Molecular Mechanism Of Nucleoporin96 Involved In Flowering And Circadian Clock Regulation

The nuclear pore complexes(NPCs)are embedded in the nuclear envelope(NE)and serve as the sole channel for nucleocytoplasmic transportation of macromolecules.NPCs profoundly affect the timing of flowering and the circadian period,but the underlying mechanisms are poorly understood.Here we reported that Nucleoporin96(Nup96)acts as a negative regulator of long-day photoperiodic flowering and the circadian period in Arabidopsis thaliana.Through multiple approaches,we identified Nup96 could interact with an E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 1(HOS1)in vitro and their interactions specially occur on nuclear membrane in vivo.Loss of function of Nup96 leads to degradation of HOS1 proteins without a change in the mRNA abundance;conversely,mutation of HOS1 also strikingly damped Nup96 protein level without changing its transcript level,suggesting that Nup96 and HOS1 are mutually stabilized.In analysis of phenotypes,the nup96 and hos1 single and the nup96 hos1 double mutants have highly similar early-flowering times and long circadian periods.And transcriptome results showed that the nup96 and hos1 single mutants share more than 50%genes with significant changes.The data above indicated Nup96 and HOS1 may work together as a functional module(Nup96-HOS1).Next,we identified two important targets of the Nup96-HOS1 module,CO and ELF3 proteins.CO is the time measurer of day length in photoperiodic flowering pathway and ELF3 is the time taker(zeitnehmer)of circadian clock period.Furthermore,we found ELF3 was ubiquitylated by HOS1,whereas CO has previously been reported as a HOS1 substrate,implying the Nup96-HOS1 module interacts with CO and ELF3 and regulates their stability.Nup96 and HOS1 mutation leads to over-accumulation of CO and ELF3 proteins in the morning,which subsequently results in early flowering mediated by over expression of FT gene and long period mediated by repression of PSEUDO-RESPONSE REGULATOR9(PRR9)transcription,respectively.Genetic analysis of the nup96 and hos1 single mutants and the nup96 hos1 double mutant suggests Nup96 and HOS1 coordinately regulate photoperiodic flowering and the circadian period.In conclusion,we speculated the Nup96-HOS1 module performs as a degradation platform on nuclear pore and functions in the morning.It participates in the measurement of day length of photoperiod as well as recording circadian period.Our results did not only uncover a novel mechanism of photoperiodic flowering regulation and circadian period determination,but also revealed that protein homeostasis on NPC is an important novel-function of nuclear pore complex.It is well known that the spatio-temporal regulation is critical to gene functions.Our data provide a line of robust evidence for this theory and proclaim fine-tuning protein homeostasis in the morning on the nuclear membrane is significant for controlling photoperiodic flowering and circadian period.