Transcriptomes And Dna Methylomes In Apomictic Cells Delineate Nucellar Embryogenesis Initiation In Citrus

In apomictic plants,asexual embryos form without meiosis and gamete fusion that are required for sexual reproduction.As a typical form of sporophytic apomixes,nucellar embryogenesis in citrus is unique and stable.The nucellar cells surrounding the embryo sac with thickened cell wall and condensed cytoplasm,namely nucellar embryo initial（NEI）cells,spontaneously develop into asexual embryos alongside the zygotic embryo.Nuceller embryos are genetically identical to the maternal parent.Nucellar polyembryony（NPE）is widely used for clonal rootstock production and has the potential to fix hybrid vigor in crops,but hinders the efficiency of citrus cross breeding because the nucellar embryos compete for nutrients with the zygote during seed development.The candidate NPE-controlling gene Cit RWP has been identified from The NPE locus of the citrus genus previously.In this study,to profile the active genes and epigenetic modifications involved in initiation of citrus NPE,the RNA-Seq and BS-Seq analyses were applied on the apomictic cells and sporophytic cells that were accurately collected by laser microdissection,soon before（earlier NEI stage,3DBA）and after（later NEI stage,7 DAA）the NEI cells are observable in ovules under microscope.The results are as follows:1.Differential gene transcription levels and key gene mining between apomictic cells and sporophytic cells.At earlier NEI stage,a total of 532 DEGs were identified,among which 444were up-regulated in Poly-NC（AM cells in earlier NEI stage）relative to Mono-NC（SO cells in earlier NEI stage）and 88 were down-regulated.At later NEI stage,2,859DEGs were identified,among which 1,483 were up-regulated in NEI cells relative to SO cells.GO enrichment of up-regulated genes in AM cells at two NEI stages displayed processes involved protein biogenesis（translation,ribosome biogenesis）and degradation（ubiquitin-dependent protein catabolic process）.Histochemical staining method showed a higher level of O₂^-and H₂O₂ in poly-embryonic ovules than mono-embryonic one at earlier NEI stage,and a higher level of H₂O₂ in poly-embryonic ovules at later NEI stage.Response to oxidative stress was enriched in NEI cell up-regulated genes.In situ RNA hybridization showed that stress-responsive genes RD21 and NAD（P）-linked oxidoreductase superfamily protein were specifically expressed in NEI cells,which indicates that stress response may play a role in nucellar embryo initiation.In addition,embryonic development and callose deposition during defense response were enriched among the up-regulated genes in NEI cells,which specified the embryogenic fate of NEI cells.In situ RNA hybridization showed that Cit RWP expressed in polyembryonic ovules and in NEI cells,but not expressed in monoembryonic ovules.A C2H2 domain containing gene Cg1g000740 showed the similar expression model with Cit RWP.The expression level of C2H2 in ovules seems positively associated with the NPE capacity of citrus.The protein subcellular localization assay showed C2H2 was localized in nucleus.A split firefly luciferase complementation assay（Bi FC）demonstrated that Cit RWP interacts with C2H2.Expression of C2H2 was suppressed in poly-embryonic mini-citrus（Fortunella hindsii）by RNAi.Ten transgenic lines were obtained,with the phenotype to be observed.2.DNA methylation profiles in the apomictic cells.The global DNA methylation level of apomictic cells and sporophytic cells were52.5-56.1%for CG context,31.8-34.1%for CHG context,and 1.0-2.1%for CHH context.The DNA methylation levels in the three contexts were all higher in the chromosomal regions that are dense with transposable elements（TEs）,relative to regions with high gene density.The global DNA methylation level was lower in apomictic cells relative to sporophytic cells at both NEI stages,especially in CHH context,indicated an overall loss of DNA methylation during NPE initiation.Together,we suggest that the global reduction of CHH methylation levels in apomictic cells may derepress retrotransposons and thus,alter the expression of adjacent genes that regulate apomictic cell development.In contrast,a hyper-methylated DMR was identified in the Cit RWP gene body in the Poly-NC,which might have caused the up-regulation of expression in apomictic cellsWe propose a potential working model for the initiation of NPE in citrus.The up-regulated Cit RWP and C2H2 may induce increases in the levels of reactive oxygen species（ROS）in poly-embryonic nucellus,which may serve as a signal that activates epigenetic regulation and therefore,regulates the expression of particular genes and biological processes that induce a cell fate transition that leads to NEI cell identity.