**1. Introduction**

The plant cell wall is a dynamic composite structure with diverse functions including mechanical and structural support for plant growth, compartmentalization of specialized cells, and the integration of cell-to-cell communication and interactions with the environment [1,2]. Given these essential roles, plants have evolved intricate mechanisms to assemble, modify, and dismantle the diverse wall components. β-glucans including cellulose, callose, xyloglucan, and mixed-linkage glucan (MLG) are prevalent wall structural constituents in most plant cell types. These polysaccharides share a backbone composed of D-glucopyranosyl building blocks linked by β-1,4 and/or β-1,3 bonds, which can contain additional sidechain substitutions. The abundance, length, and associations of these glucan structures are modified during development and in response to environmental cues [3,4]. The diversification of glucan structures found in plant walls has been accompanied by the coevolution of specific hydrolases allowing for their modification/degradation. Plant genomes encode diverse types of enzymes able to hydrolyse β-glucans. Endo-β-glucanases, the most abundant type in plants, randomly cleave internal β-D-glucosidic linkages in the glucan backbone, while exo-β-glucanases act processively on both ends of the glucan chain, releasing oligosaccharides. Finally, β-glucosidases hydrolyse terminal β-D-glucosyl residues releasing β-D-glucose. These enzymes can be further classified in many ways depending on the reaction mechanism used, the chemical reaction they catalyse, or amino acid sequence-related aspects. Based on common amino acid sequences and protein fold

**Citation:** Perrot, T.; Pauly, M.; Ramírez, V. Emerging Roles of β-Glucanases in Plant Development and Adaptative Responses. *Plants* **2022**, *11*, 1119. https://doi.org/ 10.3390/plants11091119

Academic Editors: Penélope García-Angulo and Asier Largo-Gosens

Received: 28 March 2022 Accepted: 18 April 2022 Published: 20 April 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

structures, β-glucanases are typically distributed in multiple glycoside hydrolase (GH) families according to the classification of Carbohydrate-Active enZymes (CAZy) [5]. Frequently, Enzyme Commission (EC) number(s) are also used to designate the biochemical reaction(s) catalysed by these proteins [6]. Depending on the glycoside bond they hydrolyse, three main types of β-glucanases can be found in plants: β-1,4-glucanases, β-1,3-glucanases and β-1,3-1,4-glucanases.

1,4-β-glucanases are enzymes able to hydrolyse the 1,4-glycosidic bond between two contiguous D-glucopyranose units (Figure 1). This bond is found in the structure of cellulose, the most abundant polysaccharide in plant walls. 1,4-β-glucanases, initially termed "cellulases", include several types of enzyme activities needed to degrade cellulose, such as endoglucanases (EC 3.2.1.4), cellobiohydrolases (EC 3.2.1.91), and glucosidases (EC 3.2.1.21) [7–9]. Plant cellulases are classified in the GH9 family [5,10]. Although most of them exhibit only limited activity on crystalline cellulose, they are able to hydrolyse amorphous regions of cellulose and soluble cellulose derivatives such as carboxymethyl cellulose. There is some evidence suggesting that these enzymes are also able to cleave other non-cellulosic polysaccharides containing contiguous (1,4)-β-glucosyl residues in their backbone, including MLG, xyloglucan, and glucomannan, although characterization of the hydrolytic activities on different substrates has been limited to a handful of plant GH9 proteins [11]. GH9 β-1,4-glucanases have been implicated in several aspects of cell wall metabolism in higher plants, including cellulose biosynthesis and degradation, modification of the association of cellulose microfibrils with other wall polysaccharides, or wall loosening during cell elongation [12–14].

The term β-1,4-glucanase includes additional enzyme activities such as those involved in the hydrolysis of xyloglucans, heavily xylose-substituted 1,4-β-glucans [15]. Xyloglucan endohydrolases (XEH; EC 3.2.1.151) cleave the xyloglucan chain, releasing oligosaccharides. However, some of these enzymes also exhibit transglucosylase activity (XET; EC 2.4.1.207) being able to covalently link these oligosaccharides onto the non-reducing terminal end of the glucose moiety of other xyloglucan and cellulose acceptors [16–19]. This group of enzymes, generally termed xyloglucan endotransglucosylase/hydrolases (XTH) has important roles in wall polymer rearrangement, polymer integration into the wall, and cell expansion, and has also been implicated in plant responses towards abiotic and biotic stresses. Several reviews have recently addressed this transglycosylase class of plant β-1,4-glucanases [20,21], and they will not be further considered in this review.

β-1,3-glucanases catalyse the hydrolysis of glucans containing contiguous β-1,3-linked glucosyl residues (Figure 1; [22]). β-1,3-glucanases are responsible for the degradation of callose in plants, but they can also hydrolyse the β-1,3- and β-1,3-1,6-glucans found in the walls of intruding fungi [23,24]. They are also called laminarinases, as they are able to cleave laminarin, a linear β-1,3-glucan displaying occasional β-1,6-branches found in brown algae [25]. Laminarin and the structurally similar β-1,3-glucans paramylon and pachyman are usually employed to characterize in vitro activities of those enzymes. Plant β-1,3-glucanases are classified together with β-1,3-1,4-glucanases in the GH17 family of glycosyl hydrolases [26].

β-1,3-1,4-glucanases only hydrolyse β-1,4-glucosidic linkages if an adjacent β-1,3 glucosyl linkage is present towards the non-reducing end of the substrate (Figure 1). These enzymes are highly specific, and they are not able to hydrolyse β-1,3- or β-1,4 glucans [8,27,28]. In plants, these enzymes are involved in the degradation of MLG, a hemicellulosic polysaccharide prevalent in the walls of grass species. β-1,3-1,4-glucanases are also known as lichenases or licheninases, named after their activity on lichenin (moss starch), a complex glucan occurring in Parmeliaceae lichens [29,30]. This β-1,3-1,4-glucan differs from those characterized in grasses in having a much higher proportion of cellotriosyl to cellotetraosyl units [31]. In addition, despite having similar substrate specificities, plant β-1,3-1,4-glucanases have quite distinct amino acid sequences and 3D structures compared with microbial licheninases; thus, the term β-1,3-1,4-glucanase will be used [8,32,33].

**Figure 1.** Activity of the three types of β-glucanases found in plants and their main physiological substrates. (1,3)- and (1,4)-β-glycosidic linkages are depicted as 3 and 4, respectively. Arrows indicate the glycosidic linkage hydrolysed in each case.

This review will encompass the evolutionary relationships, classification, and biological roles proposed for the following three types of plant endo-β-glucanases (referred as β-glucanases from here): β-1,4-glucanase ("cellulase" EC 3.2.1.4), β-1,3-glucanase (EC 3.2.1.39) and β-1,3-1,4-glucanase (EC 3.2.1.73). The specificities of these enzymes on their biological β-glucan substrates are shown in Figure 1.
