Study On The Molecular Mechanism Of Petal Fading Of Brunfelsia Acuminata And The Technology Of Adjusting Light And Temperature

Brunfelsia acuminata is a perennial evergreen shrub belonging to the genus Brunfelsia in the Solanaceae family.After the petals opened,the flower color changed from blue-purple to pure white due to the degradation of petal anthocyanins resulting in the presence of degrading enzymes in vivo.The molecular mechanism of flower color change is still unclear.In this study,the petals from different periods of Brunfelsia acuminata were used as test materials to explore the molecular mechanism of flower color changes through RNA-sequencing and widely-targeted metabolomics analysis,and clarified the relationship between color changes and environmental factors through light quality and temperature processing.It can not only lay the foundation for further research on color changes,also can provide a data reference for changing the color through facility regulation in practice.The main results of this study were as follows:1 Changes in morphology and physiological and biochemical indexes of Brunfelsia acuminata petals during developmentVarious morphological and physiological and biochemical indexes of the petals were tested,and the results showed that the pigment of the petals mainly distributes on the upper epidermis of the petalsand as the number of days increases,the petal pigment decreases accordingly when the petals open from deep purple to white.The components of anthocyanins detected in the petals by high performance liquid chromatography（HPLC）were malvidin-3-O-glucoside（Mv）,petunidin-3-glucoside（Pt）and delphinidin-3-glucoside（Dp）.Among them,Mv was the main component,which leaded to the blue-purple petals in Brunfelsia acuminata.2 Transcriptome analyses of petals of Brunfelsia acuminataPetals from three periods were used to analysize the transcriptome sequencingby the BGISEQ-500 platform.After assembly and de-redundancy,117,253 Unigenes were obtained and 2,026 Unigenes encoding transcription factors were predicted.Through the analysis of differential genes,the total number of differential genes（DEGs）obtained in the 1d VS 3d comparison was 9,823,of which 4334 were up-regulated and 5489 were down-regulated.The total number of differential genes（DEGs）obtained in the 1d VS 5d comparison was 22855,11299 were up-regulated and 11556 were down-regulated.The total number of differential genes（DEGs）obtained in the 3d VS 5d comparison was16,607,with 8,024 up-regulated and 8,583 down-regulated.Based on the analysis of KEGG pathway enrichment,it showed that the most significant changes of flavone and flavonol biosynthesis（ko00944）were observed.Based on the results of transcriptome analysis,all differential structural genes（DEG）and transcription factors related to flavonoid（ko00941）and anthocyanin biosynthesis（ko00942）were identified.The expression levels of 10 structural genes（CHS,CHI,F3H,F3’H,F3’5’H,DFR,ANS,UGT,GT1,OMT）and two transcription factors（MYB,b HLH）were confirmed by the high reliability of the RNA-seq data obtained at three different stages.Except for F3H,GT1 and F3’H,the expression of other genes were down-regulated.Among them,CHS,F3’5’H,DFR,UGT,OMT and MYB75 were significantly down-regulated,and may lead to the change of petals from purple to white.In addition,the expression levels of 12 related genes in other pathways related to flower color were analyzed by q RT-PCR.The results showed that COMT,C4H,FLS,PAL,4CL,SAMT,C3’H,CCo AOMT,LAR,HCT,POD genes and transcription factor MYB4 were significantly up-regulated in the three different flower color periods.F3’5’H and DFR genes are the key structural genes in the late stages of the anthocyanin biosynthetic pathway.Their expression levels in white petals（5d）were significantly lower than that in purple petals（1d）and MYB may regulate these gene changes as the main transcription factor.Therefore,according to the results of transcriptome sequencing analysis,F3’5’H,DFR gene and MYB transcription factor are candidate genes that cause petal fading.3 Widely-Targeted Metabolomics analysis of petals of Brunfelsia acuminataIn this study,Widely-Targeted Metabolomics were used to detect metabolites in three periods（1d,3d,5d）.A total of 724 metabolites were detected.Among them,there were 644 metabolites at day 1（1d）,710metabolites at day 3（3d）,and 708 metabolites at day 5（5d）.The identified metabolites include 132 flavonoids including 48 flavone,15flavanone,10 anthocyanins,20 flavonoids,32 flavonols and 7 isoflavones,among of which 292 different metabolites were detected between 1d and3d comparison,331 different metabolites between 1d and 5d and 130different metabolites between 3d and 5d.According to functional classification,the types of differential metabolites were mostly flavonoids,Organic acids and derivatives,and Amino acid and derivatives.Based on KEGG function annotation and enrichment analysis,the main pathways with the most differences in metabolites were the flavonoid and phenylpropanoid metabolic pathway.This shows that there is a relationship between petal fading and these two pathways,laying the foundation for the subsequent omics joint analysis.4 Combined analyses of transcriptome