Chloroplast Ca Fluxes into and across Thylakoids Revealed by Thylakoid-Targeted Aequorin Probes.

Chloroplasts require a fine-tuned control of their internal Ca concentration, which is crucial for many aspects of photosynthesis and for other chloroplast-localized processes. Increasing evidence suggests that calcium regulation within chloroplasts also may influence Ca signaling pathways in the cytosol. To investigate the involvement of thylakoids in Ca homeostasis and in the modulation of chloroplast Ca signals in vivo, we targeted the bioluminescent Ca reporter aequorin as a YFP fusion to the lumen and the stromal surface of thylakoids in Arabidopsis (). Thylakoid localization of aequorin-based probes in stably transformed lines was confirmed by confocal microscopy, immunogold labeling, and biochemical analyses. In resting conditions in the dark, free Ca levels in the thylakoid lumen were maintained at about 0.5 μm, which was a 3- to 5-fold higher concentration than in the stroma. Monitoring of chloroplast Ca dynamics in different intrachloroplast subcompartments (stroma, thylakoid membrane, and thylakoid lumen) revealed the occurrence of stimulus-specific Ca signals, characterized by unique kinetic parameters. Oxidative and salt stresses initiated pronounced free Ca changes in the thylakoid lumen. Localized Ca increases also were observed on the thylakoid membrane surface, mirroring transient Ca changes observed for the bulk stroma, but with specific Ca dynamics. Moreover, evidence was obtained for dark-stimulated intrathylakoid Ca changes, suggesting a new scenario for light-to-dark-induced Ca fluxes inside chloroplasts. Hence, thylakoid-targeted aequorin reporters can provide new insights into chloroplast Ca storage and signal transduction. These probes represent novel tools with which to investigate the role of thylakoids in Ca signaling networks within chloroplasts and plant cells.