Dihydroflavonol 4-reductase Gene Research Articles

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.

Insertion of a novel transposable element disrupts the function of an anthocyanin biosynthesis-related gene in Echinacea purpurea

Echinacea purpurea is a popular medical and ornamental flower. The ray florets in E. purpurea are naturally reddish purple, and several colored varieties of ray florets have been produced. The main components responsible for flower coloration in wild-type E. purpurea are two cyanidin-derived anthocyanins. The E. purpurea cultivar ‘Virgin,’ however, possesses white ray florets and is thought to be an anthocyanin-devoid mutant. To determine the mechanisms underlying ray floret pigmentation between E. purpurea wild-type and ‘Virgin,’ we carried out a comprehensive comparison of the flavonoid composition of their tissues. Moreover, we isolated two genes, encoding dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS), that are related to anthocyanin biosynthesis in E. purpurea. Structural and expression analyses showed that both the nucleotide sequences and expression of DFR genes were similar between wild-type and ‘Virgin’ plants. However, we observed insertion of a novel 349-bp non-autonomous miniature-inverted repeat transposable element in the ANS coding region of the ‘Virgin’ cultivar only. This inserted element may interrupt gene expression, as it was succeeded by a premature stop codon, and could form secondary structures in hairpin RNA that could then be processed into a mature miRNA. Collectively, these results indicate that the insertion in the ANS gene was responsible for the generation of anthocyanin-devoid ray florets in E. purpurea.

Adaptability variations and differences in photoprotection of young leaves of two tree species of subtropical forest to the light environment

In the high-light environment, young leaves accumulate anthocyanins as a photoprotection strategy. However, anthocyanin biosynthesis-related enzymes gene sequence is still unknown in the leaves of subtropical forest plants. There are thus few reports on the relationship between the expression level of these genes and photoprotection. In this study, Machilus chinensis and Castanopsis chinensis were taken as plant material in a subtropical forest. Non-full-length nucleotide sequences of chalcone synthase, dihydroflavonol 4-reductase, anthocyanin synthase, and β-tubulin gene were obtained by homologous and electronic cloning. The expression of those genes was verified and analyzed, and some physiological indicators were measured. The anthocyanin content and anthocyanin synthesis-related gene expression in the young leaves of C. chinensis were significantly higher than that of M. chinensis. Although young leaves of M. chinensis did not accumulate anthocyanins, they showed higher antioxidants and nonphotochemical quenching (NPQ). This study indicates that anthocyanins, antioxidants, and NPQ together mediate the positive effects on photoadaptation in young leaves.

Cloning and Function Identification of Dihydroflavonol 4-Reductase Gene BoaDFR in Chinese Kale

Cloning and Function Identification of Dihydroflavonol 4-Reductase Gene BoaDFR in Chinese Kale

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

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 in planta 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.

Identification and comparative expression analysis of chalcone synthase, flavanone 3-hydroxylase and dihydroflavonol 4-reductase genes in wild pomegranate (Punica granatum L.) organs

Flavonoids are the predominant polyphenolic compounds which are ubiquitously present in plants, responsible for the colour development in different plant parts, thereby playing an important role in plant physiology. In order to understand chemo-diversity and biological functions of flavonoids, it is important to understand flavonoid biosynthetic pathway that involves various structural genes. Three structural genes of flavonoid biosynthetic pathway, chalcone synthase (PgCHS; GenBank accession no: KY498075), flavanone 3-hydroxylase (PgF3H; GenBank accession no: MG012857) and dihydroflavonol 4-reductase (PgDFR; GenBank accession no: MG366127) were isolated and characterized from the wild Punica granatum. Molecular modelling indicated that the secondary structures of PgCHS, PgF3H and PgDFR are mainly α-helix and random coils. HPLC analysis of flavonoids showed its accumulation in different tissues and fruits, thus corroborating well with the expression profile of PgCHS, PgF3H and PgDFR genes. PgF3H and PgDFR genes exhibited high transcripts levels in red fruits while expression of Pg CHS gene was highest in flowers. Further, it is speculated that expression of all these structural genes is independent of each other. This is the first study that provides a comparative expression pattern of flavonoid genes playing a significant role in fruit ripening in wild pomegranate.

