Acylation Of Anthocyanins Research Articles

A vacuolar protein MaSCPL1 mediates anthocyanin acylation modifications in blue-flowered grape hyacinth

The grape hyacinth is renowned for its profuse blue flowers, which confer substantial scientific and ornamental significance as well as considerable potential for industrial applications. The serine carboxypeptidase-like acyltransferases (SCPL-ATs) family is crucial for the blue flower coloration. To elucidate SCPL-ATs involved in anthocyanin modification in grape hyacinth, we performed a transcriptomic analysis of grape hyacinth SCPL-ATs. Through gene expression profiling, we identified a promising candidate gene, MaSCPL1, whose expression patterns corresponded with variations in anthocyanin content throughout petal coloration. Subsequently, the functional role of the MaSCPL1 gene was validated using the native petal regeneration system, and the silencing of MaSCPL1 led to a decreased total anthocyanin content and Dp3MG content in grape hyacinth petals. Furthermore, we employed yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), and dual-luciferase assays to explore the regulatory interactions between the anthocyanin biosynthesis transcription factor MaMybA and the MaSCPL1 promoter. Our findings indicate that MaMybA can bind to the MaSCPL1 promoter and significantly activate its expression. Furthermore, the MaMybA-RNAi resulted in a substantial multifold reduction in the expression of MaSCPL1, implying that the regulation of MaSCPL1 expression is mediated by MaMybA. This study revealed the MaSCPL1 gene has been associated with anthocyanin acylated modification in grape hyacinth and elucidated the important role of the MaMybA-MaSCPL1 module in colouration grape hyacinth.

CONTROL OF THE SELECTIVITY OF THE SEPARATION OF GRAPE ANTHOCYANIDIN 3-GLUCOSIDES AND 3,5-DIGLUCOSIDES: DETERMINATION OF ANTHOCYANINS IN GRAPE FRUIT GROWN IN THE BELGOROD REGION

For the first time, a new version of the separation of 3-glucosides and 3,5-diglucosides of the five main anthocyanidins for grape fruits - delphinidin, cyanidin, petunidin, peonidin and malvidin under reverse-phase HPLC conditions was proposed. It has been shown that the replacement of the traditionally used acetonitrile with environmentally more acceptable ethanol upon acidification with ortho-phosphoric rather than formic acid makes it possible to significantly change the selectivity of the separation of two types of glucosides at their joint presence. The proposed separation option allows differentiating between Vitis vinifera grapes, in the fruit peel of which only 3-glucosides of the above anthocyanidins are synthesized, and grapes of other species or hybrid grape varieties. For a complete review of the anthocyanin composition, it is necessary to use a gradient mode, since the acylation of anthocyanins with acetic and p-coumaric acids significantly changes the lipophilicity of anthocyanins. The type of anthocyanins was analyzed according to the previously proposed system, which takes into account the activity of three types of enzymes: 1) 5-О-glucosyltransferase that leads to 3,5-diglucosides synthesis; 2) 3',5'-hydroxylase responsible for ring B hydroxylation; 3) anthocyanin O-methyltransferase for converting cyanidin into peonidin derivatives as well as delphinidine into petunidin and malvidin glycosides. The method was used to determine anthocyanins in 43 grape varieties grown in Belgorod on farms and private farms. Among the studied grapes, varieties were found with grape fruits with the accumulation of only 3-glucosides, as well as 3-glucosides and 3,5-diglucosides simultaneously, with delphinidin and cyanidin types of anthocyanins, and with different degrees of methylation. The calculated parameters for all grape varieties are presented in two tables and a brief discussion of some varieties is given.

