**1. Introduction**

The production of second-generation ethanol (E2G) provides an additional source of energy in the sugar and ethanol sector by increasing the biofuel yield without expanding the crop area, thus leading to a sustainable production system. However, for the process to become financially competitive in comparison with first-generation ethanol (E1G), it is necessary to reduce the costs related to the lignocellulosic biomass processing required to recover and break the sugars present in plant cell walls [1]. The principal barrier to the conversion of lignocellulosic biomass into bioethanol or chemicals is the insoluble lignin network that surrounds and shields the cellulose microfibrils from degrading enzymes. The high energy and environmental costs of the treatments necessary to overcome these drawbacks constitute major hurdles to commercial E2G production [2]. To overcome these limitations, many studies have focused on the identification of enzymes related to biomass pretreatment and hydrolysis processes. The majority of the enzymes that compose enzymatic cocktails are proteins prospected from fungi that belong to different glycoside hydrolase (GH) families [3].

and pectin acetylesterases control the degree of homogalacturonan methylesterification and acetylation, respectively [15]. Class III peroxidases (Prxs) can either form covalent bonds by oxidizing aromatic compounds such as monolignols or aromatic amino acids or produce reactive oxygen species that participate in non-enzymatic breakage of covalent bonds of polysaccharides [16]. All these proteins belong to multigene families and their genes are differentially

Glycoside Hydrolases in Plant Cell Wall Proteomes: Predicting Functions That Could Be Relevant…

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GHs are of special interest, since they can hydrolyze the glycosidic bonds from two carbohydrates or from a carbohydrate and a non-carbohydrate moiety, thus actively contributing to cell wall polymer rearrangements. In *Arabidopsis thaliana*, about 379 GHs have been identified, belonging to 29 different families, among which approximately 52% were predicted to be cell wall GHs [17]. A great number of plant cell wall GH families have been identified so far in cell wall proteomes (see [18, 19]). Associations between structure and function can be predicted to point to candidate genes prone to manipulation. Among others, GHs comprise β-glucosidases, β-galactosidases, β-mannosidases, β-glucuronidases, β-xylosidases, β-Dfucosidases, exo-β-1,4-glucanases, lactases, β-glycosidases, α-L-arabinofuranosidases, glucan

In this chapter, we will describe cell wall GHs identified in the cell wall proteomes of sugarcane and *Brachypodium distachyon*, two monocots from the grass family. Sugarcane is already largely used for E1G production and could be used for E2G production [21], whereas *B. distachyon* is a model plant amenable for genetic transformation [22]. Up to now, there were 49 and 114 GHs identified in sugarcane and *B. distachyon* cell wall proteomes, respectively (see [18]). Based on their amino acid sequences, we have made bioinformatic predictions of functional domains and phylogenic analyses. GH families possibly relevant for improving

In plants, several strategies have been used in order to extract and identify CWPs with a high number of GHs, such as vacuum-infiltration protocol with saline solution and identification of proteins predicted by bioinformatics to be targeted to the secretory pathway [23–25]. In *A. thaliana*, around 200 GHs, belonging to 13 families, are assumed to be involved in polysac-

Conversely, in the monocot rice, GH17 is the one that presents the highest number of members, followed by GH28 [26]. The cell wall particularities of dicots (e.g., *A. thaliana*) vs. monocots, among which grasses, are also reflected in the distribution of GH families. For example, the lower proportion of pectins in monocot cell walls relates to a lower number of polygalacturonases (GH28) [17]. Thus, the range of GH families depends on plant species, and each of

By bioinformatic analyses of amino acid sequences, it is possible to classify newly identified GHs into families. We have done it for the GHs identified in the sugarcane and the *B. distachyon* cell wall proteomes. However, assigning a GH to its family as defined in the CAZy database [20] does not necessarily provide a clear picture of its function, since proteins from a given GH

regulated during plant development and in response to environmental changes.

1,3- and 1,4-β-glucosidases, β-amylases (for a complete repertoire, see [20]).

biomass transformation processes to E2G are highlighted.

charide modification and cell wall reorganization [8].

**2. Plant GH families**

them has to be studied separately.

