Identification And Functional Verification Of Key Genes For Petaloid Stamens In Paeonia Lactiflora

Paeonia lactiflora is known as the ‘Prime Minister of flowers’ in China.It has a variety of flower patters,including 13 types.It is rich in colors and can be divided into 9 major colors.Paeonia lactiflora has a rising trend in the cut flower market due to its diverse flower types and colors,and double-petaled cultivars have higher ornamental and economic value in the cut flower market.Petaloid Stamen is a common phenomenon in double-petaled flowers of herbaceous peony,and it is one of the important forms of double-petaled flowers.However,the cause and molecular mechanism of Paeonia lactiflora petaloid stamens are still unclear,and the key genes regulating petaloid stamens have not been reported.Therefore,it is of great significance to identify the key genes of petaloid stamens in herbaceous peony and verify the gene function.In this study,seven flower types of herbaceous peony,including ’Fen Yu Nu’,’Lian Tai’ and other cultivars were used as the research materials.We observed the differentiation process of their flower buds,summarized their stamen primordium differentiation law,determined the key time node of stamen primordium petaloid of herbaceous peony.Finally,the three flower bud development stages of ’Fen Yu Nu’(Single flower)and ’Lian Tai’(Petaloid stamens)were selected as transcriptome sequencing materials.Through transcriptome sequencing,key genes related to petaloid stamens of herbaceous peony were identified,and related transcription factors(TFs)were cloned and the gene function was verified.At the same time,proteome study was performed on the flower buds of the three flower bud development stages of ’Lian Tai’,and the molecular mechanism of petaloid stamens of herbaceous peony was preliminary explored from the protein level.The main findings are as follows:(1)The flower bud differentiation process and differentiation morphology of 7 flower types of herbaceous peony were observed.The key time node of stamen primordium differentiation of herbaceous peony was determined,and the differentiation process of stamen primordium of petaloid stamens cultivars were divided into three stages: Stamen primordium,Stamen primordium partly petaloid,Stamen primordium completely petaloid.Observation found that the three forms of double petaled flowers of Paeonia lactiflora(petals increase naturally,stamens petaloid and pistils petaloid).Stamens petaloid is an important form of double flower formation.(2)Transcriptome sequencing was performed on the flower buds of ’Fen Yu Nu’ and ’Lian Tai’ in three differentiation stages.A total of 89,393 Unigenes were identified,of which 40,951 Unigenes were annotated.Trend analysis and Weighted gene co-expression network analysis(WGCNA)were performed on the transcriptome data of ’Fen Yu Nu’ and ’Lian Tai’.A total of seven MADS-box genes Pl AP3,Pl DEFA,Pl PI2,Pl AG,Pl SEP3,Pl SEP1,Pl SEP1-2,and 11 TFs Pl TCP2,Pl TCP4,Pl TCP9,Plb HLH36,Pl ICE1,Pl LBD38,Pl NAC083,Pl BLH11,Pl PDF2,Pl GBF1,Pl IIIA were screened out,which may be closely related to the petaloid stamens process of herbaceous peony.At the same time,the relationship between these genes was analyzed,and the gene regulation network model of the petaloid stamens process of Paeonia lactiflora was speculated.(3)The C class gene Pl AG was isolated from the Paeonia lactiflora ‘Lian Tai’.The CDS is 735 bp in length and encodes 244 amino acids and a termination codon.Pl AG protein shows high homology with AG protein sequences of other species.The ectopic expression of the Pl AG gene in Arabidopsis thaliana can promote the growth of rosette leaves and increase the number of leaves.The stamens of the floral organs of the obtained transgenic Arabidopsis thaliana plants appeared petal-like structure(petaloid stamens);the number of stamens was reduced,at the same time,the petals were missing or the number of stamens were reduced.(4)The E class gene Pl SEP1 was isolated from the Paeonia lactiflora ‘Lian Tai’.The CDS is 732 bp in length and encodes 243 amino acids and a termination codon.Pl SEP1 protein has high homology with SEP1 proteins sequences of other species.There are two conservative motifs SEPI and SEPII at the C-terminal.Overexpression of the Pl SEP1 gene in Arabidopsis thaliana can promote the growth of lateral branch and petiole length increased,and at the same time,the stamens of Arabidopsis thaliana were shortened or the number of stamens decreased.(5)Proteome study were conducted on the flower buds of the three flower bud differentiation stages of Paeonia lactiflora ’Liantai’,and a total of 8,288 proteins were identified.The combined analysis of transcriptome and proteome showed that the number of differentially expressed genes(DEGs)and differentially expressed proteins with the same trend among the L1 vs.L2,L1 vs.L3,and L2 vs.L3 groups were 396,511,and 92,respectively.After analysis,the "polysaccharide metabolism process","carbohydrate metabolism process","starch and sucrose metabolism","fructose and mannose metabolism",and "photosynthesis" metabolic pathways may be involved in the petaloid stamens of Paeonia lactiflora.