To determine the role of alpha-amylase isoform I-1 in the degradation of starch in rice leaf chloroplasts, we generated a series of transgenic rice plants with suppressed expression or overexpression of alpha-amylase I-1. In the lines with suppressed expression of alpha-amylase I-1 at both the mRNA and protein levels, seed germination and seedling growth were markedly delayed in comparison with those in the wild-type plants. However, the growth retardation was overcome by supplementation of sugars. Interestingly, a significant increase of starch accumulation in the young leaf tissues was observed under a sugar-supplemented condition. In contrast, the starch content of leaves was reduced in the plants overexpressing alpha-amylase I-1. In immunocytochemical analysis with specific anti-alpha-amylase I-1 antiserum, immuno-gold particles deposited in the chloroplasts and extracellular space in young leaf cells. We further examined the expression and targeting of alpha-amylase I-1 fused with the green fluorescent protein in re-differentiated green cells, and showed that the fluorescence of the expressed fusion protein co-localized with the chlorophyll autofluorescence in the transgenic cells. In addition, mature protein species of alpha-amylase I-1 bearing an oligosaccharide side chain were detected in the isolated chloroplasts. Based on these results, we concluded that alpha-amylase I-1 targets the chloroplasts through the endoplasmic reticulum-Golgi system and plays a significant role in the starch degradation in rice leaves.

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...