Studies On The Molecular Mechanism Of Paeonia Delavayi Flower Color Formation

Tree peony(Paeonia suffruticosa Andrews)is a very famous traditional ornamental plant in China.P.delavayi is a species endemic to Southwest China and has aroused great interest from researchers as a precious genetic resource for flower color breeding.In this study,we investigated the pigments and the regulation mechanism of flavonoid biosynthesis of P.delavayi yellow and purple-red petals by the method of HPLC pigment analysis,transcriptome sequencing and qRT-PCR expression analysis,which will provide a valuable resource for future association studies aimed at better understanding the molecular mechanisms underlying flower pigmentation in tree peonies.The main results are as follows:(1)The analysis of pigments in the petals of yellow-colored and purple-red colored individuals by HPLC showed that in the yellow flower petals,the content of Chalcone 2'G was' the highest,which was the major cause of the yellow color,and color parameters were mainly related to Km3G7 G,Ap7Neo,Is3 G and Chalcone2'G;In the purple-red individuals,Pn3G5 G,Pn3G,Cy3 G were the the main causes for color formation.The total content of yellow petals was rising at the early developmental stage,peaked at stage 3 and then dropped;the total flavone / chalcone content was rising and peaked at stage 4,and then decreased,while the total content of anthocyanin has been rising.(2)We conducted a large-scale transcriptome analysis based on Illumina HiSeq sequencing of cDNA libraries generated from P.delavayi yellow and purple-red petal.A total of 90,202 Unigenes were obtained by de novo assembly,with an average length of 721 nt.Using Blastx,44,811 Unigenes(49.68%)were found to have significant similarity with accessions in NR,NT,and Swiss-Prot databases.We also examined COG,GO and KEGG annotations to better understand the functions of these Unigenes.Further analysis of the two digital transcriptomes revealed that 6,855 Unigenes were differentially expressed between yellow and purple-red flower petals,with 3,430 up-regulated and 3,425 down-regulated.Subsequently,according to the RNA-Seq data and qRT-PCR analysis,we proposed that four up-regulated key structural genes,including F3 H,DFR,ANS and 3GT,might play an important role in the purple-red petal pigmentation;while high co-expression of THC2'GT,CHI and FNSII ensured the accumulation of those pigments contributing to the yellow color.On the other hand,we also found 50 differentially expressed transcript factors might involved in the biosynthesis of flavonoid.(3)The expression stability of twelve candidate reference genes in different tissues and five flower developmental stages of tree peony with six different colors were evaluated by three algorithms: geNorm,NormFinder and Bestkeeper.The results showed that PP2 A and UPL were suggested as the most stable genes across all samples and among different tissues.Helicase and UPL exhibited the highest expression stability in samples with different colors.As for each colored petals at different developmental stages,PP2 A and ACT2 were identified as the most stable genes in white,pink and yellow-green petals,helicase and TUA emerged as the most stably expressed in yellow petals,while UBC and UBQ were most appropriate for normalization in orange and purple-red petals.(4)Based on the analysis of transcriptome of P.delavayi petals,the key structure genes related on the flavonoid biosynthesis were selected from the DEGs between Pl and Pd.The results showed that PlCHS3,PlCHI1,PlDFR1,PlF3H1,PlANS1 were mainly responsible for that of purple-red colored individuals of P.delavayi;PlCHS1,PlCHI2,PlTHC2'GT and PlFNS1 mainly participated in that of the yellow-colored individuals of P.delavayi;Pl FLS1,Pl3GT1 and Pl7GT1 participated in both that of the yellow-colored and purple-red colored individuals of P.delavayi.The flavonoid biosynthesis pathway in P.delavayi was inferred according to the anthocyanin detected and the expression levels of the key structural genes.Yellow and purple-red individuals were involved in two different pathways.(5)Based on the transcriptome database of P.delavayi,PlbHLH3,PlWDR3,PlWDR18 and PlMYB1 were screened out and isolated,and were considered to be related on the regulation of the flavonoid biosynthesis of P.delavayi by regulating the expression of the downstream gene,such as PlDFR1,PlF3H1,Pl3GT1 and PlANS1.(6)10 Pl GSTs were isolated from the petals of yellow-colored individuals of P.delavayi,of which PlGST5 was confirmed as a relevant transporter related to anthocyanidins.In conclusion,this study firstly analyzed the trends of the pigment content of yellow-colored and purple-red colored individuals of P.delavayi during the developmental stages.Meanwhile,based on the correlation analysis of color values and pigments,a large number of genetic information on P.delavayi was obtained using Illumina high-throughput transcriptome sequencing technique,including functional genes and transcription factors related on the flavonoids biosynthesis pathway that have not been developed.The key structural genes,transcription factors and transportation gene were selected and confirmed to be related on the flavonoid biosynthesis pathway of yellow-colored and purple-red colored individuals using qRT-PCR technology.All the results will continue to provide valuable resources and substantial basis for better understanding the molecular mechanisms controlling P.delavayi flower pigmentation,and will eventually accelerate the progress of genetically engineered flower color-modified tree peony.