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

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.

**3. GHs identified in sugarcane and** *B. distachyon* **cell wall proteomes**

or differential accessibility as a consequence of differences in cell wall structure.

and seeds, GH17 was the most populated family.

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In sugarcane, 49 GHs have been identified in cell wall proteomes [28, 29]. They are distributed in 16 GH families. The GH3 family is the best represented (~20%), followed by GH17 (~16%), GH18 (~12%), and GH1 (~8%) (**Figure 1**). This distribution varies according to organ and developmental stage. In cell suspension cultures, only 4 GH families were identified among which GH3 was the most populated [28]. In 2-month-old stems, 7 GH families were found, GH3 also being the most represented [29]. Leaves recovered few GHs, from families 19, 27 (young leaves only), 28, and 31 (young leaves only). Apical internodes mainly contained GH3 members, whereas mostly GH17 members were found in basal ones [30]. Noteworthy, it should be mentioned that the absence of some GH families in a given cell wall proteomes could be due to technical limitations

In *B. distachyon*, 114 CW GHs were identified in cell wall proteomes [31–34]. They are distributed into 21 families. The most populated one was GH17 (~17%), followed by GH28 (~13%), GH1 (~9%), GH3 (~8%), and GH35 (~8%) (**Figure 1**). GH28, followed by GH1, GH3, and GH16, had the highest number of members in young leaves. In mature leaves, GH17, GH18, and GH28 were the most represented. In internodes, they were GH28 and GH17. In seedlings

**Figure 1.** GH family distribution of presently known cell wall proteomes in sugarcane (a) and *B. distachyon* (b). The number of family members identified in each of them is indicated. Same colors indicate same GH families in both species.

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

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 β-glucosidase activity (GO:0008422).

Ten GH3s have been identified in sugarcane [28–30] and nine GH3s have been identified in *B. distachyon* [31, 32, 34]. Half of the sugarcane GH3 are predicted to have a β-glucosidase activity (GO:0008422) (e.g., SCEZLB1007A09, SCEQLR1093F09, and SCQSLR1089A04). However, some GH3 are predicted to have xylosidase (e.g., Bradi5g23470) or α-L-arabinofuranosidase (AFase) activity (e.g., SCCCCL4009F05, SCCCSB1003H06, and Bradi3g59020).

biomass hydrolysis, since this material is particularly rich in arabinoxylans, which need to be

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To compare plant and microorganism GH1 and GH3, we have performed phylogenic analyses. Some GH1 and GH3 identified in cell wall proteomes of sugarcane and *B. distachyon* have been selected. For plants, we have chosen species at critical evolutionary nodes since terrestrialization like a moss (*Physcomitrella patens*), the common ancestor to higher plants (*Amborella trichopoda*), *Sorghum bicolor* as the closest plant to sugarcane having a sequenced genome, and two dicots (*Lycopersicon esculentum* and *A. thaliana*). The *B. distachyon* and sugarcane sequences of several GHs identified in cell wall proteomes have been included in this analysis. Regarding microorganisms, we have retained GHs usually used in enzymatic cocktails for cell wall deconstruction. They belong to bacteria like *Bacillus licheniformis*, or to fungi like *Aspergillus nidulans* or *Trichoderma reesei* [52, 53]. As expected, the selected GH1 and GH3 share common ancestors. The two *B. licheniformis* GH1 root the GH1 tree (**Figure 2**). *A. nidu-*

**Figure 3.** Phylogenic tree of the GH3 subfamily comprising the GH3 identified in sugarcane and *B. distachyon* cell wall proteomes. All the plant sequences have been retrieved from Phytozome v12.1 (phytozome.jgi.doe.gov), whereas the microorganism sequences originate from the NCBI website (www.ncbi.nlm.nih.gov/protein/). The MEGA6 software (www.megasoftware.net) was used to generate the tree with the following options: MUSCLE for amino acid sequence alignments and maximum likelihood tree with 500 bootstraps. Visualization was done with FigTree (tree.bio.ed.ac.uk/

degraded with AFases in addition to endoglucanases [51].

*lans* and *T. reesei* GH1 come next, prior to the *P. patens* GH1.

software/figtree/).

