Molecular cloning of a bifunctional β-xylosidase/α-L- arabinosidase from alfalfa roots

Heterologous expression in Medicago truncatula and substrate specificity of the purified enzyme

Jin Song Xiong, Maud Balland-Vanney, Zhi Ping Xie, Michael Schultze, Adam Kondorosi, É. Kondorosi, Christian Staehelin

Research output: Contribution to journalArticle

30 Citations (Scopus)

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 (β-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-β-D-xyloside, PNP-α-L-arabinofuranoside, and PNP-α-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 β-1,4-linked D-xylo-oligosaccharides and α-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 β-xylosidase/α-L-arabinofuranosidase/α-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.

Original languageEnglish
Pages (from-to)2799-2810
Number of pages12
JournalJournal of Experimental Botany
Volume58
Issue number11
DOIs
Publication statusPublished - Sep 2007

Fingerprint

Xylosidases
Medicago truncatula
alpha-N-arabinofuranosidase
Medicago sativa
Glycoside Hydrolases
Molecular Cloning
substrate specificity
Concanavalin A
Substrate Specificity
Cell Wall
glycosides
alfalfa
molecular cloning
Arabinose
Meristem
concanavalin A
hydrolases
Xylose
Oligosaccharides
Carrier Proteins

Keywords

  • α-L-arabinofuranosidase
  • α-L-arabinopyranosidase
  • β-xylosidase
  • Alfalfa (Medicago sativa)
  • Glycoside hydrolase family 3
  • Heterologous expression
  • Medicago truncatula

ASJC Scopus subject areas

  • Plant Science

Cite this

Molecular cloning of a bifunctional β-xylosidase/α-L- arabinosidase from alfalfa roots : Heterologous expression in Medicago truncatula and substrate specificity of the purified enzyme. / Xiong, Jin Song; Balland-Vanney, Maud; Xie, Zhi Ping; Schultze, Michael; Kondorosi, Adam; Kondorosi, É.; Staehelin, Christian.

In: Journal of Experimental Botany, Vol. 58, No. 11, 09.2007, p. 2799-2810.

Research output: Contribution to journalArticle

Xiong, Jin Song ; Balland-Vanney, Maud ; Xie, Zhi Ping ; Schultze, Michael ; Kondorosi, Adam ; Kondorosi, É. ; Staehelin, Christian. / Molecular cloning of a bifunctional β-xylosidase/α-L- arabinosidase from alfalfa roots : Heterologous expression in Medicago truncatula and substrate specificity of the purified enzyme. In: Journal of Experimental Botany. 2007 ; Vol. 58, No. 11. pp. 2799-2810.
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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 (β-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-β-D-xyloside, PNP-α-L-arabinofuranoside, and PNP-α-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 β-1,4-linked D-xylo-oligosaccharides and α-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 β-xylosidase/α-L-arabinofuranosidase/α-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.",
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AU - Balland-Vanney, Maud

AU - Xie, Zhi Ping

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AB - 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 (β-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-β-D-xyloside, PNP-α-L-arabinofuranoside, and PNP-α-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 β-1,4-linked D-xylo-oligosaccharides and α-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 β-xylosidase/α-L-arabinofuranosidase/α-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.

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KW - Glycoside hydrolase family 3

KW - Heterologous expression

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