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Plant Cell, Vol. 12, 1229-1238, July 2000, Copyright © 2000, American Society of Plant Physiologists

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

Kris Vissenberga, Immaculada M. Martinez-Vilchezb, Jean-Pierre Verbelena, Janice G. Millerc, and Stephen C. Fryc
a University of Antwerp UIA, Department of Biology, Universiteitsplein 1, B-2610 Wilrijk, Belgium
b Department of Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0116
c Edinburgh Cell Wall Group, Institute of Cell and Molecular Biology, University of Edinburgh, Daniel Rutherford Building, The King's Buildings, Edinburgh EH9 3JH, United Kingdom

Correspondence to: Stephen C. Fry, S.Fry{at}Ed.Ac.UK (E-mail), 44-131-650-5392 (fax)

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 (XGO–SRs), 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.




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