Kotake Toshihisa, Tsuchiya Koji, Aohara Tsutomu, Konishi Tomoyuki, Kaneko Satoshi, Igarashi Kiyohiko, Samejima Masahiro, Tsumuraya Yoichi
Department of Biochemistry and Molecular Biology, Faculty of Science, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan.
J Exp Bot. 2006;57(10):2353-62. doi: 10.1093/jxb/erj206.
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.
阿拉伯半乳聚糖蛋白（AGPs）的碳水化合物部分对其生理功能至关重要，且在体内会快速周转。AGPs碳水化合物部分的降解似乎是由几种糖苷酶协同作用发生的，其中包括α-L-阿拉伯呋喃糖苷酶、β-D-半乳糖苷酶和β-D-葡萄糖醛酸苷酶。在此，通过逆转录酶PCR从萝卜（Raphanus sativus L.）未成熟种子中克隆出一种双功能α-L-阿拉伯呋喃糖苷酶/β-D-木糖苷酶，该酶可水解AGPs碳水化合物部分的α-L-阿拉伯呋喃糖基残基。该基因命名为RsAraf1，包含一个2343 bp的开放阅读框（780个氨基酸），在N端包括一个推定的信号序列（33个氨基酸）。RsAraf1与大麦α-L-阿拉伯呋喃糖苷酶/β-D-木糖苷酶高度相似，基于序列同源性属于糖苷水解酶家族3。Southern杂交分析表明萝卜基因组中存在几个相关基因。RsAraf1在种子发育过程中均有表达，在幼苗中表达较弱。研究发现，在RsAraf1表达增强的转基因拟南芥植物制备的细胞壁蛋白组分中，α-L-阿拉伯呋喃糖苷酶活性显著高于野生型蛋白组分；粗酶制剂可从萝卜AGPs以及α-(1→5)-阿拉伯聚糖和阿拉伯木聚糖中释放出L-阿拉伯糖。因此，转基因植物细胞壁中L-阿拉伯糖基残基的量显著减少。这些结果表明RsAraf1编码一种双功能α-L-阿拉伯呋喃糖苷酶/β-D-木糖苷酶，并表明RsAraf1参与未成熟萝卜种子中AGPs碳水化合物部分的水解。
