Darwin Review Biochemistry and physiological roles of enzymes that cut and paste plant cell-wall polysaccharides

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1 Journal of Experimental Botany, Vol. 64, No. 12, pp , 2013 doi: /jxb/ert201 Darwin Review Biochemistry and physiological roles of enzymes that cut and paste plant cell-wall polysaccharides Lenka Franková and Stephen C. Fry* The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, School of Biological Sciences, The University of Edinburgh, The King s Buildings, Mayfield Road, Edinburgh E9 3J, UK * To whom correspondence should be addressed. s.fry@ed.ac.uk Received 15 April 2013; Revised 30 May 2013; Accepted 4 June 2013 Abstract The plant cell-wall matrix is equipped with more than 20 glycosylhydrolase activities, including both glycosidases and glycanases (exo- and endo-hydrolases, respectively), which between them are in principle capable of hydrolysing most of the major glycosidic bonds in wall polysaccharides. Some of these enzymes also participate in the cutting and pasting (transglycosylation) of sugar residues enzyme activities known as transglycosidases and transglycanases. Their action and biological functions differ from those of the UDP-dependent glycosyltransferases (polysaccharide synthases) that catalyse irreversible glycosyl transfer. Based on the nature of the substrates, two types of reaction can be distinguished: homo-transglycosylation (occurring between chemically similar polymers) and heterotransglycosylation (between chemically different polymers). This review focuses on plant cell-wall-localized glycosylhydrolases and the transglycosylase activities exhibited by some of these enzymes and considers the physiological need for wall polysaccharide modification in vivo. It describes the mechanism of transglycosylase action and the classification and phylogenetic variation of the enzymes. It discusses the modulation of their expression in plants at the transcriptional and translational levels, and methods for their detection. It also critically evaluates the evidence that the enzyme proteins under consideration exhibit their predicted activity in vitro and their predicted action in vivo. Finally, this review suggests that wall-localized glycosylhydrolases with transglycosidase and transglycanase abilities are widespread in plants and play important roles in the mechanism and control of plant cell expansion, differentiation, maturation, and wall repair. Key words: Cell expansion, cell wall, glycanases, glycosidases, hemicelluloses, hydrolysis, oligosaccharides, pectins, remodelling, transglycanases, transglycosidases, transglycosylation, xyloglucan. Introduction A primary wall layer is (or was, at the time of its formation) susceptible to plastic extension: i.e. able to accommodate the cell s irreversible expansion. Secondary wall layers, in contrast, which may be deposited internal to the primary wall after cell expansion has ceased and have quite distinct chemical compositions, do not subsequently increase in area. The primary wall thus serves the key role of defining the shape and size of the plant cell. Plant primary cell walls are not rigid or inert boxes but constitute a flexible and metabolically active extraprotoplasmic compartment; they control cell expansion by varying their extensibility. This ability to change biophysically is conferred by biochemical reactions and molecular rearrangements that occur within the walls (in muro). The constituent polysaccharides of plant cell walls are synthesized by the protoplast, mostly within Golgi bodies except that cellulose and callose are produced at the plasma membrane. After synthesis in the Golgi body, polysaccharides are carried in vesicles to the plasma membrane to be deposited by exocytosis on the inner face of the existing wall. In muro, the polysaccharides may undergo many interesting and physiologically relevant modifications, The Author [2013]. Published by Oxford University Press on behalf of the Society for Experimental Biology. For permissions, please journals.permissions@oup.com

2 3520 Franková and Fry including transglycosylation ( cutting and pasting molecules), cross-linking, and hydrolysis. Other proposed wallremodelling reactions include transacylation (of polymers possessing carboxy groups) and non-enzymic scission caused by hydroxyl radicals. By means of this repertoire of polysaccharide modifications, the cell has the subtlety to manipulate the nuts and bolts of development, especially the direction and rate of cell growth, by remodelling wall polysaccharides. Physiological roles of the primary cell wall that can be finetuned by the in-muro metabolic reactions discussed in this review include not only dictating cell shape and size but also causing programmed leaf abscission, pod dehiscence for seed dispersal, seed-coat bursting during germination, becoming perforated in the case of xylem and phloem, supplying food reserves for seedling growth, generating and subsequently inactivating oligosaccharin signals, governing the wall s porosity and ability to adsorb metal ions, and defending the cell against microbial ingress. Chemical composition of the primary cell wall To understand wall remodelling, we need a summary of primary cell-wall chemistry. In land plants, polysaccharides constitute the bulk of this wall s dry mass, grouped into three broad classes: cellulose, hemicelluloses, and pectins (Scheller and Ulvskov, 2010; Albersheim et al., 2011; Fry, 2011). In dicot primary walls, these three classes occur at very roughly 1:1:1 by weight. Cellulose forms partially crystalline microfibrils, the wall s skeleton, devoid of internal water; hemicelluloses hydrogen-bond strongly to the cellulose and may also become locally trapped within microfibrils; and pectins plus hemicelluloses together constitute a hydrated matrix occupying the space between microfibrils (Fig. 1). In a widely adopted (but speculative) model, long rope-like hemicellulose chains adhere to multiple microfibrils and tether them, restraining cell expansion (Fry, 1989; ayashi, 1989). Recent critical evaluation (Park and Cosgrove, 2012) suggests that, although correct in outline, this model is an oversimplification. Fig 1. Model of the polysaccharide framework in a plant cell wall, generalized for poalean and non-poalean walls. 1, Cellulose: cellulose microfibrils; 2 6, hemicelluloses: 2, xyloglucan; 3, mixed-linkage glucan; 4, xylan and related heteroxylans; 5, callose; 6, mannan and related heteromannans; 7 11, Pectins: 7, galactan; 8, arabinan; 9, homogalacturonan; 10, rhamnogalacturonan I; 11, rhamnogalacturonan II; 12, boron bridge; 13, egg-box with calcium bridges; 14 16, Non-polysaccharide components: 14, enzymes and structural proteins; 15, cellulose synthase complex; 16, transport vesicles.

3 Plant cell-wall enzymes 3521 Cellulose is a chain of (1 4)-linked β-glc residues lacking side chains. 1 An elementary microfibril (produced by a single rosette of cellulose synthases) consists of ~16 18 cellulose molecules lying in parallel (Guerriero et al., 2010). Cellulose cannot be extracted from the wall into aqueous solution except by aggressive complexing agents such as cadoxen (10% cadmium oxide in aqueous 30% 1,2-diaminoethane). emicelluloses are polymers with a backbone of β-glc, β-xyl, or β-man (or β-man and β-glc), mainly or entirely (1 4)-linked (except callose), and usually possessing short side chains; most are neutral. ighly significant hemicelluloses of the primary walls of all vascular plants are xyloglucans (backbone β-(1 4)-Glc; side chains mainly α-xyl, and often also β-gal-(1 2)-α-Xyl and α-fuc-(1 2)-β-Gal-(1 2)-α- Xyl, attached at position 6 of some of the Glc residues) and xylans sensu lato (backbone β-(1 4)-Xyl; side chains usually α-araf and/or α-glca attached at positions 2 and/or 3 of some of the Xyl residues (the polysaccharides may then be more precisely referred to as arabinoxylans, glucuronoarabinoxylans, etc., some being acidic). Xyloglucans predominate in noncommelinid species; xylans in commelinids. Sugar sequences within xyloglucan chains can be specified by a series of code letters, comparable to those used in reporting sequences of proteins or DNA; for details see Fry et al. (1993). For example, G = β-glc of the backbone, not further substituted; X = α-xyl-β-glc; L = β-gal-(1 2)-α-Xyl-β-Glc; F = α-fuc- (1 2)-β-Gal-(1 2)-α-Xyl-β-Glc. All 18 xyloglucan code-letters currently in use are listed by Franková and Fry (2012b). Frequently observed sequences in dicot xyloglucans include XXXG, XXLG, XXFG, and XLFG. A comparable system of abbreviations is in use for xylan sequences (Fauré et al., 2009). Relatively minor primary wall hemicelluloses in most land plants, but predominant in eusporangiate ferns, are mannans sensu lato (backbone β-(1 4)-Man, often with interspersed β-(1 4)-Glc; sidechains often α-gal on position 6 of some of the Man residues). An additional vascular land-plant hemicellulose, restricted to but abundant in commelinids (grasses, cereals, etc.) and Equisetum (horsetails) (see also the section Phylogenetic variation ), is mixed-linkage glucan (MLG) (backbone β-(1 4)-Glc interspersed with a minority of β-(1 3)-Glc residues; no side chains). Although chemically diverse, hemicelluloses generally share an ability to hydrogen-bond to cellulose and are extractable from the cell wall in alkali (optimally 6 M NaO at 37 C; Edelmann and Fry, 1992). Pectins (Fig. 1) are α-gala-rich polysaccharides built up of distinct domains, the simplest of which is homogalacturonan, a linear homopolymer of (1 4)-linked α-gala residues, partially methyl-esterified and often also partially O-acetylated. More complex pectic domains are the rhamnogalacturonans (RG-I and RG-II) and xylogalacturonan. It is widely thought that these pectic domains are glycosidically bonded, end-to-end, into a complete polysaccharide, conveniently described simply as pectin. Details of pectin structure are given by Albersheim et al. (2011) and Fry (2011). In brief, RG-I has a backbone of the repeating disaccharide,...(1 4)-GalA-(1 2)-Rha-..., with some of the Rha residues carrying neutral oligosaccharide side chains rich in Gal and/or Araf. RG-II is a small domain with a backbone of (1 4)-linked α-gala residues with highly complex oligosaccharides linked to the backbone via Apif residues; in the cell wall, RG-II domains are usually cross-linked to each other via borate diester groups attached to one of the Api residues. Primary wall polysaccharides may possess, in addition to sugar residues, non-carbohydrate substituents: for example, acetyl, feruloyl, and methyl esters. Other non-carbohydrate components of certain primary walls include (glyco)proteins, lignin, cutin, suberin, and silica. It is often incorrectly stated that primary walls do not lignify; however, lignification of xylem vessels begins in their primary walls. The ratio of the various primary wall polysaccharides differs between taxa and between tissues, and varies developmentally within a given cell. For example, during maturation, MLG increases in Equisetum (Fry et al., 2008b) but decreases in commelinids (Buckeridge et al., 2004). Pectins are major components of all fast-growing cells except in commelinids, which are particularly rich in xylans (Carpita and Gibeaut, 1993). Mannans are highly abundant in some fern allies and are also present in many algal species, some of which completely lack cellulose (Popper and Fry, 2004). Several other important shifts in wall polysaccharide chemistry accompanied major events in plant evolution. For example, xyloglucans are present in all land plants examined but have not been chemically demonstrated in any algae including the charophytes, 2 from which all land plants originated implying that the invention of xyloglucan accompanied (and may have enabled) the invasion by plants of the land. Wall-remodelling enzymes targeting the major wall polysaccharides thus need to be tailored to taxa, tissues, and developmental stages. The physiological need for wall polysaccharide modification in vivo Cell expansion: hydrolysis of wall components The primary wall confers the cell s ability to define its own shape and size. Cell expansion, an irreversible increase in cell volume often exceeding 1000-fold, is a dramatic and unique feature of plants, with no equivalent in animals or most micro-organisms. Controlled plant cell expansion demands the reversible loosening of the cellulose hemicellulose pectin primary 1 Sugar abbreviations: Api, d-apiose; Ara, l-arabinose; Fuc, l-fucose; Gal, d-galactose; GalA, d-galacturonic acid; Glc, d-glucose; GlcA, d-glucuronic acid; Man, d-mannose; Rha, l-rhamnose; Xyl, d-xylose. All sugars are in the six-membered pyranose ring form unless marked f for furanose. 2 The status of xyloglucan in charophytes is controversial. Immunological evidence supports the presence of a xyloglucan-like polymer in Chara, Coleochaete, Cosmarium, and Netrium (Van Sandt et al., 2007; Sørensen et al., 2011), but enzymic digestion of Chara and Coleochaete cell walls has consistently failed to yield xyloglucan s diagnostic disaccharide, isoprimeverose (e.g. Popper and Fry, 2003). Methylation analysis of Spirogyra cell walls demonstrated 4-linked and 4,6-linked Glc and terminal Xyl residues (Ikegaya et al., 2008), but these residues anomerism was not determined and the Glc residues could have been α-glc of starch (which is often difficult to completely remove by amylase digestion) rather than β-glc of xyloglucan, and the terminal Xyl residues could have been β-xyl from xylans rather than α-xyl of xyloglucan. Resolution of these discrepancies requires further research.

