Figure 5.
MYB40 modulates thiol-peptide accumulation and PCS1 expression in the Arabidopsis response to As stress.
(A) HPLC–MS chromatograms of OE26, myb40-2 mutant, and WT plants exposed to 200 μM As(V) for 7 d.
(B and C) Thiol-peptide measurements. Seven-d-old seedlings of different genotypes were transferred to ½ MS medium containing 200 μM As(V) for 7 d, then harvested for thiol-peptide measurement. The levels of PC2(B) and GSH (C) in whole seedlings were measured by fluorescent HPLC. Data are shown as mean ± SE; n = 3.
(D) qRT–PCR analysis of PCS1 expression in MYB40-overexpressing lines. Seven-d-old plants were transferred to ½ MS medium containing 200 μM As(V) and harvested at the indicated time. The qRT–PCR was performed with three technical replicates, and the experiment was repeated at least three times with similar results.
(E) qRT–PCR analysis of PCS1 in the myb40 mutants under As(V) stress. The 7-d-old seedlings were transferred to ½ MS medium with 200 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.
(F) Diagram of the PCS1 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 PCS1 promoter fragments are based on this number. The gray box indicates the UTR.
(G) ChIP–qPCR assay of MYB40 binding to the PCS1 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 assay using anti-Myc antibody. Data are shown as mean ± SE; n = 3.
(H) EMSA of MYB40 binding to the P4 fragment of the PCS1 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. Asterisks in (B, C, D, and E) indicate significant differences compared with WT plants (#) by Student's t-test: ∗P < 0.05, ∗∗P < 0.01.