Abstract
BACKGROUND: Inducing embryogenic callus with regenerative potential is a pivotal step in barley transformation. Our previous research suggests that epigenetic regulatory factors might influence barley callus formation and regeneration capacity, though the exact mechanisms remain unclear. RESULTS: In this study, we utilized RNA sequencing (RNA-seq) and whole-genome bisulfite sequencing (WGBS) to examine transcriptional and DNA methylome alterations during callus induction from immature embryos of the barley cultivar Golden Promise. Our findings revealed a slight decline in overall DNA methylation content and distinct 5-methylcytosine (5mC) enrichment patterns in CG, CHG, and CHH sequence contexts within genes and transposable elements. By integrating DNA methylation and transcriptome data, we identified differentially expressed genes (DEGs) associated with differentially methylated regions (DMRs) in the CG (879 DEGs), CHG (229 DEGs), and CHH (2020 DEGs) contexts. Notably, DMRs linked to 210, 94, and 1,214 DEGs were located in the 2 kb upstream regions in the CG, CHG, and CHH contexts, respectively. A negative correlation was observed between promoter methylation levels and transcript abundances of key regeneration-associated genes, such as HvKRP4, HvCYCD1;1, HvSCR, HvRAP2.6L/ERF113, HvWIND4, HvWOX5, HvE2Fa, HvPHV, and HvLBD16. This indicates a regulatory function of DNA methylation in transcriptional regulation during callus induction. Furthermore, treatment with the DNA methylation inhibitor 5-Aza-2'-deoxycytidine (5-Aza-dC) suppressed callus formation. Comparative RNA sequencing analysis between control and treated groups revealed 2,628 and 1,224 DEGs potentially regulated by DNA methylation, at 2- and 9-days post-induction, respectively. These genes were primarily associated with cell cycle and abscisic acid signalling pathways, influenced directly and indirectly by the global reduction in DNA methylation induced by 5-Aza-dC treatment. CONCLUSIONS: This study provides insights into the intricate relationship between DNA methylation and gene expression during barley callus formation. It could inform future efforts to enhance regeneration and transformation in this significant crop species. CLINICAL TRIAL NUMBER: Not applicable.