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

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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.

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