PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) negatively regulates anthocyanin accumulation by inhibiting PAP1 transcription in Arabidopsis seedlings.

Anthocyanin accumulation is a striking symptom of plant environmental response and plays an important role in plant adaptation to adverse stimuli. PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) is a member of the PIFs family that directly interacts with light-activated phytochromes, and it can not only regulate various light responses but also optimize growth as a key integrator of multiple signaling pathways. However, the mechanism by which PIF4 participates in the regulation of anthocyanin accumulation remains to be elucidated. In this study, we found that anthocyanin accumulation was effectively induced by white light in Arabidopsis Col-0, but such an effect was impaired in the overexpression line PIF4OX. Consistently, the transcript level of PAP1 that encodes a key transcript factor involved in regulating anthocyanin biosynthesis was significantly decreased in PIF4OX compared with Col-0. Moreover, the expression of PAP1 was markedly lower in pap1-D/PIF4OX than pap1-D, as a result, the phenotype that highly accumulates anthocyanins in leaves of pap1-D caused by PAP1 overexpressing was almost eliminated in pap1-D/PIF4OX. Analyses through chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) and electrophoretic mobility shift assay (EMSA) revealed that PIF4 could directly bind to the G-box motif present in the promoter of PAP1. Furthermore, transient transcriptional expression analysis showed that PIF4 could weaken the transcriptional activity of the PAP1 promoter, and the G-box motif is necessary for the effect of PIF4. Subsequently, when the seedlings shifted from darkness to light and grew under constant red light and short-day photoperiod, it was found that the PAP1 transcription level and anthocyanin content in pif4-2/pap1-D were significantly higher than pap1-D, implying that PIF4 mutation can strengthen PAP1's effect on anthocyanin biosynthesis under these conditions. Taken together, the results indicate that PIF4 negatively regulates anthocyanin accumulation in Arabidopsis through transcriptional suppression of PAP1 by directly binding to the G-box motif of the promoter.