Anthocyanin Composition Research Articles

Genome-wide identification of the AOMT gene family in wax apple and functional characterization of SsAOMTs to anthocyanin methylation.

Anthocyanins are major pigments in the peels of red-series wax apple fruits, and two principal components of them, namely, the cyanin and the peonidin, are non-methoxylated and methoxylated anthocyanins, respectively. Anthocyanin O-methyltransferases (AOMTs) are an important group of enzymes that have the ability to catalyze anthocyanins methylation to promote the solubility, stability, and bioactivity of anthocyanins. Although AOMT genes have been studied in a variety of plants, the function of them in wax apple is generally not well understood. The anthocyanin composition in peels of two wax apple cultivars was determined by High Performance Liquid Chromatography Tandem Mass Spectrometry (HPLS-MS). The genome-wide analysis of the AOMT genes was performed with bioinformatics technology, and the expression patterns of different plant tissues, cultivars, fruit ripening stages, and exogenous abscisic acid (ABA) treatments were analyzed by transcriptome sequencing analysis and real-time quantitative PCR verification. An initial functional evaluation was carried out in vitro using recombinant the Anthocyanin O-methyltransferase Gene 5 of S. samarangense (SsAOMT5) protein. Only two main compositions of anthocyanin were found in peels of two wax apple cultivars, and it was worth noting that Tub Ting Jiang cultivar contained non-methoxylated anthocyanin (Cy3G) only, whereas Daye cultivar contained both non-methoxylated and methoxylated (Pn3G) anthocyanins. A total of six SsAOMT genes were identified in the whole genome of wax apple, randomly distributing on three chromosomes. A phylogenic analysis of the protein sequences divided the SsAOMT gene family into three subgroups, and all SsAOMTs had highly conserved domains of AOMT family. In total, four types of stress- related and five types of hormone- related cis-elements were discovered in the promoter region of the SsAOMTs. Expression pattern analysis showed that SsAOMT5 and SsAOMT6 were expressed in all tissues to varying degrees; notably, the expression of SsAOMT5 was high in the flower and fruit and significantly higher in Daye peels than those of other cultivars in the fruit ripening period. Exogenous ABA treatment significantly increased anthocyanin accumulation, but the increase of methoxylated anthocyanin content did not reach significant level compared with those without ABA treatment, whereas the expression of SsAOMT5 upregulated under ABA treatment. We identified two homologous SsAOMT5 genes from Daye cultivar (DSsAOMT5) and Tub Ting Jiang cultivar (TSsAOMT5); the results of functional analyses to two SsAOMT5 recombinant proteins in vitro demonstrated that DSsAOMT5 showed methylation modification activity, but TSsAOMT5 did not. In conclusion, SsAOMT5 was responsible for methylated anthocyanin accumulation in the peels of wax apple and played an important role in red coloration in wax apple peels.

Phytochemical profiling, antioxidant capacities and anthocyanin compositions of the pigmented rice (Oryza sativa) of north-east India

The association between the consumption of pigmented rice (Oryza sativa L.) and the improvement of human health is generating a great deal of interest among the researchers. An experiment was conducted during 2020 and 2021 at Assam Agricultural University, Jorhat, Assam to study the phytochemical profiling, antioxidant capacities and anthocyanin compositions of the 14 pigmented rice genotypes. The results were compared with the well-known traditional pigmented rice variety from Manipur, Poreiton Chakhao. The total phenolic content, total flavonoid content and total anthocyanin content ranged from 37.101 to 493.611 mg gallic acid equivalent/100 g, 53.316 to 151.667 mg quercetin equivalent/100 g and 1.006 to 13.904 mg cyanidin-3-chloride equivalent/100 g in the studied rice genotypes. The amount of rice showing 50% DPPH free radical scavenging activity (IC 50 value) ranged from 6.610 to 29.376 mg. The HPLC analysis revealed presence of both cyanidin-3-glucoside and peonidin-3-glucoside in the black pigmented rice genotypes, viz. TTB Black Rice 7, Chakhao-1, Chakhao-2 and TTB Black Rice 11. However, in the red rice genotypes analysed (Balam and Nepali Chakuwa), these two anthocyanins were not detected.

