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.

Glutathione S-transferases (GSTs) are members of a protein superfamily with diverse physiological functions, including cellular detoxification and protection against oxidative damage. However, there is limited research on GSTs responding to cadmium (Cd) stress. This study classified 46 GST genes in Dendrobium officinale (D. officinale) into nine groups using model construction and domain annotation. Evolutionary analysis revealed nine subfamilies with diverse physical and chemical properties. Prediction of subcellular localization revealed that half of the GST members were located in the cytoplasm. According to the expression analysis of GST family genes responding to Cd stress, DoGST5 responded significantly to Cd stress. Transient expression of DoGST5-GFP in tobacco leaves revealed that DoGST5 was localized in the cytoplasm. DoGST5 overexpression in Arabidopsis enhanced Cd tolerance by reducing Cd-induced H₂O₂ and O₂⁻ levels. These findings demonstrate that DoGST5 plays a critical role in enhancing Cd tolerance by balancing reactive oxygen species (ROS) levels, offering potential applications for improving plant adaptability to heavy metal stress.

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.