Morphogenesis of flattened unifacial leaves in Juncus prismatocarpus (Juncaceae).

To reveal the mode of morphogenesis of flattened unifacial leaves, we analysed the cell division direction and distribution on the leaf blade of Juncus prismatocarpus. Using the pulse-chase 5-ethynyl-2'-deoxyuridine method, we quantified and mapped the cell division direction on the leaf blade of J. prismatocarpus and compared the distribution of thickening cell divisions with the expression pattern of DROOPING LEAF (DL), a key gene involved in leaf blade thickening. Thickening cell divisions were the most abundant (> 45%) among all cell division directions on the leaf blade of J. prismatocarpus from the early plastochron 2 stage through the plastochron 3 stage. Mapping of cell divisions indicated that cell divisions in a particular direction were not restricted to a particular domain but were distributed diffusely throughout the entire cross-sectional area of the leaf blade. Gradient analysis indicated that the distribution of thickening cell divisions of the adaxial domain was denser than that of the abaxial domain. Contrary to the prolonged and diffuse distribution of thickening cell divisions, DL expression was transient and restricted in a narrow band. Our results suggest that a diffuse 'thickening meristem' plays the key role in the development of flattened unifacial leaves.

A pulse-chase strategy for EdU labelling assay is able to rapidly quantify cell division orientation.

Measurement of the direction of cell division is an important, yet difficult, task to analyse how a plant organ acquires its final shape from an initially small group of cells. We introduce a method that rapidly and easily quantifies cell division direction and is applicable to all plant species. A pulse-chase strategy for 5-ethynyl-2'-deoxyuridine (EdU) labelling assay was established and was shown to be successful for leaves of Arabidopsis thaliana (Arabidopsis) and Juncus prismatocarpus. By optimization of the pulse and chase periods, most of the signals obtained were sets of daughter nuclei. For Arabidopsis, the optimal time was a 45-min pulse and a 7-h chase. For J. prismatocarpus, the optimal time was a 2-h pulse and a 13.5-h chase. The positions of the daughter nuclei were used to quantify cell division direction in the Arabidopsis leaf primordia. Overall, cell division along the proximal-distal axis was more frequent than along the medial-lateral axis. In petiole, major vein, minor vein and margin areas, the major cell division direction seemed to be coincident with the direction of auxin flow. The advantages of our method over the few methods used previously are discussed. We anticipate that it will provide opportunities to study plant development in the near future.

A Pulse-chase EdU Method for Detection of Cell Division Orientation in Arabidopsis and Juncus prismatocarpus Leaf Primordia.

In plants, the morphological diversity of leaves is largely determined by cell division, especially cell division orientation. Whereas cell division itself is easily monitored, the detection and quantification of cell division orientation are difficult. The few existing methods for detection and quantification of cell division orientation are either inefficient or laborious. Here, we describe a pulse-chase strategy using a 5-ethynyl-2'-deoxyuridine (EdU) labeling assay. Plant tissues are first incubated with EdU for a short period (pulse), followed by a long incubation without EdU (chase). Using this method, the positions of daughter cells are easily detected and can be used to quantify cell division orientation. Our protocol is rapid and very efficient for quantitative analysis of cell division orientation, and can be applied to both model and non-model plant species. Graphic abstract: Plant cell division pairs clearly visualized by a pulse-chase EdU method.