Anthocyanin Biosynthetic Pathway Research Articles

Metabolome and transcriptome reveal the biosynthesis of flavonoids and amino acids in Isatis indigotica fruit during development.

Isatis indigotica Fort. is a famous medicinal plant that is also used as a natural dye and functional vegetable. The characteristics of the I. indigotica fruit during development are largely unknown, information that is essential for the exploitation and seedlings cultivation of I. indigotica. In this study, the biochemical, metabolite characteristics and gene expression profiling of I. indigotica at four developmental stages were investigated. A total of 428 metabolites were detected and categorized into 17 categories. High contents of anthocyanins, especially cyanidin 3-glucoside, might contribute to the purple colouration of I. indigotica fruits. Moreover, dozens of flavonoid components, including taxifolin, quercetin, astragalin and isovitexin 2″-O-beta-D-glucoside, and several other active components were also up-regulated in mature fruits. The abundance of antioxidants might endow a significantly stronger antioxidant activity of mature I. indigotica fruits compared to many other reported species. Enrichment analyses revealed that flavonoid and anthocyanin biosynthesis genes were mostly enriched in up-regulated gene sets during fruit development. The up-regulated structural genes, including IiCHS, IiCHI, IiF3H, IiDFR, IiANS, IiFLS, IiUGT, and transcription factors such as IiMYBs, IibHLHs and IiNACs were identified as candidate regulators of flavonoid and anthocyanin biosynthetic pathway. Furthermore, biosynthesis of amino acids was enriched in all pairwise comparisons of metabolites in fruits at four developmental stages. The differential accumulation of amino acids might result from the differentially expressed genes involved in amino acid biosynthesis. Taken together, these findings provide a comprehensive understanding of metabolite profiling and gene expression patterns in I. indigotica fruit during maturity, which is useful for pharmaceutical extractions and seedling cultivation of I. indigotica.

A R2R3-MYB transcription factor, FeR2R3-MYB, positively regulates anthocyanin biosynthesis and drought tolerance in common buckwheat (Fagopyrum esculentum)

The R2R3-MYB transcription factors (TFs) play a crucial role in regulating plant secondary metabolism and abiotic stress responses, yet they are still poorly understood in common buckwheat (Fagopyrum esculentum), a valuable minor grain crop resource. In this study, a candidate gene, FeR2R3-MYB, was cloned from the anthocyanin-rich common buckwheat variety ‘QZZTQ’. FeR2R3-MYB was found to contain two MYB DNA-binding domains and be located at the nucleus with transcriptional activation activity. Molecular analysis indicated that FeR2R3-MYB is predominantly expressed in flowering tissue and is highly responsive to environmental factors such as light, drought, and cold. In addition, the promoter of FeR2R3-MYB showed a positive correlation with fragment length. Further functional analysis suggested that FeR2R3-MYB not only participates in the anthocyanin biosynthetic pathway by interacting with leucoanthocyanidin reductase (FeLAR), but also enhances drought tolerance in common buckwheat. To sum up, FeR2R3-MYB exhibits positive effects on both pigment production (e.g., anthocyanin) and abiotic stress resistance, providing valuable insights for future research in buckwheat molecular breeding and resource development.

The NAC activator, MdNAC77L, regulates anthocyanin accumulation in red flesh apple

Anthocyanin is widely acknowledged as a crucial component contributing to apple fruit quality. However, research on the influence of NAC transcription factors on anthocyanin accumulation in red flesh apple is limited. In this study, a NAC transcription factor MdNAC77L was identified as highly related to the change of anthocyanin content, based on a combined anthocyanin metabolome and transcriptome analysis. Overexpression of MdNAC77L significantly increased the accumulation of anthocyanin in both apple and strawberry fruits, along with the expression of several anthocyanin-related enzymes. Molecular-level analyses demonstrated that MdNAC77L was directly combined with the promoters of three pivotal enzymes (dihydroflavonol 4-reductase, anthocyanin synthase, and UDP-flavonoid glucosyltransferase) of anthocyanin biosynthetic pathway. We found that ABA strongly induced the expression of MdNAC77L, which activated the expression of three downstream target genes. In addition, MdNAC77L showed higher transcript accumulation in flesh with lower anthocyanin content than that in red peel. We revealed the new core factor, MdNAC77L, involved in anthocyanin accumulation in apple red flesh. This research provides a fresh perspective on the intricate regulatory mechanisms governing anthocyanin biosynthesis in red-fleshed apples.

