**1. Introduction**

The cell wall is a protective layer that is located around the cell membrane, which is found in plant, fungi, bacteria, and archaea cells. The structure and functions of the cell wall were interconnected during the process of evolution in spite of the fact that its chemical structure, and to a lesser extent, its functions are diverse in different groups of organisms. In plants, its most fundamental function is to provide structural support and protection to the cell with some additional specific functions that may occur during plant development and cell differentiation [1].

The plant cell wall is a dynamic and highly specialised network that is formed by a heterogeneous mixture of cellulose, hemicelluloses, pectins and, to some extent, proteins and phenolic compounds. The cell wall that is formed during cell division is called the primary cell wall. In many plants, as/after

the cell completes its growth, additional layers of cellulose fibres are deposited on the inner surface of the primary cell wall, which produces a secondary cell wall. The cell wall composition in vascular plants is approximately 30% cellulose, 30% hemicellulose, and 35% pectins, and 1–5% structural proteins, on a dry weight basis [2,3]. However, the precise proportions of these compounds may differ significantly at different stages of plant development. For example, grass coleoptiles consist of 60–70% hemicelluloses, 20–25% celluloses, and 10% pectin substances and the grass endosperm cell wall may contain up to as much as 85% hemicelluloses. By contrast, the secondary cell walls are generally more cellulose-rich [4–7].

*Brachypodium distachyon* (Brachypodium) belongs to the Pooideae subfamily and is a wellestablished model species for the grasses. It has several features and advantages that make it useful for gaining a better understanding of the genetic, cellular and molecular biology of temperate climate zone cereals and forage crops [8]. There are many studies, which are often connected with the chemical composition of the Brachypodium cell wall [9–12]. A comparative study of the primary cell wall in the seedlings of Brachypodium, barley and wheat demonstrated similar relative levels and developmental changes of hemicelluloses [10]. Analyses of the Brachypodium proteome facilitate better understanding of the enzymes that are involved in cell wall remodelling during seed development; such research is of great importance for gaining better understanding of these processes in grasses and for finding the key components that are responsible for the size and weight of grass grains [9]. However, there is a dearth of information about the localisation of specific cell wall components at different stages of Brachypodium development.

Here, we characterise the chemical composition of the cell walls in Brachypodium embryos and describe the differences in the number of nucleoli that were observed in the cell nuclei in different parts of an embryo. We used light and transmission electron microscopy (TEM), histological and immunolocalisation techniques to analyse the distribution of selected pectins, arabinogalactan proteins (AGP), extensins, and hemicelluloses in the cell walls, internal cell compartments, and on the embryo surface.

#### **2. Results and Discussion**

#### *2.1. The Morphological and Histological Features of Brachypodium Embryos*

In their study, Wolny et al. [13] demonstrated that Brachypodium embryos are small in size, which makes their initial examination possible only by the use of a dissecting microscope. In this study, we distinguished the main parts of the embryo, such as scutellum, V scale, coleoptile, first and second leaf, shoot apex, mesocotyl, epiblast, radicula, root cap, and coleorhiza (Figure 1). The coleoptile and coleorhiza are two organs that are found exclusively in grass species [14]. A comparison of the cell nuclei in different parts of Brachypodium embryos demonstrated that the majority contained only one nucleolus (Figure 1). However, some cells of the shoot apex, mesocotyl, radicula and root cap were characterised by the presence of a round nuclei that contained two nucleoli (Figure 1; nucleoli indicated by red arrows). TEM analysis of the selected embryo parts confirmed these observations and demonstrated the presence of a centrally positioned nucleus with one or two large nucleoli as well as a high nucleus:cytoplasm ratio (Figure 2a,b). The cytoplasm of these cells was dense and contained lipid droplets and starch granules around the nucleus. Interestingly, we also found cells in the embryo with nuclei that were extended in their shape but that also contained two nucleoli (Figure 2c). The architecture of these cells is typical for the initial vascular tissue [15]. According to Verdeil et al. [16], pluripotent plant stem cells, which are located within the root and shoot meristems, are isodiametric, have a dense cytoplasm, a high nucleus:cytoplasm ratio, a fragmented vacuome, contain granules of starch, and have a spherically-shaped nucleus with one or two nucleoli. Both of the meristematic cells of the oil palm (*Elaeis guineensis*) [17], maize (*Zea mays*) [18], and onion (*Allium cepa*) [19] have similar characteristics to those described by Verdeil et al. [16], which appear to be universal for monocots.

**Figure 1.** Schematic representation of Brachypodium embryo. Asterisks in different colours represent nuclei from histological sections of specified parts of the embryo. Red arrows mark two nucleoli. Purple arrows show respective parts of the embryo: V scale, coleoptile, first leaf, second leaf, shoot apex, mesocotyl, epiblast, coleorhiza, radicula, and root cap. Bars: 5 μm.

**Figure 2.** TEM (transmission electron microscopy) of radicula nuclei (**a**–**c**). Abbreviations: *CW*—cell wall, *LD*—lipid droplets, *N*—nucleus, *Nu*—nucleolus, *Pl*—plastid. Bars: (**a**) 3 μm; (**b**) 1.5 μm; and (**c**) 2.5 μm.
