Dihydroflavonol 4-Reductase Genes from Freesia hybrida Play Important and Partially Overlapping Roles in the Biosynthesis of Flavonoids.
Dihydroflavonol 4-Reductase Genes from Freesia hybrida Play Important and Partially Overlapping Roles in the Biosynthesis of Flavonoids.
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小苍兰二氢黄酮醇 4-还原酶基因在类黄酮生物合成中发挥重要且部分重叠的作用
DOI：
10.3389/fpls.2017.00428
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发表时间：
2017
影响因子：
5.6
通讯作者：
Gao X
中科院分区：
文献类型：
--
作者：
Li Y;Liu X;Cai X;Shan X;Gao R;Yang S;Han T;Wang S;Wang L;Gao X
二氢黄酮醇-4还原酶（DFR）是花青素生物合成和原花青素积累过程中二氢黄酮醇还原为白花青素的关键酶。在许多植物物种中，它是由一个基因家族编码的，然而，不同的拷贝是如何进化的，在不同的组织或不同的时间发挥作用，或专门使用不同但相关的底物，需要进一步研究，特别是在单子叶植物中。本研究首次从小苍兰（Freesia hybrida）中克隆了8个疑似dfr样基因。系统发育分析表明，FhDFR1、FhDFR2和FhDFR3聚在DFR亚组，其余聚在含有肉桂酰辅酶a还原酶（CCR）蛋白的亚组。然后，进一步表征三个FhDFR基因的功能。观察到不同时空的转录模式和水平，表明重复的FhDFR基因可能具有不同的功能。将其引入拟南芥dfr （tt3-1）突变体植株后，观察到花青素衍生物合成损失的部分互补，这表明FhDFRs可以将植物中的二氢槲皮素转化为花青素。生化实验也表明，FhDFR1、FhDFR2和FhDFR3可以利用二氢杨梅素生成白花色素，而FhDFR2也可以催化二氢杨梅素生成白花色素。相反，无论是转基因还是生化分析，均未证明FhDFR蛋白能将二氢山奈酚还原为白蜡精素。这些结果与小苍兰花青素谱一致，其中以飞燕苷衍生物为主，花青素衍生物含量较少，天竺葵苷衍生物未检出。由此可以推断，DFRs的底物特异性是决定杂花草中花青素苷元种类的决定因素。此外，我们还发现FhDFR基因表达模式的差异可能在转录水平上受到控制，因为FhDFR1/FhDFR2和FhDFR3的表达受到具有不同激活效率的潜在MBW调控复合物的控制。综上所述，杂交菜DFR样基因在进化过程中发生了分化，在类黄酮生物合成中发挥了部分重叠的作用，这一结果将有助于被子植物，特别是单子叶植物DFR基因家族的进化研究。
Dihydroflavonol-4-reductase (DFR) is a key enzyme in the reduction of dihydroflavonols to leucoanthocyanidins in both anthocyanin biosynthesis and proanthocyanidin accumulation. In many plant species, it is encoded by a gene family, however, how the different copies evolve either to function in different tissues or at different times or to specialize in the use of different but related substrates needs to be further investigated, especially in monocot plants. In this study, a total of eight putative DFR-like genes were firstly cloned from Freesia hybrida. Phylogenetic analysis showed that they were classified into different branches, and FhDFR1, FhDFR2, and FhDFR3 were clustered into DFR subgroup, whereas others fell into the group with cinnamoyl-CoA reductase (CCR) proteins. Then, the functions of the three FhDFR genes were further characterized. Different spatio-temporal transcription patterns and levels were observed, indicating that the duplicated FhDFR genes might function divergently. After introducing them into Arabidopsis dfr (tt3-1) mutant plants, partial complementation of the loss of cyanidin derivative synthesis was observed, implying that FhDFRs could convert dihydroquercetin to leucocyanidin in planta. Biochemical assays also showed that FhDFR1, FhDFR2, and FhDFR3 could utilize dihydromyricetin to generate leucodelphinidin, while FhDFR2 could also catalyze the formation of leucocyanidin from dihydrocyanidin. On the contrary, neither transgenic nor biochemical analysis demonstrated that FhDFR proteins could reduce dihydrokaempferol to leucopelargonidin. These results were consistent with the freesia flower anthocyanin profiles, among which delphinidin derivatives were predominant, with minor quantities of cyanidin derivatives and undetectable pelargonidin derivatives. Thus, it can be deduced that substrate specificities of DFRs were the determinant for the categories of anthocyanins aglycons accumulated in F. hybrida. Furthermore, we also found that the divergence of the expression patterns for FhDFR genes might be controlled at transcriptional level, as the expression of FhDFR1/FhDFR2 and FhDFR3 was controlled by a potential MBW regulatory complex with different activation efficiencies. Therefore, it can be concluded that the DFR-like genes from F. hybrida have diverged during evolution to play partially overlapping roles in the flavonoid biosynthesis, and the results will contribute to the study of evolution of DFR gene families in angiosperms, especially for monocot plants.