Anthocyanins in Floral Colors: Biosynthesis and Regulation in Chrysanthemum Flowers.
ABSTRACT: Chrysanthemum (Chrysanthemum morifolium) is an economically important ornamental crop across the globe. As floral color is the major factor determining customer selection, manipulation of floral color has been a major objective for breeders. Anthocyanins are one of the main pigments contributing to a broad variety of colors in the ray florets of chrysanthemum. Manipulating petal pigments has resulted in the development of a vast range of floral colors. Although the candidate genes involved in anthocyanin biosynthesis have been well studied, the genetic and transcriptional control of floral color remains unclear. Despite advances in multi-omics technology, these methods remain in their infancy in chrysanthemum, owing to its large complex genome and hexaploidy. Hence, there is a need to further elucidate and better understand the genetic and molecular regulatory mechanisms in chrysanthemum, which can provide a basis for future advances in breeding for novel and diverse floral colors in this commercially beneficial crop. Therefore, this review describes the significance of anthocyanins in chrysanthemum flowers, and the mechanism of anthocyanin biosynthesis under genetic and environmental factors, providing insight into the development of novel colored ray florets. Genetic and molecular regulatory mechanisms that control anthocyanin biosynthesis and the various breeding efforts to modify floral color in chrysanthemum are detailed.
Project description:<h4>Background</h4>Chrysanthemum morifolium is one of the most economically important and popular floricultural crops in the family Asteraceae. Chrysanthemum flowers vary considerably in terms of colors and shapes. However, the molecular mechanism controlling the development of chrysanthemum floral colors and shapes remains an enigma. We analyzed a cut-flower chrysanthemum variety that produces normal capitula composed of ray florets with normally developed pistils and purple corollas and mutant capitula comprising ray florets with green corollas and vegetative buds instead of pistils.<h4>Results</h4>We conducted a whole-transcriptome analysis of the differentially expressed genes (DEGs) in the mutant and normal capitula using third-generation and second-generation sequencing techniques. We identified the DEGs between the mutant and normal capitula to reveal important regulators underlying the differential development. Many transcription factors and genes related to the photoperiod and GA pathways, floral organ identity, and the anthocyanin biosynthesis pathway were differentially expressed between the normal and mutant capitula. A qualitative analysis of the pigments in the florets of normal and mutant capitula indicated anthocyanins were synthesized and accumulated in the florets of normal capitula, but not in the florets of mutant capitula. These results provide clues regarding the molecular basis of the replacement of Chrysanthemum morifolium ray florets with normally developed pistils and purple corollas with mutant ray florets with green corollas and vegetative buds. Additionally, the study findings will help to elucidate the molecular mechanisms underlying floral organ development and contribute to the development of techniques for studying the regulation of flower shape and color, which may enhance chrysanthemum breeding.<h4>Conclusions</h4>The whole-transcriptome analysis of DEGs in mutant and normal C. morifolium capitula described herein indicates the anthocyanin deficiency of the mutant capitula may be related to the mutation that replaces ray floret pistils with vegetative buds. Moreover, pistils may be required for the anthocyanin biosynthesis in the corollas of chrysanthemum ray florets.
Project description:Flavonoids, carotenoids, betalains, and chlorophylls are the plant pigments responsible for floral color. Anthocyanins, a class of flavonoids, are largely responsible for the red, purple, pink, and blue colors. <i>R2R3-MYB</i> genes belonging to subgroup 6 (SG6) are the upstream regulatory factors of the anthocyanin biosynthetic pathway. The canonical members of these genes in <i>Arabidopsis</i> include <i>AtMYB75</i>, <i>AtMYB90</i>, <i>AtMYB113</i>, and <i>AtMYB114</i>. The Aristolochiaceae is an angiosperm lineage with diverse floral groundplans and perianth colors. <i>Saruma henryi</i> exhibits a biseriate perianth with green sepals and yellow petals. All other genera have sepals only, with colors ranging from green (in <i>Lactoris</i>) to a plethora of yellow to red and purple mixtures. Here, we isolated and reconstructed the SG6 <i>R2R3-MYB</i> gene lineage evolution in angiosperms with sampling emphasis in Aristolochiaceae. We found numerous species-specific duplications of this gene lineage in core eudicots and local duplications in Aristolochiaceae for <i>Saruma</i> and <i>Asarum</i>. Expression of SG6 <i>R2R3-MYB</i> genes examined in different developmental stages and plant organs of four Aristolochiaceae species, largely overlaps with red and purple pigments, suggesting a role in anthocyanin and flavonoid synthesis and accumulation. A directed RNA-seq analysis corroborated our RT-PCR analyses, by showing that these structural enzymes activate during perianth development in <i>Aristolochia fimbriata</i> and that the regulatory genes are expressed in correlation with color phenotype. Finally, the reconstruction of the flavonoid and anthocyanin metabolic pathways using predicted peptides from transcriptomic data show that all pivotal enzymes are present in the analyzed species. We conclude that the regulatory genes as well as the biosynthetic pathway are largely conserved across angiosperms. In addition, the Aristolochiaceae emerges as a remarkable group to study the genetic regulatory network for floral color, as their members exhibit an outstanding floral diversity with elaborate color patterns and the genetic complement for SG6 <i>R2R3-MYB</i> genes is simpler than in core eudicot model species.
