イネ・アリューロン細胞におけるα-アミラーゼ合成誘導を指標とした活性型ジベレリン濃度の測定
穀類アリューロン細胞の活性化に伴う貯蔵デンプン分解の分子機構を理解するうえで, 発芽種子内に含まれる活性型ジベレリン (GA) の濃度を把握しておくことはきわめて重要である. そこで, アリューロン細胞におけるα-アミラーゼ合成誘導を指標に, イネの発芽種子に含まれる活性型GA濃度の簡易測定を試みた. さまざまな濃度のGAをアリューロン細胞に加えた場合に合成誘導されるα-アミラーゼの酵素活性を測定し, それらを指標に種子内の活性型GA濃度を推測した. また, 抗α-アミラーゼ抗体を用いた免疫ブロッティングにより, α-アミラーゼタンパク質の発現量をニトロセルロース膜上で検出し, それらを利用し...
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| Published in | Journal of Applied Glycoscience Vol. 52; no. 4; pp. 399 - 402 |
|---|---|
| Main Authors | , , , |
| Format | Journal Article |
| Language | Japanese |
| Published |
日本応用糖質科学会
2005
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| Online Access | Get full text |
| ISSN | 1344-7882 1880-7291 |
| DOI | 10.5458/jag.52.399 |
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| Abstract | 穀類アリューロン細胞の活性化に伴う貯蔵デンプン分解の分子機構を理解するうえで, 発芽種子内に含まれる活性型ジベレリン (GA) の濃度を把握しておくことはきわめて重要である. そこで, アリューロン細胞におけるα-アミラーゼ合成誘導を指標に, イネの発芽種子に含まれる活性型GA濃度の簡易測定を試みた. さまざまな濃度のGAをアリューロン細胞に加えた場合に合成誘導されるα-アミラーゼの酵素活性を測定し, それらを指標に種子内の活性型GA濃度を推測した. また, 抗α-アミラーゼ抗体を用いた免疫ブロッティングにより, α-アミラーゼタンパク質の発現量をニトロセルロース膜上で検出し, それらを利用したGA濃度の測定も行った. その結果, 発芽種子内における活性型GA濃度を, 0.1から1nMの範囲に特定することができた. また, GA濃度が1nM以上に上昇したか否かを判断するための指標となりうる42kDaのα-アミラーゼアイソフォームの存在を, 同時に見出すことができた. このアイソフォームは, イネのアリューロン細胞に存在する高濃度GAシグナリングを解析するために有効であると考えられる. |
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| AbstractList | 穀類アリューロン細胞の活性化に伴う貯蔵デンプン分解の分子機構を理解するうえで、発芽種子内に含まれる活性型ジベレリン(GA)の濃度を把握しておくことはきわめて重要である。そこで、アリューロン細胞におけるα-アミラーゼ合成誘導を指標に、イネの発芽種子に含まれる活性型GA濃度の簡易測定を試みた。さまざまな濃度のGAをアリューロン細胞に加えた場合に合成誘導されるα-アミラーゼの酵素活性を測定し、それらを指標に種子内の活性型GA濃度を推測した。また、抗α-アミラーゼ抗体を用いた免疫ブロッティングにより、α-アミラーゼタンパク質の発現量をニトロセルロース膜上で検出し、それらを利用したGA濃度の測定も行った。その結果、発芽種子内における活性型GA濃度を、0.1から1nMの範囲に特定することができた。また、GA濃度が1nM以上に上昇したか否かを判断するための指標となりうる42kDaのα-アミラーゼアイソフォームの存在を、同時に見出すことができた。このアイソフォームは、イネのアリューロン細胞に存在する高濃度GAシグナリングを解析するために有効であると考えられる。 |
| Author | 三ツ井, 敏明 川上, 修 山口, 淳二 光永, 伸一郎 |
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| References | 16) Y. Ogawa: Effects of various factors on the increase of α-amylase activity in rice endosperm induced by gibberellin A3. Plant Cell Physiol., 7, 509-517 (1966). 20) S. Mitsunaga, T. Tashiro and J. Yamaguchi: Identification and characterization of gibberellin-insensitive mutants selected from among dwarf mutants of rice. Theor. Appl. Genet., 87, 705-712 (1994). 4) P.J. Davies: Gibberellins: Regulators of plant height. in Plant Physiology, L. Taiz and E. Zeiger, eds., 3 rd Ed., Sinauer Associates, Inc., Sunderland, Massachusetts, pp. 461-492 (2002). 12) J. Sambrook, E.F. Fritsch and T. Maniatis: Molecular Cloning: A Laboratory Manual, 2 nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). 2) R.L. Jones and J.E. Varner: The bioassay of gibberellins. Planta, 72, 155-161 (1967). 11) F.J. Woodger, F. Gubler, B.J. Pogson and J.V. Jacobsen: A Mak-like kinase is a repressor of GAMYB in barley aleurone. Plant J., 33, 707-717 (2003). 15) S. Mitsunaga, O. Kawakami, T. Numata, J. Yamaguchi, K. Fukui and T. Mitsui: Polymorphism in rice amylases at an early stage of seed germination. Biosci. Biotechnol. Biochem., 65, 662-665 (2001). 8) N.E.J. Appleford and J.R. Lenton: Hormonal regulation of α-amylase gene expression in germinating wheat (Triticum aestivum) grains. Physiol. Plant., 100, 534-542 (1997). 1) R. Hooley: Gibberellins: Perception, transduction, and responses. Plant Mol. Biol., 26, 1529-1555 (1994). 14) T. Mitsui, J. Yamaguchi and T. Akazawa: Physicochemical and serological characterization of rice α-amylase isoforms and identification of their corresponding genes. Plant Physiol., 110, 1395-1404 (1996). 