Expression analysis of PDC and ADH gene family members and their relationship with the accumulation of acetaldehyde and ethanol during fruit ripening and packaging treatments of Chinese bayberry

Abstract:【Objective】Chinese bayberry (Morella rubra Sieb. et Zucc.) is a characteristic fruit crop with important medicinal and economic value in China. The fruits develop obvious wine smell during storage and even at harvest. The wine smell is mainly derived from accumulation of acetaldehyde and ethanol. Pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) are the key enzymes in the ethanolic fermentation pathway, where pyruvate is decarboxylated to acetaldehyde through the action of PDC, and thereafter, acetaldehyde is converted to ethanol by ADH. It was shown in other fruits that PDC and ADH played important roles in the formation of acetaldehyde a nd ethanol during fruit ripen-ing and storage. However, identification and expression profiling of the PDC and ADH gene family members, as well as the correlation analysis between the gene expression and the accumulation of acetaldehyde and ethanol have not been studied in Chinese bayberry.【Methods】Based on the genome database of Chinese bayberry, the PDC and ADH genes were identified by Hidden Markov Model (HMM) searches using the HMM profiles of PDC and ADH domain (PDC: PF00205, ADH: PF08240) downloaded from Pfam database (http://pfam.xfam.org/). The expression data, by Fragments per Kilobase of exon model per Million mapped fragments (FPKM), were analyzed to identify highly expressed members of the PDC and ADH based on previously available transcriptome data. In addition, the expression of the MrPDC1, MrPDC2 and MrADH1, MrADH2, MrADH3 was determined by RT-qPCR technology. The acetaldehyde and ethanol content were determined by gas chromatography (GC).【Results】In this study, a total of 8 MrPDCs and 22 MrADHs were identified from genome database of Chinese bayberry. The MrPDC1 and MrPDC2, as well as the MrADH1, MrADH2 and MrADH3 were identified as highly expressed members and hence used for further analysis. The expression patterns of the MrPDCs and MrADHs during fruit ripening were differential among 12 cultivars. The expression of the MrPDC1, MrADH1 and MrADH3 in most cultivars increased as fruit ripened, while the MrPDC2 and MrADH2 had higher expression levels at mature green stage (S1). For example, the expression of the MrPDC1 in many cultivars was the highest at ripe stage (S3), which was 11.30 and 4.70 times higher than those of S1 and breaker stage (S2), respectively, in Longhaishuijing. The MrPDC2 expression was highest in most cultivars at S1. There were also differences in the expression patterns of the three MrADHs during fruit ripening in different cultivars; the expression level of the MrADH1 in Tezaomei was highest at S3, which was 43.77 and 4.12 times higher than those at S1 and S2. The expression level of the MrADH2 at S1 of Wumei was 2.59 and 6.30 times higher than those at S2 and S3, respectively. The expression level of the MrADH3 at S3 of Tezaomei was 9.87 and 50.64 times higher than those at S1 and S2, respectively. The content of acetaldehyde did not vary much throughout fruit ripening and the variation across cultivars was minimal as well. Ethanol significantly accumulated at S3 in most cultivars, the content of ethanol at S3 of Tezaomei was 12.37 times higher than that at S2. The difference of the ethanol content across cultivars was also mainly reflected at S3, with the largest variation reaching 15.19 times. Overall, the acetaldehyde and ethanol contents had no significant correlation with the expression levels of the MrPDCs, but had significant and positive correlation with the expression levels of the MrADH1 and MrADH3. In addition, the result of packaging storage experiments showed that compared with ordinary packaging and single fruit packaging, hypobaric packaging resulted in obvious offflavor and a tremendous accumulation of acetaldehyde and ethanol during storage, among which the acetaldehyde content of Biqi increased by 3.73 times in 10 days and the ethanol content increased by 1.86 times in 6 days. Both the acetaldehyde and ethanol contents of Dongkui increased, by 2.29 and 1.89 times, respectively, in 4 days. RT-qPCR results showed that the expression of the MrPDC1, MrPDC2, MrADH1 and MrADH3 in the fruits of hypobaric packaging were significantly higher than those of ordinary packaging and single fruit packaging in both Biqi and Dongkui. The expression level of the MrADH3 in Biqi was highest on 2nd day in hypobaric packaging, while there was little difference in the expression levels of each gene between the fruit of ordinary packaging and the single fruit packaging. Meanwhile, the expression levels of the MrPDC1 and MrPDC2 as well as MrADH1 and MrADH3 were significantly enhanced as storage time prolonged, and being positively correlated with the acetaldehyde and ethanol accumulation.【Conclusion】Among the 8 MrPDCs and 22 MrADHs, the MrADH1 and MrADH3 play an important role in the moderate accumulation of acetaldehyde and ethanol during fruit ripening in Chinese bayberry. The hypobaric packaging induced the expression of the MrPDC1, MrPDC2, MrADH1, MrADH2 and MrADH3, resulting in excessive accumulation of acetaldehyde and ethanol and the production of fruit off-flavor or wine smell.