Production of species-specific anthocyanins through an inducible system in plant hairy roots

Anthocyanins are widely distributed pigments in flowering plants with red, purple or blue colours. Their properties in promoting heath make anthocyanins perfect natural colourants for food additives. However, anthocyanins with strong colour and stability at neutral pH, suitable as food colourants are relatively rare in nature. Acylation increases anthocyanin stability and confers bluer colour. In this study, we isolated two anthocyanin regulators SbMyb75 and SbDel from S. baicalensis, and showed that constitutive expression of the two TFs led to accumulation of anthocyanins at high levels in black carrot hairy roots. However, these hairy roots had severe growth problems. We then developed a β-estradiol inducible system using XVE and a Lex-35S promoter, to initiate expression of the anthocyanin regulators and induced this system in hairy roots of black carrot, tobacco and morning glory. Anthocyanins with various decorations were produced in these hairy roots without any accompanying side-effects on growth. We further produced highly acylated anthocyanins with blue colour in a 5 L liquid culture in a bioreactor of hairy roots from morning glory. We provide here a strategy to produce highly decorated anthocyanins without the need for additional engineering of any of the genes encoding decorating enzymes. This strategy could be transferred to other species, with considerable potential for natural colourant production for the food industries.

Characterization of Purple Carrot Germplasm for Antioxidant Capacity and Root Concentration of Anthocyanins, Phenolics, and Carotenoids.

The present study characterized a genetically and phenotypically diverse collection of 27 purple and two non-purple (one orange and one yellow) carrot accessions for concentration of root anthocyanins, phenolics, and carotenoids, and antioxidant capacity estimated by four different methods (ORAC, DPPH, ABTS, FRAP), in a partially replicated experimental design comprising data from two growing seasons (2018 and 2019). Broad and significant (p < 0.0001) variation was found among the accessions for all the traits. Acylated anthocyanins (AA) predominated over non-acylated anthocyanins (NAA) in all the accessions and years analyzed, with AA accounting for 55.5-100% of the total anthocyanin content (TAC). Anthocyanins acylated with ferulic acid and coumaric acid were the most abundant carrot anthocyanins. In general, black or solid purple carrots had the greatest TAC and total phenolic content (TPC), and the strongest antioxidant capacities, measured by all methods. Antioxidant capacity, estimated by all methods, was significantly, positively, and moderately-to-strongly correlated with the content of all individual anthocyanins pigments, TAC, and TPC, in both years (r = 0.59-0.90, p < 0.0001), but not with the carotenoid pigments lutein and β-carotene; suggesting that anthocyanins and other phenolics, but not carotenoids, are major contributors of the antioxidant capacity in purple carrots. We identified accessions with high concentration of chemically stable AA, with potential value for the production of food dyes, and accessions with relatively high content of bioavailable NAA that can be selected for increased nutraceutical value (e.g., for fresh consumption).

Ultrasonic assisted extraction of anthocyanins from rose flower petal in DES system and enzymatic acylation

Herein, the anthocyanins from rose were extracted, purified and identified by ultrasound-assisted deep eutectic solvents (DES), the resin, and ESI-Q-TOF-MS, respectively. The purified anthocyanins were modified through acylation by means of enzymatic catalysis method to improve the stability of anthocyanins. Compared with traditional extraction agents (ethanol), anthocyanins extraction rate was significantly improved when choline chloride/lactic acid was chosen as extraction solvent, and reached 8.265 mg g−1. The ESI-Q-TOF-MS detection results showed that the main component of the anthocyanins was peonidin-3,5-di-O-glucoside with 65.93% content. Meanwhile, the maximal acylation rate was 95.13% under optimal conditions (0.25 g of anthocyanin, 2.5 mL of pyridine, 4 mL of methyl benzoate; 400 mg of Novocain 435 lipase addition; a water bath at 50 °C under negative pressure of 0.09 Mpa for 9 h). Furthermore, the loss rate of acylated anthocyanins was significantly lower than that of natural anthocyanins under the same temperature or light conditions, and the acylation of anthocyanin could significantly improve stability against temperature, light and pH. The acylated anthocyanin exhibited excellent free radical scavenging activities for DPPH and ABTS+, and IC50 values (4.451 μg mL−1 for DPPH and 7.760 μg mL−1 for ABTS+) displayed its high antioxidant activity.

