Dihydroflavonol 4-reductase genes encode enzymes with contrasting substrate specificity and show divergent gene expression profiles in Fragaria species.
Dihydroflavonol 4-reductase genes encode enzymes with contrasting substrate specificity and show divergent gene expression profiles in Fragaria species.
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DOI：
10.1371/journal.pone.0112707
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Halbwirth H
中科院分区：
文献类型：
--
作者：
Miosic S;Thill J;Milosevic M;Gosch C;Pober S;Molitor C;Ejaz S;Rompel A;Stich K;Halbwirth H
在果实成熟期间，草莓积累的类黄酮类发生了明显的变化，从未成熟果实中形成的黄烷3-醇和黄酮醇转变为成熟果实中积累的花青素。在普通庭园草莓(Fragaria×Ananassa)中，伴随着羟化模式的明显变化，表现为几乎排他地积累了以天葵花素为基础的色素。在花青素中，花色苷在B环中显示一个(天葵花青素)和两个(花青素)羟基的比例几乎相等。我们从草莓果实中分离到两个二氢黄酮醇4-还原酶(DFR)基因克隆，序列相似性为82%。编码的酶在底物专一性上表现出很高的变异性。一个酶变体不接受DHK(B环上有一个羟基)，而另一个强烈支持DHK作为底物。这似乎是一种具有新的底物特异性的未鉴定的DFR变体。花托和瘦果中均有DFR的表达，DFR2的表达与果实发育密切相关，而DFR1的表达相对稳定。然而，它们的相对表达率存在显著差异。花蕾中DFR1/DFR2的表达比例较高，花蕾中的酶制剂对DHK的转化能力明显高于花蕾中的酶制剂。花青素含量是花青素的两倍多，花青素/天葵花青素的比值仅为0.05，而花青素为0.51。这两种植物果实颜色的差异可以用DFR1在F.×ananassa中的高表达、较高的酶效率(Kcat/Km值)以及F.×ananassa果实发育后期F3‘H活性的丧失来解释。
During fruit ripening, strawberries show distinct changes in the flavonoid classes that accumulate, switching from the formation of flavan 3-ols and flavonols in unripe fruits to the accumulation of anthocyanins in the ripe fruits. In the common garden strawberry (Fragaria×ananassa) this is accompanied by a distinct switch in the pattern of hydroxylation demonstrated by the almost exclusive accumulation of pelargonidin based pigments. In Fragaria vesca the proportion of anthocyanins showing one (pelargonidin) and two (cyanidin) hydroxyl groups within the B-ring is almost equal. We isolated two dihydroflavonol 4-reductase (DFR) cDNA clones from strawberry fruits, which show 82% sequence similarity. The encoded enzymes revealed a high variability in substrate specificity. One enzyme variant did not accept DHK (with one hydroxyl group present in the B-ring), whereas the other strongly preferred DHK as a substrate. This appears to be an uncharacterized DFR variant with novel substrate specificity. Both DFRs were expressed in the receptacle and the achenes of both Fragaria species and the DFR2 expression profile showed a pronounced dependence on fruit development, whereas DFR1 expression remained relatively stable. There were, however, significant differences in their relative rates of expression. The DFR1/DFR2 expression ratio was much higher in the Fragaria×ananassa and enzyme preparations from F.×ananassa receptacles showed higher capability to convert DHK than preparations from F. vesca. Anthocyanin concentrations in the F.×ananassa cultivar were more than twofold higher and the cyanidin:pelargonidin ratio was only 0.05 compared to 0.51 in the F. vesca cultivar. The differences in the fruit colour of the two Fragaria species can be explained by the higher expression of DFR1 in F.×ananassa as compared to F. vesca, a higher enzyme efficiency (K cat/K m values) of DFR1 combined with the loss of F3’H activity late in fruit development of F.×ananassa.
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