**2. Plant GH families**

**1. Introduction**

166 Advances in Biofuels and Bioenergy

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

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

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

from fungi that belong to different glycoside hydrolase (GH) families [3].

built up to improve the biomass deconstruction processes [4, 5].

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 polysaccharide modification and cell wall reorganization [8].

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 them has to be studied separately.

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 family can display different roles. For example, GH1 family members can be involved in cell wall metabolism, lignification, signaling, or defense [17]. Phylogenic analyses can help getting more information regarding the functions of specific plant GHs. Since monocots could be a major source of raw material for E2G production, it is interesting to study their cell wall metabolism and outline possible strategies to increase biomass production or lower cell wall recalcitrance to deconstruction [27]. In addition, it might be possible to find interesting analogies by comparing the plant cell wall assembly/disassembly mechanisms to those of microorganisms.

The large size of the GH1, GH17, and GH28 families is probably linked to their roles in the assembly and in the rearrangement of cell wall polysaccharides [17]. Usually the GH1, GH16, GH17, and GH35 families are less represented in dicots than in monocots [31]. GH17 display glucan-1,3-β-glucosidase activity and possible substrates could be mixed (1,3)(1,4)-β-D-glucans [8]. This is consistent with the fact that only type II grass cell walls present this kind of hemicellulose.

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

http://dx.doi.org/10.5772/intechopen.73181

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After a survey of the cell wall proteomes described so far and collecting information regarding microorganism enzymes used for biomass deconstruction, we decided to focus this review on the GH1, GH3, GH17, GH27, GH35, and GH51 families. We have predicted functional and structural domains in newly identified CWPs using the PredictProtein bioinformatic software and grouping them in families [35]. Since plant cells perform cell expansion themselves by involving cell wall polysaccharide rearrangements, the plant mechanisms could be mimicked by the enzymes used in cocktails. The comparison of plant and microorganisms enzymes presently used for biomass hydrolysis could contribute to determining their common characteristics and which specificities of plant enzymes could be copied in order to improve industrial cell wall deconstruction processes. Conversely, this comparative study could help in identifying which of the characteristics of microorganism enzymes could be engineered in

The GH1 family mainly comprises β-glucosidases, which are found in several organisms performing different functions. In plants, they are involved in cell wall catabolism, signaling, lignification, defense, symbiosis, and secondary metabolism. Putative β-glucosidase genes have been shown to be induced by biotic and abiotic stresses and they were considered critical for the success of plant development in stressful environments [36–40]. Accordingly, plants are the organisms that have the highest number of GH1s, e.g., 48 in *A. thaliana* [41] and 40 in *Oryza sativa* [42]. Over the years, various β-glucosidases hydrolyzing cell wall oligosaccharides have been characterized mainly in monocots, such as in germinating seedlings of barley where they show preference for manno-oligosaccharides in endosperm cell walls [43], and in rice seedlings, where they hydrolyze different oligosaccharides [42]. Extracellular β-glucosidases can also contribute to the production of toxic compounds, such as hydroxamic acids and cyanide [44–46]. For this process to occur correctly, the defense molecules are stored in nonactive glucosylated forms in the vacuole, while the β-glucosidases are stored in the apoplast or in protein bodies in dicots or in plastids in monocots. The enzyme and its substrate get into

The plant cell wall is a large polysaccharide repository that contains a large amount of glucosyl residues. β-glucosidases play important roles in cell wall formation and plant development, because they participate in cell wall polysaccharide turnover [47]. In sugarcane [30] and *B. distachyon* [31], more GH1 were found in cell wall proteomes of growing organs, such as young leaves and apical internodes, than in mature organs. In addition, as suggested by bioinformatic predictions, several of the identified GH1 (e.g., SCCCCL3001B10.b, SCJFLR1017E03, SCEQLB1066E08, Bradi1g10940, Bradi1g10930, Bradi1g10940, and Bradi2g59650) have a

plant species in order to obtain biomass with less recalcitrant cell walls.

contact when the cell is damaged during plant-microorganism interaction.

**4. GH1, GH3, and GH51**

β-glucosidase activity (GO:0008422).
