Anthocyanin Synthesis In Apple Research Articles

The regulatory module MdBZR1-MdCOL6 mediates brassinosteroid- and light-regulated anthocyanin synthesis in apple.

The anthocyanin content is an important indicator of the nutritional value of most fruits, including apple (Malus domestica). Anthocyanin synthesis is coordinately regulated by light and various phytohormones. In this study on apple, we revealed the antagonistic relationship between light and brassinosteroid (BR) signaling pathways, which is mediated by BRASSINAZOLE-RESISTANT 1 (MdBZR1) and the B-box protein MdCOL6. The exogenous application of brassinolide inhibited the high-light-induced anthocyanin accumulation in red-fleshed apple seedlings, whereas increases in the light intensity decreased the endogenous BR content. The overexpression of MdBZR1 inhibited the anthocyanin synthesis in apple plants. An exposure to a high-light intensity induced the degradation of dephosphorylated MdBZR1, resulting in functional impairment. MdBZR1 was identified as an upstream repressor of MdCOL6, which promotes anthocyanin synthesis in apple plants. Furthermore, MdBZR1 interacts with MdCOL6 to attenuate its ability to activate MdUFGT and MdANS transcription. Thus, MdBZR1 negatively regulates MdCOL6-mediated anthocyanin accumulation. Our study findings have clarified the molecular basis of the integration of light and BR signals during the regulation of anthocyanin biosynthesis, which is an important process influencing fruit quality.

Transcriptome analysis provides StMYBA1 gene that regulates potato anthocyanin biosynthesis by activating structural genes.

Anthocyanin biosynthesis is affected by light, temperature, and other environmental factors. The regulation mode of light on anthocyanin synthesis in apple, pear, tomato and other species has been reported, while not clear in potato. In this study, potato RM-210 tubers whose peel will turn purple gradually after exposure to light were selected. Transcriptome analysis was performed on RM-210 tubers during anthocyanin accumulation. The expression of StMYBA1 gene continued to increase during the anthocyanin accumulation in RM-210 tubers. Moreover, co-expression cluster analysis of differentially expressed genes showed that the expression patterns of StMYBA1 gene were highly correlated with structural genes CHS and CHI. The promoter activity of StMYBA1 was significantly higher in light conditions, and StMYBA1 could activate the promoter activity of structural genes StCHS, StCHI, and StF3H. Further gene function analysis found that overexpression of StMYBA1 gene could promote anthocyanin accumulation and structural gene expression in potato leaves. These results demonstrated that StMYBA1 gene promoted potato anthocyanin biosynthesis by activating the expression of structural genes under light conditions. These findings provide a theoretical basis and genetic resources for the regulatory mechanism of potato anthocyanin synthesis.

Ultraviolet-B-induced MdWRKY71-L expression regulates anthocyanin synthesis in apple

Anthocyanin plays critical role in plant secondary metabolism. Although many transcription factors (TFs) that participate in the anthocyanin biosynthesis pathway have been characterized, many have yet to be reported. In the study, we cloned a WRKY Group IIc TF in apple ( Malus × domestica ), MdWRKY71-L, which localized to the nucleus. The overexpression of MdWRKY71-L promoted the accumulation of anthocyanin in apple calli. MdWRKY71-L mainly stimulated this accumulation through interacting with the promoters of MdMYB1 and MdUFGT . In addition to plant genotype, the synthesis of anthocyanin is also affected by external factors, such as ultraviolet-B radiation. Ultraviolet-B exposure induced MdWRKY71-L expression and MdWRKY71-L promoted the expression of MdHY5 by interacting with its promoter. Our research provided new insights into how WRKY TFs participated in the ultraviolet-B signaling pathway to regulate the accumulation of anthocyanin in apple. • Transgenic MdWRKY71-L regulated apple anthocyanin synthesis. • MdWRKY71-L mainly promoted anthocyanin accumulation by binding to the promoters of MdMYB1 and MdUFGT . • MdWRKY71-L could participate in UV-induced anthocyanin synthesis in apple. • MdWRKY71-L regulated the biosynthesis of anthocyanin through MdHY5-MdMYB1 transcriptional regulatory cascade.

Apple MdMYB306-like inhibits anthocyanin synthesis by directly interacting with MdMYB17 and MdbHLH33.

