ClF3'5'H drives blue flower color differentiation in clematis interspecific hybrids

Flavonoid 3',5'-hydroxylase (F3'5'H) is the key enzyme directing metabolic flux toward delphinidin biosynthesis, which is essential for blue flower coloration. However, functional validation of F3'5'H in Clematis-a genus renowned as the "Queen of Climbers"-has not been reported. Here, an interspecific cross between white-flowered Clematis tiantaiensis and blue-purple-flowered C. lanuginosa produced F1 progeny exhibiting color segregation. Two representative offspring-blue-flowered F1-B and blue-purple-flowered F1-PB-were selected for integrated metabolomic and transcriptomic analyses. UPLC-QTOF-MS identified 13 anthocyanins comprising eight delphinidin, four cyanidin, and one petunidin derivatives. At the initial flowering stage, delphinidin derivatives accounted for 71.33% and 54.06% of total anthocyanins as relative percentages of peak area in F1-B and F1-PB, respectively (P < 0.05), whereas cyanidin derivatives showed the inverse pattern. qRT-PCR analysis revealed that ClF3'5'H expression in F1-B was significantly higher than in F1-PB during the critical pigmentation stages (P < 0.01), while ClF3'H showed the opposite trend. Spearman correlation analysis indicated that ClF3'5'H showed the strongest positive association with total anthocyanin content and all delphinidin derivatives (P < 0.0001), consistent with a key role in directing metabolic flux toward delphinidin biosynthesis; however, correlation alone does not imply causation, and functional evidence is provided by the transgenic experiments below. The full-length ClF3'5'H open reading frame (1533 bp, encoding 510 amino acids, CYP75A subfamily) was cloned. Heterologous overexpression in tobacco (Nicotiana tabacum 'NC89') enabled de novo accumulation of delphinidin-3-O-rutinoside, with delphinidin derivatives reaching 17.29% of total anthocyanins. Transgenic tobacco also showed significant upregulation of NtDFR and NtF3'H and downregulation of NtCHS, NtCHI, NtF3H, and NtANS, indicating systemic remodeling of the anthocyanin pathway. These results establish the ClF3'5'H/ClF3'H expression ratio as a molecular determinant of blue-versus-purple divergence in Clematis hybrids and provide key genetic resources for blue flower breeding.