Anthocyanin Accumulation In Apple Research Articles

Transcription factor MdNAC33 is involved in ALA-induced anthocyanin accumulation in apples

5-Aminolevulinic acid (ALA), as a new natural plant growth regulator, has a significant function in promoting anthocyanin accumulation in many species of fruits. However, the mechanisms underlying remain obscure. In a transcriptome study of our group, it was found that many transcription factors (TFs) including NACs responsive to ALA treatment during anthocyanin accumulation. In the present study, we found a NAC of apple, MdNAC33 was coordinatively expressed with anthocyanin accumulation after ALA treatment in the apple fruits and leaves, suggesting that this TF may be involved in anthocyanin accumulation induced by ALA. We found that the MdNAC33 protein was localized in the nucleus and exhibited strong transcriptional activity in both yeast cells and plants, where its C-terminal contributed to the transcriptional activity. Functional analysis showed that overexpression of MdNAC33 promoted the accumulation of anthocyanin, while the silencing vector of MdNAC33 (RNAi) significantly impaired the anthocyanin accumulation induced by ALA. Yeast one-hybrid (Y1H), luciferase assay and electrophoretic mobility shift assay (EMSA) indicated that MdNAC33 could bind to promoters of MdbHLH3, MdDFR and MdANS to activate the gene expressions. In addition, MdNAC33 specifically interacts with MdMYB1, a positive regulator of anthocyanin biosynthesis, which was then in turn binding to its target genes MdUFGT and MdGSTF12, to promote anthocyanin accumulation in apples. Taken together, our data indicate that MdNAC33 plays multiple roles in ALA-induced anthocyanin biosynthesis. It provides new insights into the mechanisms of anthocyanin accumulation induced by ALA.

The E3 ubiquitin ligase SINA1 and the protein kinase BIN2 cooperatively regulate PHR1 in apple anthocyanin biosynthesis.

PHR1 (PHOSPHATE STARVATION RESPONSE1) plays key roles in the phosphate (Pi) starvation response and in Pi-deficiency-induced anthocyanin biosynthesis in plants. However, the post-translational regulation of PHR1 is unclear, and the molecular basis of PHR1-mediated anthocyanin biosynthesis remains elusive. In this study, we determined that MdPHR1 was essential for Pi-deficiency-induced anthocyanin accumulation in apple (Malus × domestica). MdPHR1 interacted with MdWRKY75, a positive regulator of anthocyanin biosynthesis, to enhance the MdWRKY75-activated transcription of MdMYB1, leading to anthocyanin accumulation. In addition, the E3 ubiquitin ligase SEVEN IN ABSENTIA1 (MdSINA1) negatively regulated MdPHR1-promoted anthocyanin biosynthesis via the ubiquitination-mediated degradation of MdPHR1. Moreover, the protein kinase MdBIN2 phosphorylated MdPHR1 and positively regulated MdPHR1-mediated anthocyanin accumulation by attenuating the MdSINA1-mediated ubiquitination degradation of MdPHR1. Taken together, these findings not only demonstrate the regulatory role of MdPHR1 in Pi-starvation induced anthocyanin accumulation, but also provide an insight into the post-translational regulation of PHR1. This article is protected by copyright. All rights reserved.

Mdm-miR858 targets MdMYB9 and MdMYBPA1 to participate anthocyanin biosynthesis in red-fleshed apple.

Anthocyanins are important secondary metabolites in plants. They are important for human health because of their antioxidant activities and because their dietary intake reduces the incidence of cardiovascular and cerebrovascular diseases and tumors. The biosynthesis of anthocyanins and its regulation in fruits and vegetables is a global research hotspot. Compared with cultivated apples, the red-fleshed apple is a relatively new and popular commodity in the market. Previous studies on red-fleshed apples have focused on the basis for the high anthocyanin content and the transcriptional regulation of anthocyanin synthesis. In the present study, we focused on the mechanism of microRNA-mediated post-transcriptional regulation of anthocyanin synthesis in red-fleshed apples. We identified a microRNA (miRNA), designated mdm-miR858, that is specifically expressed in the flesh of apple fruit. The expression level of miR858 was significantly lower in red-fleshed apples than in white-fleshed apples. The overexpression of mdm-miR858 significantly inhibited anthocyanin accumulation, whereas the silencing of mdm-miR858 promoted anthocyanin synthesis in STTM858 transgenic apple calli. Further analyses showed that mdm-miR858 targets the transcription factor genes MdMYB9 and MdMYBPA1 to participate anthocyanin accumulation in apple. Our results also show that MdHY5, a transcription factor in the light signaling pathway, can bind to the promoter of mdm-miR858 to inhibit its transcription, thereby regulating anthocyanin synthesis. Based on our results, we describe a novel HY5-miR858-MYB loop involved in the modulation of anthocyanin biosynthesis. These findings provide new information about how plant miRNAs regulate anthocyanin anabolism and provide a basis for breeding new anthocyanin-rich, red-fleshed apple varieties.