An in-depth study of anthocyanin synthesis in the exocarp of virescens and nigrescens oil palm: metabolomic and transcriptomic analysis.

Oil palm (Elaeis guineensis Jacq.) is a very important tropical woody oil plant with high commercial and ornamental value. The exocarp of the oil palm fruit is rich in anthocyanosides and proanthocyanidins, which not only give it a bright colour, but also mark the maturity of the fruit. The study of the dynamic change pattern of anthocyanoside content and important anthocyanoside metabolism-related regulatory genes during oil palm ripening is conducive to the improvement of the ornamental value of oil palm and the determination of the optimal harvesting period of the fruits. We analyzed the virescens oil palm (AS) and nigrescens oil palm (AT) at 95 days (AS1, AT1), 125 days (AS2, AT2) and 185 days (AS3, AT3) after pollination were used as experimental materials for determining the changes in the total amount of anthocyanins as well as their metabolomics and transcriptomics studies by using the LC-MS/MS technique and RNA-Seq technique. The results showed that the total anthocyanin content decreased significantly from AS1 (119µg/g) to AS3 (23µg/g), and from AT1 (1302µg/g) to AT3 (170µg/g), indicating a clear decreasing trend during fruit development. Among them, the higher flavonoids in AS and AT included anthocyanins such as peonidin-3-O-rutinoside (H35), pelargonidin-3-O-rutinoside (H21), and cyanidin-3-O-glucoside (H7), as well as condensed tannins such as procyanidin B2 (H47), procyanidin C1 (H49), and procyanidin B3 (H48). Notably, nine genes involved in the anthocyanin biosynthetic pathway exhibited up-regulated expression during the pre-development stage of oil palm fruits, particularly during the AS1 and AT1 periods. These genes include: Chalcone synthase (CHS; LOC105036364); Flavanone 3-hydroxylase (F3H; LOC105054663); Dihydroflavonol 4-reductase (DFR; LOC105040724, LOC105048473); Anthocyanidin synthase (ANS; LOC105035842), UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT; LOC105039612); Flavonoid 3',5'-hydroxylase (F3'5'H; LOC105036086, LOC105044124, LOC105045493). In contrast, five genes demonstrated up-regulated expression as the fruits developed, specifically during the AS3 and AT3 periods. These genes include: Chalcone synthase (CHS; LOC105036921, LOC105035716); Chalcone isomerase (CHI; LOC105045978); UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT; LOC105046326); Flavonoid 3'-hydroxylase (F3'H; LOC105036086). Most of differentially expressed genes exhibited up-regulation during the early stages of fruit development, which may contribute to the elevated anthocyanin content observed in oil palm fruits of both types during the pre-developmental period. Furthermore, the expression levels of most genes were found to be higher in the AT fruit type compared to the AS fruit type, suggesting that the differential expression of these genes may be a key factor underlying the differences in anthocyanoside production in the exocarp of oil palm fruits from these two fruit types. The findings of this study provide a theoretical foundation for the identification and characterization of genes involved in anthocyanin synthesis in oil palm fruits, as well as the development of novel variations using molecular biology approaches.

De novo biosynthesis of anthocyanins in Saccharomyces cerevisiae using metabolic pathway synthases from blueberry

BackgroundAnthocyanins are water-soluble flavonoids in plants, which give plants bright colors and are widely used as food coloring agents, nutrients, and cosmetic additives. There are several limitations for traditional techniques of collecting anthocyanins from plant tissues, including species, origin, season, and technology. The benefits of using engineering microbial production of natural products include ease of use, controllability, and high efficiency.ResultsIn this study, ten genes encoding enzymes involved in the anthocyanin biosynthetic pathway were successfully cloned from anthocyanin-rich plant materials blueberry fruit and purple round eggplant rind. The Yeast Fab Assembly technology was utilized to construct the transcriptional units of these genes under different promoters. The transcriptional units of PAL and C4H, 4CL and CHS were fused and inserted into Chr. XVI and IV of yeast strain JDY52 respectively using homologous recombination to gain Strain A. The fragments containing the transcriptional units of CHI and F3H, F3’H and DFR were inserted into Chr. III and XVI to gain Strain B1. Strain B2 has the transcriptional units of ANS and 3GT in Chr. IV. Several anthocyanidins, including cyanidin, peonidin, pelargonidin, petunidin, and malvidin, were detected by LC-MS/MS following the predicted outcomes of the de novo biosynthesis of anthocyanins in S. cerevisiae using a multi-strain co-culture technique.ConclusionsWe propose a novel concept for advancing the heterologous de novo anthocyanin biosynthetic pathway, as well as fundamental information and a theoretical framework for the ensuing optimization of the microbial synthesis of anthocyanins.

