Study On The Mechanism Of Arabidopsis Blue Light Receptor Cry2-mediated Phase Separation To Regulate The Circadian Clock

The circadian clock is not only a heritable time-keeping mechanism to synchronize biological activities with the 24 h periodic changes of environmental factors,but also a significant guarantee of the surviving of most living creatures.Studies have shown that the circadian clock is involved in the process of the growth,development,and metabolism of plants,enabling them to predict and enhance their adaptability.Plant circadian clock is composed of input ways,the oscillator and output ways.Environmental signals are transduced to the oscillator via input ways,resulting in the synchoronization of oscillation frequency.The signals are then transmitted to downstream genes via output ways to trigger a series of physiological and biochemical reactions,maintaining the regular growth and development of plants.In such transduction,light signals in the environment are captured by series of photoreceptors,including five red/far red light receptors(phy A,phy B,phy C,phy D and phy E),seven blue light receptors(CRY1,CRY2,PHOTO1,PHOTO2,ZTL,FKF and LKP2)and an UV-B receptor(UVR8).At present,the molecular mechanism related to plant light signal transduction has been deeply studied.But little is known about the molecular mechanisms of how light signals regulate the circadian clock because the bridge proteins linking the light signal to the circadian rhythm have not been discovered.In previous studies,it is found that CRY2 regulates plant growth and development through the blue light dependent interaction with other signal proteins,and simultaneously adjusts the circadian clock by interacting with other proteins.However,the bridge proteins interacting with CRY2 and regulating the biological clock have not been found.Therefore,in this study,the protein interacting with the blue light receptor CRY2 was first identified from Arabidopsis by mass spectrometry,and the molecular mechanism of CRY2 regulating the biological clock was analyzed through a series of biochemical and molecular biology experiments.The specific research results are as follows:1.CRY2 and TCP22 can form liquid-liquid phase separation in a blue light dependent manner.A series of proteins interacting with CRY2 were identified and obtained fromArabidopsis thaliana by mass spectrometry,and a transcription factor TCP22 that regulates the circadian clock was screened and found.By yeast two-hybrid liquid assays and Co-IP experiments in HEK293T cells,it was proved that their interaction was a constitutive manner.Bi FC experiments further found that CRY2 formed blue light-dependent photobodies with TCP22,and the photobodies were fused with each other,proving that the photobodies are fluid.The fluorescence intensity was quickly restored to the original after photobleaching,indicating that photobodies can rapidly exchange internal and external substance.After the hydrophobic inhibitor(1,6-hexanediol)treated the protoplasts,photobodies could not be formed.The above experiments prove that the blue light-dependent photobodies formed by CRY2/TCP22 are liquid-liquid phase separation.CRY2 can form blue light-dependent phase separation in vivo and in vitro,while TCP22 can only form phase separation dependent on CRY2 in vivo.Comparing the phase separation of CRY2/TCP22 and CRY2/CRY2,it was found that the circularity and distribution rate of CRY2/TCP22 were significantly higher than those of CRY2/CRY2,indicating that TCP22 can regulate the state of CRY2 phase separation.Furthermore,TCP22 can inhibit the transition of CRY2 phase separation from liquid phase to colloidal phase.Recombination experiments in vitro once again proved that TCP22 promotes the formation of CRY2 phase separation.2.TCP22 regulates the expression of CCA1 in a CRYs-dependent manner.Ch IP experiments showed that CRY2 could interact with TBS in the CCA1promoter region;Phase separation experiments in vitro showed that TBS could co-phase with CRY2/TCP22 and could promote the phase separation of CRY2/TCP22.The p CCA1::LUC experiment found that both TCP22 and CRY2 could up-regulate the expression of CCA1 under blue light.When TCP22 was overexpressed in the cry1cry2mutant,the up-regulation of the expression of CCA1 disappeared.The blue-light pulse experiment found that blue light regulated the expression phase of CCA1,and blue light can regulate the phase of CCA1.The expression phase of wild-type CCA1 move forward under blue light,while the phase of CCA1 in TCP22OX moves forward to a greater extent.When TCP22 was overexpressed in the cry1cry2 mutant,the phase-forward phenomenon of CCA1 disappeared,indicating that the regulation of CCA1 by TCP22 was dependent on CRYs.The circadian rhythms of cry1cry2 and