Citric acid gives lemons their unique flavor, which impacts their sensory traits and market value. However, the intricate process of citric acid accumulation during lemon fruit growth remains incompletely understood. Here, we achieved a chromosomal-level genome assembly for the ‘Xiangshui’ lemon variety, spanning 364.85 Mb across nine chromosomes. This assembly revealed 27 945 genes and 51.37% repetitive sequences, tracing the divergence from citron 2.85 million years ago. DNA methylome analysis of lemon fruits across different developmental stages revealed significant variations in DNA methylation. We observed decreased CG and CHG methylation but increased CHH methylation. Notably, the expression of RdDM pathway-related genes increased with fruit development, suggesting a connection with elevated CHH methylation, which is potentially influenced by the canonical RdDM pathway. Furthermore, we observed that elevated CHH DNA methylation within promoters significantly influenced the expression of key genes, critically contributing to vital biological processes, such as citric acid accumulation. In particular, the pivotal gene phosphoenolpyruvate carboxykinase (ClPEPCK), which regulates the tricarboxylic acid cycle, was strikingly upregulated during fruit development, concomitant with increased CHH methylation in its promoter region. Other essential genes associated with citric acid accumulation, such as the MYB transcription factor (ClPH1/4/5) and ANTHOCYANIN 1 (ClAN1), were strongly correlated with DNA methylation levels. These results strongly indicate that DNA methylation crucially orchestrates the metabolic synthesis of citric acid. In conclusion, our study revealed dynamic changes in DNA methylation during lemon fruit development, underscoring the significant role of DNA methylation in controlling the citric acid metabolic pathway.

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