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Flower color is the main character throughout the plant kingdom. Though substantial information exists regarding the structural and regulatory genes involved in anthocyanin and flavonol biosynthesis, little is known that what make a diverse white vs. red color flower in natural species. Here, the contents of pigments in seven species from varied phylogenetic location in plants with red and white flowers were determined. Flavonols could be detected in red and white flowers, but anthocyanins were almost undetectable in the white cultivar. Comparisons of expression patterns of gene related to the flavonoid biosynthesis indicated that disequilibrium expression of flavonol synthase (FLS) and dihydroflavonol-4-reductase (DFR) genes determined the accumulation of flavonols and anothcyanins in both red and white flowers of seven species. To further investigate the role of such common regulatory patterns in determining flower color, FLS genes were isolated from Rosa rugosa (RrFLS1), Prunus persica (PpFLS), and Petunia hybrida (PhFLS), and DFR genes were isolated from Rosa rugosa (RrDFR1) and Petunia hybrida (PhDFR). Heterologous expression of the FLS genes within tobacco host plants demonstrated conservation of function, with the transgenes promoting flavonol biosynthesis and inhibiting anthocyanin accumulation, so resulting in white flowers. Conversely, overexpression of DFR genes in tobacco displayed down-regulation of the endogenous NtFLS gene, and the promotion of anthocyanin synthesis. On this basis, we propose a model in which FLS and DFR gene-products compete for common substrates in order to direct the biosynthesis of flavonols and anthocyanins, respectively, thereby determining white vs. red coloration of flowers.

Background: Dihydroflavonol 4-reductase (DFR) is a key enzyme in the flavonoid biosynthetic pathway that regulates anthocyanin and proanthocyanidin accumulation in plants. Although DFR genes have been studied in various species, their origin of the DFR gene family, its distribution across the plant kingdom, and the reasons behind the emergence of different DFR subtypes Methods: This study performed a whole-genome analysis of DFR genes in 237 plant species, including algae, mosses, ferns, gymnosperms, and angiosperms, integrating phylogeny, conserved motifs, duplication mechanisms, positive selection, and expression pattern analyses. Results: These results indicate that the DFR gene family originated from the common ancestor of extant ferns and seed plants, and the emergence of asparagine (Asn)-type and aspartic (Asp)-type DFRs is associated with gymnosperms. Notably, we report for the first time the presence of Asn-type, Asp-type, and arginine (Arg)-type DFRs in some species, which breaks the previous notion that Arg-type DFRs are exclusive to ferns. Tandem duplication is considered the primary driving force behind the expansion of the DFR family and is associated with the formation of different DFR subtypes. Furthermore, Asn-type DFRs were highly expressed during the early stages of seed development, suggesting their important role in seed development. Conclusions: Overall, this study revealed the dynamic evolutionary trajectory of the DFR gene family in plants, providing a theoretical foundation for future research on DFR genes.

Petal spots are widespread in angiosperms and are often implicated in pollinator attraction. Clarkia gracilis petals each have a single red-purple spot that contrasts against a pink background. The position and presence of spots in C. gracilis are determined by the epistatic interaction of alleles at two as yet unidentified loci. We used HPLC to identify the different pigments produced in the petals, and qualitative and quantitative RT-PCR to assay for spatio-temporal patterns of expression of different anthocyanin pathway genes. We found that spots contain different pigments from the remainder of the petal, being composed of cyanidin/peonidin-based, instead of malvidin-based anthocyanins. Expression assays of anthocyanin pathway genes showed that the dihydroflavonol-4-reductase 2 (Dfr2) gene has a spot-specific expression pattern and acts as a switch for spot production. Co-segregation analyses implicated the gene products of the P and I loci as trans-regulators of this switch. Spot pigments appear earlier in development as a result of early expression of Dfr2 and the flavonoid 3′ hydroxylase 1 (F3′h1) gene. Pigments in the background appear later, as a result of later expression of Dfr1 and the flavonoid 3′-5′ hydroxylase 1 (F3′5′h1) genes. The evolution of this spot production mechanism appears to have been facilitated by duplication of the Dfr gene and to have required substantial reworking of the anthocyanin pathway regulatory network.

