Phylogenetic variation in glycosidases and glycanases acting on plant cell wall polysaccharides, and the detection of transglycosidase and trans‐β‐xylanase activities

Abstract

SUMMARYWall polysaccharide chemistry varies phylogenetically, suggesting a need for variation in wall enzymes. Although plants possess the genes for numerous putative enzymes acting on wall carbohydrates, the activities of the encoded proteins often remain conjectural. To explore phylogenetic differences in demonstrable enzyme activities, we extracted proteins from 57 rapidly growing plant organs with three extractants, and assayed their ability to act on six oligosaccharides 'modelling' selected cell-wall polysaccharides. Based on reaction products, we successfully distinguished exo-and endo-hydrolases and found high taxonomic variation in all hydrolases screened:The results, as GHATAbase, a searchable compendium in Excel format, also provide a compilation for selecting rich sources of enzymes acting on wall carbohydrates. Four of the hydrolases were accompanied, sometimes exceeded, by transglycosylase activities, generating products larger than the substrate. For example, during b-xylosidase assays on (1fi4)-b-Dxylohexaose (Xyl 6 ), Marchantia, Selaginella and Equisetum extracts gave negligible free xylose but approximately equimolar Xyl 5 and Xyl 7 , indicating trans-b-xylosidase activity, also found in onion, cereals, legumes and rape. The yield of Xyl 9 often exceeded that of Xyl 7-8 , indicating that b-xylanase was accompanied by an endotransglycosylase activity, here called trans-b-xylanase, catalysing the reaction 2Xyl 6 fi Xyl 3 + Xyl 9 . Similar evidence also revealed trans-a-xylosidase, trans-a-arabinosidase and trans-a-arabinanase activities acting on xyloglucan oligosaccharides and (1fi5)-a-L-arabino-oligosaccharides. In conclusion, diverse plants differ dramatically in extractable enzymes acting on wall carbohydrate, reflecting differences in wall polysaccharide composition. Besides glycosidase and glycanase activities, five new transglycosylase activities were detected. We propose that such activities function in the assembly and re-structuring of the wall matrix.

AtBGAL10 Is the Main Xyloglucan β-Galactosidase in Arabidopsis, and Its Absence Results in Unusual Xyloglucan Subunits and Growth Defects

“…Xyloglucan b-galactosidases from nasturtium and C. langsdorfii were shown to remove Gal residues from XLXG, but less efficiently or not at all from XXLG (Crombie et al, 1998;de Alcântara et al, 1999). The same specificity has been found for Lepidium sativum b-galactosidase and is likely to be conserved in many other plant species (Franková and Fry, 2011). The low level of activity of AtBGAL10 on polymeric xyloglucan (Fig.…”

“…1, C and D). This can be explained by the transglycosylating activity of Arabidopsis a-xylosidase, which can use XXXG as both acceptor and donor (Sampedro et al, 2010;Franková and Fry, 2011). The excess of b-glucosidase over a-xylosidase activity is inconsistent with the accumulation of GLLG and GLG in digestions of XLLG with bgal10 extracts (Fig.…”

“…The appearance of a xyloglucan-specific b-galactosidase could have happened after the divergence of these basal lineages, possibly linked to more intense xyloglucan recycling. This could also explain why nonseed plants have low b-galactosidase activity against xyloglucan (Franková and Fry, 2011).…”

XTH31, Encoding an in Vitro XEH/XET-Active Enzyme, Regulates Aluminum Sensitivity by Modulating in Vivo XET Action, Cell Wall Xyloglucan Content, and Aluminum Binding Capacity in Arabidopsis

“…The reaction mixture (final volume 60 µL) contained (final concentrations) 1.7 mg/mL nonradioactive tamarind xyloglucan, 1.26 µM [1-3 H]XXLGol (containing a small proportion of [1-3 H] XLXGol; specific activity 66 MBq/µmol; synthesized by reacting tamarind xyloglucan oligosaccharides with NaB 3 H 4 and purified by preparative thin layer chromatography), 67 mM succinate (Na + , pH 5.5), 0.5% chlorobutanol, and 20 mL enzyme preparation (added last; XTH31 from P. pastoris, protein from wild-type or empty vector P. pastoris, a crude grass extract [extracted by the method of Franková and Fry (2011) from young shoots of the grass Holcus lanatus], or protein-free blank). At timed intervals, 10-mL aliquots were stopped with 10 mL formic acid, the acidified solution was dried onto Whatman 3MM paper, washed in running tap water for 24 h and redried, and any 3 H-polysaccharide remaining on the paper was assayed in OptiScint HiSafe (Perkin-Elmer).…”

AUXIN BINDING PROTEIN1 Links Cell Wall Remodeling, Auxin Signaling, and Cell Expansion in Arabidopsis

“…A large set of apoplastic glycosidases or trans-glycosidases (b-glucosidases, a-xylosidases, b-galactosidases, and a-fucosidases) act specifically on XyGs. These enzymes modify XyG structure by modulating the length of side chains, which might change their susceptibility to XTH activity (Augur et al, 1993;Iglesias et al, 2006;Franková and Fry, 2011) and/or their cellulose binding properties (Levy et al, 1997). Modifications of XyGcellulose interaction or remodeling of XyG by metabolizing enzymes may modulate cell wall extensibility and thus facilitate cell expansion.…”