The flower of the safflower (Carthamus tinctorius L.) has been widely used in traditional Chinese medicine for the ability to improve cerebral blood flow. Flavonoids are the primary bioactive components in safflower, and their biosynthesis has attracted widespread interest. Previous studies mostly used second-generation sequencing platforms to survey the putative flavonoid biosynthesis genes. For a better understanding of transcription data and the putative genes involved in flavonoid biosynthesis in safflower, we carry our study. High-quality RNA was extracted from six types of safflower tissue. The RNAs of different tissues were mixed equally and used for multiple size-fractionated libraries (1–2, 2–3 and 3-6 k) library construction. Five cells were carried (2 cells for 1–2 and for 2-3 k libraries and 1 cell for 3-6 k libraries). 10.43Gb clean data and 38,302 de-redundant sequences were captured. 44 unique isoforms were annotated as encoding enzymes involved in flavonoid biosynthesis. The full length flavonoid genes were characterized and their evolutional relationship and expressional pattern were analyzed. They can be divided into eight families, with a large differences in the tissue expression. The temporal expressions under MeJA treatment were also measured, 9 genes are significantly up-regulated and 2 genes are significantly down-regulated. The genes involved in flavonoid synthesis in safflower were predicted in our study. Besides, the SSR and lncRNA are also analyzed in our study. Full-length transcriptome sequences were used in our study. The genes involved in flavonoid synthesis in safflower were predicted in our study. Combined the determination of flavonoids, CtC4H2, CtCHS3, CtCHI3, CtF3H3, CtF3H1 are mainly participated in MeJA promoting the synthesis of flavonoids. Our results also provide a valuable resource for further study on safflower.

Background: Safflower (Carthamus tinctorius L.) is an important cash crop, of which the dried tube flower is not only an important raw material for dyes and cosmetics but also an important herb widely used in traditional Chinese medicine. The pigment and bioactive compounds are composed of flavonoids (mainly quinone chalcones), and studies have reported that MeJA can promote the biosynthesis of quinone chalcones, but the mechanism underlying the effect of MeJA in safflower remains unclear. Here, we attempt to use metabolomics and transcriptome technologies to analyse the molecular mechanism of flavonoid biosynthesis under MeJA treatment in safflower. Results: Based on a UHPLC-ESI-MS/MS detection platform and a self-built database (including hydroxysafflor yellow A, HSYA), a total of 209 flavonoid metabolites were detected, and 35 metabolites were significantly different after treatment with MeJA. Among them, 24 metabolites were upregulated upon MeJA treatment, especially HSYA. Eleven metabolites were downregulated after MeJA treatment. Integrated metabolomics and transcriptome analysis showed that MeJA might upregulate the expression of upstream genes in the flavonoid biosynthesis pathway (such as CHSs, CHIs and HCTs) and downregulate the expression of downstream genes (such as F3Ms, ANRs and ANSs), thus promoting the biosynthesis of quinone chalcones, such as HSYA. The transcription expressions of these genes were validated by real-time PCR. In addition, the promoters of two genes (CtCHI and CtHCT) that were significantly upregulated under MeJA treatment were cloned and analysed. 7 and 3 MeJA response elements were found in the promoters, respectively. Conclusions: MeJA might upregulate the expression of the upstream genes in the flavonoid biosynthesis pathway and downregulate the expression of the downstream genes, thus promoting the biosynthesis of quinone chalcones. Our results provide insights and basic data for the molecular mechanism analysis of flavonoid synthesis in safflower under MeJA treatment.

Safflower is widely used in dying and in traditional medicine, and C-glucosylquinochalcones are the main metabolic species in the red color of safflower. Various safflower cultivars have flowers with different colors. However, the metabolic and transcriptional differences among safflower cultivars with different-colored flowers and the genes participating in C-glucosylquinochalcone biosynthesis are largely unknown. To provide insights on this issue, we performed integrated metabolomics and transcriptome analyses on the flavonoid biosynthesis of flowers of different colors in safflower (white-W, yellow-Y, light red-LR, and deep red-DR). The metabolic analysis showed that flavonoid metabolites showed great differences among the different colors of safflower. More flavonoid metabolic species were detected in Y and W, while C-glucosylquinochalcones were not detected in W. The content of C-glucosylquinochalcones increased with increasing color. Transcriptional analysis showed that most of the annotated flavonoid biosynthesis genes were significantly increased in W. The expression of genes related to flavonoid biosynthesis decreased with increasing color. We analyzed the candidate genes associated with C-glucosylquinochalcones, and an integration of the metabolic and transcriptional analyses indicated that the differential expression of the chalcone synthase (CHS) gene is one of the main reasons for the difference in flavonoid species and content among the different colors of safflower. Combined with the expression pattern analysis, these results indicated that HH_035319, HH_032689, and HH_018025 are likely involved in C-glucosylquinochalcones biosynthesis. In addition, we found that their expression showed greatly increased after the methyl jasmonate (MeJA) treatment. Therefore, HH_035319, HH_032689, and HH_018025 might participate in C-glucosylquinochalcone biosynthesis, which ultimately leads to the red color in safflower.

