JXB Advance Access published online on July 5, 2007
Journal of Experimental Botany, doi:10.1093/jxb/erm133
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RESEARCH PAPER |
Molecular cloning of a bifunctional ß-xylosidase/
-L-arabinosidase from alfalfa roots: heterologous expression in Medicago truncatula and substrate specificity of the purified enzyme
1State Key Laboratory of Biocontrol, 135, Xingangxi Road, School of Life Sciences, SunYat-Sen (Zhongshan) University, Guangzhou 510275, China
2Institut des Sciences du Végétal, Centre National de la Recherche Scientifique, Unité Propre de Recherche 2355, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
To whom correspondence should be addressed. E-mail: cst{at}mail.sysu.edu.cn
Glycoside hydrolases are often members of a multigene family, suggesting individual roles for each isoenzyme. Various extracellular glycoside hydrolases have an important but poorly understood function in remodelling the cell wall during plant growth. Here, MsXyl1, a concanavalin A-binding protein from alfalfa (Medicago sativa L.) belonging to the glycoside hydrolase family 3 (ß-D-xylosidase branch) is characterized. Transcripts of MsXyl1 were detected in roots (particularly root tips), root nodules, and flowers. MsXyl1 under the control of the CaMV 35S promoter was expressed in the model legume Medicago truncatula (Gaertner). Concanavalin A-binding proteins from the transgenic plants exhibited 5–8-fold increased activities towards three p-nitrophenyl (PNP) glycosides, namely PNP-ß-D-xyloside, PNP-
-L-arabinofuranoside, and PNP--L-arabinopyranoside. An antiserum raised against a synthetic peptide recognized MsXyl1, which was processed to a 65 kDa form. To characterize the substrate specificity of MsXyl1, the recombinant protein was purified from transgenic M. truncatula leaves by concanavalin A and anion chromatography. MsXyl1cleaved ß-1,4-linked D-xylo-oligosaccharides and -1,5-linked L-arabino-oligosaccharides. Arabinoxylan (from wheat) and arabinan (from sugar beet) were substrates for MsXyl1, whereas xylan (from oat spelts) was resistant to degradation. Furthermore, MsXyl1 released xylose and arabinose from cell wall polysaccharides isolated from alfalfa roots. These data suggest that MsXyl1 is a multifunctional ß-xylosidase/-L-arabinofuranosidase/-L-arabinopyranosidase implicated in cell wall turnover of arabinose and xylose, particularly in rapidly growing root tips. Moreover, the findings of this study demonstrate that stable transgenic M. truncatula plants serve as an excellent expression system for purification and characterization of proteins.
Key words:
Alfalfa (Medicago sativa), -L-arabinofuranosidase, -L-arabinopyranosidase, glycoside hydrolase family 3, heterologous expression, Medicago truncatula, ß-xylosidase
* Present address: Université de la Réunion, 15 avenue René Cassin, 97715 Saint Denis Messag, Cedex 9, La Réunion, France.
Present address: Department of Biology, University of York, PO Box 373, York YO10 5YW, UK.
Received 5 March 2007; Revised 27 April 2007 Accepted 9 May 2007
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