Department of Cellular and Molecular Biology, Nicolaus Copernicus University, Lwowska 1, 87-100, Toruń, Poland.
Centre For Modern Interdisciplinary Technologies, Nicolaus Copernicus University, Wileńska 4, 87-100, Torun, Poland.
BMC Plant Biol. 2022 Jan 3;22(1):9. doi: 10.1186/s12870-021-03323-9.
Despite the frequent use of protoplast-to-plant system in in vitro cultures of plants, the molecular mechanisms regulating the first and most limiting stages of this process, i.e., protoplast dedifferentiation and the first divisions leading to the formation of a microcallus, have not been elucidated.
In this study, we investigated the function of miRNAs in the dedifferentiation of A. thaliana mesophyll cells in a process stimulated by the enzymatic removal of the cell wall. Leaf cells, protoplasts and CDPs (cells derived from protoplasts) cultured for 24, 72 and 120 h (first cell division). In protoplasts, a strong decrease in the amount of AGO1 in both the nucleus and the cytoplasm, as well as dicing bodies (DBs), which are considered to be sites of miRNA biogenesis, was shown. However during CDPs division, the amounts of AGO1 and DBs strongly increased. MicroRNA transcriptome studies demonstrated that lower amount of differentially expressed miRNAs are present in protoplasts than in CDPs cultured for 120 h. Then analysis of differentially expressed miRNAs, selected pri-miRNA and mRNA targets were performed.
This result indicates that miRNA function is not a major regulation of gene expression in the initial but in later steps of dedifferentiation during CDPs divisions. miRNAs participate in organogenesis, oxidative stress, nutrient deficiencies and cell cycle regulation in protoplasts and CDPs. The important role played by miRNAs in the process of dedifferentiation of mesophyll cells was confirmed by the increased mortality and reduced cell division of CDPs derived from mutants with defective miRNA biogenesis and miR319b expression.
尽管原生质体-植物系统在植物的体外培养中经常被使用，但调节这个过程的最初和最关键阶段的分子机制，即原生质体去分化和导致微愈伤组织形成的第一次分裂，尚未被阐明。
在这项研究中，我们研究了 miRNA 在拟南芥叶肉细胞去分化过程中的功能，该过程是由酶法去除细胞壁刺激的。研究了培养 24、72 和 120 小时的叶片细胞、原生质体和 CDP（原生质体衍生的细胞）（第一次细胞分裂）。在原生质体中，AGO1 的核内和细胞质中的含量以及 dicing 体（DBs），这被认为是 miRNA 生物发生的部位，均显示出强烈减少。然而，在 CDP 分裂过程中，AGO1 和 DBs 的含量强烈增加。miRNA 转录组研究表明，与培养 120 小时的 CDP 相比，原生质体中存在的差异表达 miRNA 数量较少。然后对差异表达 miRNA、选定的 pri-miRNA 和 mRNA 靶标进行了分析。
这一结果表明，miRNA 功能不是在 CDP 分裂过程中原生质体去分化初始阶段，而是在后期阶段基因表达的主要调控因子。miRNA 参与了原生质体和 CDP 中的器官发生、氧化应激、营养缺乏和细胞周期调控。miRNA 在叶肉细胞去分化过程中起着重要作用，这一点得到了以下事实的证实：miR319b 表达缺陷和 miRNA 生物发生缺陷的突变体衍生的 CDP 的死亡率增加和细胞分裂减少。