4 3522 Franková and Fry wall. Thus, plants will require a battery of wall-manipulating enzymes not found in other organisms (Labavitch, 1981; Fry, 1995, 2004; de la Torre et al., 2002; Minic, 2008). Besides the intrinsic interest of these wall enzymes for understanding plant growth, they are also exciting subjects for genetic manipulation and targets for novel herbicides. Enzymes that loosen the primary wall, for example by catalysing its partial hydrolysis, may be expected to step up the growth rate. Wall assembly: recruiting new polysaccharides Roles of polysaccharide-remodelling enzymes are not confined to cell expansion. For example, enzyme activities that cut and paste glycosidic bonds (transglycanases, e.g. xyloglucan endotransglucosylase, XET) can create new polysaccharide polysaccharide linkages and thus play a role in recruiting newly secreted polysaccharides into the wall fabric, contributing to wall assembly. In addition, hetero-transglycanases can graft part of one polysaccharide to a qualitatively different one, which may also contribute to wall assembly. Wall loosening and/or strengthening: rewiring glycosidic bonds in old polysaccharide chains The cutting/pasting of polysaccharide chains by transglycanases occurs not only at the moment of secretion but also between pairs of polysaccharides which have already been part of the wall architecture for some time. It is difficult to deduce whether such reactions contribute predominantly to wall loosening and thus growth promotion (Thompson and Fry, 2001) or to wall strengthening by stitching polymers together, (e.g. in xylem cell walls; Nishikubo et al., 2007). Other homo-transglycanase reactions include the cutting/pasting of xylans to xylans and of mannans to mannans; precise physiological roles for these processes are unclear, but may include reserve mobilization after germination of mannan-rich seeds and wall loosening during growth and fruit softening. In addition, a hetero-transglycanase activity, MLG:xyloglucan endotransglucosylase (MXE), can link a portion of MLG to a xyloglucan chain, thus creating a chimaeric polymer with MLG at one end and xyloglucan at the other. This may contribute to strengthening the Equisetum stem in mature plants, helping it to resist wind damage or herbivory. Abscission/dehiscence Another role for wall polysaccharide modification concerns enzymes that disrupt tissue cohesion by lysing the primary wall and/or middle lamella, thus permitting cell cell separation (Le Cam et al., 1994; González-Carranza et al., 2007; Zhang et al., 2007). This enables: leaf abscission in deciduous trees; dehiscence of pod-like fruits, e.g. follicles, legumes, siliquae, and capsules; softening in drupes and berries; and the spatially and temporally targeted rupture of the endosperm and/or seed coat at the moment of germination. Perforations On a highly localized scale, plant enzymes can bore holes in cell walls, for example during the formation of xylem perforation plates, phloem sieve plates, and plasmodesmata, enabling the intercellular transport of xylem sap, sucrose, and RNAs, respectively (Bollhöner et al., 2012; Tilsner and Oparka, 2012). Wall polysaccharide mobilization Enzymic lysis also occurs in the walls of certain seeds, whose enormous stockpiles of specific polysaccharides such as xyloglucans or galactomannans are hydrolysed, ultimately to monosaccharides, providing a carbon and energy source for the seedling until it attains photosynthetic self-sufficiency. Related to seed reserves, it would a priori appear energy-efficient for deciduous trees to degrade some leaf polysaccharides shortly before abscission and to salvage the resultant sugars by basipetally transporting them via the phloem (och, 2007); more work is required, however, to test this idea. Making oligosaccharins On a much smaller scale, but nevertheless qualitatively significant, some wall polysaccharides undergo enzymic turnover to release biologically active oligosaccharides ( oligosaccharins ) with putative signalling roles (McDougall and Fry, 1991; Darvill et al., 1992; Aldington and Fry, 1993; Beňová- Kákošová et al., 2006). For example, fragments of xyloglucan possessing an α-fuc residue can at concentrations of 1 nm antagonize the cell expansion that is induced by 1 μm auxin. Conversely, higher concentrations ( μm) of several xyloglucan oligosaccharides can promote cell expansion, and such concentrations are indeed present in the apoplast in vivo, at least in cell-suspension cultures (Fry, 1986). Glucomannan-related oligosaccharins have been reported to promote cell elongation in roots but inhibit it in hypocotyls (Richterová-Kučerová et al., 2012). Other oligosaccharins, produced at least in vitro from homogalacturonan, antagonize auxin-induced growth (Ferrari et al., 2008) and trigger wound-hormone release; however, such oligogalacturonides appear more likely to be generated by the action of microbial enzymes than by the plant s own repertoire. Removing oligosaccharins In addition to enzymes generating oligosaccharins from polysaccharides, plants also possess enzymes that degrade oligosaccharins, either by hydrolysis or by grafting large polysa ccharides to them (Baydoun and Fry, 1989; Darvill et al., 1992; García-Romera and Fry, 1995). This may be important since biological messages need to be inactivated when the information that they carry is no longer relevant to the plant s environmental or developmental situation. Wall porosity Partial degradation of certain cell-wall polysaccharides, especially pectins, can finetune the molecular pore size of the wall fabric (Carpita et al., 1979; Baron-Epel et al., 1988), thereby modulating what sizes of molecules, such as arabinogalactanproteins, can potentially move intercellularly as signals.

5 Plant cell-wall enzymes 3523 Cell defence Finally, the wall contains enzymes that may render it impenetrable by potential pathogens or indigestible by herbivores. Such enzymes would be mainly those involved in cross-linking, for example peroxidases and phenol oxidases (laccases). To the list of wall-bolstering wall enzymes could be added the newly discovered acyltransferase, cutin synthase (Yeats et al., 2012). Glycosidic bonds and enzyme activities that act on them In this work, the term enzyme follows the Enzyme Commission usage: it is defined as an activity, which, however, may be shared by multiple isozymes encoded by different genes. Glycosidase and glycanase activities An enzyme catalysing the hydrolysis of poly- and/or oligosaccharides will attack either at terminal sugar residues (almost always non-reducing termini) or at mid-chain residues, not both. Such exo- and endo-glycosyl hydrolase activities are termed glycosidases and glycanases, respectively. Note also the distinction between glyc-, referring to an unspecified sugar residue and gluc-, referring to a glucose residue. Examples of glycosidases are α-xylosidase, β-xylosidase, and β-glucosidase; examples of glycanases include (1 4)- β-xylanase and (1 4)-β-glucanase (cellulase). In the following sugar sequences, the reducing terminus is on the right. An enzyme that splits off the non-reducing terminal β-xyl residue from the model substrate (1 4)-β-xylohexaose (Xyl 6 ) is a β-xylosidase (Fig. 2A): Fig. 2. Diagrammatic representations of the activities of glycosidases (A, B), glycanases (C), transglycosidases (D, E), and transglycanases (F), Each circle represents a sugar residue;, bond cleaved.

6 3524 Franková and Fry Xyl-Xyl-Xyl-Xyl-Xyl-Xyl + 2 O Xyl + Xyl-Xyl-Xyl-Xyl-Xyl. An enzyme that cleaves a mid-chain linkage in the same substrate is a β-xylanase (Fig. 2C), for example: Xyl-Xyl-Xyl-Xyl-Xyl-Xyl + 2 O Xyl-Xyl-Xyl-Xyl + Xyl-Xyl. The terms glycosidase and exo do not imply that the attacked terminal residue is necessarily located at the end of the backbone; side chains are also non-reducing termini, for example certain β-gal residues of xyloglucan attacked by β-galactosidase (Fig. 2B). Glycanases catalyse the hydrolysis of glycosidic bonds within the backbone of the polysaccharide (Fig. 2C) or mid-chain bonds within a lengthy side chain. Glycosidases generally have high specificity for the glycosyl group attacked, but there is often a lower specificity towards the nature of the aglycone (for explanation, see Fig. 3A). For example, β-xylosidase may release monomeric xylose from the non-reducing terminus of xylan (Xyl n ), xylohexaose (Xyl 6 ; Fig. 3c), and even p-nitrophenyl β-xyloside (Fig. 3B). owever, there are important cases of tighter specificity: for example, plant α-xylosidases release xylose from the 1st but not the 2nd or 3rd (counting from the non-reducing end) Xyl residue of the xyloglucan heptasaccharide XXXG and do not attack p-nitrophenyl α-xyloside; Fanutti et al., 1991). Transglycosidase and transglycanase activities: readily reversible Transglycosylation is a cutting and pasting reaction in which a glycosidic bond is cleaved, but not by hydrolysis. Instead, the broken bond s energy is conserved in forming a new glycosidic linkage (Fig. 2D F). The substrates are a donor and an acceptor; the products are a hybrid and a leaving group (Fig. 2D). We will show the reactants and products in the order: donor + acceptor hybrid product + leaving group. It is helpful to distinguish readily reversible transglycosylation reactions, in which the bond cleaved has an energy (ΔG 0 ; i.e. free energy of hydrolysis) similar to that of the bond formed, from essentially irreversible ones (as will be discussed in the next section). Reversible transglycosylation is appropriate for wall remodelling, in which the reaction does not proceed in any defined direction. By analogy with hydrolase names, we use the terms transglycosidase and transglycanase for exo- and endo-enzymes respectively, referring to the glycosidic bond that is cleaved. Thus, the reactions in Fig. 2D F represent trans-β-xylosidase, trans-β-galactosidase, and trans-β-xylanase, respectively (an alternative nomenclature uses xylan endotransglycosylase instead of trans-β-xylanase; Johnston et al., 2013). When the donor is qualitatively similar to the acceptor, the reaction is homo-transglycosylation. Examples include the activities of trans-β-mannanase, trans-β-xylanase, and xyloglucan endotransglucosylase (XET, 3 trans-xyloglucanase ; Fig 4). When the donor and acceptor are qualitatively different (hetero-transglycosylation), the nomenclature is more complex since both the donor and the acceptor have to be specified. A convenient system for naming such activities takes the form donor:acceptor endotransglycosylase, e.g. MLG:xyloglucan endotransglucosylase (MXE; Fig 4). Irreversible transglycosylation Essentially irreversible transglycosylation reactions make the glycosidic bonds during de-novo polysaccharide synthesis. O a b c Aglycone NO 2 Aglycone (p-nitrophenol) (general) Glycosidic R oxygen O O O Glycosyl group (β-xylosyl) O O O O O Glycosyl group (β -Xylosyl) O O O O O Glycosyl group (β-xylosyl) Aglycone (oligosaccharide) O O O O O O n Glycosidic bond to be cleaved Anomeric centre O O O Fig. 3. Glycosyl groups versus aglycones, and the bonds cleaved by glycosidases and transglycosidases. In each case, the aglycone is released (with an atom in place of the red bond). Green arrow, glycosidic oxygen atom; red line, glycosidic bond to be cleaved by the glycosidase; black arrow, anomeric centre of the glycosyl group under consideration. Note that an aglycone can itself be a sugar. 3 XET was defined by Rose et al. (2002) as xyloglucan endotransglucosylase activity, which is a more precise and informative term that the previously used xyloglucan endotransglycosylase. Since then, endotransglucosylase has been criticized (Eklöf and Brumer, 2010) on the grounds that the enzyme transfers a whole length of the polysaccharide chain, not just a single Glc residue. owever, this is not a valid criticism because the distinction between gluco and glyco does not concern the number of residues transferred but their identity. Transglucosylase specifies that a Glc X bond is cleaved and reformed; a transglycosylase could act on a Xyl X or Gal X bond, and is thus a term that might be used when the nature of the cleaved/reformed bond is uncertain.

