Transcriptome mining of key genes involved in seasonal changes of total flavonoids in Eucommia ulmoides leaves

<i>Eucommia ulmoides</i> Oliv. (EU) is a traditional medicinal and edible tree species, and <i>Eucommia ulmoides</i> leaves (EUL) are widely exploited for their economic value owing to their high flavonoid content. In EUL, flavonoids contribute to pharmacological activities relevant to human health and to plant functions, but how flavonoid accumulation is coordinated with gene expression during leaf development remains unclear. In this study, we collected leaves at seven developmental stages from April to October, quantified total flavonoid content and profiled the corresponding transcriptomes by RNA sequencing. Transcriptomic analysis identified 14,827 genes, and differentially expressed genes (DEGs) were identified by pairwise comparisons between developmental stages. Their expression profiles were further analyzed using K-means clustering and weighted gene coexpression network analysis (WGCNA) to identify gene sets whose temporal patterns were closely aligned with changes in total flavonoid content. Among the resulting modules, the red module showed a positive correlation with total flavonoid content. Within this module, two key genes, <i>EU0113959</i> (CCR1D, cinnamoyl-CoA reductase) and <i>EU0125338</i> (PTS1, pterocarpan synthase 1), were most representative, and their expression changes broadly mirrored the seasonal dynamics of total flavonoid content. These two genes were therefore regarded as core candidate genes involved in flavonoid metabolism. Analysis of developmental dynamics revealed that the module eigengene peaked at stage T2 and then gradually decreased, whereas total flavonoid content followed a biphasic pattern, with peaks at T2 and T4 and a modest increase again at later stages. This temporal mismatch between transcriptional activity in the flavonoid-related module and flavonoid accumulation suggests that additional layers of control, such as post-transcriptional regulation, enzyme activity and metabolite transport, are likely to play key roles in shaping flavonoid metabolic profiles in EUL. Overall, this study provides a framework for understanding the developmental regulation of flavonoid biosynthesis in EUL and lays a foundation for optimizing leaf harvest timing and functionally characterizing key regulatory genes.