Metabolomic analysis combined with machine learning algorithms enables the evaluation of postharvest pecan color stability

Nut kernel color is a crucial quality indicator affecting the consumers first impression of the product. While growing evidence suggests that plant phenolics and their derivatives are linked to nut kernel color, the compounds (biomarkers) responsible for kernel color stability during storage remain elusive. Here, pathway-based metabolomics with machine learning algorithms were employed to identify key metabolites of postharvest pecan color stability. Metabolites in phenylpropanoid, flavonoid, and anthocyanin biosynthetic pathways were analyzed in the testa of nine pecan cultivars using liquid chromatography–mass spectrometry. With color measurements, different machine learning models were compared to find relevant biomarkers of pecan color phenotypes. Results revealed potential marker compounds that included flavonoid precursors and anthocyanidins as well as anthocyanins (e.g., peonidin, delphinidin-3-O-glucoside). Our findings provide a foundation for future research in the area, and will help select genes/proteins for the breeding of pecans with stable and desirable kernel color.

Comparative transcriptome analysis reveals transcriptional regulation of anthocyanin biosynthesis in purple radish (Raphanus sativus L.)

Radish exhibits significant variation in color, particularly in sprouts, leaves, petals, fleshy roots, and other tissues, displaying a range of hues such as green, white, red, purple, and black. Although extensive research has been conducted on the color variation of radish, the underlying mechanism behind the variation in radish flower color remains unclear. To date, there is a lack of comprehensive research investigating the variation mechanism of radish sprouts, leaves, fleshy roots, and flower organs. This study aims to address this gap by utilizing transcriptome sequencing to acquire transcriptome data for white and purple radish flowers. Additionally, the published transcriptome data of sprouts, leaves, and fleshy roots were incorporated to conduct a systematic analysis of the regulatory mechanisms underlying anthocyanin biosynthesis in these four radish tissues. The comparative transcriptome analysis revealed differential expression of the anthocyanin biosynthetic pathway genes DFR, UGT78D2, TT12 and CPC in the four radish tissues. Additionally, the WGCNA results identified RsDFR.9c and RsUGT78D2.2c as hub genes responsible for regulating anthocyanin biosynthesis. By integrating the findings from the comparative transcriptome analysis, WGCNA, and anthocyanin biosynthetic pathway-related gene expression patterns, it is hypothesized that genes RsDFR.9c and RsUGT78D2.2c may serve as pivotal regulators of anthocyanins in the four radish tissues. Furthermore, the tissue-specific expression of the four copies of RsPAP1 is deemed crucial in governing anthocyanin synthesis and accumulation. Our results provide new insights into the molecular mechanism of anthocyanin biosynthesis and accumulation in different tissues of radish.

Stimulation of secondary metabolism in grape berry exocarps by a nature-based strategy of foliar application of polyols