Project description:Various colored cultivars of ornamental flowers have been bred by hybridization and mutation breeding; however, the generation of blue flowers for major cut flower plants, such as roses, chrysanthemums, and carnations, has not been achieved by conventional breeding or genetic engineering. Most blue-hued flowers contain delphinidin-based anthocyanins; therefore, delphinidin-producing carnation, rose, and chrysanthemum flowers have been generated by overexpression of the gene encoding flavonoid 3',5'-hydroxylase (F3'5'H), the key enzyme for delphinidin biosynthesis. Even so, the flowers are purple/violet rather than blue. To generate true blue flowers, blue pigments, such as polyacylated anthocyanins and metal complexes, must be introduced by metabolic engineering; however, introducing and controlling multiple transgenes in plants are complicated processes. We succeeded in generating blue chrysanthemum flowers by introduction of butterfly pea UDP (uridine diphosphate)-glucose:anthocyanin 3',5'-O-glucosyltransferase gene, in addition to the expression of the Canterbury bells F3'5'H. Newly synthesized 3',5'-diglucosylated delphinidin-based anthocyanins exhibited a violet color under the weakly acidic pH conditions of flower petal juice and showed a blue color only through intermolecular association, termed "copigmentation," with flavone glucosides in planta. Thus, we achieved the development of blue color by a two-step modification of the anthocyanin structure. This simple method is a promising approach to generate blue flowers in various ornamental plants by metabolic engineering.
Project description:Chrysanthemums (Chrysanthemum morifolium Ramat.) exhibit a variety of flower colors due to their differing abilities to accumulate anthocyanins. One MYB member, CmMYB6, has been verified as a transcription regulator of chrysanthemum genes involved in anthocyanin biosynthesis; however, the co-regulators for CmMYB6 remain unclear in chrysanthemum. Here, the expression pattern of CmbHLH2, which is clustered in the IIIf bHLH subgroup, was shown to be positively correlated with the anthocyanin content of cultivars with red, pink and yellow flower colors, respectively. CmbHLH2 significantly upregulated the CmDFR promoter and triggered anthocyanin accumulation when co-expressed with CmMYB6. Yeast one-hybrid analyses indicated that CmbHLH2 was able to bind directly to the CmDFR promoter. Moreover, yeast two-hybrid assays indicated protein-protein interaction between CmbHLH2 and CmMYB6. These results suggest that CmbHLH2 is the essential partner for CmMYB6 in regulating anthocyanin biosynthesis in chrysanthemum.
