Homologous Gene Analysis Reveals ACR3 Expansion as a Key Driver of Arsenic Hyperaccumulation in Plants

<i>Pteris vittata</i> is a well-known arsenic (As) hyperaccumulator, yet the genetic basis of its exceptional ability remains unclear. In this study, we constructed a comparative transcriptome database from seven <i>Pteris</i> species, including four As hyperaccumulators and three nonhyperaccumulators, to identify key genes driving As hyperaccumulation. We found that <i>P. vittata</i> uniquely possesses both plasma membrane-localized (Type-I) and tonoplast-localized (Type-II) ACR3s, facilitating AsIII translocation and sequestration. Additionally, <i>P. vittata</i> shows an expansion of HAC and ACR3 gene families, supporting enhanced AsV reduction and AsIII transport. Evolutionary analyses revealed strong positive selection on Type-II PvACR3s, with specific adaptive amino acid sites identified. Functional assays showed that coexpression of Type-I (<i>PvACR3</i>) and Type-II (<i>PvACR3;1</i>) ACR3s in an <i>Arabidopsis thaliana</i> <i>hac1</i> mutant increased shoot As contents by 5.5-6.3-fold compared to wild-type plants, further supporting the important role of synergistic interaction between two ACR3 types in As hyperaccumulation. This study reveals unique genetic components that constitute the As hyperaccumulation pathway, providing valuable insights for enhancing phytoremediation of As-contaminated soils.