In grapes under drought stress, polyols accumulate through tight coordination at the molecular level between increased membrane transport of polyols and inhibition of polyol oxidation. Here, the effects on grape metabolism of an exogenous foliar application of polyols as a potential sustainable viticultural practice to increase grapevine performance and berry quality were thoroughly assessed. Grapevines were pulverised with a polyol solution containing 2 mM mannitol and 2 mM sorbitol, and the metabolome of grape berry exocarps and important metabolic pathways associated with berry quality and polyol metabolism were analysed at véraison and mature stages. By combining metabolomics analysis using UPLC-MS, enzyme activity assays and targeted transcriptional analyses, it was demonstrated that foliar application of polyols stimulated by 3.5-fold and 6-fold abscisic acid (ABA) biosynthesis at véraison and mature grape berries, respectively. It also stimulated the concentration of anthocyanins, stilbenes and total phenolics in exocarps through the upregulation of phenylpropanoid, stilbenoid and anthocyanin biosynthetic pathways shown by increases in phenylalanine-ammonia lyase (PAL) activity (3-fold) and VviPAL1 expression, stilbene synthase 1 (VviSTS1) transcripts (ca. 5-fold), UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) activity and VviUFGT1 expression, among other results, at the mature stage, when these changes were most noticeable. Many secondary metabolites synthesised in these pathways identified by UPLC-MS analysis were present at higher quantities in exocarps from polyol-treated plants such as fertaric acid, E-resveratrol, E-piceatannol, piceid, pallidol, E-ε-viniferin, myricetin-hexoside 1, cyanidin-3-O-glucoside and malvidin-3-O-(6-p-coumaroyl)-glucoside. Foliar application of low-concentration polyols is, therefore, a promising biostimulant-based strategy to improve grape berry quality and nutritional value in the current context of climate change.

Melatonin foliar application promoted anthocyanin accumulation boosted reactive oxygen scavenging by upregulating the key enzymes activities and genes expression of anthocyanin biosynthesis pathway in green and purple mustard under vanadium stress

Heavy metal vanadium (V) is poisonous to both plants and animals. Plant resistance to heavy metals stress can be conferred by the multifunctional signaling molecule melatonin, although the underlying mechanisms are still largely understood. We elucidated the significant contribution of the secondary metabolite anthocyanin to the enhancement of V stress tolerance by melatonin in both green and purple mustard genotypes. The effects of excessive V were leaf yellowing, slowed development, decreased photosynthetic activity, and an increase in the buildup of reactive oxygen species and malondialdehyde in both mustard genotypes. It interesting to note that during V stress, key anthocyanin biosynthetic pathway enzymes activities and leaf anthocyanin concentration increased while exogenous melatonin applied as foliar further boosted activities of key anthocyanin biosynthetic pathway enzymes and leaf anthocyanin concentration in both green and purple mustards. Furthermore, the application of exogenous melatonin increased the levels of endogenous melatonin and mitigated V toxicity. Conversely, the suppression of melatonin intensified V-induced phytotoxicity and delayed the accumulation of anthocyanin by downregulating the transcript levels of essential structural genes. Under V stress conditions, the genes expression related to anthocyanin production, antioxidant enzymes activities, and stress resistance was significantly higher in purple mustard than green mustard genotype. Additionally, melatonin decreased V deposition in roots and leaves. The results recommended that melatonin foliar application to mustard plants inhibited the availability of V to plant and promote V stress tolerance.

Metabolome and Transcriptome Reveal Chlorophyll, Carotenoid, and Anthocyanin Jointly Regulate the Color Formation of Triadica sebifera.

The Chinese tallow tree (Triadica sebifera) is an economically important plant on account of its ornamental value and oil-producing seeds. Leaf colour is a key characteristic of T. sebifera, with yellow-, red- and purple-leaved varieties providing visually impressive displays during autumn. In this study, we performed metabolomic and transcriptomic analyses to gain a better understanding of the mechanisms underlying leaf colour development in purple-leaved T. sebifera at three stages during the autumnal colour transition, namely, green, hemi-purple, and purple leaves. We accordingly detected 370 flavonoid metabolites and 10 anthocyanins, among the latter of which, cyanidin-3-xyloside and peonidin-3-O-glucoside were identified as the predominant compounds in hemi-purple and purple leaves. Transcriptomic analysis revealed that structural genes associated with the anthocyanin biosynthetic pathway, chlorophyll synthesis pathway and carotenoid synthesis pathway were significantly differential expressed at the three assessed colour stages. Additionally, transcription factors associated with the MYB-bHLH-WD40 complex, including 22 R2R3-MYBs, 79 bHLHs and 44 WD40 genes, were identified as candidate regulators of the anthocyanin biosynthetic pathway. Moreover, on the basis of the identified differentially accumulated anthocyanins and key genes, we generated genetic and metabolic regulatory networks for anthocyanin biosynthesis in T. sebifera. These findings provide comprehensive information on the leaf transcriptome and three pigments of T. sebifera, thereby shedding new light on the mechanisms underlying the autumnal colouring of the leaves of this tree.

