Abstract
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.
Highlights
The three-dimensional (3D) arrangement of cells in tissues provides an anatomical reference for monitoring the development and function of organisms [1]
In the shoot apical meristem (SAM), the plant tissue that generates the above-ground organs, stem cells are located at the tip and differentiated cells are arranged around the peripheral zone that surrounds the stem cells [2]
In the root apical meristem (RAM), located at the root tip, stem cells are arranged around the quiescent center (QC) and differentiate into diverse cell types including the columella, stele, and endodermis [3]
Summary
The three-dimensional (3D) arrangement of cells in tissues provides an anatomical reference for monitoring the development and function of organisms [1]. The 3D arrangement of cells is important for understanding how the developmental processes of multicellular organisms function. Mechanical stress in the cell wall is caused by the 3D arrangement of cells, which is essential for proper organ differentiation [6]. These studies provided important insights about the topology and anisotropy of developing organs in plants; these studies have been limited to the analysis of two-dimensional sections. Since plant tissues are organized in three dimensions, a three-dimensional analysis is necessary to understand topology and anisotropy precisely
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