The Effect of Cinnamon and Ginger Spices on Anthocyanins in Sweetened Roselle Beverages

This study explores the potential benefits of spices (cinnamon and ginger) on Roselle anthocyanins within a sweetened Roselle beverage matrix. Anthocyanins and other related properties of the beverage (colour, antioxidant capacity, total phenolics, and pH) were observed from the start and monitored for 30 days at accelerated storage conditions (40 °C). The sweeteners at the amounts used (80 g/L granulated sugar and 0.32 g/L Stevia Reb A) did not have a significant effect on the initial anthocyanin content in the beverage and did not significantly impact degradation. Upon the addition of spices to the sweetened beverage, ginger (1 g/L) did not result in significant changes, initially or during storage. However, following the addition of cinnamon (1 g/L) to the beverages (unsweetened and sweetened), an initial increase in the total phenolic and FRAP antioxidant activity in the Roselle beverages was observed; furthermore, it reduced the degradation of anthocyanins and improved colour stability during storage. This effect is postulated to be due to a co-pigmentation reaction or the acylation of anthocyanins with a complex formed from the reaction of glucose with the phenolic compounds contained in cinnamon.

Overhead photoselective shade films mitigate effects of climate change by arresting flavonoid and aroma composition degradation in wine.

Overhead photoselective shade films installed in vineyards improve berry composition in hot grape-growing regions. The aim of the study was to evaluate the flavonoid and aroma profiles and composition of wines from Cabernet Sauvignon grapes (Vitis vinifera L.) treated with partial solar radiation exclusion. Experimental design consisted in a randomized experiment with four shade films (D1, D3, D4, D5) with differing solar radiation spectra transmittance and compared to an uncovered control (C0) performed over two seasons (2021 and 2022) in Oakville (CA, USA). Berries were collected by hand at harvest and individual vinifications for each treatment and season were conducted in triplicates. Then, wine chemical composition, flavonoid and aromatic profiles were analyzed. The wines from D4 treatment had greater color intensity and total phenolic index due to co-pigmentation with anthocyanins. Shade film wines D5 and D1 from the 2020 vintage demonstrated increased total anthocyanins in the hotter of the two experimental years. In 2021, reduced cluster temperatures optimized total anthocyanins in D4 wines. Reduced cluster temperatures modulated anthocyanin acylation, methylation and hydroxylation in shade film wines. Volatile aroma composition was analyzed using gas chromatography mass spectroscopy (GCMS) and D4 wines exhibited a more fruity and pleasant aroma profile than C0 wines. Results provided evidence that partial solar radiation exclusion in the vineyard using overhead shade films directly improved flavonoid and aroma profiles of resultant wines under hot vintage conditions, providing a tool for combatting air temperatures and warmer growing conditions associated with climate change.

Orthology-based analysis helps map evolutionary diversification and predict substrate class use of BAHD acyltransferases.

Large enzyme families catalyze metabolic diversification by virtue of their ability to use diverse chemical scaffolds. How enzyme families attain such functional diversity is not clear. Furthermore, duplication and promiscuity in such enzyme families limits their functional prediction, which has produced a burgeoning set of incompletely annotated genes in plant genomes. Here, we address these challenges using BAHD acyltransferases as a model. This fast-evolving family expanded drastically in land plants, increasing from one to five copies in algae to approximately 100 copies in diploid angiosperm genomes. Compilation of >160 published activities helped visualize the chemical space occupied by this family and define eight different classes based on structural similarities between acceptor substrates. Using orthologous groups (OGs) across 52 sequenced plant genomes, we developed a method to predict BAHD acceptor substrate class utilization as well as origins of individual BAHD OGs in plant evolution. This method was validated using six novel and 28 previously characterized enzymes and helped improve putative substrate class predictions for BAHDs in the tomato genome. Our results also revealed that while cuticular wax and lignin biosynthetic activities were more ancient, anthocyanin acylation activity was fixed in BAHDs later near the origin of angiosperms. The OG-based analysis enabled identification of signature motifs in anthocyanin-acylating BAHDs, whose importance was validated via molecular dynamic simulations, site-directed mutagenesis and kinetic assays. Our results not only describe how BAHDs contributed to evolution of multiple chemical phenotypes in the plant world but also propose a biocuration-enabled approach for improved functional annotation of plant enzyme families.

