Increased Flavonol Levels in Tobacco Expressing AcFLS Affect Flower Color and Root Growth

The onion (Allium cepa L.) flavonol synthase (AcFLS-HRB) gene, encoding an enzyme responsible for flavonol biosynthesis in yellow onion, was recently identified and enzymatically characterized. Here, we performed an in vivo feeding assay involving bacterial expression of AcFLS-HRB and observed that it exhibited both flavanone 3-hydroxylase (F3H) and FLS activity. Transgenic tobacco (Nicotiana tabacum) expressing AcFLS-HRB produced lighter-pink flowers compared to wild-type plants. In transgenic petals, AcFLS-HRB was highly expressed at the mRNA and protein levels, and most AcFLS-HRB protein accumulated in the insoluble microsomal fractions. High-performance liquid chromatography (HPLC) analysis showed that flavonol levels increased but anthocyanin levels decreased in transgenic petals, indicating that AcFLS-HRB is a functional gene in planta. Gene expression analysis showed the reduced transcript levels of general phenylpropanoid biosynthetic genes and flavonoid biosynthetic genes in AcFLS-HRB overexpressed tobacco petals. Additionally, transgenic tobacco plants at the seedling stages showed increased primary root and root hair length and enhanced quercetin signals in roots. Exogenous supplementation with quercetin 3-O-rutinoside (rutin) led to the same phenotypic changes in root growth, suggesting that rutin is the causal compound that promotes root growth in tobacco. Therefore, augmenting flavonol levels affects both flower color and root growth in tobacco.