Conserved HAIKU2 Function But Divergent Epigenetic Regulation Reveals Routes Of Endosperm Developmental Evolution In Four Species

Angiosperm seeds consist of embryo,endosperm and seed coat with a vast difference in late endosperm proliferation fate.Endosperm constitutes the major volume of mature seeds in Poales and some dicots.In other dicots such as Arabidopsis,seed development involves an early phase of synthanial endosperm proliferation followed by a second phase in which cellularization begans and marks the end of endosperm proliferation,and then embryo grows to replace endosperm.The molecular mechanisms underlying the two extreme ontologies of seed morphology remain largely unknown.This study was carried out in four species,Arabidopsis thaliana,Brachypodium distachyon,Oryza Sative and Glycine max,and focused on conserved HAIKU2(IKU2)function but divergent epigenetic regulation revealing two different endosperm development fate.AtIKU2 encodes a leucine-rich repeat(LRR)receptor-like kinase and is a key regulator of Arabidopsis endosperm proliferation.Atiku2 mutant causes precocious cellularization,reduced number of endosperm nuclei and mature seed size.Arabidopsis has three major Polycomb repressive complexes 2(PRC2)complexes,EMBRYONIC FLOWER(EMF)PRC2,VERNALISATION(VRN)PRC2 and FERTILISATION INDEPENDENT SEED(FIS)PRC2.FIS PRC2 funtions in reproduction development specifically.MEDEA(MEA)is the histone methyltransferase of FIS PRC2 and is reported existing in Brassicaceae family.Its SET domain can catalyze H3K27me3 marks and conduce silence of target genes.Atmea mutant produced over-proliferation endosperm and leathal seeds.In this study,we analyzed the mechanism by which MEDEA-IKU2 pathway regulated two different endosperm development fate from the molecular,biochemical and genetic perspectives,and the following results were achieved.(1)We found AtIKU2 homologous genes,BdIKU2,Os IKU2 and GmIKU2,from Brachypodium distachyon,Oryza Sative and Glycine max,and the function in endosperm proliferation was conserved in Arabidopsis.We obtained the following transgenic plants,pAtIKU2::AtIKU2/Atiku2,pAtIKU2::BdIKU2/Atiku2,pAtIKU2::Os IKU2/Atiku2 and pAtIKU2::GmIKU2/Atiku2,and the IKU2 s can functionally rescued Atiku2 seed phenotype to Col at seed mass,transgene expression pattern,embryo development stage and cellularization progression.Conserved IKU2 s existed in these four species.(2)BdIKU2 regulated endosperm development.Knockouts of BdIKU2 caused precocious cellularization,reduced number of endosperm nuclei and produced small seeds.The cell size was not altered in Bdiku2 seeds.Both pBdIKU2::BdIKU2 and pBdIKU2::AtIKU2 complemented the Bdiku2 mutant seed development phenotype.The function of AtIKU2 and BdIKU2 in endosperm proliferation was also conserved in Brachypodium distachyon.(3)Transcription repression existed at AtIKU2 loci during cellularization stage.qRT-PCR results showed that the m RNA expression level of both pAtIKU2::AtIKU2(in pAtIKU2::AtIKU2/Atiku2 transgenic plants)and pAtIKU2::BdIKU2(in pAtIKU2::BdIKU2/Atiku2 transgenic plants)declined from 3 day after pollination(DAP).In situ hybridization results showed that both pAtIKU2::AtIKU2(in pAtIKU2::AtIKU2/Atiku2 transgenic plants)and pAtIKU2::BdIKU2(in pAtIKU2::BdIKU2/Atiku2 transgenic plants)could detected extremely low m RNA signals mainly in developing syncytial endosperm at 1 to3 DAP.Arabidopsis IKU2 was suppressed in late development phase after 3 DAP.(4)At MEDEA repressed AtIKU2 at 4 to 5 DAP and endosperm development.At MEDEA could not be recruited to AtIKU2 loci at 2 to 3 DAP,AtIKU2 was actively expressed and endosperm cells went on continous dividing.At 4 to 5 DAP(cellularization stage),At MEDEA could be recruited to AtIKU2 promoter region,enrichment of H3K27me3 marks over the AtIKU2 loci(mainly around the TSS region)deposited by FIS PRC2 was sufficient to repress AtIKU2 transcription and further endosperm proliferation.Arabidopsis seed growth transits to embryo development after.