Functional and correlation analyses of dihydroflavonol-4-reductase genes indicate their roles in regulating anthocyanin changes in Ginkgo biloba

Anthocyanins, a class of flavonoids, are synthesized in a branch of the flavonoid pathway. Dihydroflavonol-4-reductase (DFR) catalyzes the first reaction leading to anthocyanins, and it is considered a key regulatory enzyme of anthocyanin biosynthesis in plants. In this research, we isolated three new DFR genes by searching the whole-genomic sequence data of Ginkgo biloba. The genes have similar structures and are clustered in the same scaffold. Sequence analyses of GbDFR proteins revealed that these GbDFRs have a closer relationship to Arabidopsis thaliana DFRs than previously identified GbDFRs. Expression analyses revealed that GbDFR4 and GbDFR6 were preferentially expressed in leaves and fruit, respectively. Young and mature leaves shared similar gene expression patterns, while fruit harvested in May had significantly higher expression levels of GbDFRs than fruit harvested in June. Transgenic tobacco plants ectopically overexpressing GbDFR genes showed higher DFR enzymatic activity levels, higher anthocyanin contents and darker flowers than the control. Ginkgo seedlings treated with NaCl showed increased total flavonoid and anthocyanin contents, and the expression levels of GbDFR genes were also induced. In contrast, UV-B irradiation increased the total flavonoid content but decreased both the anthocyanin content and GbDFR expression levels. A salicylic acid treatment induced and inhibited the total flavonoid content with and without light, respectively. In contrast, both the GbDFR expression levels and the anthocyanin content were inhibited by the salicylic acid treatment regardless of the light conditions. This research validated the functions of GbDFRs and expands our understanding of the environmental stress-response mechanisms of ginkgo trees.

Construction of an Onion (Allium cepa L.) Genetic Linkage Map using Genotyping-by-Sequencing Analysis with a Reference Gene Set and Identification of QTLs Controlling Anthocyanin Synthesis and Content.

Anthocyanins, the pigmented flavonoids responsible for red and blue colors in horticultural products, promote human health by preventing cancers and lowering the risk of cardiovascular disease. Red onions contain several cyanidin- and peonidin-based anthocyanins. In this study, we constructed a single-nucleotide polymorphism (SNP)-based genetic linkage map in an F2 segregating population derived from a cross between the inbred line ‘SP3B’ (yellow bulb) and the doubled haploid line ‘H6′ (red bulb) to identify quantitative trait loci (QTLs) for total anthocyanin content of onion bulbs using a genotyping-by-sequencing (GBS) analysis based on a reference gene set. A total of 101.9 Gbp of raw sequences were generated using an Illumina HiSeq 2500 system and a total of 1625 SNP loci were identified with the criteria of three minimum depths, lower than 30% missing rate, and more than 5% minor allele frequency. As a result, an onion genetic linkage map consisting of 319 GBS-based SNP loci and 34 high-resolution melting (HRM) markers was constructed with eight linkage groups and a total genetic distance of 881.4 cM. In addition, the linkage groups were assigned to corresponding chromosomes by comparison with the reference genetic map OH1×5225 through marker development based on common transcripts. The analysis revealed one major QTL, qAS7.1, for anthocyanin synthesis and two significant QTLs, qAC4.1 and qAC4.2, for anthocyanin content. The QTL qAS7.1, located on chromosome 7 with a phenotypic variation of 87.61%, may be a dihydroflavonol 4-reductase (DFR) gene that determines whether the bulb color is red or yellow. The QTLs qAC4.1 and qAC4.2 are separately positioned on chromosome 4 with R2 values of 19.43% and 26.28%, respectively. This map and QTL information will contribute to marker development and breeding for high anthocyanin content in bulb onion.

Arg-type dihydroflavonol 4-reductase genes from the fern Dryopteris erythrosora play important roles in the biosynthesis of anthocyanins.