The Transcription Factor MdERF78 Is Involved in ALA-Induced Anthocyanin Accumulation in Apples.

As a friendly plant growth regulator to the environment, 5-aminolevulinic acid (ALA) has been widely used in plant production, such as fruit coloration, stress resistance, and so on. Previous studies have identified some genes that have a function in the anthocyanin accumulation induced by ALA. However, the regulatory mechanism has not been well revealed. In the current study, we proposed that an ALA-responsive transcription factor, MdERF78, regulated anthocyanin accumulation. MdERF78, overexpressed in apple peels or calli, resulted in a significant increase of anthocyanins, while MdERF78 interference had an opposite trend. Furthermore, the anthocyanin accumulation induced by MdERF78 overexpression was enhanced by exogenous ALA treatment, suggesting that MdERF78 was involved in the ALA-induced anthocyanin accumulation. Yeast one-hybrid and dual luciferase reporter assays revealed that MdERF78 bound to the promoters of MdF3H and MdANS directly and activated their expressions. Additionally, MdERF78 interacted with MdMYB1 and enhanced the transcriptional activity of MdMYB1 to its target gene promoters. Based on these, it can be concluded that MdERF78 has a positive function in ALA-induced anthocyanin accumulation via the MdERF78-MdF3Hpro/MdANSpro and MdERF78-MdMYB1-MdDFRpro/MdUFGTpro/MdGSTF12pro regulatory network. These findings provide new insights into the regulatory mechanism of ALA-promoted anthocyanin accumulation.

Transcriptome Analysis of Apples in High-Temperature Treatments Reveals a Role of MdLBD37 in the Inhibition of Anthocyanin Accumulation.

Coloring in apple fruit due to anthocyanin accumulation is inhibited by high temperature; however, the underlying mechanism remains unclear. In the present study, total anthocyanin and cyanidin 3-galactoside contents were determined and compared between cv. ‘Redchief Delicious’ apple fruits at 25 °C and 35 °C treatments. The high temperature (35 °C) treatment substantially decreased total anthocyanin and cyanidin 3-galactoside contents. The transcriptomes of 25 °C- and 35 °C-treated apples were analyzed by high-throughput RNA sequencing. A total of 8354 differentially expressed genes (DEGs) were detected at four time points corresponding to the two temperature treatments. The up-regulated DEGs were annotated using GO as well as KEGG databases. A network module of 528 genes (including 21 transcription factors) most associated with the total anthocyanin and cyanidin 3-galactoside contents was constructed by weighted correlation network analysis (WGCNA). In the WGCNA module, we unearthed a LOB domain-containing gene designated as MdLBD37. The expression of MdLBD37 was sharply up-regulated by high temperature and negatively correlated with the total anthocyanin and cyanidin 3-galactoside contents. Overexpression of MdLBD37 in apple fruit and calli decreased the expression of anthocyanin biosynthetic genes, such as MdCHI, MdCHS, MdF3H, MdANS, MdDFR, and MdUFGT, along with anthocyanin accumulation. Our results suggested that MdLBD37 significantly influenced the high-temperature inhibition of anthocyanin accumulation in apples. The findings shed more light on the mechanism of anthocyanin inhibition during high-temperature stress in apples.

A long noncoding RNA functions in high-light-induced anthocyanin accumulation in apple by activating ethylene synthesis.