MsMYB62-like as a negative regulator of anthocyanin biosynthesis in Malus spectabilis

ABSTRACT Crabapple is a valuable tree species in gardens due to its captivating array of flower and leaf colors, rendering it a favored choice in landscaping. The economic and ornamental values of Malus crabapple are closely associated with the biosynthesis of anthocyanin, a pigment responsible for its vibrant hues. The intricate regulation of anthocyanin biosynthesis involves the concerted activity of various genes. However, the specific mechanism governing this process in crabapple warrants in-depth exploration. In this study, we explored the inhibitory role of MsMYB62-like in anthocyanin biosynthesis. We identified MsDFR and MsANS as two downstream target genes of MsMYB62-like. These genes encode enzymes integral to the anthocyanin biosynthetic pathway. The findings demonstrate that MsMYB62-like directly binds to the promoters of MsDFR and MsANS, resulting in the downregulation of their expression levels. Additionally, our observations indicate that the plant hormone cytokinins exert a suppressive effect on the expression levels of MsMYB62-like, while concurrently upregulating MsDFR and MsANS. This study reveals that the MsMYB62-like-MsDFR/MsANS module plays an important role in governing anthocyanin levels in Malus crabapple. Notably, the regulatory interplay is modulated by the plant hormone cytokinins.

Transcriptomic and metabolic analysis unveils the mechanism behind leaf color development in Disanthus cercidifolius var. longipes.

Introduction: Leaf coloration in Disanthus cercidifolius var. longipes results from the interplay of various pigments undergoing complex catalytic reactions. Methods: We aimed to elucidate the mechanisms of pigment biosynthesis affecting leaf color transition in D. cercidifolius var. longipes by analyzing variations in pigment accumulation and levels of gene expression. Results: We identified 468, 577, and 215 differential metabolites in green leaves (GL), gradual-color-changing leaves (GCCL), and red leaves (RL), respectively, with 94 metabolites shared across all comparisons. Metabolite accumulation patterns were similar among GL, GCCL, and RL, with flavonoids being the main differential metabolites. Delphinidin, malvidin, and petunidin derivatives were mostly accumulated in GCCL, whereas cyanidin, pelargonidin, and peonidin derivatives accumulated in RL. Transcriptome sequencing was used to identify differentially expressed genes. The expression of anthocyanin biosynthetic pathway genes was associated with anthocyanin accumulation patterns. Discussion: Our findings reveal that the content of delphinidin, malvidin, petunidin, and carotenoids collectively determines the gradual transition of leaf color from green in spring and summer to green, purple, and orange-yellow in early autumn, whereas the content of cyanidin, peonidin, pelargonidin, and carotenoids together causes the autumnal transition to red or orange-red colors as leaves of D. cercidifolius var. longipes age.

CstMYB1R1, a REVEILLE-8-like transcription factor, regulates diurnal clock-specific anthocyanin biosynthesis and response to abiotic stress in Crocus sativus L.

CstMYB1R1 acts as a positive regulator of Crocus anthocyanin biosynthesis and abiotic stress tolerance which was experimentally demonstrated through molecular analysis and over-expression studies in Crocus and Nicotiana. Regulatory mechanics of flavonoid/anthocyanin biosynthesis in Crocus floral tissues along the diurnal clock has not been studied to date. MYB proteins represent the most dominant, functionally diverse and versatile type of plant transcription factors which regulate key metabolic and physiological processes in planta. Transcriptome analysis revealed that MYB family is the most dominant transcription factor family in C. sativus. Considering this, a MYB-related REVEILLE-8 type transcription factor, CstMYB1R1, was explored for its possible role in regulating Crocus flavonoid and anthocyanin biosynthetic pathway. CstMYB1R1 was highly expressed in Crocus floral tissues, particularly tepals and its expression was shown to peak at dawn and dusk time points. Anthocyanin accumulation also peaked at dawn and dusk and was minimum at night. Moreover, the diurnal expression pattern of CstMYB1R1 was shown to highly correlate with Crocus ANS/LDOX gene expression among the late anthocyanin pathway genes. CstMYB1R1 was shown to be nuclear localized and transcriptionally active. CstMYB1R1 over-expression in Crocus tepals enhanced anthocyanin levels and upregulated transcripts of Crocus flavonoid and anthocyanin biosynthetic pathway genes. Yeast one hybrid (Y1H) and GUS reporter assay confirmed that CstMYB1R1 interacts with the promoter of Crocus LDOX gene to directly regulate its transcription. In addition, the expression of CstMYB1R1 in Nicotiana plants significantly enhanced flavonoid and anthocyanin levels and improved their abiotic stress tolerance. The present study, thus, confirmed positive role of CstMYB1R1 in regulating Crocus anthocyanin biosynthetic pathway in a diurnal clock-specific fashion together with its involvement in the regulation of abiotic stress response.