Effects of phenolic compounds of colored wheats on colorectal cancer cell lines

In this study, the different colored wheat brans were analyzed and compared for phenolic content (PC), phenolic compositions, and the total antioxidant capacity (TEAC) with methods based on the ability to eliminate radicals of 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH), and anthocyanin compositions. This study also aims to characterize the bioactive components of wheat grain genotypes as well as to test the protective and rescuer effects of their extracts on colorectal cancer (CRC) cell lines. PCs in the bound insoluble fraction of red wheat bran, blue wheat bran, and purple wheat bran were determined as 369.60, 446.95, and 486.79 mg gallic acid equivalents (GAE)/100 g wheat bran, respectively, while strong relationships were detected between PC and antioxidant activity (DPPH and ABTS) results. HPLC anal-ysis of phenolic extracts demonstrated that ferulic acid was determined as the dominant phenolic acid in the bound fractions of red, purple, and blue wheats. In the free fractions, p-coumaric acid (11.55 µg/100 g wheat bran) was the dominant phenolic acid for red wheat bran, whereas ellagic acid (14.72 and 11.55 µg/100 g wheat bran) was the highest phenolic acid for purple and blue wheat brans, respectively. In bound fractions, ferulic acid was the highest phenolic acid for red (988.39 µg/100 g wheat bran), purple (1948.76 µg/100 g wheat bran), and blue (2263.96 µg/100 g wheat bran) wheat brans. On the other hand, Cyanidin-3-O-glucoside chloride was the predominant anthocyanin in free extracts of purple and blue wheat brans. In line with the antioxidant activities and phenolic acid concentrations, the blue wheat bran extracts increased CRC cell viability nonsignificantly in HT-29 and HCT-116 cell lines, whereas purple wheat bran extract had a significantly higher (P = 0.0361) rescuer effect compared to vehicle control under 50 µM H2O2 concentration. In conclusion, the in vitro data here show that blue and purple wheat brans are posing a novel means to increase the defense of cells against oxidative stress and cell death.

Content, Composition, and Biosynthesis of Anthocyanin in Fragaria Species: A Review

Anthocyanins are responsible for fruit coloration and are beneficial to human health. The fruits of cultivated strawberry (Fragaria ×ananassa) varieties are colorful, a trait that attracts consumers. The fruits of wild Fragaria species, close relatives of the cultivated strawberry, vary in color. In this review, we describe the content and composition of anthocyanins in cultivated and wild strawberry varieties. We also explore the biosynthetic pathway of anthocyanins, including their transcriptional regulation mechanisms. Additionally, we discuss the effect of environmental factors on anthocyanin accumulation. This review will inform further studies toward developing anthocyanin-rich strawberries via environmental control and exogenous application of compounds.

Effect of Applied Shading During Fruit Ripening on Tannin Structure-Activity Relationships of Grape Skin Extracts.

Historically, the effect of wine grape shading on flavonoids have investigated the impact of light incidence on proanthocyanidin (PA), flavonol, or anthocyanin concentration. In addition to concentration, the current experiment was designed to look at changes in proanthocyanidin composition, size and tannin activity through ripening. Tannin activity is a methodology for assessing the impact of structure and size on the affinity of tannin towards a hydrophobic surface and is considered to be a proxy for predicted astringency descriptive quality. In 2016 a shade cloth study was imposed on Cabernet Sauvignon on Mt. Veeder, within the larger Napa Valley viticultural area. A control, which was unshaded, and two treatments consisting of 40% and 80% shade were applied at the onset of veraison. Results showed significant differences in the composition and concentration of anthocyanins throughout ripening. Compositional differences in proanthocyanidin were also observed, where shaded treatments had a significantly higher proportion of galloylated subunits. The molecular mass of the extracted tannin was significantly lower in the unshaded control than in the 80% shade treatment. These factors led to a lower measured tannin activity in extracts from exposed fruit. This work suggests that manipulation of canopy architecture, such as artificial shading, leads to changes in berry pigmentation, tannin composition and activity. These results show that astringency and mouthfeel characteristics of a wine may be altered by vineyard management practices. This article is protected by copyright. All rights reserved.

