Background

Plant cells are attached to each other by cell walls and cannot migrate. Therefore, organized, oriented cell division during the early stages of leaf development largely determines the shapes of mature leaves. Thus far, no method for efficient and rapid detection and quantification of cell division orientation has been reported. Existing methods include visualization of daughter nuclei (in telophase) using a pCYCB1;1::GUS (β-glucuronidase) reporter line (Horiguchi et al., 2011) or visualization of spindle equators (in metaphase) using 4’,6-diamidino-2-phenylindole (DAPI) staining (Fukushima et al., 2015). However, these methods require considerable processing time and effort to identify cells from a particular phase. Recently, 5-ethynyl-2’-deoxyuridine (EdU), a thymidine analog, has become the agent of choice for monitoring cell division activity (Kotogany et al., 2010; Ichihashi et al., 2011; Nakayama et al., 2012, 2014 and 2015; Furuya et al., 2018; Koga et al., 2020). By combining a pulse-chase strategy and EdU staining, we developed a method that can rapidly detect large numbers of cell pairs (two daughter cells after a cell division event) over a short period of time (Yin and Tsukaya, 2016). In brief, plant tissues are first incubated with EdU for a short period (pulse), during which EdU is incorporated in cells in S phase. The pulse period is followed by a long incubation without EdU (chase), allowing EdU-carrying cells to proceed through the subsequent G₂ and M phases. By optimization of the lengths of pulse and chase periods, pairs of daughter cells can be detected, because they contain identical amounts of EdU. Cell division orientation can be deduced according to the positions of the two daughter cells. Using this method, we showed that cell division orientation is locally non-stochastic in leaf primordia of Arabidopsis thaliana (Arabidopsis), a model plant species for which leaf morphogenesis has been extensively studied (Yin and Tsukaya, 2016). Furthermore, we combined this method with conventional paraffin sectioning and analyzed cell division orientation in leaves with more complex structures such as those of Juncus prismatocarpus, a non-model plant species (Yin and Tsukaya, 2019).