Reasearch Of Re-blooming Molecular Mechanism In Tree Peony ’High Noon’

Tree peony(Paeonia suffruticosa Andr.)is one of the local plant germplasm resource in China,which is known as the’king of flowers’with ornamental,medicinal and oil value.However,the feature of annual flowering and short flowering period has greatly restricted the commercial application of tree peony.Therefore,it has become an urgent problem to breed new cultivars of tree peony with improved flowering characteristic.’High Noon’is a cultivar with a re-blooming feature of tree peony,which is important for molecular mechanism research of re-flowering regulation in tree peony.This study researched the developmental feature of flowering bud in annually flowering cultivar’Fendan’and re-blooming cultivar’High Noon’and revealed the molecular mechanism of re-blooming in’High Noon’through analysis of ISO-seq and RNA-seq.The function of MADS-box genes played important roles in the flowering regulation were verified through molecular methods.The main results are as follows:(1)The anatomical observation of’Fengdan’and’High Noon’in the different developmental stages of flower bud during flowering showed that the first developmental flowering bud of both cultivars was similar.The apical meristem in vegetative stage S1 was slightly tip.When entered the differentiation stage S2,the apical meristem turned flat,and the cells in flank differentiated into floral organ primordium cells.As the external temperature drops,the peony enters the dormant stage S3,during which the flower buds no longer grow,but continue to complete the flower bud differentiation.In March of the next year,the external temperature rose,and the flower buds entered the germination stage S4.During this period,the flower buds grew rapidly,and differentiated into flower organs.Then the flower entered the flowering stage.The floral bud in the reblooming of’High Noon’went through only the vegetative stage S5 and the flower bud differentiation stage S6,without the dormant stage.The developmental characteristics of the vegetative stage S5 are consistent with that of the first vegetative stage S1,which is in the shape of a bulge.After S5,the bud turned into the differentiation stage S6,during which the floral buds differentiated rapidly and formed the flower bud primordium.(2)The ISO-sequencing of was performed with the mixed flower buds of’High Noon’during its six development period,and a total of 31.77 Gb data was generated.After correction,a total of 81,753 full-length transcripts with an average length of 2,060 bp were obtained.Among them,a total of 60,964 transcripts were annotated,and a total of 75,646 transcripts with an average CDS length of 708 bp were predicted.The integrity of the transcripts sequence generated in ISO-sequencing was evaluated and the results showed that 85%of the genes were intact,which suggested a relatively complete transcript sequence.In the same way,The ISO-seq of mixed samples from four development stages of flower buds of’Fengdan’was conducted,and a total of18.44 Gb sequencing data was obtained.After sequence correction,85,371 full-length transcriptome sequences with an average length of 3,738bp were obtained.Among them,a total of 76,748 transcripts were functionally annotated,and a total of 83,456 transcripts with an average CDS length of 955 bp were predicted.The complete gene accounts for 79.9%,which is a relatively complete transcript sequence.(3)To obtain the gene expression level of each stage,the next-generation transcriptome was sequened for the six typical stages of flower bud development of’High Noon’,and then was mapped to the full-length transcriptome.A total of 21 400 differentially expressed genes(DEGs)were obtained by comparing the expression levels of flower buds in each two adjacent periods.The most differentially expressed genes(10 658)were found between S3 and S4,while the least was found between S5 and S6.The DEGs between the adjacent periodwas conducted with GO term and KEGG pathway enrichment and expression trends analysis.The results showed that pathways including cell division,biological rhythms and plant hormone were involved in flower bud differentiation of’High Noon’.Among these plant hormone,auxin and cytokinin played a positive role in the floral bud diffirentiation,wherase brassinolide played nagetive role.In addition,the developmental feature of the second differentiation in’High Noon’floral bud without dormancy was closely related with the low expression level in ABA signaling pathway.(4)A total of 338 and 280 genes related to flowering time regulation was identified in’High Noon’and’Fengdan’,respectively.It is classified to five flowering time pathways,flowering integrators and flowering repressors.Gene expression analysis showed that pathways including photoperiod pathway,autonomous pathway and age pathway,GA pathway were involved in the development of the first floral bud of’Fengdan’and’High Noon’,While vernalization mainly promoted floral organ development.For the second floral bud formation in’High Noon’,the main participants was photoperiod pathway,autonomous pathway and GA pathway.(5)A total of 50 and 37 MADS-box gene were identified in the full-length transcriptome of’Fengdan’and’High Noon’.The phylogenetic and structural analysis classified them into Mα、Mβ、Mγ、MIKC~*和MIKC~C types.Among the MIKC~C,genes related to FLC,SVP and SOC and the floral organ development genes,like AGL6 and AG,were identified.Genes regulating the different development stages of floral bud in’High Noon’and’Fengdan’were identified through the expression analysis with transcriptome data.It provided effective supporting data for subsequent gene function verification.(6)The functions in flowering time regulation of PlMADS39 and PlMADS48 in’High Noon’were researched.The results showed that both PlMADS39 and PlMADS48 were located in the nucleus and PlMADS48 had transcriptional activation activity.Overexpression of PlMADS39and overexpression of PlMADS48 in Arabidopsis resulted early flowering.PlMADS48 binded to the promoter of Pl FT,while PlMADS39 interact with Pl FT protein.Therefore,it was inferred that PlMADS48 promotes flowering by regulating the expression of Pl FT through binding to its promoter,while PlMADS39 regulate flowering by interacting with Pl FT.In summary,this paper analyzes the developmental characteristics of flower buds in’Fengdan’and’High Noon’during flowering.The regulation pathways of flower bud development in different stages were explored.The genes of flowering time regulation pathway were screened,and the functions of each pathway in the flowering process of the two cultivars were analyzed.PlMADS39 and PlMADS48 were identified to involve in the regulation of flowering time.This study provides evidence for further elucidating the regulation mechanism of flowering time and multiple flowering of peony,and also provides new candidate genes for improving the flowering time directionally in the subsequent breeding of tree peony.