Identification Of MiRNA Involved In Citrus Nucellar Embryo Initiation And Regulatory Mechanism Of Csi-miR156a In Somatic Embryogenesis

Citrus is one of the most important economical fruit tree crops widely grown in the world.Hybrid breeding in citrus has long been hindered by nucellar embryony.However,nucallar embryony enables the fixation of desirable traits as it could produce offsprings genetically identical to the female parent.Therefore,studies on the mechanism of nucellar embryogenesis would provide important insights for citrus breeding.microRNA(miRNA)is a vital regulator of plant growth and development.To identify miRNA associated with nucellar embryogenesis,we performed small RNA sequencing and compared the miRNA abundance in ovules between mono-and polyembryonic cultivars,which would be of value for exploring the miRNA-mediated regulatory mechanism of nucellar embryo initiation.In addition to hybrid breeding,biotechnological approaches are widely applied in citrus breeding.Somatic embryogenesis(SE)is commonly used for plant regeneration and in vitro germplasm conservation of citrus.It has been considered as the key step in biotechnological breeding.However,the capabilities of SE in most of citrus genotypes gradually decrease and was even lost after long term subculture.In this study,miR156,a highly conserved miRNA in plant,was selected for further investigate its potential role in regulating SE.The main results are showed as follows:1.Identificaiton of mi RNA involved in citrus nucellar embryogenesis by small RNA sequencing analysisSmall RNA sequencing was carried out in the ovules of two pairs of mono-and polyembryonic cultivars prior to and at the stage of nucellar embryo initiation.A total of 91 known miRNAs and 60 novel miRNAs were identified in the mandarin pair,with 695 and 163 targets predicted correspondingly.In the pummelo/grapefruit pair,93 known miRNAs and 66 novel miRNAs were identified and the correspondent numbers of predicted targets were 417 and 152.Only 13 miRNAs showed differentially expression at each stage within the mandarin pair,while 31 and 41 miRNAs were differentially expressed at two stages within pummelo/grapefruit pair.However,only two overlapped differentially expressed miRNAs were identified,including conserved mi R1446 and novel miRN23-5p.Six miRNAs were selected to verify by qRT-PCR,most of which showed similar expression pattern with sequencing data.RNA-seq data of the same two pairs of poly-and mono-embryonic ovules was retrieved from the previous study and reanalyzed with optimized methodologies.A total of 305 overlapped differentially expressed genes(DEGs)were identified between the two pairs of poly-and mono-embryonic ovules.Gene ontology(GO)analysis was performed based on the DEGs in each pair and the DEGs overlapped between pairs.As a result,stress response process was shown to be significantly enriched among the upregulated genes in polyembryonic ovules.Particularly,most of the genes in the stress response category were related with oxidative stress,suggesting that oxidative stress was important for citrus nucellar embryogenesis.The expression level of miRN23-5p was lower at six time points during ovules development in the two polyembryonic ovules than in the monoembryonic counterpart,whereas the predicted targets showed the opposite expression pattern as expected.Moreover,miRN23-5p mediated cleavage of the target transcript Cs9g06920 was verified by tobacco transient assay,implying the interaction between miRN23-5p and Cs9g06920 might play a role in nucellar embryo initiation.2.Functional characterization of csi-miR156 a in SEThe full-length sequence of csi-miR156 a coding gene CsMIR156 A was obtained by RACE.Overexpression vector containing csi-miR156 a precursor MIR156 a was transformed into the wild Fortunella hindsii callus with weak SE capability,and transgenic lines were obtained successfully.The csi-miR156 a overexpressed callus lines showed significantly higher SE competence than wild type.Expression of two targets of SPL were downregualted,and reversely correlated with SE capability in various citrus genotypes,implying that SPL might negatively regulated SE.The subcellular localization analysis showed the nucleus localization of the two SPLs.CsSPL14 exhibited transactivation activity which could active the downstream genes expression in yeast,whereas CsSPL3 did not.Suppression of CsSPL3 and CsSPL14 by RNAi in wild Fortunella hindsii callus also significantly enhanced SE capability,resembling the phenotype of csi-miR156 a overexpressed callus lines.The digital gene expression profiles were performed in csi-miR156 a overexpressed callus lines and wild type.In csi-miR156 a overexpressed callus lines,the biological processes of stress response and hormone-mediated signaling transduction were overrepresented among the upregulated genes,whereas methylation related processes and cell cycle were significantly enriched among the downregulated genes.The expression level of eight of the DEGs involved in stress response were detected in miR156 a overexpressed lines and the RNAi lines against the two SPL targets,showing similar pattern as indicated by digital profile.Two candidate proteins,which included CsARK1 and SnRK1 catalytic subunit alpha KIN10(CsAKIN10),were identified to interact with CsSPL14 by yeast two hybrid screening and validated by point to point verification.Bimolecular fluorescence complementation(BiFC)assays were applied to confirm the interaction between CsSPL14 and CsARK1 or CsAKIN10.The subcellular distribution analysis suggested that CsAKIN10 was localized in nucleus.In summary,we profiled the known and novel miRNA in two pairs of mono-/polyembryonic ovules,from which a potential ‘miRNA-target’ module was suggested to be associated with nucellar embryo initiation.The function of csi-miR156a-SPL module in somatic embryogenesis was validated,with proteins downstream SPL screened,and their interactions involved in somatic embryogenesis were discussed.