MeNADP-ME3 Confers Salt and Drought Tolerance in Arabidopsis and Drives Functional Diversification of the NADP-ME Family in Cassava

As a typical C3-C4 intermediate plant, cassava (<i>Manihot esculenta</i> Crantz) exhibits high photosynthetic efficiency and low photorespiration. NADP-malic enzyme (NADP-ME) is a key enzyme in the C4 photosynthetic pathway that provides elevated CO2 concentrations for Rubisco. However, research on NADP-ME in C3-C4 intermediate species remains limited. In this study, we identified four <i>NADP-ME</i> genes in the cassava genome, with segmental duplication serving as the primary driving force for gene evolution. Cis-acting element analysis indicated potential roles of <i>MeNADP-ME</i> genes in environmental adaptation, stress responses, and growth regulation. Expression profiling using bulk RNA sequencing and single-cell RNA sequencing revealed distinct expression patterns in different tissues and cell subsets. Comparative analysis with <i>Arabidopsis</i> (<i>Arabidopsis thaliana</i>) and maize (<i>Zea mays</i>) NADP-ME families demonstrated that <i>MeNADP-ME3</i> exhibits bundle sheath cell-specific expression analogous to <i>ZmchlC4NADP-ME</i> in maize. Notably, photosynthetic genes and plasmodesmata (PD)-related genes exhibited high co-expression within mesophyll subcluster 13 and bundle sheath cells, providing molecular evidence for a limited C4 photosynthetic pathway in cassava. Protein-protein interaction predictions implicated MeNADP-ME3 in photosynthetic carbon metabolism and photorespiration regulation. Furthermore, qRT-PCR revealed significant responsiveness of <i>MeNADP-ME3</i> to various abiotic stresses, and confocal imaging confirmed its chloroplast localization. Functional validation demonstrated that <i>Arabidopsis</i> overexpressing <i>MeNADP-ME3</i> exhibited 30-120% enhanced antioxidant enzyme activities (SOD, POD, CAT) and 20-32% reduced oxidative damage markers (MDA, H2O2) under drought and salt stresses. These findings reveal the evolutionary trajectory of <i>NADP-ME</i> genes in C3-C4 intermediate species and provide genetic resources for developing stress-tolerant cassava cultivars.