7 Fig. 4. Proposed biological roles of enzymes with transglycosidase and transglycanase activities that catalyse transglycosylation reactions between oligosaccharides and/or polysaccharides of the plant cell wall. Plant cell-wall enzymes 3525

8 3526 Franková and Fry ere, the bond broken is more energy-rich (has a larger negative ΔG 0 of hydrolysis) than the one made. Irreversibility is appropriate for biosynthesis of major metabolic end products such as wall polysaccharides. The high-energy donors are usually NDP-sugars (e.g. UDP-xylose), the acceptor is the nascent polysaccharide, and the enzymes are polysaccharide synthases or NDP-sugar : glycan glycosyltransferases. This type of enzyme is membrane-bound and not discussed in detail in this review. Carbohydrate-Active enzyme proteins and their genes Browsing for plant cutting-and-pasting enzymes We will now describe specific examples of potential wallremodelling enzymes, focusing on transglycosidases and transglycanases. Thanks mainly to recent progress in proteomics, genomics, and metabolomics, numerous enzyme databases have been created and are now available online. owever, this enormous resource is significantly reduced when one is searching for plant enzyme databases, especially when focusing on those that provide information on carbohydrate-active catalytic proteins. Some glycosidases and glycanases have the ability to catalyse transglycosylation in addition to hydrolysis; however, extracting the data on such enzymes that cut and paste plant cell-wall polysaccharides can be laborious, especially when an initial search for what the user considers the appropriate keyword fails. For example, transglycosidase, transglycosylase, or glycosyltransferase have not been incorporated into the headline entries under glycosidases but are mentioned only marginally, if at all. On the other hand, many databases provide entries which are not directly related to the term sought, so users have to go through numerous categories manually. Nevertheless, more than 19 databases that to some extent provide information on plant wall-modifying enzymes and/or their genes are currently available online (Table 1). One of the most browsed enzyme databases is that of the International Union of Biochemistry and Molecular Biology (IUBMB), covering enzyme nomenclature, published online in This is a list of recommended names for enzyme activities, classified into classes and sub-classes according to Enzyme Commission (EC) numbers. It is based on the enzymes specific substrate preferences and is therefore guided by the reactions they catalyse. As this concept does not take into account the structural topology and stereochemical mechanistic features of enzymes, it can be applied only to those proteins whose functions have been biochemically identified. The same principle (i.e. classification based on EC numbers) was employed in constructing the BRENDA and IntEnz/ENZYME databases (Table 1), which, in addition to nomenclature, provide detailed information about enzyme features (such as kinetics, stability, substrate specificity, products formed, cofactors, subunits, etc.) and gene sequences, respectively. Unlike many other databases, BRENDA is highly searchable and enables immediate access to helpful data on enzyme sources and localization, reaction and specificity, stability, and structure. owever, despite BRENDA s virtues, there are a few cases in which the relevant records are supported by inappropriate references or are misleading. For example, the endo-1,5-α-l-arabinanase entry lists Arabidopsis thaliana and Gossypium hirsutum under the Organism item, wrongly implying that α-arabinanase was detected in those two species. In fact, α-arabinanase activity was never detected either in Arabidopsis or Gossypium. Instead, a commercial (non-plant) α-arabinanase was used by the cited authors to digest Arabidopsis cytosolic heteroglycans (Fettke et al., 2006) and cotton wall polysaccharides (Zheng and Mort, 2008). To avoid any misinterpretation, users should cross-reference to verify the accuracy of data extracted from any online resources. The Carbohydrate-Active enzyme database As an alternative to the EC system, Carbohydrate-Active enzyme (CAZy) classifies carbohydrate-acting enzymes and carbohydrate-binding proteins into families based on their sequence and structural folding features (enrissat, 1991; enrissat and Bairoch, 1993). In contrast to the EC-IUBMB classification, this structure-based system does not assign EC numbers (i.e. the substrate specificity and the type of reaction being catalysed). Thus a single CAZy family can include enzymes which act on various substrates. It also allows inclusion of proteins of unknown function, avoiding any premature prediction of possible enzyme activity (Cantarel et al., 2009). The CAZy classification is based on structurally related catalytic and carbohydrate-binding domains of proteins and comprises five classes: (i) glycosyl hydrolases (Gs, including the enzymes discussed above as glycosidases and glycanases); (ii) glycosyltransferases (GTs, principally those mentioned as catalysing irreversible transglycosylation); (iii) polysaccharide lyases (PLs); (iv) carbohydrate esterases (CEs); and (v) carbohydrate-binding modules (CBMs). Sometimes the folding of proteins provides a better basis for classification than a simple one-dimensional sequence and allows hierarchical categorization (clustering) of different families whose members seem to be structurally related. This phenomenon can be observed in the class of glycanohydrolases, where some families are grouped into clans (clan G-A to G-N) defined by similarities in 3D structure (fold) and a highly conserved catalytic domain and catalytic mechanism, despite differences in complete amino acid sequences (enrissat and Bairoch, 1996). The CAZy database provides numerous options for searching through the individual categories such as family, organism, or protein name, EC number, and mechanism. Despite the availability of these options, an initial search for required data may fail after the relevant category has been selected. For example, searching for enzyme activity defined by the protein name β-xylosidase will reveal 501 hits, but displaying only 10 results per page. Clicking on the next 10 records yields the error message résultats de la recherche. A user would then intuitively use another option, such as entering the EC number (in this case EC ), which, however, provides the same hits and the same problem. As an alternative, a user can try the Glycoside ydrolase (G) family classification,

9 Plant cell-wall enzymes 3527 Table 1. Synopsis of databases providing information on plant cell wall polysaccharide-modifying enzymes and/or their genes Database name Description Web link Data available on: Notes PS PE GO EF IUBMB Enzyme Enzyme Commission (EC) + Includes brief description of the enzyme-catalysed reaction Nomenclature classification of enzymes by the reactions they catalyse ac.uk/iubmb/enzyme BRENDA Comprehensive enzyme information system Summarizes detailed characteristics of enzymes/functional gene products abstracted from the literature; enzymes classified according to the EC list. CAZy Carbohydrate Active enzyme database New sequence-based classification system introduced; enzymes categorized into the families; information on sequences through the GenBank and UniProtKB link IntEnz/ENZYME Integrated relational enzyme database/swissprot enzyme intenz; Describes type of enzyme based on the recommendations of the EC; gene sequences and ontology provided through the link to UniProtKB nomenclature database expasy.org ExPASy Sib bioinformatics resource portal basic Lists many scientific databases such as PROSIT, ENZYME, OMA etc., easily searchable; provides access to software tools PDB Protein Data Bank database Provides information on 3D structures and similarities, ligands, methods, etc. KEGG Kyoto Encyclopedia of Genes Consists of sub-databases categorized according to the information and Genomes database available on systems, genomics and chemistry EMBL-EBI European Bioinformatics Institute databases services Provides access to biological databases such as ENA, IntAct, InterPro UniProtKB Annotated protein sequence Easily searchable, most information linked to the European database Nucleotide Archive (ENA) OrthoDB ierarchical catalogue of eukaryotic orthologues orthodb Lists eukaryotic orthologous protein-coding genes; no record on plant proteins but many on fungal carbohydrate-active enzymes NCBI National Center for Biotechnology Information database gov Supports access to a variety of enzyme and nucleotide databases, genome-specific resources etc.; provides tools for sequence analysis and 3D structure display. PPDB Plant Proteome Database Stores experimental data from proteome and mass spectrometry for Arabidopsis thaliana and maize (Zea mays) edu basic analysis, curated information about protein function, protein properties and subcellular localization; predicted protein can be searched for experimental information. PMN Plant Metabolic Network database + Contains curated information from the literature and computational analyses about the genes, enzymes, compounds, reactions and pathways. XT World Database of XTs from Arabidopsis, tomato and rice cornell.edu/xt + Web page proposes and standardizes the XT nomenclature; a list of new gene names with links to the NCBI sequences also provided. CWN Cell-Wall Navigator database edu/cellwall + Contains gene families that are involved in sugar substrate generation and primary cell-wall metabolism; linked to sources of the complete genome sequences of Arabidopsis thaliana and Oryza sativa and to those of UniProt and NCBI. AmiGO Gene ontology database Includes all manual gene product annotations and electronic annotations from all databases other than UniProtKB; possibility to set up a filter. TAIR The Arabidopsis Information Resource database Includes the complete genome sequence of Arabidopsis in addition to gene product information, metabolism, genome maps, genetic and physical markers and seed stocks. PlantTribes Floral Genome Project database edu/tribedb/10_ genomes/index.pl + + A classification system for plant proteins based on cluster analyses of the inferred proteomes of 9 sequenced angiosperms; includes information about domains, traditional gene family names and unified common terms pdawg An integrated database for Contains 19 complete plant genomes including 12 from algae; linked plant cell-wall genes edu/pdawg/species. php to the Pfam database (includes annotations and additional family information); provides data on subcellular localization and phylogeny. GATAbase Glycosyl ydrolase And Transglycosylase Activity database homepages.ed.ac.uk/ sfry/gatabase.html + + A list of individual enzyme activities for which evidence was obtained in plant protein extracts; readily searchable; valuable resource for selecting plant organs from whichto extract and study enzymes of interest. Provides notes and comments about the reaction products. EF, Enzyme features; GO, Gene ontology/annotations; PE, plant enzymes; PS, protein sequences.

