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.

De novo transcriptome assembly of the Chinese pearl barley, adlay, by full-length isoform and short-read RNA sequencing.

Adlay (Coix lacryma-jobi) is a tropical grass that has long been used in traditional Chinese medicine and is known for its nutritional benefits. Recent studies have shown that vitamin E compounds in adlay protect against chronic diseases such as cancer and heart disease. However, the molecular basis of adlay's health benefits remains unknown. Here, we generated adlay gene sets by de novo transcriptome assembly using long-read isoform sequencing (Iso-Seq) and short-read RNA-Sequencing (RNA-Seq). The gene sets obtained from Iso-seq and RNA-seq contained 31,177 genes and 57,901 genes, respectively. We confirmed the validity of the assembled gene sets by experimentally analyzing the levels of prolamin and vitamin E biosynthesis-associated proteins in adlay plant tissues and seeds. We compared the screened adlay genes with known gene families from closely related plant species, such as rice, sorghum and maize. We also identified tissue-specific genes from the adlay leaf, root, and young and mature seed, and experimentally validated the differential expression of 12 randomly-selected genes. Our study of the adlay transcriptome will provide a valuable resource for genetic studies that can enhance adlay breeding programs in the future.

Comparative Analysis of Two Flavonol Synthases from Different-Colored Onions Provides Insight into Flavonoid Biosynthesis.

We isolated cDNAs encoding flavonol synthase (FLS) from the red onion "H6" (AcFLS-H6) and the yellow onion "Hwangryongball" (AcFLS-HRB). We found three amino acid variations between the two sequences. Kinetic analysis with recombinant proteins revealed that AcFLS-HRB exhibited approximately 2-fold higher catalytic efficiencies than AcFLS-H6 for dihydroflavonol substrates and that both proteins preferred dihydroquercetin to dihydrokaempferol. The expression patterns of flavonoid biosynthesis genes corresponded to the accumulation patterns of flavonoid aglycones in both onions. Whereas the other flavonoid biosynthesis genes were weakly expressed in the HRB sheath compared to that of H6, the expression of FLS was similar in both onions. This relatively enhanced FLS expression, along with the higher activity of AcFLS-HRB, could increase the quercetin production in the HRB sheath. The quercetin content was approximately 12-fold higher than the cyanidin content in the H6 sheath, suggesting that FLS has priority in the competition between FLS and dihydroflavonol 4-reductase (DFR) for their substrate dihydroquercetin.