Dihydroflavonol 4-reductase Gene Research Articles

CRISPR/Cas editing of a <i>CPC gene in Arabidopsis thaliana</i>

BACKGROUND: Identification of target genes responsible for visible phenotypic effect may contribute to the development of transgene-free bioengineering strategies and application of crop varieties with edited genome. CAPRICE (CPC) is a single-repeat R3 MYB transcription factor, involved in anthocyanin biosynthesis and trichome formation. It is assumed that CPC controls the expression of Dihydroflavonol-4-reductase (DFR), a key gene of anthocyanin biosynthesis. AIM: The aim of the study was to determine whether knockout of the CPC gene using CRISPR/Cas9 results in visible anthocyanin accumulation. MATERIALS AND METHODS: Three guide RNAs were designed to excise a MYB domain from the CPC gene of Arabidopsis thaliana. Anthocyanin content and expression of CPC and DFR genes were studied in edited plants. RESULTS: The expected 662 bp deletion was detected in 2,7% of glufosinate-resistant plants, however none of the mutations were homozygous. Four edited lines were studied in four generations. An upregulation of the DFR gene was observed in edited lines, however CPC gene expression, anthocyanin content and trichome development were not significantly different from those in control plants. Moreover, in A. thaliana pigmentation did not directly depend on DFR or CPC gene expression. CONCLUSIONS: Our results suggest that CPC gene is involved in regulation of DFR gene expression and anthocyanin biosynthesis pathway, however in case of mutations plants might utilize other transcription factors to maintain homeostasis. Therefore, CPC gene is not a suitable target for CRISPR/Cas studies in Arabidopsis.

Haplotype-resolved chromosomal-level genome assembly reveals regulatory variations in mulberry fruit anthocyanin content.

Understanding the intricate regulatory mechanisms underlying the anthocyanin content (AC) in fruits and vegetables is crucial for advanced biotechnological customization. In this study, we generated high-quality haplotype-resolved genome assemblies for two mulberry cultivars: the high-AC 'Zhongsang5801' (ZS5801) and the low-AC 'Zhenzhubai' (ZZB). Additionally, we conducted a comprehensive analysis of genes associated with AC production. Through genome-wide association studies (GWAS) on 112 mulberry fruits, we identified MaVHAG3, which encodes a vacuolar-type H+-ATPase G3 subunit, as a key gene linked to purple pigmentation. To gain deeper insights into the genetic and molecular processes underlying high AC, we compared the genomes of ZS5801 and ZZB, along with fruit transcriptome data across five developmental stages, and quantified the accumulation of metabolic substances. Compared to ZZB, ZS5801 exhibited significantly more differentially expressed genes (DEGs) related to anthocyanin metabolism and higher levels of anthocyanins and flavonoids. Comparative analyses revealed expansions and contractions in the flavonol synthase (FLS) and dihydroflavonol 4-reductase (DFR) genes, resulting in altered carbon flow. Co-expression analysis demonstrated that ZS5801 displayed more significant alterations in genes involved in late-stage AC regulation compared to ZZB, particularly during the phase stage. In summary, our findings provide valuable insights into the regulation of mulberry fruit AC, offering genetic resources to enhance cultivars with higher AC traits.

Integrative Metabolomic and Transcriptomic Analyses Reveal the Mechanism of Petal Blotch Formation in Rosa persica.