Effects of four drying methods on the quality, antioxidant activity and anthocyanin components of blueberry pomace

The effects of drying on the quality, antioxidant activity and anthocyanin components of blueberry pomace were investigated using hot air drying (HAD), freeze-drying (FD), microwave vacuum drying (MVD) and microwave freeze vacuum drying (MFD). The quality, antioxidant activity, and individual anthocyanin compositions of blueberry pomace were evaluated and compared with the non-dried control. MVD produced the highest levels of total phenols, total anthocyanins, total sugars and color values in blueberry pomace while HAD gave the lowest. The ABTS +and DPPH radical scavenging abilities of blueberry pomace were significantly high when MVD was used. Moreover, FD, MVD, and MFD resulted in the retention of significantly more anthocyanin species than HAD as determined by HPLC coupled to the mass spectrometer. Overall, MVD proved to be the best technique for preservation of antioxidant capacity and natural color. Therefore, MVD of blueberry pomace can improve efficiency and productivity of the blueberry fruit processing industry while reducing the environmental burden.Graphical

Integrated Analysis of Transcriptome and Metabolome Reveals New Insights into the Molecular Mechanism Underlying the Color Differences in Wolfberry (Lycium barbarum)

Wolfberry (Lycium barbarum L.) is a small Solanaceae shrub with medicinal and edible homology, and widely used as ethnobotanical medicine and nutraceutical food. The wolfberry fruits mainly have red, purple, and yellow phenotypes. Wolfberries are rich in flavonoids, which are natural water-soluble pigments that endow a variety of colors in plants There are very few investigations on mechanism of flavonoids biosynthesis and fruit coloring reported about wolfberry. The widely targeted metabolome and transcriptome analysis were performed to obtain metabolite and gene expression profiles of red, yellow, and purple wolfberries and to explain the underlying molecular mechanism of the color differences in wolfberry. As result, metabolomics analysis revealed that the bluish anthocyanins Malvidin and petunidin trended to accumulate in purple wolf-berry, while red and yellow wolfberries trended to accumulate more yellowish flavonoids. And transcriptome analysis showed that flavonoid synthesis-related genes, such as CHS, F3H, ANS and DFR, and several MYB and bHLH genes were differentially expressed among wolfberries in different colors: most of them were more highly expressed in purple wolfberries than in red and yellow ones. In conclusion, the different flavonoids’ accumulation patterns may result in the different fruit colors of wolfberry, and the MYB or bHLH transcription factors could regulate the expression of flavonoids biosynthesis related genes to change the composition of flavonoids or anthocyanins in wolfberry fruits and result in varied fruit colors. These findings provide new insights into the underlying molecular mechanism of the fruit color differences in wolfberry and provide new ideas for molecular breeding of wolfberry.

Investigation of the complex of phenolic compounds in fruits of the domestic plum (Prunus domestica L.) in conditions of Tambov Region

