Transcriptome Mining of Key Genes Involved in Seasonal Changes of Total Flavonoidsin Eucommia ulmoides leaves

Siran WangFei DuanJun Qing^*Yu-e Bai^*

• Inner Mongolia Agricultural University, Hohhot, China

Eucommia ulmoides Oliv. (EU) is a traditional medicinal and edible tree 7 species, and Eucommia ulmoides leaves (EUL) are widely exploited for their economic 8 value owing to their high flavonoid content. In EUL, flavonoids contribute to 9 pharmacological activities relevant to human health and to plant functions, but how 10 flavonoid accumulation is coordinated with gene expression during leaf development 11 remains unclear. In this study, we collected leaves at seven developmental stages from 12 April to October, quantified total flavonoid content and profiled the corresponding 13 transcriptomes by RNA sequencing. Transcriptomic analysis identified 14,827 genes, 14 and differentially expressed genes (DEGs) were identified by pairwise comparisons 15 between developmental stages. Their expression profiles were further analysed using 16 K-means clustering and weighted gene coexpression network analysis (WGCNA) to 17 identify gene sets whose temporal patterns were closely aligned with changes in total 18 flavonoid content. Among the resulting modules, the red module showed a positive 19 correlation with total flavonoid content. Within this module, two key genes, 20 EU0113959 (CCR1D, cinnamoyl-CoA reductase) and EU0125338 (PTS1, pterocarpan 21 synthase 1), were most representative, and their expression changes broadly mirrored 22 the seasonal dynamics of total flavonoid content. These two genes were therefore 23 regarded as core candidate genes involved in flavonoid metabolism. Analysis of 24 developmental dynamics revealed that the module eigengene peaked at stage T2 and 25 then gradually decreased, whereas total flavonoid content followed a biphasic pattern, 26 with peaks at T2 and T4 and a modest increase again at later stages. This temporal 27 mismatch between transcriptional activity in the flavonoid-related module and 50 flavonoid accumulation suggests that additional layers of control, such as post-51 transcriptional regulation, enzyme activity and metabolite transport, are likely to play 52 key roles in shaping flavonoid metabolic profiles in EUL. Overall, this study provides 53 a framework for understanding the developmental regulation of flavonoid biosynthesis 54 in EUL and lays a foundation for optimising leaf harvest timing and functionally 55 characterising key regulatory genes.