Dihydroflavonol 4-reductase (DFR), a key enzyme involved in the biosynthesis of anthocyanins, has been cloned from various species. However, little research has been conducted on this enzyme in ferns, which occupy a unique evolutionary position. In this study, we isolated two novel DFR genes from the fern Dryopteris erythrosora. In vitro enzymatic analysis revealed that DeDFR1 and DeDFR2 enzymes can catalyze dihydrokaempferol and dihydroquercetin but cannot catalyze dihydromyricetin. Amino acid sequence analysis showed that DeDFR1 and DeDFR2 have an arginine at the same substrate-specificity-determining site as that in the ferns Salvinia cucullata and Azolla filiculoides. Thus, we speculate that the Arg-type DFR is a new DFR functional type. To further verify the substrate preferences of the Arg-type DFR, an amino acid substitution assay was conducted. When N133 was mutated to R133, Arabidopsis DFR protein completely lost its catalytic activity for dihydromyricetin, as observed for DeDFR1 and DeDFR2. Additionally, heterologous expression of DeDFR2 in the Arabidopsis tt3-1 mutant resulted in increasing anthocyanin accumulation. In summary, DeDFR1 and DeDFR2 are considered to be a new type of DFR with unique structures and functions. The discovery of the Arg-type DFR provides new insights into the anthocyanin biosynthesis pathway in ferns.

Application of cucumber mosaic virus to efficient induction and long-term maintenance of virus-induced gene silencing in spinach.

Virus-induced gene silencing (VIGS) is a useful tool for functional genomics in plants. In this study, we tried to apply cucumber mosaic virus (CMV) to efficient induction of VIGS in spinach. Although VIGS for spinach had been previously developed based on two viruses (beet curly top virus and tobacco rattle virus), they still have some problems with systemic movement and long-term maintenance of VIGS in spinach. Although ordinary CMV strains infect spinach inducing distinct mosaic symptoms, using a CMV pseudorecombinant, we can modify the viral pathogenicity to attenuate viral symptoms that may mask the silencing phenotype. We here successfully demonstrated the viral ability to silence the phytoene desaturase (PDS) and the dihydroflavonol 4-reductase (DFR) genes in spinach. Because CMV could quickly induce VIGS even at 7-10 days postinoculation and the virus did not disappear even at the flowering stage, this CMV-based VIGS system would contribute to functional genomics in spinach and especially to the elucidation of molecular mechanisms for some properties unique to spinach such as plasticity of sex expression; the CMV-induced VIGS can last until the flowering stage after the virus was inoculated onto the seedling.

Cloning and Characterization of a Tryptophan–Aspartic Acid Repeat Gene Associated with the Regulation of Anthocyanin Biosynthesis in Oriental Hybrid Lily

Anthocyanins are major pigments responsible for the color of lily (Lilium sp.) flowers. Anthocyanin synthesis is part of the flavonoid metabolic pathway. Numerous transcription factors, including R2R3-MYBs, basic helix-loop-helix (bHLH), and tryptophan–aspartic acid repeat (also known as WD40 or WD repeat) proteins, known to regulate flavonoid biosynthesis have been identified in various plant species. However, there is limited information available on WD repeat proteins in lilies. In this study, we identified a WD repeat gene in the Oriental hybrid lily ‘Sorbonne’ (Lilium hybrid WD repeat, LhWDR). LhWDR contains no introns, and has a 1100–base pair open reading frame, encoding a putative protein of 370 amino acids. LhWDR was found to be localized in the cytoplasm of transgenic Arabidopsis thaliana root cells. Expression patterns of LhWDR in different organs and at different periods of lily tepal growth revealed that the expression levels of this gene are closely associated with anthocyanin accumulation. A yeast two-hybrid assay demonstrated that full-length LhWDR interacts with the 420 N-terminal amino acids of Lilium hybrid bHLH2. Interestingly, overexpression of LhWDR in A. thaliana led to an upregulation of the dihydroflavonol 4-reductase gene, which is an important structural gene downstream of the anthocyanin pathway. These results indicate that the WD repeat protein LhWDR might interact with a bHLH transcription factor to regulate anthocyanin biosynthesis.

Color characteristics, pigment accumulation and biosynthetic analyses of leaf color variation in herbaceous peony (Paeonia lactiflora Pall.).

