**Mehdi Cherkaoui 1, Virginie Lollier 1, Audrey Geairon 1, Axelle Bouder 1, Colette Larré 1, Hélène Rogniaux 1, Elisabeth Jamet 2, Fabienne Guillon <sup>1</sup> and Mathilde Francin-Allami 1,\***


Received: 15 November 2019; Accepted: 23 December 2019; Published: 29 December 2019

**Abstract:** The cell wall is an important compartment in grain cells that fulfills both structural and functional roles. It has a dynamic structure that is constantly modified during development and in response to biotic and abiotic stresses. Non-structural cell wall proteins (CWPs) are key players in the remodeling of the cell wall during events that punctuate the plant life. Here, a subcellular and quantitative proteomic approach was carried out to identify CWPs possibly involved in changes in cell wall metabolism at two key stages of wheat grain development: the end of the cellularization step and the beginning of storage accumulation. Endosperm and outer layers of wheat grain were analyzed separately as they have different origins (maternal and seed) and functions in grains. Altogether, 734 proteins with predicted signal peptides were identified (CWPs). Functional annotation of CWPs pointed out a large number of proteins potentially involved in cell wall polysaccharide remodeling. In the grain outer layers, numerous proteins involved in cutin formation or lignin polymerization were found, while an unexpected abundance of proteins annotated as plant invertase/pectin methyl esterase inhibitors were identified in the endosperm. In addition, numerous CWPs were accumulating in the endosperm at the grain filling stage, thus revealing strong metabolic activities in the cell wall during endosperm cell differentiation, while protein accumulation was more intense at the earlier stage of development in outer layers. Altogether, our work gives important information on cell wall metabolism during early grain development in both parts of the grain, namely the endosperm and outer layers. The wheat cell wall proteome is the largest cell wall proteome of a monocot species found so far.

**Keywords:** cell wall; grain; remodeling; polysaccharide; proteome; wheat

#### **1. Introduction**

The wheat grain is a caryopsis composed of an embryo separated from the starchy endosperm and aleurone cells by the scutellum. The progeny is surrounded by the maternal tissues, which are composed of several outer layers [1]. They consist of the pericarp, the testa, and the nucellar epidermis [2]. The pericarp includes epidermal cells, parenchyma cells, and cross and tube cells. Cytological and physiological features of wheat endosperm and pericarp were characterized all along the grain development [3–5].

At the early developmental stage, the outer layers constitute most of the grain volume [3]. By around 150 growing degree days (GDD), the layers of the outer integument are the first to degenerate, whereas endosperm cells continue to divide. The beginning of grain filling occurs around 220 GDD. The aleurone layer becomes recognizable. Endosperm cells stop dividing and the grains

reach their maximum fresh mass at around 440 GDD. Concomitantly with the fusion of the pericarp with the maternal epidermis, the expanding endosperm compresses the outer layers. At grain maturity, except for the aleurone cells and embryo, all tissues degenerate, and only cell walls remain.

Cell walls play key roles during grain development. They have various compositions depending on the cell type and developmental stages. Due to their importance in nutrient transport, as well as cell size, shape, and stiffness, cell walls are essential for the determination of the final grain size and the nutritional quality of cereal grain [6–8]. They account for 3% of the endosperm, and around 70% of the outer layers in the mature wheat grain [9]. The cell walls are the primary contributors to cereal dietary fiber with important implications for human nutrition and disease prevention [10]. Cell walls also have important effects on cereal processes, such as milling, brewing, and breadmaking quality [11]. They are mostly composed of polysaccharides, together with a smaller amount of proteins, lipids, and phenolic compounds that participate in the cell wall metabolism.

Cell walls of developing endosperm have recently been described in detail [12–17]. Despite their important roles, both in grain physiology and from a nutritional point of view, cell walls of the outer layers of cereal grain have been poorly studied so far. Callose was the first polysaccharide to be detected during the development of wheat grain [18]. Arabinoxylans (AXs) and mixed β-glucans (MLGs) are the main polysaccharides of wheat grain, and of grasses more broadly. The remaining polysaccharides consist of cellulose, mannans, xyloglucans, and pectins [14,15,18]. MLGs are deposited early during the cellularization step and throughout the grain development. AXs are detected at the beginning of the differentiation in the endosperm cells. Spatial and temporal variations of the AX structure were observed in wheat grain. This structural heterogeneity is mainly explained by the variation in the substitution degree of AXs by arabinose, but also by ferulic acid, and to a lesser extent, p-coumaric acid, which binds to the arabinose residues [12,19]. Although less abundant than in dicot plants, cellulose was observed not only in the outer layers, but also in endosperm cells in a proportion that was not expected [20]. Recently, pectins were detected in the endosperm and the outer layers of wheat grain [14]. An impressive deposition of homogalacturonans (HGs) in the testa was reported at 250 GDD, with a decrease in their methylation degree during grain development. Rhamnogalacturonans of type I (RGI) were mainly detected in the endosperm and mostly in aleurone cells. Spatial and temporal variations of the RGI structure have been observed [14].

In addition to variability of the polysaccharide distribution and structures, the cell walls of the outer layers contain significant amounts of hydrophobic and polyphenolic polymers that compose the lignin and/or cutins. Lignification occurs earlier than previously reported in the outer layers and long before the grain reaches its final size [2]. Several cuticles are formed early in the development and persist at grain maturity. They are considered to be physically associated with the cell walls and have some overlapping functions. These cuticles are mainly composed of cutins and soluble waxes. Polysaccharides are also embedded into the cuticular layers [21]. Recently, xyloglucans were detected in the cuticle above the testa in the grain of the wild grass *Brachypodium distachyon* [22].

As described above, the composition and structure of cell wall polysaccharides vary during grain development and according to grain tissue to answer to the physiological needs of plants. Cell wall polysaccharide modifications imply numerous modifying enzymes, among which, glycosyl hydrolases (GHs) act on polysaccharides and oxidoreductases. They are mainly located in the cell wall and participate in the cell elongation, cuticle deposition, and cell wall structural variation that occurs during grain development. They may be transported by vesicles from the cytoplasm to the extracellular compartment [23]. The identification and functional understanding of these protein actors are essential to gain access to cell wall dynamics. To date, only a few experiments have been reported on grass cell wall proteomes: *Brachypodium distachyon* [24–26], *Saccharum* spp (sugarcane) [27–29], *Oryza sativa* (rice) [30–33], and *Triticum aestivum* (wheat) [34]. Transcriptomic data on the developing wheat grain allowed for the identification of GH genes [15], and recently, we reported the first data on the cell wall proteome of endosperm and the outer layers of wheat grain at a given developmental stage [34]. More than 600 cell wall proteins (CWPs) were identified, half of which were present in both tissues, suggesting common and specific tissue remodeling activities.

In this study, we investigated the cell wall proteome targeting both the endosperm and outer layers of the wheat grain at two key developmental stages in order to report on the cell wall dynamics during grain development. We identified proteins annotated as CWPs that are potentially involved in cell wall polysaccharide remodeling and cell wall assembly. A quantitative analysis allowed for describing the cell wall proteome content during the grain developmental stage for two tissues: the endosperm and the outer layers.
