Ca2+ transfer between chloroplast and ER is perturbed by high light

Plants rely on water and light for photosynthesis, but water droplets on leaves can focus light into high-intensity spots, risking photodamage. Excessive light can impair growth or induce cell death, making it essential for plants to detect and respond to light fluctuations. While Ca signaling has been linked to high light (HL) acclimation, the subcellular dynamics remain unclear. Here, we investigate Ca responses to HL exposure in Arabidopsis thaliana. Using a glass bead to simulate light-focusing by water droplets, a biphasic increase of Ca concentration was detected in the chloroplast stroma by the genetically encoded calcium indicator YC3.6 and confirmed using a newly established stroma-localized R-GECO1 (NTRC-R-GECO1). The stromal response was largely independent of light wavelength and unaffected in phot1 phot2 and cry1 cry2 mutants. Chemical inhibition of photosynthetic electron transport, microscopy-based Fv/Fm experiments, and measurement of the reactive oxygen species (ROS)-redox balance with roGFP-based reporters and Singlet Oxygen Sensor Green (SOSG) chemical dye suggested that photodamage and singlet oxygen contribute to the stromal Ca response. While blue and white light also triggered a Ca response in the cytosol and nucleus, pharmacological inhibition with cyclopiazonic acid (CPA) and loss-of-function mutants of the Ca transporters BIVALENT CATION TRANSPORTER 2 (BICAT2) and endoplasmic reticulum (ER)-type Ca-ATPase (ECA) suggested that the HL response depends on a Ca exchange between the ER and chloroplast stroma. The response was primarily light dependent but accelerated by increasing external temperature. This study implicates a novel Ca-mediated acclimation mechanism to HL stress, a process of growing relevance in the context of climate change.