Acylation of Anthocyanins and Their Applications in the Food Industry: Mechanisms and Recent Research Advances.

Anthocyanins are extensively used as natural non-toxic compounds in the food industry due to their unique biological properties. However, the instability of anthocyanins greatly affects their industrial application. Studies related to acylated anthocyanins with higher stability and increased solubility in organic solvents have shown that the acylation of anthocyanins can improve the stability and fat solubility of anthocyanins. However, relevant developments in research regarding the mechanisms of acylation and applications of acylated anthocyanins are scarcely reviewed. This review aims to provide an overview of the mechanisms of acylation and the applications of acylated anthocyanins in the food industry. In the review, acylation methods, including biosynthesis, semi-biosynthesis, and chemical and enzymatic acylation, are elaborated, physicochemical properties and biological activities of acylated anthocyanins are highlighted, and their application as colourants, functionalizing agents, intelligent indicators, and novel packaging materials in the food industry are summarized. The limitations encountered in the preparation of acylated anthocyanins and future prospects, their applications are also presented. Acylated anthocyanins present potential alternatives to anthocyanins in the food industry due to their functions and advantages as compared with non-acylated analogues. It is hoped that this review will offer further information on the effective synthesis and encourage commercialization of acylated anthocyanins in the food industry.

Improvement of stability and lipophilicity of pelargonidin-3-glucoside by enzymatic acylation with aliphatic dicarboxylic acid

Dicarboxylic acids derived acylated-anthocyanins are common in nature, which can also be obtained by enzymatic acylation of anthocyanins. However, little research have focused on the properties of anthocyanins with dicarboxylic acid derivatives due to the complexity of isolation, detection, and identification. In this work, pelargonidin-3-glucoside (Pg3G) was acylated with various dicarboxylic acids. The conversion yields of acylated Pg3G were positively associated with carbon chain lengths of dicarboxylic acids. The primary acylated products were identified as pelargonidin-3-(6″-malonyl) glucoside, pelargonidin-3-(6″-succinyl) glucoside, and pelargonidin-3-(6″-glutaryl) glucoside using LC-MS and NMR. Furthermore, the three acylated Pg3G derivatives exhibited improved thermostability and enhanced lipophilicity compared with Pg3G. The improved thermostability was attributed to the influence of dicarboxylic acids substituent on the distribution of flavylium cation, quinoidal base, hemiketal, cis-chalcone, and trans-chalcone at the equilibrium condition. Overall, our research provided insights about the improved stability and lipophilicity of pelargonidin-3-glucoside following enzymatic acylation with aliphatic dicarboxylic acids.

Physicochemical properties, degradation kinetics, and antioxidant capacity of aqueous anthocyanin-based extracts from purple carrots compared to synthetic and natural food colorants

As a means to evaluate the potential of carrot anthocyanins as food colorants and nutraceutical agents, we investigated the physicochemical stability and antioxidant capacity of purple carrot extracts under different pH (2.5–7.0) and temperature (4–40 °C) conditions, in comparison to a commercial synthetic (E131) and a natural grape-based (GRP) colorant. During incubation, the colorants were weekly-monitored for various color parameters, concentration of anthocyanins and phenolics, and antioxidant capacity. Carrot colorants were more stable than GRP; and their thermal stability was equal (at 4 °C) or higher than that of E131 (at 25–40 °C). Carrot anthocyanins had lower degradation rate at low pH and temperature, with acylated anthocyanins (AA) being significantly more stable than non-acylated anthocyanins (NAA). Anthocyanins acylated with feruloyl and coumaroyl glycosides were the most stable carrot pigments. The higher stability of carrot colorants is likely due to their richness in AA and -to a lesser extent- copigmentation with other phenolics.

