Spatiotemporal Dynamics of Cytosolic NADPH in Living Arabidopsis thaliana

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.