Herbaceous peony (Paeonia lactiflora Pall.) is one of the color-leaved ornamental spring plants, with graceful appearance and splendid color. However, the underlying mechanism of this coloration variation from purple to green has not been studied in P. lactiflora. In th study, the leaves in purple, purple-green, and green stages were compared in terms of anatomical, physiological, and molecular. We found that the variation of leaf color from purple to green was mainly determined by the change in pigments distributed in the leaf surface. Physiological experiments showed a significant increase in chlorophyll contents and a notable reduction in anthocyanin contents in leaves from the purple to green stages. We further found that the anthocyanin biosynthesis-related dihydroflavonol 4-reductase (DFR) gene and anthocyanin synthase (ANS) gene as well as chlorophyll biosynthesis-related glutamyl-tRNA reductase (HEMA) gene showed a decreased trend in leaves from purple to green stages, whereas the chlorophyll degradation-related chlorophyll b reductase (NYC) gene showed a rising trend. Alteration of DFR and ANS gene expression might reduce anthocyanin accumulation, whereas increased HEMA gene expression would enhance chlorophyll biosynthesis and reduced NYC gene expression would inhibit chlorophyll degradation. Consequently, reduction in anthocyanins and enhanced deposition of chlorophylls resulted in leaf coloration variation from purple to green in P. lactiflora, which could improve our understanding of its mechanism for further studies.

Comprehensive analysis of putative dihydroflavonol 4-reductase gene family in tea plant.

One identified dihydroflavonol 4-reductases (DFR) encoding gene (named as CsDFRa herein) and five putative DFRs (named as CsDFRb1, CsDFRb2, CsDFRb3, CsDFRc and CsDFRd) in tea (Camellia sinensis) have been widely discussed in recent papers concerning multi-omics data. However, except for CsDFRa, their function and biochemical characteristics are not clear. This study aims to compare all putative CsDFRs and preliminarily evaluate their function. We investigated the sequences of genes (coding and promoter regions) and predicted structures of proteins encoded, and determined the activities of heterologously expressed CsDFRs under various conditions. The results showed that the sequences of five putative CsDFRs were quite different from CsDFRa, and had lower expression levels as well. The five putative CsDFRs could not catalyze three dihydroflavonol substrates. The functional CsDFRa had the strongest affinity with dihydroquercetin, and performed best at pH around 7 and 35°C but was not stable at lower pHs or higher temperatures. Single amino acid mutation at position 141 modified the preference of CsDFRa for dihydroquercetin and dihydromyricetin, and also weakened its stability. These data suggest that only CsDFRa works in the pathway for generating anthocyanidins and catechins. This study provides new insights into the function of CsDFRs and may assist to develop new strategies to manipulate the composition of tea flavonoids in the future.

Cloning and Functional Characterization of Dihydroflavonol 4-Reductase Gene Involved in Anthocyanidin Biosynthesis of Grape Hyacinth.

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.

Differential Regulation of Anthocyanins in Green and Purple Turnips Revealed by Combined De Novo Transcriptome and Metabolome Analysis.

Purple turnip Brassica rapa ssp. rapa is highly appreciated by consumers but the metabolites and molecular mechanisms underlying the root skin pigmentation remain open to study. Herein, we analyzed the anthocyanin composition in purple turnip (PT) and green turnip (GT) at five developmental stages. A total of 21 anthocyanins were detected and classified into the six major anthocynanin aglycones. Distinctly, PT contains 20 times higher levels of anthocyanins than GT, which explain the difference in the root skin pigmentation. We further sequenced the transcriptomes and analyzed the differentially expressed genes between the two turnips. We found that PT essentially diverts dihydroflavonols to the biosynthesis of anthocyanins over flavonols biosynthesis by strongly down-regulating one flavonol synthase gene, while strikingly up-regulating dihydroflavonol 4-reductase (DFR), anthocyanidin synthase and UDP-glucose: flavonoid-3-O-glucosyltransferase genes as compared to GT. Moreover, a nonsense mutation identified in the coding sequence of the DFR gene may lead to a nonfunctional protein, adding another hurdle to the accumulation of anthocyanin in GT. We also uncovered several key members of MYB, bHLH and WRKY families as the putative main drivers of transcriptional changes between the two turnips. Overall, this study provides new tools for modifying anthocyanin content and improving turnip nutritional quality.