SUMMARYAnthocyanin is one of the most important pigments and nutrients in fruits. Genes encoding R2R3‐MYB transcription factors (TFs) are key to anthocyanin regulation. R2R3‐MYB activators have been widely studied in the anthocyanin synthesis pathway. However, the mechanism by which R2R3‐MYB repressors negatively regulate anthocyanin synthesis remains poorly understood. In the present study, we characterized a subfamily 1 R2R3‐MYB anthocyanin repressor gene, MYB306‐like, from apple (Malus × domestica) and demonstrated its significance in anthocyanin regulation. The MdMYB306‐like protein activates the expression of an anthocyanin repressor gene, MdMYB17, and inhibits the expression of MdDFR (an anthocyanin structural gene) by directly binding to the corresponding promoters. The MdMYB306‐like protein interacts with MdMYB17 and MdbHLH33 through its N‐terminus. In addition, MdMYB306‐like interacts with MdbHLH33 and MdMYB17 to enhance its regulatory activities on MdMYB17 and MdDFR, respectively. Our results revealed that the MdMYB306‐like protein, in conjunction with MdMYB17 and MdbHLH33, forms a key regulatory module to fine‐tune anthocyanin synthesis in apple.

Ethylene precisely regulates anthocyanin synthesis in apple via a module comprising MdEIL1, MdMYB1, and MdMYB17.

Ethylene regulates anthocyanin synthesis in ripening apple fruit via the antagonistic activities of the R2R3-MYB repressors and activators. However, the molecular mechanism underlying this process remains unknown. In this study, ethylene significantly induced the expression of the R2R3-MYB gene MdMYB17 in apple fruit. Moreover, MdMYB17 was revealed to be an important repressor of anthocyanin synthesis. Specifically, MdMYB17 binds directly to the promoters of the ethylene-induced genes MdMYB1 and MdEIL1, which encode positive regulators of anthocyanin synthesis, and represses their expression. Additionally, MdMYB1 and MdEIL1 bind to the MdMYB17 promoter to activate its expression. Thus, MdMYB17, MdMYB1, and MdEIL1 form a regulatory module that controls the expression of the corresponding genes. MdMYB17 interacts with MdEIL1. The interaction between MdMYB17 and MdEIL1 attenuates the regulatory effects of MdMYB17 on MdMYB1 and MdEIL1 as well as the regulatory effects of MdEIL1 on MdMYB17. Overall, our results reveal the molecular mechanisms by which MdMYB17, MdMYB1, and MdEIL1 finely mediate ethylene-regulated anthocyanin synthesis in apple fruit.

MdERF1B-MdMYC2 module integrates ethylene and jasmonic acid to regulate the biosynthesis of anthocyanin in apple.

Ethylene and jasmonic acid (JA) are crucial hormones that promote anthocyanin synthesis in apple (Malus × domestica). However, the mechanism by which these hormones cooperate to modulate anthocyanin production in apple is unclear. According to our results, MdERF1B expression was strongly induced by ethylene and JA. Physiological phenotypes and the results of molecular biological analyses indicated that MdERF1B encodes a positive regulator of anthocyanin synthesis. Specifically, MdERF1B was capable of combining directly with the MdMYC2 promoter to promote gene expression. Additionally, MdERF1B interacted with two JA signaling pathway inhibitors, namely MdJAZ5 and MdJAZ10. The MdERF1B–MdJAZ5/10 protein complex decreased the ability of MdERF1B to activate the MdMYC2 promoter. Furthermore, MdEIL1, which is a crucial protein for ethylene signal transduction, was observed to bind directly to the MdERF1B promoter, thereby upregulating gene expression. These results suggest that MdERF1B is a core gene responsive to JA and ethylene signals. The encoded protein, together with MdMYC2, MdJAZ5/10, and MdEIL1, modulates anthocyanin synthesis in apple. This study clarifies the synergistic mechanism by which JA and ethylene regulate anthocyanin production in apple.

Mdm-miR828 Participates in the Feedback Loop to Regulate Anthocyanin Accumulation in Apple Peel.

Anthocyanins are responsible for the red pigmentation in the peel of apple (Malus × domestica Borkh.) fruit. Relatively few studies have investigated anthocyanins at the posttranscriptional level. MicroRNAs play an important role in plant growth and development by regulating gene expression at the posttranscriptional level. In this study, mdm-miR828 showed a relatively low expression level during the rapid fruit coloration period. However, the mdm-miR828 expression level increased in the late fruit coloration stage. Overexpression of mdm-miR828 inhibited anthocyanin synthesis in apple and Arabidopsis. Dual-luciferase and yeast one-hybrid assays showed that MdMYB1 is capable of binding to the promoter of mdm-MIR828b to promote its expression. The results indicate that mdm-miR828 is involved in a feedback regulatory mechanism associated with anthocyanin accumulation in apple. In addition, mdm-miR828 is involved in the inhibition of anthocyanin accumulation in response to high temperature.

