Data_Sheet_1_Tartary buckwheat FtF3′H1 as a metabolic branch switch to increase anthocyanin content in transgenic plant.docx

Tartary buckwheat (TB) is a pseudocereal rich in flavonoids, mainly including flavonols and anthocyanins. The flavonoid 3′-hydroxylase (F3′H) is a key enzyme in flavonoid biosynthesis and is encoded by two copies in TB genome. However, its biological function and effects on flavonol and anthocyanin synthesis in TB have not been well validated yet. In this study, we cloned the full-length FtF3′H1 gene highly expressed in all tissues (compared with FtF3′H2) according to TB flowering transcriptome data. The corresponding FtF3′H1 protein contains 534 amino acids with the molecular properties of the typical plant F3′H and belongs to the CYP75B family. During the flowering stage, the FtF3′H1 expression was highest in flowers, and its expression pattern showed a significant and positive correlation with the total flavonoids (R2 > 0.95). The overexpression of FtF3′H1 in Arabidopsis thaliana, Nicotiana tabacum and TB hairy roots resulted in a significant increase in anthocyanin contents (p < 0.05) but a decrease in rutin (p < 0.05). The average anthocyanin contents were 2.94 mg/g (fresh weight, FW) in A. thaliana (about 135% increase), 1.18 mg/g (FW) in tobacco (about 17% increase), and 1.56 mg/g (FW) TB hairy roots (about 44% increase), and the rutin contents were dropped to about 53.85, 14.99, 46.31%, respectively. However, the expression of genes involved in anthocyanin (DFRs and ANSs) and flavonol (FLSs) synthesis pathways were significantly upregulated (p < 0.05). In particular, the expression level of DFR, a key enzyme that enters the anthocyanin branch, was upregulated thousand-fold in A. thaliana and in N. tabacum. These results might be attributed to FtF3′H1 protein with a higher substrate preference for anthocyanin synthesis substrates. Altogether, we identified the basic biochemical activity of FtF3′H1 in vivo and investigated its involvement in anthocyanin and flavonol metabolism in plant.

苦荞（Tartary buckwheat, 简称TB）是一类富含黄酮类物质的假禾谷作物，其黄酮类成分主要包括黄酮醇与花色苷两类。黄酮类3'-羟化酶（flavonoid 3′-hydroxylase, F3′H）是黄酮类生物合成途径中的关键酶，在TB基因组中由两个拷贝编码。然而，其生物学功能以及对TB中黄酮醇与花色苷合成的调控作用尚未得到充分验证。本研究基于苦荞开花期转录组数据，克隆得到在各组织中表达量均显著高于FtF3′H2的全长FtF3′H1基因。该基因编码的FtF3′H1蛋白包含534个氨基酸，具备典型植物F3′H的分子特性，隶属于CYP75B家族。开花期内，FtF3′H1在花中的表达量最高，其表达模式与总黄酮含量呈显著正相关（决定系数R²>0.95）。在拟南芥（Arabidopsis thaliana）、普通烟草（Nicotiana tabacum）以及苦荞毛状根中过表达FtF3′H1，可使花色苷含量显著上升（p<0.05），但芦丁（rutin）含量显著下降（p<0.05）。转基因拟南芥、普通烟草及苦荞毛状根的平均花色苷含量分别为2.94 mg/g（鲜重，fresh weight, FW）、1.18 mg/g（鲜重，FW）及1.56 mg/g（鲜重，FW），较对照组分别提升约135%、17%与44%；而芦丁含量则分别降至对照组的53.85%、14.99%与46.31%。与此同时，参与花色苷合成途径的二氢黄酮醇4-还原酶（Dihydroflavonol 4-reductase, DFRs）与花青素合酶（Anthocyanidin synthase, ANSs），以及参与黄酮醇合成途径的黄酮醇合酶（Flavonol synthase, FLSs）的编码基因表达量均显著上调（p<0.05）。尤为值得注意的是，作为进入花色苷合成分支的关键酶，DFR在拟南芥与普通烟草中的表达量甚至上调了千倍以上。上述结果可能归因于FtF3′H1蛋白对花色苷合成底物具有更高的底物偏好性。综上，本研究明确了FtF3′H1在体内的基本生化活性，并解析了其在植物体内参与花色苷与黄酮醇代谢的调控机制。