Abstract
Abiotic stress can reprogram the gametophytic pathway; the mechanisms by which floral bud pre-treatment influences microspore embryogenesis initiation remain unclear. In this study, we use bisulfite sequencing, sRNA-seq, and RNA-seq to analyze the dynamic changes in rice microspores under different cold treatment durations. Our results showed that a 10-day cold treatment is essential for CXJ microspore embryogenesis initiation. DNA methylation levels showed a slight change at CG, CHG, and CHH sites under cold treatment. The number of both hyper- and hypomethylated DMRs increased over cold treatment, with more hypermethylated DMRs at 5 and 10 dpt. Hypermethylated DMRs were more frequently in the TSS region compared to hypomethylated DMRs. The proportion of 24 nt sRNAs increased upon cold stress, with more downregulated than upregulated sRNAs at 10 dpt. The number of DMR target DEGs increased from 5 to 10 dpt. Promoter hypomethylation at the CHH site was more frequently associated with DEGs. These outcomes suggested that the RdDM pathway participates in the initiation of rice ME. GO analysis indicated that DMR target DEGs at 10 dpt were enriched in responses to chemical stimuli, biological processes, and stress responses. An auxin-related gene, OsHOX28, was further identified. Its upregulation, potentially mediated by the RdDM pathway, may play a crucial role in the initiation of rice ME. This study provides more information on epigenetic mechanisms during rice ME.