The fruits of Prunus domestica L. are a valuable source of phenolic compounds, in particular hydroxycinnamic acids and their esters. Hybrid origin of Prunus domestica hexaploid species, a huge variety of varieties and di erent growing areas do not allow scientists to make a de nitive conclusion about certain phenolic compounds in plum fruits. The purpose of our research is to determine and identify the phenolic composition of fruits of Prunus domestica L. growing in the Tambov region of the Russian Federation. Eight varieties of universal and table plum were taken as objects of research. Experimental plantations were planted in 2014 according to the scheme of 6x3 m. SVG 11-19 was used as a rootstock. The study of phenolic components of plum fruits was performed by HPLC using a Thermo Ultimate 3000 liquid chromatograph equipped with a DAD-3000. Separation was performed on a Hypersil Gold C18 column (4.6 × 250 mm, 5 μm). The binary mobile phase gradient was phosphate and acetonitrile. For the analysis, plum fruits were extracted with 70 % aqueous methanol. The levels of hydroxycinnamic acids in the plum fruits studied were determined: 4-ca eoylquinic acid-5.33…181.54 mg/100 g, 5- ca eoylquinic acid - 1.69…31.73 mg/100 g, 3-p-coumaroylquinic acid - 0.54…5.77 mg/100 g, 3- ca eoylquinic acid-0.04…0.63 mg/100 g. Among avonols, the predominant component is quercitin-3-rutinoside - 0.77…9.17 mg/100 g. Three anthocyanins were identi ed in the studied plum fruits: cyanidin-3-glucoside, cyanidin-3-rutinoside and peonidin-3-glucoside. In the structural composition of plum fruit anthocyanins, cyanidin-3-glucoside dominates, accounting for 60 to 90 % of the total anthocyanin content. The obtained data on the content of phenolic components in fruits of isolated plum varieties (Prunus domestica L.) indicate the promise of their further study for breeding studies and as a source of hydroxycinnamic acids, anthocyanins and avonols in the diet of the population

Influence of Different Drying Methods on Anthocyanins Composition and Antioxidant Activities of Mangosteen (Garcinia mangostana L.) Pericarps and LC-MS Analysis of the Active Extract.

Mangosteen pericarps (MP) often end up as agricultural waste despite being rich in powerful natural antioxidants such as anthocyanins and xanthones. This study compared the effect of different drying processes and times on phenolic compounds and antioxidant activities of MP. Fresh MP were subjected to 36 and 48 h of freeze-drying (-44 ± 1 °C) and oven-drying (45 ± 1 °C), and 30 and 40 h of sun-drying (31 ± 3 °C). The samples were analyzed for anthocyanins composition, total phenolic content (TPC), total flavonoid content (TFC), antioxidant activities, and color characteristics. Analysis of liquid chromatography-mass spectrometry (LC-MS) with electrospray ionization identified two anthocyanins in MP: cyanidin-3-O-sophoroside and cyanidin-3-O-glucoside. Overall, the drying process, time, and their interactions significantly (p < 0.05) influenced the phenolic compounds, antioxidant activities, and color in MP extracts. Both freeze-drying after 36 h (FD36) and 48 h (FD48) possessed significantly (p < 0.05) higher total anthocyanins (2.1-2.2 mg/g) than other samples. However, FD36 was associated with significantly (p < 0.05) higher TPC (~94.05 mg GAE/g), TFC (~621.00 mg CE/g), and reducing power (~1154.50 μmol TE/g) compared to FD48. Moreover, FD36 is more efficient for industrial applications due to less time and energy consumption. Subsequently, obtained dried MP extracts could be further utilized as an alternative to synthetic food colorants.

Phytochemical Composition of Cranberry (Vaccinium oxycoccos L.) Fruits Growing in Protected Areas of Lithuania.

The fruits of Vaccinium oxycoccos L. are an important source of bioactive compounds with antibacterial, antifungal, and anti-inflammatory effects. Studies on the phytochemical analysis of cranberry fruit samples showed that the qualitative and quantitative composition of biologically active compounds varied in cranberry fruit samples collected from different types of wetland sites: the total anthocyanin content was 698 ± 24-8352 ± 200 µg/g, the total flavonol content-518 ± 16-2811 ± 31 µg/g, the total content of triterpene compounds-4060 ± 122-6542 ± 157 µg/g, the content of chlorogenic acid-17 ± 0.4 µg/g to 1224 ± 41 µg/g, and the total content of proanthocyanidins-919 ± 19 µg EE/g to 3038 ± 137 µg EE/g. The percentage composition of anthocyanins in cranberry fruit varied between the different wetland sites: in some cranberry fruit samples, four anthocyanins (cyanidin-3-galactoside, cyanidin-3-arabinoside, peonidin-3-galactoside, and peonidin-3-arabinoside) were predominant, while in other samples, six anthocyanins (cyanidin-3-galactoside, cyanidin-3-arabinoside, peonidin-3-galactoside, peonidin-3-arabinoside, cyanidin-3-glucoside, and peonidin-3-glucoside) predominated. The results of these studies showed the differences in the composition of secondary metabolites in the studied cranberry samples and prove that the standardization of the qualitative and quantitative composition of cranberry fruit raw materials and the application of routine tests are necessary for the expansion of the use of botanical raw materials in the production of functional foods and phytopreparations.

