The burning glass effect of water droplets triggers a high light-induced calcium response in the chloroplast stroma

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 Ca2+ signaling has been linked to high light (HL) acclimation, the subcellular dynamics remain unclear. Here, we investigate Ca2+ responses to HL exposure in Arabidopsis thaliana. Using a glass bead to simulate light-focusing by water droplets, a biphasic increase of Ca2+ 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 Ca2+ response. While blue and white light also triggered a Ca2+ response in the cytosol and nucleus, pharmacological inhibition with cyclopiazonic acid (CPA) and loss-of-function mutants of the Ca2+ transporters BIVALENT CATION TRANSPORTER 2 (BICAT2) and endoplasmic reticulum (ER)-type Ca2+-ATPase (ECA) suggested that the HL response depends on a Ca2+ exchange between the ER and chloroplast stroma. The response was primarily light dependent but accelerated by increasing external temperature. This study implicates a novel Ca2+-mediated acclimation mechanism to HL stress, a process of growing relevance in the context of climate change.