Integration of Mitoflash and Time-Series Transcriptomics Facilitates Energy Dynamics Tracking and Substrate Supply Analysis of Floral Thermogenesis in Lotus.

The high biosynthetic and energetic demands of floral thermogenesis render thermogenic plants the ideal systems to characterize energy metabolism in plants, but real-time tracking of energy metabolism in plant cells remains challenging. In this study, a new method was developed for tracking the mitochondrial energy metabolism at the single mitochondria level by real-time imaging of mitochondrial superoxide production (i.e., mitoflash). Using this method, we observed the increased mitoflash frequencies in the receptacles of Nelumbo nucifera Gaertn. at the thermogenic stages. This increase, combined with the higher expression of antioxidant response-related genes identified through time-series transcriptomics at the same stages, shows us a new regulatory mechanism for plant redox balance. Furthermore, we found that the upregulation of respiratory metabolism-related genes during the thermogenic stages not only correlates with changes in mitoflash frequency but also underscores the critical roles of these pathways in ensuring adequate substrate supply for thermogenesis. Metabolite analysis revealed that sugars are likely one of the substrates for thermogenesis and may be transported over long distances by sugar transporters. Taken together, our findings demonstrate that mitoflash is a reliable tool for tracking energy metabolism in thermogenic plants and contributes to our understanding of the regulatory mechanisms underlying floral thermogenesis.