Group III-A XTH Genes of Arabidopsis Encode Predominant Xyloglucan Endohydrolases That Are Dispensable for Normal Growth 1[C][W][OA] Nomchit Kaewthai 2 , Delphine Gendre 2 , Jens M. Eklöf, Farid M. Ibatullin, Ines Ezcurra, Rishikesh P. Bhalerao, and Harry Brumer* Division of Glycoscience, School of Biotechnology, Royal Institute of Technology, AlbaNova University Centre, S–106 91 Stockholm, Sweden (N.K., J.M.E., F.M.I., I.E., H.B.); Umeå Plant Science Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, SE–901 83 Umea, Sweden (D.G., R.P.B.); Biophysics Division, Petersburg Nuclear Physics Institute, National Research Center Kurchatov Institute, Gatchina 188300, Russia (F.M.I.); and Michael Smith Laboratories and Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada (H.B.) The molecular basis of primary wall extension endures as one of the central enigmas in plant cell morphogenesis. Classical cell wall models suggest that xyloglucan endo-transglycosylase activity is the primary catalyst (together with expansins) of controlled cell wall loosening through the transient cleavage and religation of xyloglucan-cellulose cross links. The genome of Arabidopsis (Arabidopsis thaliana) contains 33 phylogenetically diverse XYLOGLUCAN ENDO-TRANSGLYCOSYLASE/ HYDROLASE (XTH) gene products, two of which were predicted to be predominant xyloglucan endohydrolases due to clustering into group III-A. Enzyme kinetic analysis of recombinant AtXTH31 confirmed this prediction and indicated that this enzyme had similar catalytic properties to the nasturtium (Tropaeolum majus) xyloglucanase1 responsible for storage xyloglucan hydrolysis during germination. Global analysis of Genevestigator data indicated that AtXTH31 and the paralogous AtXTH32 were abundantly expressed in expanding tissues. Microscopy analysis, utilizing the resorufin b-glycoside of the xyloglucan oligosaccharide XXXG as an in situ probe, indicated significant xyloglucan endohydrolase activity in specific regions of both roots and hypocotyls, in good correlation with transcriptomic data. Moreover, this hydrolytic activity was essentially completely eliminated in AtXTH31/AtXTH32 double knockout lines. However, single and double knockout lines, as well as individual overexpressing lines, of AtXTH31 and AtXTH32 did not demonstrate significant growth or developmental phenotypes. These results suggest that although xyloglucan polysaccharide hydrolysis occurs in parallel with primary wall expansion, morphological effects are subtle or may be compensated by other mechanisms. We hypothesize that there is likely to be an interplay between these xyloglucan endohydrolases and recently discovered apoplastic exo-glycosidases in the hydrolytic modification of matrix xyloglucans. The molecular basis of primary wall extension endures as one of the central enigmas in plant cell morphogenesis (Cosgrove, 2005; Geitmann and Ortega, 2009; Jarvis, 2009; Torres et al., 2009). Classical models of the primary cell wall in land plants describe the wall as a composite structure composed of load-bearing, semicrystalline cellulose fibrils surrounded by an amorphous matrix of cross-linking glycans (hemicelluloses and pectins), structural (glyco)proteins, and, in some cases, polyphe- nolics (Carpita and Gibeaut, 1993; Carpita and McCann, 2000; Cosgrove, 2005; Jarvis, 2009). Moreover, primary plant cell walls have a high water content (approxi- mately 80% in primary walls; Jarvis, 2009), which is responsible for maintaining the structure in a dynamic, hydrogel-like state (Ha et al., 1997; Zwieniecki et al., 2001; Jarvis, 2009). Contemporary structural analysis, using increasingly sophisticated probes, is refining our understanding of the exquisite spatial localization of cell wall polymers (Knox, 2008; Yarbrough et al., 2009). Genetic and biochemical approaches are at present uncovering the individual enzymes responsible for building these macromolecules, and models of coordi- nated supramolecular assembly at the plasma membrane continue to evolve (Geisler et al., 2008; Liepman et al., 2010). Lastly, there is a growing interest in the roles of endogenous glycoside hydrolases in primary plant cell wall remodeling (Vicente et al., 2007; Lopez-Casado 1 This work was supported by the Ministry of Science and Tech- nology, Thailand (fellowship to N.K.), the Swedish Research Council, Vetenskapsrådet, the Swedish Research Council Formas, and the Swedish Foundation for Strategic Research (via Biomime, the Swed- ish Center for Biomimetic Fiber Engineering). 2 These authors contributed equally to the article. * Corresponding author; e-mail brumer@msl.ubc.ca. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy de- scribed in the Instructions for Authors (www.plantphysiol.org) is: Harry Brumer (brumer@msl.ubc.ca). [C] Some figures in this article are displayed in color online but in black and white in the print edition. [W] The online version of this article contains Web-only data. [OA] Open Access articles can be viewed online without a subscrip- tion. www.plantphysiol.org/cgi/doi/10.1104/pp.112.207308 440 Plant Physiology Ò , January 2013, Vol. 161, pp. 440–454, www.plantphysiol.org Ó 2012 American Society of Plant Biologists. All Rights Reserved. Downloaded from https://academic.oup.com/plphys/article/161/1/440/6110726 by guest on 30 June 2022