Proteomic Identification of α-Amylase Isoforms Encoded by<i>RAmy3B/3C</i>from Germinating Rice Seeds

Nanjo, Yohei; Asatsuma, Satoru; Itoh, Kazuyoshi; Hori, Hidetaka; Mitsui, Toshiaki

doi:10.1271/bbb.68.112

Cited by 23 publications

(24 citation statements)

References 19 publications

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“…20) α Amylase II 5 6 encoded by RAmy3B C was also detected in the germinating seeds. 5) Similar to the other α amylase isoforms, the expression of α amylase II 5 6 was suggested be regulated by gibberellin and abscisic acid in germinating seeds. 5) As described above, α amylase is a key enzyme for germination and seedling growth of rice seeds.…”

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.ALPHA.-Amylase Affects Starch Accumulation in Rice Grains

Asatsuma¹,

Sawada²,

Kitajima³

et al. 2006

J. Appl. Glycosci.

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α Amylases (EC 3.2.1.1) in cereal are polymorphic enzymes. Ten distinct α amylase genes have been cloned and sequenced, 1 3) and more than 20 native α amylase isoforms have been identified and characterized in rice. 4 6) α Amylase I 1 encoded by RAmy1A is a glycoprotein bearing typical N linked oligosaccharide chains. 7,8) This isoform is heat stable compared with the other isoforms. The conjugation of an oligosaccharide side chain to polypeptide was thought to be involved in its heat stability.9,10) In germinating rice seeds, the expression of α amylase I 1 is regulated by gibberellin at both transcriptional and post transcriptional steps. 11,12) The enzyme is synthesized in the scutellar epithelium and the aleurone layer, and subsequently secreted to the starchy endosperm.6) The experimental data obtained by employing the transgenic rice plant with suppressed expression of α amylase I 1 revealed that this isoform plays an important role during seed germination and seedling growth. 13)α Amylase II 4 encoded by RAmy3D (Amy3) is actively expressed in suspension cultured cells derived from the rice embryo. The α amylase II 4 expression is regulated by sugars transcriptionally and post transcriptionally.14 18) At an early stage of germination, α amylase II 4 is a predominant isoform expressed in the coleoptile tissues of rice seed. This isoform is also expressed and secreted from the scutellar epithelium at the early stage of germination and the aleurone layer at the later stage.6) The expression of α amylase II 3 encoded by RAmy3E (Amy8) was detected in germinating seeds and suspension cultured cells derived from the embryo in rice. 3,5,19) The transcription of the α amylase II 3 gene has been reported to be regulated by sugars and hormones through Myb transacting factors. 20) α Amylase II 5 6 encoded by RAmy3B C was also detected in the germinating seeds.5) Similar to the other α amylase isoforms, the expression of α amylase II 5 6 was suggested be regulated by gibberellin and abscisic acid in germinating seeds. 5)As described above, α amylase is a key enzyme for germination and seedling growth of rice seeds. However, we found the typical α amylase isoforms I 1 and II 4 in ripening seeds. We examined the function of α amylase in grain during the ripening period in transgenic rice plants overexpressing the enzyme isoforms. MATERIALS AND METHODS Plant materials.The rice variety (Oryza sativa L. cv. Nipponbare) used in this study was supplied from the Niigata Agricultural Research Institute (Niigata, Japan).Assays. Starch and protein contents were determined as described by Matsukura et al . 21) and Bradford, 22) respectively. α Amylase activity was determined using β limit dextrin as the substrate. β limit dextrin was prepared by hydrolyzing potato starch solution (2% w v in 50 mM acetate buffer, pH 5.3), using crystalline potato β amylase free from α amylase (24 h, 30 C). 0.3% β limit dextrin dissolved in 50 mM acetate buffer (pH 5.3) containing 1 mM CaCl2 served as the substrate. The reaction mixture contain...

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confidence: 89%

“…3,5,19) The transcription of the α amylase II 3 gene has been reported to be regulated by sugars and hormones through Myb transacting factors. 20) α Amylase II 5 6 encoded by RAmy3B C was also detected in the germinating seeds.…”

mentioning

confidence: 99%

.ALPHA.-Amylase Affects Starch Accumulation in Rice Grains

Asatsuma¹,

Sawada²,

Kitajima³

et al. 2006

J. Appl. Glycosci.

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“…The several α-amylase isoforms identified in rice cells are grouped into two major classes, AmyI and AmyII, by their enzyme properties (Mitsui et al, 1996;Nanjo et al, 2004a;Kitajima et al, 2009). AmyI-1, II-3, II-4, II-5, and II-6 were the major isoforms in rice seedlings and suspensioncultured cells derived from embryos (Mitsui et al, 1996;Nanjo et al, 2004a). AmyI-1 expression levels were much greater in germinated seeds of all three cultivars than in non-germinating seeds (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…After electrophoresis, the separated proteins were transferred to polyvinylidene fluoride membranes (Hybond-P; GE Healthcare, USA) (Towbin et al, 1979). The membranes were incubated in 15 mM phosphate-buffered saline (pH 6.8) containing 0.1% Tween-20 and 5% skim milk for blocking, and then incubated with specific antibodies: antirice α-amylase isoform AmyI-1 (rabbit serum; 1:10 000), AmyII-3 (mouse serum; 1:500), AmyII-4 (rabbit serum; 1:5000) (Mitsui et al, 1996), and AmyII-6 (rabbit serum; 1:5000) (Nanjo et al, 2004a). Horseradish peroxidaseconjugated anti-rabbit IgG (Nacalai Tesque, Japan) and antimouse IgG (MP Biomedicals, USA) were used as secondary antibodies.…”

Section: Western Blotting Analysismentioning

confidence: 99%