Metabolomics and transcriptomics provide insights into the molecular mechanisms of anthocyanin accumulation in the seed coat of differently colored mung bean (Vigna radiata L.)

Black mung bean is rich in anthocyanin, however, the accumulation and the molecular mechanism of anthocyanin synthesis in black mung bean are unclear. In this study, anthocyanin metabolomics and transcriptomics on the seed coats of two different colors of mung bean were performed to clarify the composition of anthocyanins, and identify transcription factors involved in regulating anthocyanin biosynthesis. In the mature stage, 23 kinds of anthocyanin compounds were identified. All anthocyanin components contents were significantly higher in seed coat of black mung bean compare with green mung bean. Transcriptome analysis suggested that most of the structural genes for anthocyanin biosynthesis and some potential regulatory genes were significantly differentially expressed. WGCNA suggested VrMYB90 was an important regulatory gene in anthocyanin biosynthesis. Arabidopsis thaliana overexpressing VrMYB90 showed significant accumulation of anthocyanins. PAL, 4CL, DFR, F3′5′H, LDOX, F3′H and UFGT were up-regulated in 35S:VrMYB90 Arabidopsis thaliana. These findings provide valuable information for understanding the synthesis mechanism of anthocyanins in black mung bean seed coats.

Effect of Growth Stages on Anthocyanins and Polyphenols in the Root System of Sweet Potato.

The storage roots of purple-fleshed sweet potato contain a variety of anthocyanins and polyphenols. Little is known about changes in the total content and composition of anthocyanins and polyphenols in the early growth stages of the root system. In this study, we investigated the changes in anthocyanins and polyphenols in the root system of purple-fleshed sweet potato cultivars at 15, 30, 45, and 60 days after transplant (DAT). Unexpectedly, the highest percentage of acylated anthocyanins in three purple-fleshed cultivars among all growth stages was at 15 DAT. On the other hand, the total polyphenol content in the early growth stages of the root system increased rapidly toward 45 DAT, just before the beginning of storage root enlargement, and then decreased rapidly as the storage roots began to enlarge. These data indicate that the early growth stage of the root system is a critical time. This timing may present a strategy to maximize the accumulation of polyphenols with high antioxidant activity, as well as acylated anthocyanins, to protect against abiotic and biotic stresses.

Application of salicylic acid to cv. Muscat Hamburg grapes for quality improvement: Effects on typical volatile aroma compounds and anthocyanin composition of grapes and wines

Aromas and anthocyanins are important factors affecting grapes and wines quality. Monoterpenes are regarded as typical volatile aroma compounds in Muscat Hamburg grapes. In this study, we determined the effects of exogenous salicylic acid (SA) on anthocyanins and monoterpene components of Muscat Hamburg (Vitis vinifera) berries and wines. Berries were treated with aqueous solutions of SA (0.0, 50, 100 and 200 mg/L) at E-L 34 (berry color change stage) during 2018 and 2019 seasons. 50 mg/L salicylic acid (SA50) significantly increased the sum of anthocyanins contents in ripe berries, while 100 mg/L and 200 mg/L salicylic acid had the opposite effect. Additionally, SA50 significantly promoted the free or bound monoterpenes accumulation in mature grapes. The concentrations of geraniol, neral, geranic acid, nerol, nerol oxide, and phellandrene were higher in the wine produced from SA50 treatment grapes than in the wine produced from control groups. The SA50 treatments increased the transcription of some key genes related to monoterpene biosynthesis, namely, VviPNLinNer1, VviPNLinNer2, VviPNLNGl2, VviPNLNGl4, VviTer, and VviGT14, which accelerated the synthesis and accumulation of monoterpene components in grapes. Overall, these study findings can provide fundamental knowledge for future improvements to grape aromas and anthocyanins.

