Figure 4.

ARF8.4 Expression Restores Normal AUX/IAA19 Transcript Levels in arf8-7 Inflorescences, and ARF8.4 Directly Binds More Efficiently Than ARF8.2 to the AUX/IAA19 Promoter.

(A) Comparative analysis by qRT-PCR of AUX/IAA19 transcript levels in wild-type and arf8-7 stamens at stages 10-12, and in mock- and estradiol-treated arf8-7 inflorescences expressing ARF8.4 (8.4ox), ARF8.2 (8.2ox), or ARF8.1 (8.1ox). AUX/IAA19 cDNA levels are relative to actin cDNA. Values are means ± se of nine data points obtained from three biological replicates that were each analyzed in triplicate. Biological replicates were obtained by pooling either stamens at stages 10-12, wild-type inflorescences, mock- or estradiol-treated inflorescences from five independent plants for each genotype. Asterisks indicate a significant difference from the wild-type value: *P < 0.05 and **P < 0.01. Circles indicate a significant difference from the mock-treated value: °°P < 0.01 and °°°P < 0.001. est, estradiol.

(B) Schematic diagram of putative ARF binding sites in the AUX/IAA19 promoter (1500 bp from the transcription start site). The upper black lines indicate fragments amplified in ChIP-qPCR assays.

(C) Chromatin immunoprecipitation analysis of ARF8.4 and ARF8.2 binding to the AUX/IAA19 promoter. Enrichment was observed in all four regions in ARF8.4-overexpressing inflorescences and in regions 3, 4, and 5 in ARF8.2-overexpressing inflorescences. ChIP-qPCR values are means ± se of nine data points obtained from three biological replicates that were each analyzed in triplicate. Biological replicates were obtained by pooling inflorescences isolated from 50 independently grown plants for each genotype. Asterisks indicate a significant difference from the -Ab control value: *P < 0.05 and ***P < 0.001. Ab, antibody.