Anthocyanin production in apple (Malus domestica) fruit and their consequent coloration can be induced by high-light treatment. The hormone ethylene is also essential for this coloration, but the regulatory relationships that link ethylene and light with anthocyanin-associated coloration are not well defined. In this study, we observed that high-light treatment of apple fruit increased anthocyanin accumulation more than moderate-light treatment did and was the main contributor of induced ethylene production and activation of anthocyanin biosynthesis. A transcriptome study of light-treated apple fruit suggested that a long noncoding RNA (lncRNA), MdLNC610, the corresponding gene of which is physically located downstream from the 1-aminocyclopropane-1-carboxylate oxygenase (ACO) ethylene biosynthesis gene MdACO1, likely affects anthocyanin biosynthesis under high-light treatment. Expression and promoter β-glucuronidase reporter analyses further showed that MdLNC610 upregulates expression of MdACO1 and so likely participates in high-light-induced ethylene biosynthesis. Overexpression of MdACO1 and MdLNC610 in apple fruit and calli indicated that a major increase in MdLNC610 expression activates MdACO1 expression, thereby causing an increase in ethylene production and anthocyanin levels. These results suggest that MdLNC610 participates in the regulation of high-light-induced anthocyanin production by functioning as a positive regulator to promote MdACO1 expression and ethylene biosynthesis. Our study provides insights into the relationship between mRNA and lncRNA networks in the ethylene biosynthetic pathway and anthocyanin accumulation in apple fruit.

ROS1 promotes low temperature-induced anthocyanin accumulation in apple by demethylating the promoter of anthocyanin-associated genes.

Low temperature can affect the growth and development of plants through changes in DNA demethylation patterns. Another known effect of low temperature is the accumulation of anthocyanin pigments. However, it is not known whether the two phenomena are linked, specifically whether DNA demethylation participates in anthocyanin accumulation in response to low-temperature stress. The ROS1 gene is involved in plant DNA demethylation and influences methylation levels in response to low-temperature stress. In this study, using RNA sequencing, we detected the transcription levels of MdROS1, as well as those of anthocyanin biosynthesis-related genes, correlate with the anthocyanin content in apple (Malus domestica), at low temperature. Genomic bisulfite sequencing showed that the methylation levels of the promoters of the anthocyanin-related genes MdCHS, MdCHI, MdF3′H, MdANS, MdUFGT, and MdMYB10 decreased in apple leaves after low-temperature treatment. Similar expression and methylation results were found in apple fruit. Transiently silencing MdROS1 in the leaves and fruit of apple cultivars inhibited the accumulation of anthocyanins and led to decreased expression of anthocyanin biosynthetic genes, and the opposite results were detected in MdROS1-overexpressing leaves and fruit. A promoter binding assay showed that the conserved RRD-DME domains of MdROS1 bind directly to the promoters of MdF3′H and MdUFGT. Taken together, these results suggest that ROS1 affects the anthocyanin biosynthetic pathway by decreasing the methylation level of anthocyanin-related gene promoters, thereby increasing their expression and increasing anthocyanin accumulation.

MicroRNA172 targets APETALA2 to regulate flavonoid biosynthesis in apple (Malus domestica).

MicroRNA172 (miR172) plays a role in regulating a diverse range of plant developmental processes, including flowering, fruit development and nodulation. However, its role in regulating flavonoid biosynthesis is unclear. In this study, we show that transgenic apple plants over-expressing miR172 show a reduction in red coloration and anthocyanin accumulation in various tissue types. This reduction was consistent with decreased expression of APETALA2 homolog MdAP2_1a (a miR172 target gene), MdMYB10, and targets of MdMYB10, as demonstrated by both RNA-seq and qRT-PCR analyses. The positive role of MdAP2_1a in regulating anthocyanin biosynthesis was supported by the enhanced petal anthocyanin accumulation in transgenic tobacco plants overexpressing MdAP2_1a, and by the reduction in anthocyanin accumulation in apple and cherry fruits transfected with an MdAP2_1a virus-induced-gene-silencing construct. We demonstrated that MdAP2_1a could bind directly to the promoter and protein sequences of MdMYB10 in yeast and tobacco, and enhance MdMYB10 promotor activity. In Arabidopsis, over-expression of miR172 reduced flavonoid (including anthocyanins and flavonols) concentration and RNA transcript abundance of flavonoid genes in plantlets cultured on medium containing 7% sucrose. The anthocyanin content and RNA abundance of anthocyanin genes could be partially restored by using a synonymous mutant of MdAP2_1a, which had lost the miR172 target sequences at mRNA level, but not restored by using a WT MdAP2_1a. These results indicate that miR172 inhibits flavonoid biosynthesis through suppressing the expression of an AP2 transcription factor that positively regulates MdMYB10.

