Functional analysis of a dihydroflavonol 4-reductase gene in Ophiorrhiza japonica (OjDFR1) reveals its role in the regulation of anthocyanin

Dihydroflavonol 4-reductase (DFR), a key regulatory enzyme, participated in the biosynthesis of anthocyanins, proanthocyanidins and other flavonoids that essential for plant survival and human health. However, the role of this enzyme in Ophiorrhiza japonica is still unknown. Here, three putative DFR-like genes were firstly isolated from O. japonica. Phylogenetic analysis indicated that OjDFR1 was classified into DFR subgroup, while the rest two were clustered into other NADPH-dependent reductases. Then, functions of the three genes were further characterized. Expression analysis showed that OjDFR1 transcripts had strong correlations with the accumulation pattern of anthocyanin during the flower developmental, whereas other two were not, this suggested the potential roles of OjDFR1 in anthocyanin biosynthesis. Subsequently, all three clones were functionally expressed in Escherichia coli, but confirming that only OjDFR1 encode active DFR proteins that catalyzed the reduction of dihydroflavonols to leucoanthocyanidin. Consistant with the biochemical assay results, overexpressing OjDFR1 in Arabidopsis tt3-1 mutant successfully restored the deficiency of anthocyanin and proanthocyanidin, hinting its function as DFR in planta. Additionally, heterologous expression of OjDFR1 in transgenic tobacco contributed to darker flower color via up-regulating the expressions of endogenous NtANS and NtUFGT, which suggested that OjDFR1 was involved in flower color development. In summary, this study validates the functions of OjDFR1 and expands our understanding of anthocyanin biosynthesis in O. japonica.

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Cloning and Functional Characterization of Dihydroflavonol 4-Reductase Gene Involved in Anthocyanin Biosynthesis of Chrysanthemum

International Journal of Molecular Sciences ◽

10.3390/ijms21217960 ◽

2020 ◽

Vol 21 (21) ◽

pp. 7960

Author(s):

Sun-Hyung Lim ◽

Bora Park ◽

Da-Hye Kim ◽

Sangkyu Park ◽

Ju-Hee Yang ◽

...

Keyword(s):

Developmental Stages ◽

Anthocyanin Biosynthesis ◽

Functional Characterization ◽

Flower Color ◽

Chrysanthemum Morifolium ◽

C Terminus ◽

Reductase Gene ◽

The Difference ◽

Chrysanthemum Morifolium Ramat

Dihydroflavonol 4-reductase (DFR) catalyzes a committed step in anthocyanin and proanthocyanidin biosynthesis by reducing dihydroflavonols to leucoanthocyanidins. However, the role of this enzyme in determining flower color in the economically important crop chrysanthemum (Chrysanthemum morifolium Ramat.) is unknown. Here, we isolated cDNAs encoding DFR from two chrysanthemum cultivars, the white-flowered chrysanthemum “OhBlang” (CmDFR-OB) and the red-flowered chrysanthemum “RedMarble” (CmDFR-RM) and identified variations in the C-terminus between the two sequences. An enzyme assay using recombinant proteins revealed that both enzymes catalyzed the reduction of dihydroflavonol substrates, but CmDFR-OB showed significantly reduced DFR activity for dihydrokaempferol (DHK) substrate as compared with CmDFR-RM. Transcript levels of anthocyanin biosynthetic genes were consistent with the anthocyanin contents at different flower developmental stages of both cultivars. The inplanta complementation assay, using Arabidopsis thaliana dfr mutant (tt3-1), revealed that CmDFR-RM, but not CmDFR-OB, transgenes restored defective anthocyanin biosynthesis of this mutant at the seedling stage, as well as proanthocyanidin biosynthesis in the seed. The difference in the flower color of two chrysanthemums can be explained by the C-terminal variation of CmDFR combined with the loss of CmF3H expression during flower development.

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Transcriptomic Analysis of the Anthocyanin Biosynthetic Pathway Reveals the Molecular Mechanism Associated with Purple Color Formation in Dendrobium Nestor

Life ◽

10.3390/life11020113 ◽

2021 ◽

Vol 11 (2) ◽

pp. 113

Author(s):

Xueqiang Cui ◽

Jieling Deng ◽

Changyan Huang ◽

Xuan Tang ◽

Xianmin Li ◽

...

Keyword(s):