Petal blotch is a specific flower color pattern commonly found in angiosperm families. In particular, Rosa persica is characterized by dark red blotches at the base of yellow petals. Modern rose cultivars with blotches inherited the blotch trait from R. persica. Therefore, understanding the mechanism for blotch formation is crucial for breeding rose cultivars with various color patterns. In this study, the metabolites and genes responsible for the blotch formation in R. persica were identified for the first time through metabolomic and transcriptomic analyses using LC-MS/MS and RNA-seq. A total of 157 flavonoids were identified, with 7 anthocyanins as the major flavonoids, namely, cyanidin 3-O-(6″-O-malonyl) glucoside 5-O-glucoside, cyanidin-3-O-glucoside, cyanidin 3-O-galactoside, cyanidin O-rutinoside-O-malonylglucoside, pelargonidin 3-O-glucoside, pelargonidin 3,5-O-diglucoside, and peonidin O-rutinoside-O-malonylglucoside, contributing to pigmentation and color darkening in the blotch parts of R. persica, whereas carotenoids predominantly influenced the color formation of non-blotch parts. Zeaxanthin and antheraxanthin mainly contributed to the yellow color formation of petals at the semi-open and full bloom stages. The expression levels of two 4-coumarate: CoA ligase genes (Rbe014123 and Rbe028518), the dihydroflavonol 4-reductase gene (Rbe013916), the anthocyanidin synthase gene (Rbe016466), and UDP-flavonoid glucosyltransferase gene (Rbe026328) indicated that they might be the key structural genes affecting the formation and color of petal blotch. Correlation analysis combined with weighted gene co-expression network analysis (WGCNA) further characterized 10 transcription factors (TFs). These TFs might participate in the regulation of anthocyanin accumulation in the blotch parts of petals by modulating one or more structural genes. Our results elucidate the compounds and molecular mechanisms underlying petal blotch formation in R. persica and provide valuable candidate genes for the future genetic improvement of rose cultivars with novel flower color patterns.

Biochemical, Molecular Characterization, Antioxidant, And Cytotoxicity Of Dicliptera Bupleuroides With Potential Drug Discovery: In Vitro And In Silico Analysis

AbstractDicliptera is a genus belonging to family Acanthaceae. Dicliptera bupleuroides is a small perennial herb commonly known as roxburg's foldwing. It is locally used in folkloric medicines for the treatment of different diseases and as a general tonic for wound healing. The current investigational study was to optimize solvent extraction protocol, to identify quantitative and qualitative biochemical composition, antioxidant and cytotoxic activities of D. bupleuroides and to characterize the gene associated with biosynthesis of flavonol. The plant of D. bupleuroides was collected from district Abbottabad and separated into its four parts including leaf, stem, flower, and root. To extract the biochemical compounds, five different solvents were used such as methanol, acidified methanol, 80 % methanol, ethyl acetate, and n‐butanol. Total phenolic, flavonoids, flavonol, flavanol (catechin), and anthocyanin contents were determined and antioxidant potential of the extracts was evaluated using various antioxidant assays. The methanolic extracts of D. bupleuroides showed promising antioxidant potential towards DPPH, ABTS, H2O2, ferric, phosphomolybdate and hydroxyl radical scavenging capabilities.The extracts obtained from the solvents mentioned above were tested for cytotoxicity using the 3T3 cell line by using mouse fibroblast cells. The results indicated that none of the extracts demonstrated any toxic effects on the cells, suggesting that they are non‐toxic. The RNA was extracted and cDNA was synthesized using reverse transcriptase enzyme. Degenerate primers for dihydroflavonol reductase (DFR) gene were synthesized to amplify the desired product. The high quality of cDNA amplification showed the presence of the DFR gene in the Dicliptera bupleuroides leaf for the first time with a product size of 419 bp respectively. The DFR gene isolated from Dicliptera bupleuroides is involved in flavonol biosynthesis regulation. In addition, Glide molecular docking results proposed that the constituents within the n‐hexanic extract of Dicliptera bupleuroides not only furnish an updated approach to better understand the antioxidant potential of its various constituents to interact with an antioxidant target protein (PDB ID:3MNG) but also ensure promise in addressing these potential therapeutic modalities. The active compounds of D. bupleuroides could be suitable for pharmaceutical industries to develop stress related drugs.

The Characterization of R2R3-MYB Genes in Ammopiptanthus nanus Uncovers That the miR858-AnaMYB87 Module Mediates the Accumulation of Anthocyanin under Osmotic Stress.