Antimicrobial activity of cyanidin-3-O-glucoside–lauric acid ester against Staphylococcus aureus and Escherichia coli

Enzymatic acylation of anthocyanin with fatty acid improves its lipophilic solubility and application potential. Nevertheless, evaluation of functional properties of product is premise for application. This study investigated the antimicrobial potential and the underlying mechanisms of an acylated anthocyanin, namely, cyanidin-3-O-glucoside–lauric acid ester (C3G-LA), to provide guidelines for its application. C3G-LA exhibited outstanding antibacterial activity against Staphylococcus aureus [minimum inhibitory concentration (MIC) = 0.3125 mg/mL] and modest activity against Escherichia coli (MIC = 5 mg/mL). Moreover, C3G-LA manifested bactericide ability against S. aureus at 0.625 mg/mL. Decreases in membrane integrity (by 96% and 92% at MIC in S. aureus and E. coli, respectively), intracellular ATP concentration (by 96% and 92%) and intracellular pH (by 11% and 9%) and changes in cellular morphology altogether indicated the dysfunction of cell membrane under C3G-LA treatment. These findings demonstrated that C3G-LA could be adopted as an alternative food preservative against foodborne pathogens.

Inhibitory effect of acylated anthocyanins on heterocyclic amines in grilled chicken breast patty and its mechanism

Heterocyclic amines (HCAs) are a group of carcinogenic substances produced in protein-rich poultry meat under high-temperature. Enzymatic acylation of anthocyanins (ACNs) is a reliable way to improve their stability, and we recently found the acylated cyaniding-3-O-glucose (cyanidin-3-6-cinnamoyl-glucoside, C3(6C)G) could effective inhibit the HCAs formation, but the underline mechanism was still obscure. Thus, the present study investigated the inhibitory effect ofC3(6C)G on HCAs formation in the food system (chicken breast) and to explore the potential mechanism. The results showed that C3(6C)G with different concentrations (0.1, 0.5 and 1.0 mg/mL) could significantly inhibit lipid oxidation and decrease the total HCAs content (P<0.05) in chicken breast meat patty after roasting. The samples with 0.1 mg/mL C3(6C)G had the best inhibition effect on total HCAs, with an inhibition rate of 28%, and the inhibition rates for IQ, Harman, TRP-P-2, PhIP and AαC were 34%, 46%, 100%, 54% and 41%, respectively.

The Genetic Basis of Anthocyanin Acylation in North American Grapes (Vitis spp.).

Hydroxycinnamylated anthocyanins (or simply ‘acylated anthocyanins’) increase color stability in grape products, such as wine. Several genes that are relevant for anthocyanin acylation in grapes have been previously described; however, control of the degree of acylation in grapes is complicated by the lack of genetic markers quantitatively associated with this trait. To characterize the genetic basis of anthocyanin acylation in grapevine, we analyzed the acylation ratio in two closely related biparental families, Vitis rupestris B38 × ‘Horizon’ and ‘Horizon’ × Illinois 547-1, for 2 and 3 years, respectively. The acylation ratio followed a bimodal and skewed distribution in both families, with repeatability estimates larger than 0.84. Quantitative trait locus (QTL) mapping with amplicon-based markers (rhAmpSeq) identified a strong QTL from ‘Horizon’ on chromosome 3, near 15.85 Mb in both families and across years, explaining up to 85.2% of the phenotypic variance. Multiple candidate genes were identified in the 14.85–17.95 Mb interval, in particular, three copies of a gene encoding an acetyl-CoA-benzylalcohol acetyltransferase-like protein within the two most strongly associated markers. Additional population-specific QTLs were found in chromosomes 9, 10, 15, and 16; however, no candidate genes were described. The rhAmpSeq markers reported here, which were previously shown to be highly transferable among the Vitis genus, could be immediately implemented in current grapevine breeding efforts to control the degree of anthocyanin acylation and improve the quality of grapes and their products.