Metabolome combined with transcriptome profiling reveals the dynamic changes in flavonoids in red and green leaves of Populus × euramericana 'Zhonghuahongye'.

Flavonoids are secondary metabolites that have economic value and are essential for health. Poplar is a model perennial woody tree that is often used to study the regulatory mechanisms of flavonoid synthesis. We used a poplar bud mutant, the red leaf poplar variety 2025 (Populus × euramericana 'Zhonghuahongye'), and green leaves as study materials and selected three stages of leaf color changes for evaluation. Phenotypic and biochemical analyses showed that the total flavonoid, polyphenol, and anthocyanin contents of red leaves were higher than those of green leaves in the first stage, and the young and tender leaves of the red leaf variety had higher antioxidant activity. The analyses of widely targeted metabolites identified a total of 273 flavonoid metabolites (114 flavones, 41 flavonols, 34 flavonoids, 25 flavanones, 21 anthocyanins, 18 polyphenols, 15 isoflavones, and 5 proanthocyanidins). The greatest difference among the metabolites was found in the first stage. Most flavonoids accumulated in red leaves, and eight anthocyanin compounds contributed to red leaf coloration. A comprehensive metabolomic analysis based on RNA-seq showed that most genes in the flavonoid and anthocyanin biosynthetic pathways were differentially expressed in the two types of leaves. The flavonoid synthesis genes CHS (chalcone synthase gene), FLS (flavonol synthase gene), ANS (anthocyanidin synthase gene), and proanthocyanidin synthesis gene LAR (leucoanthocyanidin reductase gene) might play key roles in the differences in flavonoid metabolism. A correlation analysis of core metabolites and genes revealed several candidate regulators of flavonoid and anthocyanin biosynthesis, including five MYB (MYB domain), three bHLH (basic helix-loop-helix), and HY5 (elongated hypocotyl 5) transcription factors. This study provides a reference for the identification and utilization of flavonoid bioactive components in red-leaf poplar and improves the understanding of the differences in metabolism and gene expression between red and green leaves at different developmental stages.

A transient expression tool box for anthocyanin biosynthesis in Nicotiana benthamiana.

Transient expression in Nicotiana benthamiana offers a robust platform for the rapid production of complex secondary metabolites. It has proven highly effective in helping identify genes associated with pathways responsible for synthesizing various valuable natural compounds. While this approach has seen considerable success, it has yet to be applied to uncovering genes involved in anthocyanin biosynthetic pathways. This is because only a single anthocyanin, delphinidin 3-O-rutinoside, can be produced in N. benthamiana by activation of anthocyanin biosynthesis using transcription factors. The production of other anthocyanins would necessitate the suppression of certain endogenous flavonoid biosynthesis genes while transiently expressing others. In this work, we present a series of tools for the reconstitution of anthocyanin biosynthetic pathways in N. benthamiana leaves. These tools include constructs for the expression or silencing of anthocyanin biosynthetic genes and a mutant N. benthamiana line generated using CRISPR. By infiltration of defined sets of constructs, the basic anthocyanins pelargonidin 3-O-glucoside, cyanidin 3-O-glucoside and delphinidin 3-O-glucoside could be obtained in high amounts in a few days. Additionally, co-infiltration of supplementary pathway genes enabled the synthesis of more complex anthocyanins. These tools should be useful to identify genes involved in the biosynthesis of complex anthocyanins. They also make it possible to produce novel anthocyanins not found in nature. As an example, we reconstituted the pathway for biosynthesis of Arabidopsis anthocyanin A5, a cyanidin derivative and achieved the biosynthesis of the pelargonidin and delphinidin variants of A5, pelargonidin A5 and delphinidin A5.

