Figure 2.

Other ERFs Function Redundantly with ORA59 to Regulate NRT1.8 Expression.

(A) Transient transcriptional activity assay of P_NRT1.8:LUC. The P_NRT1.8:LUC-35S:REN reporter construct was transiently expressed in Arabidopsis protoplasts together with the control vector, 35S:ORA59, 35S:ERF15, 35S:ERF1B, or 35S:ERF104 effector. REN was used as an internal control. The LUC:REN ratio represents the relative activity of the NRT1.8 promoter.

(B) and (C) EMSA of ERF1B (B) or ERF104 (C) binding to the NRT1.8 promoter regions. Wild-type and mutant probes as indicated in Figure 1A were amplified. Competition for the labeled wild-type probe was performed by adding 400-/800-fold excess of unlabeled wild-type probe. C, DNA-protein complex; F, free probe.

(D) and (E) ChIP assay using IgG beads to precipitate ERF1B-TAP protein (D) or anti-GFP antibody against ERF104-GFP protein (E), followed by qPCR detection of P1, P2, and the negative control P3 regions as indicated in Figure 1A. TUB2 and PDF1.2 were used as internal and positive controls, respectively.

(F) Quantitative RT-PCR analysis of NRT1.8 expression in roots of 4-week-old wild-type, ora59-1 erf104, tri-1, and tri-2 plants exposed to 200 μM CdCl₂, 150 mM NaCl, or 20 μM ACC for 6 h. Data were normalized to that of SAND.

Values are means ± sd; n = 3.