R2R3-MYB transcription factors (TFs) participate in the modulation of plant development, secondary metabolism, and responses to environmental stresses. Ammopiptanthus nanus, a leguminous dryland shrub, tolerates a high degree of environmental stress, including drought and low-temperature stress. The systematic identification, structural analysis, evolutionary analysis, and gene profiling of R2R3-MYB TFs under cold and osmotic stress in A. nanus were performed. Up to 137 R2R3-MYB TFs were identified and clustered into nine clades, with most A. nanus R2R3-MYB members belonging to clade VIII. Tandem and segmental duplication events drove the expansion of the A. nanus R2R3-MYB family. Expression profiling revealed that multiple R2R3-MYB genes significantly changed under osmotic and cold stress conditions. MiR858 and miR159 targeted 88 R2R3-MYB genes. AnaMYB87, an miR858-targeted clade VIII R2R3-MYB TF, was up-regulated under both osmotic and cold stress. A transient expression assay in apples showed that the overexpression of AnaMYB87 promoted anthocyanin accumulation. A luciferase reporter assay in tobacco demonstrated that AnaMYB87 positively affected the transactivation of the dihydroflavonol reductase gene, indicating that the miR858-MYB87 module mediates anthocyanin accumulation under osmotic stress by regulating the dihydroflavonol reductase gene in A. nanus. This study provides new data to understand the roles of R2R3-MYB in plant stress responses.

Molecular evolution and expression assessment of DFRs in apple

BackgroundAnthocyanins are the secondary metabolites of flavonoids in plants. As a key enzyme in the biosynthetic pathway of anthocyanin, dihydroflavonol 4-reductase (DFR) act as an important regulatory point, but DFR family genes has not been systematically characterized in apple (Malus domestica Borkh.).MethodsThe members of DFR genes in apple were identified and their gene structure, chromosome distribution, evolutionary relationships, collinearity, cis-component and protein interaction relationships were predicted using bioinformatics methods. The expression patterns of MdDFRs in various organs, such as leaves, fruit flushes, fruits, ripe fruit peels, flowers and stems were analyzed using GeneChip expression array analysis. qRT-PCR was employed to analyze the expression levels of MdDFRs in different apple varieties with varying levels of fruit skin at maturity.ResultsThe apple database revealed 96 DFR genes, which are distributed on 17 chromosomes and can be divided into 3 subfamilies. These 96 DFR genes were mostly composed of α-helix and random coil according to secondary structure prediction, and were mainly expressed in chloroplasts and cytoplasm. MYB binding site involved in flavonoid biosynthetic genes regulation element (MBSI) was identified in the promoter of MdDFR15/76/81/89/90/91/93/94. Lignin/flavonoid synthesis-related elements of MYB recognition site and MYB-binding site were identified in the promoters of MdDFR05/09/13/19/22/24/26/30/31/33/34/46/50/52/54/64/65/69/75/76/79/86. The internal collinearity analysis of the apple MdDFR genome revealed a total of 34 pairs of duplicated gene pairs. Interspecific collinearity analysis showed that there were 66 and 57 homologous gene pairs in apple/tomato and apple/grape, respectively. GeneChip expression array analysis showed that MdDFR72 and MdDFR96 were higher expressed in ripe fruit fleshes and peel, MdDFR01/06/67/49/54/91 were higher expressed in flowers, MdDFR64 was higher expressed in ripe fruit peels and flowers than those of other tissues. Besides, 75 MdDFR proteins interacted directly or indirectly with anthocyanidin synthesis related proteins MdANS, MdF3H, MdMYB1, MdMYBPA1 to form a protein interaction network. Interestingly, MdDFR69 and MdDFR87 had direct interactions with these four proteins, MdDFR64 had direct interactions with MdANS and MdF3H. qRT-PCR analysis showed that the expression levels of MdDFR01/05/31/53/64/69/73/84/87/94/96 were up-regulated with the accumulation of anthocyanins.ConclusionsThis study lays a foundation for further research on the function of DFR genes in apple.Graphical

Genome-wide identification of putative dihydroflavonol 4-reductase (DFR) gene family in eight Solanaceae species and expression analysis in Solanum lycopersicum