Comparative analysis of the quality and health-promoting compounds of two-shaped fruits of wild Lycium ruthenicum Murr. from the Qinghai–Tibet Plateau

Lycium ruthenicum Murr. is an econo-ecological tree species that abounds in the Qinghai–Tibet Plateau, which is located in the Chaka Salt Lake region of Qinghai Province, China. This study compared the phenotypes and main inclusions in two-shaped berries (flat peach and sphere types) at different developmental stages of L. ruthenicum Murr. to provide first-hand information for targeted breeding toward higher quality and stronger stress resistance of the fruit. Results showed the distinct advantage of the quality characters of flat peach-type fruits over the sphere-type with regard to the length of bearing shoot, internode, peduncle, single fruit weight, and 100-pod weight. In terms of inclusions, the anthocyanin contents in two-shaped fruits showed no significant difference at S5. Anthocyanin accumulated in the developing fruits, and the amount peaked at S4 and then gradually decreased. The expression profiles of the key genes in anthocyanin biosynthesis were correlated with anthocyanin accumulation during fruit development, and the high expressions of flavonoid 3′,5′-hydroxylase gene and dihydroflavonol 4-reductase gene were found to be the critical factors of specific delphinidin accumulation. Sphere-type mature fruits had considerable higher total polyphenol level than flat peach fruits, but the opposite result was observed for polysaccharides. The contents of Ca, Co, Cr, Cu, K, Li, Mn, P, Ti, V, and Al presented no considerable differences, whereas B, Mg, Na, Si, and Zn exhibited evident advantages in sphere-type fruits. α-Toc was the most abundant component in both fruits and no evident difference in its content was observed. The data on nutritional contents provide a theoretical basis for different breeding goals of L. ruthenicum and its further utilization as health food.

An R3-MYB gene of Phalaenopsis, MYBx1, represses anthocyanin accumulation

Anthocyanins are secondary metabolites that are involved in providing flower petal pigmentation. In plants, anthocyanin synthesis is regulated positively by a conserved MYB-bHLH-WD40 (MBW) complex and negatively by repressors. We isolated a repressor of anthocyanin synthesis, MYBx1, from a moth orchid Phalaenopsis cv. Big Chili. MYBx1 encodes a typical R3-MYB protein and exhibited diverse expression patterns in four Phalaenopsis cultivars, Big Chili, Fuller’s Sunset, Sogo Yukidian “V3”, and Sogo Lit-Sunny. A yeast two-hybrid assay analysis indicated that MYBx1 can also participate in the MBW complex by interacting with the transcription factors ANTHOCYANIN (AN) 1 and AN11. Over-expressing MYBx1 in the petunia hybrid M1 × R27 resulted in reduced accumulations of flower petal pigments through the repression of anthocyanin activities. A quantitative real-time PCR analysis of the expression patterns of anthocyanin synthesis-related structural genes in the transgenic petunia flower petals showed that the expression levels of chalcone synthase, flavanone 3-hydroxylase, and dihydroflavonol reductase genes were considerably down-regulated. However, no significant changes in the expression levels of the MBW complex members, AN1, AN2, and AN11, were observed. This study improves our understanding of anthocyanin synthesis-related regulatory networks in plants and also provides an important foundation for the further genetic engineering of ornamental plant flower colors.

Identification of Two Novel R2R3-MYB Transcription factors, PsMYB114L and PsMYB12L, Related to Anthocyanin Biosynthesis in Paeonia suffruticosa.

