Figure 4.

MYB40 represses PHT1;1 expression in the Arabidopsis response to As stress.

(A) qRT–PCR analysis of PHT1;1 expression in MYB40-overexpressing lines and WT plants. The qRT–PCR was performed with three technical replicates, and the experiment was repeated at least three times with similar results.

(B) qRT–PCR analysis of PHT1;1 in the myb40 mutants under As(V) stress. Seven-d-old seedlings were transferred to ½ MS medium with 0, 300, and 400 μM As(V) for 2 d, then harvested for RNA extraction. The qRT–PCR was performed with three technical replicates, and the experiment was repeated at least three times with similar results. Asterisks in (A and B) indicate significant differences compared with WT plants (^#) by Student's t-test: ∗∗P < 0.01.

(C) Immunoblot analysis of MYB40 protein in the MYB40-Myc transgenic line. Protein extract was analyzed by immunoblotting using anti-Myc antibody. Actin was used as the loading control.

(D) Diagram of the PHT1;1 promoter showing relative positions and sizes of different fragments. The adenine residue of the translational start codon ATG was assigned position +1, and the numbers flanking the sequences of the PHT1;1 promoter fragments are based on this number. The gray box indicates the untranslated region (UTR).

(E) ChIP–qPCR assay of MYB40 binding to the PHT1;1 promoter in vivo. Seven-d-old MYB40-Myc seedlings were transferred to ½ MS medium containing 200 μM As(V) for 2 d, then harvested for ChIP–qPCR using anti-Myc antibody. Data are shown as mean ± SE; n = 3.

(F) EMSA of MYB40 binding to the PHT1;1 promoter in vitro. Each biotin-labeled DNA probe was incubated with SUMO–His–MYB40 protein. An excess of unlabeled probe with the same sequence was added to compete with the labeled probe.

(G) The expression of MYB40 and PHT1;1 was tested by qRT–PCR in OE26, OE26 Super:PHT1;1 co-overexpression, Super:PHT1;1, and WT plants. The qRT–PCR was performed with three technical replicates, and the experiment was repeated at least three times with similar results.

(H) Phenotypic comparison. Plants were germinated and grown on ½ MS medium containing 0 or 200 μM arsenate for 7 d, and then representative photographs were taken. Scale bar corresponds to 0.5 cm.