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.

中文翻译：

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植物色素相互作用因子 4 (PIF4) 通过抑制拟南芥幼苗中的 PAP1 转录来负调节花青素的积累

花青素积累是植物环境反应的显着症状，在植物对不利刺激的适应中起着重要作用。光敏色素相互作用因子 4 (PIF4) 是 PIFs 家族的成员，可直接与光激活光敏色素相互作用，它不仅可以调节各种光反应，而且作为多种信号通路的关键整合者，还可以优化生长。然而，PIF4 参与调控花青素积累的机制仍有待阐明。在这项研究中，我们发现白光在拟南芥 Col-0 中有效诱导了花青素的积累，但在过表达系 PIF4OX 中这种作用减弱。一贯地，与 Col-0 相比，PIF4OX 中编码参与调节花青素生物合成的关键转录因子的 PAP1 的转录水平显着降低。此外，PAP1 在 pap1-D/PIF4OX 中的表达明显低于 pap1-D，因此，在 pap1-D/PIF4OX 中几乎消除了由 PAP1 过度表达引起的 pap1-D 叶片中花青素高度积累的表型。通过染色质免疫沉淀定量 PCR (ChIP-qPCR) 和电泳迁移率变化测定 (EMSA) 进行的分析表明，PIF4 可以直接与 PAP1 启动子中存在的 G-box 基序结合。此外，瞬时转录表达分析表明 PIF4 可以削弱 PAP1 启动子的转录活性，并且 G-box 基序是 PIF4 作用所必需的。随后，当幼苗由暗转光并在恒定红光和短日光周期下生长时，发现 pif4-2/pap1-D 中 PAP1 的转录水平和花青素含量显着高于 pap1-D，说明 PIF4在这些条件下，突变可以加强 PAP1 对花青素生物合成的影响。总之，结果表明 PIF4 通过直接结合启动子的 G-box 基序对 PAP1 的转录抑制来负调节拟南芥中的花青素积累。在这些条件下对花青素生物合成的影响。总之，结果表明 PIF4 通过直接结合启动子的 G-box 基序来抑制 PAP1 的转录，从而负调节拟南芥中花青素的积累。在这些条件下对花青素生物合成的影响。总之，结果表明 PIF4 通过直接结合启动子的 G-box 基序来抑制 PAP1 的转录，从而负向调节拟南芥中的花青素积累。