Dataset Information

Dihydroflavonol 4-Reductase Genes from Freesia hybrida Play Important and Partially Overlapping Roles in the Biosynthesis of Flavonoids.

ABSTRACT: 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.

SUBMITTER: Li Y

PROVIDER: S-EPMC5368250 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Dihydroflavonol 4-Reductase Genes from <i>Freesia hybrida</i> Play Important and Partially Overlapping Roles in the Biosynthesis of Flavonoids.

Li Yueqing Y Liu Xingxue X Cai Xinquan X Shan Xiaotong X Gao Ruifang R Yang Song S Han Taotao T Wang Shucai S Wang Li L Gao Xiang X

Frontiers in plant science 20170328

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 puta ...[more]

PMID: 28400785

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Project description:Starch and sucrose metabolism plays a crucial role in the formation and development of bulbs in bulbous plants. However, these mechanisms remain unclear and unexplored in the corms of Freesia hybrida. Herein, we investigated the dynamics of the major form of carbohydrates and related enzyme activities and profiled the transcriptome of freesia corms at four developmental stages with the aim to reveal the relation between the expression of genes involved in the metabolism of carbohydrates and the accumulation of carbohydrates in corm developmental stages for further exploring the mechanism on the starch and sucrose metabolism regulating the formation and development of corms in F. hybrida. The content of starch, sucrose and soluble sugars followed an overall upward trend across the corm developmental stages. Activities of the adenosine diphosphoglucose pyrophosphorylase, starch branching enzyme and β-amylase generally followed the pattern of the starch and sucrose levels. Activities of sucrose phosphate synthase increased from corm formation till the initial swelling stage and subsequently reached a plateau. Activities of invertase and sucrose synthase peaked at the later rapid swelling stage. These suggested that the starch and sucrose dynamics paralleled corm swelling under the action of metabolic enzymes. A total of 100,999 unigenes were assembled in the transcriptomic analysis, and 44,405 unigenes of them were annotated. Analysis based on Clusters of Orthologous Groups suggested that carbohydrate transport and metabolism (9.34% of the sequences) was prominent across the corm developmental process. In total 3,427 differentially expressed genes (DEGs) were identified and the enrichment analysis detected starch and sucrose metabolism as a critical pathway in corm development, especially at the rapid swelling stage. Further, DEGs encoding key carbohydrate-metabolizing enzymes were identified and correlated to enzyme activities and carbohydrate accumulation. The results construct a valuable resource pool for further molecular-level studies, which are helpful for metabolic regulation of carbohydrates and improvement in F. hybrida.

Project description:Grape hyacinth (Muscari spp.) is a popular ornamental plant with bulbous flowers noted for their rich blue color. Muscari species have been thought to accumulate delphinidin and cyanidin rather than pelargonidin-type anthocyanins because their dihydroflavonol 4-reductase (DFR) does not efficiently reduce dihydrokaempferol. In our study, we clone a novel DFR gene from blue flowers of Muscari. aucheri. Quantitative real-time PCR (qRT-PCR) and anthocyanin analysis showed that the expression pattern of MaDFR had strong correlations with the accumulation of delphinidin, relatively weak correlations with cyanidin, and no correations with pelargonidin. However, in vitro enzymatic analysis revealed that the MaDFR enzyme can reduce all the three types of dihydroflavonols (dihydrokaempferol, dihydroquercetin, and dihydromyricetin), although it most preferred dihydromyricetin as a substrate to produce leucodelphinidin, the precursor of blue-hued delphinidin. This indicated that there may be other functional genes responsible for the loss of red pelargonidin-based pigments in Muscari. To further verify the substrate-specific selection domains of MaDFR, an assay of amino acid substitutions was conducted. The activity of MaDFR was not affected whenever the N135 or E146 site was mutated. However, when both of them were mutated, the catalytic activity of MaDFR was lost completely. The results suggest that both the N135 and E146 sites are essential for the activity of MaDFR. Additionally, the heterologous expression of MaDFR in tobacco (Nicotiana tabacum) resulted in increasing anthocyanin accumulation, leading to a darker flower color, which suggested that MaDFR was involved in color development in flowers. In summary, MaDFR has a high preference for dihydromyricetin, and it could be a powerful candidate gene for genetic engineering for blue flower colour modification. Our results also make a valuable contribution to understanding the basis of color variation in the genus Muscari.

Dataset Information

Dihydroflavonol 4-Reductase Genes from Freesia hybrida Play Important and Partially Overlapping Roles in the Biosynthesis of Flavonoids.

Publications

Dihydroflavonol 4-Reductase Genes from <i>Freesia hybrida</i> Play Important and Partially Overlapping Roles in the Biosynthesis of Flavonoids.

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