Dihydroflavonol 4-reductase (DFR; EC1.1.1.219) is an important rate-limiting enzyme in the plant flavonoid pathway toward both anthocyanins and proanthocyanidins. Although DFR genes have been isolated from multiple plants and their functions have been well characterized in some plants, little is known about DFRs in Solanaceae species. Therefore, in this study, we performed genome-wide analysis and identified 6, 5, 4, 5, 5, 6, 6 and 5 DFR gene family members in eight Solanaceae species (S. lycopersicum, S. pennellii, S. tuberosum, S. melongena, C. annuum, N. tabacum, P. inflata, and P. axillaris) respectively. The putative DFR genes were systematically identified using bioinformatics to predict their protein properties, cellular location, phylogenetic relationships, gene structure, conserved motifs, and cis-acting elements in the promoters. Furthermore, quantitative real-time PCR (qRT-PCR) was used to identify the expression pattern of DFRs in tomato. We classified all DFRs into five groups based on their phylogenetic features. Sequence analysis showed that all encoded DFR protein sequences possess a highly conserved NAD-dependent epimerase/dehydratase. In addition, almost all the members of each group displayed similar gene structures and motif distributions, which might be related to their identical executive functions. All 42 DFRs possess a series of light-responsive, phytohormone-responsive, MYB-responsive, stress-responsive, and tissue-specific expression-related cis-elements in the promoter sequences. qRT-PCR analysis showed that tomato DFRs were expressed in many different organs. This study will provide a theoretical basis for further investigation of the function of DFRs in Solanaceae.

Production of yellow-flowered gentian plants by genetic engineering of betaxanthin pigments.

Genetic engineering of flower color provides biotechnological products such as blue carnations or roses by accumulating delphinidin-based anthocyanins not naturally existing in these plant species. Betalains are another class of pigments that in plants are only synthesized in the order Caryophyllales. Although they have been engineered in several plant species, especially red-violet betacyanins, the yellow betaxanthins have yet to be engineered in ornamental plants. We attempted to produce yellow-flowered gentians by genetic engineering of betaxanthin pigments. First, white-flowered gentian lines were produced by knocking out the dihydroflavonol 4-reductase (DFR) gene using CRISPR/Cas9-mediated genome editing. Beta vulgaris BvCYP76AD6 and Mirabilis jalapa MjDOD, driven by gentian petal-specific promoters, flavonoid 3',5'-hydroxylase (F3'5'H) and anthocyanin 5,3'-aromatic acyltransferase (AT), respectively, were transformed into the above DFR-knockout white-flowered line; the resultant gentian plants had vivid yellow flowers. Expression analysis and pigment analysis revealed petal-specific expression and accumulation of seven known betaxanthins in their petals to c. 0.06-0.08 μmol g FW-1 . Genetic engineering of vivid yellow-flowered plants can be achieved by combining genome editing and a suitable expression of betaxanthin-biosynthetic genes in ornamental plants.

Transcriptomic analysis reveals the mechanism underlying the anthocyanin changes in Fragaria nilgerrensis Schlecht. and its interspecific hybrids

BackgroundFragaria nilgerrensis (FN) provides a rich source of genetic variations for strawberry germplasm innovation. The color of strawberry fruits is a key factor affecting consumer preferences. However, the genetic basis of the fruit color formation in F. nilgerrensis and its interspecific hybrids has rarely been researched.ResultsIn this study, the fruit transcriptomes and flavonoid contents of FN (white skin; control) and its interspecific hybrids BF1 and BF2 (pale red skin) were compared. A total of 31 flavonoids were identified. Notably, two pelargonidin derivatives (pelargonidin-3-O-glucoside and pelargonidin-3-O-rutinoside) were revealed as potential key pigments for the coloration of BF1 and BF2 fruits. Additionally, dihydroflavonol 4-reductase (DFR) (LOC101293459 and LOC101293749) and anthocyanidin 3-O-glucosyltransferase (BZ1) (LOC101300000), which are crucial structural genes in the anthocyanidin biosynthetic pathway, had significantly up-regulated expression levels in the two FN interspecific hybrids. Moreover, most of the genes encoding transcription factors (e.g., MYB, WRKY, TCP, bHLH, AP2, and WD40) related to anthocyanin accumulation were differentially expressed. We also identified two DFR genes (LOC101293749 and LOC101293459) that were significantly correlated with members in bHLH, MYB, WD40, AP2, and bZIP families. Two chalcone synthase (CHS) (LOC101298162 and LOC101298456) and a BZ1 gene (LOC101300000) were highly correlated with members in bHLH, WD40 and AP2 families.ConclusionsPelargonidin-3-O-glucoside and pelargonidin-3-O-rutinoside may be the key pigments contributing to the formation of pale red fruit skin. DFR and BZ1 structural genes and some bHLH, MYB, WD40, AP2, and bZIP TF family members enhance the accumulation of two pelargonidin derivatives. This study provides important insights into the regulation of anthocyanidin biosynthesis in FN and its interspecific hybrids. The presented data may be relevant for improving strawberry fruit coloration via genetic engineering.

