Feng Yang

Chrysanthemum indicum L. capitulum is an enriched source of flavonoids with broad-ranging biological activities, mainly due to their anti-inflammatory, anti-cancer, immune regulation, anti-microbial activity, hepatoprotective, and neuroprotective effects. The O-methylation of various secondary metabolites has previously been demonstrated to be mainly catalyzed by S-adenosyl-L-methionine-dependent O-methyltransferase (OMT) proteins encoded by the OMT gene family. However, limited comprehensive study was published on the OMT gene family, especially the CCoAOMT subfamily, involved in the O-methylation of flavonoids in Chrysanthemum. Here, we analyzed the spatiotemporal expression patterns of C. indicum OMT genes in leaf and flower at different developmental stages. Transcriptome sequencing and qRT-PCR analysis showed that COMTs were mainly highly expressed in capitulum, especially in full bloom, while CCoAOMTs were mainly highly expressed in leaves. Correlation analysis of OMT gene expression and flavonoids accumulation revealed that four OMTs (CHR00029120, CHR00029783, CHR00077404, and CHR00078333) were putatively involved in most methylated flavonoids biosynthesis in the capitulum. Furthermore, we identified a true CCoAOMT enzyme, CiCCoAOMT1, and found that it catalyzed O-methylation of quercetin and luteolin at the 3′-OH position. In summary, this work provides an important theoretical basis for further research on the biological functions of OMTs in C. indicum.

Key message Chrysanthemum morifolium MYB3 factors are transcriptional activators for the regulation of flavonol biosynthesis. Abstract Flavonol was not only the critical secondary metabolite participating in the growth and development of plants but also the main active ingredient in medicinal chrysanthemum. However, few pieces of research revealed the transcriptional regulation of flavonol biosynthesis in Chrysanthemum morifolium. Here, we isolated two CmMYB3 transcription factors (CmMYB3a and CmMYB3b) from the capitulum of Chrysanthemum morifolium cv ‘Hangju’. According to the sequence characteristics, the CmMYB3a and CmMYB3b belonged to the R2R3-MYB subgroup 7, whose members were often reported to regulate flavonol biosynthesis positively. CmMYB3a and CmMYB3b factors were identified to localize in the nucleus by subcellular localization assay. Besides, both of them have obvious transcriptional self-activation activity in their C-terminal. After the overexpression of CmMYB3 genes in Nicotiana benthamiana and Arabidopsis thaliana, the flavonol contents in plants were increased, and the expression of AtCHS, AtCHI, AtF3H, and AtFLS genes in A. thaliana was also improved. Interestingly, the CmMYB3a factor had stronger functions in improving flavonol contents and related gene expression levels than CmMYB3b. The interaction analysis between transcription factors and promoters suggested that CmMYB3 could bind and activate the promoters of CmCHI and CmFLS genes in C. morifolium, and CmMYB3a also functioned more powerfully. Overall, these results indicated that CmMYB3a and CmMYB3b work as transcriptional activators in controlling flavonol biosynthesis.

Chrysanthemum morifolium. cv “Hangju” is an important medicinal material with many functions in China. Flavonoids as the main secondary metabolites are a major class of medicinal components in “Hangju” and its composition and content can change significantly after flooding. This study mimicked the flooding stress of “Hangju” during flower bud differentiation and detected its metabolites in different growth stages. From widely targeted metabolomics data, 661 metabolites were detected, of which 46 differential metabolites exist simultaneously in the different growth stages of “Hangju”. The top three types of the 46 differential metabolites were flavone C-glycosides, flavonol and flavone. Our results demonstrated that the accumulation of flavonoids in different growth stages of “Hangju” was different; however, quercetin, eriodictyol and most of the flavone C-glycosides were significantly enhanced in the two stages after flooding stress. The expression of key enzyme genes in the flavonoid synthesis pathway were determined using RT-qPCR, which verified the consistency of the expression levels of CHI, F3H, DFR and ANS with the content of the corresponding flavonoids. A regulatory network of flavonoid biosynthesis was established to illustrate that flooding stress can change the accumulation of flavonoids by affecting the expression of the corresponding key enzymes in the flavonoid synthesis pathway.