The Experts below are selected from a list of 1413 Experts worldwide ranked by ideXlab platform
Enrico Martinoia - One of the best experts on this subject based on the ideXlab platform.
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A rice ABC transporter, OsABCC1, reduces arsenic accumulation in the grain
'Indiana University Press (Project Muse)', 2018Co-Authors: Song, Won Yong, Enrico Martinoia, Yamaki T, Yamaji N, Ko D, Kh Jung, Fujii-kashino M, An G, Lee YAbstract:Arsenic (As) is a chronic poison that causes severe skin lesions and cancer. Rice (Oryza sativa L.) is a major dietary source of As; therefore, reducing As accumulation in the rice grain and thereby diminishing the amount of As that enters the food chain is of critical importance. Here, we report that a member of the Oryza sativa C-type ATP-binding cassette (ABC) transporter (OsABCC) family, OsABCC1, is involved in the detoxification and reduction of As in rice grains. We found that OsABCC1 was expressed in many organs, including the roots, leaves, nodes, peduncle, and rachis. Expression was not affected when plants were exposed to low levels of As but was up-regulated in response to high levels of As. In both the basal nodes and upper nodes, which are connected to the panicle, OsABCC1 was localized to the Phloem region of vascular bundles. Furthermore, OsABCC1 was localized to the tonoplast and conferred phytochelatin-dependent As resistance in yeast. Knockout of OsABCC1 in rice resulted in decreased tolerance to As, but did not affect cadmium toxicity. At the reproductive growth stage, the As content was higher in the nodes and in other tissues of wild-type rice than in those of OsABCC1 knockout mutants, but was significantly lower in the grain. Taken together, our results indicate that OsABCC1 limits As transport to the grains by sequestering As in the vacuoles of the Phloem Companion Cells of the nodes in rice.open17318
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a rice abc transporter osabcc1 reduces arsenic accumulation in the grain
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Won Yong Song, Tomohiro Yamaki, Donghwi Ko, Miho Fujiikashino, Gynheung An, Kihong Jung, Naoki Yamaji, Enrico MartinoiaAbstract:Arsenic (As) is a chronic poison that causes severe skin lesions and cancer. Rice (Oryza sativa L.) is a major dietary source of As; therefore, reducing As accumulation in the rice grain and thereby diminishing the amount of As that enters the food chain is of critical importance. Here, we report that a member of the Oryza sativa C-type ATP-binding cassette (ABC) transporter (OsABCC) family, OsABCC1, is involved in the detoxification and reduction of As in rice grains. We found that OsABCC1 was expressed in many organs, including the roots, leaves, nodes, peduncle, and rachis. Expression was not affected when plants were exposed to low levels of As but was up-regulated in response to high levels of As. In both the basal nodes and upper nodes, which are connected to the panicle, OsABCC1 was localized to the Phloem region of vascular bundles. Furthermore, OsABCC1 was localized to the tonoplast and conferred phytochelatin-dependent As resistance in yeast. Knockout of OsABCC1 in rice resulted in decreased tolerance to As, but did not affect cadmium toxicity. At the reproductive growth stage, the As content was higher in the nodes and in other tissues of wild-type rice than in those of OsABCC1 knockout mutants, but was significantly lower in the grain. Taken together, our results indicate that OsABCC1 limits As transport to the grains by sequestering As in the vacuoles of the Phloem Companion Cells of the nodes in rice.
Tomoyuki Yamaya - One of the best experts on this subject based on the ideXlab platform.
