Functional Analysis of Genes GlaDFR1 and GlaDFR2 Encoding Dihydroflavonol 4-Reductase (DFR) in Gentiana lutea L. Var. Aurantiaca (M. Lainz) M. Lainz.
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SUBMITTER: Yu T
PROVIDER: S-EPMC8763498 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Gentiana lutea L. is widely used in bitter beverages and in medicine; Gentianae Radix is the pharmaceutical name of the root of G. lutea. These uses have generated a high demand. The wild populations of Gentiana lutea var. aurantiaca (M. Laínz) M. Laínz have been decimated; it is necessary to establish guidelines for its cultivation. Gentian as most alpine species has dormant seeds. Dormancy can be removed by cold and by means of a gibberellic acid (GA3) treatment. However, cold treatments produce low germination percentages and GA3 treatments may produce off-type seedlings. So, the objective was to determine, for the first time, the presowing treatments that allow high germination rate and good seedling morphology. The best pregerminative doses of GA3 to break seed dormancy were 100, 500, and 1000 ppm, while the best doses to optimize the seedling habit were 50 and 100 ppm. This study provides, for the first time, a 100 ppm GA3 dose that led to a high germination rate and good seedling morphology, as the starting point for gentian regular cultivation.
| S-EPMC4106047 | biostudies-other
Project description:Flower color is an important characteristic that determines the commercial value of ornamental plants. Gentian flowers occur in a limited range of colors because this species is not widely cultivated as a cut flower. Gentiana lutea L. var. aurantiaca (abbr, aurantiaca) is characterized by its orange flowers, but the specific pigments responsible for this coloration are unknown. We therefore investigated the carotenoid and flavonoid composition of petals during flower development in the orange-flowered gentian variety of aurantiaca and the yellow-flowered variety of G. lutea L. var. lutea (abbr, lutea). We observed minor varietal differences in the concentration of carotenoids at the early and final stages, but only aurantiaca petals accumulated pelargonidin glycosides, whereas these compounds were not found in lutea petals. We cloned and sequenced the anthocyanin biosynthetic gene fragments from petals, and analyzed the expression of these genes in the petals of both varieties to determine the molecular mechanisms responsible for the differences in petal color. Comparisons of deduced amino acid sequences encoded by the isolated anthocyanin cDNA fragments indicated that chalcone synthase (CHS), chalcone isomerase (CHI), anthocyanidin synthase 1 (ANS1) and ANS2 are identical in both aurantiaca and lutea varieties whereas minor amino acid differences of the deduced flavonone 3-hydroxylase (F3H) and dihydroflavonol 4-reductase (DFR) between both varieties were observed. The aurantiaca petals expressed substantially higher levels of transcripts representing CHS, F3H, DFR, ANS and UDP-glucose:flavonoid-3-O-glucosyltransferase genes, compared to lutea petals. Pelargonidin glycoside synthesis in aurantiaca petals therefore appears to reflect the higher steady-state levels of pelargonidin synthesis transcripts. Moreover, possible changes in the substrate specificity of DFR enzymes may represent additional mechanisms for producing red pelargonidin glycosides in petals of aurantiaca. Our report describing the exclusive accumulation of pelargonidin glycosides in aurantiaca petals may facilitate the modification of gentian flower color by the production of red anthocyanins.
