National Institute of Genetics, Shizuoka, Japan.
Department of Genetics, School of Life Science, Sokendai (Graduate University for Advanced Studies), Shizuoka, Japan.
PLoS One. 2022 Jun 3;17(6):e0269374. doi: 10.1371/journal.pone.0269374. eCollection 2022.
The shoot apical meristem (SAM) is composed of a population of stem cells giving rise to the aboveground parts of plants. It maintains itself by controlling the balance of cell proliferation and specification. Although knowledge of the mechanisms maintaining the SAM has been accumulating, the processes of cellular specification to form leaves and replenishment of unspecified cells in the SAM during a plastochron (the time interval between which two successive leaf primordia are formed) is still obscure. In this study, we developed a method to quantify the number of specified and unspecified cells in the SAM and used it to elucidate the dynamics of cellular specification in the SAM during a plastochron in rice. OSH1 is a KNOX (KNOTTED1-like homeobox) gene in rice that is expressed in the unspecified cells in the SAM, but not in specified cells. Thus, we could visualize and count the nuclei of unspecified cells by fluorescent immunohistochemical staining with an anti-OSH1 antibody followed by fluorescein isothiocyanate detection. By double-staining with propidium iodide (which stains all nuclei) and then overlaying the images, we could also detect and count the specified cells. By using these measurements in combination with morphological observation, we defined four developmental stages of SAM that portray cellular specification and replenishment of unspecified cells in the SAM during a plastochron. In addition, through the analysis of mutant lines with altered size and shape of the SAM, we found that the number of specified cells destined to form a leaf primordium is not affected by mild perturbations of meristem size and shape. Our study highlights the dynamism and flexibility in stem cell maintenance in the SAM during a plastochron and the robustness of plant development.
茎尖分生组织（SAM）由一群干细胞组成，这些干细胞产生植物的地上部分。它通过控制细胞增殖和特化的平衡来维持自身。尽管维持 SAM 的机制的知识一直在积累，但在一个成叶周期（两个连续的叶原基形成之间的时间间隔）中，细胞特化形成叶片和补充 SAM 中未特化细胞的过程仍然不清楚。在这项研究中，我们开发了一种定量 SAM 中特化和未特化细胞数量的方法，并利用它阐明了在水稻 SAM 中一个成叶周期内细胞特化的动态。OSH1 是水稻中的一个 KNOX（KNOTTED1 类同源盒）基因，在 SAM 中的未特化细胞中表达，但不在特化细胞中表达。因此，我们可以通过用抗 OSH1 抗体进行荧光免疫组织化学染色，然后用异硫氰酸荧光素检测来可视化和计数未特化细胞的核。通过与碘化丙啶（染色所有核）双重染色，然后覆盖图像，我们也可以检测和计数特化细胞。通过将这些测量值与形态观察相结合，我们定义了 SAM 的四个发育阶段，描绘了一个成叶周期中 SAM 中的细胞特化和未特化细胞的补充。此外，通过对 SAM 大小和形状改变的突变体系的分析，我们发现，形成叶原基的特化细胞的数量不受分生组织大小和形状的轻微扰动的影响。我们的研究强调了在一个成叶周期中 SAM 中干细胞维持的动态性和灵活性，以及植物发育的稳健性。
