Molecular characterization of rice arsenic-induced RING finger E3 ligase 2 (OsAIR2) and its heterogeneous overexpression in Arabidopsis thaliana

Abstract

Arsenic (As) accumulation adversely affects the growth and productivity of plants and poses a serious threat to human health and food security. In this study, we identified one As-responsive Really Interesting New Gene (RING) E3 ubiquitin ligase gene from rice root tissues during As stress. We named it Oryza sativa As-Induced RING E3 ligase 2 (OsAIR2). Expression of OsAIR2 was induced under various abiotic stress conditions, including heat, salt, drought and As exposure. Results of an in vitro ubiquitination assay showed that OsAIR2 possesses an E3 ligase activity. Within the cell, OsAIR2 was found to be localized to the Golgi apparatus. Using yeast two-hybrid (Y2H) assay, the 3-ketoacyl-CoA thiolase (KAT) protein was identified as an interaction partner. We found that the O. sativa KAT1 (OsKAT1) is localized to the cytosol and peroxisomes. Moreover, in vitro pull-down assay verified the physical interaction between OsAIR2 and OsKAT1. Interestingly, in vitro ubiquitination assay and in vivo proteasomal degradation assay revealed that OsAIR2 ubiquitinates OsKAT1 and promotes the degradation of OsKAT1 via the 26S proteasome degradation pathway. Heterogeneous overexpression of OsAIR2 in Arabidopsis improved the seed germination and increased the root length under arsenate stress conditions. Therefore, these results suggest that OsAIR2 may be associated with the plant response to As stress and acts as a positive regulator of As stress tolerance.