Economic Forest Germplasm Improvement and Comprehensive Utilization of Resources of Hubei Key Laboratories, Hubei Huanggang, China ; College of Chemistry and Life Science, Huanggang Normal University, Huanggang, China ; College of Forest Resources and Environment, Nanjing Forestry University, Nanjing, China.
PLoS One. 2013 Aug 26;8(8):e72017. doi: 10.1371/journal.pone.0072017. eCollection 2013.
Dihydroflavonol-4-reductase (DFR, EC1.1.1.219) catalyzes a key step late in the biosynthesis of anthocyanins, condensed tannins (proanthocyanidins), and other flavonoids important to plant survival and human nutrition. Three DFR cDNA clones (designated GbDFRs) were isolated from the gymnosperm Ginkgo biloba. The deduced GbDFR proteins showed high identities to other plant DFRs, which form three distinct DFR families. Southern blot analysis showed that the three GbDFRs each belong to a different DFR family. Phylogenetic tree analysis revealed that the GbDFRs share the same ancestor as other DFRs. The expression of the three recombinant GbDFRs in Escherichia coli showed that their actual protein sizes were in agreement with predictions from the cDNA sequences. The recombinant proteins were purified and their activity was analyzed; both GbDFR1 and GbDFR3 could catalyze dihydroquercetin conversion to leucocyanidin, while GbDFR2 catalyzed dihydrokaempferol conversion to leucopelargonidin. qRT-PCR showed that the GbDFRs were expressed in a tissue-specific manner, and transcript accumulation for the three genes was highest in young leaves and stamens. These transcription patterns were in good agreement with the pattern of anthocyanin accumulation in G.biloba. The expression profiles suggested that GbDFR1 and GbDFR2 are mainly involved in responses to plant hormones, environmental stress and damage. During the annual growth cycle, the GbDFRs were significantly correlated with anthocyanin accumulation in leaves. A fitted linear curve showed the best model for relating GbDFR2 and GbDFR3 with anthocyanin accumulation in leaves. GbDFR1 appears to be involved in environmental stress response, while GbDFR3 likely has primary functions in the synthesis of anthocyanins. These data revealed unexpected properties and differences in three DFR proteins from a single species.
二氢黄酮醇 4-还原酶（DFR，EC1.1.1.219）催化花色苷、缩合单宁（原花青素）和其他对植物生存和人类营养重要的类黄酮生物合成后期的一个关键步骤。从裸子植物银杏中分离出三个 DFR cDNA 克隆（命名为 GbDFRs）。推断的 GbDFR 蛋白与其他植物 DFR 具有高度的同一性，这些 DFR 形成三个不同的 DFR 家族。Southern blot 分析表明，这三个 GbDFR 各自属于不同的 DFR 家族。系统发育树分析表明，GbDFRs 与其他 DFR 具有相同的祖先。三个重组 GbDFRs 在大肠杆菌中的表达表明，它们的实际蛋白大小与 cDNA 序列的预测相符。重组蛋白被纯化并分析其活性；GbDFR1 和 GbDFR3 均可催化二氢槲皮素转化为莱菔氰定，而 GbDFR2 可催化二氢山奈酚转化为莱菔素。qRT-PCR 显示 GbDFRs 以组织特异性方式表达，三个基因的转录物在幼叶和雄蕊中积累最高。这些转录模式与银杏中花色苷的积累模式非常吻合。表达谱表明，GbDFR1 和 GbDFR2 主要参与对植物激素、环境胁迫和损伤的反应。在年度生长周期中，GbDFRs 与叶片中花色苷的积累呈显著正相关。拟合线性曲线显示，GbDFR2 和 GbDFR3 与叶片中花色苷积累的关系最好。GbDFR1 似乎参与了环境胁迫反应，而 GbDFR3 可能主要参与了花色苷的合成。这些数据揭示了来自单个物种的三个 DFR 蛋白的意想不到的性质和差异。
