Oryza sativa heavy metal ATPase (OsHMA) transporter-mediated cadmium uptake in rice: functional insights for low-Cd breeding

Cadmium (Cd) is a hazardous and non-essential heavy metal that enters the environment through anthropogenic activities and natural emissions, polluting crops and threatening human health. We hypothesized that OsHMA transporters play distinct and functionally specialized roles in Cd uptake and transport in rice, and their expression could reduce Cd accumulation in shoots, while enhance plant tolerance to Cd-induced toxicity. This study identified and functionally characterized OsHMA family members involved in this process. Heterologous expression in the Cd-sensitive yeast strain ∆ycf1 showed that OsHMA1/2/3/4/5/7/8/9 possess Cd-specific functional activity. Under Cd treatment, rice overexpressing OsHMA1/3/4/5/7/8/9 exhibited improved growth and reduced Cd accumulation in shoots, while CRISPR-Cas9 knockouts of OsHMA1/3/4/7/9 accumulated more Cd in shoots and showed greater sensitivity. Notably, KO- OsHMA5 and KO- OsHMA8 mutants exhibited higher Cd content in roots and lower in shoots and improved growth performance than wild type. Subcellular localization revealed a diverse distribution of OsHMA proteins; OsHMA1, OsHMA7 and OsHMA8 co-localized with chloroplasts; OsHMA5 and OsHMA9 were localized to the plasma membrane; OsHMA2, OsHMA4, and OsHMA6 were localized both to the plasma membrane and endoplasmic reticulum; and OsHMA3 had fluorescence signals in the vacuolar (tonoplast) membrane. These findings confirm that OsHMA genes are functionally specialized in Cd uptake, supporting our hypothesis, and demonstrate that targeted manipulation of these genes through CRISPR/Cas9 tool may provide further insight into their roles in regulating Cd accumulation in rice.