Anthocyanin Biosynthesis In Apple Research Articles

A viroid-derived small interfering RNA targets bHLH transcription factor MdPIF1 to regulate anthocyanin biosynthesis in Malus domestica.

Fruit colour is a critical determinant for the appearance quality and commercial value of apple fruits. Viroid-induced dapple symptom severely affects the fruit coloration, however, the underlying mechanism remains unknown. In this study, we identified an apple dimple fruit viroid (ADFVd)-derived small interfering RNA, named vsiR693, which targeted the mRNA coding for a bHLH transcription factor MdPIF1 (PHYTOCHROME-INTERACTING FACTOR 1) to regulate anthocyanin biosynthesis in apple. 5' RLM-RACE and artificial microRNA transient expression system proved that vsiR693 directly targeted the mRNA of MdPIF1 for cleavage. MdPIF1 positively regulated anthocyanin biosynthesis in both apple calli and fruits, and it directly bound to G-box element in the promoter of MdPAL and MdF3H, two anthocyanin biosynthetic genes, to promote their transcription. Expression of vsiR693 negatively regulated anthocyanin biosynthesis in both apple calli and fruits. Furthermore, co-expression of vsiR693 and MdPIF1 suppressed MdPIF1-promoted anthocyanin biosynthesis in apple fruits. Infiltration of ADFVd infectious clone suppressed coloration surrounding the injection sites in apple fruits, while a mutated version of ADFVd, in which the vsiR693 producing region was mutated, failed to repress fruit coloration around the injection sites. These data provide evidence that a viroid-derived small interfering RNA targets host transcription factor to regulate anthocyanin biosynthesis in apple.

The SMXL8-AGL9 module mediates crosstalk between strigolactone and gibberellin to regulate strigolactone-induced anthocyanin biosynthesis in apple.

Although the strigolactone (SL) signaling pathway and SL-mediated anthocyanin biosynthesis have been reported, the molecular association between SL signaling and anthocyanin biosynthesis remains unclear. In this study, we identified the SL signal transduction pathway associated with anthocyanin biosynthesis and the crosstalk between gibberellin (GA) and SL signaling in apple (Malus × domestica). ELONGATED HYPOCOTYL5 (HY5) acts as a key node integrating SL signaling and anthocyanin biosynthesis, and the SL response factor AGAMOUS-LIKE MADS-BOX9 (AGL9) promotes anthocyanin biosynthesis by activating HY5 transcription. The SL signaling repressor SUPPRESSOR OF MAX2 1-LIKE8 (SMXL8) interacts with AGL9 to form a complex that inhibits anthocyanin biosynthesis by downregulating HY5 expression. Moreover, the E3 ubiquitin ligase PROTEOLYSIS1 (PRT1) mediates the ubiquitination-mediated degradation of SMXL8, which is a key part of the SL signal transduction pathway associated with anthocyanin biosynthesis. In addition, the GA signaling repressor REPRESSOR-of-ga1-3-LIKE2a (RGL2a) mediates the crosstalk between GA and SL by disrupting the SMXL8-AGL9 interaction that represses HY5 transcription. Taken together, our study reveals the regulatory mechanism of SL-mediated anthocyanin biosynthesis and uncovers the role of SL-GA crosstalk in regulating anthocyanin biosynthesis in apple.

Apple SINA11-JAZ2 module is involved in jasmonate signaling response.

The E3 ubiquitin ligase MdSINA11 targets the jasmonate ZIM domain protein MdJAZ2 for ubiquitination and degradation through the 26S proteasome pathway, thereby initiating jasmonate signaling and jasmonic acid-triggered anthocyanin biosynthesis in apple.

MdbHLH162 connects the gibberellin and jasmonic acid signals to regulate anthocyanin biosynthesis in apple.

