Background: Cassava (Manihot esculenta Crantz) ranks as the sixth largest food crop worldwide and serves as an important cash and energy crop. Heavy-metal-associated isoprenylated plant proteins (HIPPs) are metallochaperones involved in metal homeostasis and stress adaptation in vascular plants. However, research on the identification and function of HIPPs in cassava has been poorly explored. Methods: This study conducted a pan-genome-wide investigation to identify and characterize MeHIPPs in 31 cassava accessions. Subsequent analyses examined their physicochemical properties, subcellular localization, phylogeny, Ka/Ks, chromosomal localization, synteny, gene structure, and cis-acting elements. Additionally, the expression profiles of MeHIPPs in different tissues and cell subsets and under different stress conditions were analyzed using transcriptome data and quantitative real-time polymerase chain reaction (qRT-PCR). Results: A total of 59 MeHIPP pan-genes were identified, including five core genes, 22 softcore genes, 17 dispensable genes, and 15 private genes, which were unevenly distributed on chromosomes. Based on phylogenetic analysis, these genes were classified into five major subgroups. Evolutionary analyses indicated that segmental duplication predominated in family expansion and that most members may be subjected to purifying selection. Cis-element analysis highlighted the importance of MeHIPPs in plant adaptation to environmental stress. The expression profiles suggested widespread involvement of MeHIPP genes in response to Xanthomonas phaseoli pv. manihotis (Xpm) infection and drought stress. Different MeHIPP genes exhibited varying transcript levels in different tissues and cell subsets. qRT-PCR analysis revealed that the selected MeHIPP genes had distinct expression patterns under Cd stress. Conclusions: This study provides valuable insights into the functional characteristics of MeHIPP genes and their evolutionary relationships, laying a theoretical foundation for further functional research on stress resistance.

As a typical C3-C4 intermediate plant, cassava (Manihot esculenta 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 ${\mathrm{CO}}_2$ concentrations for Rubisco. However, research on NADP-ME in C3-C4 intermediate species remains limited. In this study, we identified four NADP-ME 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 MeNADP-ME 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 Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) NADP-ME families demonstrated that MeNADP-ME3 exhibits bundle sheath cell-specific expression analogous to ZmchlC4NADP-ME 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 MeNADP-ME3 to various abiotic stresses, and confocal imaging confirmed its chloroplast localization. Functional validation demonstrated that Arabidopsis overexpressing MeNADP-ME3 exhibited 30–120% enhanced antioxidant enzyme activities (SOD, POD, CAT) and 20–32% reduced oxidative damage markers (MDA, ${\mathrm{H}}_2{\mathrm{O}}_2$) under drought and salt stresses. These findings reveal the evolutionary trajectory of NADP-ME genes in C3-C4 intermediate species and provide genetic resources for developing stress-tolerant cassava cultivars.