Kiełkowska Agnieszka, Brąszewska Agnieszka
Department of Plant Biology and Biotechnology, Faculty of Biotechnology and Horticulture, University of Agriculture in Krakow, Al. 29-Listopada 54, Krakow, 31-425, Poland.
Institute of Biology, Biotechnology and Environmental Protection, Faculty of Natural Sciences, University of Silesia in Katowice, 28 Jagiellonska Street, Katowice, 40-032, Poland.
BMC Plant Biol. 2025 Apr 11;25(1):463. doi: 10.1186/s12870-025-06473-2.
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.
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.
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.
甲基化是一种主要的DNA修饰，有助于核基因表达的表观遗传调控和基因组稳定性。DNA甲基转移酶（DNMT）抑制剂广泛应用于表观遗传学和癌症研究，但它们在植物中的生物学效应及其作用机制尚未得到充分认识。本研究着重比较两种DNMT抑制剂，即5-氮杂胞苷（AZA）和zebularine（ZEB）对细胞过程的影响，包括体外器官发生。原生质体是分析植物生物学过程的独特单细胞系统；因此在我们的研究中，两种抑制剂都应用于原生质体培养基或用于原生质体来源愈伤组织再生的培养基。
AZA诱导原生质体活力呈剂量依赖性降低，延迟细胞壁重建，并降低有丝分裂活性，而低浓度（2.5 µM）的ZEB促进有丝分裂并刺激原生质体来源愈伤组织的发育。与原生质体来源愈伤组织处理相比，将药物直接应用于原生质体时观察到更高的芽再生效率。我们的研究结果表明，两种药物都通过诱导多倍体化影响所获得再生植株的基因组稳定性。两种药物均诱导低甲基化并调节原生质体来源愈伤组织中甲基化DNA的分布模式。
与ZEB相比，AZA对植物原生质体的毒性更大。两种抑制剂都影响原生质体来源再生植株的倍性状态。将全基因组甲基化水平数据与再生效率进行比较表明，甘蓝的器官发生部分受DNA甲基化水平变化的控制。