Anthocyanins are secondary metabolites induced by environmental stimuli and developmental signals. The positive regulators of anthocyanin biosynthesis have been reported, whereas the anthocyanin repressors have been neglected. Although the signal transduction pathways of gibberellin (GA) and jasmonic acid (JA) and their regulation of anthocyanin biosynthesis have been investigated, the cross-talk between GA and JA and the antagonistic mechanism of regulating anthocyanin biosynthesis remain to be investigated. In this study, we identified the anthocyanin repressor MdbHLH162 in apple and revealed its molecular mechanism of regulating anthocyanin biosynthesis by integrating the GA and JA signals. MdbHLH162 exerted passive repression by interacting with MdbHLH3 and MdbHLH33, which are two recognized positive regulators of anthocyanin biosynthesis. MdbHLH162 negatively regulated anthocyanin biosynthesis by disrupting the formation of the anthocyanin-activated MdMYB1-MdbHLH3/33 complexes and weakening transcriptional activation of the anthocyanin biosynthetic genes MdDFR and MdUF3GT by MdbHLH3 and MdbHLH33. The GA repressor MdRGL2a antagonized MdbHLH162-mediated inhibition of anthocyanins by sequestering MdbHLH162 from the MdbHLH162-MdbHLH3/33 complex. The JA repressors MdJAZ1 and MdJAZ2 interfered with the antagonistic regulation of MdbHLH162 by MdRGL2a by titrating the formation of the MdRGL2a-MdbHLH162 complex. Our findings reveal that MdbHLH162 integrates the GA and JA signals to negatively regulate anthocyanin biosynthesis. This study provides new information for discovering more anthocyanin biosynthesis repressors and explores the cross-talk between hormone signals.

E3 ubiquitin ligases SINA4 and SINA11 regulate anthocyanin biosynthesis by targeting the IAA29-ARF5-1-ERF3 module in apple.

Auxin/indole-3-acetic acid (AUX/IAA) and auxin response factor (ARF) proteins are important components of the auxin signalling pathway, but their ubiquitination modification and the mechanism of auxin-mediated anthocyanin biosynthesis remain elusive. Here, the ARF MdARF5-1 was identified as a negative regulator of anthocyanin biosynthesis in apple, and it integrates auxin and ethylene signals by inhibiting the expression of the ethylene response factor MdERF3. The auxin repressor MdIAA29 decreased the inhibitory effect of MdARF5-1 on anthocyanin biosynthesis by attenuating the transcriptional inhibition of MdERF3 by MdARF5-1. In addition, the E3 ubiquitin ligases MdSINA4 and MdSINA11 played negative and positive regulatory roles in anthocyanin biosynthesis by targeting MdIAA29 and MdARF5-1 for ubiquitination degradation, respectively. MdSINA4 destabilized MdSINA11 to regulate anthocyanin accumulation in response to auxin signalling. In sum, our data revealed the crosstalk between auxin and ethylene signals mediated by the IAA29-ARF5-1-ERF3 module and provide new insights into the ubiquitination modification of the auxin signalling pathway.

The transcription factor MdMYB2 influences cold tolerance and anthocyanin accumulation by activating SUMO E3 ligase MdSIZ1 in apple.

Conjugation of the small ubiquitin-like modifier (SUMO) peptide to target proteins is an important post-translational modification. SAP AND MIZ1 DOMAIN-CONTAINING LIGASE1 (MdSIZ1) is an apple (Malus domestica Borkh). SUMO E3 ligase that mediates sumoylation of its targets during plant growth and development under adverse environmental conditions. However, it is unclear how MdSIZ1 senses the various environmental signals and whether sumoylation is regulated at the transcriptional level. In this study, we analyzed the MdSIZ1 promoter and found that it contained an MYB binding site (MBS) motif that was essential for the response of MdSIZ1 to low temperature (LT) and drought. Subsequently, we used yeast one-hybridization screening to demonstrate that a MYB transcription factor, MdMYB2, directly bound to the MBS motif in the MdSIZ1 promoter. Phenotypic characterization of MdMYB2 and MdSIZ1 suggested that the expression of both MdMYB2 and MdSIZ1 substantially improved cold tolerance in plants. MdMYB2 was induced by LT and further activated the expression of MdSIZ1, thereby promoting the sumoylation of MdMYB1, a key regulator of anthocyanin biosynthesis in apple. MdMYB2 promoted anthocyanin accumulation in apple fruits, apple calli, and Arabidopsis (Arabidopsis thaliana) in an MdSIZ1-dependent manner. In addition, the interaction of MdMYB2 and the MdSIZ1 promoter substantially improved plant tolerance to cold stress. Taken together, our findings reveal an important role for transcriptional regulation of sumoylation and provide insights into plant anthocyanin biosynthesis regulation mechanisms and stress response.

