NtFLS1 disruption modulates flavonoid metabolic partitioning and promotes anthocyanin accumulation in tobacco

Flower color in tobacco is largely determined by flavonoid and anthocyanin accumulation. Flavonol synthase (FLS) is a key branch-point enzyme in the flavonoid biosynthetic pathway that competes with dihydroflavonol 4-reductase for common substrates, thereby influencing the biosynthesis of flavonols and anthocyanins. However, the role of NtFLS1 in tobacco flower pigmentation remains unclear. To investigate the function of NtFLS1 , CRISPR/Cas9-mediated mutagenesis was performed in Nicotiana tabacum . Compared with wild-type K326 plants, ntfls1 mutants exhibited markedly darker flowers at the full-bloom stage, with floral pigmentation changing from light pink to deep purple. Transcriptome analysis identified 7,052 differentially expressed genes between mutant and wild-type flowers, with significant enrichment in phenylpropanoid and flavonoid biosynthesis pathways. Expression analysis showed that CHS and CHI were downregulated, whereas COMT and F3’H were upregulated in ntfls1 flowers. Metabolomic analysis further revealed substantial accumulation of ( +)-trans-Taxifolin, Scopoletin, and multiple anthocyanins, including Cyanidin 3-O-glucoside and Pelargonidin-derived compounds. Integrated transcriptomic and metabolomic analyses suggested that NtFLS1 disruption altered flavonoid pathway partitioning and promoted anthocyanin accumulation in tobacco flowers. NtFLS1 is involved in the regulation of flavonoid biosynthesis and flower pigmentation in tobacco. Disruption of NtFLS1 was associated with increased anthocyanin accumulation and enhanced floral pigmentation, likely through redistribution of flavonoid pathway intermediates. These findings provide new insights into the molecular basis of flower color formation in tobacco and may facilitate molecular breeding of ornamental traits in Solanaceous crops.