Fig. 5.
bZIP63 directly binds the ARF19 promoter and controls ARF19 transcription. (A and B) ChIPseq was performed with roots of 10-d-old seedlings upon 4 h of uD comparing bzip63 and a complementation line expressing a functional bZIP63:YFP under control of the native promoter. Chromatin was immunoprecipitated using anti-GFP antibodies, and genomic fragments were subjected to high-throughput DNA sequencing. A total of 821 enriched binding fragments (peaks) corresponding to 500 target genes were identified (Dataset S1). (A) Nucleotide logo displaying the predicted, enriched cis-element matching the experimentally defined bZIP63 specific binding site (G/C-box; C/GACGTG). (B) Reads from bZIP63:YFP bound DNA fragments mapped against selected known bZIP63 target promoters as controls (MCCA and ProDH) and ARF19 as a potential target in LR development. No strong binding was observed for ARF7 or GATA23. Blue color bars represent the 5′ end of the respective open reading frames. The ARF19 promoter is marked for G-Box-1 binding region (black box). (C) ChIPPCR of roots treated with 4-h uD was used to verify binding of bZIP63:YFP to the ARF19 promoter. Using the primer pairs indicated, significant binding was determined around G-Box-1, whereas no significant enrichment was observed for the nonbinding control (ACTIN7 and ACT7) or G-Box-2 and -3. Enrichment of promoter sequences derived from WT (gray bars) and bZIP63:YFP (red bars) are indicated. Presented are mean values ± SD from three independent plant pools relative to input (determined by ProACTIN8 abundance). Student’s t test, *P < 0.05. (D–F) bZIP63 loss-of-function mutants are impaired in induced ARF19 transcription upon uD. The 8-d-old Arabidopsis WT and bzip63 seedlings were cultivated under control conditions or treated with 1 and 4 h of uD or 4 h uD plus 8 h of light recovery (R) before harvesting. RT-qPCR of roots at the time points indicated for bZIP63 (D), DIN6/ASN1 (E), and ARF19 (F). Given are mean values ± SEM derived from roots of three independent plant pools relative to EF1A. Student’s t test, *P < 0.05, ***P < 0.001. (G–I) ARF19 is required for uD-induced LR initiation. eLRD of WT and arf19 mutant analyzed in the setup described in Fig. 1E. (eLRD [G]; LR number [H]; and primary root length [I]). Seedlings were grown in long day regime (16 h light/8 h dark at 100 μmol ⋅ m−2 ⋅ s−1) on solidified 1/2 MS media. Statistically significant differences between control (C) and treated (uD) samples were determined by Mann–Whitney U test. *P < 0.05, **P < 0.01, and ***P < 0.001.