The Research On The Regulation Mechanism Of The Formation Of Pigment In Sterile Flowers Of Hydrangea Macrophylla

Flower color is one of the most important ornamental traits of plants,which has an important influence on the ornamental value of plants.It is also one of the hotspots of long-term research by plant breeders and scientists.Hydrangea macrophylla is loved by people because of its large and gorgeous flowers and changeable colors.It has become one of the flowers with great development potential in the world.The inflorescence of H.macrophylla is composed of infertile flowers and sterile flowers.The color of infertile flowers determines the color of the inflorescence.Previous studies have shown that anthocyanins are the main reason for the color differences formation of infertile flower in H.macrophylla.However,the molecular regulation mechanism of the formation of anthocyanins in sterile flowers of H.macrophylla is still unclear.In this study,white H.macrophylla ’Emilia’ infertile flower in full bloom(EMF3),red H.macrophylla ’Fengdai’ infertile flower in full bloom(HMF3),and blue H.macrophylla ’Forever Summer’ infertile flower in bud stage,color change and full bloom(numbered FSF1,FSF2,and FSF3 in sequence)were studied using targeted metabolomics and transcriptomics.The purpose is to reveal the molecular regulation mechanism of anthocyanin formation in sterile hydrangea flowers of different varieties and different periods from the physiological,biochemical and molecular levels,and to provide references for the cultivation of hydrangea varieties with new colors.The main findings are as follows:(1)162 kinds of flavonoid metabolites were detected from HMF3,EMF3 and FSF3,of which 68 kinds of differential metabolites,including 6 kinds of anthocyanins.Apigeninidin,pelargonidin-3-O-(6"-acetylglucoside)and a small amount of delphinidin-3-O-glucoside were detected in EMF3.Cyanidin 3-(6"-caffeylsophoroside)-5-glucoside and peonidin O-hexoside,apigeninidin were detected in HMF3.In addition to cyanidin detected in FSF3,a large amount of delphinium-3-O-glucoside was also detected.The difference in the type and content of anthocyanins in sterile flowers of H.macrophylla may be the main reason for the difference in flower color.In addition,the high accumulation of dihydroflavonoids and flavonols in FSF3 may have a color-increasing effect on the formation of blue infertile flower.(2)100 kinds of flavonoid metabolites were detected from FSF1.FSF2 and FSF3,of which 44 kinds of differential metabolites.Among the 7 different metabolites of anthocyanins,there are 3 kinds of delphiniums and 4 kinds of cyanidins.The relative content of delphinium-3-O-glucoside in FSF3 is 2.47 times and 369.16 times of FSF2 and FSF1,respectively,which is consistent with the trend of blue sterile flower color formation.The relative content of the remaining six anthocyanin substances all showed a trend of first decreasing and then increasing.Delphirin-3-O-glucoside is the most important factor in the formation of the blue sterile flowers of H.macrophylla ’Forever Summer’.(3)Transcriptome sequencing was performed on FSF1,FSF2,FSF3,EMF3 and HMF3,and a total of 132.23G clean bases were obtained.The number of transcripts and unigenes were 394,287 and 288,026.The assembled lengths were 298,418,344 nt and 269,165,629 nt,and the average lengths were 757 nt and 935 nt,respectively.The transcriptome sequencing and assembly results were good.Through DEGs screening and functional annotation,a large number of DEGs related to flower color synthesis were obtained,which provided basic data for studying the molecular regulation mechanism of anthocyanin formation in infertile flowers of H.macrophylla.All raw data have been saved in the NCBI SRA database under the accession number PRJNA588557.(4)Through the analysis of flower color-related metabolic pathways in HMF3,EMF3 and FSF3,it is found that the anthocyanin biosynthetic pathway is the most important metabolic pathway that regulates the accumulation of anthocyanin substances in FSF3 and HMF3.The flavonoids and flavonols biosynthetic pathway in HMF3 may have a substrate competition mechanism with the anthocyanin biosynthetic pathway.The transcription factors MYB,bHLH and WD40 related to flower color formation were screened through protein-protein interaction network and phylogenetic tree analysis,and their target gene loci were HymCYP73A and HymDFR,respectively.Co-expression analysis showed that there was a very significant correlation between transcription factors and anthocyanins,and their correlation coefficients were all over 0.81.It is speculated that the transcription factors HymMYB308,HymbHLH and HymWDR6 regulate the synthesis of anthocyanins by regulating the expression of the target genes HymCYP73A and HymDFR,and ultimately regulate the flower color formation of infertile flowers in H.macrophylla varieties during their full bloom.(5)Analyzing the DEGs in the flower color-related metabolic pathways of FSF1,FSF2 and FSF3,the up-regulated expression of CHS and CHI promoted the synthesis and accumulation of flavonoids in FSF1,and the up-regulated expression of F3H,C3’5’H,CYP75B1,DFR and ANS in FSF2 and FSF3 promoted the synthesis and accumulation of anthocyanins.The DEGs of the carotenoid biosynthetic pathway showed a downward-regulated expression trend,and it has l ittle contribution to the flower color formation of the infertile flower of H.macrophylla ’Forever Summer’.The transcription factors HymWER-like,HymMYB114 and HymWDR68,which are related to the flower color formation of the blue infertile flowers of H.macrophylla’Forever Summer’,were screened using protein-protein interaction network analysis,and the predicted target gene loci were all HymDFR.The transcription factors HymWER-like.HymMYB114 and HymWDR68 have significant negative correlations with HymF3H,HymDFR,HymANS and Delphidin-3-O-glucoside.It is inferred that the transcription factors HymWER-like,HymMYB114 and HymWDR68 regulate the synthesis and accumulation of delphinidin-3-O-glucoside through negative regulation of the expression of the target gene HymDFR,and ultimately regulate the blue sterile flower formation of H.macrophylla ’Forever Summer’.(6)The ORF sequence of HymWER-like was obtained,which was 598 bp in length and encoded 198 amino acids.It is a typical R2R3 type MYB transcription factor and has high homology with the homologous sequences of Camellia and Chinese Actinidia.The results of qRT-PCR analysis showed that the expression level of HymWER-like in the bud was the highest,and the expression level in the full bloom stage was lower than that in the bud stage,and it was opposite to the trend of blue infertile flower color formation of H.macrophylla ’Forever Summer’.Correlation analysis showed that the relative expression of HymWER-like and the content of anthocyanins and delphinium-3-O-glucoside in sterile blue hydrangea flowers were extremely significantly negatively correlated,and the correlation coefficients were respectively-0.983 and-0.979,and are also very significantly negatively correlated with key genes HymF3H,HymC3’5’H,HymDFR,and HymANS in the anthocyanin biosynthetic pathway,and the correlation coefficients all excwed 0.87.This is basically consistent with the transcriptome data of infertile flowers of blue H.macrophylla ’Forever Summer’.The results of the study not only prove the accuracy of the transcriptome data,but also prove that the transcription factor HymWER-like has a negative regulatory effect on the infertile flower color formation of the blue H.macrophylla ’Forever Summer’.In this paper,a regulatory model of transcription factors regulating the flower color differences formation in infertile flowers of H.macrophylla is constructed,and the mechanism of the flower color differences formation in infertile flowers of H.macrophylla is revealed.This provides theoretical reference and genetic resources for the flower color regulation in infertile flowers of H.macrophylla.