The investigation on metabolites, genes and open chromatins involved in colored leaves ofEucommia ulmoides‘Ziye’

SUMMARY Eucommia ulmoides Oliver ‘Ziye’ has unique purple-red leaves, which contain a variety of flavonoids, so it has high ornamental and medicinal value. However, the categories of flavonoids and molecular mechanism of specific accumulation of flavonoids in ‘Ziye’ leaves is still unclear. Here, differences in metabolic level, gene expression level, chromatin accessibility and cis-regulatory elements were compared between ‘Ziye’ and ‘Huazhong 1’ with green leaf color by metabolome profiling, RNA-seq, and ATAC-seq. A total of 205 flavonoids were identified from these two varieties using ultraperformance liquid chromatography–mass spectrometry (UPLC-MS). The accumulation of most delphinidin, cyaniding, quercetin, myricetin, and isorhamnetin derivatives peaked in old leaves of ‘Ziye’. Single-molecule long-read sequencing indicated that genes in the phenylpropanoid biosynthesis and flavonoid biosynthetic pathway, as well as many transcription factors including MYB, ERF, and WRKY were highly expressed in ‘Ziye’ leaves. ATAC-seq revealed the presence of cell preferentially enriched peaks, which annotated to 6114 genes. Analysis of the genomic regions preferentially accessible in each cell type identified hundreds of overrepresented TF-binding motifs, highlighting sets of TFs such as MYB, ERF, and WRKY that are probably important for color formation of ‘Ziye’ cell. Interestingly, the TFs within each of these cell type-enriched sets also showed evidence of extensively co-regulating each other. Our work demonstrated how chromatin accessibility and TF expression level influenced the expression of flavonoid biosynthesis associated genes, resulted in flavonoids accumulation in ‘Ziye’ leaf. Our results could lay a foundation for further studies of gene expression and functional genomics in E. ulmoides .