Figure 5.

MYB49 regulates genes involved in metal accumulation. A, Hierarchical clustering analysis diagram showing the DEGs in OX49 and SRDX49 seedlings. B, RT-qPCR analysis of the genes involved in heavy metal accumulation and tolerance in 7-d-old Arabidopsis Col-0, OX49, SRDX49, and myb49 seedlings germinated on 1/2 MS medium and then transferred to fresh medium containing 30 μm Cd for 12 h. The expression levels of the indicated genes in wild-type Col-0 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). The blue line represents a value of relative gene expression of 1.5. C, A Y1H assay showed that MYB49 bound to the promoters of bHLH38, bHLH101, HIPP22, and HIPP44. Transformed yeast cells were grown on SD-Leu/aureobasidin A (AbA) medium. The assay was performed three times with the same result. D, ChIP-qPCR assays of MYB49-DNA complexes. The scheme of the primer design for each gene is shown at top. The black boxes represent the gene-coding regions, and the black lines represent the promoter regions. Red lines mark the locations of amplicons amplified in the ChIP-qPCR. The promoter fragment enrichment following ChIP-qPCR was performed in the absence (IgG) or presence (anti-GFP) of anti-GFP antibody. Error bars represent the sd (n = 3). Asterisks indicate significant differences from the control (Student’s t test, P < 0.01). E, EMSA binding of MYB49 to the HIPP22 promoter region harboring MYB49-binding sites. Two biological experiments were performed with similar results.