Identification and Expression Analysis of DFR Gene Family in Brassica napus L.

Dihydroflavonol 4-reductase (DFR) is a key enzyme in the flavonoid biosynthetic pathway and is essential for the formation of plants' color. In this study, 26 BnDFR genes were identified using 6 Arabidopsis DFR genes as reference. The physicochemical properties, subcellular localization, and conserved structure of BnDFR proteins were analyzed; the evolutionary relationship, collinearity analysis, and expression characteristics of BnDFR genes were studied; and the correlation between the expression level of BnDFR genes and anthocyanin content in rape petals were analyzed. The results showed that the 26 BnDFRs were located in chloroplasts, cytoplasm, nuclei, and mitochondria, distributed on 17 chromosomes, and divided into 4 groups; members of the same group have a similar function, which may be related to the environmental response elements and plant hormone response elements. Intraspecific collinearity analysis showed 51 pairs of collinear genes, and interspecific collinearity analysis showed 30 pairs of collinear genes. Analysis of the expression levels of BnDFRs and anthocyanin content in different color rape petals showed that BnDFR6 and BnDFR26 might play an important role in the synthesis of anthocyanins in rape petals. This provides theoretical guidance for further analysis of the anthocyanin anabolism mechanism involved in the DFR gene in Brassica napus.

Overexpression of the Wheat TaPsb28 Gene Enhances Drought Tolerance in Transgenic Arabidopsis

Psb28 is a soluble protein in the photosystem II (PSII) complex, but its role in the drought stress response of wheat remains unclear. Here, we functionally characterized the TaPsb28 gene, which positively regulates drought tolerance in wheat. When the full-length 546-bp TaPsb28 cDNA was transferred into Arabidopsis thaliana, it was located in the guard cell chloroplast around the stroma. Overexpression of TaPsb28 conferred drought tolerance, as exhibited by the increases in the survival rate. Transgenic plants maintained lower MDA content and higher chlorophyll content by inducing chlorophyll synthase (ChlG) gene transcription. The content of abscisic acid (ABA) and zeatin increased significantly in wild-type (WT) plants under drought stress, and the transcriptional expression levels of RD22, dihydroflavonol 4-reductase (DFR) and anthocyanin reductase (ANR) genes were induced, thus enhancing the contents of endogenous cyanidin, delphinidin, and proanthocyanidins. However, in transgenic plants, although anthocyanins were further aggregated, the ABA increase was inhibited, zeatin was restored to the control level under drought stress, and stomatal closure was promoted. These findings indicate ABA and zeatin have opposite synergistic effects in the process of drought tolerance caused by TaPsb28 because only after the effect of zeatin is alleviated can ABA better play its role in promoting anthocyanin accumulation and stomatal closure, thus enhancing the drought tolerance of transgenic plants. The results suggest that overexpression of TaPsb28 exerts a positive role in the drought response by influencing the functional metabolism of endogenous hormones. The understanding acquired through the research laid a foundation for further in-depth investigation of the function of TaPsb28 in drought resistance in wheat, especially its relationship with anthocyanidin accumulation.

Competition between FLS and DFR regulates the distribution of flavonols and proanthocyanidins in Rubus chingii Hu.