Red leaf lettuce (Lactuca sativa L. cv. ‘Red Fire’) is a preferred crop in plant factories with artificial light (PFALs) due to its short cultivation cycle and high anthocyanin content, which increases both its nutritional value and visual appeal. However, anthocyanins strongly influence leaf coloration and antioxidant profiles, and their levels are highly responsive to the light environment. Therefore, targeted editing of flavonoid biosynthesis may provide a breeding strategy to diversify pigment composition and associated functional traits under PFAL conditions. In this study, we used CRISPR/Cas9 to knock out DFR (dihydroflavonol 4-reductase), a key enzyme in the anthocyanin pathway. Genome-edited lines were generated via a dual-guide RNA system, resulting in a successfully edited red leaf genotype. The DFR-knockout lines displayed a complete loss of red pigmentation and a visibly distinct green phenotype. Metabolite profiling revealed a significant decrease in anthocyanin levels, accompanied by an increase in total flavonoid levels in some lines. Growth traits, including shoot dry weight and leaf number, were not significantly affected, suggesting that DFR knockout does not compromise growth under PFAL conditions. These findings highlight DFR as a promising target for creating pigment-altered lettuce lines for controlled-environment cultivation, including PFAL systems.

Dihydroflavonol-4 reductase (DFR) is a key enzyme in plant anthocyanin synthesis and can affect the synthesis of anthocyanin. In order to explore the role of DFR in anthocyanin synthesis of capsicum, the sequence of the DFR gene family was downloaded from the Arabidopsis genome database. The study showed the presence of nine members of the DFR gene family in pepper, which are primarily localized in the cytoplasm and chloroplasts. The majority of these proteins exhibit hydrophilic characteristics, with their secondary structure predominantly consisting of α-helices and random coils. Furthermore, these genes can be categorized into four distinct subfamilies, with the gene structure and conserved motifs of CaDFR members within the same subfamily providing robust support for this classification. The promoter regions of CaDFRs are enriched with numerous light-responsive elements, phytohormone-responsive elements, stress-responsive elements, and elements associated with plant growth and development, in addition to binding sites for 35 different transcription factors. According to the results of qRT-PCR analysis, the expression level of CaDFR5 was consistent with the change of anthocyanin content, and the full length of the CaDFR5 gene was further cloned. This study provides an important reference for improving fruit colour and lays a foundation for further exploring the function of the DFR gene family.

CRISPR/Cas, one of the most rapidly developing technologies in the world, has been applied successfully in plant science. To test new nucleases, gRNA expression systems and other inventions in this field, several plant genes with visible phenotypic effects have been constantly used as targets. Anthocyanin pigmentation is one of the most easily identified traits, that does not require any additional treatment. It is also associated with stress resistance, therefore plants with edited anthocyanin genes might be of interest for agriculture. Phenotypic effect of CRISPR/Cas editing of PAP1 and its homologs, DFR, F3H and F3′H genes have been confirmed in several distinct plant species. DFR appears to be a key structural gene of anthocyanin biosynthesis, controlled by various transcription factors. There are still many promising potential model genes that have not been edited yet. Some of them, such as Delila, MYB60, HAT1, UGT79B2, UGT79B3 and miR156, have been shown to regulate drought tolerance in addition to anthocyanin biosynthesis. Genes, also involved in trichome development, such as TTG1, GLABRA2, MYBL2 and CPC, can provide increased visibility. In this review successful events of CRISPR/Cas editing of anthocyanin genes are summarized, and new model genes are proposed. It can be useful for molecular biologists and genetic engineers, crop scientists, plant genetics and physiologists.

