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.

Keywords: Cabbage; DNA hypomethylation; Methyltransferase inhibitors; Mitotic divisions; Ploidy; Protoplasts; Regeneration.

Fig. 1

Development in protoplast cultures of cabbage treated with 5-azacytidine (AZA) and zebularine (ZEB) and in controls (─). Viability of protoplasts in 1st (a) and 5th day of culture (b). FDA-stained viable cells express yellow-green fluorescence after excitation with UV light. White arrow indicates cell colony. (c) Mean effect of accession, AZA and ZEB on the cell viability at 1st (green points) and 5th (red points) day of culture. Points are means ± SEM (n = 60). Points marked with the same letter are not significantly different (P ≤ 0.05, MRT). Statistical grouping for green and red points should be considered separately. Protoplasts stained with calcofluor and observed in white (d) and UV (e) light after 48 h of culture. Both pictures show the same field of view. Complete resynthesis (yellow arrows); partial resynthesis (green arrows); lack of cell wall synthesis (red arrows). Mean effect of accession, AZA and ZEB on the regeneration of cellulose in protoplasts (f). Bars are means ± SEM (n = 30). The division frequency of protoplast-derived cells in 5th day (g) and 15th day of culture (h). g: Enlarged cell with reorganized cytoplasm and chloroplasts before mitosis (blue arrow); cells after second mitosis (red arrow); few-celled colony (yellow arrow). h: Multiple-celled colonies; (i) Mean effect of accession, AZA and ZEB and its concentration on the division frequency of protoplast-derived cells in 5th and 15th day of culture. Bars are means ± SEM (n = 60). Bars denoted with the same letter are not significantly different (P ≤ 0.05, MRT). Statistical grouping for blue and green bars should be considered separately

Fig. 2

Micro-callus production and organogenesis from protoplast-derived cells of cabbage treated with 5-azacytidine (AZA) and zebularine (ZEB) and in controls (─). (a) Petri dishes with alginate layers overgrown with protoplast-derived callus after four weeks of culture. (b) Mean effect of accession, AZA, and ZEB and their concentrations on micro-callus production from protoplast cultures. Bars are means ± SEM (n = 45). Blue-colored bars represent controls. Bars denoted with the same letter are not significantly different (P ≤ 0.05, MRT). (c) Close up on alginate layer with micro-callus colonies. (d) Mean effect of accession, AZA, and ZEB and their concentrations on shoot regeneration. Bars are means ± SEM (n = 32). Blue-colored bars represent controls. Bars denoted with the same letter are not significantly different (P ≤ 0.05, MRT). (e) Shoot organogenesis (marked with arrows) from the protoplast-derived callus

Fig. 3

Flow cytometry histograms showing the relative nuclear DNA content of leaves of cabbage regenerants developed from control and drug-treated protoplast cultures, and exemplary pictures of regenerants of certain ploidy being approximately 3 months after acclimatization. On histograms, 2 C and 4 C peaks are marked by red dotted lines and refer to the DNA content of the control sample. Pictures from top: diploid (2x), tetraploid (4x), mixoploid (2x + 4x), and aneuploid (4x aneuploid) regenerants, respectively

Fig. 4

Development and shoot regeneration from protoplast-derived callus treated with 5-azacytidine (AZA) and zebularine (ZEB) and in controls (─). (a) The mean effect of accession and drugs on the weight of the callus. Bars are means ± SEM (n = 30). Blue-colored bars represent controls. Bars denoted with the same letter are not significantly different (P ≤ 0.05, MRT). (b) 90-mm petri dishes with calluses undergoing organogenesis in roots (blue arrows) and shoots (red arrows) and callus with no signs of organogenesis (yellow arrow). (c) Mean effect of accession and drugs on shoot regeneration after 16 weeks of culture. Bars are means ± SEM (n = 32). Blue-colored bars represent controls. Bars denoted with the same letter are not significantly different (P ≤ 0.05, MRT)

Fig. 5

Quantification of global levels of DNA methylation and immunolocalization of 5-methyl-cytidine (5mC) in nuclei of 4-week-old protoplast-derived calluses of cabbage treated with 5-azacytidine (AZA) and zebularine (ZEB) and in controls (─). Mean effect of: the inhibitor (a), the inhibitor × concentrations (b); the inhibitor × concentrations × accession (c) on global levels of DNA methylation. Bars are means ± SEM (n = 8). Blue-colored bars represent controls. Bars denoted with the same letters are not significantly different (P ≤ 0.05, MRT)

Fig. 6

Immunolocalization of 5-methyl-cytidine (5mC) in nuclei of callus samples treated with 5-azacytidine (AZA) and zebularine (ZEB) and in controls (─). (a) Qualitative analysis of 5mC immunosignals. Bars are means ± SEM (n = 12). Miniatures show a representative distribution of 5mC patterns (green signals) in nuclei in each observed category. (b) In situ localization of 5mC immunosignals (green in merge). Nuclei were counterstained with DAPI (red in merge). All bars in the merge = 5 μm