Advanced illumination-imaging reveals photosynthesis-triggered pH, ATP and NAD redox signatures across plant cell compartments

Zheng, K.; Elsässer, M.; Niemeier, J.-O.; Barreto, P.; Cislaghi, A. P.; Hoang, M.; Feitosa-Araujo, E.; Wagner, S.; Giese, J.; Kotnik, F.; Martinez, M. d. P.; Buchert, F. E.; Ugalde, J. M.; Armbruster, U.; Hippler, M.; Meyer, A. J.; Kunz, H.-H.; Maurino, V. G.; Finkemeier, I.; Schallenberg-Rüdinger, M.; Schwarzländer, M.

doi:10.1101/2025.06.16.659786

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bioRxiv · 10.1101/2025.06.16.659786

Advanced illumination-imaging reveals photosynthesis-triggered pH, ATP and NAD redox signatures across plant cell compartments

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Abstract

Photosynthesis provides energy and organic substrates to most life. In plants, 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 energy physiology within and beyond the chloroplast. We developed a new standard for the live-monitoring of subcellular energy physiology by combining confocal imaging of genetically encoded fluorescent protein biosensors with advanced on-stage illumination technology to investigate pH, MgATP2- and NADH/NAD+ dynamics at dark-light transitions in Arabidopsis mesophyll cells. Our findings reveal a stromal alkalinization signature induced by photosynthetic proton pumping, extending to the cytosol and mitochondria as an alkalinization wave. Photosynthesis leads to increased MgATP2- levels in both the stroma and cytosol. Additionally, we observed reduction of the NAD pool driven by photosynthesis-derived electron export. Arabidopsis lines defective in chloroplast NADP- and mitochondrial NAD-dependent malate dehydrogenases show more reduced cytosolic NAD redox status even in darkness, highlighting the involvement of chloroplasts and mitochondria in shaping cytosolic redox metabolism via malate metabolism. Our study sets a novel methodological standard for precision live-monitoring of photosynthetic cell physiology. Applying this technology reveals signatures of photosynthetic physiology within and beyond the chloroplast with unprecedented resolution. Those signatures link photosynthetic activity and the fundamental biochemical functions of phototrophic cells. Significance statementBy applying novel live microscopy monitoring using fluorescent protein biosensors in plant cells, we reveal that dark-light transitions trigger profound re-orchestration of subcellular pH, ATP and NAD redox physiology not limited to chloroplasts but extending into the cytosol and the mitochondria.

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Zheng, K., Elsässer, M., Niemeier, J.-O., Barreto, P., Cislaghi, A. P., Hoang, M., Feitosa-Araujo, E., Wagner, S., Giese, J., Kotnik, F., Martinez, M. d. P., Buchert, F. E., Ugalde, J. M., Armbruster, U., Hippler, M., Meyer, A. J., Kunz, H.-H., Maurino, V. G., Finkemeier, I., Schallenberg-Rüdinger, M., Schwarzländer, M.. 2025-06-21. Advanced illumination-imaging reveals photosynthesis-triggered pH, ATP and NAD redox signatures across plant cell compartments. https://doi.org/10.1101/2025.06.16.659786

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