Effect of Paper-Bagging on Apple Skin Patterning Associated with MdMYB10 Promoter Methylation.

Paper-bagging is an efficient method to maximize apple skin color, but a relationship between this technique and fruit skin patterning has not been demonstrated. Here, the ‘Fuji’ fruit with red-striped skin changed to red-blushed skin under re-exposure to light after bag treatment. Higher expression of MdMYB10, a transcription factor that regulates anthocyanin biosynthesis in apples, correlated with increased anthocyanin concentration in bag removal fruit. At the mature stage, a comparison of methylation status in the MdMYB10 promoter revealed that the methylation level in the region from −2585 to −2117 bp was reduced in bag removal fruit, especially for CHG context. It can be regulated by the downregulated expression of DNA methyltransferases such as MdMET, MdCMT, and MdDRM. Our results suggest that the bag removal treatment in this cultivar causes a change in skin patterning from striped to blushed pigmentation by inducing DNA demethylation of MdMYB10.

Phytochrome interacting factor MdPIF7 modulates anthocyanin biosynthesis and hypocotyl growth in apple.

Light affects many physiological and developmental processes of plants by regulating the expression and activity of light-responsive proteins. Among them, phytochrome interacting factors (PIFs) play pivotal roles in the regulation of anthocyanin accumulation and hypocotyl growth. However, the molecular mechanism is not well understood, especially in woody plants, such as apple (Malus × domestica). In this study, we identified a light-responsive PIF protein, MdPIF7, in apple and investigated the molecular mechanism of its regulation of anthocyanin biosynthesis and hypocotyl growth. We found that overexpression of MdPIF7 decreased anthocyanin accumulation in transgenic apple materials and promoted hypocotyl elongation in ectopically expressed Arabidopsis (Arabidopsis thaliana). Further investigation showed that MdPIF7 functioned by interacting with B-box 23 (MdBBX23), a positive regulator of anthocyanin biosynthesis in apple and hypocotyl growth inhibition in ectopically expressed Arabidopsis, and attenuating the transcriptional activation of MdBBX23 on LONG HYPOCOTYL 5 (MdHY5). In addition, MdPIF7 interacted with basic region leucine zipper 44 (MdbZIP44) and ethylene response factor 38 (MdERF38), two positive regulators of anthocyanin biosynthesis, and it negatively regulated MdbZIP44- and MdERF38-promoted anthocyanin accumulation by interfering with the interaction between MdbZIP44/MdERF38 and MdMYB1. Taken together, our results reveal that MdPIF7 regulates anthocyanin biosynthesis in apple and hypocotyl growth in ectopically expressed Arabidopsis through MdPIF7-MdBBX23-MdHY5 and MdPIF7-MdbZIP44/MdERF38-MdMYB1 modules. Our findings enrich the functional studies of PIF proteins and provide insights into the molecular mechanism of PIF-mediated anthocyanin biosynthesis and hypocotyl growth.

MdBBX21, a B-Box Protein, Positively Regulates Light-Induced Anthocyanin Accumulation in Apple Peel.

The red coloration of apple (Malus × domestica Borkh.) is due to the accumulation of anthocyanins in the fruit peel. Light is essential for anthocyanin biosynthesis in apple. In this study, we performed a transcriptome sequencing (RNA-seq) analysis of apple fruit exposed to light after unbagging. The identified differentially expressed genes included MdBBX21, which is homologous to Arabidopsis BBX21, suggesting it may be involved in light-induced anthocyanin biosynthesis. Additionally, MdBBX21 was localized in the nucleus and its gene was expressed earlier than MdMYB1 in apple peel treated with light. Overexpressing MdBBX21 in Arabidopsis and apple calli under light increased anthocyanin accumulation. Dual-luciferase and yeast one-hybrid assays confirmed that MdBBX21 binds to the MdHY5, MdBBX20, and MdBBX22-1/2 promoters and induces expression. At the same time, MdHY5 can also activate the expression of MdBBX21. Furthermore, bimolecular fluorescence complementation and yeast two-hybrid assays demonstrated that MdBBX21 can interact with MdHY5. This interaction can significantly enhance MdMYB1 promoter activity. These findings clarify the molecular mechanism by which MdBBX21 positively regulates light-induced anthocyanin accumulation in apple.

