Real-time monitoring of stromal NADPH levels in Arabidopsis using a metagenome-derived NADPH-binding fluorescent protein.

The light irradiation to the plant chloroplasts drives NADPH and ATP synthesis in the stroma via the electron transport chains within the thylakoid membranes. Conventional methods for assessing photosynthetic light reactions are often invasive or require specific conditions. While detection markers do not significantly affect plant growth itself, developing a method for the real-time and non-invasive detection of NADPH is a highly impactful and important research area in plant physiology and biochemistry. This study introduces a genetically encoded NADPH-binding blue fluorescent protein (mBFP) targeted to the chloroplast stroma or thylakoid membrane in Arabidopsis thaliana and Nicotiana benthamiana. Using two-photon microscopy, we monitored real-time stromal NADPH levels in transgenic leaves of Arabidopsis in response to light exposure. A mutant mBFP construct targeted to the thylakoid membrane allowed us to detect the stromal NADPH levels in real time under different light conditions. This in planta biosensor provides a non-invasive tool for studying photosynthetic responses to light more quantitatively and holds potential for optimizing light conditions in controlled-environment agriculture, such as indoor vertical farms, to improve crop productivity.