Function of Anthocyanin and Chlorophyll Metabolic Pathways in the Floral Sepals Color Formation in Different Hydrangea Cultivars

Hydrangea (<i>Hydrangea macrophylla</i>) is distinguished by having sepals instead of real petals, a trait that facilitates color diversity. Floral color is largely predetermined by structural genes linked to anthocyanin production, but the genetic factors determining floral hue in this non-model plant remain unclear. Anthocyanin metabolites, transcriptome, and the CIE<i>L*a*b*</i> hue system were employed to elucidate the biochemical and molecular mechanisms of floral color formation in three hydrangea cultivars: 'DB' (deep blue), 'LB' (light blue), and 'GB' (green blue). UPLC-MS/MS identified 47 metabolites, with delphinidin, cyanidin, malvidin, petunidin, pelargonidin, and peonidin being prominent. Delphinidins were 90% of the primary component in 'DB'. The dataset identifies 51 and 31 DEGs associated with anthocyanin, flavonoid, and chlorophyll biosynthesis, with <i>CHS</i>, <i>CHI</i>, <i>F3H</i>, <i>F3'5'H</i>, <i>DFR</i>, <i>ANS</i>, <i>BZ1</i>, and <i>3AT</i> displaying the highest expression in 'DB'. Notably, <i>DFR</i> (cluster-46471.3) exhibits high expression in 'DB' while being down-regulated in 'LB' and 'GB', correlating with higher anthocyanin levels in floral pigmentation. Comparative analyses of 'LB' vs. 'DB', 'DB' vs. 'GB', and 'LB' vs. 'GB' revealed 460, 490, and 444 differentially expressed TFs, respectively. WRKY, ERF, bHLH, NAC, and AP2/ERF showed the highest expression in 'DB', aligning with the color formation and key anthocyanin biosynthesis-related gene expression. The findings reveal the molecular mechanisms behind floral pigmentation variations and lay the groundwork for future hydrangea breeding programs.