Xie De-Yu, Jackson Lisa A, Cooper John D, Ferreira Daneel, Paiva Nancy L
Plant Biology Division, The Samuel Roberts Noble Foundation, Inc., 2510 Sam Noble Parkway, Ardmore, Oklahoma 73402, USA.
Plant Physiol. 2004 Mar;134(3):979-94. doi: 10.1104/pp.103.030221. Epub 2004 Feb 19.
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.
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）以及其他对植物生存和人类营养至关重要的类黄酮生物合成后期的关键步骤。从模式豆科植物蒺藜苜蓿品种Jemalong中分离出两个DFR cDNA克隆（MtDFR1和MtDFR2）。这两个克隆都在大肠杆菌中实现了功能表达，证实它们都编码有活性的DFR蛋白，能够轻易地将紫杉叶素（二氢槲皮素）还原为无色花青素。已证明蒺藜苜蓿叶片中的花青素是花青素 - 葡萄糖苷衍生物，种皮原花青素是已知的儿茶素和表儿茶素衍生物，它们均由无色花青素生物合成而来。尽管这两种重组DFR蛋白的氨基酸相似度很高（79%相同），但它们表现出不同的pH和温度谱以及不同的相对底物偏好性。虽然在蒺藜苜蓿中未鉴定出天竺葵素衍生物，但MtDFR1能轻易还原二氢山奈酚，这与其他DFR中已知决定底物特异性的位置上存在天冬酰胺残基一致，而MtDFR2在同一位置含有天冬氨酸残基，对二氢山奈酚仅有微弱活性。两种重组DFR蛋白都能非常有效地还原5 - 脱氧二氢黄酮醇底物紫铆因和二氢刺槐素，这两种物质以前未被报道为蒺藜苜蓿的成分。这两个基因的转录物积累在幼嫩种子和花朵中最高，这与这些组织中缩合单宁和无色花青素的积累情况一致。发育叶片中MtDFR1的转录水平与叶片花青素积累密切平行。在转基因烟草（烟草）中过表达MtDFR1导致花朵中花青素积累明显增加，而MtDFR2则没有。这些数据揭示了来自同一物种的两种DFR蛋白具有意想不到的特性和差异。