Regulatory Mechanisms of Anthocyanin Biosynthesis in Apple and Pear.

Anthocyanins contribute to the quality and flavour of fruits. They are produced through the phenylpropanoid pathway, which is regulated by specific key genes that have been identified in many species. The dominant anthocyanin forms are reversibly transformed at different pH states, thus forming different colours in aqueous solutions. In plants, anthocyanins are controlled by specific factors of the biosynthetic pathway: light, temperature, phytohormones and transcription factors. Although great progress in research on anthocyanin structures and the regulation of anthocyanin biosynthesis has been made, the molecular regulatory mechanisms of anthocyanin biosynthesis in different plants remain less clear. In addition, the co-regulation of anthocyanin biosynthesis is poorly understood. In this review, we summarise previous findings on anthocyanin biosynthesis, including the biochemical and biological features of anthocyanins; differences in anthocyanin biosynthesis among fruit species, i.e., apple, red pear, and the model plant Arabidopsis thaliana; and the developmental and environmental regulation of anthocyanin accumulation. This review reveals the molecular mechanisms underlying anthocyanin biosynthesis in different plant species and provides valuable information for the development of anthocyanin-rich red-skinned and red-fleshed apple and pear varieties.

FcMADS9 of fig regulates anthocyanin biosynthesis

MADS-box proteins have been shown to regulate fruit coloration, however, no information is available on MADS-box proteins in fig (Ficus carica L.). In this study, a full-length MADS-box gene was cloned from fig peels, namely FcMADS9, and its open reading frame was 759 bp, encoding 252 amino acids. Phylogenetic analysis suggested that it belonged to MIKCC family and SEPALLATA subfamily. Multiple sequence alignment showed that FcMADS9 contained the MADS-box, the Intervening, the Keratin and the C-terminal domains, and it composed a close relationship with MdMADS1, which regulates anthocyanin biosynthesis in apple. Overexpression of FcMADS9 upregulated the expressions of several anthocyanin biosynthetic genes and promoted anthocyanin accumulation in apple peels and calli, indicating its regulatory role in anthocyanin biosynthesis. The expression of FcMADS9 in fig peels was significantly enhanced by 8.24 times by 150 mg L−1 ethephon, a generator of ethylene, indicating that FcMADS9-induced anthocyanin accumulation may have a close relationship with ethylene. Then, anthocyanin accumulation in FcMADS9-overexpressed apple calli was further significantly increased by ethephon treatment, but was partially inhibited by 1-methylcyclopropene, an antagonist of ethylene for receptor binding sites, indicating that FcMADS9 regulates anthocyanin biosynthesis and ethylene is involved in this effect.

BTB/TAZ protein MdBT2 integrates multiple hormonal and environmental signals to regulate anthocyanin biosynthesis in apple.

BT2 is a BTB/TAZ domain protein with key roles in multiple stress responses and the plant development of Arabidopsis (Figueroa et al. 2005; Ren et al. 2007; Mandadi et al. 2009). Recent studies have demonstrated that apple MdBT2 functions as a negative regulator in diverse hormonal and environmental signal-induced anthocyanin biosynthesis, suggesting that MdBT2 integrates stress signals and anthocyanin biosynthesis.

Methylation of MdMYB1 locus mediated by RdDM pathway regulates anthocyanin biosynthesis in apple.