The fungi β-glucosidases can degrade cellulose together with other kinds of enzymes, like endoglucanases and cellobiohydrolases. They separate the molecules of glucose from cellobiose, thus being used in enzymatic cocktails to produce cellulosic bioethanol [48]. In barley, the structure of the GH3 β-D-glucan exohydrolase ExoI was determined through X-ray crystallography, showing a two-domain globular structure being different from that of GH1 [49]. Besides the catalytic site, this enzyme has another binding site for (1 → 3, 1 → 4)-β-D-glucans only identified in monocots. Xylan 1,4-β-D-xylosidases hydrolyze xylose from xylo-oligosaccharides. These enzymes have several uses, such as in the industrial processes related to bread dough, animal feed digestibility, and deinking of recycled papers. In enzymatic cocktails, they are considered the most efficient enzymes to break glycosidic bonds of hemicelluloses [50]. The few GH51 members identified in sugarcane and *B. distachyon* were also assumed to be AFases (EC 3.2.1.55, GO:0046556). AFases catalyze the hydrolysis of α-L-arabinofuranoside in α-L-arabinosides. They act together with hemicellulases and pectinolytic enzymes to achieve hemicellulose and pectin hydrolysis. Several AFases used commercially belong to the GH51 family, generally originating from fungi. Such enzymes are of special interest for monocots

**Figure 2.** Phylogenic tree of the GH1 subfamily comprising the GH1 identified in sugarcane and *B. distachyon* cell wall proteomes. All the plant sequences have been retrieved from Phytozome v12.1 (phytozome.jgi.doe.gov), whereas the microorganism sequences originate from the NCBI website (www.ncbi.nlm.nih.gov/protein/). The MEGA6 software (www. megasoftware.net) was used to generate the tree with the following options: MUSCLE for amino acid sequence alignments and maximum likelihood tree with 500 bootstraps. Visualization was done with FigTree (tree.bio.ed.ac.uk/software/figtree/).

biomass hydrolysis, since this material is particularly rich in arabinoxylans, which need to be degraded with AFases in addition to endoglucanases [51].

Ten GH3s have been identified in sugarcane [28–30] and nine GH3s have been identified in *B. distachyon* [31, 32, 34]. Half of the sugarcane GH3 are predicted to have a β-glucosidase activity (GO:0008422) (e.g., SCEZLB1007A09, SCEQLR1093F09, and SCQSLR1089A04). However, some GH3 are predicted to have xylosidase (e.g., Bradi5g23470) or α-L-arabinofuranosidase

The fungi β-glucosidases can degrade cellulose together with other kinds of enzymes, like endoglucanases and cellobiohydrolases. They separate the molecules of glucose from cellobiose, thus being used in enzymatic cocktails to produce cellulosic bioethanol [48]. In barley, the structure of the GH3 β-D-glucan exohydrolase ExoI was determined through X-ray crystallography, showing a two-domain globular structure being different from that of GH1 [49]. Besides the catalytic site, this enzyme has another binding site for (1 → 3, 1 → 4)-β-D-glucans only identified in monocots. Xylan 1,4-β-D-xylosidases hydrolyze xylose from xylo-oligosaccharides. These enzymes have several uses, such as in the industrial processes related to bread dough, animal feed digestibility, and deinking of recycled papers. In enzymatic cocktails, they are considered the most efficient enzymes to break glycosidic bonds of hemicelluloses [50]. The few GH51 members identified in sugarcane and *B. distachyon* were also assumed to be AFases (EC 3.2.1.55, GO:0046556). AFases catalyze the hydrolysis of α-L-arabinofuranoside in α-L-arabinosides. They act together with hemicellulases and pectinolytic enzymes to achieve hemicellulose and pectin hydrolysis. Several AFases used commercially belong to the GH51 family, generally originating from fungi. Such enzymes are of special interest for monocots

**Figure 2.** Phylogenic tree of the GH1 subfamily comprising the GH1 identified in sugarcane and *B. distachyon* cell wall proteomes. All the plant sequences have been retrieved from Phytozome v12.1 (phytozome.jgi.doe.gov), whereas the microorganism sequences originate from the NCBI website (www.ncbi.nlm.nih.gov/protein/). The MEGA6 software (www. megasoftware.net) was used to generate the tree with the following options: MUSCLE for amino acid sequence alignments and maximum likelihood tree with 500 bootstraps. Visualization was done with FigTree (tree.bio.ed.ac.uk/software/figtree/).

(AFase) activity (e.g., SCCCCL4009F05, SCCCSB1003H06, and Bradi3g59020).

170 Advances in Biofuels and Bioenergy

To compare plant and microorganism GH1 and GH3, we have performed phylogenic analyses. Some GH1 and GH3 identified in cell wall proteomes of sugarcane and *B. distachyon* have been selected. For plants, we have chosen species at critical evolutionary nodes since terrestrialization like a moss (*Physcomitrella patens*), the common ancestor to higher plants (*Amborella trichopoda*), *Sorghum bicolor* as the closest plant to sugarcane having a sequenced genome, and two dicots (*Lycopersicon esculentum* and *A. thaliana*). The *B. distachyon* and sugarcane sequences of several GHs identified in cell wall proteomes have been included in this analysis. Regarding microorganisms, we have retained GHs usually used in enzymatic cocktails for cell wall deconstruction. They belong to bacteria like *Bacillus licheniformis*, or to fungi like *Aspergillus nidulans* or *Trichoderma reesei* [52, 53]. As expected, the selected GH1 and GH3 share common ancestors. The two *B. licheniformis* GH1 root the GH1 tree (**Figure 2**). *A. nidulans* and *T. reesei* GH1 come next, prior to the *P. patens* GH1.

