Populus euphratica R2R3-MYB transcription factor RAX2 binds ANN1 promoter to increase cadmium enrichment in Arabidopsis

The expression of R2R3-MYB transcription factor PeRAX2 increased transiently upon CdCl₂ exposure (100 μM, 48 h) in leaves and roots of Populus euphratica. We observed that overexpression of PeRAX2 increased Cd²⁺ concentration in Arabidopsis root cells and Cd²⁺ amount in whole plant, which was due to the increased Cd²⁺ influx into root tips. However, the Cd²⁺ influx facilitated by PeRAX2 overexpression was substantially reduced by LaCl₃ (an inhibitor of Ca²⁺-channels), suggesting that PeRAX2 could promote the Cd²⁺ entering through PM Ca²⁺-permeable channels (CaPCs) in the roots. It is noting that the expression of annexin1 (AtANN1), which mediates the influx of divalent cations through the PM calcium channels, was upregulated by Cd²⁺ in PeRAX2-transgenic Arabidopsis. Bioinformatic analysis revealed that the AtANN1 promoter (AtANN1-pro) contains four cis-elements for MYB binding. The PeRAX2 interaction with AtANN1-pro was validated by LUC reporter assay, EMSA, and Y1H assay. Our data showed that PeRAX2 binds to the AtANN1 promoter region to regulate gene transcription and that AtANN1 mediates the Cd²⁺ entry through CaPCs in the PM, leading to a Cd²⁺ enrichment in transgenic plants. The PeRAX2-stimulated Cd²⁺ enrichment consequently resulted in high H₂O₂ production in root cells of transgenic plants. The expression of AtSOD and AtPOD and activities of CAT, SOD, POD increased in the transgenic lines under Cd²⁺ stress. However, the Cd²⁺-upregulated expression and activity of antioxidative enzymes were less pronounced in the PeRAX2-overexpressed lines, compared to the wildtype and vector controls. As a result, root length and plant growth were more suppressed by Cd²⁺ in the transgenic lines. Our data suggest that transcriptional regulation of AtANN1 by PeRAX2 can be utilized to improve Cd²⁺ enrichment and phytoremediation, although the enriched Cd²⁺ affected antioxidant defense system and plant growth in the model species.