Molecular Mechanism ◽

Developmental Stages ◽

Anthocyanin Biosynthesis ◽

Transcriptome Profiling ◽

Flower Color ◽

Flower Bud ◽

Color Formation ◽

Color Development ◽

Purple Coloration ◽

Purple Color

Dendrobium nestor is a famous orchid species in the Orchidaceae family. There is a diversity of flower colorations in the Dendrobium species, but knowledge of the genes involved and molecular mechanism underlying the flower color formation in D. nestor is less studied. Therefore, we performed transcriptome profiling using Illumina sequencing to facilitate thorough studies of the purple color formation in petal samples collected at three developmental stages, namely—flower bud stage (F), half bloom stage (H), and full bloom stage (B) in D. nestor. In addition, we identified key genes and their biosynthetic pathways as well as the transcription factors (TFs) associated with purple flower color formation. We found that the phenylpropanoid–flavonoid–anthocyanin biosynthesis genes such as phenylalanine ammonia lyase, chalcone synthase, anthocyanidin synthase, and UDP-flavonoid glucosyl transferase, were largely up-regulated in the H and B samples as compared to the F samples. This upregulation might partly account for the accumulation of anthocyanins, which confer the purple coloration in these samples. We further identified several differentially expressed genes related to phytohormones such as auxin, ethylene, cytokinins, salicylic acid, brassinosteroid, and abscisic acid, as well as TFs such as MYB and bHLH, which might play important roles in color formation in D. nestor flower. Sturdy upregulation of anthocyanin biosynthetic structural genes might be a potential regulatory mechanism in purple color formation in D. nestor flowers. Several TFs were predicted to regulate the anthocyanin genes through a K-mean clustering analysis. Our study provides valuable resource for future studies to expand our understanding of flower color development mechanisms in D. nestor.

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Comparative Transcriptome Analysis Reveals Effects of Exogenous Hematin on Anthocyanin Biosynthesis during Strawberry Fruit Ripening

International Journal of Genomics ◽

10.1155/2016/6762731 ◽

2016 ◽

Vol 2016 ◽

pp. 1-14 ◽

Cited By ~ 5

Author(s):

Yi Li ◽

Huayin Li ◽

Fengde Wang ◽

Jingjuan Li ◽

Yihui Zhang ◽

...

Keyword(s):

Transcriptome Analysis ◽

Regulatory Networks ◽

Anthocyanin Biosynthesis ◽

Anthocyanin Synthesis ◽

Control Group ◽

Anthocyanin Content ◽

Color Development ◽

Strawberry Fruit Ripening ◽

Positive Effect

Anthocyanin in strawberries has a positive effect on fruit coloration. In this study, the role of exogenous hematin on anthocyanin biosynthesis was investigated. Our result showed that the white stage of strawberries treated with exogenous hematin had higher anthocyanin content, compared to the control group. Among all treatments, 5 μM of hematin was the optimal condition to promote color development. In order to explore the molecular mechanism of fruit coloring regulated by hematin, transcriptomes in the hematin- and non-hematin-treated fruit were analyzed. A large number of differentially expressed genes (DEGs) were identified in regulating anthocyanin synthesis, including the DEGs involved in anthocyanin biosynthesis, hormone signaling transduction, phytochrome signaling, starch and sucrose degradation, and transcriptional pathways. These regulatory networks may play an important role in regulating the color process of strawberries treated with hematin. In summary, exogenous hematin could promote fruit coloring by increasing anthocyanin content in the white stage of strawberries. Furthermore, transcriptome analysis suggests that hematin-promoted fruit coloring occurs through multiple related metabolic pathways, which provides valuable information for regulating fruit color via anthocyanin biosynthesis in strawberries.

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DNA Restriction and Modification in Escherichia coli: Functional Analysis of the Role of the dnaC(D) Gene Product

Microbiology ◽

10.1099/00221287-119-1-231 ◽

1980 ◽

Vol 119 (1) ◽

pp. 231-238

Author(s):

J. HUBACEK ◽

M. WEISEROVA

Keyword(s):

Escherichia Coli ◽

Functional Analysis ◽

Gene Product ◽

Dna Restriction

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Identification and Functional Analysis of a Flavonol Synthase Gene from Grape Hyacinth

Molecules ◽

10.3390/molecules24081579 ◽

2019 ◽

Vol 24 (8) ◽

pp. 1579 ◽

Cited By ~ 1

Author(s):

Hongli Liu ◽

Beibei Su ◽

Han Zhang ◽

Jiaxin Gong ◽

Boxiao Zhang ◽

...

Keyword(s):

Anthocyanin Biosynthesis ◽

Expression Patterns ◽

Flower Color ◽

Pcr Analysis ◽

Anthocyanin Content ◽

In Planta ◽

Total Anthocyanin ◽

Flavonol Synthase ◽

Total Anthocyanin Content ◽

Grape Hyacinth

Flavonols are important copigments that affect flower petal coloration. Flavonol synthase (FLS) catalyzes the conversion of dihydroflavonols to flavonols. In this study, we identified a FLS gene, MaFLS, expressed in petals of the ornamental monocot Muscari aucheri (grape hyacinth) and analyzed its spatial and temporal expression patterns. qRT-PCR analysis showed that MaFLS was predominantly expressed in the early stages of flower development. We next analyzed the in planta functions of MaFLS. Heterologous expression of MaFLS in Nicotiana tabacum (tobacco) resulted in a reduction in pigmentation in the petals, substantially inhibiting the expression of endogenous tobacco genes involved in anthocyanin biosynthesis (i.e., NtDFR, NtANS, and NtAN2) and upregulating the expression of NtFLS. The total anthocyanin content in the petals of the transformed tobacco plants was dramatically reduced, whereas the total flavonol content was increased. Our study suggests that MaFLS plays a key role in flavonol biosynthesis and flower coloration in grape hyacinth. Moreover, MaFLS may represent a new potential gene for molecular breeding of flower color modification and provide a basis for analyzing the effects of copigmentation on flower coloration in grape hyacinth.

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