Root Zn sequestration transporter heavy metal ATPase 3 from Odontarrhena chalcidica enhance Cd tolerance and accumulation in Arabidopsis thaliana

The Ni hyperaccumulator Odontarrhena chalcidica (formerly Alyssum murale), exhibits a significant capacity to accumulate Zn in the roots. However, the molecular mechanisms underlying the variation in Ni and Zn accumulation are poorly understood. Here, we isolated a homolog of heavy metal ATPase 3 from O. chalcidica (OcHMA3) and characterized its functions using heterologous systems. Phylogenetic analysis revealed that OcHMA3 protein shares 87.6 % identity with AtHMA3, with similar metal binding sites to other HMA3 proteins. Heterologous expression of OcHMA3 in yeast increased sensitivity to Cd, Ni and Zn, suggesting it functions as a broad-specificity transporter. Further investigation showed OcHMA3 is constitutively expressed in the roots and localized to the tonoplast. Overexpression of OcHMA3 in A. thaliana shoots increased its roots Zn concentrations by 41.9 % - 74.1 %. However, overexpression of OcHMA3 in roots enhanced its tolerance to Cd and increased roots Cd concentrations by 50.9 % - 90.6 %. Our findings indicated OcHMA3 is responsible for Zn sequestration in root vacuoles, likely leading to Zn retention in roots and subsequent Ni hyperaccumulation in shoots. This study elucidates the molecular mechanism of Ni and Zn accumulation in O. chalcidica, and identifies OcHMA3 as a potential gene for developing Zn-rich plants and for phytoextraction in Cd-contaminated soils.