Heavy metal transmembrane transporter CaNRAMP3 enhances cadmium accumulation in pepper fruit

• CaNRAMP3 functions as an influx transporter at the plasma membrane, bringing Cd²⁺ into the cytosol of fruit cells. • Hap3 (double reference genotype) was a key marker for the phenotype of low Cd accumulation in pepper fruits. • Silencing CaNRAMP3 via VIGS reduced Cd contents in pepper fruits. • CaNRAMP3 formed a co-expression network with CaE2, CaPP2 , and CaCOP9, CaUXT, CaLPP2, CaDERL2 , and HRABI1 . Cadmium (Cd) contamination in pepper ( Capsicum annuum L.) fruits is a significant concern in China, limiting the sustainability of pepper production. To identify key genetic loci for Cd accumulation, we performed a combined transcriptome sequencing analysis with selective sweep detection in pepper cultivars with high and low levels of fruit Cd accumulation. In this study, we identified CaNRAMP3 ( LOC107860226 ), a natural resistance-associated macrophage protein gene. CaNRAMP3 functions as an influx transporter at the plasma membrane, bringing Cd 2+ into the cytosol of fruit cells, thereby contributing to Cd accumulation. Haplotype analysis discovered two SNP sites in the promoter of CaNRAMP3 , which could be categorized into five distinct haplotypes across 186 pepper populations. The Hap3 (double reference genotype) was a key marker for the phenotype of low Cd accumulation in pepper fruits. Furthermore, following the silencing of CaNRAMP3 in pepper, the Cd content in the fruits was reduced by 50.90%. Collectively, our findings not only establish a theoretical foundation for the development of pepper varieties with reduced Cd accumulation but also identify potential molecular targets for breeding applications.