and metabolomics of Brunfelsia acuminataIn this study,the combined analysis of differential metabolites and differential genes explored through RNA-sequencing and widely-targeted metabolomics analysis based on the functional annotation and KEGG enrichment.It showed that the pathways for the most differential genes and differential metabolites in the process of flower color change were flavonoid and phenylpropanoid biosynthetic pathway.After screening,the differential metabolites（DEMs）,differential genes and transcription factors related to petal color changes and established differential metabolites and gene network diagrams.In the constructed network diagram,it was found that genes and metabolites in the anthocyanin synthesis pathway were down-regulated,but genes and corresponding differential metabolites in other pathways that shared the same precursor material were up-regulated.Especially in the downstream part of anthocyanin biosynthesis（FLS,LAR genesand were MYB4 significantly up-regulated,and DFR,ANS and MYB75 genes were significantly down-regulated）.They may compete for common substance precursors,causing the expression level of anthocyanin synthase genes to be affected.If inhibited,the synthetic precursor substances flow to other pathways and promote the increase of flavonols and other substances,which causes the color of petals to change from purple to white.Therefore,the results of the comprehensive joint analysis concluded that the initiation genes DFR and ANS in the downstream of anthocyanin synthesis may be the key structural genes for petal fading,and the MYB75 and MYB4transcription factors are the key to the down-regulation of these genes.5 Analysis of the flower color change of Brunfelsia acuminata petals with different light quality and temperature treatmentsIn order to study the effect of light quality on the pigment in the petals of Brunfelsia acuminata,the separated petals and the petals on the plant were used as experimental materials,and the lighting intelligent control system was used to modulate different ratios of red light,blue light,and green light.the ratio were:（white light,W,1:1:1）,（dark processing,An,0:0:0）,（red light,R,10:0:0）,（green light,G,0:10:0）,（blue light,B,0:0:10）,（Y,8:2:0）,（P,7:0:3）,（F,3:0:7）,（L,5:5:0）,respectively.The temperature was maintained at 25℃and the light intensity was 100μmol·m^-2·s^-1for three experiment methods to explore.The results showed that the anthocyanin content gradually decreased with the increase of the treatment time after the exposure of different light quality,whatever it was the petals in vitro or on the plant.In the comparison of light quality,the red light（R）treatment has the highest anthocyanin content and the slowest degradation rate;the blue light（B）treatment has the lowest anthocyanin content and the fastest degradation rate.At the same time,the enzymes and genes of the petals changed correspondingly after light quality treatment.On the whole,the enzyme activities of CHI,DFR and ANS were significantly higher in red light treatment than other treatments.Moreover,the expression levels of CHI,F3’5’H,ANS and AOMT genes were significantly higher than other treatments.Among them,the expression levels of ANS and UGT genes at48h and 96h under red light were higher than other genes.This may be due to the ANS,UGT and other genes were expressed late in the petals open,resulting in the main reason for delaying the red anthocyanin degradation.In addition,the expression of LAR,POD and MYB4 genes suddenly increased after 48h of blue light treatment,and their expression levels were significantly higher than the other two groups.It was speculated that blue light accelerates the degradation of anthocyanins and may be related to them.The three different light qualities of white light,red light and blue light were treated at three temperatures（15℃,15℃,35℃）.The results showed that the lower the temperature,the higher the anthocyanin content.Furthermore,the highest anthocyanin content in red light treatment and the lowest anthocyanin content in blue light treatment.Therefore,it was believed that red light treatment（R）is beneficial to delay the degradation of anthocyanins,and the lower the temperature,the slower the degradation rate.In summary,in this study,the main reason for the fading of petals of Brunfelsia acuminata petals was that the anthocyanin synthesis pathway competes with other pathways.The down-regulated expression of key anthocyanin synthesis genes（DFR,ANS）and transcription factors MYB75and up-regulated expression of MYB4,leads to the flow of precursors for anthocyanin synthesis to volatile substances and other flavonoid pathways（up-regulated expression of genes such as PAL、4CL、C3’H、CCo AOMT、FLS、LAR and the increase of related metabolites）.Therefore,DFR and ANS genes,as the most critical genes in the late stage of anthocyanin biosynthesis,were most likely the target structural genes that directly cause petal fading,and MYB75 and MYB4 were transcription factors that regulate their changes.In addition,the expression of these genes were related to light quality,and red light and low temperature can effectively delay the degradation of anthocyanins and affect the change of flower color.