SummaryMethylation at the MdMYB1 promoter in apple sports has been reported as a regulator of the anthocyanin pathway, but little is known about how the locus is recognized by the methylation machinery to regulate anthocyanin accumulation. In this study, we analysed three differently coloured ‘Fuji’ apples and found that differences in the transcript levels of MdMYB1, which encodes a key regulator of anthocyanin biosynthesis, control the anthocyanin content (and therefore colour) in fruit skin. The CHH methylation levels in the MR3 region (−1246 to −780) of the MdMYB1 promoter were found to be negatively correlated with MdMYB1 expression. Thus, they were ideal materials to study DNA methylation in apple sports. The protein of RNA‐directed DNA methylation (RdDM) pathway responsible for CHH methylation, MdAGO4, was found to interact with the MdMYB1 promoter. MdAGO4s can interact with MdRDM1 and MdDRM2s to form an effector complex, fulfilling CHH methylation. When MdAGO4s and MdDRM2s were overexpressed in apple calli and Arabidopsis mutants, those proteins increase the CHH methylation of AGO4‐binding sites. In electrophoretic mobility shift assays, MdAGO4s were found to specifically bind to sequence containing ATATCAGA. Knockdown of MdNRPE1 did not affect the binding of MdAGO4s to the c3 region of the MdMYB1 promoter in 35S::AGO4 calli. Taken together, our data show that the MdMYB1 locus is methylated through binding of MdAGO4s to the MdMYB1 promoter to regulate anthocyanin biosynthesis by the RdDM pathway.

The ERF transcription factor MdERF38 promotes drought stress-induced anthocyanin biosynthesis in apple.

Drought stress induces anthocyanin biosynthesis in many plant species, but the underlying molecular mechanism remains unclear. Ethylene response factors (ERFs) play key roles in plant growth and various stress responses, including affecting anthocyanin biosynthesis. Here, we characterized an ERF protein, MdERF38, which is involved in drought stress-induced anthocyanin biosynthesis. Biochemical and molecular analyses showed that MdERF38 interacted with MdMYB1, a positive modulator of anthocyanin biosynthesis, and facilitated the binding of MdMYB1 to its target genes. Therefore, MdERF38 promoted anthocyanin biosynthesis in response to drought stress. Furthermore, we found that MdBT2, a negative modulator of anthocyanin biosynthesis, decreased MdERF38-promoted anthocyanin biosynthesis by accelerating the degradation of the MdERF38 protein. In summary, our data provide a mechanism for drought stress-induced anthocyanin biosynthesis that involves dynamic modulation of MdERF38 at both transcriptional and post-translational levels.

MdWRKY40 promotes wounding-induced anthocyanin biosynthesis in association with MdMYB1 and undergoes MdBT2-mediated degradation.

Wounding stress leads to anthocyanin accumulation. However, the underlying molecular mechanism remains elusive. In this study, MdWRKY40 was found to promote wounding-induced anthocyanin biosynthesis in association with MdMYB1 and undergo MdBT2-mediated degradation in apple. We found that MdMYB1, a positive regulator of anthocyanin biosynthesis, was essential for the wounding-induced anthocyanin biosynthesis in apple. MdWRKY40 was identified as an MdMYB1-interacting protein, and enhanced the binding of MdMYB1 to its target genes in response to wounding. We found that MdBT2 interacted physically with MdWRKY40 and was involved in its degradation through the 26S proteasome pathway. Our results demonstrate that MdWRKY40 is a key modulator in the wounding-induced anthocyanin biosynthesis, which provides new insights into the regulation of wounding-induced anthocyanin biosynthesis at both the transcriptional and post-translational levels in apple.

The R2R3-MYB transcription factor MdMYB24-like is involved in methyl jasmonate-induced anthocyanin biosynthesis in apple

