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Aya Kitajima, Satoru Asatsuma,
Hisao Okada,
Yuki Hamada,
Kentaro Kaneko,
Yohei Nanjo,
Yasushi Kawagoe,
Kiminori Toyooka,
Ken Matsuoka,
Masaki Takeuchi,
Akihiko Nakano,
Toshiaki Mitsui
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ABSTRACT: The well-characterized secretory glycoprotein, rice (Oryza sativa) alpha-amylase isoform I-1 (AmyI-1), was localized within the plastids and proved to be involved in the degradation of starch granules in the organelles of rice cells. In addition, a large portion of transiently expressed AmyI-1 fused to green fluorescent protein (AmyI-1-GFP) colocalized with a simultaneously expressed fluorescent plastid marker in onion (Allium cepa) epidermal cells. The plastid targeting of AmyI-1 was inhibited by both dominant-negative and constitutively active mutants of Arabidopsis thaliana ARF1 and Arabidopsis SAR1, which arrest endoplasmic reticulum-to-Golgi traffic. In cells expressing fluorescent trans-Golgi and plastid markers, these fluorescent markers frequently colocalized when coexpressed with AmyI-1. Three-dimensional time-lapse imaging and electron microscopy of high-pressure frozen/freeze-substituted cells demonstrated that contact of the Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids occur within the cells. The transient expression of a series of C-terminal-truncated AmyI-1-GFP fusion proteins in the onion cell system showed that the region from Trp-301 to Gln-369 is necessary for plastid targeting of AmyI-1. Furthermore, the results obtained by site-directed mutations of Trp-302 and Gly-354, located on the surface and on opposite sides of the AmyI-1 protein, suggest that multiple surface regions are necessary for plastid targeting. Thus, Golgi-to-plastid traffic appears to be involved in the transport of glycoproteins to plastids and plastid targeting seems to be accomplished in a sorting signal-dependent manner.
The Plant Cell 09/2009; 21(9):2844-58. · 8.99 Impact Factor
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ABSTRACT: Secretory proteins and extracellular glycans are transported to the extracellular space during cell growth. These materials are carried in secretory vesicles generated at the trans-Golgi network (TGN). Analysis of the mammalian post-Golgi secretory pathway demonstrated the movement of separated secretory vesicles in the cell. Using secretory carrier membrane protein 2 (SCAMP2) as a marker for secretory vesicles and tobacco (Nicotiana tabacum) BY-2 cell as a model cell, we characterized the transport machinery in plant cells. A combination of analyses, including electron microscopy of quick-frozen cells and four-dimensional analysis of cells expressing fluorescent-tagged SCAMP2, enabled the identification of a clustered structure of secretory vesicles generated from TGN that moves in the cell and eventually fuses with plasma membrane. This structure was termed the secretory vesicle cluster (SVC). The SVC was also found in Arabidopsis thaliana and rice (Oryza sativa) cells and moved to the cell plate in dividing tobacco cells. Thus, the SVC is a motile structure involved in mass transport from the Golgi to the plasma membrane and cell plate in plant cells.
The Plant Cell 05/2009; 21(4):1212-29. · 8.99 Impact Factor
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ABSTRACT: We found that appropriate treatment with a highly potent and long-lasting abscisic acid analog enhanced the tissue expansion of scutellum during early seedling development of rice, accompanied by increases of protein and starch accumulation in the tissue. A comparative display of the protein expression patterns in the abscisic acid analog-treated and non-treated tissues on two dimensional gel electrophoretogram indicated that approximately 30% of the scutellar proteins were induced by abscisic acid. The abscisic acid-induced proteins included sucrose metabolizing, glycolytic, and ATP-producing enzymes. Most of these enzyme proteins also increased during the seedling growth. In addition, the expression of some isoforms of UDP-glucose pyrophosphorylase, 3-phosphoglycerate kinase, and mitochondrial ATP synthase beta chain was stimulated in the scutellum, with suppressed expression of alpha-amylase. We concluded that abscisic acid directly and indirectly stimulates the expression of numerous proteins, including carbohydrate metabolic enzymes, in scutellar tissues.
Bioscience Biotechnology and Biochemistry 06/2007; 71(5):1260-8. · 1.28 Impact Factor
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ABSTRACT: 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.
Plant and Cell Physiology 07/2005; 46(6):858-69. · 4.70 Impact Factor
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ABSTRACT: Hormonal regulation of expression of alpha-amylase II-4 that lacks the gibberellin-response cis-element (GARE) in the promoter region of the gene was studied in germinating rice (Oryza sativa L.) seeds. Temporal and spatial expression of alpha-amylase II-4 in the aleurone layer were essentially identical to those of alpha-amylase I-1 whose gene contains GARE, although these were distinguishable in the embryo tissues at the early stage of germination. The gibberellin-responsible expression of alpha-amylase II-4 was also similar to that of alpha-amylase I-1. However, the level of alpha-amylase II-4 mRNA was not increased by gibberellin, indicating that the transcriptional enhancement of alpha-amylase II-4 expression did not occur in the aleurone. Gibberellin stimulated the accumulation of 45Ca2+ into the intracellular secretory membrane system. In addition, several inhibitors for Ca2+ signaling, such as EGTA, neomycin, ruthenium red (RuR), and W-7 prevented the gibberellin-induced expression of alpha-amylase II-4 effectively. While the gibberellin-induced expression of alpha-amylase II-4 occurred normally in the aleurone layer of a rice dwarf mutant d1 which is defective in the alpha subunit of the heterotrimeric G protein. Based on these results, it was concluded that the posttranscriptional regulation of alpha-amylase II-4 expression by gibberellin operates in the aleurone layer of germinating rice seed, which is mediated by Ca2+ but not the G protein.
