An alpha-L-arabinofuranosidase/beta-D-xylosidase from immature seeds of radish (Raphanus sativus L.).
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 alpha-L-arabinofuranosidase, beta-D-galactosidase, and beta-D-glucuronidase. Here, a bifunctional alpha-L-arabinofuranosidase/beta-D-xylosidase from immature seeds of radish (Raphanus sativus L.), which hydrolyses alpha-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 alpha-L-arabinofuranosidase/beta-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 alpha-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 alpha-(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 alpha-L-arabinofuranosidase/beta-D-xylosidase and suggest that RsAraf1 is involved in the hydrolysis of the carbohydrate moieties of AGPs in immature radish seeds.
Kotake
Toshihisa
T
Department of Biochemistry and Molecular Biology, Faculty of Science, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan. kotake@molbiol.saitama-u.ac.jp
Tsuchiya
Koji
K
Aohara
Tsutomu
T
Konishi
Tomoyuki
T
Kaneko
Satoshi
S
Igarashi
Kiyohiko
K
Samejima
Masahiro
M
Tsumuraya
Yoichi
Y
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics; D002473:Cell Wall / Q000378:metabolism; D018076:DNA, Complementary; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009088:Mucoproteins / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000201:enzymology; D011134:Polysaccharides / Q000378:metabolism; D031224:Raphanus / Q000201:enzymology* / Q000235:genetics; D012639:Seeds / Q000201:enzymology*; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic; D014995:Xylosidases / Q000378:metabolism*
D018076:DNA, Complementary; D009088:Mucoproteins; D010940:Plant Proteins; D011134:Polysaccharides; C006619:arabinogalactan proteins; D006026:Glycoside Hydrolases; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase; C019713:alpha-N-arabinofuranosidase