Molecular And Physiological Basis Of Ornamental Flower Development And Flowering Delay In Tree Peony

Paeonia spp.is a traditional famous flower in our country with a cultivation history of more than 1,600 years.The flowers are graceful,luxurious and distinguished,and have the reputation crowned the ‘king of flower’.Tree peony ornamental cultural festivals are held every year in Luoyang in Henan province,Heze in Shandong province and other places.Visitors from home and abroad are bustling in an endless stream.However,short flowering period and concentrated flowering time of the tree peony cannot meet the growing expectations and eliminate the economic and cultural development of the tree peony flower industry.In this century,a new resources food of tree peony seed oil has been developed.Paeonia ostii T.Hong et J.X.Zhang var.lishizhenii B.A.Shen has develomed as a multi-purpose peony material assembled ornamental,medicinal and food.In this study,P.ostii var.lishizhenii and other cultivars of tree peony were used as materials to explored transcriptome gene expression regulation of ornamental tree peony flower types(single petal,semi-double petal and double petal),and to reveal the molecular and physiological basis for prolonging the ornamental period.Four regulators,salicylic acid(SA),naphthalene acetic acid(NAA),melatonin(MT)and brassinolide(BR),used in experiments.By analyzing the changes of flowering period,physiological indicators,endogenous hormones,and DNA methylation(MSAP),the treatment was optimized,and the miRNAs and their target genes related to flowering period regulation were discovered,which would provide theoretical basis and technical approach for revealing the molecular genetic mechanism of flowering regulation and prolonging ornamental flowering period of tree peony.The main results are as follows:1.The transcriptome differentially expressed genes of the three flower types development transcriptome from the same plant were enriched in plant hormone signal transduction and other pathways,and discoverd2 genes of floral organ formation.It was found that 360.19 Gb of clean data were obtained from pedicels,receptacles,calyx,petals,stamens and stigmas.Splicing by Trinity software and de-redundancy by D-HIT software clustering,a total of 55,934 unigenes were obtained with an average length of 1108.5 bp.Of these,31,910 unigenes were annotated into NR,KOG,GO,Swissprot,egg NOG,KEGG,Pfam databases.Alternation of vegetative to reproductive growth,differentially expressed genes screened between pedicel and receptacle,receptacle and calyx,calyx and petal,petal and stamen,and stamen and stigma,were 3783,89,4747,444 and 4235,respectively.The differentially expressed genes between single petal and semidouble petal,semi-double petal and double petal,single petal and double petal were 4257,2959 and 3786,respectively.GO and KEGG analysis showed that the differentially expressed genes involved mainly in the pathways of plant hormone signal transduction,starch and sucrose metabolism.5 unigenes annotated as homologous genes of AGAMOUS(AG)that associated with stamen and carpel formation,and 1 unigene annotated as a homologous gene of PISTILLATA 1(PI 1)that associated with petal and stamen formation.2.Four plant growth regulators sprayed on the leaves of plants with bud incoming blooming in P.ostii var.lishizhenii was affected delaying blooming respectively.BR 0.050 mg/L delayed flowering to 3 days and overall flowering period remained unchanged,which was the best treatment effect.SA 1000 mg/L delayed best 3 days to bloom and shorten the overall flowering period by 1 day.MT 40 mg/L delayed flowering time to 2 days and overall flowering period remained unchanged.NAA 1000 mg/L delayed 2 days for flowering and shorten the overall flowering period by 2 days.The activities of SOD,POD and CAT increased by43.54%-139.10%,24.91%-54.11% and 42.86%-52.33%,respectively.MDA content decreased by 30.29%-38.31%.Soluble sugar and soluble protein contents did not change regularly.After sprayed SA,NAA and MT,the contents of endogenous hormones gibberellin,kinetin and indole-3-acetic acid in P.ostii var.lishizhenii decreased by 18.73%-76.38%,6.23%-57.34% and 26.77%-65.29%,respectively.The acid content increased by 35.30%-221.05% before flowering,and the gibberellin/abscisic acid decreased by 13.61%-82.34%.The salicylic acid content changed irregularly.Meanwhile,the full methylation rate and the hemimethylation rate of MSAP decreased by 7.02%-10.82% and 11.17%-27.26%,respectively.3.BR delays the flowering by affecting the expression of miRNAs and their target genes in two pathways in P.ostii var.lishizhenii.