10 3528 Franková and Fry under which the EC numbers are associated with individual G families. In the case of EC , ten G families are attributed to this enzyme activity and users are left with their own manual search to find the necessary information, such as the organism source. Each G family contains entries categorized into archea, bacteria, eukaryota. The problem may occur when users wish to find plant-specific entries that are included simply under eukaryota. Based on structural similarities, each G family can include not only proteins with defined enzyme activity, but also peptide fragments and predicted or unknown proteins. These data are supported by references (available through NCBI or UniProtKB), which are sometimes unpublished data or direct submissions (to the online database). Even enzyme activities with defined name (and/or EC number) are sometimes only predicted, so there is no evidence at transcript or protein level for the corresponding protein. Such information can be misleading since a user would intuitively trust an entry bearing a given enzyme name or EC number, believing that enzyme s existence has been proven at the protein level (in vivo or in vitro). Nevertheless, CAZy remains the only complex database on enzymes which form, cleave, or reconstitute bonds in carbohydrates. From the whole range of CAZy groups, approximately 22 families seem to be associated with enzymes that may postsynthetically modify the plant cell wall (Table 2; note that families or enzyme activities that do not include any plant member are omitted). Plant glycosidases (Fig. 2A, B) are mostly grouped in G families 1, 2, 3, 27, 29, 31, 35, 36, 38, 51, and 95, while plant glycanases (Fig. 2C) fall into G families 2, 5, 9, 10, 16, 17, 28, and 81. owever, the boundaries between glycosidase and glycanases G families is not always strictly determined; for example, family G2 includes both exo- and endo-acting members. Dedicated reversible transglycanases are restricted to family G16. In contrast, dedicated transglycosidases do not seem to exist in any G family, although families G1 and 31 contain bifunctional enzymes with both glycosidase and transglycosidase activity (Table 2). Transglycosylases or transglycosylating glycosyl hydrolases? Most enzyme databases use the term glycosyltransferase for enzymes that catalyse the transfer of sugar residues, usually one at a time, from an activated donor substrate to a specific acceptor substrate, forming a new glycosidic bond. Such enzymes can also be described as aglycone-glycoside synthases, oligosaccharide synthases, and polysaccharide synthases. The donor substrate is usually a nucleoside diphospho- or monophospho-sugar or a sugar 1-phosphate (Lairson et al., 2008; Palcic, 2011), and since the bond broken in the donor is more energetic than the newly formed one, the transglycosylation reaction is usually essentially irreversible. The reaction may affect the acceptor s mass, solubility, transport, and bioactivity (Ross et al., 2001). A second group of enzymes catalysing (reversible) transglycosylation, which is not prominently distinguished in enzyme databases and calls for an individual search (and a lot of patience), comprises glycosyl hydrolases that also possess appreciable transglycosylation activity. Such transglycosidase and transglycanase activities are known mostly from fungi and bacteria. If one searches for plant transglycanases and transglycosidases (not NDP-sugar-dependent), one would find fructan:fructan 1-fructosyltransferase (a transβ-fructanase) and sucrose:fructan 6-fructosyltransferase (a trans-β-fructosidase), disproportionating enzyme (D-enzyme or 4-α-glucanotransferase) and amylo-(1,4 1,6)-transglucosylase (branching or Q-enzyme, a trans-α-glucanase which converts amylose to amylopectin) (ExPASy, BRENDA). A more intensive search for cell-wall-modifying transglycanases and transglycosidases reveals entries on XET (xyloglucan endotransglucosylase) and trans-β-mannanase (mannan endotransglycosylase), mostly on the CAZy and BRENDA servers. Both trans-β-mannanase and XET are classified among glycosyl hydrolases (families G5 and G16, respectively; because their mechanism of action and structural affiliations are different from those classed as glycosyltransferases (GT families). Other than XET and transβ-mannanase, no records on plant wall-related enzymes that catalyse transglycosylation are yet available in online database directories, so data on transglycosylation activities (discussed further in the sections Inverting matters and Predicted activities vs. biological roles of G families ) are currently available only in original articles. Nevertheless, further new homo-transglycosidase and homo- and hetero-transglycanase activities are being discovered in plants (rmova et al., 1998, 2006, 2007; Fry et al., 2008a; Kosík et al., 2010; Franková and Fry, 2011) although the sequences of the corresponding proteins are not always known. Mechanisms of enzymic hydrolysis and transglycosylation The enzyme-catalysed hydrolysis of glycosidic bonds can take place via either of two reaction mechanisms: single- or double-displacement. The single-displacement mechanism proceeds in one step through an oxocarbenium ion-like transition state with the assistance of two carboxylic acids at the active site (usually glutamic and/or aspartic acid; McCarter and Withers, 1994). One carboxylic acid (acting as a catalytic base) is required for nucleophilic attack on water, while the second (acting as a catalytic acid) brings about the cleavage of the glycosidic bond (Koshland, 1953; Sinnott, 1990; Withers, 2001). The result of such a mechanism is the inversion of anomeric configuration (e.g. bond cleaved = α-lfucosyl R; initial products = β-l-fucose + R O), and this defines inverting glycosidases. Regardless of the initial product formed, the sugar released (in aqueous solution) soon ends up as an equilibrium mixture of, for example, α-l-fucose and β-l-fucose, as a result of mutarotation. The double-displacement mechanism is achieved in two steps: (i) the formation of an intermediate containing a glycosyl enzyme ester bond; and (ii) its hydrolysis (Sinnott, 1990; Davies and enrissat, 1995). Both steps proceed via an oxocarbenium ion-like transition state and also require

11 Plant cell-wall enzymes 3529 Table 2. Distribution of plant cell-wall-remodelling enzymes in CAZy families Data on plant enzymes were laboriously extracted from the very long list of plant, fungal, and animal CAZymes, all of which were included in the one group Eukaryota. Unnamed/predicted protein products with unknown function and fragments are not included; families or enzyme activities that do not include any plant member are omitted. Family Catalytic domain Enzyme activity Mechanism Transglycosylation activity Reference on transglycosylation EC number Plant sources of the protein with known function/activity G1 (β/α) 8 β-glucosidase Retaining + Crombie et al. (1998); Opassiri et al. (2003) Arabidopsis thaliana, Avena sativa, Brassica napus, Carapichea ipecuanha, Carica papaya, Cicer arietinum, Consolida orientalis, Corbicula japonica, ordeum vulgare, Lotus japonicus, Malus domestica, Manihot esculenta, Medicago truncatula, Olea europea, Oryza sativa, Pinus contorta, Solanum lycopersicum, Trifolium repens, Vitis vinifera, Zea mays β-mannosidase Retaining ND ordeum vulgare, Oncidium Gower Ramsey, Oryza sativa, Solanum lycopersicum G2 (β/α) 8 β-mannosidase Retaining ND Brassica oleracea Mannosylglycoprotein Retaining ND Arabidopsis thaliana, Lilium longiflorum endo-β-mannosidase β-galactosidase Retaining ND Arabidopsis thaliana G3 α-arabinofuranosidase /β-1,4-xylosidase Retaining ND / Actinidia deliciosa, Arabidopsis thaliana, Solanum lycopersicum, Fragaria ananassa, ordeum vulgare, Malus domestica, Medicago sativa ssp. varia, Medicago truncatula, Pyrus pyrifolia, Raphanus sativus β-1,4-xylosidase Retaining ND Arabidopsis thaliana, Camellia sinensis, ordeum vulgare, Medicago truncatula, Populus tremula alba, Solanum lycopersicum, Zea mays β-glucosidase Retaining ND Gossypium hirsutum, Nicotiana tabacum, Tropaeolum majus β-glucosidase Retaining ND ordeum vulgare (preferred substrates are polysaccharides, thus exo-β-glucanase ) G5 (β/α) 8 β-mannanase, trans-β-mannanase Retaining + rmova et al. (2006); Schröder et al. (2006) / ordeum vulgare, Coffea arabica, Daucus carota, Glycine max, Lactuca sativa, Solanum lycopersicum G9 (α /α) 6 β-1,4-glucanase (cellulase) Inverting ND Arabidopsis thaliana, Brassica napus, Capsella rubella, Capsicum annuum, Citrus sinensis, Colocasia esculenta, Cucumis melo, Dimocarpus longan, Fragaria ananassa, Glycine max, Gossypium barbadense, Gossypium herbaceum, Gossypium hirsutum, ordeum vulgare, Malus domestica Mangifera inica, Medicago truncatula, Nicotiana tabacum, Oryza officinalis, Oryza sativa, Persea americana, Phaseolus vulgaris, Picea glauca, Picea sitchensis, Pinus radiata, Pinus taeda, Pisum sativum, Populus alba, Populus alba grandidentata, Populus tremuloides, Prunus persica, Pyrus communis, Saccharum hybrid cultivar R570, Triticum aestivum, Vitis vinifera, Sambucus nigra, Solanum lycopersicum, Sorghum bicolor G10 (β/α) 8 β-1,4-xylanase, trans-β-xylanase Retaining +. Johnston et al. (2013) Arabidopsis thaliana, Carica papaya, ordeum vulgare, Nicotiana tabacum, Oryza sativa, Zea mays

12 3530 Franková and Fry Table 2. (Continued) Family Catalytic domain Enzyme activity Mechanism Transglycosylation activity Reference on transglycosylation EC number Plant sources of the protein with known function/activity G16 β-jelly roll Xyloglucan endotransglucosylase Xyloglucan endohydrolase Retaining + Xu et al. (1995); Campbell and Braam, 1999) Retaining + De Silva et al. (1993); Fanutti et al. (1993); Tabuchi et al. (2001); Baumann et al. (2007); Zhu et al. (2012) Actinidia deliciosa, Annona cherimola, Arabidopsis thaliana, Asparagus officinalis, Beta vulgaris, Betula pendula, Brassica oleracea var. botrytis, Brassica rapa, Capsicum annuum, Carica papaya, Cenchrus americanus, Chrysanthemum morifolium, Dahlia pinnata, Daucus carota, Fagus sylvatica, Festuca pratensis, Gerbera hybrid cultivar, ordeum vulgare, Litchi chinensis, Medicago truncatula, Musa acuminate, Nicotiana tabacum, Oryza sativa, Pisum sativum, Populus euphratica, Pyrus communis, Pyrus pyrifolia, Rosa chinensi, Shorea parvifolia, Solanum lycopersicum, Striga asiatica, Triticum aestivum, Vitis labrusca vinifera Arabidopsis thaliana, Tropaeolum majus, Vigna angularis G17 (β/α) 8 β-1,3-glucanase Retaining ND Arabidopsis thaliana, Atropa belladonna, Avena sativa, Beta vulgaris ssp. vulgaris, Brassica rapa, Cicer arietinum, Cichorium intybus endivia, Citrus clementina reticulata, Citrus jambhiri, Citrus sinensis, Glycine max, Gossypium hirsutum, evea brasiliensis, ordeum vulgare, Medicago sativa, Musa acuminata, Musa paradisiaca, Nicotiana tabacum, Olea europaea, Oryza sativa, Phaseolus vulgaris, Pisum sativum, Salix gilgiana, Solanum lycopersicum, Solanum tuberosum, Triticum aestivum, Vitis vinifera, Zea mays Lichenase (MLG-specific Retaining ND Avena sativa, ordeum vulgare, Nicitiana plumbaginifolia, Oryza sativa, Triticum aestivum β-1,4-glucanase) G 27 (β/α) 8 α-galactosidase Retaining ND Coffea arabica, Coffea canephora, Cucumis sativus, Glycine max, elianthus annuus, Oryza sativa, Pisum sativum G28 β-helix α-galacturonidase ( exopolygalacturonase ) Inverting ND Arabidopsis thaliana, Brassica rapa ssp. campestris, Oryza brachyantha, Oryza coarctata, Oryza minuta, Zea mays Galacturonanase ( endopolygalacturonase, pectinase) Inverting ND Arabidopsis thaliana, Brassica napus, Brassica rapa ssp. campestris, Carica papaya, Cucumis melo, Daucus carota, Eucalyptus globulus, Fragaria chiloensis, Fragaria ananassa, Glycine max, Gossypium barbadense, Gossypium hirsutum, ypericum perforatum, Lilium longiflorum, Medicago sativa, Musa acuminata, Nicotiana tabacum, Oncidium Gower Ramsey, Oryza brachyantha, Platanus acerifolia, Prunus armeniaca, Prunus domestica ssp. insititia, Prunus persica, Pyrus communis, Salix gilgiana, Solanum lycopersicum, Vitis vinifera G29 α-1,3-fucosidase, Retaining ND Arabidopsis thaliana α-1,4-fucosidase G31 (β/α) 8 α-glucosidase, Retaining + Sampedro et al Arabidopsis thaliana α-xylosidase (2010) / α-xylosidase Retaining ND Oryza sativa, Tropaeolum majus

13 Plant cell-wall enzymes 3531 Table 2. (Continued) Family Catalytic domain Enzyme activity Mechanism Transglycosylation activity Reference on transglycosylation EC number Plant sources of the protein with known function/activity G35 (β/α) 8 β-galactosidase Retaining (inferred) ND Arabidopsis thaliana, Asparagus officinalis, Brassica oleracea, Capsicum annuum, Carica papaya, Cicer arietinum, Citrus sinensis, Coffea arabica, Fragaria ananassa, Glycine max, Gossypium hirsutum, ordeum vulgare, Mangifera indica, Nicotiana tabacum, Oryza sativa, Persea americana, Petunia hybrida, Prunus persica, Pyrus communis, Pyrus pyrifolia, Solanum lycopersicum, Triticum monococcum, Vigna radiata, Vitis vinifera, Ziziphus jujuba G36 (β/α) 8 α-galactosidase Retaining ND Arabidopsis thaliana, Cucumis melo, Cucumis sativus, Oryza sativa, Pisum sativum, Zea mays G38 (β/α) 7 α-mannosidase Retaining ND Arabidopsis thaliana, Capsicum annuum, Medicago truncatula, Oryza sativa G51 (β/α) 8 α-arabinofuranosidase Retaining ND Arabidopsis thaliana, Carica papaya, Fragaria ananassa,gunnera manicata, ordeum vulgare, Malus domestica, Medicago truncatula, Prunus persica, Pyrus communis, Pyrus pyrifolia, Solanum lycopersicum α-arabinofuranosidase, Retaining ND Arabidopsis thaliana β-xylosidase / G81 ND β-1,3-glucanase Inverting ND Arabidopsis thaliana, Glycine max G95 (α/α) 6 α-1,2-fucosidase Inverting ND Arabidopsis thaliana, Lilium longiflorum, Oryza sativa CE6 (α/β/α)- Xylan acetylesterase Deacetylation Arabidopsis thaliana, ordeum vulgare Sandwich CE8 β-elix Pectin methylesterase Demethyl esterification Allium cepa, Arabidopsis halleri ssp. halleri, Arabidopsis thaliana, Brassica napus, Brassica oleracea, Brassica rapa ssp. pekinensis, Capsicum annuum, Citrus sinensis, Fragaria ananassa, Linum usitatissimum, Lycoris aurea, Medicago truncatula, Nicotiana benthamiana, Nicotiana plumbaginifolia, Nicotiana tabacum, Olea europea, Oncidium Gower Ramsey, Oryza rufipogon, Oryza sativa, Petunia integrifolia subsp. inflata, Phaseolus vulgaris, Physcomitrella patens, Picea abies, Pisum sativum, Populus tremula tremuloides, Prunus persica, Pyrus communis, Salix gilgiana, Sesbania rostrata, Silene latifolia ssp. alba, Solanum lycopersicum, Solanum tuberosum, Vitis riparia, Vitis vinifera CE13 (α/β/α)- Sandwich Pectin acetylesterase Deacetylation Lactuca sativa, Litchi chinensis, Medicago truncatula, Oryza sativa, Populus trichocarpa, Vitis vinifera, Vigna radiata var. radiata, Sorghum bicolor PL1 Parallel β-helix Pectate lyase β-elimination Arabidopsis thaliana, Carica papaya, Dianthus caryophyllus, Fragaria chiloensis, Gossypium barbadense, Gossypium herbaceum, Gossypium hirsutum, Gossypium raimondii, evea brasiliensis, Lilium longiflorum, Malus domestica, Mangifera indica, Medicago sativa, Musa acuminate, Nicotiana tabacum, Populus tremula Populus tremuloides, Prunus persica, Rosa borboniana, Salix gilgiana, Solanum lycopersicum, Zinnia violacea ND, not determined.