Project description:BACKGROUND:Elucidating the candidate genes and key metabolites responsible for pulp and peel coloration is essential for breeding pitaya fruit with new and improved appeal and high nutritional value. Here, we used transcriptome (RNA-Seq) and metabolome analysis (UPLC-MS/MS) to identify structural and regulatory genes and key metabolites associated with peel and pulp colors in three pitaya fruit types belonging to two different Hylocereus species. RESULT:Our combined transcriptome and metabolome analyses suggest that the main strategy for obtaining red color is to increase tyrosine content for downstream steps in the betalain pathway. The upregulation of CYP76ADs is proposed as the color-breaking step leading to red or colorless pulp under the regulation by WRKY44 transcription factor. Supported by the differential accumulation of anthocyanin metabolites in red pulped pitaya fruit, our results showed the regulation of anthocyanin biosynthesis pathway in addition to betalain biosynthesis. However, no color-breaking step for the development of anthocyanins in red pulp was observed and no biosynthesis of anthocyanins in white pulp was found. Together, we propose that red pitaya pulp color is under the strict regulation of CYP76ADs by WRKYs and the anthocyanin coexistence with betalains is unneglectable. We ruled out the possibility of yellow peel color formation due to anthocyanins because of no differential regulation of chalcone synthase genes between yellow and green and no detection of naringenin chalcone in the metabolome. Similarly, the no differential regulation of key genes in the carotenoid pathway controlling yellow pigments proposed that the carotenoid pathway is not involved in yellow peel color formation. CONCLUSIONS:Together, our results propose several candidate genes and metabolites controlling a single horticultural attribute i.e. color formation for further functional characterization. This study presents useful genomic resources and information for breeding pitaya fruit with commercially attractive peel and pulp colors. These findings will greatly complement the existing knowledge on the biosynthesis of natural pigments for their applications in food and health industry.
Project description:Anthocyanin pigments are responsible for many of the vivid pink, purple, red, and blue flower colors across angiosperms and frequently vary within and between closely related species. While anthocyanins are well known to influence pollinator attraction, they are also associated with tolerance to abiotic stressors such as extreme temperatures, reduced precipitation, and ultraviolet radiation. Using a comparative approach, we tested whether abiotic variables predict floral anthocyanin in monkeyflowers (Phrymaceae) across western North America. Within two polymorphic species, we found that abiotic variables predicted flower color across their geographic ranges. In <i>Erythranthe discolor</i>, the frequency of pink flowered (anthocyanin producing) individuals was greater in populations with reduced precipitation. In <i>Diplacus mephiticus</i>, the frequency of pink flowered individuals was greater at higher elevations that had reduced precipitation and lower temperatures but less ultraviolet radiation. At the macroevolutionary scale, across two parallel radiations of North American monkeyflowers, species with floral anthocyanins (pink, purple, or red corollas) occupied areas with reduced precipitation in <i>Erythranthe</i> but not <i>Diplacus</i>. However, after accounting for phylogenetic relatedness, we found no evidence for the joint evolution of flower color and environmental affinity in either clade. We conclude that although abiotic stressors may play a role in the evolution of flower color within polymorphic species, we found no evidence that these processes lead to macroevolutionary patterns across monkeyflowers.
Project description:The generation of chrysanthemum (Chrysanthemum × morifolium) flower color is mainly attributed to the accumulation of anthocyanins. In the anthocyanin biosynthetic pathway in chrysanthemum, although all of the structural genes have been cloned, the regulatory function of R2R3-MYB transcription factor (TF) genes, which play a crucial role in determining anthocyanin accumulation in many ornamental crops, still remains unclear. In our previous study, four light-induced R2R3-MYB TF genes in chrysanthemum were identified using transcriptomic sequencing. In the present study, we further investigated the regulatory functions of these genes via phylogenetic and alignment analyses of amino acid sequences, which were subsequently verified by phenotypic, pigmental, and structural gene expression analyses in transgenic tobacco lines. As revealed by phylogenetic and alignment analyses, CmMYB4 and CmMYB5 were phenylpropanoid and flavonoid repressor R2R3-MYB genes, respectively, while CmMYB6 was an activator of anthocyanin biosynthesis, and CmMYB7 was involved in regulating flavonol biosynthesis. Compared with wild-type plants, the relative anthocyanin contents in the 35S:CmMYB4 and 35S:CmMYB5 tobacco lines significantly decreased (p < 0.05), while for 35S:CmMYB6 and 35S:CmMYB7, the opposite result was obtained. Both in the 35S:CmMYB4 and 35S:CmMYB5 lines, the relative expression of several anthocyanin biosynthetic genes in tobacco was significantly downregulated (p < 0.05); on the contrary, several genes were upregulated in the 35S:CmMYB6 and 35S:CmMYB7 lines. These results indicate that CmMYB4 and CmMYB5 negatively regulate anthocyanin biosynthesis in chrysanthemum, while CmMYB6 and CmMYB7 play a positive role, which will aid in understanding the complex mechanism regulating floral pigmentation in chrysanthemum and the functional divergence of the R2R3-MYB gene family in higher plants.