(1)By full-length transcriptomic sequencing petals of P.ostii var.lishizhenii,achieved a total of 22.25 G of raw data.21.27 G of clean data,13,978,732 subreads and 694,572 ROI obtained after filtration,respectively.After clustering correction of reads,there were 135,541 isoforms with Quvier>0.99,and 62,229 isoforms were obtained by remove redundancy de-redundancy.Of these,58,218 isoforms were annotated into NR,NT,Swissprot,KEGG,KOG,Interpro and GO databases.The results of enrichment analysis showed that 54 GO terms and 20 KEGG pathways were enriched.By comparing with the database,a total of 53,760 CDS were obtained with a maximum length of 6,375 bp.(2)miRNA and transcriptome sequencing were performed on the BR 0.050 mg/L treatment in P.ostii var.lishizhenii.60,442 isoforms,22 known miRNAs and 84 novel miRNAs were identified.Through the combined analysis of miRNA and transcriptome,18 known miRNAs were predicted to correspond to 376 target genes,and 23 novel miRNAs were predicted to correspond to 177 target genes.GO analysis of these target genes were annotated into 29 functional groups,including biological processes,cellular components and molecular functions.These target genes in KEGG analysis were annotated into 62 pathways,including cellular processes,environmental information processing,genetic information processing and metabolism and biological systems.Through the analysis and annotation of miRNA and its target gene expression,6miRNAs and corresponding 3 target genes were screened out.Among them,the target genes of 2 miRNAs(Po-miR156 b and Po-miR172a)were annotated as the homologous gene of SQUAMOSA PROTEIN BINDING LIKE 9(SPL9)and the seventh homologous gene(AP2 7)of the APETALA 2(AP2)family,respectively.The target gene of four novel miRNAs(Po-novel＿miR2,Po-novel＿miR36,Po-novel＿miR60 and Po-novel＿miR79)corresponding was annotated as homologous genes of SUPPRESSOR 1(SPA1).Cloning and bioinformatics analysis of the structural features of miRNAs and their target genes revealed that Po-miR156 b and Po-miR172 a formed stable secondary stem-loop structure,and the mature sequence was generated on the 5’ end arm of the gene precursor sequence.The open reading frame of Po SPL9 was1128 bp,coding 375 amino acids and containing the SBP conserved domain.It belonged to SPL transcription factor.The open reading frame of Po AP2 7 was 987 bp,coding 328 amino acids and containing the AP2 conserved domain.It belonged to AP2/ERF transcription factor.The secondary structures of Po-novel＿miR2,Po-novel＿mir36,Po-novel＿mir60,and Po-novel＿mir79 were predicted to form stable secondary stem-loop structures,and the mature sequences were generated on the 5’ end arm of the gene precursor sequence.(3)The response to BR mechanism of miRNAs and their target genes was analyzed by RT-q PCR,and it was found that the expression trend of 6 miRNAs and their target genes was opposite.Combined analysis of miRNA and transcriptome showed that responded to BR in two pathways.(1)Increasing Po-miR156 b to inhibited Po SPL9 expression,thereby promoted FUL and LFY expression and promoted expression of downstream Po-miR172 a to inhibit Po AP2 7 expression.(2)Increasing the expression of Po-novel＿miR2,Po-novel＿mir36,Po-novel＿mir60 and Po-novel＿mir79 to inhibited the expression Po SPA1.In short,in P.ostii var.lishizhenii five unigenes annotated as AG homologous genes,and one unigene annotated as PI 1 homologous genes related to flower organ development.BR 0.050 mg/L sprayed on leaves when the flower buds grow as the flat peach shape to regulate flowering delay by promoting 6 miRNAs,including Po-miR56b/Po-miR72 a and Po-novel＿miR2、Po-novel＿mir36、Po-novel＿mir60、Ponovel＿mir79,and inhibited the expression of thair target genes.It provides a basis to reveal molecular regulation mechanism of flower type and a way for developing technical measures to prolong the ornamental flowering in tree peony.