Investigating photosynthesis by in planta ATP, NADPH and NADH/NAD+ sensors

Abstract

During photosynthesis, ATP and NADPH generated from photosystems are consumed by various anabolic metabolisms in different ratios. Previous in vitro methods (e.g., enzyme assays, bioluminescence, LC-MS, etc.) for measuring these molecules required prior tissue extraction and thus cannot provide real-time or subcellular information. We have introduced three novel sensors for the detection of ATP (AT1.03), NADPH (iNAP) and NADH/NAD+ ratio (SoNar) in Arabidopsis thaliana to monitor real-time dynamic changes of the levels of these energy carriers in different subcellular compartments (e.g., cytosol and chloroplasts) of various tissues (e.g., leaves, hypocotyl, roots, etc.). By employing the ATP sensor, we showed that the stromal ATP concentration is always lower than the cytosolic concentration. Cytosolic ATP does not enter mature chloroplasts efficiently and therefore is unlikely to support the Calvin-Benson-Bassham cycle. We propose that, during the evolution of unicellular to multicellular photosynthetic organisms, the entry of cytosolic ATP into mature chloroplasts was restricted by the down-regulation of NTT transporter so as to reduce energy expenditure in the dark. Hence, chloroplasts have to export surplus reducing equivalents during photosynthesis to balance the ATP/NADPH ratio. In addition, there was a confusion on whether malate is imported into or exported from the mitochondria during photosynthesis. By employing NADPH and NADH/NAD+ sensors and various inhibitors, we showed that photorespiration generates more NADH than the NADH-dissipating capacity of mETC, therefore, surplus NADH is exported from mitochondria to the cytosol in the form of malate via the malate-OAA shuttle. These works show the usefulness of these ratiometric sensors in plant investigations.