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Additional file 2: of Reduction in sucrose contents by downregulation of fructose-1,6-bisphosphatase 2 causes tiller outgrowth cessation in rice mutants lacking glutamine synthetase1;2
2018Co-Authors: Miwa Ohashi, Toshihiko Hayakawa, Keiki Ishiyama, Soichi Kojima, Miyako Kusano, Atsushi Fukushima, Tomoyuki YamayaAbstract:Figure S2. In situ hybridization of OscFBP1 in the basal portions of the shoots. Longitudinal sections of the basal portions of the shoots were prepared from wild-type (WT) (a-c, g-i) and gs1;2 mutant (gs1;2) (d-f) rice grown hydroponically in the presence of 1 mM NH4Cl until the fourth leaf stage. The antisense probe for the OscFBP1 transcript was hybridized with the longitudinal sections from the shoot basal portions of wild-type (a-c) and gs1;2 mutant (d-f) rice. The sense probe for the OscFBP1 transcript was hybridized with the sections from the shoot basal portions of the wild type (g-i) as a negative control. The Phloem Companion Cells of the nodal vascular anastomoses (a, d, g), the internodal parenchyma Cells (b, e, h), and the leaf sheath (c, f, i) are shown. The red arrowhead in (a, d) indicates the hybridization signal of the OscFBP1 transcript in the Phloem Companion Cells of the nodal vascular anastomoses. Abbreviation: cc, Companion cell. Scale bars: 20 μm (a, b, d, e, g, h) and 50 μm (c, f, i). (TIF 5981 kb
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Outgrowth of Rice Tillers Requires Availability of Glutamine in the Basal Portions of Shoots
'Springer Science and Business Media LLC', 2018Co-Authors: Miwa Ohashi, Toshihiko Hayakawa, Keiki Ishiyama, Soichi Kojima, Noriyuki Konishi, Kazuhiro Sasaki, Mitsue Miyao, Tomoyuki YamayaAbstract:Abstract Background Our previous studies concluded that metabolic disorder in the basal portions of rice shoots caused by a lack of cytosolic glutamine synthetase1;2 (GS1;2) resulted in a severe reduction in the outgrowth of tillers. Rice mutants lacking GS1;2 (gs1;2 mutants) showed a remarkable reduction in the contents of both glutamine and asparagine in the basal portions of shoots. In the current study, we attempted to reveal the mechanisms for this decrease in asparagine content using rice mutants lacking either GS1;2 or asparagine synthetase 1 (AS1). The contributions of the availability of glutamine and asparagine to the outgrowth of rice tillers were investigated. Results Rice has two AS genes, and the enzymes catalyse asparagine synthesis from glutamine. In the basal portions of rice shoots, expression of OsAS1, the major species in this tissue, was reduced in gs1;2 mutants, whereas OsAS2 expression was relatively constant. OsAS1 was expressed in Phloem Companion Cells of the nodal vascular anastomoses connected to the axillary bud vasculatures in the basal portions of wild-type shoots, whereas cell-specific expression was markedly reduced in gs1;2 mutants. OsAS1 was up-regulated significantly by NH4 + supply in the wild type but not in gs1;2 mutants. When GS reactions were inhibited by methionine sulfoximine, OsAS1 was up-regulated by glutamine but not by NH4 +. The rice mutants lacking AS1 (as1 mutants) showed a decrease in asparagine content in the basal portions of shoots. However, glutamine content and tiller number were less affected by the lack of AS1. Conclusion These results indicate that in Phloem Companion Cells of the nodal vascular anastomoses, asparagine synthesis is largely dependent on glutamine or its related metabolite-responsive AS1. Thus, the decrease in glutamine content caused by a lack of GS1;2 is suggested to result in low expression of OsAS1, decreasing asparagine content. However, the availability of asparagine generated from AS1 reactions is apparently less effective for the outgrowth of tillers. With respect to the tiller number and the contents of glutamine and asparagine in gs1;2 and as1 mutants, the availability of glutamine rather than asparagine in basal portions of rice shoots may be required for the outgrowth of rice tillers
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Reduction in sucrose contents by downregulation of fructose-1,6-bisphosphatase 2 causes tiller outgrowth cessation in rice mutants lacking glutamine synthetase1;2
'Springer Science and Business Media LLC', 2018Co-Authors: Miwa Ohashi, Toshihiko Hayakawa, Keiki Ishiyama, Soichi Kojima, Miyako Kusano, Atsushi Fukushima, Tomoyuki YamayaAbstract:Abstract Background Our previous transcriptomic analysis revealed that downregulation of nitrogen and carbon metabolism in the basal portions of the shoots inhibited cytosolic glutamine synthetase1;2 (GS1;2), which severely reduced rice tiller number. In the present study, we used rice mutants lacking GS1;2 (gs1;2 mutants) to determine the contribution of carbon metabolism to tiller growth. Results Metabolomic analysis indicated the effects of carbon metabolism disorder such as reductions in the levels of sugar metabolites (e.g., sucrose and glucose 6-phosphate) in the shoot basal portions of the gs1;2 mutant seedlings. Decrease in sucrose caused by the lack of GS1;2 was successfully restored to the wild-type levels by introducing OsGS1;2 cDNA into the mutants. In the basal portions of the