Figure 5.

The ARE Mediates WRKY6 Repression of the Pi Transporter PHT1;1 and Confers the As(V) Tolerance Phenotype.

(A) Kinetic analysis of transient LUC activity in N. benthamiana leaf discs agroinfiltrated with PHT1;1-LUC wild type or the mutated versions alone or with a WRKY6-GFP–overexpressing construct. Leaf discs were incubated in medium with 30 μM As(V) (1.5 h). Values show mean ± sd.

(B) ChIP assay of WRKY6-GFP followed by qPCR of the PHT1;1 promoter. ChIP assays were performed in N. benthamiana leaf discs agroinfiltrated with PHT1;1-LUC wild type and the PHT1;1-LUC mutated version (PHT1;1p-Im/IIm), with WRKY6-GFP or GFP-overexpressing constructs. Values represent the x-fold enrichment of WRKY6-bound DNA of the PHT1;1 promoter in immunoprecipitated samples relative to total input DNA. ARE (PHT1;1p-I/II-LUC) or mutated ARE-containing fragments (PHT1;1p-Im/IIm-LUC) in the PHT1;1 promoter were amplified by qPCR using specific primers. Values show mean ± sd. *P < 0.01 (Student’s t test). Values show mean ± sd.

(C) As(V) tolerance phenotype of wild-type (Col-0), OXWRKY6-GFP–overexpressing line (OXWRKY6), and wrky6-TDNA plants grown on 15 μM Pi supplemented with 15 μM As(V) for 7 d.

(D) Intracellular arsenic concentration in Col-0, OXWRKY6, and wrky6-TDNA plants exposed to 5 μM As(V) (1 h). Values show mean ± sd. *p < 0.05 (Student’s t test). DW, dry weight.