DNA methylation-mediated ROS production contributes to seed abortion in litchi

Although there is increasing evidence suggesting that DNA methylation regulates seed development, the underlying mechanisms remain poorly understood. Therefore, we aimed to shed light on this by conducting whole-genome bisulfite sequencing using seeds from the large-seeded cultivar 'HZ' and the abortive-seeded cultivar 'NMC'. Our analysis revealed that the 'HZ' seeds exhibited a hypermethylation level compared to the 'NMC' seeds. Furthermore, we found that the genes associated with differentially methylated regions (DMRs) and differentially expressed genes (DEGs) were mainly enriched in the reactive oxygen species (ROS) metabolic pathway. To investigate this further, we conducted nitroblue tetrazolium (NBT) and 2,7-Dichlorodihydrofluorescein (DCF) staining, which demonstrated a significantly higher amount of ROS in the 'NMC' seeds compared to the 'HZ' seeds. Moreover, we identified that the gene LcGPX6, involved in ROS scavenging, exhibited hypermethylation levels and parallelly lower expression levels in 'NMC' seeds compared to 'HZ' seeds. Interestingly, the ectopic expression of LcGPX6 in Arabidopsis enhanced ROS scavenging and resulted in lower seed production. Together, we suggest that DNA methylation-mediated ROS production plays a significant role in seed development in litchi, during which hypermethylation levels of LcGPX6 might repress its expression, resulting in the accumulation of excessive ROS and ultimately leading to seed abortion.