Enhancing soluble sugar accumulation in banana (Musa acuminata) through 5-azacytidine-mediated reinforcement of starch degradation involving DNA methylation-dependent and independent pathways

INTRODUCTION: The banana industry faces challenges in maintaining sweetness during postharvest handling, as conventional ethylene ripening often compromises starch-to-sugar conversion. Current technologies lack modulation of this metabolic pathway. Emerging epigenetic tools offer promise for sugar enhancement. OBJECTIVES: This study investigates how the DNA demethylating agent 5-azacytidine (AZ) epigenetically reprograms the starch-to-sugar conversion in banana pulp under ethylene. The further goal is to advance the understanding of fruit epigenetic regulation, inspiring a molecular-level strategy to optimize postharvest fruit traits. METHODS: Bananas underwent optimized vacuum infiltration with 1 mmol/L AZ or control solution (-0.08 MPa, 30 min), followed by ethylene-induced ripening. Assays included biochemical measurements (Spectrophotometry), key gene expression (qRT-PCR), promoter-specific DNA methylation (McrBC-PCR), and translational regulation (polysome profiling). At least 3 biological replicates were used and significance (p < 0.05) was analyzed by two‑way ANOVA or t‑tests with Bonferroni/FDR correction. RESULTS: Only combined with ethylene fumigation, AZ significantly elevated total soluble solids and soluble sugars (sucrose, glucose, fructose) with up to a 2.1-fold increase over the control (ethylene only), correlating with accelerated starch degradation. The activation of 70.27% (26/37) starch-degrading genes and increased enzyme activity (α-amylase, β-amylase, debranching enzymes) were observed. On one hand, AZ induced global DNA demethylation and promoter hypomethylation for key starch-degrading genes (e.g., MaGWD1, MaBAM11, MaAMY3). This DNA hypomethylation-dependent mechanism strongly correlated with gene activation (p＜0.001). On the other hand, polysome profiling demonstrated that AZ directly enhanced the total translation efficiency and specific translation efficiency of key starch-degrading transcripts such as MaBAM3, MaBAM11, and MaISA2. CONCLUSION: In the context of ethylene-induced post-ripening, AZ operated through dual epigenetic regulatory mechanisms, DNA methylation-dependent and independent pathways (post-transcriptional translation regulation), leading to the acceleration of starch-to-sugar conversion in banana pulp for enhancing the flavor. This provides a novel direction for future strategies on postharvest quality control.