14 3532 Franková and Fry two acidic amino acid residues one acting as a nucleophile and the second as an acid/base catalyst. In the first (glycosylation) step, a nucleophilic residue attacks the anomeric centre (defined in Fig. 3) allowing displacement of the aglycone and formation of the glycosyl enzyme complex. At the same time, the carboxylic group (functioning as an acid catalyst) protonates the glycosidic oxygen (defined in Fig. 3), cleaving the original glycosidic bond in the substrate. The glycosyl enzyme ester bond is then hydrolysed by water in the second (deglycosylation) step. The same carboxylic group (now acting as a base) deprotonates the water molecule, forming a new O group. Enzymes operating via this double-displacement mechanism are called retaining as overall they maintain the initial conformation at the anomeric carbon (e.g. bond cleaved β-d-glucosyl R; products β-d-glucose + R O). Retaining hydrolases of interest in connection with plant cell walls include those in CAZy families G1 3, 5, 10, 16, 17, 27, 29, 31, 35, 36, 38, and 51 (Table 2). Inverting G families of interest include G9, 28, 81, and 95 ( Inverting matters Unlike inverting hydrolases, which catalyse only hydrolysis, some retaining glycosylhydrolases can also participate appreciably in transglycosylation reactions (Koshland, 1953; Sinnott, 1990; Scigelova et al., 1999; Moracci et al., 2001; Tramice et al., 2007). This is due to their ability to form a glycosyl enzyme complex which can then be attacked by an acceptor substrate other than water, generating a new glycosidic bond instead of releasing a reducing sugar as hydrolysis product. This knowledge has been applied to the design and development of biotechnologically improved enzymes. New mutants of retaining glycosylhydrolases (including those from plants; rmova et al., 2002; ommalai et al., 2007; Piens et al., 2007) were created by selective intervention in their hydrolytic domain such as the replacement of a catalytic nucleophile (e.g. Glu 231 or Glu 235 of the barley β-1,3-glucanase and Cellulomonas β-1,4-xylanase respectively) by an inert, nonnucleophilic residue (e.g. Ser, Gly, Ala, or Cys; Withers, 2001; rmova et al., 2002; Kim et al., 2006). These modified glycosylhydrolases possessed hitherto unknown synthetic abilities, generating novel glycoconjugates, and have been termed glycosynthases (Mackenzie et al., 1998; Moracci et al., 2001). The mutant glycosynthase itself cannot form a glycosyl enzyme intermediate complex because it lacks a catalytic nucleophile. owever, the formation of a glycosyl enzyme intermediate can be mimicked by the use of glycosyl fluorides as donors, which possess an anomeric configuration opposite to that of the natural substrate and a fluorine atom as a good leaving group (comparable to the UDP in UDPglucose; Withers, 2001; Kim et al., 2006; Kang et al., 2007). Oligosaccharide fluorides were successfully employed in the synthesis of long oligosaccharides by mutant versions of plant hydrolases such as rice β-glucosidase, Populus XET, and barley β-1,3-d-glucanases (rmova et al., 2002; ommalai et al., 2007; Piens et al., 2007). Owing to the low cost of glycoconjugate synthesis, the application of glycosylhydrolases modified by genetic engineering has been put into practice and often prevails over the traditional chemical synthesis and glycosyltransferase approach (the use of natural enzymes and expensive sugar-nucleotides; Withers, 2001; Piens et al., 2007). owever, screens for natural plant hydrolases capable of significant transglycosidase or transglycanase activity at low substrate concentrations are also of interest as they are accessible from a wide diversity of plant taxa and are relatively cheap. Thus, glycosynthases together with cheap and widespread native retaining hydrolases represent a powerful synthetic tool for preparing new compounds with possible application in the carbohydrate and pharmaceutical industries. Transglycosylation catalysed by retaining hydrolases may in many cases be observed only at high acceptor substrate concentrations, capable of competing with water (the acceptor substrate in hydrolysis). Such transglycosylation is often described as mechanistic, and may be denigrated if unphysiologically high substrate concentrations are required, or even overlooked because hydrolysis (which is irreversible) will, in the end, inevitably exceed reversible transglycosylation during prolonged enzyme assays by tapping off constituents of the interconverting glycoside pool: Of the 156 CAZy families, only 22 contain plant enzymes that appear likely to post-synthetically modify plant cellwall polysaccharides (Table 2), namely families G1, 2, 3, 5, 9, 10, 16, 17, 27, 28, 29, 31, 35, 36, 38, 51, 81, and 95, CE6, 8, and 13, and PL1. As mentioned above in the section Transglycosylases or transglycosylating glycosyl hydrolases?, the CAZy database reports the transglycosylating ability of only two plant G families: G16 and G5, represented by XET and trans-β-mannanase activities, respectively. The sequences and 3D structures of these two types of wall-acting enzyme place them in G (not GT) families, but their activities are associated solely or primarily with transglycosylation (Table 2). The ability to catalyse transglycosylation is also recorded for members of some fungal and bacterial G families but for no other plant G families. owever, some additional retaining plant G CAZymes listed in Table 2 have been found experimentally to catalyse not only hydrolysis but also transglycosylation reactions in the presence of moderate acceptor substrate concentrations, e.g. 1 5 mm (Crombie et al., 1998; Opassiri et al., 2003; Schröder et al., 2006; Sampedro et al., 2010; Johnston et al., 2013), although this is not recorded in the CAZy database. Such concentrations are on the verge of being low enough to be considered physiological, and the enzymes involved

15 Plant cell-wall enzymes 3533 might also perform transglycosylation within the wall matrix. Examples include soyabean β-glucosidase, fenugreek endoβ-mannanases, and clover α-galactosidase, which catalyse in-vitro transglycosylation reactions at ~ mm acceptor substrate concentrations (Williams et al., 1977; Coulombel et al., 1981; Nari et al., 1983a,b). Moreover, recently reported trans-β-xylosidase, trans-β-xylanase, trans-β-galactosidase, and trans-α-xylosidase activities were detected with 0.5, 0.5, 1, and mm oligosaccharide substrates respectively (Franková and Fry, 2011, 2012a). These concentrations can be regarded as low and close to (or even lower than) those occurring in muro, since cellulose and hemicelluloses constitute about 20 30% and up to 20%, respectively, of the primary cell-wall dry weight (Varner and Lin, 1989). The discovery of novel transglycosylase activities in plants suggests (Popper and Fry, 2008) that hemicelluloses and pectins in the wall matrix may not be linked only by non-covalent bonds, as was assumed in earlier cell-wall models (Northcote, 1972; Monro et al., 1976). The plant cell wall itself is not equipped with either a pool of activated substrates (e.g. NDP-sugars) or the enzymic machinery (glycosyltransferases) to synthesize polysaccharides de novo (Schröder et al., 2009). Therefore, the manufacture of new glycosidic bonds within the cell wall (e.g. during wall integration of new polymers, restructuring of existing material, and bonding of polysaccharides to each other) can only be accomplished by means of transglycosylation reactions. It might be only a matter of time before other transglycosylation activities are discovered in plants and accepted as being non- mechanistic in the context of their possible biological roles in vivo. Plant-centred, but no plant-specific, CAZy families No pioneer CAZy family found only in plants All the G and CE families that include plant cell-wallremodelling CAZymes also have representatives in archaea, bacteria, viruses, protists, and animals. In other words, there is no protein family containing only plant CAZymes. owever, regarding the metabolism of xyloglucan (which is unique to plants), two enzyme activities (XET and XE) seem to be highly conserved in plants. Both activities fall into family G16, which also includes keratin-sulphate endo- 1,4-β-galactosidase (EC ), endo-1,3-β-glucanase (EC ), endo-(1,3-1,4)-β-glucanase ( cellulase ; EC ), lichenase (EC ), β-agarase (EC ), and κ-carrageenase (EC ). Despite having the activities listed above, the XT branch of family G16 can be regarded as a plant-specific CAZy family, since XET- and XE-active G16 members are found only in plants. Nevertheless, a few members of other families (mainly G5, 7, 12, 44 and 74) show XE activity (Gilbert et al., 2008; Vlasenko et al., 2010; Ariza et al., 2011). These 1,4-β-glucan-degrading enzymes (called cellulases, albeit sometimes highly xyloglucan-specific) are bacterial or fungal and their possible ability to catalyse transfer with xyloglucan was not reported. Other CAZy families which are best represented among plant wallrelated enzymes are the carbohydrate esterase families CE8 and CE13. The sequences falling into these two families were predicted pectin methylesterases and pectin acetylesterases. The importance of pectin methylesterase (PME) in planta is implied by the fact that Arabidopsis contains at least 79 putative PME genes (Markovič and Janeček, 2004). For example, the CAZy database lists about 65 records for Arabidopsis PMEs and 2 for fragments thereof ( Even though all these sequences are included in the PME family, only a few of them have been screened for a functional PME product and possible biophysicochemical properties (for details see Richard et al., 1994, 1996; Francis et al., 2006). Likewise, out of 66 entries for Arabidopsis available on the UniProtKB server, 14 represent putative and 44 probable PMEs, indicating that the actual enzyme activity was tested in very few cases. Predicted activities vs. biological roles of G families Both the spatial and temporal regulation of gene expression and variation in enzyme activities are routinely monitored by: (i) quantification of mrnas (by in-situ hybridization and gene-specific microarrays); (ii) measurement of protein steady-state levels; (iii) tissue printing (hybridization, immunohistochemistry); and (iv) the study of mutants with overexpressed or knocked-down genes. Sometimes mrna levels do not faithfully predict actual enzyme activities (because there may be post-transcriptional regulation), which makes the interpretation of biological roles more difficult. Likewise, overexpressing or silencing of selected genes may not produce any morphological phenotype in transgenic plants, especially when the gained/lost function can be compensated for by non-affected isoforms or other regulatory mechanisms. In such cases, a reliable wet biochemical approach (e.g. sensitive enzyme assays, histochemistry, polysaccharide content and composition analysis, reducing sugar assays) becomes an indispensable complement to functional genomics. The activity of an archetypal plant transglycanase XET is associated with various key biological functions (cellwall loosening and expansion, fruit softening, germination, reserve mobilization, secondary wall deposition, wall assembly and strengthening) and is attributed to multiple isoforms. For example, the Glycine max, Arabidopsis lyrata, A. thaliana, and Zea mays genomes are equipped with 64, 39, 33, and 32 XT genes, respectively, that were predicted to encode functional gene products (Michel et al., 2001; Nishitani, 2005; Eklöf and Brumer, 2010). Based on the available genome data, the XT enzymes and their genes have been grouped into a phylogenetic tree which is divided into three clades (I, II, and IIIb) expected to exhibit only XET activity (Eklöf and Brumer, 2010), and a fourth (IIIa) assumed to work predominantly as XE. Their ability to fulfil this role will discussed in the section Xyloglucan endotransglucosylase/hydrolase. Apart from the action of dedicated transglycosylases (XTs with XET activity), many other part-time transglycosylation activities have been reported in plants. So far, only six plant glycosyl hydrolases with known protein sequences have been demonstrated to catalyse glycosyl transfer: rice Bglu β-glucosidase (Opassiri et al., 2003) and nasturtium