**Figure 3.** Phylogenic tree of the GH3 subfamily comprising the GH3 identified in sugarcane and *B. distachyon* cell wall proteomes. All the plant sequences have been retrieved from Phytozome v12.1 (phytozome.jgi.doe.gov), whereas the microorganism sequences originate from the NCBI website (www.ncbi.nlm.nih.gov/protein/). The MEGA6 software (www.megasoftware.net) was used to generate the tree with the following options: MUSCLE for amino acid sequence alignments and maximum likelihood tree with 500 bootstraps. Visualization was done with FigTree (tree.bio.ed.ac.uk/ software/figtree/).

Finally, the tree is split into two distinct clades, each containing either one or two closely related *A. trichopoda* GH1. Monocot and dicot GH1 are finally separated in sub-clades. Regarding the GH3, the situation is more complex (**Figure 3**). Two clades are separated at the basis of the tree: clade A is rooted by three *B. licheniformis* GH3, followed by an *A. nidulans* GH3, whereas clade B is only rooted by an *A. nidulans* GH3. As for the GH1 tree, each sub-clade comprises an *A. trichopoda* GH3, whereas monocot and dicot GH3 form distinct groups. Similar results could be obtained for the other GH families. This phylogenic analysis shows the close relationships between microorganism and plant GH1 or GH3. Additional work is required to define precisely their specificities with the aim of generating new tools for industrial processes of biomass deconstruction.

β clade lost it through evolution. It is thought that the loss of this domain facilitates the extracellular secretion induced by biotic stresses, thus improving the response to pathogens [61, 62]. Other studies also revealed the antifungal effects of plant extracellular chitinases (GH18 and GH19) in combination with those of GH17 [69]. Indeed, fungi cell walls are composed of chitin and of branched β-(1,3):β-(1,6) glucans [57, 70–73]. Thereby, transgenic plants overexpressing a chitinase and/or a ß-l,3 glucanase became less susceptible to fungal attack [74, 75].

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The GH27 identified in cell wall proteomes of both sugarcane and *B. distachyon* was predicted to have α-galactosidase activity (EC 3.2.1.22, GO:0004557). α-galactosidases break galactosidic linkages in galactose-containing oligosaccharides, galactolipids, and galactomannans [76]. Since galactomannans are hemicelluloses, α-galactosidases could be used in enzymatic

GH35 are mainly β-galactosidases (EC 3.2.1.23), but exo-1,4-β-D-glucosaminidase (E.C 3.2.1.165) and exo-β-1,4-galactanases (EC 3.2.1.-) also belong to this family. β-galactosidases are found in microorganisms such as bacteria, fungi, and yeast, as well as in animals and plants [77]. They catalyze the hydrolysis of terminal non-reducing β-D-galactose residues in different molecules, like glycoproteins, oligosaccharides, glycolipids and lactose (www.cazy. org). β -galactosidases are classified in two families: GH2 are predominantly found in micro-

GH35 can be distributed into two main groups according to their preferred substrates: hydrolysis of pectic β-1,4-galactans, cleavage of β-1,3- and β-1,6-galactosyl linkages of *O*-glycans of arabinogalactan proteins [80]. In plants, they are associated with secondary metabolism or polysaccharide degradation, performing important roles in physiological events, including cell wall degradation and expansion during plant development, and turnover of signaling molecules [79–83]. They were also shown to be involved in ripening and abscission of mango, papaya, and orange fruits [84–86]. The GH35 found in the cell wall proteomes of sugarcane [30] and *B. distachyon* [31] is predicted to have a β-galactosidase activity (GO:0004565). In *B. distachyon*, they were only identified in leaves and in seedlings, whereas they were mostly found in sugarcane internodes. GH14 are very close to GH35 due to sequence similarity, perhaps playing similar roles, and they have only been identified in sugarcane internodes. Interestingly, the SCUTAM2089E05 GH14 was found to be differentially expressed in ancestral genotypes of sugarcane showing differential carbon allocation to lignin or sucrose [87].

Microorganisms use an arsenal of GHs to degrade plant cell walls, in order to establish themselves in their host. Similar mechanisms are thought to be used in their own plant cell wall modification, since plant cell walls embrace several types of carbohydrates with a variety of structures and biological functions. For sugarcane biomass deconstruction, the first step

cocktails to enhance the cell wall hydrolysis process by acting as a hemicellulase.

organisms (around 70%), and GH35 are found in plants [78, 79].

**6. GH27 and GH35**

**7. Concluding remarks**