| S-EPMC5010251 | biostudies-literature
Project description:Accumulation of intracellular sorbitol due to increased aldose reductase (ALR2) activity has been implicated in the development of various secondary complications of diabetes. Thus, ALR2 inhibition could be an effective strategy in the prevention or delay of certain diabetic complications. Gentiana lutea grows naturally in the central and southern areas of Europe. Its roots are commonly consumed as a beverage in some European countries and are also known to have medicinal properties. The water, ethanol, methanol, and ether extracts of the roots of G. lutea were subjected to in vitro bioassay to evaluate their inhibitory activity on the ALR2. While the ether and methanol extracts showed greater inhibitory activities against both rat lens and human ALR2, the water and ethanol extracts showed moderate inhibitory activities. Moreover, the ether and methanol extracts of G. lutea roots significantly and dose-dependently inhibited sorbitol accumulation in human erythrocytes under high glucose conditions. Molecular docking studies with the constituents commonly present in the roots of G. lutea indicate that a secoiridoid glycoside, amarogentin, may be a potential inhibitor of ALR2. This is the first paper that shows G. lutea extracts exhibit inhibitory activity towards ALR2 and these results suggest that Gentiana or its constituents might be useful to prevent or treat diabetic complications.
| S-EPMC3403369 | biostudies-literature
Project description:Corolla color in Gentiana lutea L. exhibits a yellow/orange variation. We previously demonstrated that the orange petal color of G. lutea L. var. aurantiaca is predominantly caused by newly synthesized pelargonidin glycosides that confer a reddish hue to the yellow background color, derived from the carotenoids. However, the anthocyanin molecules of these pelargonidin glycosides are not yet fully identified and characterized. Here, we investigated the regulation, content and type of anthocyanins determining the petal coloration of the orange-flowered G. lutea L. var. aurantiaca. Anthocyanins from the petals of G. lutea L. var. aurantiaca were characterized and quantified by HPLC-ESI-MS/MS (High-performance liquid chromatography-electrospray ionization-tandem mass spectrometry) coupled with a diode array detector in flowers at three different stages of development (S1, S3 and S5). Eleven pelargonidin derivatives were identified in the petals of G. lutea L. var. aurantiaca for the first time, but quantitative and qualitative differences were observed at each developmental stage. The highest levels of these pelargonidin derivatives were reached at the fully open flower stage (S5) where all anthocyanins were detected. In contrast, not all the anthocyanins were detected at the budlet stage (S1) and mature bud stage (S3) and those corresponded to more complex pelargonidin derivatives. The major pelargonidin derivatives found at all the stages were pelargonidin 3-O-glucoside, pelargonidin 3,5-O-diglucoside and pelargonidin 3-O-rutinoside. Furthermore, the expression of DFR (dihydroflavonol 4-reductase), ANS (anthocyanidin synthase), 3GT (UDP-glucose:flavonoid 3-O-glucosyltransferase), 5GT (UDP-glucose:flavonoid 5-O-glucosyltransferase) and 5AT (anthocyanin 5-aromatic acyltransferase) genes was analyzed in the petals of three developmental stages, showing that the expression level of DFR, ANS and 3GT parallels the accumulation of the pelargonidin glucosides. Overall, this study enhances the knowledge of the biochemical basis of flower coloration in Gentiana species, and lays a foundation for breeding of flower color and genetic variation studies on Gentiana varieties.
| S-EPMC6370212 | biostudies-literature
Project description:Dihydroflavonol 4-reductase (DFR, EC 1.1.1.219) is a rate-limited enzyme in the biosynthesis of anthocyanins and condensed tannins (proanthocyanidins) that catalyzes the reduction of dihydroflavonols to leucoanthocyanins. In this study, two full-length transcripts encoding for PtrDFR1 and PtrDFR2 were isolated from Populus trichocarpa. Sequence alignment of the two PtrDFRs with other known DFRs reveals the homology of these genes. The expression profile of PtrDFRs was investigated in various tissues of P. trichocarpa. To determine their functions, two PtrDFRs were overexpressed in tobacco (Nicotiana tabacum) via Agrobacterium-mediated transformation. The associated color change in the flowers was observed in all 35S:PtrDFR1 lines, but not in 35S:PtrDFR2 lines. Compared to the wild-type control, a significantly higher accumulation of anthocyanins was detected in transgenic plants harboring the PtrDFR1. Furthermore, overexpressing PtrDFR1 in Chinese white poplar (P. tomentosa Carr.) resulted in a higher accumulation of both anthocyanins and condensed tannins, whereas constitutively expressing PtrDFR2 only improved condensed tannin accumulation, indicating the potential regulation of condensed tannins by PtrDFR2 in the biosynthetic pathway in poplars.