16 3534 Franková and Fry β-glucosidase (Crombie et al., 1998), both showing trans-βglucosidase activity; Arabidopsis AtXyl1 α-xylosidase (with trans-α-xylosidase activity; Sampedro et al., 2010), barley vman1 β-mannanase and tomato LeMan4 mannanase (with trans-β-mannanase activity; rmova et al., 2006; Schröder et al., 2006); and papaya CpaEXY1 β-xylanase (with trans-β-xylanase activity; Johnston et al., 2013). Their function, regulation, and activity were examined not only at the transcriptional level but also at the protein level (enzyme assays and reaction product analysis). Based on structural features, they were all predicted to function as glycosyl hydrolases (Table 2). owever, the importance of cutting and pasting glycosidic bonds in vitro and its relevance in vivo was highlighted. Fig. 4 depicts proposed biological functions of plant transglycosylases, including those whose protein sequence is not known (section Inverting matters ). Gene expression does not guarantee enzyme action in vivo Newly discovered enzyme activities call for subsequent protein purification and genetic studies which might reveal the identity of proteins responsible for the reactions catalysed. Equally, the discovery of expressed genes calls for enzymological and biological studies of the reactions which their translation products may catalyse. The finding that a given gene is transcribed such that its mrna can be detected, via cdna analysis, in a particular cell at a particular time, and that the gene in question fits in a particular CAZy class, does not prove that that gene s product is capable of catalysing the CAZy-predicted reaction, still less that it actually does so in vivo. These issues require testing experimentally. There is an important distinction between enzyme activity (e.g. assayed in vitro, under optimized conditions, with substrates arbitrarily chosen by the experimenter) and enzyme action (as occurring in muro, with natural substrates). A protein might fail to exhibit its CAZy-predicted activity in vitro for any of several reasons, such as: (i) badly chosen conditions (p, ionic strength, cofactor availability), not accurately mimicking those occurring in vivo; (ii) enzyme denaturation during extraction; (iii) heterologously produced protein (e.g. in Escherichia coli or Pichia) lacking correct post-translational modifications, e.g. N-glycosylation; and (iv) the true substrate of the enzyme may not be as predicted by CAZy. This last uncertainty can readily be explored by assays on pure (e.g. heterologously expressed) protein. In many cases, the enzyme is indeed active, although there are exceptions. For example the protein encoded by a putative plant α-fucosidase gene showed no α-fucosidase activity in vitro (Tarragó et al., 2003). As a second example, plant bifunctional α-arabinofuranosidase/β-xylosidase (e.g. MsXyl1 of Medicago, ARA-I/XYL of barley, and AtBXL1 of Arabidopsis; Lee et al., 2003; Xiong et al., 2007; Arsovski et al., 2009; see section The mutation/rnai approach ) is placed in CAZy family G3, which mainly contains β-glucosidases, β-glucanases and β-xylosidases. In this case, the unexpected α-arabinofuranosidase activity could have been easily overlooked as plant α-arabinosidases belong to G51. A third example, Arabidopsis AtFuc1 α-fucosidase, was primarily thought to be acting on α-1,2 fucosyl linkages other than those of xyloglucan (de la Torre et al., 2002). Later, it was reported that AtFuc1 hydrolyses both 3- and 4-linked fucoses but not 2-linked α-fucose nor the α-fucose that is 1,3-linked to the innermost GlcNAc residue of glycoproteins (Zeleny et al., 2006). Thus the enzyme was moved from family G95 to G29. The true α-1,2-fucosidase (G95) associated with xyloglucan metabolism is encoded by AtFXG1 (also AXY8 or AtFuc95A) and exhibits activity against XXFG and 2 -fucosyllactose (de la Torre et al., 2002; Günl et al., 2011). Like AtFuc1, it is inactive on pnp-α-fuc. More worryingly, even if a protein does possess a given enzymic activity on soluble substrates in vitro, it might fail to exert any corresponding action in vivo owing to multiple reasons such as: (i) the endogenous substrate may be inaccessible to endogenous enzyme, for example because the putative substrate (e.g. mannan) is shrouded by some other polysaccharide (e.g. pectin) ( masking ; Marcus et al., 2010); (ii) the enzyme may be localized in different cells, or in different parts of the cell, from the putative polysaccharide substrate; (iii) the apoplast of the cells in question may have an inappropriate p for action of the enzyme; (iv) activators, for example cofactors, may be absent in vivo; and (v) inhibitors may be present in vivo. For any of these reasons, in-vitro assays of enzyme action may not correctly predict in-vivo action, and one may be led to false conclusions about the biological role of a gene. A successful combination of classical wet biochemistry, applied both in vitro and in vivo, plus a functional genomic approach may bring new and reliable insights into the roles of polysaccharide-modifying enzymes in plants, as will be discussed further in the section Experimentally investigating wall enzyme action in vivo. Assaying polysaccharide-restructuring enzyme activities Numerous enzymes are extractable from plant cell walls in aqueous buffers (sometimes assisted by high salt), including glycanases, glycosidases, esterases, proteinases, transglycanases, transglycosidases, transacylases, peroxidases, oxidases, and lyases. Often, their substrate specificities suggest physiological significance in modifying wall components (Minic, 2008). ere are discussed methods for assaying glycanases, glycosidases, transglycanases, and transglycosidases. Glycanases and glycosidases Glycanase and glycosidase activities are assayed on substrates in which a glycosidic bond is hydrolysed. Methods for glycanase-catalysed endo-hydrolysis of polysaccharides include (in approximate order of decreasing sensitivity): (i) Loss of a polysaccharide solution s viscosity, measured in a simple viscometer (Farkaš and Maclachlan, 1988).

17 Plant cell-wall enzymes 3535 Polysaccharides are also cleaved non-enzymically by ascorbate-generated hydroxyl radicals ( O) (Fry, 1998); therefore, in these highly sensitive assays, enzyme extracts should be freed of ascorbate etc., for example by dialysis. (ii) Increase in the number of reducing termini (oxo groups, assayed colorimetrically). It should be checked that the new reducing termini formed are not monosaccharides released by glycosidases, nor mid-chain oxo groups introduced by O (Fry et al., 2001; Vreeburg and Fry, 2005). (iii) Release of radioactive oligosaccharides from a reducingend-labelled polysaccharide, e.g. [galactitol- 3 ]galactan (Fry, 1983) or [glucitol- 3 ]xyloglucan (Zhu et al., 2012). Since most glycosidases attack at the non-reducing end, this method is unlikely to be compromised by contaminating glycosidases. (iv) Increase in products insoluble in a precipitant (e.g. ethanol). For convenience, the substrate can be prelabelled with fluorescent or coloured tags (as in azo-xylan). (v) Release of water-soluble dyed products from artificially cross-linked polysaccharides (e.g. azurine-crosslinked polysaccharides). Glycanases and glycosidases can also be assayed on fully defined oligosaccharides (Fig. 2A C) that model biologically relevant polysaccharides. Products generated are analysed by TLC or PLC: monosaccharides are diagnostic of glycosidase activities; oligosaccharides smaller than the starting material but unaccompanied by monosaccharides indicate glycanases. Alternative assays for certain glycosidases employ fluorogenic or chromogenic model substrates (e.g. p-nitrophenyl or for example, a Tropaeolum XT has K m values for [ 3 ]XLLG, XLLG SR, XLLG FITC, and XLLG AA of 60, 81, 130, and 530 μm respectively, indicating a low affinity for XLLG AA; in contrast, turnover numbers of the enzyme for the same substrates are respectively 20, 400, 1300, and 79 molecules of substrate per molecule of enzyme per hour (Kosík et al., 2011). Thus absolute reaction rates measured on fluorescent substrates may be misleading, albeit useful for screening purposes. The labelled high-m r hybrid product formed in such transglycanase assays is separated from remaining unreacted acceptor by a size-dependent method, for example paperbinding (if the acceptor, e.g. [ 3 ]XXXGol, is easily washed off paper whereas the donor, e.g. xyloglucan or MLG, has an affinity for cellulose and remains bound), paper chromatography, gel-permeation chromatography, and ethanol precipitation. With paper chromatography, all polysaccharides will remain immobile in solvent mixtures such as ethyl acetate/acetic acid/water, while many oligosaccharides tested as acceptors migrate satisfactorily away from the origin; however, cello-, xylo-, manno- and MLG-oligosaccharides (MLGOs) remain partially or completely at the origin owing to their own affinity for cellulose, so paper chromatography is not recommended for these. Transglycosidases transfer only a single sugar residue, so a labelled acceptor substrate would not increase greatly in size. A convenient alternative is dual-labelling: a donor substrate radiolabelled in an appropriate sugar residue ( ) reacts with an acceptor substrate that is physically separable, for example by virtue of a cationic label ( ). The product of interest carries both labels: 4-methylumbelliferyl β-galactoside; NP- and 4-MU-β-Gal, respectively). owever, such substrates are not always suitable: plant extracts that efficiently hydrolyse XGOs do not recognize NP-α-Xyl or NP-α-Fuc (Fanutti et al., 1991; Léonard et al., 2008). Transglycanases and transglycosidases In transglycosylation reactions, the reactants and products may be indistinguishable, for example (where,, and are chemically identical sugar residues as, for example, depicted for homo-transglycanases in Fig. 4), so some kind of labelling is often used. For transglycanases, the acceptor substrate can often (perhaps always) be an oligosaccharide, even if the donor must be a polysaccharide. A radiochemically or fluorescently tagged oligosaccharide is incubated with a nonlabelled polysaccharide and the diagnostic reaction product is recognized by its label and large size. Sensitive labels include tritium ( 3 ), sulphorhodamine (SR), anthranilic acid (AA), and fluorescein isothiocyanate (FITC). Some transglycanases discriminate between differently labelled acceptor substrates: As with hydrolases, transglycosylases can be assayed on a nonlabelled oligosaccharide (as both donor and acceptor), e.g. by TLC analysis. The products proving transglycosylation are those larger than the substrate; smaller products are less informative because they could be either hydrolysis products or leaving groups formed by transglycosylation. Fluorescently and radiolabelled products can be also assayed by capillary electrophoresis and high-voltage paper electrophoresis respectively. Other techniques such as PLC, nuclear magnetic resonance spectroscopy, or matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass-spectrometry are an alternative to a quick semi-quantitative TLC, but not applicable when radiolabelled substrates are used. Specific examples of polysacchariderestructuring activities ydrolases Plant cell walls contain more than 12 glycosidase activities (e.g. β-glucosidase, β-galactosidase, β-xylosidase, α-xylosidase) and more than nine glycanases (e.g. β-mannanase, β-xylanase) (reviewed by Labavitch, 1981;