Anthocyanins in apple species are important secondary metabolites that are beneficial for human health. Previous studies revealed that methyl jasmonate (MeJA) promotes anthocyanin accumulation by up-regulating the transcription of related genes. In this study, we isolated a jasmonate (JA)-induced apple MYB gene, MdMYB24-like (MdMYB24L). The encoded nuclear protein contains a conserved R2R3 domain and is homologous to Arabidopsis thaliana AtMYB24. Additionally, MdMYB24L was observed to interact with JA signaling factors (MdJAZ8, MdJAZ11, and MdMYC2) in yeast and in planta. The MdMYC2 protein was also targeted by MdJAZ8 and MdJAZ11, which are rapidly degraded under MeJA treatment. The overexpression of MdMYB24L resulted in higher anthocyanin contents in the transgenic apple ‘Orin’ calli than in the wild-type control calli. Moreover, the expression levels of the anthocyanin biosynthesis structural genes MdUFGT and MdDFR were up-regulated in the transgenic calli. Furthermore, MdMYB24L positively regulated the transcription of MdDFR and MdUFGT by binding to the MYB-binding site motifs in their promoters. Interestingly, the interaction between MdMYC2 and MdMYB24L further enhanced the transcription of MdUFGT, whereas MdJAZ8 and MdJAZ11 attenuated this effect. We herein provide new details regarding the molecular mechanism by which MYB transcription factors help regulate anthocyanin biosynthesis via JA signaling pathways.

Identification of new regulators through transcriptome analysis that regulate anthocyanin biosynthesis in apple leaves at low temperatures.

Anthocyanin pigments play many roles in plants, including providing protection against biotic and abiotic stresses. To identify new regulatory genes in apple (Malus domestica) that may be involved in regulating low temperature induced anthocyanin biosynthesis, we performed RNA-seq analysis of leaves from the ‘Gala’ apple cultivar following exposure to a low temperature (16 °C). A visible red color appeared on the upper leaves and the anthocyanin content increased significantly after the low temperature treatment. Genes from the flavonoid biosynthesis pathway were significantly enriched among the differentially expressed genes, and the expression of several transcription factors was shown by WGCNA (weighted gene co-expression network analysis) to correlate with anthocyanin accumulation, including members of the MYB, MADS, WRKY, WD40, Zinc Finger and HB-ZIP families. Three MYB transcription factors (MdMYB12, MdMYB22 and MdMYB114), which had several CBF/DREB response elements in their promoters, were significantly induced by low temperature exposure and their expression also correlated highly with anthocyanin accumulation. We hypothesize that they may act as regulators of anthocyanin biosynthesis and be regulated by CBF/DREB transcription factors in apple leaves under low temperature conditions. The analyses presented here provide insights into the molecular mechanisms underlying anthocyanin accumulation during low temperature exposure.

The effect of promoter methylation on MdMYB1 expression determines the level of anthocyanin accumulation in skins of two non-red apple cultivars

BackgroundFruit color in apple (Malus domestica Borkh.) is ascribed mainly to the accumulation of anthocyanin pigments, and is an important trait for determining fruit market acceptance. Bagging is a commonly used treatment to enhance the red pigmentation in apple skin. The MdMYB1 transcription factor gene plays an important role in the biosynthesis of anthocyanin in apple after bag removal, but little is known about how MdMYB1 transcription is regulated.ResultsIn this study, we investigated pigmentation in the non-red skinned cultivars ‘Granny Smith’ and ‘Golden Delicious’ after bag removal. The fruit skins of the two cultivars showed red/pink pigmentation after bag treatment. Transcript levels of MdMYB1, the master regulator of anthocyanin biosynthesis in apple, increased, and showed a correlation with anthocyanin content in both cultivars after bag removal. The MdMYB1 genomic sequences were compared in the two cultivars, which showed that the green-fruited cultivar ‘Granny Smith’ harbors the MdMYB1–1 and MdMYB1–2 alleles, while the yellow-fruited cultivar ‘Golden Delicious’ harbors only MdMYB1–2. A comparison of methylation levels in the 2 kb region upstream of the MdMYB1 ATG between the bag-treated fruits after removal from the bags and the unbagged fruits showed a correlation between hypomethylation and the red-skin phenotype in ‘Granny Smith’. Moreover, ‘Granny Smith’ fruits responded to treatment with 5-aza-2′-deoxycytidine, an inducer of DNA demethylation. An investigation of the MdMYB1 promoter in ‘Granny Smith’ showed reduced methylation in the regions − 2026 to − 1870 bp, − 1898 to − 1633 bp, and − 541 to − 435 bp after bag removal and 5-aza-2′-deoxycytidine treatments.ConclusionsDifferences in anthocyanin levels between ‘Granny Smith’ and ‘Golden Delicious’ can be explained by differential accumulation of MdMYB1-specific mRNA. Different levels of MdMYB1 transcripts in the two cultivars are associated with methylation levels in the promoter region. Hypomethylation of the MdMYB1 promoter is correlated with the formation of red pigmentation in ‘Granny Smith’ fruit skins. As a result, red pigmentation in Granny Smith’ was more intense than in ‘Golden Delicious’ fruits after bag removal.

