Germination and the Early Stages of Seedling Development in Brachypodium distachyon

Abstract

Successful germination and seedling development are crucial steps in the growth of a new plant. In this study, we investigated the course of the cell cycle during germination in relation to grain hydration in the model grass Brachypodium distachyon (Brachypodium) for the first time. Flow cytometry was performed to monitor the cell cycle progression during germination and to estimate DNA content in embryo tissues. The analyses of whole zygotic embryos revealed that the relative DNA content was 2C, 4C, 8C, and 16C. Endoreplicated nuclei were detected in the scutellum and coleorhiza cells, whereas the rest of the embryo tissues only had nuclei with a 2C and 4C DNA content. This study was accompanied by a spatiotemporal profile analysis of the DNA synthetic activity in the organs of Brachypodium embryos during germination using EdU labelling. Upon imbibition, nuclear DNA replication was initiated in the radicle within 11 h and subsequently spread towards the plumule. The first EdU-labelled prophases were observed after 14 h of imbibition. Analysis of selected genes that are involved in the regulation of the cell cycle, such as those encoding cyclin-dependent kinases and cyclins, demonstrated an increase in their expression profiles.

Keywords: Brachypodium distachyon embryo; EdU; cell cycle; germination; replication.

Figure 1

Brachypodium distachyon (Brachypodium) grains at key time points of imbibition (A) and the progress of Brachypodium embryo size growth during 46 h of imbibition (B), data are means ± SE. Abbreviations: Cr—coleorhiza, R—radicle, Cl—coleoptile. Scale bar represents 1 mm.

Figure 2

Flow cytometry analysis of DNA content pattern in the Brachypodium embryos that were isolated at 4 h after imbibition (HAI) (A). Histogram showing the ploidy distribution in whole embryos. (B) Percentage of cells with a 2C, 4C, 8C, and 16C DNA content in a whole embryo (E) and in specific embryo organs, (Scut) scutellum, (R) radicle, (Cr + R) coleorhiza and radicle, (Cl + P) coleoptile with a shoot apex and primary leaves.

Figure 3

Percentage of nuclei in the G1, S, and G2 phases and the 4C/2C ratio in the cells from Brachypodium embryos during the first 24 h of growth.

Figure 4

Detection of EdU (green fluorescence) incorporated into actively replicating nuclei in Brachypodium embryos during imbibition. Fragments of cross sections through embryos 11 HAI (A–C), 12 HAI (D–F), 13 HAI (G–I), and 14 HAI (J–L). Cross sections through the shoot apex (A,D,G,J), radicle (B,E,H,K), and radicle with a visible root cap (C,F,I,L). Inset in (K) presents an enlargement with the nucleus at prophase. Abbreviations: SA—shoot apex, FL—first leaf, Mes—mesocotyl, Rad—radicle, RC—root cap. The slides were counterstained with DAPI (grey fluorescence). The grey colour of cell walls was caused by their autofluorescence. Scale bar represents 20 µm; all photomicrographs were taken at the same magnification.

Figure 5

Expression of the CDKA, CDKB1, CDKB2, CDKD, CYCB1, CYCD3, CYCD4-1, WEE1, and protein kinase genes (A) and the CYCA3 gene (B) that were involved in the cell cycle activity in Brachypodium embryos at 0, 4, 12, and 24 HAI. The relative expression levels were normalised to an internal control (AK437296, gene encoding for ubiquitin) and calibrated to the control (the embryos from dried seeds). The data from RT-PCR (mean ± SD of three replicates) are given in arbitrary units.