18 3536 Franková and Fry Fischer and Bennet, 1991; rmova and Fincher, 2001; Fry, 2004; Libertini et al., 2004; Baumann et al., 2007; Gilbert et al., 2008; Minic, 2008; Schröder et al., 2009). We recently surveyed 57 species for such activities using simple oligosaccharides that model cell-wall polysaccharides (Franková and Fry, 2011). The extensive results (exemplified in Table 3) are available in GATAbase (Table 1). Tables 4 and 5 give fuller lists of plant glycanases and glycosidases, respectively, potentially attacking wall polysaccharides (Labavitch, 1981; Fry, 1995, 2004; de la Torre et al., 2002; Minic, 2008). Known activities are, together, theoretically capable of hydrolysing most of the major glycosidic bonds in wall polysaccharides (except RG-II). This, however, is not to imply that all, or any, of them are present in vivo at sufficiently high activity to completely lyse the wall. Nevertheless, the numerous hydrolase activities certainly contribute to the diverse examples of wall restructuring occurring during normal plant growth and development, as already discussed. Transglycanases Trans-β-xylanase The GATAbase study, using fully defined oligosaccharide substrates, revealed not only hydrolases but also transglycanase and transglycosidase activities, several of which were new (Franková and Fry, 2011). For example, trans-β-xylanase activity converts Xyl 6 to Xyl 9 plus Xyl 3 : (Johnston et al., 2013). ere the donor was high-m r xylan, the acceptor was [ 3 ]Xyl 5 -ol, and the large radioactive hybrid product was recognized by its immobility on paper chromatography (albeit slightly contaminated by unreacted [ 3 ]Xyl 5 -ol). The authors attributed the trans-β-xylanase activity to a protein previously characterized as β-xylanase; thus the same protein can catalyse both endo-hydrolysis and endo-transglycosylation, the ratio between these depending on the acceptor-substrate concentration. Xyloglucan endotransglucosylase/hydrolase (XT) Some transglycanases are undetectable on simple, well-defined oligosaccharides because the donor needs to be a polysaccharide. The first in-vitro demonstrations of XET activity were in plant enzyme extracts incubated with xyloglucan (donor) plus a labelled XGO (acceptor), for example [ 3 ]XXFG (Fry et al., 1992) or XGO PA (pyridylamino; Nishitani and Tominaga, 1992). The reaction generated a large polysaccharide XGO conjugate. The enzymes had high affinity for XXFG (K m 50 μm) and higher affinity for XXXG (K m 33 μm) and XLLG (K m 19 μm). Polysaccharides other than xyloglucan had little, if any, donor ability (Fry et al., 1992). Nishitani and Tominaga (1992) showed that the enzyme required the donor to be a polysaccharide of M r > for appreciable activity. Certain XTs can, however, utilize oligosaccharides as both donor and acceptor: for example: XXX XXX + XXXXXX XXXXXXXXX + XXX ( indicates the cleaved bond) ; whereas trans-β-xylosidase activity transfers one residue at a time: X XXXXX + XXXXXX XXXXXXX + XXXXX In a crude extract, these two activities are distinguishable by their largest products: trans-β-xylanase immediately starts making Xyl 9, whereas trans-β-xylosidase initially produces Xyl 7 and would not begin to make Xyl 9, if any, until after a lag period during which Xyl 7 and Xyl 8 were sequentially generated. An alternative assay, modelled on one widely used for XET, also revealed trans-β-xylanase activity in plant extracts where is a fluorescent label (Saura-Valls et al., 2006), albeit with low affinity (K m 0.4 mm for donor and 1.9 mm for acceptor). Besides assays on extracted plant proteins, a genomic approach can be adopted. Plants have numerous cell-wall genes (Mao et al., 2009): Arabidopsis has 730 open reading frames encoding putative glycosyltransferases and -hydrolases (enrissat et al., 2001), 33 of which are XTs. Work is in progress to test experimentally the predicted activities of encoded XT-like proteins produced heterologously, e.g. in the yeast Pichia, and the following Arabidopsis XTs do exhibit XET activity in vitro: XT22 (formerly TC4; Purugganan et al., 1997), 14, and 26 (Maris et al., 2009); and Table 3. Some hydrolase and transglycosylase activities detected in a survey of land plants, assayed on well-defined model substrates Substrate cleaved Bond broken Glycosidase (exo) Glycanase (endo) Transglycosidase (exo)transglycanase (endo) Mannohexaose β-man β-mannosidase (+) β-mannanase (±) Arabinohexaose α-ara α-arabinosidase (+)? Trans-α-arabinosidase (±) Trans-α-arabinanase (±) Xylohexaose β-xyl β-xylosidase (+) β-xylanase (+) Trans-β-xylosidase (+) Trans-β-xylanase (+) XXXG α-xyl α-xylosidase (+) NA Trans-α-xylosidase (+) NA XXFG α-fuc α-fucosidase (+) NA NA XLXG and 1st Gal of XLXG β-gal β-galactosidase (+) NA Trans-β-galactosidase (+) NA XXLG β-gal β-galactosidase (±) NA Trans-β-galactosidase (+) NA +, activity detected in most or all land plants; ±, activity detected in few land plants;, activity not detected in land plants;?, data uncertain; NA, not applicable. Data from Franková and Fry (2011).

19 Plant cell-wall enzymes 3537 Table 4. Range of cell-wall-related glycanase activities reported in plant extracts Cleaved bond (bold, underlined) Polysaccharide whose backbone could potentially be hydrolysed Name for the enzyme activity Reference...(1 4)-α-GalA-(1 4)-α-GalA-(1 4)... Pectic homogalacturonan...(1 4)-β-Gal-(1 4)-β-Gal-(1 4)... Galactan/arabinogalactan domains of RG-I Pectinase, galacturonanase, Taylor et al. (1993); Ghiani et al. (2011) endo-polygalacturonase Galactanase Lazan et al. (2004)...(1 4)-β-Glc-(1 4)-β-Glc-(1 4)... Cellulose, MLG; sometimes xyloglucan Cellulase, β-1,4-glucanase Truelsen and Wyndaele (1991); Ohmiya et al. (1995)...(1 4)-β-Glc-(1 4)-β-Glc-(1 3)......(1 3)-β-Glc-(1 4)-β-Glc-(1 4)... MLG Lichenase (MLG-specific rmova and Fincher (2001) β-1,4-glucanase)...(1 4)-β-Glc-(1 4)-β-Glc-(1 4)... Xyloglucan Xyloglucan endo-hydrolase (XE), Tabuchi et al. (2001); Zhu et al. (2012) with α-xyl on O-6 of 2nd Glc xyloglucan endo-glucanase (XEG)...(1 3)-β-Glc-(1 3)-β-Glc-(1 3)... (with β-glc on O-6 in laminarin) Callose, laminarin Laminarinase, β-1,3-glucanase rmova and Fincher (1993); Martin and Somers (2004)...(1 4)-β-Man-(1 4)-β-Man-(1 4)... Mannan Mannanase Dahal et al. (1997); Schröder et al. (2006)...(1 4)-β-Xyl-(1 4)-β-Xyl-(1 4)... Xylan Xylanase Ronen et al. (1991); Johnston et al. (2013)...(1 4)-β-GlcNAc-(1 4)-β- GlcNAc-(1 4)... Chitin (of pathogenic organism) Chitinase (defence-induced) Cota et al. (2007); Mizuno et al. (2008)...(1 4)-β-GlcN-(1 4)-β-GlcN-(1 4)... Chitosan Chitosanase (constitutively present) Ouakfaoui and Asselin (1992); ung et al. (2002) XT12, 13, 17, 18, and 19 (Maris et al., 2011). In addition, XET activity has been confirmed for: ZmXT1 of maize (Genovesi et al., 2008); PttXET16A of poplar (Johansson et al., 2004); vxet5 of barley (rmova et al., 2007); and BRU1 of soyabean (Oh et al., 1998). All putative XT translation-products tested to date have exhibited XET activity with tamarind xyloglucan as donor. Relatively subtle differences in substrate preference exist, e.g. between fucosylated and non-fucosylated xyloglucans (Purugganan et al., 1997) and between contrasting acceptor XGOs (Maris et al., 2009). More pronounced variation in p preference was found: AtXT12 and AtXT17 have p optima of 5.0 and 7.5 respectively (Maris et al., 2011), possibly indicating physiological differences in role. owever, the main differences between XTs appear to be in the genes promoters, such that XET activity can be induced in different tissues and in response to different environmental stimuli (Nishitani, 2005; Becnel et al., 2006). Among the group IIIa XTs, examples from Tropaeolum (Fanutti et al., 1996; Baumann et al., 2007), Vigna (Tabuchi et al., 2001) and Arabidopsis (AtXT31; Zhu et al., 2012; Kaewthai et al., 2013) are the only gene products for which endo-hydrolytic activity has so far been demonstrated in addition to low or very low XET activity. Two Tropaeolum XTs, TmNXG1 and TmNXG2, produced in the seed after germination, catalyse both hydrolysis (XE activity) and transglycosylation (XET activity, especially at higher substrate concentrations) of xyloglucan (Fanutti et al., 1996). ydrolysis may fulfil the need to mobilize seed-reserve xyloglucans for the nutrition of the young seedling. Structural features indicate that these two enzymes acquired XE by loss-of-function mutations of an XET-active ancestor (Baumann et al., 2007). By comparison with these features, two of the 33 Arabidopsis XT genes (XT31 and XT32) were predicted to encode XE-active enzymes. Recently, this prediction has been verified for XT31 (AtXT31; Zhu et al., 2012; Kaewthai et al., 2013). When produced in Pichia cells, XT31 exhibited very slight XET activity, but >5000-fold greater XE activity (Zhu et al., 2012). Trans-β-mannanase In tomato fruit, trans-β-mannanase activity was detected with high-m r mannan as donor and [ 3 ]manno-oligosaccharides as acceptor (Schröder et al., 2006). The activity was attributed to a previously characterized β-mannanase protein which even at low acceptor substrate concentrations (0.18 mm) also effected transglycosylation. Since this enzyme s β-mannanase activity can be assayed on Man6, it would be expected that its trans-β-mannanase activity would also be detectable with Man6 as donor (and acceptor) substrate. owever, the GATAbase survey revealed no trans-β-mannanase activity on 1.6 mm Man6 in any of the 57 extracts studied. Although the list of 57 did not include tomato, this observation may indicate that trans-β-mannanase activity is not widespread in the plant kingdom even though β-mannosidase is (Franková and Fry, 2011) and β-mannanase was found to be evolutionarily ancient and involved in diverse biological processes (Yuan et al., 2007). MLG:xyloglucan endotransglucosylase (MXE) Several studies have explored the possibility that non-xyloglucan polysaccharides might serve as donors in conjunction with XGOs as acceptors hetero -transglycanase activities. rmova et al. (2007) tested the substrate specificity of a purified