Project description:Anthocyanins extracted from black carrots have received increased interest as natural colorants in recent years. The reason is mainly their high content of acylated anthocyanins that stabilizes the color and thereby increases the shelf-life of products colored with black carrot anthocyanins. Still, the main type of anthocyanins synthesized in all black carrot cultivars is cyanidin limiting their use as colorants due to the narrow color variation. Additionally, in order to be competitive against synthetic colors, a higher percentage of acylated anthocyanins and an increased anthocyanin content in black carrots are needed. However, along with the increased interest in black carrots there has also been an interest in identifying the structural and regulatory genes associated with anthocyanin biosynthesis in black carrots. Thus, huge progress in the identification of genes involved in anthocyanin biosynthesis has recently been achieved. Given this information it is now possible to attempt to modulate anthocyanin compositions in black carrots through genetic modifications. In this review we look into genetic modification opportunities for generating taproots of black carrots with extended color palettes, with a higher percentage of acylated anthocyanins or a higher total content of anthocyanins.
Project description:Anthocyanins are natural pigments that have important functions in plant growth and development. Radish taproots are rich in anthocyanins which confer different taproot colors and are potentially beneficial to human health. The crop differentially accumulates anthocyanin during various stages of growth, yet molecular mechanisms underlying this differential anthocyanin accumulation remains unknown. In the present study, transcriptome analysis was used to concisely identify putative genes involved in anthocyanin biosynthesis in radish. Spatial-temporal transcript expressions were then profiled in four color variant radish cultivars. From the total transcript sequences obtained through illumina sequencing, 102 assembled unigenes, and 20 candidate genes were identified to be involved in anthocyanin biosynthesis. Fifteen genomic sequences were isolated and sequenced from radish taproot. The length of these sequences was between 900 and 1,579 bp, and the unigene coverage to all of the corresponding cloned sequences was more than 93%. Gene structure analysis revealed that RsF3'H is intronless and anthocyanin biosynthesis genes (ABGs) bear asymmetrical exons, except RsSAM. Anthocyanin accumulation showed a gradual increase in the leaf of the red radish and the taproot of colored cultivars during development, with a rapid increase at 30 days after sowing (DAS), and the highest content at maturity. Spatial-temporal transcriptional analysis of 14 genes revealed detectable expressions of 12 ABGs in various tissues at different growth levels. The investigation of anthocyanin accumulation and gene expression in four color variant radish cultivars, at different stages of development, indicated that total anthocyanin correlated with transcript levels of ABGs, particularly RsUFGT, RsF3H, RsANS, RsCHS3 and RsF3'H1. Our results suggest that these candidate genes play key roles in phenotypic and spatial-temporal anthocyanin accumulation in radish through coordinated regulation and the major control point in anthocyanin biosynthesis in radish is RsUFGT. The present findings lend invaluable insights into anthocyanin biosynthesis and may facilitate genetic manipulation for enhanced anthocyanin content in radish.
Project description:Anthocyanins are natural water-soluble pigments that are important in plants because they endow a variety of colors to vegetative tissues and reproductive plant organs, mainly ranging from red to purple and blue. The colors regulated by anthocyanins give plants different visual effects through different biosynthetic pathways that provide pigmentation for flowers, fruits and seeds to attract pollinators and seed dispersers. The biosynthesis of anthocyanins is genetically determined by structural and regulatory genes. MYB (v-myb avian myeloblastosis viral oncogene homolog) proteins are important transcriptional regulators that play important roles in the regulation of plant secondary metabolism. MYB transcription factors (TFs) occupy a dominant position in the regulatory network of anthocyanin biosynthesis. The TF conserved binding motifs can be combined with other TFs to regulate the enrichment and sedimentation of anthocyanins. In this study, the regulation of anthocyanin biosynthetic mechanisms of MYB-TFs are discussed. The role of the environment in the control of the anthocyanin biosynthesis network is summarized, the complex formation of anthocyanins and the mechanism of environment-induced anthocyanin synthesis are analyzed. Some prospects for MYB-TF to modulate the comprehensive regulation of anthocyanins are put forward, to provide a more relevant basis for further research in this field, and to guide the directed genetic modification of anthocyanins for the improvement of crops for food quality, nutrition and human health.