Purple Rehmannnia : investigation of the activation of R2R3-MYB transcription factors involved in anthocyanin biosynthesis.

Engineering anthocyanin biosynthesis in herbs could provide health-promoting foods for improving human health. Rehmannia glutinosa is a popular medicinal herb in Asia, and was a health food for the emperors of the Han Dynasty (59 B.C.). In this study, we revealed the differences in anthocyanin composition and content between three Rehmannia species. On the 250, 235 and 206 identified MYBs in the respective species, six could regulate anthocyanin biosynthesis by activating the ANTHOCYANIDIN SYNTHASE (ANS) gene expression. Permanent overexpression of the Rehmannia MYB genes in tobacco strongly promoted anthocyanin content and expression levels of NtANS and other genes. A red appearance of leaves and tubers/roots was observed, and the total anthocyanin content and the cyanidin-3-O-glucoside content were significantly higher in the lines overexpressing RgMYB41, RgMYB42 and RgMYB43 from R. glutinosa,as well as RcMYB1 and RcMYB3 in R. chingii and RhMYB1 from R. henryi plants. Knocking out of RcMYB3 by CRISPR/Cas9 gene editing resulted in the discoloration of the R. chingii corolla lobes, and decreased the content of anthocyanin. R. glutinosa overexpressing RcMYB3 displayed a distinct purple color in the whole plants, and the antioxidant activity of the transgenic plants was significantly enhanced compared to WT. These results indicate that Rehmannia MYBs can be used to engineer anthocyanin biosynthesis in herbs to improve their additional value, such as increased antioxidant contents. This article is protected by copyright. All rights reserved.

Extraction and Compositional Analysis of Anthocyanins and Polysaccharides from Aronia Melanocarpa Fruit

We have examined the basic composition of anthocyanins and polysaccharides, the active components in Aronia melanocarpa fruit. To this end, microwave-assisted ethanol extraction and cellulase enzymatic hydrolysis were employed to extract anthocyanins and crude polysaccharides, respectively. Aronia melanocarpa fruit polysaccharides were separated and purified by DEAE-52 cellulose and Sephadex G-100 glucan gel column chromatography, followed by high-performance liquid chromatography. The results demonstrated that the optimal extraction parameters of anthocyanin from Aronia melanocarpa fruit were a 60% ethanol volume fraction, a liquid-to-solid ratio of 30:1, a microwave time of 60 s, and a microwave power of 210 W. The extracted anthocyanins reached 200.47 mg/100 g, while the yield of polysaccharides was 3.82%. Anthocyanins in Aronia melanocarpa fruit were mainly composed of cyanidin, delphinidin, and mallow pigment, among which cyanidin was the main component, with a content as high as 90.79%. There were two distinct fractions of polysaccharides. The fraction-I polysaccharides were mainly composed of rhamnose, galacturonic acid, glucose, galactose, and arabinose, with a molar ratio of 8.80: 8.97: 2.38: 27.46: 47.94. In contrast, the fraction-II polysaccharides were primarily composed of rhamnose, glucuronic acid, galactose, xylose, and arabinose, with a molar ratio of 1.67: 9.22: 8.47: 70.69: 6.16. The results of this study can provide a reference for the in-depth development of Aronia melanocarpa resources

Improved Phenolic Profile, Sensory Acceptability, and Storage Stability of Strawberry Decoction Beverages Added with Blueberry Decoctions

Blueberries are rich in polyphenols, anthocyanins, and proanthocyanidins; however, they are expensive. In contrast, strawberries have a lower cost and are rich in ellagitannins. Therefore, a strawberry-blueberry blend decoction could produce a low-cost beverage with a rich and diverse phytochemical profile. In this study, we developed three berry-based beverages: blend strawberry-blueberry (SBB), strawberry (SB), and blueberry (BB). The polyphenol profile of the beverages was characterized by UPLC-ESI-Q-ToF MSE, an acceptability test was carried out with potential consumers, and a stability analysis was performed under commercial storage conditions (4, 25, and 34 °C). The SBB exhibited a good sensorial preference (score of 81.3) and showed high contents and a diverse composition of anthocyanins and proanthocyanidins, which were up to 3.0- and 1.2-fold higher compared to the SB, respectively. Regarding the storage stability, the SBB showed better retention of lightness (97.9%) and red color (66.7%) at the end of the storage under refrigerated conditions (4 °C) compared to the SB. Therefore, these results demonstrate that using blended berry fruits leads to the creation of a functional beverage that has higher nutraceutical potential than single-berry-based beverages.

