title: FRET-based genetically encoded sensors allow high-resolution live cell imaging of Ca²⁺ dynamics.
abstract: Temporally and spatially defined calcium signatures are integral parts of numerous signalling pathways. Monitoring calcium dynamics with high spatial and temporal resolution is therefore critically important to understand how this ubiquitous second messenger can control diverse cellular responses. Yellow cameleons (YCs) are fluorescence resonance energy transfer (FRET)-based genetically encoded Ca(2+)  -sensors that provide a powerful tool to monitor the spatio-temporal dynamics of Ca(2+)  fluxes. Here we present an advanced set of vectors and transgenic lines for live cell Ca(2+)  imaging in plants. Transgene silencing mediated by the cauliflower mosaic virus (CaMV) 35S promoter has severely limited the application of nanosensors for ions and metabolites and we have thus used the UBQ10 promoter from Arabidopsis and show here that this results in constitutive and stable expression of YCs in transgenic plants. To improve the spatial resolution, our vector repertoire includes versions of YCs that can be targeted to defined locations. Using this toolkit, we identified temporally distinct responses to external ATP at the plasma membrane, in the cytosol and in the nucleus of neighbouring root cells. Moreover analysis of Ca(2+)  dynamics in Lotus japonicus revealed distinct Nod factor induced Ca(2+)  spiking patterns in the nucleus and the cytosol. Consequently, the constructs and transgenic lines introduced here enable a detailed analysis of Ca(2+)  dynamics in different cellular compartments and in different plant species and will foster novel approaches to decipher the temporal and spatial characteristics of calcium signatures.
authors.affiliation: Department of Developmental Biology, Centre for Organismal Studies (COS), University of Heidelberg, Im Neuenheimer Feld 230, 69120 Heidelberg, Germany.
authors.forename: Melanie
authors.initials: M
authors.lastname: Krebs
authors.forename: Katrin
authors.initials: K
authors.lastname: Held
authors.forename: Andreas
authors.initials: A
authors.lastname: Binder
authors.forename: Kenji
authors.initials: K
authors.lastname: Hashimoto
authors.forename: Griet
authors.initials: G
authors.lastname: Den Herder
authors.forename: Martin
authors.initials: M
authors.lastname: Parniske
authors.forename: Jörg
authors.initials: J
authors.lastname: Kudla
authors.forename: Karin
authors.initials: K
authors.lastname: Schumacher
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D017360:Arabidopsis / Q000235:genetics*; D015374:Biosensing Techniques / Q000379:methods; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000032:analysis; D017796:Caulimovirus / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D018548:Cotyledon / Q000235:genetics / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005822:Genetic Vectors; D000070116:Lotus / Q000166:cytology* / Q000378:metabolism; D018517:Plant Roots / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D019076:Transgenes
chemical_list: D002135:Calcium-Binding Proteins; C495639:yellow cameleon; D000255:Adenosine Triphosphate; D002118:Calcium