Changes in the VOC of Fruits at Different Refrigeration Stages of ‘Ruixue’ and the Participation of Carboxylesterase MdCXE20 in the Catabolism of Volatile Esters

Aroma is a crucial quality attribute of apple fruit, which significantly impacts its commercial value and consumer choice. Despite its importance the volatile aroma substances produced by the new variety ‘Ruixue’ after harvest remain unclear. In this study, we utilized headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) to investigate the changes in volatile substances, fruit hardness, crispness, and related aroma synthase activity of commercially mature ‘Ruixue’ apples during cold storage. Our findings revealed a gradual decline in fruit firmness and brittleness of ‘Ruixue’ apples during cold storage, with hexyl acetate, hexyl caproate, and hexyl thiocyanate being the main hexyl esters detected. To gain a better understanding of the metabolic pathway of esters, we identified 42 MdCXE gene members that are associated with ester degradation. Through RT-qPCR analysis, we discovered that carboxylesterase MdCXE20 exhibited higher expression levels compared to other MdCXE genes during cold storage. To confirm the role of MdCXE20, we conducted a transient injection of apple fruits and observed that overexpression of MdCXE20 led to the degradation of esters such as hexyl hexanoate, butyl hexanoate, butyl 2-methylbutyrate, hexyl butyrate, and hexyl 2-methylbutyrate. The results of the study showed that the virus-induced gene silencing of MdCXE20 found the opposite results. Additionally, the esters of OE-MdCXE20 callus showed a lower content of ester VOC than the control callus, according to the homologous stable transformation of ‘Wanglin’ callus. Overall, these findings suggest that the MdCXE20 gene plays a crucial role in the decrease of esters in ‘Ruixue’ apples, which ultimately affects their flavor.