A role of age-dependent DNA methylation reprogramming in regulating the regeneration capacity of Boea hygrometrica leaves

Plants can regenerate new individuals under appropriate culture conditions. Although the molecular basis of shoot regeneration has steadily been unraveled, the role of age-dependent DNA methylation status in the regulation of explant regeneration remains practically unknown. Here, we established an effective auxin/cytokinin-induced shoot regeneration system for the resurrection plant Boea hygrometrica via direct organogenesis and observed that regeneration was postponed with increasing age of donor plants. Global transcriptome analysis revealed significant upregulation of genes required for hormone signaling and phenylpropanoid biosynthesis and downregulation of photosynthetic genes during regeneration. Transcriptional changes in the positive/negative regulators and cell wall-related proteins involved in plant regeneration, such as ELONGATED HYPOCOTYL5 (HY5), LATERAL ORGAN BOUNDARIES DOMAIN, SHOOT-MERISTEMLESS, and WUSCHEL, were associated with the regeneration process. Comparison of DNA methylation profiling between leaves from young seedlings (YL) and mature plants (ML) revealed increased asymmetrical methylation in ML, which was predominantly distributed in promoter regions of genes, such as HY5 and a member of ABA-responsive element (ABRE) binding protein/ABRE binding factor, as well as genes encoding glycine-rich cell wall structural protein, CENTRORADIALIS-like protein, and beta-glucosidase 40-like essential for shoot meristem and cell wall architecture. Their opposite transcription response in ML explants during regeneration compared with those from YL demonstrated the putative involvement of DNA methylation in regeneration. Moreover, a significant lower expression of DNA glycosylase-lyase required for DNA demethylation in ML was coincident with its postponed regeneration compared with those in YL. Taken together, our results suggest a role of promoter demethylation in B. hygrometrica regeneration.