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.

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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.

BackgroundFruit peel colour is an important agronomic trait for fruit quality. Cytosine methylation plays an important role in gene regulation. Although the DNA methylation level of a single gene is important to affect the phenotype of mutation, there are large unknown of difference of the DNA methylation in plant and its mutants.ResultsUsing bisulfite sequencing (BS-Seq) and RNA-sequencing (RNA-Seq), we analysed three deep-red-skinned apple (Malus × domestica) mutants (Yanfu 3, YF3; Yanfu 8, YF8; Shannonghong, SNH) and their lighter-skinned parents (Nagafu 2, NF2; Yanfu 3, YF3; Ralls, RL) to explore the different changes in methylation patterns associated with anthocyanin concentrations. We identified 13,405, 13,384, and 10,925 differentially methylated regions (DMRs) and 1987, 956, and 1180 differentially expressed genes (DEGs) in the NF2/YF3, YF3/YF8, and RL/SNH comparisons, respectively. And we found two DMR-associated DEGs involved in the anthocyanin pathway: ANS (MD06G1071600) and F3H (MD05G1074200). These genes exhibited upregulated expression in apple mutants, and differences were observed in the methylation patterns of their promoters. These results suggested that both the regulatory and structural genes may be modified by DNA methylation in the anthocyanin pathway. However, the methylation of structural genes was not the primary reason for expression-level changes. The expression of structural genes may be synergistically regulated by transcription factors and methylation changes. Additionally, the expression of the transcription factor gene MYB114 (MD17G1261100) was upregulated in the deep-red-skinned apple.ConclusionThrough the analysis of global methylation and transcription, we did not find the correlation between gene expression and the DNA methylation. However, we observed that the upregulated expression of ANS (MD06G1071600) and F3H (MD05G1074200) in apple mutants results in increased anthocyanin contents. Moreover, MYB114 (MD17G1261100) is likely another regulatory gene involved in apple coloration. Our data provided a new understanding about the differences in formation of apple colour mutants.Electronic supplementary materialThe online version of this article (10.1186/s12864-019-5499-2) contains supplementary material, which is available to authorized users.

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