17) M. Ueguchi-Tanaka, Y. Fujisawa, M. Kobayashi, M. Ashikari, Y. Iwasaki, H. Kitano and M. Matsuoka: Rice dwarf mutant d1, which is defective in the α subunit of the heterotrimeric G protein, affects gibberellin signal transduction. Proc. Natl. Acad. Sci. USA, 97, 11638-11643 (2000). 7) Y.-H. Choi, M. Kobayashi and A. Sakurai: Endogenous gibberellin A1 level and α-amylase activity in germinating rice seeds. J. Plant Growth Regul., 15, 147-151 (1996). 19) F. Panabieres, F. Kerhardy, A. Montembault, J. Daussant and M. Delseny: Induction of α-amylase isoenzymes by gibberellic acid in imbibed rice half-seeds. Plant Sci., 64, 15-23 (1989). 13) S. Mitsunaga and J. Yamaguchi: Induction of α-amylase is repressed by uniconazole, an inhibitor of the biosynthesis of gibberellin, in a dwarf mutant of rice, Waito-C. Plant Cell Physiol., 34, 243-249 (1993). 3) M. Kaneko, H. Itoh, M. Ueguchi-Tanaka, M. Ashikari and M. Matsuoka: The α-amylase induction in endosperm during rice seed germination is caused by gibberellin synthesized in epithelium. Plant Physiol., 128, 1264-1270 (2002). 21) A. Dill and T.-P. Sun: Synergistic derepression of gibberellin signaling by removing RGA and GAI function in Arabidopsis thaliana. Genetics, 159, 777-785 (2001). 9) A. Sasaki, H. Itoh, K. Gomi, M. Ueguchi-Tanaka, K. Ishiyama, M. Kobayashi, D.-H. Jeong, G. An, H. Kitano, M. Ashikari and M. Matsuoka: Accumulation of phosphorylated repressor for gibberellin signaling in an F-box mutant. Science, 299, 1896-1898 (2003). 18) Y. Fujisawa, H. Kato and Y. Iwasaki: Structure and function of heterotrimeric G proteins in plants. Plant Cell Physiol., 42, 789-794 (2001). 6) M. Kobayashi, M. Gomi, J. Agematsu, T. Asami, S. Yoshida and A. Sakurai: Fluctuation of endogenous gibberellin and abscisic acid levels in germinating seeds of barley. Biosci. Biotechnol. Biochem., 59, 1969-1970 (1995). 5) Y.-H. Choi, M. Kobayashi, S. Fujioka, T. Matsuno, T. Hirosawa and A. Sakurai: Fluctuation of endogenous gibberellin levels in the early development of rice. Biosci. Biotechnol. Biochem., 59, 285-288 (1995). 10) K. Washio: Functional dissections between GAMYB and Dof transcription factors suggest a role for protein-protein associations in the gibberellin-mediated expression of the RAmy 1 A gene in the rice aleurone. Plant Physiol., 133, 850-863 (2003). 22) A.L. Silverstone, H.-S. Jung, A. Dill, H. Kawaide, Y. Kamiya and T.-P. Sun: Repressing a repressor: Gibberellin-induced rapid reduction of the RGA protein in Arabidopsis. Plant Cell, 13, 1555-1565 (2001). |
| References_xml | – reference: 2) R.L. Jones and J.E. Varner: The bioassay of gibberellins. Planta, 72, 155-161 (1967). – reference: 17) M. Ueguchi-Tanaka, Y. Fujisawa, M. Kobayashi, M. Ashikari, Y. Iwasaki, H. Kitano and M. Matsuoka: Rice dwarf mutant d1, which is defective in the α subunit of the heterotrimeric G protein, affects gibberellin signal transduction. Proc. Natl. Acad. Sci. USA, 97, 11638-11643 (2000). – reference: 15) S. Mitsunaga, O. Kawakami, T. Numata, J. Yamaguchi, K. Fukui and T. Mitsui: Polymorphism in rice amylases at an early stage of seed germination. Biosci. Biotechnol. Biochem., 65, 662-665 (2001). – reference: 3) M. Kaneko, H. Itoh, M. Ueguchi-Tanaka, M. Ashikari and M. Matsuoka: The α-amylase induction in endosperm during rice seed germination is caused by gibberellin synthesized in epithelium. Plant Physiol., 128, 1264-1270 (2002). – reference: 12) J. Sambrook, E.F. Fritsch and T. Maniatis: Molecular Cloning: A Laboratory Manual, 2 nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). – reference: 18) Y. Fujisawa, H. Kato and Y. Iwasaki: Structure and function of heterotrimeric G proteins in plants. Plant Cell Physiol., 42, 789-794 (2001). – reference: 1) R. Hooley: Gibberellins: Perception, transduction, and responses. Plant Mol. Biol., 26, 1529-1555 (1994). – reference: 7) Y.