Figure 6.

ABI5 negatively regulates MYB49-mediated Cd accumulation. A, Transient dual-luciferase reporter assays show that the activation of bHLH38, bHLH101, HIPP22, and HIPP44 expression by MYB49 is repressed by ABI5. Protoplast transient expression assays used the 1.5-kb promoter fragments of the bHLH38, bHLH101, HIPP22, and HIPP44 genes. The luciferase luminescence intensities were quantitated following transfection with different vectors: 62SK represents empty pGreenII 62-SK vector; MYB49 represents the pGreenII 62-SK-MYB49 vector; ABI5 represents the pGreenII 62-SK-AtABI5 vector; pbHLH38, pbHLH101, pHIPP22, and pHIPP44 represent pGreenII 0800-LUC-pbHLH38, pGreenII 0800-LUC-pbHLH101, pGreenII 0800-LUC-pHIPP22, and pGreenII 0800-LUC-pHIPP44 vectors, respectively. Renilla luciferase (REN) was used for normalization. Error bars represent the sd (n = 3). Asterisks indicate significant differences from the control (Student’s t test, P < 0.01). B, The binding of MYB49 to the HIPP22 promoter region harboring MYB49-binding sites was analyzed using EMSA in the presence of ABI5. Two biological experiments were performed with similar results. C and D, RT-qPCR analysis of the expression of bHLH38, bHLH101, HIPP22, and HIPP44 genes in 7-d-old Arabidopsis Col-0, ABI5OX, Ws, abi5-1, OX49, and OX49/ABI5OX seedlings germinated on 1/2 MS medium and then transferred to fresh medium without (C) or with (D) 30 μm Cd for 1 d. The expression levels of the indicated genes in wild-type Col-0 or Ws were set to 1. Error bars represent the sd (n = 3). Asterisks indicate significant differences from the control (Student’s t test, P < 0.01). E, Four-week-old hydroponics-grown Col-0, OX49, ABI5OX, and OX49/ABI5OX seedlings were transferred to fresh medium containing 10 µm CdCl₂ for 2 d, and Cd contents were then determined by ICP-AES. Error bars represent the sd (n = 6). Different letters indicate that values were significantly different at P < 0.01 according to Tukey’s test. DW, Dry weight.