Kihara Institute for Biological Research, Yokohama City University, Maioka 641-12, Totsuka, Yokohama 244-0813, Kanagawa, Japan.
Imaging Frontier Center, Organization for Research Advancement, Tokyo University of Science, 2641 Yamazaki, Noda 278-8510, Chiba, Japan.
Int J Mol Sci. 2021 Dec 21;23(1):40. doi: 10.3390/ijms23010040.
The three-dimensional (3D) arrangement of cells in tissues provides an anatomical basis for analyzing physiological and biochemical aspects of plant and animal cellular development and function. In this study, we established a protocol for tissue clearing and 3D imaging in rice. Our protocol is based on three improvements: clearing with iTOMEI (clearing solution suitable for plants), developing microscopic conditions in which the Z step is optimized for 3D reconstruction, and optimizing cell-wall staining. Our protocol successfully 3D imaged rice shoot apical meristems, florets, and root apical meristems at cellular resolution throughout whole tissues. Using fluorescent reporters of auxin signaling in rice root tips, we also revealed the 3D distribution of auxin signaling events that are activated in the columella, quiescent center, and multiple rows of cells in the stele of the root apical meristem. Examination of cells with higher levels of auxin signaling revealed that only the central row of cells was connected to the quiescent center. Our method provides opportunities to observe the 3D arrangement of cells in rice tissues.
组织中细胞的三维（3D）排列为分析动植物细胞发育和功能的生理和生化方面提供了解剖学基础。在本研究中，我们建立了一个用于水稻组织透明化和 3D 成像的方案。我们的方案基于三个改进：使用 iTOMEI（适合植物的透明溶液）进行透明化、开发优化 Z 步以进行 3D 重建的微观条件，以及优化细胞壁染色。我们的方案成功地对水稻茎尖分生组织、小花和根尖分生组织进行了 3D 成像，在整个组织中达到了细胞分辨率。使用水稻根尖中生长素信号的荧光报告基因，我们还揭示了在根顶端分生组织的中柱、静止中心和多个茎细胞列中激活的生长素信号事件的 3D 分布。对具有更高水平生长素信号的细胞的检查表明，只有中央列的细胞与静止中心相连。我们的方法为观察水稻组织中细胞的 3D 排列提供了机会。
