In vivo biosensing of subcellular pyruvate pools reveals photosynthesis-dependent metabolite dynamics in Nicotiana benthamiana.

Pyruvate is central to metabolism across biology. It acts as a metabolic hub linking key 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 void in our understanding of how pyruvate levels behave within cells, where pyruvate is distributed across different compartments, and 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 establish in vivo biosensing of pyruvate in plants. We provide advanced characterisation of the biosensor properties and demonstrate 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 cytosolic 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 the context of living plant tissues.