Energy metabolism, antioxidant defense system, metal transport, and ion homeostasis are key contributors to Cd tolerance in SSSL derived from wild rice.

Cadmium (Cd) toxicity poses major challenges to rice cultivation, affecting plant growth and development. Wild rice and nanoparticles offer promising strategies to enhance Cd tolerance, yet little is known about their combined effects. This study evaluates the single segment substitution line (SG004) from Oryza glumaepatula (wild rice) and its response to Cd stress compared to cultivated rice (HJX74). Both genotypes were also treated with calcium oxide nanoparticles (np-CaO). Results showed that Cd exposure disrupts reactive oxygen species (ROS) metabolism in both lines, such as malondialdehyde (MDA) increases by 57 % in HJX74 compared to SG004. Moreover, SG004 exhibited a 26 % reduction in shoot length compared to 41 % in HJX74 and a 42 % decline in chlorophyll ab content versus 53 % in HJX74. Antioxidant activity such as glutathione (GSH) decreased 25 % more in HJX74 than SG004 under Cd toxicity. Additionally, SG004 had lower Cd accumulation in roots (70 %) and shoots (85 %) than HJX74, indicating its enhanced tolerance to Cd toxicity. The root cell cytology reveals several deformations in different organelles of HJX74 but less in SG004. RNAseq analysis identifies key pathways, including energy metabolism, antioxidant defense, metal transport, and ion homeostasis, which may be critical for SG004 enhanced tolerance. Notably, two distinct metallothionein-like genes (BGIOSGA019338, BGIOSGA035982), a peroxidase (BGIOSGA019133), ammonium (BGIOSGA008640, BGIOSGA008641, and potassium transporters (BGIOSGA030867), NRAMP1 (BGIOSGA025476), and an aluminum-activated malate transporter (BGIOSGA014531), showed differential expressions in SG004 under Cd stress. Genes within the substituted fragment, including those for peroxidase 25 (BGIOSGA002866), metallothionein (BGIOSGA002389), and reductase (BGIOSGA002387), are also upregulated in SG004, reinforcing the role of antioxidant and ion homeostasis pathways. The utilization of np-CaO alleviates Cd-induced stress in both genotypes, hence reinforcing the application of wild rice and nanoparticles to improve Cd tolerance.