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.

Genome-Wide Analysis of the Glutathione S-Transferase (GST) Genes and Functional Identification of MdGSTU12 Reveals the Involvement in the Regulation of Anthocyanin Accumulation in Apple.

Anthocyanins have essential biological functions, affecting the development of horticultural production. They are synthesized in the cytoplasm through flavonoid metabolic pathways and finally transported into vacuoles for storage. Plant glutathione S-transferases (GSTs) are multifunctional enzymes involved in anthocyanin transportation. In this study, we identified 38 GSTs from the apple (Malus domestica) genome (HFTH1 Whole Genome v1.0) based on the sequence similarity with the GST family proteins of Arabidopsis. These MdGST genes could be grouped into nine chief subclasses: U, F, L, Z, T, GHR, EF1Bγ, TCHQD, and DHAR. The structures, motifs, three-dimensional models, and chromosomal distribution of MdGST genes were further analyzed. Elements which are responsive for some hormones and stress, and others that involve genes related to flavonoid biosynthesis were forecast in the promoter of MdGST. In addition, we identified 32 orthologous gene pairs between apple and Arabidopsis. These genes indicated that numerous apple and Arabidopsis counterparts appeared to be derived from a common ancestor. Amongst the 38 MdGST genes, MdGSTU12 was considerably correlated with anthocyanin variation in terms of extracting expression profiles from reported. Finally, further functional identification in apple transgenic calli and subcellular localization confirmed that MdGSTU12 was of great significance in anthocyanin accumulation in apple.

Apple MPK4 mediates phosphorylation of MYB1 to enhance light-induced anthocyanin accumulation.

Anthocyanins are plant pigments with diverse biological functions that contribute to fruit quality and are beneficial to human health. Anthocyanin accumulation can be influenced by environmental signals, such as light, and plants have developed sophisticated systems to receive and transduce these signals. However, the associated molecular mechanisms are not well understood. In this study, we investigated the potential function of mitogen-activated protein kinases, which are members of the light signaling pathway, during light-induced anthocyanin accumulation in apple (Malus domestica) fruit peels. An antibody array and yeast two-hybrid screen indicated that proteins encoded by two MdMPK4 genes are light-activated and interact with the transcription factor and anthocyanin biosynthesis regulator MdMYB1. A phosphorylation assay showed that the MdMPK4 proteins phosphorylate MdMYB1, thereby increasing its stability under light conditions. Transient MdMPK4 and MdMYB1 overexpression assays further revealed that light-induced anthocyanin accumulation relies on MdMPK4 kinase activity, which is required for maximum MdMYB1 activity. Based on the expression of the chromosome 6 allele MdMPK4-06G under light conditions and the presence of light response elements in the MdMPK4-06G promoter, we concluded that it is more responsive to light than the chromosome 14 allele MdMPK4-14G. These results suggest a potential biotechnological strategy for increasing fruit anthocyanin content via light induction.

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.

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.

Genome-wide analysis of auxin response factor (ARF) genes and functional identification of MdARF2 reveals the involvement in the regulation of anthocyanin accumulation in apple

ABSTRACT Auxin response factors (ARFs) mediate auxin response gene transcription by directly binding with AuxREs. However, whether ARF family genes involved in anthocyanin accumulation in apple is largely unexplored. In this study, thirty-three MdARF family members were identified in the apple (Malus × domestica) genome (HFTH1 Whole Genome v1.0) based on the sequence similarity with ARF family members of Arabidopsis. Phylogenetic analysis revealed that thirty-three MdARF members were clustered into Class I, Class II and Class III. Functional domain analysis showed that MdARF proteins have some typical domains such as B3-like DNA-binding domain and Auxin_resp domain, as well as motif III and motif IV. Chromosome localisation analysis demonstrated that apple ARF members were widely distributed in the genome. Among these MdARF genes, MdARF2 was significantly correlated with anthocyanin variation according to extracting expression profiles from published RNA-seq data. Further functional identification in apple calli suggested that MdARF2 functions as a transcriptional repressor of anthocyanin biosynthesis. These findings provide evidence that MdARF family members play a role in the regulation of anthocyanin accumulation in apple.

