<i>In vivo</i> biosensing of subcellular pyruvate pools reveals photosynthesis-dependent metabolite dynamics in <i>Nicotiana benthamiana</i>

Pyruvate is central to metabolism across biology. It acts as a metabolic hub linking major pathways including glycolysis, the Krebs cycle, fermentation, and synthesis of amino acids, fatty acids, isoprenoids, and nucleotides. Even though the central role of pyruvate is well established biochemically, there is a remarkable gap in our understanding of how pyruvate levels behave within cells, where pyruvate is distributed across different compartments. Moreover, differential changes in pyruvate pools may occur rapidly upon changes in metabolic fluxes. Recently, this problem has been addressed by the development of a genetically encoded pyruvate biosensor to provide first insights into the pyruvate dynamics in animal cells. Here, we established in vivo biosensing of pyruvate in plants. We provided advanced characterization of the biosensor properties and demonstrated the functionality of the sensor in the cytosol, the mitochondria, and the chloroplasts of Nicotiana benthamiana epidermal cells. Finally, we harnessed the tool to investigate the impact of photosynthesis on pyruvate with unprecedented spatial and temporal resolution, revealing pronounced changes in subcellular pyruvate pools. While highlighting the current limitations of the biosensor, this study provides proof-of-concept for how the dynamics and regulation of central carbon metabolites can be revealed in living plant tissues.