Figure 7.

Constitutive ERF-VIIs Directly Bind to the 33-bp Region of the LBD41 Promoter.

(A) RT-qPCR analysis of LUC in a stable 3x33bpmin:LUC transgenic line in mesophyll protoplasts transiently expressing either HA-GFP or (MA)RAP2.2-HBD. Chx or DMSO was applied 30 min before Dex or ethanol, and protoplasts were incubated for an additional 4 h under long-day conditions. Transcript levels were normalized to EF1a. Data are means ± sd from three biological replicates (each with three technical replicates). Different letters indicate values that vary significantly at P < 0.05 (Tukey HSD test).

(B) Y1H assay with stable 3x33bp:HIS 3x33bp:LacZ yeast strains transiently expressing AD-ERF-VII fusions. Water and an AD-only expressing vector were used as negative controls. Panels show equally concentrated yeast transformants and 1:10- and 1:100-fold dilutions after 5 d of growth on selective medium in the absence and presence of 40 mM of the HIS gene inhibitor 3-AT. Blue circles indicate β-galactosidase (LacZ) activity as double positive control.

(C) ChIP of RAP2.2- and RAP2.12-associated promoter regions. FLAG-tagged overexpression lines were used (Figure 1), and HRPE-containing promoter regions of LBD41 and PCO1 as well as the 5′-ATCTA-3′-containing region of PSY were tested by subsequent RT-qPCR. Fold enrichment was calculated in comparison to Col-0 wild type. Data are means ± sd from three biological replicates, each with technical duplicates.