Demethylating drugs alter protoplast development, regeneration, and the genome stability of protoplast-derived regenerants of cabbage

Abstract

Background: Methylation is a major DNA modification contributing to the epigenetic regulation of nuclear gene expression and genome stability. DNA methyltransferases (DNMT) inhibitors are widely used in epigenetic and cancer research, but their biological effects and the mechanisms of their action are not well recognized in plants. This research focuses on comparing the effects of two DNMT inhibitors, namely 5-azacytidine (AZA) and zebularine (ZEB), on cellular processes, including organogenesis in vitro. Protoplasts are a unique single-cell system to analyze biological processes in plants; therefore in our study, both inhibitors were applied to protoplast culture medium or the medium used for the regeneration of protoplast-derived calluses. Results: AZA induced a dose-dependent reduction in protoplast viability, delayed cell wall reconstruction, and reduced mitotic activity, while ZEB in low concentration (2.5 µM) promoted mitoses and stimulated protoplast-derived callus development. The higher effectiveness of shoot regeneration was observed when drugs were applied directly to protoplasts compared to protoplast-derived callus treatments. Our findings reveal that both drugs affected the genome stability of the obtained regenerants by inducing polyploidization. Both drugs induced hypomethylation and modulated the distribution patterns of methylated DNA in the protoplast-derived callus. Conclusion: AZA was more toxic to plant protoplasts compared to ZEB. Both inhibitors affect the ploidy status of protoplast-derived regenerants. A comparison of the data on global methylation levels with the regeneration efficiency suggests that organogenesis in cabbage is partially controlled by variations in DNA methylation levels.