20 3538 Franková and Fry Table 5. Range of cell-wall-related glycosidase activities reported in plant extracts Non-reducing terminal residue cleaved Polysaccharide possessing such termini (backbone) Polysaccharide possessing such termini (side chains) Names for the enzyme activity Reference α-araf-( Pectic arabinan/arabinogalactan Pectic arabinan and arabinogalactan, arabinoxylans, α-arabinosidase Lee et al. (2003); Rosli et al. (2009) and glucuronoarabinoxylans α-araf-( α-fuc-( F of XXFG at non-reducing terminus of xyloglucan α-1,2-fucosidase Léonard et al. (2008); Franková and Fry (2011); Günl et al. (2011) α-fuc-( Wall glycoproteins and glycolipids? α-1,3 1,4-Fucosidase de la Torre et al. (2002); Zeleny et al. (2006) α-fuc-( α-gal-( Galactomannans/ α-galactosidase Appukuttan and Basu (1987) galactoglucomannans β-gal-( Galactan/arabinogalactan domains of RG-I Pectic β-galactan and arabinogalactan domains of β-galactosidase De-Veau et al. (1993); Kaneko and Kobayashi (2003) RG-I, some xylans β-gal-( β-gal-( in XLXG and 1st L of XLLG Xyloglucan β-galactosidase (widespread activity) de Alcântara et al. (1999); Sampedro et al. (2012) β-gal-( in XXLG - Xyloglucan β-galactosidase (rare activity) Buckeridge et al. (1997); Franková and Fry (2011) α-gala-( Pectic homogalacturonan of DP 5 α-galacturonidase, exo-polygalacturonase Pressey and Reger (1989); García-Romera and Fry (1994); Tanaka et al. (2002) β-glc-( Cellulose, MLG, xyloglucan, callose, laminarin Laminarin β-glucosidase, exo-β-glucanase Crombie et al. (1998); rmova et al. (1996); Opassiri et al. (2003) β-glc-( β-glc-( β-man( Mannans β-mannosidase rmova et al. (2006); Franková and Fry (2011) α-xyl-( Single Xyl at non-reducing termi-α-xylosidasnus Fanutti et al. (1991); Günl and of xyloglucan Pauly (2011) β-xyl-( Xylans Xylans (xylogalacturonans?) β-xylosidase Martínez et al. (2004); Minic et al. (2004); Franková and Fry (2011) β-xyl-(1 3?.. barley XT (vxet5). With XGO SR as acceptor, the preferred donor was xyloglucan, but certain other water-soluble, substituted (1 4)-β-d-glucans were also effective donors: reaction rates relative to that with xyloglucan as donor (rate 100 ) were hydroxyethylcellulose, 44; sulphocellulose, 5; and carboxymethylcellulose, 0.4. Probably the hydroxyethyl ether and sulphate ester groups (both mainly linked to O-6 of β-glc residues) sufficiently resembled the 6-O-linked α-xyl residues of xyloglucan to fit in the enzyme s donor site. Water-soluble β-glucans with different backbone linkages were much less effective: e.g. barley MLG, relative rate 0.2; Cetraria MLG ( lichenan ), 0; (1 3)-β-glucan, 0; and glucomannan, 0. Furthermore, this purified XT was 1000-times more effective with XGO SR as acceptor substrate than with cello-oligosaccharide SRs; it thus has a very high but not absolute specificity for xyloglucan and certain artificial (1 4)-β-glucans with hydrophilic side chains, whereas its MXE reaction rate (with MLG as donor substrate) is 0 0.2% of its XET rate. rmova et al. (2007) suggested that other, untested XTs might exhibit higher MXE activity. A good test of this suggestion is to assay crude plant extracts, which will contain multiple isoenzymes. In fact, Ait Mohand and Farkaš (2006) had conducted such assays in Tropaeolum extracts. In agreement with the vxet5 data, they showed that the XET donor preferences were xyloglucan > EC > CMC. They also detected heterotransglycanase activities (donor, xyloglucan; acceptor cello- or laminari-oligosaccharide SRs, these acceptors lacking side chains) and suggested that the results indicate the plant s ability to covalently link polysaccharides to qualitatively different ones, forming, for example, xyloglucan cellulose bonds. Following up the work on vxet5, Fry et al. (2008a) surveyed extracts from diverse land-plants and algae for MXE activity. Curiously, one evolutionarily isolated genus, Equisetum, gave extracts with very high MXE activity, often exceeding its XET activity. Equisetum extracts acting on MLG as donor showed several significant differences from when acting on xyloglucan as donor, indicating that the MXE-active protein is different from the major XET-active XTs. For example, (i) when XGOs were used as acceptor substrates, different Equisetum extracts showed a consistent pattern of preferences among side chain-substituted (1 4)-β-glucans (xyloglucan > water-soluble cellulose acetate > EC > CMC), whereas the rate with MLG as

21 Plant cell-wall enzymes 3539 donor varied independently; (ii) MXE and XET activities peaked in old and young Equisetum stems respectively; (iii) MXE had a higher affinity for XXXGol (K m ~4 μm) than any known XT; and (iv) MXE and XET activities differed in their oligosaccharide acceptor-substrate preferences, for example XET activity 3-fold preferred XLLGol over XXXGol, whereas MXE activity slightly preferred XXXGol (Fry et al., 2008a). Other possible homo- and hetero-transglycanases The GATAbase survey also indicated the presence in certain plant extracts (broad bean, pea, and cauliflower) of trans-α-arabinanase activity, converting (1 5)-α-arabinooctaose (Ara 8 ) to both smaller and larger products such as Ara (Franková and Fry, 2011). The reaction may be of the type AAAA AAAA + AAAAAAAA AAAAAAAAAAAA + AAAA where A = arabinose bond; ʹ = bond broken. Other surveys of plant extracts returned negative data, e.g. where the donor was xyloglucan and the prospective acceptors were galacto- or arabino-oligosaccharides that represent RG-I side chains (Popper and Fry, 2008). Thus, no evidence could be found for a hetero-transglycanase that might generate the xyloglucan rhamnogalacturonan bonds detected in muro (Thompson and Fry, 2000; Popper and Fry, 2005). Negative data were also obtained with homogalacturonans as donor and/or acceptor (García-Romera and Fry, 1994); this can now be explained by the fact that all plant galacturonases are inverting hydrolases (CAZy family G28) and would thus not readily evolve transglycanase capability. An interesting high-throughput strategy for discovering transglycanases has been developed (Kosík et al., 2010). Fourteen different polysaccharides (potential donors) were printed onto a nitrocellulose surface, forming a glycochip, which was then bathed in an Arabidopsis or Tropaeolum enzyme extract containing 5 μm of an oligosaccharide SR (potential acceptor). After incubation, the chip was washed in ethanol, which removed unreacted oligosaccharide, after which any fluorescence on the chip was considered to represent a transglycanase product. With the Tropaeolum extract, numerous donor:acceptor pairs led to apparent activity, including some novel pairs, such as galactomannan:xgo, glucuronoxylan:xgo, galactomannan:mlgo, and xyloglucan:mlgo. Other pairs were also listed, for example xyloglucan:cello-oligosaccharides, xyloglucan:laminari-oligosaccharides, and glucuronoxylan:galactomannan-oligosaccharides, but data in these cases are not shown. Conversely, some substrate pairs do seem to reveal slight activity, especially at p 7, for example arabinoxylan:xgo, pectinate:xgo, arabinogalactan:xgo, and arabinan:xgo, but these examples were not mentioned. Curiously, in the arabinoxylan:xgo case, activity was only observed with arabinoxylan from wheat, not oat (supplementary figure S2 of Kosík et al., 2010), raising the possibility that a contaminating polysaccharide in the wheat preparation was responsible. Compared with Tropaeolum, Arabidopsis extracts gave far fewer hits ; for example, galactomannan:xgo activity was undetectable. Boiled-enzyme controls verified the lack of physical binding of the fluorescent oligosaccharides to the printed polysaccharides. owever, the study lacked a control testing whether polysaccharides co-extracted with the enzymes (e.g. xyloglucan in the case of Tropaeolum seedlings) might hydrogen-bond to some of the immobilized polysaccharides on the chip and give false-positives fluorescent spots that resemble novel hetero-transglycanase products but are actually attributable to XET activity. Stratilová et al. (2010) further showed that hetero-transglycanase activities of a purified pi-6.3 Tropaeolum XT represent side reactions due to relaxed substrate specificity, the favoured activity being XET. Evidence for this conclusion was that low concentrations of added unlabelled XGOs were sufficient to competitively interfere in hetero-reactions. For example, the xyloglucan:mlgo SR (MXE) reaction was 93% inhibited by 1.9 mm unlabelled XGOs but only 35% inhibited by a higher concentration (7.6 mm) of unlabelled MLGOs. Trans-α-xylosidase and other transglycosidase activities In principle, any retaining glycosidase can exhibit some transglycosidase activity at high substrate concentrations. For example, M substrates revealed trans-α-galactosidase activity in a Prunus α-galactosidase (Dey, 1979), and 100 mm nitrophenyl β-glucoside revealed trans-β-glucosidase activity in a barley β-glucosidase (rmova et al., 1998). These are unphysiologically high concentrations. Transglycosidase activities detectable at lower concentrations start to become biologically significant. For example, a purified Tropaeolum seed enzyme exhibited trans-β-glucosidase activity in a TLC-based assay on a defined oligosaccharide, cellotetraose [(1 4)-β-Glc 4 ]. This activity transiently competed with concurrent β-glucosidase (exo-hydrolytic) activity when acting on 5 mm cellotetraose, although hydrolysis strongly predominated at 2 mm, so it is uncertain whether the transglycosidase activity would operate appreciably at physiological substrate concentrations (Crombie et al., 1998). In rice, both bglu1 and bglu2 (encoding β-glucosidases) are highly expressed in young seedlings and mature nodes, bglu1 also in flowers (Opassiri et al., 2003). Purified bglu1 hydrolysed both pnp-β-glc and certain small oligosaccharides, optimally (1 3)-β-Glc 2 and (1 4)-β-Glc 4, and showed moderate trans-β-glucosidase activity when these substrates were present at 5 mm. This supports the theory that bglu1 can potentially generate new longer oligosaccharides from shorter ones in vivo and this may play a role in recycling the sugars released from the cell wall after germination or during flower expansion (Opassiri et al., 2003). owever, it is unclear what biological benefit might accrue from resizing small cello-oligosaccharides. Incubation of 1.4 mm XXXG with diverse plant extracts gave a series of products one, two, or three sugar residues larger than the heptasaccharide starting material, indicating transglycosidase activity (Fig. 5). The virtual absence of free xylose

22 3540 Franková and Fry Fig. 5. TLC evidence for trans-α-xylosidase activity. The chromatogram shows products formed by the action of plant extracts on 1.4 mm XXXG (xyloglucan heptasaccharide; for further explanation of G, L, and X, see text). Pure substrate is shown in the left-hand lane. Blue indicates smaller products remaining after transglycosylation plus any hydrolysis products; pink indicates products formed by trans-α-xylosylation, during which xylose residue(s) are added to XXXG or to one of its major smaller products (pink arrows indicating these trans-xylosylation reactions). DP, degree of polymerization (for example, DP10 indicates a decasaccharide); IP, isoprimeverose. From Franková and Fry (2012a). Trans-[α]-xylosidase and trans-[β]-galactosidase activities, widespread in plants, modify and stabilise xyloglucan structures. The Plant Journal, with permission of John Wiley & Sons. among the reaction products indicates little α-xylosidase (exohydrolase) activity (Franková and Fry, 2012a). 3 -Labelled substrates showed detectable transglycosylation at concentrations as low as 16 μm (in addition to hydrolysis, which is favoured at such low substrate concentrations). Definitive evidence for the trans-α-xylosidase reaction occurring was provided by dual-labelling experiments. With an aminolabelled (cationic) XGO as acceptor and [xylosyl- 3 ]XXXG as donor, extracts of monocots (snowdrop, asparagus) and dicots (chicory, parsley, cauliflower) all generated double-labelled (positively charged, radioactive) products (Franková and Fry, 2012a): 2012a). It is likely that polysaccharide-to-polysaccharide transfer of single xylose residues can also occur. In the case of the XGO-to- XGO reaction, the transferred α-xylose residue ends up attached to an existing xylose residue (not to a glucose residue), forming a novel xyloglucan trisaccharide unit, α-xylp-(1 4)-α-Xylp-(1 6)-Glc, assigned the sequence code-letter V (Franková and Fry, 2012b). It will be interesting to discover whether V also occurs in natural xyloglucan. A similar dual-labelling strategy using [Gal- 3 ]XXLG or [Gal- 3 ]XLLG and XGO N 2 gave evidence for trans-βgalactosidase activity in plant extracts. In contrast, [Fuc- 3 ] With 37 μm [Xyl- 3 ]XXXG as donor plus 1 mm XGO N 2 as acceptor, transglycosylation exceeded xylosyl hydrolysis 1.6- to 7.3- fold (lowest in cauliflower, highest in snowdrop), implying the presence of enzymes that favour transglycosylation. The extracts also transferred α-xylose residues from [xylosyl- 3 ]XXXG to polysaccharide acceptor substrates (xyloglucan, water-soluble cellulose acetate, MLG, glucomannan, and arabinoxylan) (Franková and Fry, XXFG gave no positively charged, radioactive products, indicating the absence of a trans-α-fucosidase activity as predicted for CAZy class G95 (inverting), which includes plant α-1,2-fucosidases. Panning for novel homo- and hetero-transglycosidase activities is now feasible, given that simple but effective assays are available.