Spatiotemporal Dynamics of Cytosolic NADPH in Living Arabidopsis thaliana

ABSTRACT

In plants, an adequate supply of Nicotinamide adenine dinucleotide phosphate (NADPH) in the cytosol is crucial for maintaining a variety of biosynthetic and antioxidant reactions. Cytosolic NADPH is mainly generated via four enzymatic pathways: NADP‐dependent malic dehydrogenase (NADP‐ME), cytosolic oxidative pentose phosphate pathway (cOPPP), cytosolic NADP‐dependent isocitrate dehydrogenase (cyNADP‐ICDH), and non‐phosphorylating glyceraldehyde‐3‐phosphate dehydrogenase (np‐GAPDH). Here, we employed the cytosolic NADPH sensor mCherry‐iNAP1 along with a GUS reporter system and enzyme assays to clarify the contributions of the four NADP(H)‐reducing pathways in Arabidopsis seedlings and mature leaves. Our results show that, in 5‐day‐old seedlings, cyNADP‐ICDH and np‐GAPDH supply cytosolic NADPH to both roots and shoots, cOPPP could contribute more cytosolic NADPH to roots than to shoots, and NADP‐ME2 primarily functions in roots. In leaves of 21‐day‐old plants, cyNADP‐ICDH and np‐GAPDH supply cytosolic NADPH throughout the day, while cOPPP could be more active at night, and NADP‐ME2 utilizes daytime‐accumulated malate to produce substantial cytosolic NADPH at night. Spatiotemporal visualization of cytosolic NADPH through in vivo imaging provides crucial information for future redox engineering of stress‐resistant crops.