L-Isoleucine (Ile) Promotes Anthocyanin Accumulation in Apples

The functions of l-isoleucine (Ile) in anthocyanin biosynthesis were investigated in apple fruit. Whole trees (Malus × domestica Bokh.) were treated with Ile or n-propyl dihydrojasmonate (PDJ), which is a jasmonic acid (JA) analog, 20 and 30 days before harvest. Both Ile and PDJ treatments stimulated anthocyanin formation at 30 and 35 days after treatment (DAT). The expressions of MdUF3GT and MdMYB1 genes under the Ile and PDJ treatments were higher than those in the untreated control at 19 and 30 DAT. Endogenous JA and jasmonoyl-l-isoleucine (JA-Ile) concentrations in Ile- and PDJ- treated fruit were increased at 19 and 30 DAT. At 30 DAT, the Ile and PDJ treatments stimulated the expressions of MdJAR1, which is related to the conjugation of JA to Ile, and MdMYC2, which is a transcription factor in the JA response. Our study suggests that Ile can induce anthocyanin formation depending on the increase of JA-Ile production and the up-regulation of MdJAR1 and MdMYC2 in apples.

Anthocyanin Biosynthesis and Methylation of the MdMYB10 Promoter Are Associated with the Red Blushed-Skin Mutant in the Red Striped-Skin "Changfu 2" Apple.

The color of apple skin, particularly anthocyanin-based coloration, is a key factor determining market acceptance. The mechanisms of anthocyanin accumulation in apples with different skin color patterns (i.e., striped and blushed) were analyzed. In total, 14 anthocyanins and 5 procyanidins were simultaneously assayed in red blushed-skin mutants (CF-B1 and CF-B2) and red striped-skin parents (CF-S1 and CF-S2), and 13 significant differences were revealed. Anthocyanin accumulation was significantly higher in the red blushed-skin apples than it was in the parents. The transcript levels of anthocyanin biosynthesis genes and regulatory factors (MdMYB10, MdbHLH3, and MdWD40) were associated with different skin color patterns during the coloring period at 4, 6, and 8 days after the fruits were debagged. The methylation levels of the MdMYB10 promoter regions -1203 to -779 bp, -1667 to -1180 bp, and -2295 to -1929 bp were associated with different skin color patterns, and there was more methylation in red striped-skin apples. These results improve our understanding of anthocyanin accumulation and its underlying molecular mechanism in apples with different skin color patterns, thereby providing valuable information for apple breeding.

A novel NAC transcription factor, MdNAC42, regulates anthocyanin accumulation in red-fleshed apple by interacting with MdMYB10.

Anthocyanin pigmentation is an important consumption trait of apple (Malus domestica Borkh.). In this study, we focused on the identification of NAC (NAM, ATAF1/2 and CUC2) proteins involved in the regulation of anthocyanin accumulation in apple flesh. A group of MdNACs was selected for comparison of expression patterns between the white-fleshed cultivar 'Granny Smith' and red-fleshed 'Redlove'. Among them, MdNAC42 was screened, which exhibited a higher expression level in red-fleshed than in white-fleshed fruit, and has a positive correlation with anthocyanin content as fruits ripened. Moreover, overexpression of MdNAC42 in apple calli resulted in the up-regulation of flavonoid pathway genes, including MdCHS, MdCHI, MdF3H, MdDFR, MdANS and MdUFGT, thereby increasing the accumulation of anthocyanins, which confirmed the roles of MdNAC42 in anthocyanin biosynthesis. Notably, MdNAC42 was demonstrated to have an obvious interaction with MdMYB10 either in vitro or in vivo by yeast two-hybrid combined with bimolecular fluorescence complementation, further suggesting that MdNAC42 is an important part of the regulatory network controlling the anthocyanin pigmentation of red-fleshed apples. To the best of our knowledge, this is the first report identifying the MdNAC gene as related to anthocyanin accumulation in red-fleshed apples. This study provides valuable information for improving the regulatory model of anthocyanin biosynthesis in apple fruit.

An apple MYB transcription factor regulates cold tolerance and anthocyanin accumulation and undergoes MIEL1-mediated degradation.

