title: Heterologous expression of tomato glycoside hydrolase family 3 α-L-arabinofuranosidase/β-xylosidases in tobacco suspension cultured cells and synergic action of a family 51 isozyme under antisense suppression of the enzyme.
abstract: Four cDNA clones (SlArf/Xyl1-4) encoding α-l-arabinofuranosidase/β-xylosidase belonging to glycoside hydrolase family 3 were obtained from tomato (Solanum lycopersicum) fruit. SlArf/Xyl1 was expressed in various organs. Its level was particularly high in flower and leaves but low in fruit. SlArf/Xyl3 was highly expressed in flower. On the contrary, SlArf/Xyl2 and 4 were expressed in early developmental stage in various organs. Comparison with SlArf/Xyl4, SlArf/Xyl2 expression was observed in earlier stages. The active recombinant proteins were obtained by using BY-2 tobacco (Nicotiana tabacum) suspension cultured cells. The SlArf/Xyl1 and 2 recombinant proteins showed a bi-functional activity of α-l-arabinofuranosidase/β-xylosidase while the SlArf/Xyl4 protein possessed a β-xylosidase activity predominantly. Neither enzyme activities were detected for the SlArf/Xyl3 protein under the same conditions. Although SlArf/Xyl2 possessed a bi-functional activity, it preferentially hydrolyzed arabinosyl residues from tomato hemicellulosic polysaccharides. Antisense suppression of SlArf/Xyl2 resulted in no apparent changes in the enzyme activities, monosaccharide composition or fruit phenotype. Increment of a family 51 α-l-arabinofuranosidase expression rather than that of family 3 resulted in a restoring the activity in SlArf/Xyl2-suppressed fruit. The ability of recombinant SlArf/Xyl2 to hydrolyze both arabinan and arabinoxylan is nearly identical to that of α-l-arabinofuranosidases belonging to family 51. Our results suggested that BY-2 cells are a useful expression system for obtaining active cell wall hydrolyzing enzymes. In addition, an α-l-arabinofuranosidase activity derived from SlArf/Xyl2 would be essential in young organ development and the action of the enzyme could be restored by the other enzyme belonging to a different family under a defective condition.
authors.affiliation: College of Bioresource Sciences, Nihon University, Kameino, Fujisawa, 252-0880, Japan; Graduate School of Bioresource Sciences, Nihon University, Kameino, Fujisawa, 252-0880, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Tateishi
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Kamiyoshihara
authors.forename: Junko
authors.initials: J
authors.lastname: Matsuno
authors.forename: Fumika
authors.initials: F
authors.lastname: Miyohashi
authors.forename: Hajime
authors.initials: H
authors.lastname: Shiba
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Kanayama
authors.forename: Keiichi
authors.initials: K
authors.lastname: Watanabe
authors.forename: Kazunari
authors.initials: K
authors.lastname: Nomura
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Inoue
mesh_terms: D001089:Arabinose / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005638:Fruit / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D018506:Gene Expression Regulation, Plant; D006026:Glycoside Hydrolases / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D018551:Solanum lycopersicum / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D010641:Phenotype; D010802:Phylogeny; D030821:Plants, Genetically Modified; D016372:RNA, Antisense / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013535:Suspensions; D014026:Nicotiana / Q000166:cytology* / Q000235:genetics*; D014994:Xylose / Q000378:metabolism; D014995:Xylosidases / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D007527:Isoenzymes; D016372:RNA, Antisense; D012333:RNA, Messenger; D013535:Suspensions; D014994:Xylose; D001089:Arabinose; D006026:Glycoside Hydrolases; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase; C019713:alpha-N-arabinofuranosidase