Rubus chingii Hu is a berry plant of the genus Rubus of the Rosaceae family, which has high nutritional and medicinal value and is rich in flavonoids. Flavonol synthase (FLS) and dihydroflavonol 4-reductase (DFR) compete for the common substrate dihydroflavonols to regulate the metabolic flux of flavonoids. However, the competition between FLS and DFR based on enzyme is rarely reported. Here, we isolated and identified two FLS genes (RcFLS1 and RcFLS2) and one DFR gene (RcDFR) from Rubus chingii Hu. RcFLSs and RcDFR were highly expressed in stems, leaves, and flowers, although the flavonol accumulation in these organs was significantly higher than that of proanthocyanidins (PAs). The recombinant RcFLSs demonstrated bifunctional activities via hydroxylation and desaturation at the C-3α position having a lower Michaelis constant (Km) for dihydroflavonols than RcDFR. We also found that a low concentration of flavonols could significantly inhibit RcDFR activity. To investigate the competitive relationship between RcFLSs and RcDFR, we used a prokaryotic expression system (E. coli) to co-express these proteins. The transgenic cells expressing recombinant proteins were incubated with substrates, and the reaction products were analyzed. Furthermore, two transient expression systems (tobacco leaves and strawberry fruits) and a stable genetic system (Arabidopsis thaliana) were used to co-express these proteins in vivo. The results showed that RcFLS1 was dominant in the competition with RcDFR. Our results demonstrated that the competition between FLS and DFR regulated the metabolic flux distribution of flavonols and PAs, which will be of great significance for the molecular breeding of Rubus plants.

Low-light intensity reprogramed flower pigmentation in Dendrobium Sonia via downregulation of dihydroflavonol 4-reductase and anthocyanidin synthase genes

Flower color is an important trait of orchids that extensively influences consumer-purchasing decisions, and thus manipulation of flower color variation is a way for orchid production to provide more options to consumers. The application of net shading is one of the simplest methods for transformation of an original orchid flower color. In this study, we report the first color modification of the commercial Dendrobium Sonia (Ds) flower through a simple light intensity adjustment by the application of net shading. A reduction of light intensity significantly decreased anthocyanin production in all stages of flower development, leading to a paler purple. The expression profiles of six flower-color associated genes, including chalcone synthase, DsCHS; chalcone isomerase, DsCHI; flavanone 3-hydroxylase, DsF3H; dihydroflavonol 4-reductase, DsDFR; anthocyanidin synthase, DsANS; and flavonol synthase, DsFLS in three flower stages (very young bud, mature bud, and fully open flower) developing under typical-light (240 μmol photons m−2 s−1) and low-light (45 μmol photons m−2 s−1) intensities were investigated. The lighter flower color produced under the low-light treatment correlated with downregulation of DsDFR and DsANS expression, while the early anthocyanin biosynthetic genes, including DsCHS, DsCHI, DsF3H, and DsFLS, were not significantly affected. This indicates that the expression of DsDFR and DsANS in D. Sonia is light dependent, while the expression of DsCHS, DsCHI, DsF3H, and DsFLS is light independent. Therefore, this low-light transformed flower color is not genetically modified but is economically and socially acceptable, and also nature- and environment-friendly. This finding will be useful for future applications with other Dendrobium species to produce flower color shade variation.

Overexpression of the Ginkgo biloba dihydroflavonol 4-reductase gene GbDFR6 results in the self-incompatibility-like phenotypes in transgenic tobacco

ABSTRACT Although flavonoids play multiple roles in plant growth and development, the involvement in plant self-incompatibility (SI) have not been reported. In this research, the fertility of transgenic tobacco plants overexpressing the Ginkgo biloba dihydroflavonol 4-reductase gene, GbDFR6, were investigated. To explore the possible physiological defects leading to the failure of embryo development in transgenic tobacco plants, functions of pistils and pollen grains were examined. Transgenic pistils pollinated with pollen grains from another tobacco plants (either transgenic or wild-type), developed full of well-developed seeds. In contrast, in self-pollinated transgenic tobacco plants, pollen-tube growth was arrested in the upper part of the style, and small abnormal seeds developed without fertilization. Although the mechanism remains unclear, our research may provide a valuable method to create SI tobacco plants for breeding.

