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.

Department of Biological and Environmental Sciences University of Gothenburg 40530 Gothenburg Sweden

Department of Biosciences University of Milan Via Giovanni Celoria 26 20133 Milan Italy

Department of Biosciences University of Milan Via Giovanni Celoria 26 20133 Milan Italy; Institute of Biophysics National Research Council of Italy 20133 Milan Italy

Department of Functional and Evolutionary Ecology University of Vienna Djerassiplatz 1 1030 Vienna Austria

Department of Materials Science and Engineering Uppsala University 75103 Uppsala Sweden; Science for Life Laboratory Uppsala University 75103 Uppsala Sweden

Department of Molecular Sciences Uppsala BioCenter Swedish University of Agricultural Sciences and Linnean Center for Plant Biology 75007 Uppsala Sweden

Department of Plant Biotechnology and Bioinformatics Ghent University 9052 Ghent Belgium; VIB Center for Plant Systems Biology 9052 Ghent Belgium

Institute of Plant Biology and Biotechnology Universität Münster Schlossplatz 8 48143 Münster Germany

Photon Systems Instruments 66424 Drasov Czech Republic

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