Flower color is a charming phenotype with very important ornamental and commercial values. Anthocyanins play a critical role in determining flower color pattern formation, and their biosynthesis is typically regulated by R2R3-MYB transcription factors (TFs). Paeonia suffruticosa is a famous ornamental plant with colorful flowers. However, little is known about the R2R3-MYB TFs that regulate anthocyanin accumulation in P. suffruticosa. In the present study, two R2R3-MYB TFs, namely, PsMYB114L and PsMYB12L, were isolated from the petals of P. suffruticosa ‘Shima Nishiki’ and functionally characterized. Sequence analysis suggested that PsMYB114L contained a bHLH-interaction motif, whereas PsMYB12L contained two flavonol-specific motifs (SG7 and SG7-2). Subsequently, the in vivo function of PsMYB114L and PsMYB12L was investigated by their heterologous expression in Arabidopsis thaliana and apple calli. In transgenic Arabidopsis plants, overexpression of PsMYB114L and of PsMYB12L caused a significantly higher accumulation of anthocyanins, resulting in purple-red leaves. Transgenic apple calli overexpressing PsMYB114L and PsMYB12L also significantly enhanced the anthocyanins content and resulted in a change in the callus color to red. Meanwhile, gene expression analysis in A. thaliana and apple calli suggested that the expression levels of the flavonol synthase (MdFLS) and anthocyanidin reductase (MdANR) genes were significantly downregulated and the dihydroflavonol 4-reductase (AtDFR) and anthocyanin synthase (AtANS) genes were significantly upregulated in transgenic lines of PsMYB114L. Moreover, the expression level of the FLS gene (MdFLS) was significantly downregulated and the DFR (AtDFR/MdDFR) and ANS (AtANS/MdANS) genes were all significantly upregulated in transgenic lines plants of PsMYB12L. These results indicate that PsMYB114L and PsMYB12L both enhance anthocyanin accumulation by specifically regulating the expression of some anthocyanin biosynthesis-related genes in different plant species. Together, these results provide a valuable resource with which to further study the regulatory mechanism of anthocyanin biosynthesis in P. suffruticosa and for the breeding of tree peony cultivars with novel and charming flower colors.

Differential accumulation of pelargonidin glycosides in petals at three different developmental stages of the orange-flowered gentian (Gentiana lutea L. var. aurantiaca).

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.

The Effect of Maturity Status on Biochemical Composition, Antioxidant Activity, and Anthocyanin Biosynthesis Gene Expression in a Pomegranate (Punica granatum L.) Cultivar with Red Flowers, Yellow Peel, and Pinkish Arils

Pomegranate (Punica granatum L.) has widely been used as a fruit and in folk medicine since ancient civilizations in the world. It is now known that bioactive compounds present in pomegranates attribute to its therapeutic potential. Harvesting at the correct maturity stage is one of the key factors deciding the quality of harvest for consumption in fresh or value-added forms. Identification of the correct maturity stage (harvesting index) is particularly difficult for cultivars having yellowish peel and pinkish arils (sarcotesta). We studied the changes in total phenolic content (TPC), antioxidant activity (AOX), punicalagin α and β contents, color indices, total soluble solids (TSS), and expression of anthocyanin biosynthetic pathway genes from flowering to harvesting in the pomegranate cultivar Nimali, having red flowers, yellow peels, and pinkish arils at maturity over two growing seasons. Interestingly, there was no seasonal variation observed in any of the parameters over two cultivation seasons. Although the β punicalagin content did not change, the TPC, AOX, punicalagin α contents in both peel and arils gradually decreased from flowering to maturity. Though the TPC of both peels and arils, AOX and total punicalagin content of peel did not change significantly 140 days after flower initiation, the TPC and the total punicalagin of arils reached a stable level at 160 days. The TSS in both peels and arils increased significantly with the maturity having the highest values at 180 days. The peel color changed from green to yellow with maturity with a significant increase in l* and b* values and significant decrease in a* value. Nevertheless, the aril color changed from pale white to pink with the maturity with significant reduction of l* and b* values and significant increase of a* value. Changes in pomegranate dihydroflavonol 4-reductase (DFR), flavanone 3-hydroxylase (F3H), and leucoanthocyanidin dioxygenase (anthocyanidin synthase-ANS) gene expression in Nimali arils correlated with its color changes during maturity. These findings support to identify the harvesting index of Nimali ensuring the maximum nutritional and health benefits of pomegranate flower, arils, and peels for different downstream uses.