Plant and Cell Physiology Advance Access originally published online on October 20, 2006
Plant and Cell Physiology 2006 47(11):1555-1571; doi:10.1093/pcp/pcl021
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Carbohydrate-Binding Module of a Rice Endo-ß-1,4-glycanase, OsCel9A, Expressed in Auxin-Induced Lateral Root Primordia, is Post-Translationally Truncated
1 Hydraulic and Bio Engineering Research Section, Technology Center, Taisei Co., 344-1 Nase-cho, Totuka-ku, Yokohama, 245-0051 Japan
2 Research Division, Japan Turfgrass Inc., 3-6-3 Akanehama, Narashino-shi, Chiba, 275-0024 Japan
3 Biological Function Division, National Food Research Institute, 2-1-12 Kannondai, Tsukuba-shi, Ibaraki, 305-8642 Japan
4 Department of Plant Biotechnology, National Institute for Agrobiological Science, 2-1-2 Kannnondai, Tsukuba-shi, Ibaraki, 305-8518 Japan
5 Department of Developmental Biology and Neurosciences, Tohoku University, Sendai, 980-8578 Japan
6 Department of Wheat and Barley Research, National Institute of Crop Science, 2-1-18 Kannondai, Tuskuba-shi, Ibaraki, 305-8518 Japan
* Corresponding author: E-mail, kouki.yoshida{at}sakura.taisei.co.jp; Fax, +81-45-814-7257
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Abstract |
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We report the cloning of a glycoside hydrolase family (GHF) 9 gene of rice (Oryza sativa L. cv. Sasanishiki), OsCel9A, corresponding to the auxin-induced 51 kDa endo-1,4-ß-glucanase (EGase). This enzyme reveals a broad substrate specificity with respect to sugar backbones (glucose and xylose) in ß-1,4-glycans of type II cell wall. OsCel9A encodes a 640 amino acid polypeptide and is an ortholog of TomCel8, a tomato EGase containing a carbohydrate-binding module (CBM) 2 sequence at its C-terminus. The expression of four rice EGase genes including OsCel9A showed different patterns of organ specificity and responses to auxin. OsCel9A was preferentially expressed during the initiation of lateral roots or subcultured root calli, but was hardly expressed during auxin-induced coleoptile elongation or in seed calli, in contrast to OsCel9D, a KORRIGAN (KOR) homolog. In situ localization of OsCel9A transcripts demonstrated that its expression was specifically up-regulated in lateral root primordia (LRP). Northern blotting analysis showed the presence of a single product of OsCel9A. In contrast, both mass spectrometric analyses of peptide fragments from purified 51 kDa EGase proteins and immunogel blot analysis of EGase proteins in root extracts using two antibodies against internal peptide sequences of OsCel9A revealed that the entire CBM2 region was post-translationally truncated from the 67 kDa nascent protein to generate 51 kDa EGase isoforms. Analyses of auxin concentration and time course dependence of accumulation of two EGase isoforms suggested that the translation and post-translational CBM2 truncation of the OsCel9A gene may participate in lateral root development.
Keywords: Auxin - Carbohydrate-binding module family 2 - Glycoside hydrolase family 9 - Lateral root formation - Processing - Rice
Abbreviations: ATCH, adrenocorticotropic hormone; CBM, carbohydrate-binding module; CTAB, cetyltrimethylammonium bromide; DEPC, diethyl pyrocarbonate; DTT, dithiothreitol; EGase, endo-1,4-ß-glucanase; GAX, glucuronoarabinoxylan; GHF, glycoside hydrolase family; Glu-C, endoproteinase Glu-C; IEF, isoelectric focusing; KLH, keyhole limpet hemocyanin; KOR, KORRIGAN; LRP, lateral root primordia; Lys-C, endoproteinase Lys-C; 2-ME, 2-mercaptoethonol; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MS, mass spectrometry; m/z, mass-to-charge ratio; nano ESI MS/MS, nano electrospray ionization tandem mass spectrometry; PMF, peptide mass fingerprinting; RT–PCR, reverse transcription–PCR; TCA, trichloroacetic acid; UTR, untranslated region
This paper is dedicated to the memory of the late Mr. Hideya Akagi, Mr. Yukio Kira, Dr. Okayama and Dr. Seitaroh Yokogi.
The ordered locus name of OsCel9A is OsOlg0220100 (http://rapdb.lab.nig.ac.jp/).
The nucleotide sequences of OsCel9A and OsCel9D have been submitted to the GenBank database with the accession numbers AB038510 and AB040817, respectively.
(Received July 17, 2006; Accepted October 6, 2006)
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