ABSTRACT
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.
ABSTRACT
NADH and NAD+ are a ubiquitous cellular redox couple. Although the central role of NAD in plant metabolism and its regulatory role have been investigated extensively at the biochemical level, analyzing the subcellular redox dynamics of NAD in living plant tissues has been challenging. Here, we established live monitoring of NADH/NAD+ in plants using the genetically encoded fluorescent biosensor Peredox-mCherry. We established Peredox-mCherry lines of Arabidopsis (Arabidopsis thaliana) and validated the biophysical and biochemical properties of the sensor that are critical for in planta measurements, including specificity, pH stability, and reversibility. We generated an NAD redox atlas of the cytosol of living Arabidopsis seedlings that revealed pronounced differences in NAD redox status between different organs and tissues. Manipulating the metabolic status through dark-to-light transitions, respiratory inhibition, sugar supplementation, and elicitor exposure revealed a remarkable degree of plasticity of the cytosolic NAD redox status and demonstrated metabolic redox coupling between cell compartments in leaves. Finally, we used protein engineering to generate a sensor variant that expands the resolvable NAD redox range. In summary, we established a technique for in planta NAD redox monitoring to deliver important insight into the in vivo dynamics of plant cytosolic redox metabolism.