Identification of CCR4C as a chloroplast-localized NADP(H) phosphatase regulating NAD(P)(H) balance in Arabidopsis

NAD(P)(H) metabolism plays a crucial role in plant development and growth. NADK2, a chloroplast-localized NAD kinase, supplies NADP⁺ to the photosynthetic electron transport chain. The Arabidopsis T-DNA insertion mutant of NADK2 (nadk2) exhibits a reduced NADP⁺/NAD⁺ ratio, slow growth, and pale green leaves. To gain further insights into NAD(P)(H) metabolism in chloroplasts, nadk2 revertant mutants (nkr) were screened from the M2 generation of EMS (ethyl methane sulfonate)-treated nadk2 seeds. Among them, nkr1 displayed greener leaves and improved growth compared to nadk2. Genetic mapping and genomic sequencing identified At3g18500 (CCR4C) as the causal gene. The nkr1 mutant carried a single nucleotide substitution, introducing a stop codon within the predicted N-terminal chloroplast localization signal, resulting in the loss of CCR4C protein function. The nadk2 ccr4c double mutant restored leaf color and growth to near wild-type levels. To investigate the function of CCR4C, recombinant CCR4C protein was purified and shown to directly convert NADP(H) to NAD(H). Localization analysis with CCR4C-GFP fusion proteins confirmed chloroplast targeting. Furthermore, ccr4c single mutants exhibited disrupted NAD(P)(H) balance and enhanced tolerance to ROS stress (e.g., H₂O₂, methyl viologen). These findings reveal CCR4C as a chloroplast-localized NADP(H) phosphatase crucial for maintaining NAD(P)(H) balance, providing insights into how plant cells manage chloroplast metabolism.