The RPA2A-PRC2 And Phosphorylation Of VIN3 Regulate Flowering Time In Arabidopsis

The heterotrimetic protein complex Replication protein A(RPA)is an essential replisome component that binds to single-stranded DNA during DNA replication.RPA2A is a subunit of RPA complex,which controls the root development and flowering time in Arabidopsis,but the underlying mechanism is not clear yet.PRC2(Polycomb Repressive Complex 2)is a conserved chromatin modification complex in multicellular organisms,which catalyzes trimethylation of lysine 27 of histone H3(H3K27me3)to inhibit gene expression.VIN3(VERNALIZATION INSENSITIVE 3)is a PHD domain-containing protein.During vernalization,VIN3 and PRC2 form the PHD-PRC2 complex,which promotes H3K27me3 deposition and inhibits FLC(FLOWERING LOCUS C)expression.However,the detailed mechanism remains to be explored.In this research,we studied the mechanism of RPA2A regulating flowering time by cooperating with PRC2 in Arabidopsis,and the effect of VIN3 phosphorylation on FLC regulation.The main results obtained are as follows:1.Gene expression pattern and DNA replication analyses displayed that RPA2A is mainly expressed in active dividing cells,and maintains proper cell cycle by promoting DNA replication.2.Molecular biology and genetic biology analyses confirmed that RPA2A delays flowering mainly through inhibiting key flowering genes including florigen gene FT.3.Combined RNA-seq and H3K27me3 Ch IP-seq analyses indicated that RPA2A inhibits the expression of some PRC2 target genes by maintaining H3K27me3 modification,such as FT,AG,and AGL71;Protein interaction and Ch IP-q PCR analyses showed that RPA2A recruits PRC2 to the chromatin of key flowering genes by interacting with PRC2.4.IP-MS analysis showed that there were many phosphorylation sites on VIN3 and SWN during vernalization;phenotypic observation and gene expression analyses showed that the mutation of VIN3 phosphorylation sites did not affect flowering time,vernalization induced VIN3 expression,and FLC inhibition in vernalization.In conclusion,these results showed that RPA2A interacts with PRC2 to recruit PRC2 to the chromatin of key flowering genes,maintains H3K27me3 modification levels during development to inhibit the expression of flowering genes,and maintain normal vegetative growth.In addition,our study of VIN3 phosphorylation function showed that these phosphorylation sites of VIN3 that we found may not play a role in vernalization.Therefore,the regulation mechanism of VIN3 in responding to vernalization still needs to be explored.