shoots, the lack of GS1;2 caused low expression of cytosolic fructose 1,6-bisphosphatase2 (OscFBP2), which is a key cytosolic sucrose synthesis enzyme; it is especially important in the Phloem Companion Cells of the nodal vascular anastomoses. NH4 + supply upregulated OscFBP2 expression in the shoot basal portions of the wild type but not in those of the gs1;2 mutants. Rice mutants lacking cFBPase2 presented with ~ 30% reduction in total cFBPase activity in the basal portions of their shoots. These mutants displayed reductions in sucrose levels of the basal portions of their shoots but not in their leaf blades. They also had relatively lower tiller numbers at the early growth stage. Conclusions Metabolomic analysis revealed that the lack of GS1;2 reduced sucrose metabolism in the basal portions of the shoots. Our results indicated that sucrose reduction was caused by the downregulation of OscFBP2 expression in the basal portions of the gs1;2 mutant shoots. The reduction in sucrose content caused by the lack of cFBPase2 resulted in lower tiller number at the early growth stage. Therefore, adequate sucrose supply via cFBPase2 may be necessary for tiller growth in the basal portions of rice shoots
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lack of cytosolic glutamine synthetase1 2 activity reduces nitrogen dependent biosynthesis of cytokinin required for axillary bud outgrowth in rice seedlings
Plant and Cell Physiology, 2017Co-Authors: Miwa Ohashi, Tomoyuki Yamaya, Keiki Ishiyama, Soichi Kojima, Mikiko Kojima, Hitoshi Sakakibara, Toshihiko HayakawaAbstract:: A mutation abolishing cytosolic glutamine synthetase1;2 (GS1;2) activity impairs assimilation of ammonium into glutamine in both roots and basal portions of shoots, and severely decreases axillary bud outgrowth (tillering) in mutant rice seedlings. Although the gs1;2 mutant phenotype is independent of strigolactone, which inhibits tillering, it also demonstrates glutamine- or related metabolite-responsive biosynthesis of cytokinin (CK), which promotes tillering. Here, we examined the connection between GS1;2 and CK biosynthesis during tillering, focusing on basal portions of the shoots as well as apical and axillary bud meristems in the gs1;2 mutant. Despite a sufficient ammonium supply, decreases in precursor CK contents and a decrease in ammonium assimilation into glutamine were observed in basal portions of mutant shoots. Reintroducing expression of OsGS1;2 cDNA driven by its own promoter restored precursor CK contents and ammonium assimilation to wild-type levels. In basal portions of the shoots, glutamine-responsive adenosine phosphate-isopentenyltransferase4 (OsIPT4), which is also predominant in rice roots, was the predominant isogene for IPT, which synthesizes CK. Cell-specific expression of OsIPT4 in Phloem Companion Cells in nodal vascular anastomoses connected to the axillary bud vasculature also decreased in the gs1;2 mutant. Expression of CK-responsive type-A response regulator genes as local indicators of active CKs was also abolished in the axillary bud meristem of the mutant. These results suggest that the lack of GS1;2 activity decreased levels of glutamine or a related metabolite required for CK biosynthesis, causing a deficiency in active CK in the axillary bud meristem necessary for tillering.
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disruption of a novel nadh glutamate synthase2 gene caused marked reduction in spikelet number of rice
Frontiers in Plant Science, 2011Co-Authors: Wataru Tamura, Toshihiko Hayakawa, Soichi Kojima, Ayako Toyokawa, Hideo Watanabe, Mayumi Tabuchikobayashi, Tomoyuki YamayaAbstract:Inorganic ammonium ions are assimilated by a coupled reaction of glutamine synthetase and glutamate synthase (GOGAT). In rice, three genes encoding either ferredoxin (Fd)-GOGAT, NADH-GOGAT1, or NADH-GOGAT2, have been identified. OsNADH-GOGAT2, a newly identified gene, was expressed mainly in fully expanded leaf blades and leaf sheaths. Although the distinct expression profile to OsNADH-GOGAT1, which is mainly detected in root tips, developing leaf blades and grains, was shown in our previous studies, physiological role of NADH-GOGAT2 is not yet known. Here, we isolated retrotransposon mediated-knockout mutants lacking OsNADH-GOGAT2. In rice grown under paddy field conditions, disruption of the OsNADH-GOGAT2 gene caused a remarkable decrease in spikelet number per panicle associated with a reductions in yield and whole plant biomass, when compared with wild-type plants. Expression of this gene was mainly detected in Phloem Companion Cells and Phloem parenchyma Cells associated with large vascular bundles in fully expanded leaf blades, when the promoter region fused with a β-glucuronidase gene was introduced into the wild-type rice. These results suggest that the NADH-GOGAT2 is important in the process of glutamine generation in senescing leaves for the remobilization of leaf nitrogen through Phloem to the panicle during natural senescence. These results also indicate that other GOGATs, i.e. NADH-GOGAT1 and ferredoxin-GOGAT are not able to compensate the function of NADH-GOGAT2.
Toshihiko Hayakawa - One of the best experts on this subject based on the ideXlab platform.
