Chloroplast-specific in vivo Ca2+ imaging using yellow Cameleon fluorescent protein sensors reveals organelle-autonomous Ca2+ signatures in the stroma

Loro G., Wagner S., Doccula F., Behera S., Weinl S., Kudla J., Schwarzländer M., Costa A., Zottini M.

Abstract

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.