COP1 And PhyB Physically Interact With PIL1 To Regulate Its Stability And Photomorphogenic Development In Arabidopsis

Light serves as a significant signal to modulate almost every aspect of plant development throughout their life cycle, such as seed germination, photomorphogenesis, floral transition and circadian rhythm. Arabidopsis, as a model organism, primarily has the cryptochrome(CRY) and phytochrome(PHY) to perceive blue and red/far red light, respectively. CONSTITUTIVELY PHOTOMORPHOGENIC 1(COP1), as a RING motif-containing E3 ligase, acts to negatively regulate photomorophogenesis. COP1 interacts with and promotes the degradation of the positive transcription factors of photomorphogenesis, such as ELONGATED HYPOCOTYL 5(HY5) and LONG HYPOCOTYL IN FAR-RED 1(HFR1), and promote their degradation. PHYTOCHROMES INTERACTING-FACTORS(PIFs), as a group of bHLH transcription factors, physically interact with phytochromes, and negatively regulate photomorphogenesis. When exposed to red light, phyB is activated and translocated into the nucleus. In nuclear, phyB promotes photomorphogensis throughout inhibiting the activity of COP1 and promoting the degradation of PIFs. However, it is unknown that whether there are additional factors that may act in cryptochromes or phytochrome-mediated signaling pathway.Combining the methods of genetics and molecular biology, our study demonstrates the molecular mechamism of the modulation photomorphogesis by PIF3-LIKE 1(PIL1) in Arabidopsis. Firstly, based on the previous demonstrations that PIL1 shares high amino acid sequence similarity to HFR1 and that the pil1 mutant shows reduced photomorphogenic phenotype, similar to the hfr1 mutant, Our study suggests that PIL1 is a new COP1-interacting proteins through combined biochemical assays. And COP1 promotes the degradation of PIL1 via the 26 S proteasome-dependent pathway. The genetic study suggests that both PIL1 and HFR1 are genetically downstream of COP1. Moreover, PIL1 physically interacts with HFR1, suggesting that PIL1 and HFR1 form a heterodimer to promote photomorphogenesis additively.Secondly, since PIL1 possesses a putative active phyB binding(APB) motif, our study demonstrates that PIL1 preferially interacts with photoactive phyB upon red light through combined biochemical assays. Western Blot analysis indicates that phyB promotes the accumulation of PIL1 upon red light. Subsequently, we study whether phyB affects the association capacity of COP1 with PIL1 through Co-IP assays, and it demonstrates that phyB acts to promote the COP1–PIL1 disassociation in response to red light exposure. In addition, our study suggests the PIL1 physically interacts with PIFs(PIF1, PIF3, PIF4 and PIF5). Genetic study demonstrates that pil1 pifq(pil1 pif1 pif3 pif4 pif5) quintuple mutant has an enhanced photomorphogenic phenotype in blue, red, and far-red light, respectively, similar to pifq(pif1 pif3 pif4 pif5), indicating that PIFs is genetically up epistatic to PIL1. Furthermore, the results of qRT-PCR and Dual-LUC experiments suggest that PIL1 and HFR1 may act together to suppress PIFs transcriptional activities and promote phomorphogenic development.In conclusion, our study reveals the molecular mode of action of PIL1 in mediating photomorphogenesis upon red light irradiation. In darkness, COP1 promotes the degradation of PIL1 and HFR1, which attenuate their inhibition of PIFs. Upon red light, phyB interacts with COP1 and PIL1, resulting in the accumulation of PIL1 presumably through different layers of regulation including repressing COP1 activity, inhibiting the COP1–PIL1 association, and promoting the translocation of COP1 from the nucleus to cytoplasm. The accumulated PIL1, together with HFR1, interact with PIFs and inhibit the transcription of their direct-target genes, and thus promote photomorphogenesis. Therefore, our study suggests that PIL1, HFR1, and PIFs constitute a subset of antagonistic bHLH factors acting downstream of phyB and COP1 to regulate photomorphogenic development, and establishes the status of PIL1, as a light transduction signaling hub, enlarging the knowledge of the mechanism of photomorphogenesis.