| S-EPMC3281835 | biostudies-literature
Project description:Dihydroflavonol 4-reductase (DFR) is the key enzyme committed to anthocyanin and proanthocyanidin biosynthesis in the flavonoid biosynthetic pathway. DFR proteins can catalyse mainly the three substrates (dihydrokaempferol, dihydroquercetin, and dihydromyricetin), and show different substrate preferences. Although relationships between the substrate preference and amino acids in the region responsible for substrate specificity have been investigated in several plant species, the molecular basis of the substrate preference of DFR is not yet fully understood.By using degenerate primers in a PCR, we isolated two cDNA clones that encoded DFR in buckwheat (Fagopyrum esculentum). Based on sequence similarity, one cDNA clone (FeDFR1a) was identical to the FeDFR in DNA databases (DDBJ/Gen Bank/EMBL). The other cDNA clone, FeDFR2, had a similar sequence to FeDFR1a, but a different exon-intron structure. Linkage analysis in an F2 segregating population showed that the two loci were linked. Unlike common DFR proteins in other plant species, FeDFR2 contained a valine instead of the typical asparagine at the third position and an extra glycine between sites 6 and 7 in the region that determines substrate specificity, and showed less activity against dihydrokaempferol than did FeDFR1a with an asparagine at the third position. Our 3D model suggested that the third residue and its neighbouring residues contribute to substrate specificity. FeDFR1a was expressed in all organs that we investigated, whereas FeDFR2 was preferentially expressed in roots and seeds.We isolated two buckwheat cDNA clones of DFR genes. FeDFR2 has unique structural and functional features that differ from those of previously reported DFRs in other plants. The 3D model suggested that not only the amino acid at the third position but also its neighbouring residues that are involved in the formation of the substrate-binding pocket play important roles in determining substrate preferences. The unique characteristics of FeDFR2 would provide a useful tool for future studies on the substrate specificity and organ-specific expression of DFRs.
| S-EPMC5725924 | biostudies-literature
Project description: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. Three DFR cDNA clones (designated GbDFRs) were isolated from the gymnosperm Ginkgo biloba. The deduced GbDFR proteins showed high identities to other plant DFRs, which form three distinct DFR families. Southern blot analysis showed that the three GbDFRs each belong to a different DFR family. Phylogenetic tree analysis revealed that the GbDFRs share the same ancestor as other DFRs. The expression of the three recombinant GbDFRs in Escherichia coli showed that their actual protein sizes were in agreement with predictions from the cDNA sequences. The recombinant proteins were purified and their activity was analyzed; both GbDFR1 and GbDFR3 could catalyze dihydroquercetin conversion to leucocyanidin, while GbDFR2 catalyzed dihydrokaempferol conversion to leucopelargonidin. qRT-PCR showed that the GbDFRs were expressed in a tissue-specific manner, and transcript accumulation for the three genes was highest in young leaves and stamens. These transcription patterns were in good agreement with the pattern of anthocyanin accumulation in G.biloba. The expression profiles suggested that GbDFR1 and GbDFR2 are mainly involved in responses to plant hormones, environmental stress and damage. During the annual growth cycle, the GbDFRs were significantly correlated with anthocyanin accumulation in leaves. A fitted linear curve showed the best model for relating GbDFR2 and GbDFR3 with anthocyanin accumulation in leaves. GbDFR1 appears to be involved in environmental stress response, while GbDFR3 likely has primary functions in the synthesis of anthocyanins. These data revealed unexpected properties and differences in three DFR proteins from a single species.
