of Biosciences, University of Milan, 20133 Milan, Italy (G.L., F.G.D., S.B., A.C.);Department of Biology, University of Padua, Italy, 35131 Padua, Italy (G.L., M.Z.);Institut für Biologie und Biotechnologie der Pflanzen, Universität Münster, 48149 Münster, Germany (S.We., J.K.);Plant Energy Biology Lab, Institute of Crop Science and Resource Conservation (INRES), University of Bonn, 53113 Bonn, Germany (S.Wa., M.S.); andInstitute of Biophysics, Consiglio Nazionale delle Ricerche, 20133 Milan, Italy (A.C.).
authors.forename: Smrutisanjita
authors.identifier: 0000-0001-8749-6130
authors.initials: S
authors.lastname: Behera
authors.affiliation: Department of Biosciences, University of Milan, 20133 Milan, Italy (G.L., F.G.D., S.B., A.C.);Department of Biology, University of Padua, Italy, 35131 Padua, Italy (G.L., M.Z.);Institut für Biologie und Biotechnologie der Pflanzen, Universität Münster, 48149 Münster, Germany (S.We., J.K.);Plant Energy Biology Lab, Institute of Crop Science and Resource Conservation (INRES), University of Bonn, 53113 Bonn, Germany (S.Wa., M.S.); andInstitute of Biophysics, Consiglio Nazionale delle Ricerche, 20133 Milan, Italy (A.C.).
authors.forename: Stefan
authors.initials: S
authors.lastname: Weinl
authors.affiliation: Department of Biosciences, University of Milan, 20133 Milan, Italy (G.L., F.G.D., S.B., A.C.);Department of Biology, University of Padua, Italy, 35131 Padua, Italy (G.L., M.Z.);Institut für Biologie und Biotechnologie der Pflanzen, Universität Münster, 48149 Münster, Germany (S.We., J.K.);Plant Energy Biology Lab, Institute of Crop Science and Resource Conservation (INRES), University of Bonn, 53113 Bonn, Germany (S.Wa., M.S.); andInstitute of Biophysics, Consiglio Nazionale delle Ricerche, 20133 Milan, Italy (A.C.).
authors.forename: Joerg
authors.initials: J
authors.lastname: Kudla
authors.affiliation: Department of Biosciences, University of Milan, 20133 Milan, Italy (G.L., F.G.D., S.B., A.C.);Department of Biology, University of Padua, Italy, 35131 Padua, Italy (G.L., M.Z.);Institut für Biologie und Biotechnologie der Pflanzen, Universität Münster, 48149 Münster, Germany (S.We., J.K.);Plant Energy Biology Lab, Institute of Crop Science and Resource Conservation (INRES), University of Bonn, 53113 Bonn, Germany (S.Wa., M.S.); andInstitute of Biophysics, Consiglio Nazionale delle Ricerche, 20133 Milan, Italy (A.C.).
authors.forename: Markus
authors.identifier: 0000-0003-0796-8308
authors.initials: M
authors.lastname: Schwarzländer
authors.affiliation: Department of Biosciences, University of Milan, 20133 Milan, Italy (G.L., F.G.D., S.B., A.C.);Department of Biology, University of Padua, Italy, 35131 Padua, Italy (G.L., M.Z.);Institut für Biologie und Biotechnologie der Pflanzen, Universität Münster, 48149 Münster, Germany (S.We., J.K.);Plant Energy Biology Lab, Institute of Crop Science and Resource Conservation (INRES), University of Bonn, 53113 Bonn, Germany (S.Wa., M.S.); andInstitute of Biophysics, Consiglio Nazionale delle Ricerche, 20133 Milan, Italy (A.C.) alex.costa@unimi.it.
authors.forename: Alex
authors.identifier: 0000-0002-2628-1176
authors.initials: A
authors.lastname: Costa
authors.affiliation: Department of Biosciences, University of Milan, 20133 Milan, Italy (G.L., F.G.D., S.B., A.C.);Department of Biology, University of Padua, Italy, 35131 Padua, Italy (G.L., M.Z.);Institut für Biologie und Biotechnologie der Pflanzen, Universität Münster, 48149 Münster, Germany (S.We., J.K.);Plant Energy Biology Lab, Institute of Crop Science and Resource Conservation (INRES), University of Bonn, 53113 Bonn, Germany (S.Wa., M.S.); andInstitute of Biophysics, Consiglio Nazionale delle Ricerche, 20133 Milan, Italy (A.C.).
authors.forename: Michela
authors.initials: M
authors.lastname: Zottini
mesh_terms: D000331:Aequorin / Q000235:genetics / Q000378:metabolism*; D017360:Arabidopsis / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins; D002736:Chloroplasts / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D018515:Plant Leaves / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D018517:Plant Roots / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D018087:Plastids / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D014617:Vacuoles / Q000378:metabolism
chemical_list: D002135:Calcium-Binding Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C495639:yellow cameleon; D000331:Aequorin; D002118:Calcium