Hydroxysafflor yellow A (HSYA) is a clinical investigational new drug for the treatment of acute ischemic stroke. It has a unique quinochalcone di-C-glycoside structure and is exclusively found in the flowers of safflower (Carthamus tinctorius). To date, little is known about the biosynthesis of HSYA. In this work, we characterize four key biosynthetic enzymes from C. tinctorius: CtF6H (6-hydroxylation of naringenin to produce carthamidin), CtCHI1 (isomerization between carthamidin and isocarthamidin), CtCGT (flavonoid di-C-glycosyltransferase), and Ct2OGD1 (2-oxoglutarate-dependent dioxygenase). Notably, Ct2OGD1 coordinates with CtCGT to convert carthamidin or isocarthamidin to HSYA. Functions of these genes are confirmed by VIGS (virus-induced gene silencing) in C. tinctorius, de novo biosynthesis of HSYA in Nicotiana benthamiana, semi-synthesis in yeast, and in vitro enzyme assays. We further find that the simultaneous presence and high expression of the above four key genes, together with the absence of F2H (flavanone 2-hydroxylase) genes, are essential for the biosynthesis of HSYA, and thus interpret mechanisms for the unique presence of HSYA in safflower. This work elucidates the biosynthetic pathway of HSYA and provides a foundation for the green and efficient production of this valuable medicinal natural product.

Background: Safflower (Carthamus tinctorius L.), well known for its flower, is widely used as a dye and traditional Chinese medicine. Flavonoids, especially flavonoid glycosides, are the main pigments and active components. However, their biosynthesis is largely unknown. Interestingly, the colour of flowers in safflower changed from yellow to red during flower development, while much of the gene and chemical bases during colour transition are unclear. Methods: In this research, widely targeted metabolomics and transcriptomics were used to elucidate the changes in flavonoid biosynthesis from the gene and chemical points of view in flowers of safflower during colour transition. The screening of differential metabolites depended on fold change and variable importance in project (VIP) value. Differential expressed genes (DEGs) were screened by DESeq2 method. RT-PCR was used to analyse relative expressions of DEGs. Results: A total of 212 flavonoid metabolites, including hydroxysafflor yellow A, carthamin and anthocyanins, were detected and showed a large difference. The candidate genes of glycosyltransferases and flavonoid hydroxylase that might participate in flavonoid glycoside biosynthesis were screened. Ten candidate genes were screened. Through integrated metabolomics and transcriptome analysis, a uridine diphosphate glucose glycosyltransferase gene, CtUGT9 showed a significant correlation with flavonoid glycosides in safflower. In addition, expression analysis showed that CtUGT9 was mainly expressed in the middle development of flowers and was significantly upregulated under MeJA treatment. Our results indicated that CtUGT9 might play an important role in flavonoid glycoside biosynthesis during colour-transition in safflower.

Background: Safflower (Carthamus tinctorius L.) is a domesticated species with a long history of cultivation and widespread distribution across the globe, and light plays an important role in controlling its distribution boundary. Flowers from safflower have been widely used in traditional Chinese medicine because of their ability to improve cerebral blood flow. Flavonoids are the main active compounds in safflower and have many pharmacological effects. In this study, we aimed to explore the relationship between different light intensities and flavonoid biosynthesis in safflower flowers cultivated in greenhouse. Methods: The transcriptome of safflower flowers grown under different light intensities were sequenced through BGISEQ-500 platform. After assembled and filtered, Unigenes were annotated by aligning with seven functional databases. Differential expression analysis of two samples was performed with the DEseq2 package. Differentially expressed genes (DEGs) related with flavonoids biosynthesis were analyzed by Real-time PCR (RT-PCR). Flavonoids accumulation in flowers were determined by high performance liquid chromatography and spectrophotometer. Results: Transcriptome analysis of safflower flowers cultivated under different light intensities was performed. A total of 99.16 Gb data were obtained, and 78,179 Unigenes were annotated. Among the DEGs, 13 genes were related to flavonoid biosynthesis. The differential expressions of seven key genes were confirmed by RT-PCR. In addition, the levels of some flavonoids were measured in safflower flowers grown under different light intensities. CtHCT3 gene expression showed a significantly negative correlation with kaempferol content in safflower grown under different light intensities. Conclusion: Our results strongly suggested that the reduction in light intensity in a suitable range promoted flavonoid biosynthesis in safflower flowers. We suggest that the expressions of HCT genes played an important role in flavonoid accumulation in safflower flowers. Our study lays a foundation for further research on the effects of light on flavonoid biosynthesis in safflower.