Dissection Of The Mechanism Of DCP5-SSF Interaction And Liquid-Liquid Phase Separation Regulating Flowering In Arabidopsis Thaliana

Flowering is a key transition process of plants from vegetative to reproductive growth,which plays an important role in plant growth and adaptation.According to reports,in vivo and in vitro factors involved in the regulation of plant flowering time are various and strikingly complex.Although the regulation of flowering time has been deeply studied,there are still many regulatory factors whose mechanisms have not been fully revealed.Processing bodies(P-bodies)are highly conserved membraneless organelles in eukaryotes,but their molecular organization in cells and biological functions are still obscure.Using molecular biology,biochemistry and genetic approaches,we analyzes the molecular mechanism of P-body involving in FLOWERING LOCUS C(FLC)transcription and flowering time control in Arabidopsis thaliana.The main results are as follows:1.Through protein co-immunoprecipitation combined with mass spectrometry(IP-MS)analysis of the flowering regulator SSF(Sister of FCA)previously identified in our laboratory,a SSF-interacting protein-DCP5 was screened,which is an important component of P bodies.The interaction between the two proteins was verified by yeast two-hybrid(Y2H),luciferase separation(LCI),bimolecular fluorescence complementation(BIFC),in vitro Pull-down and in vivo protein co-immunoprecipitation(Co-IP)techniques.DCP5 co-localized with SSF in the nucleus.Additionally,SSF not only interacts with DCP5,but also interacts with DCP2,another P-body component,in nucleus.2.During the analysis of SSF and DCP5 proteins,we found that both of them have prion-like domains(Pr D)and highly disordered regions;SSF contains one Pr D and DCP5 contains two Pr Ds.Both SSF and DCP5 exhibited typical liquid-liquid phase separation(LLPS)phenomenon under confocal laser imaging after fusing the GFP fluorescent tag.In addition,experiments with in vitro protein analysis,transgenic phenotype analysis,transcriptional activity analysis and chromatin co-immunoprecipitation(Ch IP)showed that Pr D plays important roles in the liquid-liquid separation ability of the two proteins and their biological functions.3.Compared with the wild type,the dcp5-1 mutant showed a late-flowering phenotype,and the expressions of both mature and nascent FLC m RNA were up-regulated.Through the analysis of the flowering time and FLC expression of ssf-2,dcp5-1 and the double mutant ssf-2 dcp5-1,DCP5 is genetically located at the upstream of SSF for FLC regulation.Furthermore,the gene expression and protein accumulation of SSF and DCP5 did not affect each other,and DCP5 could promote the liquid-liquid phase separation of SSF in vitro and in vivo,but in contrast,SSF had little effect on the liquid-liquid phase separation of DCP5.4.While analyzing DCP5 intracellular localization,we found that DCP5 can be detected in both nucleus and cytoplasm proportions,which suggests that it also plays a regulatory function in nucleus in addition to in cytosol.FLC m RNA capping and decapping analysis showed that DCP5 can affect the homeostasis of FLC m RNA 5? cap structure.Ch IP experiments revealed that more RNA polymerase II(RNA Pol II)was enriched at the FLC locus in the dcp5-1 mutant,transcribing more FLC RNA,and more importantly,our Ch IP analysis using transgenic plants showed that DCP5 could bind to multiple regions of the FLC gene,and this process was dependent on SSF,as at the background of ssf-2,DCP5 cannot enrich on FLC,indicating that SSF has the role to recruit DCP5 to FLC chromatin region.5.The PrD domains play important roles in the function of SSF and DCP5.Y2 H,Co-IP and transcriptional activity were analyzed,indicating that Pr D domains had important effect on the interaction between these two proteins,and SSF overexpression could promote the inhibition capacity of DCP5 on FLC transcription.In conclusion,in this thesis,the interaction protein DCP5 of the flowering transcription factor SSF was screened by IP-MS method,its molecular function in the FLC transcription and flowering time was deeply studied,and the role of LLPS in SSF and DCP5 was further revealed.Overall,the molecular mechanism of protein properties and functions of DCP5 and SSF in flowering regulation has expanded our understanding of the function of P-body components.