The raw material for E2G production is plant fiber, which is mainly composed of cell walls. Nevertheless, less work has been devoted to study plant cell wall proteins (CWPs), and thus, to understand how the plant mechanisms themselves function to loosen and tighten up back the cell wall in order to promote cell growth and adapt to their changing environment. Accordingly, a common opinion today is that it is important to understand how cell walls are built up to improve the biomass deconstruction processes [4, 5].

Plant cells are surrounded by a wall characterized by its specific structure and composition [6]. Cell walls are mainly composed of polysaccharides, lignin, suberin, waxes, proteins, calcium, boron, and water, and have the ability to self-assemble [7]. Plants have two different kinds of cell wall deposition. Primary cell walls are synthesized in still-growing cells, whose form is not definitive, and thus, they can undergo growth and expansion. Secondary cell walls are synthesized in already fully expanded cells which are differentiating to perform specific functions, like xylem and fibers cells. In lignified secondary walls, the proportion of cellulose is higher than in primary cell walls with a higher degree of polymerization and crystallinity specificities [8]. Cellulose, hemicellulose, and pectins are the cell wall polysaccharides, and the biogenesis of cell walls involves their synthesis in intracellular compartments or at the plasma membrane, secretion, assembly, and rearrangement *in muro*. The primary cell walls of grasses have specific characteristics, and they are called type II cell walls [9]. They have a low content in pectins and xyloglucans, but a high content in mixed linked β-D-glucan [also named (1 → 3, 1 → 4)-β-Dglucans] during growth and in glucuronoarabinoxylans (GAXs). They also present ferulate and p-coumarate esters formed by attachment to the arabinosyl units of GAXs that are absent in gymnosperms, dicots, and other monocots. It has been assumed that plants devote more than 10% of their genome to the biogenesis of cell walls [10]. The cell wall is a dynamic structure involved in several physiological processes such as: cell growth [11], defense against pathogens [12], or signaling [13]. In sugarcane, the cell wall also plays a key role in the distribution of sucrose [14].

Displaying roles in cell growth, enzymes are part of the cell wall proteome. Glycosidases and glycanases have exo- and endo-GH activities, respectively, while trans-glycosidases and transglycanases perform exo- and endo-transglycosylation, respectively. Pectin methylesterases and pectin acetylesterases control the degree of homogalacturonan methylesterification and acetylation, respectively [15]. Class III peroxidases (Prxs) can either form covalent bonds by oxidizing aromatic compounds such as monolignols or aromatic amino acids or produce reactive oxygen species that participate in non-enzymatic breakage of covalent bonds of polysaccharides [16]. All these proteins belong to multigene families and their genes are differentially regulated during plant development and in response to environmental changes.

GHs are of special interest, since they can hydrolyze the glycosidic bonds from two carbohydrates or from a carbohydrate and a non-carbohydrate moiety, thus actively contributing to cell wall polymer rearrangements. In *Arabidopsis thaliana*, about 379 GHs have been identified, belonging to 29 different families, among which approximately 52% were predicted to be cell wall GHs [17]. A great number of plant cell wall GH families have been identified so far in cell wall proteomes (see [18, 19]). Associations between structure and function can be predicted to point to candidate genes prone to manipulation. Among others, GHs comprise β-glucosidases, β-galactosidases, β-mannosidases, β-glucuronidases, β-xylosidases, β-Dfucosidases, exo-β-1,4-glucanases, lactases, β-glycosidases, α-L-arabinofuranosidases, glucan 1,3- and 1,4-β-glucosidases, β-amylases (for a complete repertoire, see [20]).

In this chapter, we will describe cell wall GHs identified in the cell wall proteomes of sugarcane and *Brachypodium distachyon*, two monocots from the grass family. Sugarcane is already largely used for E1G production and could be used for E2G production [21], whereas *B. distachyon* is a model plant amenable for genetic transformation [22]. Up to now, there were 49 and 114 GHs identified in sugarcane and *B. distachyon* cell wall proteomes, respectively (see [18]). Based on their amino acid sequences, we have made bioinformatic predictions of functional domains and phylogenic analyses. GH families possibly relevant for improving biomass transformation processes to E2G are highlighted.
