Transcriptome Analysis Revealed the Roles of Carbohydrate Metabolism on Differential Acetaldehyde Production Capacity in Persimmon Fruit in Response to High-CO₂ Treatment

Abstract

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-CO 2 treatment was the most effective widely used method for astringency removal. However, differential effects of high-CO 2 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-CO 2 (95% CO 2 ) 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 CO 2 . 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.

C-CorA: A Cluster-Based Method for Correlation Analysis of RNA-Seq Data

“…Three PDC genes (EVM0028451, EVM0027273 and EVM0022732) and two ADH genes (EVM0007501 and EVM0027066) were detected in the highest correlated module by a weighted gene co-expression network analysis (WGCNA) as reported in Kou et al [29]. When we applied the C-CorA method on the same dataset as Kou et al [29], two more genes (PDC, EVM0018709 and ADH, EVM0007329) were detected. The gene expressions and ethanol/acetaldehyde production are shown in Figure 8.…”

“…were detected in the highest correlated module by a weighted gene co-expression network analysis (WGCNA) as reported in Kou et al [29]. When we applied the C-CorA method on the same dataset as Kou et al [29], two more genes (PDC, EVM0018709 and ADH, EVM0007329) were detected. The gene expressions and ethanol/acetaldehyde production are shown in Figure 8.…”

“…The production of acetaldehyde depends on two key enzymes, pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) [28]. Three PDC genes (EVM0028451, EVM0027273 and EVM0022732) and two ADH genes (EVM0007501 and EVM0027066) were detected in the highest correlated module by a weighted gene co-expression network analysis (WGCNA) as reported in Kou et al [29]. When we applied the C-CorA method on the same dataset as Kou et al [29], two more genes (PDC, EVM0018709 and ADH, EVM0007329) were detected.…”

Investigating phenotypic relationships in persimmon accessions through integrated proteomic and metabolomic analysis of corresponding fruits

“…Functionally related to these enzymes are also some proteins involved in carbohydrate metabolism, cell respiration and ultimately to acetaldehyde degradation/ethanol formation, including beta-fructofuranosidase, alpha-galactosidase, galactose mutarotase, beta-glucosidase isoforms, sucrose synthase, dTDP-4-dehydrorhamnose 3,5-epimerase, UDP-D-glucose 6-dehydrogenase, trehalase, fructose kinase isoforms, phosphofructokinase, glyceraldehyde 3-phosphate dehydrogenase and alcohol dehydrogenase ( Supplementary Figure S1B ). Acetaldehyde, ethanol and some of the above-mentioned enzymes have already been reported regulating PAs degradation and astringency in persimmon ( Luo et al., 2014 ; Chen et al., 2017 ; Kou et al., 2021b ; Wu et al., 2022 ). Their differential representation could possibly account for the slight changes in total sugar content between cultivars ( Table 1 ) ( Giordani, 2002 ; Insero et al., 2002b ).…”

“…Several transcriptomic studies were accomplished on D. kaki to identify regulatory networks and protein regulators associated with tannin metabolism in cultivars showing variable PAs accumulation patterns ( Jung et al., 2017 ; Nishiyama et al., 2018 ; Zheng et al., 2021 ), or to reveal deregulated metabolic pathways in fruits at different developmental ( Zhang and Luo, 2022 ) and post-harvest ( Kou et al., 2021a ) stages, or following treatment with ethanol ( Luo et al., 2014 ), warm water ( Chen et al., 2017 ) and CO 2 ( Kou et al., 2021b ). Various key genes affecting PAs formation and removal were identified.…”