Multilayer Regulation of Yellow Flower Pigmentation in <i>Meconopsis integrifolia</i> by Competing Enzymes MiFLS2 and MiDFR6

Meconopsis integrifolia is a famous alpine flower widely admired for its striking yellow flowers, yet the molecular mechanisms underlying this unique pigmentation remain poorly understood. Through integrated metabolomic and transcriptomic analyses across three key floral developmental stages, we identified 87 flavonoids, with flavonols constituting the major differential metabolites. Kaempferol 3-β-D-glucopyranoside, quercetin 3-O-sophoroside and quercetin 3-O-galactoside were the predominant flavonols, exhibiting a progressive decrease during petal development. We further cloned two pivotal biosynthetic genes, MiFLS2 and MiDFR6, and western blot analysis and subcellular localisation revealed that both proteins are distributed in the cytoplasm and nucleus. Functional verification in transgenic tobacco revealed that MiFLS2 overexpression increased flavonol accumulation while suppressing anthocyanin biosynthesis, leading to lighter-coloured flowers. In contrast, MiDFR6 overexpression coordinately up-regulated both flavonol and anthocyanin pathways but ultimately promoted redder pigmentation, indicating distinct regulatory roles. Critically, we uncovered a possible multilayer regulatory mechanism: The expression of MiFLS2 is negatively correlated with that of upstream flavonoid genes and MiDFR6, hinting at a possible feedback inhibitory role. Conversely, MiDFR6 overexpression is associated with the coordinated upregulation of multiple structural genes in the flavonoid biosynthesis pathway, implying a putative positive regulatory function. Metabolite analysis confirmed that pelargonidin-3-O-rutinoside, cyanidin-3-O-rutinoside, and kaempferol 3-β-D-glucopyranoside are key contributors to flower colour variation. Ultimately, the high MiFLS2/MiDFR6 expression ratio in M. integrifolia suggests a key factor in controlling the biochemical fate of dihydroflavonols, conferring yellow coloration. Our findings provide novel insights into the competitive and regulatory mechanisms controlling flower colour in alpine plants.