Gibberellin (GA) is frequently used in tree peony forcing culture, but inappropriate application often causes flower deformity. Here, 5-azacytidine (5-azaC), an efficient DNA demethylating reagent, induced tree peony flowering with a low deformity rate by rapidly inducing PsFT expression, whereas GA treatment affected various flowering pathway genes with strong pleiotropy. The 5-azaC treatment, but not GA, significantly reduced the methylation level in the PsFT promoter with the demethylation of five CG contexts in a 369 bp CG-rich region, and eight light-responsive related cis-elements were also predicted in this region, accompanied by enhanced leaf photosynthetic efficiency. Through GO analysis, all methylation-closer differentially expressed genes (DEGs) were located in the thylakoid, the main site for photosynthesis, and were mainly involved in response to stimulus and single-organism process, whereas GA-closer DEGs had a wider distribution inside and outside of cells, associated with 12 categories of processes and regulations. We further mapped five candidate DEGs with potential flowering regulation, including three kinases (SnRK1, WAK2, and 5PTase7) and two bioactive enzymes (cytochrome P450 and SBH1). In summary, 5-azaC and GA may have individual roles in inducing tree peony flowering, and 5-azaC could be a preferable regulation approach; DNA demethylation is suggested to be more focused on flowering regulation with PsFT playing a core role through promoter demethylation. In addition, 5-azaC may partially undertake or replace the light-signal function, combined with other factors, such as SnRK1, in regulating flowering. This work provides new ideas for improving tree peony forcing culture technology.
Keywords: DNA methylation; McrBC PCR; Paeonia suffruticosa; photoperiod pathway; promoter; transcriptome.