-H. Choi, M. Kobayashi and A. Sakurai: Endogenous gibberellin A1 level and α-amylase activity in germinating rice seeds. J. Plant Growth Regul., 15, 147-151 (1996). – reference: 19) F. Panabieres, F. Kerhardy, A. Montembault, J. Daussant and M. Delseny: Induction of α-amylase isoenzymes by gibberellic acid in imbibed rice half-seeds. Plant Sci., 64, 15-23 (1989). – reference: 14) T. Mitsui, J. Yamaguchi and T. Akazawa: Physicochemical and serological characterization of rice α-amylase isoforms and identification of their corresponding genes. Plant Physiol., 110, 1395-1404 (1996). – reference: 6) M. Kobayashi, M. Gomi, J. Agematsu, T. Asami, S. Yoshida and A. Sakurai: Fluctuation of endogenous gibberellin and abscisic acid levels in germinating seeds of barley. Biosci. Biotechnol. Biochem., 59, 1969-1970 (1995). – reference: 10) K. Washio: Functional dissections between GAMYB and Dof transcription factors suggest a role for protein-protein associations in the gibberellin-mediated expression of the RAmy 1 A gene in the rice aleurone. Plant Physiol., 133, 850-863 (2003). – reference: 16) Y. Ogawa: Effects of various factors on the increase of α-amylase activity in rice endosperm induced by gibberellin A3. Plant Cell Physiol., 7, 509-517 (1966). – reference: 4) P.J. Davies: Gibberellins: Regulators of plant height. in Plant Physiology, L. Taiz and E. Zeiger, eds., 3 rd Ed., Sinauer Associates, Inc., Sunderland, Massachusetts, pp. 461-492 (2002). – reference: 5) Y.-H. Choi, M. Kobayashi, S. Fujioka, T. Matsuno, T. Hirosawa and A. Sakurai: Fluctuation of endogenous gibberellin levels in the early development of rice. Biosci. Biotechnol. Biochem., 59, 285-288 (1995). – reference: 21) A. Dill and T.-P. Sun: Synergistic derepression of gibberellin signaling by removing RGA and GAI function in Arabidopsis thaliana. Genetics, 159, 777-785 (2001). – reference: 22) A.L. Silverstone, H.-S. Jung, A. Dill, H. Kawaide, Y. Kamiya and T.-P. Sun: Repressing a repressor: Gibberellin-induced rapid reduction of the RGA protein in Arabidopsis. Plant Cell, 13, 1555-1565 (2001). – reference: 13) S. Mitsunaga and J. Yamaguchi: Induction of α-amylase is repressed by uniconazole, an inhibitor of the biosynthesis of gibberellin, in a dwarf mutant of rice, Waito-C. Plant Cell Physiol., 34, 243-249 (1993). – reference: 9) A. Sasaki, H. Itoh, K. Gomi, M. Ueguchi-Tanaka, K. Ishiyama, M. Kobayashi, D.-H. Jeong, G. An, H. Kitano, M. Ashikari and M. Matsuoka: Accumulation of phosphorylated repressor for gibberellin signaling in an F-box mutant. Science, 299, 1896-1898 (2003). – reference: 20) S. Mitsunaga, T. Tashiro and J. Yamaguchi: Identification and characterization of gibberellin-insensitive mutants selected from among dwarf mutants of rice. Theor. Appl. Genet., 87, 705-712 (1994). – reference: 8) N.E.J. Appleford and J.R. Lenton: Hormonal regulation of α-amylase gene expression in germinating wheat (Triticum aestivum) grains. Physiol. Plant., 100, 534-542 (1997). – reference: 11) F.J. Woodger, F. Gubler, B.J. Pogson and J.V. Jacobsen: A Mak-like kinase is a repressor of GAMYB in barley aleurone. Plant J., 33, 707-717 (2003). |
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| Snippet | 穀類アリューロン細胞の活性化に伴う貯蔵デンプン分解の分子機構を理解するうえで, 発芽種子内に含まれる活性型ジベレリン (GA) の濃度を把握しておくことはきわめて重要である. そこで, アリューロン細胞におけるα-アミラーゼ合成誘導を指標に, イネの発芽種子に含まれる活性型GA濃度の簡易測定を試みた.... ... |
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| Title | イネ・アリューロン細胞におけるα-アミラーゼ合成誘導を指標とした活性型ジベレリン濃度の測定 |
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