Effect of Acylation of Anthocyanins with Acetic Acid on the Retention of Glycosides in Reverse Phase Chromatography

The effect of acylation of anthocyanins with acetic acid on the chromatographic retention is evaluated. It is shown that the absence of correlations between the experimental data on the retention of substances and the CLOGP data is explained, in some cases of structural changes, by the specificity of the float mechanism of the retention of anthocyanins. In this case, the conformational analysis of anthocyanins (in the Chem3D software package) can explain a number of features peculiar to the effect of acylation on the retention in accordance with the Horvath–Melander model used for glycoside substituents. It is shown that the contribution of the acetyl group to the total retention value of anthocyanins depends on the composition of the mobile phase, the structures of glycosidic substituents and aglycone for a given stationary phase, and the temperature of separation. In general, acetylation leads to a substantial increase in the retention of anthocyanins, namely, to an increase by a factor of about 10 for 3-(6-acetylglucosides), and this increase is about 1.5 times lower in the case of similar acetylation of 3,5-diglucosides.

Anthocyanin 5,3′-aromatic acyltransferase from Gentiana triflora, a structural insight into biosynthesis of a blue anthocyanin

The acylation of anthocyanins contributes to their structural diversity. Aromatic acylation is responsible for the blue color of anthocyanins and certain flowers. Aromatic acyltransferase from Gentiana triflora Pall. (Gentianaceae) (Gt5,3′AT) catalyzes the acylation of glucosyl moieties at the 5 and 3′ positions of anthocyanins. Anthocyanin acyltransferase transfers an acyl group to a single position, such that Gt5,3′AT possesses a unique enzymatic activity. Structural investigation of this aromatic acyl group transfer is fundamental to understand the molecular mechanism of the acylation of double positions. In this study, structural analyses of Gt5,3′AT were conducted to identify the underlying mechanism. The crystal structure indicated that Gt5,3′AT shares structural similarities with other BAHD family enzymes, consisting of N and C terminal lobes. Structural comparison revealed that acyl group preference (aromatic or aliphatic) for the enzymes was determined by four amino acid positions, which are well conserved in aromatic and aliphatic CoA-binding acyltransferases. Although a complex structure with anthocyanins was not obtained, the binding of delphinidin 3,5,3′-triglucoside to Gt5,3′AT was investigated by evaluating the molecular dynamics. The simulation indicated that acyl transfer by Gt5,3′AT preferentially occurs at the 5-position rather than at the 3′-position, with interacting amino acids that are mainly located in the C-terminal lobe. Subsequent assays of chimeric enzymes (exchange of the N-terminal lobe and the C-terminal lobe between Gt5,3′AT and lisianthus anthocyanin 5AT) demonstrated that acyl transfer selectivity may be caused by the C-terminal lobe.

Day Temperature Has a Stronger Effect Than Night Temperature on Anthocyanin and Flavonol Accumulation in 'Merlot' (Vitis vinifera L.) Grapes During Ripening.

Flavonoids impart color and mouthfeel to grapes and wine and are very sensitive to environmental conditions. Growth chamber experiments were performed to investigate the effect of temperature regimes and the differences between day/night temperatures on anthocyanins and flavonols in Merlot grapes. Among the regimes tested, the ones with diurnal 20°C determined the highest levels of anthocyanins and flavonols. Higher diurnal temperatures decreased those levels but increased the proportion of methoxylated and acylated species. When regimes with the same day temperature but different night temperatures were compared, differences between day/night temperatures did not affect anthocyanins, unless a difference of 25°C between day and night temperatures was imposed. When regimes with the same night temperature but different day temperatures were compared, the regime with higher day temperature had a lower anthocyanin level. No relationships were observed between the effects of temperature regimes on anthocyanin level and the expression of key anthocyanin genes. However, the effects on anthocyanin acylation level were consistent with the effects on the acyltransferase expression, and the effects on flavonol level were consistent with the effects on the expression of key flavonol genes. This study indicates that, in Merlot grapes, anthocyanins and flavonols are mostly sensitive to day temperatures.

Microwave-Assisted Synthesis and Ionic Liquids: Green and Sustainable Alternatives toward Enzymatic Lipophilization of Anthocyanin Monoglucosides.