Epigenetic regulation of MdMYB1 is associated with paper bagging-induced red pigmentation of apples.

Paper-bagging treatment can transform non-transcribed MdMYB1 - 2 and MdMYB1 - 3 alleles into transcribed alleles through epigenetic regulations, resulting in the red pigmentation of a normally non-red apple cultivar 'Mutsu.' Anthocyanin biosynthesis in apples is regulated by MdMYB1/A/10, an R2R3-Type MYB gene. 'Mutsu,' a triploid apple cultivar harboring non-transcribed MdMYB1-2 and MdMYB1-3 alleles, retains green skin color under field conditions. However, it can show red/pink pigmentation under natural or artificial ultraviolet-B (UV-B) light exposure after paper-bagging and bag removal treatment. In the present study, we found that in 'Mutsu,' paper bagging-induced red pigmentation was due to the activation of non-transcribed MdMYB1-2/-3 alleles, which triggered the expression of downstream anthocyanin biosynthesis genes in a UV-B-dependent manner. By monitoring the epigenetic changes during UV-B-induced pigmentation, no significant differences in DNA methylation and histone modifications in the 5' upstream region of MdMYB1-2/-3 were recorded between the UV-B-treated fruit skin (red) and the fruit skin treated only by white light (green). In contrast, bag treatment lowered the DNA methylation in this region of MdMYB1-2/-3 alleles. Similarly, higher levels of histone H3 acetylation and trimethylation of H3 tail at lysine 4, and lower level of trimethylation of H3 tail at lysine 27 were observed in the 5' upstream region of MdMYB1-2/-3 in the skin of the fruit immediately after bag removal. These results suggest that bagging treatment can induce epigenetic changes, facilitating the binding of trans factor(s) to MdMYB1-2/-3 alleles, resulting in the activation of these MYBs after bag removal.

Expression Profiling of Several Gene Families Involved in Anthocyanin Biosynthesis in Apple (Malus domestica Borkh.) Skin During Fruit Development

Anthocyanins are secondary metabolites in higher plants that contribute to the colors of flowers and fruits. MYB, bHLH, and UFGT genes are key genes in fruit anthocyanin synthesis. We identified two MYB1 genes, two bHLH3 genes, three bHLH33 genes, and four UFGT genes in the apple (Malus domestica Borkh.) genome and analyzed the expression of these genes in apple skin at the fruitlet stage and ripening fruit stage, two peaks of anthocyanin accumulation in apple skin. Results showed that the two MYB genes, one bHLH3 gene, one bHLH33 gene, and two UFGT genes were involved in anthocyanin synthesis. MdbHLH3-1 and MdbHLH33-1 transcript levels were correlated with anthocyanin accumulation in the four cultivars during fruit development. However, there was variation in MdMYBs and MdUFGTs expression patterns at different fruit development stages among cultivars. Transcript levels of MdMYB1a and MdUFGT2-1 were higher in the red skin of young fruit, whereas transcript levels of MdMYB1, MdUFGT2-1, and MdUFGT4 were higher in the red skin of ripening fruit. Besides, MdUFGT4 was only expressed in fruit skin of red cultivars, indicating that MdUFGT4 was important for anthocyanin synthesis in red skin cultivars. Furthermore, all these genes transcripts were induced by light. Our results indicated that MdMYB1a, MdMYB1, MdbHLH3-1, MdbHLH33-1, MdUFGT2-1, and MdUFGT4 were involved in apple skin anthocyanin synthesis, but they may function in different roles at different fruit development stages in different cultivars.