In eukaryotes, subcellular compartments such as mitochondria, the endoplasmic reticulum, lysosomes, and vacuoles have the capacity for Ca2+ 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 Ca2+ regulation. So far, monitoring of stromal Ca2+ dynamics in vivo has exclusively relied on using the luminescent Ca2+ 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) Ca2+ sensor lines expressing plastid-targeted FRET-based Yellow Cameleon (YC) sensors. We demonstrate that the probes reliably report in vivo Ca2+ 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 Ca2+ dynamics to single chloroplasts, we confirm findings of gradual, sustained stromal Ca2+ increases at the tissue level after light-to-low-intensity blue light illumination transitions, but monitor transient Ca2+ spiking as a distinct and previously unknown component of stromal Ca2+ signatures. Spiking was dependent on the availability of cytosolic Ca2+ but not synchronized between the chloroplasts of a cell. In contrast, the gradual sustained Ca2+ increase occurred independent of cytosolic Ca2+, suggesting intraorganellar Ca2+ release. We demonstrate the capacity of the YC sensor toolkit to identify novel, fundamental facets of chloroplast Ca2+ dynamics and to refine the understanding of plastidial Ca2+ regulation.
Chloroplast-specific in vivo Ca2+ imaging using Yellow Cameleon fluorescent protein sensors reveals organelle-autonomous Ca2+ signatures in the stroma / G. Loro, S. Wagner, F.G. Doccula, S. Behera, S. Weinl, J. Kudla, M. Schwarzländer, A. Costa, M. Zottini. - In: PLANT PHYSIOLOGY. - ISSN 1532-2548. - 171:4(2016 Aug 03), pp. 2317-2330.
|Titolo:||Chloroplast-specific in vivo Ca2+ imaging using Yellow Cameleon fluorescent protein sensors reveals organelle-autonomous Ca2+ signatures in the stroma|
LORO, GIOVANNA (Primo)
COSTA, ALEX (Penultimo)
|Parole Chiave:||arabidopsis-thaliana; intact chloroflasts; extracellular ATP; free calcium; mediated activation; mitochondrial; dynamics; plants; oscillations; indicators|
|Settore Scientifico Disciplinare:||Settore BIO/04 - Fisiologia Vegetale|
|Progetto:||NUCLEOTIDI CICLICI NELLA RISPOSTA A STRESS BIOTICO IN PIANTA|
|Data di pubblicazione:||3-ago-2016|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.1104/pp.16.00652|
|Appare nelle tipologie:||01 - Articolo su periodico|