Figure 3.

The Regulated Relationship between EIN3 and PIF3 at the Transcription and Protein Levels.

(A) Immunoblot results showing the endogenous PIF3 protein levels in wild-type, ein3 eil1, and EIN3-ox etiolated seedlings without (MS) or with ACC treatment. The seedlings were grown in the dark for 4 d on half-strength MS medium (MS) or half-strength MS medium supplemented with 10 μM ethylene precursor ACC. The PIF3 antibody was used to detect endogenous PIF3 proteins, and pif3 was used as a negative control for the PIF3 protein band. Ponceau S staining was used as a control for loading.

(B) RT-qPCR results showing the PIF3 gene expression levels of 4-d-old etiolated wild-type, EIN3-ox, and ein3 eil1 seedlings. Each experiment was performed at least three times with similar results and the representative results were presented. Error bars represent average value ± sd (n = 3) from technical triplicates.

(C) Immunoblot results showing the effects of ethylene on the PIF3-Myc protein levels. The PIF3-ox seedlings were grown in the dark for 4 d on half-strength MS medium (MS), half-strength MS medium supplemented with 10 μM ethylene precursor ACC, 100 μM ethylene perception inhibitor Ag⁺ (AgNO₃), or 25 μM ethylene biosynthesis inhibitor AVG. Anti-Myc antibody was used to detect the PIF3-Myc proteins, and the wild type was used as a negative control for the PIF3-Myc protein band. Ponceau S staining was used as a control for loading.

(D) Immunoblot results showing the PIF3-Myc protein levels in the PIF3-ox (35S:PIF3-Myc/Col-0), PIF3-ox/ein3 eil1, and PIF3-ox/ein2 etiolated seedlings. The seedlings were grown in the dark for 4 d on half-strength MS medium. Anti-Myc antibody was used to detect the PIF3-Myc proteins and the wild type was used as a negative control for the PIF3-Myc protein band. Ponceau S staining was used as a control for loading.

(E) RT-qPCR results showing the EIN3 gene expression levels in 4-d-old etiolated wild-type (Col-0), PIF3-ox, and pif3 seedlings. Each experiment was performed at least three times with similar results and the representative results were presented. Error bars represent average value ± sd (n = 3) from technical triplicates.

(F) Immunoblot results show the EIN3-Myc protein levels in pER8-EIN3-Myc/ein3 eil1 and pER8-EIN3-Myc/pif3 ein3 eil1 etiolated seedlings. The seedlings were grown in the dark for 4 d on half-strength MS medium supplemented with 10 µM β-estradiol inducer. Anti-Myc antibody was used to detect the PIF3-Myc proteins and the wild type was used as a negative control for the PIF3-Myc protein band. Ponceau S staining was used as a control for loading.