IAA3-mediated repression of PIF proteins coordinates light and auxin signaling in Arabidopsis
Fig 5
IAA3 impairs PIFs binding to DNA and negatively regulates PIFs’ target genes.
(A) The transient dual-LUC reporter assay showing the effects of IAA3 on PIF transcriptional regulation of PIL1. The ATG18-GFP protein is as the negative control in the assay. Data are means ± SD (n = 3 experiments). Significant differences are indicated with asterisks (***P < 0.001, two-tailed Student’s t-test). (B) Western blotting for the detection of expressed PIF4-MYC, IAA3-GFP and ATG18-GFP in transformed tobacco leaves from (A) using an anti-MYC or anti-GFP antibody, respectively. (C) A fragment for PIL1 promoter containing G-box motif (CACGTG) was selected to used as probe in the EMSA experiment. (D) EMSA experiment showing IAA3 inhibition of the DNA-binding activity of PIFs. The cold probe was added as a competitor. MBP was used as a negative control. (E) Venn diagrams showing significant overlap between IAA3-regulated genes and PIFs-dependent genes. Detailed gene information is given in S1 Table. (F) Heat map of 625 DEGs coregulated by IAA3 and PIFs. (G) and (H) qRT-PCR analysis of known PIFs’ direct target genes. The transcription levels of PIFs’ direct target genes in Ler and shy2-3 mutant (G). Auxin induction of PIFs-regulated genes. Five-day Col-0 seedlings grown in light condition were treated with 5 μM IAA for 24 hours (H). The expression was normalized to the ACTIN2 (ACT2) expression level, and was relative to wild-type levels. Data represents mean ±SD from three biological replicates. Significant differences are indicated by ***P < 0.001(two-tailed Student’s t-test).