The effect of osmotic stress, lighting spectrum and temperature on growth and gene expression related to anthocyanin biosynthetic pathway in wild strawberry (Fragaria vesca L.) in vitro

ABSTRACT The goal of this research was to evaluate the effect of light, temperature, sucrose and PEG on the growth of Fragaria vesca in vitro and the expression of regulatory Myb10, WD40 and enzyme-coding genes CHI, CHS, DFR, EGL, F3H and UFGT, which are essential for anthocyanin biosynthesis. We observed plants’ response to osmotic stress, the decrease in growth and microshoot weight. A change in the expression of the investigated genes was evident under the suboptimal concentration of sucrose. The addition of PEG to the medium caused a decrease in microshoot weight and gene expression. Blue + red lights of the LED lighting system significantly affected microshoot growth in vitro. Red and blue + red + UV lights slightly reduced microshoot weight and caused a reddish colour of petioles, which indicate increased anthocyanin synthesis. Moreover, most of the studied genes’ expression tended to increase when shoots were exposed to blue, blue + red and blue + red + UV lights. A temperature of 15°C (vs 22°C) significantly reduced the mean fresh weight of microshoots while increasing CHI and CHS gene expression and decreasing WD40 gene expression. Exposure to a higher temperature (30°C) induced the vitrification of microshoots, although the fresh weight did not differ from that of the control. Gene expression also depended on the duration of exposure. In the case of CHS, gene expression remained the same or increased after exposure for 1 week and then decreased after exposure for 4 weeks.

Characterization of the varied output from the anthocyanin pathway in Phalaenopsis-type Dendrobium hybrids and its relationship with flower coloration

Floral color is an important Phalaenopsis-type Dendrobium hybrids (Den-Phals) trait in the flower markets. Anthocyanins are key pigments contributing to purple, pink, and violet floral color. However, the impact of anthocyanin composition on flower coloration has not yet been fully elucidated. In this study, the flower color characteristics of 62 Den-Phals were evaluated. Most flowers contained purple, while pure blue, orange, and red floral colors were scarce. In total 35 anthocyanins were identified and two acylated pelargonidin glycosides were tentatively characterized using ultra-performance liquid chromatography (UPLC)-mass spectrum (MS) analysis. Unfortunately, the major anthocyanin components remained unidentified. However, the hydrolysis anthocyanins to their anthocyanidins suggested that the cyanidin was the major component in most hybrids with a relative content over 85 %. Pelargonidin was almost the only pigment in reddish pink flowers. The slightly higher peonidin content was found in purple-violet flowers (up to 6.23 %). The trend indicated that with the increase of total anthocyanidin and cyanidin pigments, the floral color deepened and turned to purple. Flavones or flavonols as co-pigments contributed to brighter and yellower color. From the above results, the varied output from the anthocyanin biosynthetic pathway among Den-Phals was used to identify the rate-limiting steps involved and a strategy for the cultivation of pure red, blue, and violet flowers is proposed. The study provides a basis for screening excellent germplasm and improving floral color in Den-Phals.

Association Analysis of Transcriptome and Targeted Metabolites Identifies Key Genes Involved in Iris germanica Anthocyanin Biosynthesis.

