Genome-wide DNA methylation comparison reveals epigenetically regulated regeneration capacity of Boea hygrometrica during development

Plants are competent to regenerate new individuals from differentiated tissues under appropriate culture conditions. Although the molecular basis of shoot regeneration has steadily been unraveled, role of DNA methylation in the regulation of plant regeneration capacity remains practically unknown. Here, we established an effective auxin/cytokinin-induced regeneration system of the Chinese resurrection plant Boea hygrometrica through direct organogenesis, and observed that the potential regeneration capacity of leaf explants was gradually decreased with increased age of donor plants. Global transcriptome analysis revealed significant up-regulation of genes required for phenylpropanoid biosynthesis and phytohormone signaling while inhibition of photosynthetic activity in leaf explants during regeneration. Transcriptional changes of positive regulators HY5, STM, EMK and FLA, and negative regulators TSD and CDK involved in plant regeneration, were positively correlated with the regeneration process of B. hygrometrica, implicating their conserved functions across plant species. Comparison of global DNA methylation profiles between expanding young leaves and fully expanded mature leaves with different regeneration capability using whole-genome bisulfite sequencing revealed that increased asymmetrical methylation in mature leaves were predominant distributed in promoter regions, demonstrating their putative inhibitory function for downstream gene expression in B. hygrometrica leaves during maturation. Moreover, the predicted possible DNA methylation control for genes encoding GRCWP1 and BGS40L essential for cell wall architecture, CENL1 controlling extend shoot meristem phases, ANL2 and WRKY75 mediating root hair development, as well as HY5 and two members of ABA signaling components ABF and PP2C also provide new insights into the association of DNA methylation dynamics with regeneration capacity. To investigate the transcriptome changes during regeneration process of B. hygrometrica, we collected leaf explants cultured in induction medium for 0, 5, 10 and 15 days (YL-0d, YL-5d, YL-10d and YL-15d) to conduct the whole-genome transcriptome analysis. Each sample has three biological replicates. In order to further understand the potential role of genomic DNA methylation during shoot regeneration, high-throughput sequencing was performed on bisulfite-treated DNA fragments of young leaves (YL) and mature leaves (ML) of B. hygrometrica to determine the DNA methylation profiles at single nucleotides, yielding a total of 44.49 and 41.19 Gb of clean bases, respectively.