In Vivo Colocalization of Xyloglucan Endotransglycosylase Activity and Its Donor Substrate in the Elongation Zone of Arabidopsis Roots

Abstract

We have developed a method for the colocalization of xyloglucan endotransglycosylase (XET) activity and the donor substrates to which it has access in situ and in vivo. Sulforhodamine conjugates of xyloglucan oligosaccharides (XGOSRs), infiltrated into the tissue, act as acceptor substrate for the enzyme; endogenous xyloglucan acts as donor substrate. Incorporation of the XGO-SRs into polymeric products in the cell wall yields an orange fluorescence indicative of the simultaneous colocalization, in the same compartment, of active XET and donor xyloglucan chains. The method is specific for XET, as shown by competition experiments with nonfluorescent acceptor oligosaccharides, by negligible reaction with cello-oligosaccharide-SR conjugates that are not XET acceptor substrates, by heat lability, and by pH optimum. Thin-layer chromatographic analysis of remaining unincorporated XGO-SRs showed that these substrates are not extensively hydrolyzed during the assays. A characteristic distribution pattern was found in Arabidopsis and tobacco roots: in both species, fluorescence was most prominent in the cell elongation zone of the root. Proposed roles of XET that include cell wall loosening and integration of newly synthesized xyloglucans could thus be supported. INTRODUCTIONThe primary cell walls of flowering plants consist fundamentally of a framework of cellulose microfibrils embedded in a matrix of hemicellulose, pectins, and structural proteins (Carpita and Gibeaut, 1993; Brett and Waldron, 1996). Xyloglucan, the major hemicellulosic polysaccharide in the primary cell wall matrix of dicots, consists of a backbone of ␤ -(1 → 4)-linked D -glucose residues, the majority of which are ␣ -D -xylosylated at O-6. Some xylose residues are further substituted by galactosyl and fucosyl-galactosyl groups. Other, minor carbohydrate side chains and O-acetyl groups are also present (Fry, 1989a; Hayashi, 1989). Because xyloglucans can form tight hydrogen bonds with cellulose microfibrils (Valent and Albersheim, 1974; Hayashi et al., 1987 Hayashi et al., , 1994a Hayashi et al., , 1994b Hayashi, 1989), they may thereby tether adjacent microfibrils (Fry, 1989b). A proportion of the xyloglucan molecules are covalently attached to acidic pectins (Thompson and Fry, 2000). Xyloglucans also serve as storage polysaccharides in some seeds (Edwards et al., 1985).For plant cells to expand, cellulose microfibrils in parallel alignment need to move apart or past one another, and this movement may create the possibility for newly synthesized xyloglucan molecules to become hydrogen-bonded (Fry, 1989b). Because xyloglucan tethers are thought to be the principal tension-bearing molecules in the cell wall, breaking of the tethers has been proposed as a mechanism for achieving reversible cell wall loosening in elongating tissue without compromising strength (Fry, 1989b; Hayashi, 1989; Hoson et al., 1991). Although the cell wall contains numerous enzymes that can modify polysaccharides (Fry, 1995), xyloglucan endotransglycosylases (XETs) seem well...

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

“…S9) as well as XET activity staining (Fig. 3), with both the AtXTH31/AtXTH32 knockout and wild-type lines exhibiting similar patterns, which were equivalent to those observed previously for the wild type (Vissenberg et al, 2000(Vissenberg et al, , 2001McCartney et al, 2003). AtXTH31 is highly expressed in this zone (AEV 12,000 versus 870 for AtXTH32); however, the lack of differential signal from XXXG-Res hydrolysis implied that either sequential a-xylosidase and b-glucosidase activity (Iglesias et al, 2006), or one or more other enzyme(s) with XEH activity, is active here (Fig.…”

“…In both lines, XET activity was observed in the root elongation zone (compare with Supplemental Fig. S9) and at sites of root hair initiation, precisely as in previous studies (Vissenberg et al, 2000(Vissenberg et al, , 2001.…”

“…6). Strong XET activity was also observed in lateral root initiation zones, as has been demonstrated previously in Arabidopsis (Vissenberg et al, 2000). XEH activity was also high in wild-type cell walls in the junction region between root and hypocotyls, also known as the collet, which contains early emerging root hairs (Lin and Schiefelbein, 2001;Scheres et al, 2002;Supplemental Fig.…”

“…For comparison with previous studies, XET activity was visualized in Col-0 and xth31-1/ xth32-1 roots by monitoring the incorporation of the xyloglucan oligosaccharide-sulforhodamine conjugate XXXG-SR into the cell wall by confocal fluorescence microscopy. In this stopped assay, XXXG-SR functions as an alternate glycosyl acceptor for enzymes with XET activity, thus forming covalent xyloglucan-XXXG-SR conjugates at sites where the polysaccharide and enzyme are colocalized (Vissenberg et al, 2000;Nishikubo et al, 2007). As shown in Figure 3, the localization of XET activity was essentially identical in wild-type (Fig.…”

“…In light of the minor effects of AtXTH31 and AtXTH32 on Arabidopsis development, we directed our attention to the analysis of potential changes in XET and XEH enzyme activities in situ using specific fluorescent (Vissenberg et al, 2000) and fluorogenic (Ibatullin et al, 2009) probes, respectively. Based on the close homology of AtXTH31 and AtXTH32 as group III-A members, the double knockout line was examined directly to avoid potential complications due to functional compensation.…”