Ultraviolet B-induced MdWRKY72 expression promotes anthocyanin synthesis in apple

Ultraviolet-B (UV-B) radiation promotes anthocyanin synthesis in many plants. Although several transcription factors promote anthocyanin synthesis in response to UV-B radiation, the underlying mechanism remains unclear. In this study, the MdWRKY72 transcription factor gene was isolated from the ‘Taishanzaoxia’ apple genome. Quantitative real-time PCR analyses revealed that the genes encoding enzymes and transcription factors involved in the anthocyanin synthesis pathway (MdANS, MdDFR, MdUFGT, and MdMYB1) were more highly expressed in MdWRKY72-overexpressing transgenic calli than in the wild-type ‘Orin’ apple calli. The results indicated that MdWRKY72 increases anthocyanin synthesis in transgenic calli exposed to UV-B radiation. The results of a gel shift assay and chromatin immunoprecipitation proved that MdWRKY72 promotes MdMYB1 expression indirectly by binding to a W-box element in the MdHY5 promoter and directly by binding to a W-box element in the MdMYB1 promoter. Thus, MdWRKY72 increases anthocyanin synthesis via direct and indirect mechanisms. These findings may be useful for elucidating the molecular mechanism underlying UV-B-induced anthocyanin synthesis mediated by MdWRKY72.

MdWRKY11 Participates in Anthocyanin Accumulation in Red-Fleshed Apples by Affecting MYB Transcription Factors and the Photoresponse Factor MdHY5.

Red-fleshed apples are popular as a result of their high anthocyanin content. MdMYB10 and its homologues are known to be important regulators of anthocyanin synthesis in apple, but the roles of other transcription factors are not well-understood. Here, we explored the role of MdWRKY11 in regulating anthocyanin synthesis in apple flesh. Overexpression of MdWRKY11 in apple callus could significantly promote anthocyanin accumulation, and the expression of some MYB transcription factors and structural genes increased significantly. In binding analyses, MdWRKY11 bound to W-box cis-elements in the promoters of MdMYB10, MdMYB11, and MdUFGT. However, MdWRKY11 did not interact with MdMYB10, MdbHLH3, or MdWD40 proteins, the members of the MBW complex. Sequence analyses revealed that another W-box cis-element was present in the promoter of MdHY5 (encoding a photoresponse factor), and MdWRKY11 was able to bind to the promoter of MdHY5 and promote its activity. Our findings clarify the role of MdWRKY11 in anthocyanin synthesis in red-fleshed apple and imply that other novel genes may be involved in anthocyanin synthesis.

Anthocyanin accumulation correlates with hormones in the fruit skin of \u2018Red Delicious\u2019 and its four generation bud sport mutants

BackgroundBud sport mutants of apple (Malus domestica Borkh.) trees with a highly blushed colouring pattern are mainly caused by the accumulation of anthocyanins in the fruit skin. Hormones are important factors modulating anthocyanin accumulation. However, a good understanding of the interplay between hormones and anthocyanin synthesis in apples, especially in mutants at the molecular level, remains elusive. Here, physiological and comparative transcriptome approaches were used to reveal the molecular basis of color pigmentation in the skin of ‘Red Delicious’ (G0) and its mutants, including ‘Starking Red’ (G1), ‘Starkrimson’ (G2), ‘Campbell Redchief’ (G3) and ‘Vallee spur’ (G4).ResultsPigmentation in the skin gradually proliferated from G0 to G4. The anthocyanin content was higher in the mutants than in ‘Red Delicious’. The activation of early phenylpropanoid biosynthesis genes, including ASP3, PAL, 4CL, PER, CHS, CYP98A and F3’H, was more responsible for anthocyanin accumulation in mutants at the color break stage. In addition, IAA and ABA had a positive regulatory effect on the synthesis of anthocyanins, while GA had the reverse effect. The down-regulation of AACT1, HMGS, HMGR, MVK, MVD2, IDI1 and FPPS2 involved in terpenoid biosynthesis influences anthocyanin accumulation by positively regulating transcripts of AUX1 and SAUR that contribute to the synthesis of IAA, GID2 to GA, PP2C and SnRK2 to ABA. Furthermore, MYB and bHLH members, which are highly correlated (r=0.882–0.980) with anthocyanin content, modulated anthocyanin accumulation by regulating the transcription of structural genes, including CHS and F3’H, involved in the flavonoid biosynthesis pathway.ConclusionsThe present comprehensive transcriptome analyses contribute to the understanding of the the relationship between hormones and anthocyanin synthesis as well as the molecular mechanism involved in apple skin pigmentation.

