Molecular and Biochemical Analysis of Two cDNA Clones Encoding Dihydroflavonol-4-Reductase from Medicago truncatula1
Molecular and Biochemical Analysis of Two cDNA Clones Encoding Dihydroflavonol-4-Reductase from Medicago truncatula1
复制标题
DOI：
10.1104/pp.103.030221
复制
发表时间：
2004-03
期刊：
影响因子：
7.4
通讯作者：
D. Xie;Lisa A. Jackson;J. Cooper;D. Ferreira;N. Paiva
中科院分区：
文献类型：
--
作者：
D. Xie;Lisa A. Jackson;J. Cooper;D. Ferreira;N. Paiva
二氢黄酮醇-4-还原酶（DFR; EC1.1.1.219）催化花青素、缩合单宁（原花青素）和对植物存活和人类营养重要的其它类黄酮的生物合成后期的关键步骤。从模式豆科植物蒺藜苜蓿（Medicago truncatula cv Jemalong）中分离到两个DFR cDNA克隆（MtDFR 1和MtDFR 2）。这两个克隆在大肠杆菌中功能性表达，证实两者都编码活性DFR蛋白，其容易将花旗松素（二氢槲皮素）还原为leucocyanidin。M.蒺藜叶花色素苷被证明是花青素-葡萄糖苷衍生物，并且种皮原花色素是已知的儿茶素和表儿茶素衍生物，所有这些都是从无色花青素生物合成的。尽管氨基酸相似性高（79%相同），重组DFR蛋白表现出不同的pH值和温度曲线和不同的相对底物偏好。虽然在M.在蒺藜中，MtDFR 1容易地还原二氢山奈酚，这与在已知用于确定其它DFR中的底物特异性的位置处存在天冬酰胺残基一致，而MtDFR 2在相同位点处含有天冬氨酸残基，并且仅对二氢山奈酚具有少量活性。这两种重组DFR蛋白都非常有效地减少了5-脱氧二氢黄酮醇底物褐素和二氢胭脂碱，这些物质以前没有报道为M的成分。蒺藜这两个基因的转录积累是最高的年轻的种子和花，在这些组织中的凝聚单宁和leucoanthocyanidins的积累一致。MtDFR 1在发育叶片中的转录水平与叶片花色素苷的积累密切相关。在转基因烟草（烟草）中过表达MtDFR 1导致花中花青素积累的明显增加，而MtDFR 2没有。这些数据揭示了来自单一物种的两种DFR蛋白的意外性质和差异。
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. Two DFR cDNA clones (MtDFR1 and MtDFR2) were isolated from the model legume Medicago truncatula cv Jemalong. Both clones were functionally expressed in Escherichia coli, confirming that both encode active DFR proteins that readily reduce taxifolin (dihydroquercetin) to leucocyanidin. M. truncatula leaf anthocyanins were shown to be cyanidin-glucoside derivatives, and the seed coat proanthocyanidins are known catechin and epicatechin derivatives, all biosynthesized from leucocyanidin. Despite high amino acid similarity (79% identical), the recombinant DFR proteins exhibited differing pH and temperature profiles and differing relative substrate preferences. Although no pelargonidin derivatives were identified in M. truncatula, MtDFR1 readily reduced dihydrokaempferol, consistent with the presence of an asparagine residue at a location known to determine substrate specificity in other DFRs, whereas MtDFR2 contained an aspartate residue at the same site and was only marginally active on dihydrokaempferol. Both recombinant DFR proteins very efficiently reduced 5-deoxydihydroflavonol substrates fustin and dihydrorobinetin, substances not previously reported as constituents of M. truncatula. Transcript accumulation for both genes was highest in young seeds and flowers, consistent with accumulation of condensed tannins and leucoanthocyanidins in these tissues. MtDFR1 transcript levels in developing leaves closely paralleled leaf anthocyanin accumulation. Overexpression of MtDFR1 in transgenic tobacco (Nicotiana tabacum) resulted in visible increases in anthocyanin accumulation in flowers, whereas MtDFR2 did not. The data reveal unexpected properties and differences in two DFR proteins from a single species.