Methylome and transcriptome landscapes reveal Tetraspanin8 as a key player of cold tolerance in Citrus.

DNA methylation is a critical epigenetic mark regulating a diversity of physiological and biological processes in plants. However, the role of DNA methylation in the regulation of cold stress response remains poorly elucidated, particularly in perennial plants like citrus. In this study, 33 genes exhibited greater upregulation but more pronounced hypomethylation in Ichang papeda (Citrus ichangensis), a cold-hardy plant, relative to lemon (Citrus limon, cold-sensitive) by an integrated analysis of transcriptome and methylome. Tetraspanin8 (TET8) exhibited the most substantial changes in both expression and methylation levels. TET8, localized in the plasma membrane and endoplasmic reticulum, played a positive role in cold tolerance. TET8 was mainly methylated in the context of CpG in the exon and 3' untranslated region. 5-Azacytidine treatment led to noticeable inhibition of CpG methylation in TET8, significant upregulation of TET8 and enhanced cold tolerance. Moreover, the expression level of Methyltransferase1 (MET1) was lower in Ichang papeda than in lemon. Silencing of MET1 suppressed CpG methylation, but upregulated TET8 and conferred cold tolerance. These findings indicate that MET1-mediated CpG methylation in TET8 plays a key role in the modulation of cold tolerance, which provides valuable insights into the understanding on epigenetic regulation of cold stress response in plants.