Anthocyanins recycling and transformations into novel compounds have been of great interest of the scientific community to improve the circular economy and enhance their technological applications. The enzymatic acylation of anthocyanins into lipophilic derivatives by conjugation with fatty acids emerged as one of the approaches; however, the literature describes only the use of organic solvents and conventional heating. In this work, the production of cyanidin-3-glucoside-octanoic acid conjugate combining ionic liquids (ILs), microwave (MW) irradiation, and Candida antarctica lipase B as a biocatalyst was attempted for the first time. Overall, the use of MW irradiation could reduce drastically the reaction time, allowing the formation of an acylated product with similar concentrations to the conventional method. On the other hand, the study of the lipophilic conjugate synthesis using different ILs showed that their composition is crucial to achieve the desired enzymatic reaction, and in this case, the combination of an imidazolium derivative as the cation with the triflate as the anion was suitable for the production of this derivative. These promising results achieved through the combination of greener alternatives to those reported in the literature are a starting point for further developments to produce this kind of anthocyanin derivatives exploring such sustainable and ecofriendly conditions.

Identification of an SCPL Gene Controlling Anthocyanin Acylation in Carrot (Daucus carota L.) Root.

Anthocyanins are natural health promoting pigments that can be produced in large quantities in some purple carrot cultivars. Decoration patterns of anthocyanins, such as acylation, can greatly influence their stability and biological properties and use in the food industry as nutraceuticals and natural colorants. Despite recent advances made toward understanding the genetic control of anthocyanin accumulation in purple carrot, the genetic mechanism controlling acylation of anthocyanin in carrot root have not been studied yet. In the present study, we performed fine mapping combined with gene expression analyses (RNA-Seq and RT-qPCR) to identify the genetic factor conditioning the accumulation of non-acylated (Cy3XGG) versus acylated (Cy3XFGG and Cy3XSGG) cyanidin derivatives, in three carrot populations. Segregation and mapping analysis pointed to a single gene with dominant effect controlling anthocyanin acylation in the root, located in a 576kb region containing 29 predicted genes. Orthologous and phylogenetic analyses enabled the identification of a cluster of three SCPL-acyltransferases coding genes within this region. Comparative transcriptome analysis indicated that only one of these three genes, DcSCPL1, was always expressed in association with anthocyanin pigmentation in the root and was co-expressed with DcMYB7, a gene known to activate anthocyanin biosynthetic genes in carrot. DcSCPL1 sequence analysis, in root tissue containing a low level of acylated anthocyanins, demonstrated the presence of an insertion causing an abnormal splicing of the 3rd exon during mRNA editing, likely resulting in the production of a non-functional acyltransferase and explaining the reduced acylation phenotype. This study provides strong linkage-mapping and functional evidences for the candidacy of DcSCPL1 as a primary regulator of anthocyanin acylation in carrot storage root.

Insight into Composition of Bioactive Phenolic Compounds in Leaves and Flowers of Green and Purple Basil.

Basil (Ocimum basilicum L.) is a culinary, medicinal, and ornamental plant appreciated for its antioxidant properties, mainly attributed to high content of rosmarinic acid. This species also includes purple varieties, characterized by the accumulation of anthocyanins in leaves and flowers. In this work, we compared the main morphological characteristics, the antioxidant capacity and the chemical composition in leaves, flowers, and corollas of green (‘Italiano Classico’) and purple (‘Red Rubin’ and ‘Dark Opal’) basil varieties. The LC-ESI-MS/MS analysis of individual compounds allowed quantifying 17 (poly)phenolic acids and 18 flavonoids, differently accumulated in leaves and flowers of the three varieties. The study revealed that in addition to rosmarinic acid, basil contains several members of the salvianolic acid family, only scarcely descripted in this species, as well as, especially in flowers, simple phenolic acids, such as 4-hydroxybenzoic acid and salvianic acid A. Moreover, the study revealed that purple leaves mainly contain highly acylated anthocyanins, while purple flowers accumulate anthocyanins with low degree of decoration. Overall, this study provides new biochemical information about the presence of not yet characterized bioactive compounds in basil that could contribute to boosting the use of this crop and to gaining new knowledge about the roles of these compounds in plant physiology.