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Photosynthetic activity triggers pH and NAD redox signatures across different plant cell compartments

A characteristic feature of most plants is their ability to perform photosynthesis, which ultimately provides energy and organic substrates to most life. Photosynthesis dominates chloroplast physiology but represents only a fraction of the tightly interconnected metabolic network that spans the entire cell. Here, we explore how photosynthetic activity affects the energy physiological status in cell compartments beyond the chloroplast. We develop precision live monitoring of subcellular energy physiology under illumination to investigate pH, MgATP2− and NADH/NAD+ dynamics at dark-light transitions by confocal imaging of genetically encoded fluorescent protein biosensors in Arabidopsis leaf mesophyll. We resolve the in vivo signature of stromal alkalinisation resulting from photosynthetic proton pumping and observe a similar pH signature also in the cytosol and the mitochondria suggesting that photosynthesis triggers an ‘alkalinisation wave’ that affects the pH landscape of large parts of the cell. MgATP2− increases in the stroma at illumination, but no major effects on MgATP2− concentrations in the cytosol were resolved. Photosynthetic activity triggers a signature of substantial NAD reduction in the cytosol that is driven by photosynthesis-derived electron export. Strikingly, cytosolic NAD redox status was deregulated in mutants of chloroplastic NADP- and mitochondrial NAD-dependent malate dehydrogenases even at darkness, pinpointing the participation of the chloroplasts and mitochondria in shaping cytosolic redox metabolism in vivo with a dominant function of malate metabolism. Our data illustrate how profoundly and rapidly changes in photosynthetic activity affect the physiological and metabolic landscape throughout green plant cells. One-sentence summary: Dark-light transitions trigger profound re-orchestration of subcellular pH and NAD redox physiology not only in the chloroplast but also beyond, in the cytosol and the mitochondria, as revealed by precision live-monitoring using fluorescent protein biosensors.