Transgenic tobacco plant overexpressing ginkgo dihydroflavonol 4-reductase gene GbDFR6 exhibits multiple developmental defects.

Dihydroflavonol Q 4-reductase (DFR), a key enzyme in the flavonoid biosynthetic pathway in plants, significantly influences plant survival. However, the roles of DFR in the regulation of plant development are largely unknown. In the present study, phenotypes of transgenic tobacco plants overexpressing the Ginkgo biloba DFR gene, GbDFR6, were investigated. Transgenic tobacco seedlings exhibited relatively low fresh weights, long primary roots, decreased lateral root numbers, and impaired root gravitropic responses when compared to wild-type tobacco plants. Adult transgenic tobacco plants exhibited a considerably high percentage of wrinkled leaves when compared to the wild-type tobacco plants. In addition to the auxin-related phenotypic changes, transgenic tobacco plants exhibited delayed flowering phenotypes under short-day conditions. Gene expression analysis revealed that the delayed flowering in transgenic tobacco plants was caused by the low expression levels of NtFT4. Finally, variations in anthocyanin and flavonoid contents in transgenic tobacco plants were evaluated. The results revealed that the levels of most anthocyanins identified in transgenic tobacco leaves increased. Specifically, cyanidin-3,5-O-diglucoside content increased by 9.8-fold in transgenic tobacco plants when compared to the wild-type tobacco plants. Pelargonidin-3-O-(coumaryl)-glucoside was only detected in transgenic tobacco plants. Regarding flavonoid compounds, one flavonoid compound (epicatechin gallate) was upregulated, whereas seven flavonoid compounds (Tamarixetin-3-O-rutinoside; Sexangularetin-3-O-glucoside-7-O-rhamnoside; Kaempferol-3-O-neohesperidoside; Engeletin; 2'-Hydoxy,5-methoxyGenistein-O-rhamnosyl-glucoside; Diosmetin; Hispidulin) were downregulated in both transgenic tobacco leaves and roots. The results indicate novel and multiple roles of GbDFR6 in ginkgo and provide a valuable method to produce a late flowering tobacco variety in tobacco industry.

A Mutation in the MYBL2-1 Gene Is Associated with Purple Pigmentation in Brassica oleracea.

Anthocyanins are well-known antioxidants that are beneficial for plants and consumers. Dihydroflavonol-4-reductase (DFR) is a key gene of anthocyanin biosynthesis, controlled by multiple transcription factors. Its expression can be enhanced by mutations in the negative regulator of anthocyanin biosynthesis myeloblastosis family transcription factor-like 2 (MYBL2). The expression profiles of the DFR gene were examined in 43 purple and green varieties of Brassica oleracea L., Brassica napus L., Brassica juncea L., and Brassica rapa L. MYBL2 gene expression was significantly reduced in purple varieties of B. oleracea, and green varieties of B. juncea. The MYBL2 gene sequences were screened for mutations that can affect pigmentation. Expression of the DFR gene was cultivar-specific, but in general it correlated with anthocyanin content and was higher in purple plants. Two single nucleotide polymorphysms (SNPs) were found at the beginning of the DNA-binding domain of MYBL2 gene in all purple varieties of B. oleracea. This mutation, leading to an amino acid substitution and the formation of a mononucleotide repeat (A)8, significantly affects RNA structure. No other noteworthy mutations were found in the MYBL2 gene in green varieties of B. oleracea and other studied species. These results bring new insights into the regulation of anthocyanin biosynthesis in genus Brassica and provide opportunities for generation of new purple varieties with precise mutations introduced via genetic engineering and CRISPR/Cas.

Discovery of a DFR gene that controls anthocyanin accumulation in the spiny Solanum group: roles of a natural promoter variant and alternative splicing.