The molecular mechanism underlying anthocyanin metabolism in apple using the MdMYB16 and MdbHLH33 genes.

MdMYB16 forms homodimers and directly inhibits anthocyanin synthesis via its C-terminal EAR repressor. It weakened the inhibitory effect of MdMYB16 on anthocyanin synthesis when overexpressing MdbHLH33 in callus overexpressing MdMYB16. MdMYB16 could interact with MdbHLH33. Anthocyanins are strong antioxidants that play a key role in the prevention of cardiovascular disease, cancer, and diabetes. The germplasm of Malus sieversii f. neidzwetzkyana is important for the study of anthocyanin metabolism. To date, only limited studies have examined the negative regulatory mechanisms underlying anthocyanin synthesis in apple. Here, we analyzed the relationship between anthocyanin levels and MdMYB16 expression in mature Red Crisp 1-5 apple (M. domestica) fruit, generated an evolutionary tree, and identified an EAR suppression sequence and a bHLH binding motif of the MdMYB16 protein using protein sequence analyses. Overexpression of MdMYB16 or MdMYB16 without bHLH binding sequence (LBSMdMYB16) in red-fleshed callus inhibited MdUFGT and MdANS expression and anthocyanin synthesis. However, overexpression of MdMYB16 without the EAR sequence (LESMdMYB16) in red-fleshed callus had no inhibitory effect on anthocyanin. The yeast one-hybrid assay showed that MdMYB16 and LESMdMYB16 interacted the promoters of MdANS and MdUFGT, respectively. Yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays showed that MdMYB16 formed homodimers and interacted with MdbHLH33, however, the LBSMdMYB16 could not interact with MdbHLH33. We overexpressed MdbHLH33 in callus overexpressing MdMYB16 and found that it weakened the inhibitory effect of MdMYB16 on anthocyanin synthesis. Together, these results suggested that MdMYB16 and MdbHLH33 may be important part of the regulatory network controlling the anthocyanin biosynthetic pathway.

Reactive oxygen species produced via plasma membrane NADPH oxidase regulate anthocyanin synthesis in apple peel.

Solar ultraviolet irradiation regulates anthocyanin synthesis in apple peel by modulating the production of reactive oxygen species via plasma membrane NADPH oxidase instead of other pathways. The synthesis of anthocyanin in apple peels is dependent upon solar irradiation. Using 3-mm commercial glass to attenuate solar UV-A and UV-B light, we confirmed that solar UV irradiation regulated anthocyanin synthesis in apple peels after exposing previously bagged fruit to sunlight. During sunlight exposure, UV attenuation did not affect the expression of MdHY5, MdCOP1, or MdCRY2, but significantly lowered plasma membrane NADPH oxidase activity and superoxide anion concentrations. UV attenuation also reduced the expression levels of MdMYB10, MdPAL, MdCHS, MdF3H, MdDFR, MdANS and MdUFGT1, UDP-glycose:flavonoid 3-O-glycosyltransferase (UFGT) activity, and local concentrations of anthocyanin and quercetin-3-glycoside. In contrast, exogenous application of hydrogen peroxide could enhance anthocyanin and quercetin-3-glycoside synthesis. Xanthophyll cycle pool size on a chlorophyll basis was higher but its de-epoxidation was lower under direct sunlight irradiation than that under UV-attenuating conditions. This suggests that reactive oxygen species (ROS) produced in chloroplast are not major contributors to anthocyanin synthesis regulation. Inhibition of plasma membrane NADPH oxidase activity lowered the production of ROS through this mechanism, significantly inhibited the synthesis of anthocyanin, and increased the total production of ROS in apple peel under direct sunlight irradiation, suggesting that ROS produced via plasma membrane NADPH oxidase regulates anthocyanin synthesis. In summary, solar UV irradiation regulated anthocyanin synthesis in apple peels by modulating the production of ROS via plasma membrane NADPH oxidase.

