Xyloglucan endotransglycosylase, a new wall-loosening enzyme activity from plants

Abstract

1. Cell-free extracts of all plants tested contained a novel enzyme activity (xyloglucan endotransglycosylase, XET) able to transfer a high-Mr portion from a donor xyloglucan to a suitable acceptor such as a xyloglucan-derived nonasaccharide (Glc4Xyl3GalFuc; XG9). 2. A simple assay for the enzyme, using [3H]XG9 and based on the ability of the [3H]polysaccharide product to bind to filter paper, is described. 3. The enzyme was highly specific for xyloglucan as the glycosyl donor, and showed negligible transglycosylation of other polysaccharides, including CM-cellulose. 4. The Km for XG9 was 50 microM; certain other 3H-labelled xyloglucan oligosaccharides also acted as acceptors, and certain non-radioactive xyloglucan oligosaccharides competed with [3H]XG9 as acceptor; the minimum acceptor structure was deduced to be: [formula: see text] 5. The pH optimum was approx. 5.5 and the enzyme was less than half as active at pH 7.0. The enzyme was slightly activated by Ca2+, Mg2+, Mn2+, spermidine, ascorbate and 2-mercaptoethanol, and inhibited by Ag+, Hg2+, Zn2+ and La3+. 6. XET activity was essentially completely extracted by aqueous solutions of low ionic strength; Triton X-100, Ca2+, La3+, and Li+ did not enhance extraction. Negligible activity was left in the unextractable (cell-wall-rich) residue. 7. The enzyme differed from the major cellulases (EC 3.2.1.4) of pea in: (a) susceptibility to inhibition by cello-oligosaccharides, (b) polysaccharide substrate specificity, (c) inducibility by auxin, (d) requirement for salt in the extraction buffer and (e) activation by 2-mercaptoethanol. XET is therefore concluded to be a new enzyme activity (xyloglucan: xyloglucan xyloglucanotransferase; EC 2.4.1.-). 8. XET was detected in extracts of the growing portions of dicotyledons, monocotyledons (graminaceous and liliaceous) and bryophytes. 9. The activity was positively correlated with growth rate in different zones of the pea stem. 10. We propose that XET is responsible for cutting and rejoining intermicrofibrillar xyloglucan chains and that it thus causes the wall-loosening required for plant cell expansion.

Xyloglucan undergoes interpolymeric transglycosylation during binding to the plant cell wall in vivo: evidence from 13C/3H dual labelling and isopycnic centrifugation in caesium trifluoroacetate

“…Substrate solution [20 µl of 0.3 % w\v xyloglucan ($ 60 pmol) containing 1.6 kBq of [$H]XLLGol ($ 80 pmol)] was mixed with cXET (10 µl of a 1:10 dilution in succinate buffer) and incubated at 25 mC. At various time points, replicate assays were stopped with formic acid (100 µl, 30 % w\v) and paper-binding $H-labelled products were assayed [16].…”

“…It was therefore of interest to investigate whether xyloglucan chains undergo transglycosylation in the walls of living plant cells. Xyloglucan endotransglycosylases (XETs) are widespread in land plants [13][14][15][16][17][18][19], and numerous correlations between XET activity and wall loosening have been noted [5,7,[20][21][22][23][24]. XET cuts a xyloglucan chain (donor substrate)…”

“…and transfers the newly formed potentially reducing terminus on to the non-reducing terminus of another chain (acceptor substrate) [15][16][17]. In itro, the acceptor can be a high-M r xyloglucan or a short xyloglucan oligomer as small as a pentasaccharide [25].…”

“…Transglycosylation between xyloglucan chains in the cell wall could play a role in wall construction [8,26,27] and\or wall loosening [8,14,16,21,22,25,28]. Progress in testing these ideas has been hampered by our inability to say whether XET catalyses interpolymeric transglycosylation in i o. XETs are known to catalyse polymer-to-oligomer transglycosylation in itro [15][16][17]25], interpolymeric transglycosylation in itro [17,29,30] and polymer-to-oligomer transglycosylation in i o (although not necessarily involving wall-bound polymer) [13,14]. However, there is no direct evidence for interpolymeric transglycosylation in i o.…”

Group III-AXTHGenes of Arabidopsis Encode Predominant Xyloglucan Endohydrolases That Are Dispensable for Normal Growth

“…Since the landmark discovery of XET activity in the early 1990s (Farkas et al, 1992;Fry et al, 1992;Nishitani and Tominaga, 1992), these enzymes are now known to be encoded by large XYLOGLUCAN ENDO-TRANSGLYCOSYLASE/HYDROLASE (XTH) gene families present in diverse land plant species, including both monocots and dicots (Eklöf and Brumer, 2010). As the name suggests, a small number of XTH gene products have demonstrable xyloglucan endo-hydrolase (XEH; EC 3.2.1.151) activity (for review, see Eklöf and Brumer, 2010).…”