**6. GH27 and GH35**

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.

GH17 are encoded by large gene families in plants. In *O. sativa*, GH17 is the largest GH family [17]. In *A. thaliana* and *Populus trichocarpa*, it comprises 50 and 100 members, respectively [54]. The GH3 family includes β-1,3-glucanase (glucan endo-1,3-β-D-glucosidase, E.C 3.2.1.39), glucan 1,3-β-glucosidase (E.C 3.2.1.58), licheninase (EC 3.2.1.73), glucan 1,4-β-glucosidase (EC 3.2.1.74) activities (enzyme.expasy.org/). All the GH17 found in sugarcane and *B. distachyon* cell wall proteomes are predicted to have a glucan endo-1,3-β-D-glucosidase activity (GO:0042973).

β-1,3-glucanases have been shown to be important proteins involved in plant defense reactions against pathogens and are considered as pathogenesis-related proteins of the PR-2 family [55]. Their role is hydrolysis of the β-1,3-glucan bonds, an important structural component of fungal cell walls, resulting in their destabilization and in the release of elicitors that further stimulate defense responses [55]. This antifungal activity was shown both *in vitro* [56] and *in vivo* [57]. In sugarcane, GH17 SCQSRT2031D12 identified in basal internodes was considered similar to the *A. thaliana* At4g16260 β-1,3-endoglucanase that has been associated with increased resistance to pathogen attack [58]. Noteworthy, β-1,3-glucanases can accumulate in vacuoles of root cells or mature leaf cells in response to pathogen infection, whereas others are secreted to the extracellular space, but they can also be secreted in the absence of pathogen infection [55]. They are, thus, also important during plant development, being involved in cell division, pollen devel-

According to phylogenic analyses, the GH17 family is divided into three distinct clades (denoted α, β, and γ) [61, 62], with 10% of its members having cell wall-related functions [61]. GH17 of the α clade are more related to stem elongation, but also responsive to gibberellin, those of the β and γ clades are more related to stress response and defense against pathogens [62–65]. In addition to the GH17 domain *per se*, proteins of the GH17 family comprise other domains as shown by [61] studying the β-1,3-glucanase sequences of *A. thaliana.* They noted that all the sequences had a predicted N-terminal signal peptide linking them to the secretory pathway. Half of them had a C-terminal extension, being first classified as an X8 domain [66]. Previously, the X8 domain was identified as the cellulose binding module 43 (CBM43) responsible for the interaction with β-1,3-glucans [67]. The other GH17 had either a C-terminal glycosylphosphatidylinositol (GPI)-anchor [66, 68] or a vacuolar targeting peptide [55]. The absence or gain of these domains could be related to ancestral traits. All the γ clade members and more than half of the α clade members retained the CBM43 domain, whereas all the members of the

opment, seed germination, and maturation as well as in signaling [55, 59, 60].

**5. GH 17**

172 Advances in Biofuels and Bioenergy

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

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

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].