Summary MYB transcription factors (TFs) have been demonstrated to play diverse roles in plant growth and development through interaction with basic helix‐loop‐helix (bHLH) TFs. MdbHLH33, an apple bHLH TF, has been identified as a positive regulator in cold tolerance and anthocyanin accumulation by activating the expressions of MdCBF2 and MdDFR. In the present study, a MYB TF MdMYB308L was found to also positively regulate cold tolerance and anthocyanin accumulation in apple. We found that MdMYB308L interacted with MdbHLH33 and enhanced its binding to the promoters of MdCBF2 and MdDFR. In addition, an apple RING E3 ubiquitin ligase MYB30‐INTERACTING E3 LIGASE 1 (MdMIEL1) was identified to be an MdMYB308L‐interacting protein and promoted the ubiquitination degradation of MdMYB308L, thus negatively regulated cold tolerance and anthocyanin accumulation in apple. These results suggest that MdMYB308L acts as a positive regulator in cold tolerance and anthocyanin accumulation in apple by interacting with MdbHLH33 and undergoes MdMIEL1‐mediated protein degradation. The dynamic change in MYB‐bHLH protein complex seems to play a key role in the regulation of plant growth and development.

Transcriptome profiling of anthocyanin biosynthesis in the peel of \u2018Granny Smith\u2019 apples (Malus domestica) after bag removal

BackgroundBagging is commonly used to enhance red pigmentation and thereby improve fruit quality of apples (Malus domestica). The green-skinned apple cultivar ‘Granny Smith’ develops red pigmentation after bagging removal, but the signal transduction pathways mediating light-induced anthocyanin accumulation in apple peel are yet to be defined. The aim of this study was to identify the mechanisms underpinning red pigmentation in ‘Granny Smith’ after bag removal based on transcriptome sequencing.ResultsThe anthocyanin content in apple peel increased considerably after bag removal, while only trace amounts of anthocyanins were present in the peel of unbagged and bagged fruits. RNA sequencing identified 18,152 differentially expressed genes (DEGs) among unbagged, bagged, and bag-removed fruits at 0, 4, and 10 days after bag removal. The DEGs were implicated in light signal perception and transduction, plant hormone signal transduction, and antioxidant systems. Weighted gene co-expression network analysis of DEGs generated a module of 23 genes highly correlated with anthocyanin content. The deletion of − 2026 to − 1870 bp and − 1062 to − 964 bp regions of the MdMYB1 (LOC103444202) promoter induced a significant decrease in glucuronidase activity and anthocyanin accumulation in apple peel.ConclusionsBagging treatment can induce red pigmentation in ‘Granny Smith’ via altering the expression patterns of genes involved in crucial signal transduction and biochemical metabolic pathways. The − 2026 to − 1870 bp and − 1062 to − 964 bp regions of the MdMYB1 promoter are essential for MdMYB1-mediated regulation of anthocyanin accumulation in the ‘Granny Smith’ apple cultivar. The findings presented here provide insight into the mechanisms of coloration in the peel of ‘Granny Smith’ and other non-red apple cultivars.

MdGSTF6, activated by MdMYB1, plays an essential role in anthocyanin accumulation in apple

Anthocyanins are biosynthesized on the cytosolic surface of the endoplasmic reticulum and then transported into the vacuole for storage. Glutathione S-transferases (GSTs) are considered to be responsible for the transport of anthocyanins into the vacuole. However, the regulatory mechanisms of GSTs in plants are still unclear. Here, we performed a genome-wide analysis and identified 69 GST genes in apple. The expression of MdGSTF6 was positively correlated with the anthocyanin content (r = 0.949) during ‘Yanfu 8’ fruit development. The overexpression of MdGSTF6 in the Arabidopsis thaliana tt19 mutant resulted in seedlings of 35S::MdGSTF6-GFP/tt19 that could accumulate anthocyanin and rescue its phenotype, suggesting that MdGSTF6 was an anthocyanin transporter. The silencing of MdGSTF6 affected anthocyanin accumulation in apple fruit. Moreover, the knockdown of MdGSTF6 by RNA interference in cultured ‘Gala’ seedlings inhibited anthocyanin accumulation. The interaction experiments showed that MdMYB1 could bind directly to the MdGSTF6 promoter to transcriptionally activate its expression. Collectively, our results demonstrate that MdGSTF6 encodes an important GST transporter of anthocyanins in apple fruit and provide evidence for the associated regulatory mechanisms. Therefore, MdMYB1 can not only regulate anthocyanin synthesis, but also control the transport of anthocyanin in apples. This information may be useful for further clarifying the regulation of anthocyanin transport in apple.