Transcriptome and metabolome analyses reveal molecular mechanisms of anthocyanin-related leaf color variation in poplar (Populus deltoides) cultivars.

Colored-leaf plants are increasingly popular for their aesthetic, ecological, and social value, which are important materials for research on the regulation of plant pigments. However, anthocyanin components and the molecular mechanisms of anthocyanin biosynthesis in colored-leaf poplar remain unclear. Consequently, an integrative analysis of transcriptome and metabolome is performed to identify the key metabolic pathways and key genes, which could contribute to the molecular mechanism of anthocyanin biosynthesis in the colored-leaf cultivars poplar. In this study, integrated metabolite and transcriptome analysis was performed to explore the anthocyanin composition and the specific regulatory network of anthocyanin biosynthesis in the purple leaves of the cultivars 'Quanhong' (QHP) and 'Zhongshanyuan' (ZSY). Correlation analysis between RNA-seq data and metabolite profiles were also performed to explore the candidate genes associated with anthocyanin biosynthesis. R2R3-MYB and bHLH TFs with differential expression levels were used to perform a correlation analysis with differentially accumulated anthocyanins. A total of 39 anthocyanin compounds were detected by LC-MS/MS analysis. Twelve cyanidins, seven pelargonidins, five delphinidins, and five procyanidins were identified as the major anthocyanin compounds, which were differentially accumulated in purple leaves of QHP and ZSY. The major genes associated with anthocyanin biosynthesis, including structural genes and transcription factors, were differentially expressed in purple leaves of QHP and ZSY through RNA-sequencing (RNA-seq) data analysis, which was consistent with quantitative real-time PCR analysis results. Correlation analysis between RNA-seq data and metabolite profiles showed that the expression patterns of certain differentially expressed genes in the anthocyanin biosynthesis pathway were strongly correlated with the differential accumulation of anthocyanins. One R2R3-MYB subfamily member in the SG5 subgroup, Podel.04G021100, showed a similar expression pattern to some structural genes. This gene was strongly correlated with 16 anthocyanin compounds, indicating that Podel.04G021100 might be involved in the regulation of anthocyanin biosynthesis. These results contribute to a systematic and comprehensive understanding of anthocyanin accumulation and to the molecular mechanisms of anthocyanin biosynthesis in QHP and ZSY.

Integrated transcriptomics and metabolomics analysis provide insight into anthocyanin biosynthesis for sepal color formation in Heptacodium miconioides.

Heptacodium miconioides Rehd., commonly known as "seven-son flower," is an ornamental species with a beautiful flower pattern and persistent sepals. Its sepals are of horticultural value, turning bright red and elongating in the autumn; however, the molecular mechanisms that cause sepal color change remain unclear. We analyzed the dynamic changes in anthocyanin composition in the sepal of H. miconioides at four developmental stages (S1-S4). A total of 41 anthocyanins were detected and classified into 7 major anthocyanin aglycones. High levels of the pigments cyanidin-3,5-O-diglucoside, cyanidin-3-O-galactoside, cyanidin-3-O-glucoside, and pelargonidin-3-O-glucoside were responsible for sepal reddening. Transcriptome analysis revealed 15 differentially expressed genes involved in anthocyanin biosynthesis that were detected between 2 developmental stages. Of these, the high expression of HmANS was considered critical structural gene related to anthocyanin biosynthesis pathway in the sepal through co-expression analysis with anthocyanin content. In addition, a transcription factor (TF)-metabolite correlation analysis revealed that three HmMYB, two HmbHLH, two HmWRKY, and two HmNAC TFs exhibited a strong positive role in the regulation of the anthocyanin structural genes (Pearson's correlation coefficient > 0.90). Luciferase activity assay showed that HmMYB114, HmbHLH130, HmWRKY6, and HmNAC1 could activate the promoters of HmCHS4 and HmDFR1 genes in vitro. These findings increase our understanding of anthocyanin metabolism in the sepal of H. miconioides and provide a guide for studies involving sepal color conversion and regulation.