TCP22/cry1cry2 are down-regulated under blue light compared with continuous red light.Which proves that plants mainly input blue light signals to the core oscillator CCA1 mainly through CRYs.The dual-luciferase reporter assay in HEK293T cells found that CRY2/TCP22 could not directly activate the expression of CCA1 under blue light.3.CRY2 regulates TCP22 phosphorylation by enhancing the interaction between TCP22 and PPK1 under blue light.Experiments in vivo found that TCP22 can undergo a certain protein modification,and this protein modification can be enhanced under blue light.Through yeast two-hybrid and Bi FC experiments,it was found that TCP22 can interact with protein kinases PPKs.After HEK293T was co-transfected with TCP22 and PPKs,a modified band appeared above the TCP22 band in western detection,which could be eliminated by Lambda protein phosphatase(?-PPase).Meanwhile the modified band in TCP22/ppk124 in western detection basically disappeared.These results proved that PPKs are the protein kinases of TCP22 and can catalyze the phosphorylation of TCP22.Further research found that after the phosphorylation of TCP22/cry1cry2,the blue light response was lost,indicating that the promotion of TCP22 phosphorylation by blue light depends on CRYs.The LUC experiment also further verified this result.The phosphorylation site of TCP22 was further identified by massspectrometry,and it was found that the phosphorylation site mainly occurred on the serine threonine of the 37-50th amino acid in the N-terminal,and this serine threonine was all mutated into alanine(named m TCP22^12STA),it could not be phosphorylated by PPK1 and could not form phase separation in vitro.p CCA1::LUC experiments found that m TCP22^12STA can inhibit the expression of CCA1.Co-IP and Bi FC experiments further found that m TCP22^12STA can interact with PPK1 normally,and can also interact with CRY2 and form a colloidal phase separation,indicating that m TCP22^12STAcompetitively binds endogenous PPKs and CRYs in vivo,and then dominantly negatively regulates CCA1.4.PPK1 regulates of properties CRY2/TCP22 condensates and the expression of CCA1.Phase separation assay in vitro showed that PPK1 co-phased with TCP22 and promoted the phase separation of TCP22.Bi FC showed that CRY2/PPK1/TCP22 could form phase separation in a blue light dependent manner,FRAP proved that PPK1regulated CRY2/TCP22 phase separation and inhibited CRY2/TCP22 phase transition from liquid to a gel state.p CCA1::LUC in vivo showed that PPK1 regulates CCA1 expression under blue light.Blue light pulse assay showed that the phase of CCA1 regulated by TCP22 was dependent on PPKs.Dual luciferase reporter assay of HEK293T cells confirmed that both phosphorylated and non-phosphorylated TCP22 could not activate CCA1 directly,at the same time,CRY2/PPK1/TCP22 complex could not activate CCA1 directly under blue light.5.CRY2 recruites TCP22,PPK1 and LWD1 via blue light dependent phase separation to regulate the expression of CCA1.The dual-luciferase reporter assay in HEK293T cells found that TCP22 activated CCA1 expression only when it interacted with LWD1,and the phosphorylated TCP22and LWD1 had stronger activation ability,indicating that the direct activation of CCA1expression by TCP22 in vitro depends on LWDs.PPK1 enhanced the interaction between TCP22 and LWD1.In addition,LWD1 interacted more easily with phosphorylated TCP22,which explained why phosphorylated TCP22 and LWD1 were more capable of activating CCA1.Futhermore,CRY2/PPK1/TCP22/LWD1 directly activated the expression of CCA1 under blue light,and CRY2 interacted with LWDs in a blue light-dependent manner.Meanwhile,CRY2/PPK1 promoted the co-phase between TCP22 and LWD1.In conclusion,CRY2 and TCP22 formed blue light dependent phase separation,and CRY2 recruited PPKs and LWDs in a blue light dependent manner.PPKs catalyzed phosphoration of TCP22 and promoted the interaction between TCP22 and LWDs.In addition,CRY2 promoted the interaction between TCP22 and LWDs under blue light,and finally regulated the expression of CCA1 under blue light.Taking all together,we demonstrated that Arabidopsis blue light receptor CRY2formed LLPS with transcription factor TCP22,then recruited protein kinase PPKs and light-regulated protein LWDs into the phase,thus regulated the expression of CCA1 via various biochemical processes as protein phosphorylation modification,protein-protein interaction,and protein-DNA interaction,etc.For the first time,we have analyzed the mechanism of blue light signal regulation of the core oscillator CCA1,which has laid a foundation for the study of the mechanism of light input to the core oscillator of the circadian clock,and provided new ideas for the research of blue light signal transduction and biological clocks of other species.