The anthocyanin biosynthetic pathway is the main pathway regulating floral coloration in Iris germanica, a well-known ornamental plant. We investigated the transcriptome profiles and targeted metabolites to elucidate the relationship between genes and metabolites in anthocyanin biosynthesis in the bitone flower cultivar 'Clarence', which has a deep blue outer perianth and nearly white inner perianth. In this study, delphinidin-, pelargonidin-, and cyanidin-based anthocyanins were detected in the flowers. The content of delphinidin-based anthocyanins increased with the development of the flower. At full bloom (stage 3), delphinidin-based anthocyanins accounted for most of the total anthocyanin metabolites, whereas the content of pelargonidin- and cyanidin-based anthocyanins was relatively low. Based on functional annotations, a number of novel genes in the anthocyanin pathway were identified, which included early biosynthetic genes IgCHS, IgCHI, and IgF3H and late biosynthetic genes Ig F3'5'H, IgANS, and IgDFR. The expression of key structural genes encoding enzymes, such as IgF3H, Ig F3'5'H, IgANS, and IgDFR, was significantly upregulated in the outer perianth compared to the inner perianth. In addition, most structural genes exhibited their highest expression at the half-color stage rather than at the full-bloom stage, which indicates that these genes function ahead of anthocyanins synthesis. Moreover, transcription factors (TFs) of plant R2R3-myeloblastosis (R2R3-MYB) related to the regulation of anthocyanin biosynthesis were identified. Among 56 R2R3-MYB genes, 2 members belonged to subgroup 4, with them regulating the expression of late biosynthetic genes in the anthocyanin biosynthetic pathway, and 4 members belonged to subgroup 7, with them regulating the expression of early biosynthetic genes in the anthocyanin biosynthetic pathway. Quantitative real-time PCR (qRT-PCR) analysis was used to validate the data of RNA sequencing (RNA-Seq). The relative expression profiles of most candidate genes were consistent with the FPKM of RNA-seq. This study identified the key structural genes encoding enzymes and TFs that affect anthocyanin biosynthesis, which provides a basis and reference for the regulation of plant anthocyanin biosynthesis in I. germanica.

Fine mapping and identification of CaTTG1, a candidate gene that regulates the hypocotyl anthocyanin accumulation in Capsicum annuum L.

Pepper (Capsicum annuum L.) is a typical self-pollinating crop with obvious heterosis in hybrids. Consequently, the use of morphological markers during the pepper seedling stage is crucial for pepper breeding. The color of hypocotyl is widely used as a phenotypic marker in crossing studies of pepper. Pepper accessions generally have purple hypocotyls, which are mainly due to the anthocyanin accumulation in seedlings, and green hypocotyls are rarely observed in pepper. Here we reported the characterization of a green hypocotyl mutant of pepper, Cha1, which was identified from a pepper ethyl methanesulfonate (EMS) mutant library. Fine mapping revealed that the causal gene, CaTTG1, belonging to the WD40 repeat family, controlled the green hypocotyl phenotype of the mutant. Virus-induced gene silencing (VIGS) confirmed that CaTTG1 regulated anthocyanin accumulation. RNA-seq data showed that expression of structural genes CaDFR, CaANS, and CaUF3GT in the anthocyanin biosynthetic pathway was significantly decreased in Cha1 compared to the wild type. Yeast two-hybrid (Y2H) experiments also confirmed that CaTTG1 activated the synthesis of anthocyanin structural genes by forming a MBW complex with CaAN1 and CaGL3. In summary, this study provided a green hypocotyl mutant of pepper, and the Kompetitive Allele Specific PCR (KASP) marker developed based on the mutation site of the underlying gene would be helpful for pepper breeding.

Transcriptome analysis of genes involved in flower and leaf color of Oncidium by RNA-seq

Oncidium, an important tropical orchid, has high ornamental value due to its specific color and occupies a significant market position for the worldwide flower. Transcriptome analysis of flower and leaf color formation provides new sources for producing novel Oncidium hybridum cultivars. We sequenced 12 samples of flowers (yellow and white) and leaves (striped and regular) of O. hybridum and assembled 381,136 and 453,566 unigene sequences from RNA-seq data, respectively. Among unigenes, 662 and 1,324 differentially expressed genes were identified in flower and leaf samples, respectively. Gene ontology and pathway enrichment showed that secondary metabolite biosynthetic pathways were responsible for flower and leaf color formation. It was determined that UGT75C1, E2.4.1.115, CCD7, E2.1.1.76, and CCoAOMT are involved in regulating flower color, and UGT75C1, LHCB, UGT, RP-L18Ae, and ABCB1 play crucial roles in regulating leaf color. Kyoto Encyclopedia of Genes and Genomes analysis revealed that UGT75C1 was significantly enriched in the anthocyanin biosynthetic pathway, showing effects on flower and leaf colors. This study was the first detailed analysis of the molecular mechanisms of O. hybridum flower and leaf colors, and the results advanced the understanding of the genetic basis of flower and leaf colors; they also provided additional support for improving commercial value and producing novel cultivars of O. hybridum.