Persimmon (Diospyros kaki Thunb.) fruit is unique due to the continuous accumulation of soluble tannins during fruit development in most cultivars, which causes undesired astringency. High-CO₂ treatment was the most effective widely used method for astringency removal. However, differential effects of high-CO₂ treatment between cultivars were observed and the molecular basis remained inclusive. Previously, one cultivar ("Luoyangfangtianshengshi," LYFTSS) showed rapid deastringency, while two cultivars ("Shijiazhuanglianhuashi," SJZLHS; "Laopige," LPG) showed slow deastringency in response to high-CO₂ (95% CO₂) treatment. In this study, the metabolites (acetaldehyde and ethanol) related to deastringency were further analyzed and both acetaldehyde and ethanol were higher in SJZLHS and LYFTSS than that in LPG, where acetaldehyde was undetectable. Based on the RNA-seq data, the weighted gene coexpression network analysis (WGCNA) revealed that one module, comprised of 1773 unigenes, significantly correlated with the contents of acetaldehyde and ethanol (P < 0.001). Further analysis based on the acetaldehyde metabolism pathway indicated that the differentially expressed structural genes, including previously characterized DkADH and DkPDC and also their upstream members (e.g., PFK, phosphofructokinase), showed positive correlations with acetaldehyde production. Quantitative analysis of the precursor substances indicated that sucrose, glucose, and fructose exhibited limited differences between cultivar except for malic acid. However, the content of malic acid is much less than the total soluble sugar content. To verify the correlations between these genes and acetaldehyde production, the fruit from 14 more cultivars were collected and treated with high CO₂. After the treatment, acetaldehyde contents in different cultivars ranked in 30.4-255.5 μg/g FW. Real-time polymerase chain reaction (PCR) and correlation analysis indicated that the EVM0002315 (PFK) gene, belonging to carbohydrate metabolism, was significantly correlated with acetaldehyde content in fruit. Thus, it could be proposed that the differentially expressed carbohydrate metabolism related genes (especially PFK) are the basis for the variance of acetaldehyde production among different persimmon cultivars.

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