Plant Physiology and Biochemistry 07/2004; 42(6):477-84. · 2.84 Impact Factor
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ABSTRACT: We isolated and identified 10 alpha-amylase isoforms by using beta-cyclodextrin Sepharose affinity column chromatography and two-dimensional polyacrylamide gel electrophoresis from germinating rice (Oryza sativa L.) seeds. Immunoblots with anti-alpha-amylase I-1 and II-4 antibodies indicated that 8 isoforms in 10 are distinguishable from alpha-amylase I-1 and II-4. Peptide mass fingerprinting analysis showed that there exist novel isoforms encoded by RAmy3B and RAmy3C genes. The optimum temperature for enzyme reaction of the RAmy3B and RAmy3C coding isoforms resembled that of alpha-amylase isoform II-4 (RAmy3D). Furthermore, complex protein polymorphism resulted from a single alpha-amylase gene was found to occur not only in RAmy3D, but also in RAmy3B.
Bioscience Biotechnology and Biochemistry 02/2004; 68(1):112-8. · 1.28 Impact Factor
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ABSTRACT: We isolated and identified 10 α-amylase isoforms by using β-cyclodextrin Sepharose affinity column chromatography and two-dimensional polyacrylamide gel electrophoresis from germinating rice (Oryza sativa L.) seeds. Immunoblots with anti-α-amylase I-1 and II-4 antibodies indicated that 8 isoforms in 10 are distinguishable from α-amylase I-1 and II-4. Peptide mass fingerprinting analysis showed that there exist novel isoforms encoded by RAmy3B and RAmy3C genes. The optimum temperature for enzyme reaction of the RAmy3B and RAmy3C coding isoforms resembled that of α-amylase isoform II-4 (RAmy3D). Furthermore, complex protein polymorphism resulted from a single α-amylase gene was found to occur not only in RAmy3D, but also in RAmy3B.
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ABSTRACT: The aim of this reserch is clarify details of funtional expression of α-amylase in rice. Studies of suspension-cultured cell system provided evidences that phosphate is a potent stimulator for α-amylase secretion and starch degradation. Studies of transgenic rice plants with suppressed expression or overexpression of α-amylase showed that α-amylase is involved in degradation of plastidial starch in living cells. These results indecate that there exist multi-subcellular localization of α-amylase encoded by single gene in rice. Furthermore, α-amylase I-1 fused with green fluorescence protein was targeted into the plastids in bombarded onion epidermal cells, indicating that rice α-amylase I-1 molecule contains a plastid targeting signal common to both rice and onion cells. The cotents are follows. Novel Regulatory Factor for α-Amylase Secretion and Stach Degradation in Rice Cell Culture Effects of phosphate on the Ca2+ uptake and the sucrose-controlled secretion of α-amylase molecules in cultured cells were investigated, Phosphate markedly stimulated Ca2+ uptake into rice cells, particulary at the outer cell layer of the cell cluster. Phosphate at 10 mM was found to increse the synthesis and extracelluar liberaton of α-amylase II-4 molecules in the sucrose-supplemented cells. The distribution pattern of enzyme in rice cell clusters induced by phosphate was similar to that of Ca2+ uptake, Phosphate did not increase the level of mRNA of α-amylase II-4, indicating that phosphate stimulates the translation and posttranslational secretory processes of α-amylase II-4 molecules in the presence of sucrose. Furthermore, phosphate enhanced both the Ca2+ uptake and α-amylase II-4 synthesis in the microsomes. These results strongly suggested that the ratio of phosphate to sugar is important for regulating the Ca2+ uptake, and that phosphate and sugar precisely coordinate the Ca2+-mediated synthesis and extracelluar liberation of α-amylase II-4 molecules in rice cells. In addition, phosphate reduced the starch content in rice cells, same as the sugar-starved cells. The results may indicate that α-amylase plays a role for degrading starch in living cells. Involment of α-Amylase in Starch Degradation in Rice Plastids To determine role of α-amylase isoform I-1 in the degradation of starch in rice leaf chloroplasts, I renerated a series of transgenic rice plants with suppressed expression of α-amylase I-1. In the lines with suppressed expression or overexpression of α-amylase I-1 at both mRNA and protein levels, seed germinaton and seedling growth were markedly delayed in comparison with those in the wild-type plants. Houever, the growth retardation was overcome by supplementation of sugars. Interestingly, a significant increse 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 overexpressinf α-amylase i-1. In immunocytochemicalanalysis with specific anti-α-amylase I-1 antiserum, immuno-gold particles deposited in the choroplasts and estracellular space in young leaf cells. I further examined the expression and targeting of α-amylase I-1 fused with the green fluorescent protein in re-differentiated green cells, and showed that the fluorescence of expressed fusion protein co-localized with the chlorophyll autofluorescence in the transgenic cells. In addition, mature protein species of α-amylase I-1 bearing an oligosaccharide side chain were detected in the isolated chloroplasts. Based on these results, I concluded that α-amylase I-1 targets the chloroplasts through the ER-Golgi system and plays a significant role in the starch degradation in rice leaves. Overexpression of α-Amylase affects Starch accumulation in Ripening Seeds of Rice To determine physiological function of α-amylase isoform II-4, I generated a series transgenic rice plants with overexpression of α-amylase II-4. In the line with overexpression of α-amylase II-4 at both the mRNA and protein levels, the ability of seed germination and seedling growth was unaltered. Unlike α-amylase I-1, starch accumulation in leaf tissues overexpressing α-amylase II-4 was scarcely changed. However, the seeds produced in the transgenic rice plants exhibited an abnormal accumulation of starch, as well as that in rice overexpressing α-amylase I-1. Based on these in endosperm of ripening seed and controls accumulation of reserve stach in rice.
