RESEARCH PAPER |
An 
-L-arabinofuranosidase/ß-D-xylosidase from immature seeds of radish (Raphanus sativus L.)
1Department of Biochemistry and Molecular Biology, Faculty of Science, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan
2Biological Function Division, National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
3Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan
*To whom correspondence should be addressed. E-mail: kotake{at}molbiol.saitama-u.ac.jp
The carbohydrate moieties of arabinogalactan proteins (AGPs) are essential for their physiological functions and undergo rapid turnover in vivo. Degradation of the carbohydrate moieties of AGPs seems to occur by concerted action of several glycosidases, among them 
-L-arabinofuranosidase, ß-D-galactosidase, and ß-D-glucuronidase. Here, a bifunctional -L-arabinofuranosidase/ß-D-xylosidase from immature seeds of radish (Raphanus sativus L.), which hydrolyses -L-arabinofuranosyl residues of the carbohydrate moieties of AGPs, has been cloned by reverse transcriptase-PCR. The gene, designated RsAraf1, contained an open reading frame of 2343 bp (780 amino acids), including a putative signal sequence (33 amino acids) at the N-terminus. RsAraf1 is highly similar to barley -L-arabinofuranosidase/ß-D-xylosidases and belongs to family 3 of the glycosyl hydrolases based on sequence homology. Southern blot analysis revealed that several related genes exist in the radish genome. RsAraf1 is expressed throughout seed development and weakly expressed in young seedlings. It was found that -L-arabinofuranosidase activity in a cell-wall protein fraction prepared from transgenic Arabidopsis plants with enhanced expression of RsAraf1 was significantly higher than that in a wild-type protein fraction; the crude enzyme preparation released L-arabinose from radish AGPs as well as -(1
5)-arabinan and arabinoxylan. Accordingly, the amount of L-arabinosyl residues in the cell walls of transgenic plants was significantly decreased. These results indicate that RsAraf1 encodes a bifunctional -L-arabinofuranosidase/ß-D-xylosidase and suggest that RsAraf1 is involved in the hydrolysis of the carbohydrate moieties of AGPs in immature radish seeds.
Key words:
Arabidopsis, -L-arabinofuranosidase, arabinogalactan protein, cell wall, family 3 glycoside hydrolase, polysaccharide, radish, seed development, transgenic plant, and ß-xylosidase
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