title: Molecular cloning of a bifunctional beta-xylosidase/alpha-L-arabinosidase from alfalfa roots: heterologous expression in Medicago truncatula and substrate specificity of the purified enzyme.
abstract: 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 (beta-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-beta-D-xyloside, PNP-alpha-L-arabinofuranoside, and PNP-alpha-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 beta-1,4-linked D-xylo-oligosaccharides and alpha-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 beta-xylosidase/alpha-L-arabinofuranosidase/alpha-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.
authors.affiliation: State Key Laboratory of Biocontrol, 135, Xingangxi Road, School of Life Sciences, SunYat-Sen (Zhongshan) University, Guangzhou 510275, China.
authors.forename: Jin-Song
authors.initials: JS
authors.lastname: Xiong
authors.forename: Maud
authors.initials: M
authors.lastname: Balland-Vanney
authors.forename: Zhi-Ping
authors.initials: ZP
authors.lastname: Xie
authors.forename: Michael
authors.initials: M
authors.lastname: Schultze
authors.forename: Adam
authors.initials: A
authors.lastname: Kondorosi
authors.forename: Eva
authors.initials: E
authors.lastname: Kondorosi
authors.forename: Christian
authors.initials: C
authors.lastname: Staehelin
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000455:Medicago sativa / Q000201:enzymology* / Q000235:genetics; D046913:Medicago truncatula / Q000235:genetics*; D008969:Molecular Sequence Data; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018517:Plant Roots / Q000201:enzymology / Q000235:genetics; D030821:Plants, Genetically Modified / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D006026:Glycoside Hydrolases; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase; C019713:alpha-N-arabinofuranosidase