The RNA Directed DNA Methylation (RdDM) Pathway Regulates Anthocyanin Biosynthesis in Crabapple (<i>Malus</i> cv. spp.) Leaves by Methylating the <i>McCOP1</i> Promoter
The synthesis of anthocyanin pigments in plants is known to be regulated by multiple mechanisms, including epigenetic regulation; however, the contribution of the RNA-directed DNA methylation (RdDM) pathway is not well understood. Here, we used bisulfite sequencing and Real Time (RT)-quantitative (q...
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| Autores principales: | , , , , , , , , |
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| Formato: | article |
| Lenguaje: | EN |
| Publicado: |
MDPI AG
2021
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| Materias: | |
| Acceso en línea: | https://doaj.org/article/fac8ceb9f82d45aba18cadfe67821dde |
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| Sumario: | The synthesis of anthocyanin pigments in plants is known to be regulated by multiple mechanisms, including epigenetic regulation; however, the contribution of the RNA-directed DNA methylation (RdDM) pathway is not well understood. Here, we used bisulfite sequencing and Real Time (RT)-quantitative (q) PCR to analyze the methylation level of the promoter of constitutively photomorphogenic 1 (<i>McCOP1</i>) from <i>Malus</i> cv. spp, a gene involved in regulating anthocyanin biosynthesis. The CHH methylation level of the <i>McCOP1</i> promoter was negatively correlated with <i>McCOP1</i> RNA expression, and inhibiting DNA methylation caused decreased methylation of the <i>McCOP1</i> promoter and asymmetric cytosine CHH methylation. We observed that the <i>McCOP1</i> promoter was a direct target of the RdDM pathway argonaute RISC component 4 (McAGO4) protein, which bound to a <i>McCOP1</i> promoter GGTTCGG site. Bimolecular fluorescence complementation (BIFC) analysis showed that RNA-directed DNA methylation (McRDM1) interacted with McAGO4 and another RdDM protein, domains rearranged methyltransferase 2 (McDRM2), to regulate the CHH methylation of the <i>McCOP1</i> promoter. Detection of CHH methylation and COP1 gene expression in the <i>Arabidopsis thaliana</i><i>atago4</i>, <i>atdrm2</i> and <i>atrdm1</i> mutants showed that <i>RDM1</i> is the effector of the RdDM pathway. This was confirmed by silencing <i>McRDM1</i> in crabapple leaves or apple fruit, which resulted in a decrease in <i>McCOP1</i> CHH methylation and an increase in <i>McCOP1</i> transcript levels, as well as in anthocyanin accumulation. In conclusion, these results show that the RdDM pathway is involved in regulating anthocyanin accumulation through CHH methylation of the <i>McCOP1</i> promoter. |
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