| S-EPMC3753345 | biostudies-literature
Project description:CRISPR/Cas9 technology is a versatile tool for targeted mutagenesis in many organisms, including plants. However, this technique has not been applied to the Japanese morning glory (Ipomoea [Pharbitis] nil), a traditional garden plant chosen for the National BioResource Project in Japan. We selected dihydroflavonol-4-reductase-B (DFR-B) of I. nil, encoding an anthocyanin biosynthesis enzyme, as the target gene, and changes in the stem colour were observed during the early stages of plant tissue culture by Rhizobium [Agrobacterium]-mediated transformation. Twenty-four of the 32 (75%) transgenic plants bore anthocyanin-less white flowers with bi-allelic mutations at the Cas9 cleavage site in DFR-B, exhibiting a single base insertion or deletions of more than two bases. Thus, these results demonstrate that CRISPR/Cas9 technology enables the exploration of gene functions in this model horticultural plant. To our knowledge, this report is the first concerning flower colour changes in higher plants using CRISPR/Cas9 technology.
| S-EPMC5577235 | biostudies-literature
Project description:BACKGROUND: Flower color of soybean is primarily controlled by six genes, viz., W1, W2, W3, W4, Wm and Wp. This study was conducted to investigate the genetic and chemical basis of newly-identified flower color variants including two soybean mutant lines, 222-A-3 (near white flower) and E30-D-1 (light purple flower), a near-isogenic line (Clark-w4), flower color variants (T321 and T369) descended from the w4-mutable line and kw4 (near white flower, Glycine soja). RESULTS: Complementation tests revealed that the flower color of 222-A-3 and kw4 was controlled by the recessive allele (w4) of the W4 locus encoding dihydroflavonol 4-reductase 2 (DFR2). In 222-A-3, a single base was deleted in the first exon resulting in a truncated polypeptide consisting of 24 amino acids. In Clark-w4, base substitution of the first nucleotide of the fourth intron abolished the 5' splice site, resulting in the retention of the intron. The DFR2 gene of kw4 was not expressed. The above results suggest that complete loss-of-function of DFR2 gene leads to near white flowers. Light purple flower of E30-D-1 was controlled by a new allele at the W4 locus, w4-lp. The gene symbol was approved by the Soybean Genetics Committee. In E30-D-1, a single-base substitution changed an amino acid at position 39 from arginine to histidine. Pale flowers of T369 had higher expression levels of the DFR2 gene. These flower petals contained unique dihydroflavonols that have not yet been reported to occur in soybean and G. soja. CONCLUSIONS: Complete loss-of-function of DFR2 gene leads to near white flowers. A new allele of the W4 locus, w4-lp regulates light purple flowers. Single amino acid substitution was associated with light purple flowers. Flower petals of T369 had higher levels of DFR2 gene expression and contained unique dihydroflavonols that are absent in soybean and G. soja. Thus, mutants of the DFR2 gene have unique flavonoid compositions and display a wide variety of flower color patterns in soybean, from near white, light purple, dilute purple to pale.
| S-EPMC4015899 | biostudies-literature
Project description:In Gentiana lutea two varieties are described: G. lutea var. aurantiaca with orange corolla colors and G. lutea var. lutea with yellow corolla colors. Both color varieties co-occur in NW Spain, and pollinators select flower color in this species. It is not known whether a hybridization barrier exists between these G. lutea color varieties. We aim to test the compatibility between flower color varieties in G. lutea and its dependence on pollen vectors. Within a sympatric population containing both flower color morphs, we analyzed differences in reproductive success (number, weight, viability and germinability of seeds) depending on fertilization treatments (autogamy and xenogamy within variety and among varieties). We found a 93% reduction in number of seeds and a 37% reduction in seed weight respectively of autogamy treatments compared to xenogamy crossings. Additionally, reproductive success is higher within color varieties than among varieties, due to a 45% seed viability reduction on hybrids from different varieties. Our results show that G. lutea reproductive success is strongly dependent on pollinators and that a partial hybridization barrier exists between G. lutea varieties.
| S-EPMC4627910 | biostudies-literature