(5)BdIKU2 and Os IKU2 loci are devoid of H3K27me3 marks which are synchronous with active IKU2 transcription and endosperm cells proliferation.qRT-PCR results showed that BdIKU2 and Os IKU2 expression maintained a relatively stable level in developing seeds up to 20 day after anthysis(DAA).Ch IP results showed that no remarkable H3K27me3 changes was detected at BdIKU2 and Os IKU2 loci for any of the fragments at 2 to 3,6 to 7 and 14 to 15 DAA.In situ hybridization results showed that BdIKU2 and Os IKU2 could be detected not only in coenocyte endosperm,but also in those dividing central starchy endosperm cells and aleurone layer cells,and can also in central starchy endosperm cells during endoreduplication stage just before programmed cell death(PCD)stage.The expression of BdIKU2 and Os IKU2 were continuous in cellularization and postcellularization stage with continuously dividing endosperm cells and increased numbers.(6)Prolonged expression of pBdIKU2::BdIKU2 in 4-9 DAP produced undesirable free endosperm nuclei.High and prolonged Signals of pBdIKU2::BdIKU2(in pBdIKU2::BdIKU2/Atiku2 transgenic plants)could be detedted in endosperm tissue at 4 to 7 DAP as shown by in situ hybridization.Abnormal seeds had more endosperm nuclei in pBdIKU2::BdIKU2/Atiku2 transgenic plants.pBdIKU2::BdIKU2 was devoid of the the transcription repression system that supressed AtIKU2 in Ababidopsis.(7)GmIKU2 expression was repressed at late development stage.Soybean had a very similar seed developmental path as that of Arabidopsis as shown in paraffin sections.GmIKU2 expression was high from 1 to 6 day after flowering(DAF)and then gradually declined from 7 to 18 DAF,H3K27me3 marks were barely detected at 2 to 4 DAF but highly enriched at 7 to 9 DAF at GmIKU2 loci.GmIKU2 expression was repressed by histone modification.(8)One MEDEA-equivalent was identified in soybean.Phylogenetic tree analysis showed that At MEDEA and At SWN were clustered on one branch and At MEDEA is proposed to originate during a whole-genome duplication from At SWN.The SET proteins from Glycine max and Nymphaea colorata were clustered on one branch with At MEDEA while SETs from Poales,Brachypodium distachyon,Oryza Sative,Zea mays,Aegilops tauschii,Hordeum vulgare,Setaria viridis,Triticum aestivum and Triticum durum,together with Amborella trichopoda were clustered on one branch.(9)GmMEDEA exression could be detected in developing endosperm and repressed late endosperm development fate.One GmSWN was demonstrated as a functional equivalent of At MEA.Either p At MEDEA::At MEDEA or p At MEDEA::GmMEDEA complemented Atmea-3 phenotype.Both At MEDEA and GmMEDEA were recriuted to the AtIKU2 promoter at 4 to 5 DAP,Once H3K27me3 marks are enriched,AtIKU2 transcription was silenced.In addition,GmMEDEA exression could be detected in developing endosperm.Our results support the long-standing hypothesis that MEDEA is evolved from SWN through genome duplication and acquires function in suppressing endosperm proliferation and regulation over the growth rate of embryo and endosperm.In conclusion,AtIKU2 homologous genes exised in Brachypodium distachyon,Oryza Sative and Glycine max and the function in endosperm proliferation was conserved,however,MEDEA was divergent.In late development stage of Arabidopsis and soybean,MEDEA was recruited to IKU2 promoter,catalyzed H3K27me3 marks and conducing silence of IKU2 and endosperm proliferation.While in Brachypodium distachyon and Oryza Sative,no MEDEA homologous genes were found.BdIKU2 and Os IKU2 loci sustained a continuous expression and promoted endosperm proliferation.The expression pattern of MEDEA-IKU2 detedmined two different endosperm proliferation fate.