Postharvest Irradiation Enhances Anthocyanin Synthesis in Apples But Not in Pears

`Cripps' Pink' apples (Malus ×domestica Borkh.) subjected to 72 hours of postharvest irradiation developed a better red blush with high pressure sodium (HPS) (hue angle 56.5°) than with UV-B plus incandescent (UVB+I) lamps (hue angle 70.7°). Only HPS lamps were used in subsequent experiments. The increase in red color (hue angle decrease of 14.9°) in `Braeburn' apples held at -0.5 °C for 8 weeks prior to treatment was smaller than in fruit stored for 4 weeks (hue angle decrease of 23°). No increase in color or anthocyanin concentration was observed in `Forelle' pears (Pyrus communis L.) that were similarly treated. `Forelle' pears were harvested with or without attached stem and leaves to determine whether precursor availability restricted postharvest color development. Fruit were irradiated with HPS at 20/20 °C and 20/6 °C (day/night) for 168 hours. The absence of leaves hastened the decrease in hue angle, but this was due to yellowing and not to development of red blush. Since `Forelle' pears showed no response to light after harvest, two fully red cultivars, Bon Rouge and Red Anjou, were irradiated with HPS lamps for 72 hours. Hue angle was not affected by irradiation. Thus, anthocyanin synthesis was stimulated by postharvest irradiation with HPS lights in apples, but not in pears.

Activities of chalcone synthase and UDPGal: flavonoid-3-o-glycosyltransferase in relation to anthocyanin synthesis in apple

The activities of two enzymes, chalcone synthase (CS) and UDPGal:flavonoid-3-o-glycosyltransferase (UFGalT), and their relationships with anthocyanin synthesis in apple peel, were studied in 3 years of experiments. UFGalT activity was positively correlated with anthocyanin accumulation during fruit maturation in both ‘Delicious’ and ‘Ralls’ apples. Ethephon treatment at 110 days after full blossom (DAFB) increased UFGalT activity and also promoted anthocyanin formation. However, relatively high UFGalT activity was detected in control fruit at 90 DAFB although there was no anthocyanin in fruit peel. Also, when ethephon was applied at 70 DAFB, it induced high UFGalT activity and increased flavonoid accumulation without inducing anthocyanin formation at 90 DAFB. CS activity and flavonoid content were relatively high and constant from fruitlet to maturation stage. Fruit bagging suppressed accumulation of flavonoids and anthocyanins, but when the bags were removed at 110 DAFB, flavonoids and anthocyanins significantly increased by 130 DAFB. The data indicate that CS do not play a regulatory role in anthocyanin synthesis in apples exposed to light. UFGalT activity was correlated with anthocyanin synthesis during fruit maturation but it is not the only regulatory enzyme in this process since this enzyme was also active at 90 DAFB when no anthocyanin was present. We conclude that UFGalT probably catalyzes both quercetin glycoside and anthocyanin formations, and anthocyanin formation probably depends on cyanidin presence rather than on UFGalT activity. The controlling point in regulating anthocyanin synthesis is likely to be located at the steps from dihydroquercetin to the formation of cyanidin.

761 PB 187 DIFFERENTIAL DEVELOPMENT OF ANTHOCYANINS IN BAGGED `DELICIOUS' AND `FUJI' APPLES

Within red cultivars, highly colored apples are often preferred. In addition to being esthetically more appealing. better color often indicates riper, better tasting fruit. Anthocyanin synthesis in apples is influenced by many external factors including light, temperature, nutrition, pruning, thinning, growth regulators, and bagging. Bagging is the practice of enclosing young fruitlets in several layers of paper to promote color development after the bag is removed in the fall before harvest. In experiments related to the temperature optimum of color development in various cultivars, bagging was used to produce fruit void of anthocyanins. Double layer paper bags (black-lined outer bag, red inner bag) were placed on `Akafu-1 Fuji', `Oregon-Spur Delicious', and the early coloring `Scarlet Spur Delicious' on June 21, 1993. Bags were not removed until fruit was taken to the lab on September 22 for both `Delicious' and `Fuji'. Whereas bagged `Fuji' apples were without red pigment, bagged `Delicious' sports showed considerable red pigment development, completely covering the apple in the case of the blush-type `Scarlet Spur' and showing streaks without pigment in the snipe-type `Oregon-Spur'.