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Abstract
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.
Highlights
Somatic mutations, referred to as ‘bud sports’ or ‘sports’, represent an important method to generate superior cultivars of woody crop species
The binding of MdAGO4s was not affected by knockdown of MdNRPE1 in MdAGO4-1/2-overexpressing callus. These findings revealed that MdMYB1 methylation can be modified by the RNA-directed DNA methylation (RdDM) pathway in apple fruit and that MdAGO4s bind directly to DNA at the ATATCAGA sequence
We found that differences in CHH methylation might regulate MdMYB1 transcription; it is possible that MdAGO4 binds to the MdMYB1 promoter
Summary
Referred to as ‘bud sports’ or ‘sports’, represent an important method to generate superior cultivars of woody crop species. Sport varieties account for more than 30% of the popularized fruit varieties in China, especially those of citrus and apple (Chen et al, 2015). Fruit colour mutations are the most frequent. The famous ‘Red Delicious’ apple is a sport cultivar from ‘Delicious’ and forms a global brand (Iglesias et al, 1999). Another apple cultivar, ‘Fuji’, is very popular in China. Several red sports of ‘Fuji’ have been selected because of its poor coloration. The popularization of the red sports of ‘Fuji’ has promoted the rapid development of the apple industry in China. China is the leading apple-producing country and accounts for half of the cultivated area and yield of apples worldwide (Wang and Shi, 2017)
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