RNA-Seq Based Transcriptomic Analysis of Bud Sport Skin Color in Grape Berries

The most common bud sport trait in grapevines is the change in color of grape berry skin, and the color of grapes is mainly developed by the composition and accumulation of anthocyanins. Many studies have shown that MYBA is a key gene regulates the initiation of bud sport color and anthocyanin synthesis in grape peels. In the current study, we used berry skins of ‘Italia’, ‘Benitaka’, ‘Muscat of Alexandria’, ‘Flame Muscat’, ‘Rosario Bianco’, ‘Rosario Rosso’, and ‘Red Rosario’ at the véraison stage (10 weeks post-flowering and 11 weeks post-flowering) as research materials. The relative expressions of genes related to grape berry bud sport skin color were evaluated utilizing RNA-Seq technology. The results revealed that the expressions of the VvMYBA1/A2 gene in the three red grape varieties at the véraison stage were higher than in the three white grape varieties. The VvMYBA1/A2 gene is known to be associated with UFGT in the anthocyanin synthesis pathway. According to the results, VvMYBA1/A2 gene expression could also be associated with the expression of LDOX. In addition, a single gene (gene ID: Vitvi19g01871) displayed the highest expressions in all the samples at the véraison stage for the six varieties. The expression of this gene was much higher in the three green varieties compared to the three red ones. GO molecular function annotation identified it as a putative metallothionein-like protein with the ability to regulate the binding of copper ions to zinc ions and the role of maintaining the internal stable state of copper ions at the cellular level. High expression levels of this screened gene may play an important role in bud sport color of grape berry skin at the véraison stage.

Two genes, ANS and UFGT2, from Vaccinium spp. are key steps for modulating anthocyanin production.

Anthocyanins are a major group of red to blue spectrum plant pigments with many consumer health benefits. Anthocyanins are derived from the flavonoid pathway and diversified by glycosylation and methylation, involving the concerted action of specific enzymes. Blueberry and bilberry (Vaccinium spp.) are regarded as 'superfruits' owing to their high content of flavonoids, especially anthocyanins. While ripening-related anthocyanin production in bilberry (V. myrtillus) and blueberry (V. corymbosum) is regulated by the transcriptional activator MYBA1, the role of specific structural genes in determining the concentration and composition of anthocyanins has not been functionally elucidated. We isolated three candidate genes, CHALCONE SYNTHASE (VmCHS1), ANTHOCYANIDIN SYNTHASE (VmANS) and UDP-GLUCOSE : FLAVONOID-3-O-GLYCOSYLTRANSFERASE (VcUFGT2), from Vaccinium, which were predominantly expressed in pigmented fruit skin tissue and showed high homology between bilberry and blueberry. Agrobacterium-mediated transient expression of Nicotiana benthamiana showed that overexpression of VcMYBA1 in combination with VmANS significantly increased anthocyanin concentration (3-fold). Overexpression of VmCHS1 showed no effect above that induced by VcMYBA1, while VcUFGT2 modulated anthocyanin composition to produce delphinidin-3-galactosylrhamnoside, not naturally produced in tobacco. In strawberry (Fragaria × ananassa), combined transient overexpression of VcUFGT2 with a FLAVONOID 3´,5´-HYDROXYLASE from kiwifruit (Actinidia melanandra) modulated the anthocyanin profile to include galactosides and arabinosides of delphinidin and cyanidin, major anthocyanins in blueberry and bilberry. These findings provide insight into the role of the final steps of biosynthesis in modulating anthocyanin production in Vaccinium and may contribute to the targeted breeding of new cultivars with improved nutritional properties.