Anthocyanins are important pigments that impart color in plants. In Solanum, different species display various fruit or flower colors due to varying degrees of anthocyanin accumulation. Here we identified two anthocyanin-free mutants from an ethylmethane sulfonate-induced mutant library and naturally occurring mutants in Solanum melongena, with mutations in the 5' splicing site of the second intron of dihydroflavonol-4-reductase (DFR) - leading to altered splicing. Further study revealed that alternative splicing of the second intron was closely related to anthocyanin accumulation in 17 accessions from three cultivated species: S. melongena, Solanum macrocarpon and Solanum aethiopicum, and their wild related species. Analysis of natural variations of DFR, using an expanded population including 282 accessions belonging to the spiny Solanum group, identified a single-nucleotide polymorphism in the MYB recognition site in the promoter region, which causes differential expression of DFR and affects anthocyanin accumulation in fruits of the detected accessions. Our study suggests that, owing to years of domestication, the natural variation in the DFR promoter region and the alternative splicing of the DFR gene account for altered anthocyanin accumulation during spiny Solanum domestication.

Isolation and functional diversification of dihydroflavonol 4-Reductase gene HvDFR from Hosta ventricosa indicate its role in driving anthocyanin accumulation

ABSTRACT Anthocyanins are natural colorants are synthesized in a branch of the flavonoid pathway. Dihydroflavonol-4reductase (DFR) catalyzes dihydroflavonoids into anthocyanins biosynthesis, which is a key regulatory enzyme of anthocyanin biosynthesis in plants. Hosta ventricosa is an ornamental plant with elegant flowers and rich colorful leaves. How the function of HvDFR contributes to the anthocyanins biosynthesis is still unknown. In this study, the DFR homolog was identified from H. ventricosa and sequence analysis showed that HvDFR possessed the conserved NADPH binding and catalytic domains. A phylogenetic analysis showed that HvDFR was close to the clade formed with MaDFR and HoDFR in Asparagaceae. Gene expression analysis revealed that HvDFR was constitutive expressed in all tissues and expressed highly in flower as well as was positively correlated with anthocyanin content. In addition, the subcellular location of HvDFR showed that is in the nucleus and cell membrane. Overexpression of HvDFR in transgenic tobacco lines enhanced the anthocyanins accumulation along with the key genes upregulated, such as F3H, F3ʹH, ANS, and UFGT. Our results indicated a functional activity of the HvDFR, which provide an insight into the regulation of anthocyanins content in H. ventricosa.

Expression of the ANS, CHS and DFR genes involved in the biosynthesis of anthocyanins in Vaccinium floribundum Kunth from Ecuador, using RT-qPCR.

Vaccinium floribundum Kunth, a wild native species of berry in Ecuador, presents a lot of phenolic compounds, specifically anthocyanins; hence it is considered a natural nutraceutical due to all its nutritional properties. The comparison of the expression of genes is involved in the biosynthesis pathway of anthocyanin of several populations. The aim of the research was to analyze the expression levels of three genes involved in the biosynthesis of anthocyanin in this species collected in two areas of the province of Pichincha: Machachi population of the Mejía canton, with geographic coordinates 0 ° 31′04.8 ″ S 78 ° 37′07.4 ″ W and altitude 3200 meters above sea level, and Pintag population of the Quito cantón, with geographic coordinates 0 ° 24′00.0 ″ S 78 ° 24′00.0 ″ W and altitude 3000 meters above sea level. The gene expression analysis was performed using the quantitative polymerase chain reaction technique and reverse transcription (RT-qPCR). For the population of Machachi, the glyceraldehyde-3-phosphate dehydrogenase gene had an average concentration of 648.59 ng/µL, followed by the chalcone synthase gene with 143.71 ng/µL, then by the dihydroflavonol 4-reductase gene with 59.58 ng/µL and finally by the anthocyanin synthase gene with 39 ng/µL. For the population of Pintag, the glyceraldehyde-3-phosphate dehydrogenase gene has an average concentration of 667.32 ng/µL, followed by the chalcone synthase gene with 157.22 ng/µL, then by the dihydroflavonol 4-reductase gene with 60.42 ng/µL, and finally by the anthocyanin synthase gene with 44.40 ng/µL. Each gene has a similar expression level in both populations, but there are differences when comparing the expression level among genes. Many enzymes, structural genes, and regulatory elements have been observed as transcription factors involved in anthocyanin biosynthesis.

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.