Methylation of EjNAC5 and its interactions with other transcription factors regulate loquat fruit chilling lignification

Changes in both lignin biosynthesis and DNA methylation have been reported to be associated with chilling stress in plants. When stored at low temperatures, red-fleshed loquat is prone to lignification, with increased lignin content and fruit firmness, which has deleterious effects on taste and eating quality. Here, we found that 5 °C storage mitigated the increasing firmness and lignin content of red-fleshed 'Dahongpao' ('DHP') loquat fruit that occurred during 0 °C storage. EjNAC5 was identified by integrating RNA sequencing with whole-genome bisulfite sequencing analysis of 'DHP' loquat fruit. The transcript levels of EjNAC5 were positively correlated with changes in firmness and negatively correlated with changes in DNA methylation level of a differentially methylated region in the EjNAC5 promoter. In white-fleshed 'Baisha' ('BS') loquat fruit, which do not undergo chilling-induced lignification at 0 °C, the transcripts of EjNAC5 remained low and the methylation level of the differentially methylated region in the EjNAC5 promoter was higher, compared with 'DHP' loquat fruit. Transient overexpression of EjNAC5 in loquat fruit and stable overexpression in Arabidopsis and liverwort led to an increase in lignin content. Furthermore, EjNAC5 interacts with EjERF39 and EjHB1 and activates the transcription of Ej4CL1 and EjPRX12 genes involved in lignin biosynthesis. This regulatory network involves different transcription factors from those involved in the lignification pathway. Our study indicates that EjNAC5 promoter methylation modulates EjNAC5 transcript levels and identifies novel EjNAC5-EjERF39-Ej4CL1 and EjNAC5-EjHB1-EjPRX12 regulatory modules involved in chilling induced-lignification.