Dihydroflavonol 4-reductase (DFR) is pivotal for anthocyanin biosynthesis and plays a crucial role in plant development and stress adaptation. However, a systematic characterization of the gene family is lacking in Thunb. In the present study, based on genome and transcriptome data of , the research identified six gene family members throughout the entire genome. The genes were located on Chr.04 and Chr.09 and the full-length coding sequences of - were cloned. Subcellular localization analysis showed that LjDFRs are primarily found at the cell membrane and in the nucleus. Phylogenetic analysis showed closer clustering of genes with and . Promoter analysis linked genes to light response, hormone signaling, and stress-responses. qRT-PCR analysis demonstrated tissue-specific and stage-specific expression patterns among members. Notably, expression was significantly higher in the intensely pigmented tissues of Thunb. var. (Wats.) Bak. compared to . . Coupled with its phylogenetic proximity to the anthocyanin-related and genes, this suggests that may be positively correlated with anthocyanin accumulation. Additionally, the expression of and was markedly induced by both drought and salt stress, indicating their roles in abiotic stress responses. This research provides a foundation for further functional studies of genes in anthocyanin biosynthesis and stress resistance and offers candidate genes for molecular breeding of . .

Key message The ectopic expression of AtDFR results in increased accumulation of anthocyanins leading to enhanced salinity and drought stress tolerance in B . napus plants. Flavonoids with antioxidant effects confer many additional benefits to plants. Evidence indicates that flavonoids, including anthocyanins, protect tissues against oxidative stress from various abiotic stressors. We determined whether increases in anthocyanins increased abiotic stress tolerance in Brassica napus , because the values of B . napus L. and its cultivation area are increasing worldwide. We overexpressed Arabidopsis dihydroflavonol-4-reductase ( DFR ) in B . napus . Increased DFR transcript levels for AtDFR - OX B . shoots correlated with higher anthocyanin accumulation. AtDFR - OX Brassica shoots exhibited lower reactive oxygen species (ROS) accumulation than wild-type (WT) shoots under high NaCl and mannitol concentrations. This was corroborated by 3,3-diaminobenzidine staining for ROS scavenging activity in 1,1-diphenyl-2-picryl-hydrazyl assays. Shoots of the AtDFR - OX B . napus lines grown in a high salt medium exhibited enhanced salt tolerance and higher chlorophyll content than similarly grown WT plants. Our observations suggested that the AtDFR gene can be effectively manipulated to modulate salinity and drought stress tolerance by directing to high accumulation of anthocyanins in oilseed plants.

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.

Background Anthocyanins are important contributors to coloration across a wide phylogenetic range of plants. Biological functions of anthocyanins span from reproduction to protection against biotic and abiotic stressors. Owing to a clearly visible phenotype of mutants, the anthocyanin biosynthesis and its sophisticated regulation have been studied in numerous plant species. Genes encoding the anthocyanin biosynthesis enzymes are regulated by a transcription factor complex comprising MYB, bHLH and WD40 proteins. Results A systematic comparison of anthocyanin-pigmented vs. non-pigmented varieties was performed within numerous plant species covering the taxonomic diversity of flowering plants. The literature was screened for cases in which genetic factors causing anthocyanin loss were reported. Additionally, transcriptomic data sets from four previous studies were reanalyzed to determine the genes possibly responsible for color variation based on their expression pattern. The contribution of different structural and regulatory genes to the intraspecific pigmentation differences was quantified. Differences concerning transcription factors are by far the most frequent explanation for pigmentation differences observed between two varieties of the same species. Among the transcription factors in the analyzed cases, MYB genes are significantly more prone to account for pigmentation differences compared to bHLH or WD40 genes. Among the structural genes, DFR genes are most often associated with anthocyanin loss. Conclusions These findings support previous assumptions about the susceptibility of transcriptional regulation to evolutionary changes and its importance for the evolution of novel coloration phenotypes. Our findings underline the particular significance of MYBs and their apparent prevalent role in the specificity of the MBW complex.