| Light is not only providing energy sources for plant growth,it also acts as an important environmental signal to implicate multiple cellular processes.In the past decades,genetic and biochemical approaches have demonstrated the complexity of light signal pathways that comprised with different positive and negative regulators through different interlocked feedback loops.Multiple different type of photoreceptors,including phytochromes and cryptochromes,are utilized to monitor the dynamic changes of various kinds of light signals and then transduce these signals to the diverse downstream components.Phytochrome A（phy A）is the photoreceptor mediating far-red（FR）response througth its downstream transcriptional factors.Transposase-derived transcription factor FHY3 and its closest homolog FAR1 were originally identified as two positive regulators of FR response through activating the transcription of FHY1 directly,thus to promote the translocation of phy A from the cytosol into nuclear and mediate FR response.Genome-wide downstream targets analyses have revealed that FHY3 directly binds to over 1000 different targets,thus involved in multiple cellular processes.In the light response,FHY3 and FAR1 bind to the promoter regions of ELF4 and activate its expression,thus regulating the circadian clock and flowering time.In addition,FHY3 physically interacts with transcription regulators PIF1,EIN3,and CCA1 to integrate light signaling to the processes of chlorophyll biosynthesis,uptake of phosphorus,and maintains of the circadian clock.Although FHY3 has been identified to play essential roles in multiple light-induced cellular processes through regulating the transcription of various downstream targets,while the regulatory of FHY3 which mediated by light signaling and the mechanism of FHY3that function with photochrome is still largely unknown.In this study,we committed to research the regulation of FHY3 protein and biological processes in FR-induced interaction between FHY3 with phy A and DET1 protein and found that FHY3 participates in regulating and maintaining of FR signaling pathway by interacting with phy A and DET1.The main results are as follows:（1）The Y2H,Bi FC,and Co-IP assays were performed to further confirmed the FR-induced interaction between FHY3 and phy A,and found that multiple domains of FHY3 and phy A include FHY3-N,FHY3-C,phy A-PAS,and phy A-HKRD were involved in the protein interaction.（2）Western blot assays of FHY3 in different dark-grown transgenic lines and mutants with FR treatment were set up,and the results showed that phy A mediated the accumulation of FHY3at the beginning of FR treatment by a post-translational manner.In addition,we found that phy A promoted the degradation of FHY3 in darkness.（3）According to Ch IP-q PCR assay,transcription assays,and RT-q PCR,we demonstrated that phy A positively regulated the FHY3 mediated transcriptional activation to the circadian clock genes CCA1 and ELF4.Furthermore,research to the 35Sp:phy A-NLS-GFP transgenic lines,we showed that phy A was associated with the promoter of FHY3 downstream genes with FHY3,and coordinately regulated the transcriptional activation.（4）Transgenic lineΔMu fhy3-4 was generated and found that the expression of congregated FHY3 N-terminal and C-terminal significantly recovered the loss of photomorphogenesis in fhy3-4 mutant.Further study,two other transgenic linesΔMu No-0 and FHY3-CT No-0 were generated,and we found that the expression of the FHY3-CT domain induced a defect of photomorphogenesis in No-0.Western blot and RT-q PCR assays showed that FHY3-CT protein destroys the phy A protein,while the FHY3ΔMudidn’t.Even though the FHY3ΔMufusion protein could function as a full-length FHY3 protein to protect phy A protein to avoiding COP1-mediated degradation,it suggested that the multiple domains involved interaction between FHY3 and phy A was necessary for the FHY3-mediated phy A stabilization.（5）Arabidopsis thaliana DE-ETIOLATED1（DET1）,a key repressor of photomorphogenesis,physically interacted with FAR-RED ELONGATED HYPOCOTYL3（FHY3）after seedlings were irradiated with light,DET1 associated with FHY3 to the promoter region of FHY1 and ELF4,and repressed the transcription activation of FHY3.（6）Further,DET1 recruited HISTONE DEACETYLASE6（HDA6）to the promoter region of FHY1 and ELF4,thus reducing the abundance of H3K27ac and H3K4me3,repressing their expression,and feedback regulates the far-red responses of seedlings.（7）In addition to regulating FR signaling pathway,DET1 also represses the transcriptional activation of FHY3 to ABI5,promoting seedling greening upon etiolated seedlings reaching the soil surface.In summary,our study demonstrated that phy A induced the accumulation of FHY3 at the beginning of FR response.FHY3 recruited phy A to the promoters of downstream genes to activate those genes,while FHY3 promoting the stability of phy A via the multiple domains involved interaction;and FHY3 interacted with the negative regulators DET1 and HDA6 which represses the expression of FHY1 and ELF4 by regulating the H3K4me3 and H3K27ac at promoters of FHY1 and ELF4,thus to controlling and maintaining the FR signaling transduction. |
