Metabolomics Integrated With Transcriptomics Reveals Biosynthesis Mechanism Of Medicinal Components In Carthamus Tinctorius L.

Carthamus tinctorius L,commonly known as safflower,native to Egypt,India,and the Mediterranean coast,which is one member of the Asteraceae family.There were more than2,000 years since safflower has been introduced and cultivated in China.It is a new economic crop that integrates medicinal materials,dyes,oil and feed.The dried filigree of safflower can be used as traditional Chinese medicine with various effects such as promoting blood circulation,removing blood stasis and relieving pain.The main medicinal component of safflower is its unique flavonoid metabolite — hydroxysafflor yellow A(HSYA)which contributes the most quality and economic value of safflower.exploring the synthetic mechanism of safflower HSYA and identifying candidate genes involved in biosynthesis of flavonoid are important for safflower quality improvement.In this study,25 white strains of safflower(H8),34 red strains(F6),and 35 yellow strains of safflower(I9)which were cultivated in Jiangxia Experimental Field,Wuhan,Hubei,were using for metabolomics integrated with transcriptomics analysis which aims at discovering the metabolic differences of different flower colors of safflower,revealing the mechanism of safflower flower formation,and laying a genetic foundation for the analysis of flavonoids in safflower,especially HSYA synthesis mechanism,providing genetic resources and theoretical basis for the innovation of safflower germplasm resources and the cultivation of high-quality medicinal safflower varieties.The main findings of this study are as follows:1.Metabolomes analysis in safflower with different flower colorsThe principal component analysis of the metabolites showed that there were dramatic metabolic differences in safflower with different flower colors.The most significant difference in metabolism was observed between white flowers and other flowers.The difference between the safflowers of the red flowers and the yellow flowers was relatively small.Orthogonal partial least-squares discrimination analysis(OPLS-DA)was used to identify differential metabolites among three groups.A total of 123 differential metabolites were identified among the three groups,including 9 significantly different metabolites,of that metabolites,Luteolin7-galactoside、Kaempferol 3-alpha-D-galactoside、Luteolin-3’,7-di-O-glucoside、Astragalin were the highest content in the H8;HSYA,glucuronic acid derivatives,gossypol-8-O-glucuronide,quercetin-3,4’-O-di-β-glucopyranoside,these substances are the highest in F6,HSYA was not detected in H8;the isoquercetin was the highest in the I9.K-means cluster analysis of metabolites found that the safflower of different flower varieties has its special metabolites of which content is higher than other flower varieties.Among them,the contents of kaempferol,quercetin,coumarin,and other metabolites were higher only in the white varieties of safflower;isorhamnetin,morning glory pigment,and grass quality are only higher in yellow flowers of safflower variety;HSYA,lignans,rutin,catechins are only found in the red flower of safflower variety has a higher content.Metabolic pathway enrichment analysis found that these metabolites were significantly enriched in flavonoid biosynthetic metabolism and flavonoid and flavonol biosynthetic pathways.Metabolic pathway analysis found that different flower colors of safflower have different metabolism directions of naringenin and eriodicyol.The metabolites of red flowers in white varieties flowed more to dihydrokaempferol and downstream of flavone and flavonol,while the metabolites in red and yellow varieties flowed more to the anthocyanin metabolism pathway downstream of eriodicyol.2.Transcriptome analysis in safflower with different flower colorsAfter trimming low-quality reads and removing the adapter,30.4 GB clean data of all samples were obtained.The alignment rates of each sample that unique matches with the reference genome were higher than 94%.728 differentially expressed genes(DEGs)were identified between H8 and F6,1428 DEGs were identified between H8 and I9,1103 DEGs were identified between F6 and I9,and a total of 2772 differential genes were identified among three groups.The GO and KEGG enrichment analysis found that the top enriched KEGG term were phenylpropanoid biosynthesis and flavonoid biosynthesis.Weighted gene co-expression network analysis(WGCNA)was platformed based on all DEGs,a total of 8 gene modules were identified.By calculating the correlation between gene modules and significant difference metabolites,it was found that green modules and red modules were significantly related to significant differential metabolites.KEGG enrichment analysis of module genes found that genes in the green module were mainly concentrated in metabolic pathways such as flavonoid biosynthesis and metabolism of xenobiotics by cytochrome P450;genes in the red gene module were mainly concentrated in metabolic pathways such as anthocyanin biosynthesis and oxidative phosphorylation.Metabolomics integrated with transcriptomics analysis found that the genes involved in flavonoid metabolism pathway were differentially expressed among different flower colors safflower,which led to different metabolic flows of naringenin and eugenol in different colors flower of safflower.Flow to dihydrokaempferol and downstream flavonoids and flavonoid metabolism pathways in white varieties safflower,while metabolites in red and yellow varieties safflower flow more to anthocyanin metabolism pathway downstream of eugenol,resulting in flavonoids and flavonoids in white safflower filament The higher content of anthocyanins and the lower content of pigment-related products downstream of anthocyanins are the main reasons for the formation of white flowers.Bulked Segregant RNA-seq was platformed on different colors safflower,a total of342,303 mutation sites were found between H8 and F6,and 339,580 mutations were found between H8 and I9.Two candidate intervals that control flower color genes located in,67.15-67.41 M on chromosome 3 and 38.26-40.37 M on chromosome 11 were obtained.The genes in this interval may affect the flower color formation of safflower and HSYA synthesis.