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Growth and development of plants is ultimately driven by light energy captured through photosynthesis. ATP acts as universal cellular energy cofactor fuelling all life processes, including gene expression, metabolism, and transport. Despite a mechanistic understanding of ATP biochemistry, ATP dynamics in the living plant have been largely elusive. Here, we establish MgATP2- measurement in living plants using the fluorescent protein biosensor ATeam1.03-nD/nA. We generate Arabidopsis sensor lines and investigate the sensor in vitro under conditions appropriate for the plant cytosol. We establish an assay for ATP fluxes in isolated mitochondria, and demonstrate that the sensor responds rapidly and reliably to MgATP2- changes in planta. A MgATP2- map of the Arabidopsis seedling highlights different MgATP2- concentrations between tissues and within individual cell types, such as root hairs. Progression of hypoxia reveals substantial plasticity of ATP homeostasis in seedlings, demonstrating that ATP dynamics can be monitored in the living plant.

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<i>a. thaliana</i>, ATP, fluorescent protein sensor, hypoxia, in vivo, light sheet fluorescence microscopy (LSFM), plant biology, root hair, Adenosine Triphosphate, Arabidopsis, Biosensing Techniques, Energy Metabolism, Genes, Reporter, Homeostasis, Hypoxia, Luminescent Proteins, Plant Cells, Seedlings, Staining and Labeling