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Additional file 2: of Reduction in sucrose contents by downregulation of fructose-1,6-bisphosphatase 2 causes tiller outgrowth cessation in rice mutants lacking glutamine synthetase1;2
2018Co-Authors: Miwa Ohashi, Toshihiko Hayakawa, Keiki Ishiyama, Soichi Kojima, Miyako Kusano, Atsushi Fukushima, Tomoyuki YamayaAbstract:Figure S2. In situ hybridization of OscFBP1 in the basal portions of the shoots. Longitudinal sections of the basal portions of the shoots were prepared from wild-type (WT) (a-c, g-i) and gs1;2 mutant (gs1;2) (d-f) rice grown hydroponically in the presence of 1 mM NH4Cl until the fourth leaf stage. The antisense probe for the OscFBP1 transcript was hybridized with the longitudinal sections from the shoot basal portions of wild-type (a-c) and gs1;2 mutant (d-f) rice. The sense probe for the OscFBP1 transcript was hybridized with the sections from the shoot basal portions of the wild type (g-i) as a negative control. The Phloem Companion Cells of the nodal vascular anastomoses (a, d, g), the internodal parenchyma Cells (b, e, h), and the leaf sheath (c, f, i) are shown. The red arrowhead in (a, d) indicates the hybridization signal of the OscFBP1 transcript in the Phloem Companion Cells of the nodal vascular anastomoses. Abbreviation: cc, Companion cell. Scale bars: 20 μm (a, b, d, e, g, h) and 50 μm (c, f, i). (TIF 5981 kb
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Outgrowth of Rice Tillers Requires Availability of Glutamine in the Basal Portions of Shoots
'Springer Science and Business Media LLC', 2018Co-Authors: Miwa Ohashi, Toshihiko Hayakawa, Keiki Ishiyama, Soichi Kojima, Noriyuki Konishi, Kazuhiro Sasaki, Mitsue Miyao, Tomoyuki YamayaAbstract:Abstract Background Our previous studies concluded that metabolic disorder in the basal portions of rice shoots caused by a lack of cytosolic glutamine synthetase1;2 (GS1;2) resulted in a severe reduction in the outgrowth of tillers. Rice mutants lacking GS1;2 (gs1;2 mutants) showed a remarkable reduction in the contents of both glutamine and asparagine in the basal portions of shoots. In the current study, we attempted to reveal the mechanisms for this decrease in asparagine content using rice mutants lacking either GS1;2 or asparagine synthetase 1 (AS1). The contributions of the availability of glutamine and asparagine to the outgrowth of rice tillers were investigated. Results Rice has two AS genes, and the enzymes catalyse asparagine synthesis from glutamine. In the basal portions of rice shoots, expression of OsAS1, the major species in this tissue, was reduced in gs1;2 mutants, whereas OsAS2 expression was relatively constant. OsAS1 was expressed in Phloem Companion Cells of the nodal vascular anastomoses connected to the axillary bud vasculatures in the basal portions of wild-type shoots, whereas cell-specific expression was markedly reduced in gs1;2 mutants. OsAS1 was up-regulated significantly by NH4 + supply in the wild type but not in gs1;2 mutants. When GS reactions were inhibited by methionine sulfoximine, OsAS1 was up-regulated by glutamine but not by NH4 +. The rice mutants lacking AS1 (as1 mutants) showed a decrease in asparagine content in the basal portions of shoots. However, glutamine content and tiller number were less affected by the lack of AS1. Conclusion These results indicate that in Phloem Companion Cells of the nodal vascular anastomoses, asparagine synthesis is largely dependent on glutamine or its related metabolite-responsive AS1. Thus, the decrease in glutamine content caused by a lack of GS1;2 is suggested to result in low expression of OsAS1, decreasing asparagine content. However, the availability of asparagine generated from AS1 reactions is apparently less effective for the outgrowth of tillers. With respect to the tiller number and the contents of glutamine and asparagine in gs1;2 and as1 mutants, the availability of glutamine rather than asparagine in basal portions of rice shoots may be required for the outgrowth of rice tillers
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Reduction in sucrose contents by downregulation of fructose-1,6-bisphosphatase 2 causes tiller outgrowth cessation in rice mutants lacking glutamine synthetase1;2
'Springer Science and Business Media LLC', 2018Co-Authors: Miwa Ohashi, Toshihiko Hayakawa, Keiki Ishiyama, Soichi Kojima, Miyako Kusano, Atsushi Fukushima, Tomoyuki YamayaAbstract:Abstract Background Our previous transcriptomic analysis revealed that downregulation of nitrogen and carbon metabolism in the basal portions of the shoots inhibited cytosolic glutamine synthetase1;2 (GS1;2), which severely reduced rice tiller number. In the present study, we used rice mutants lacking GS1;2 (gs1;2 mutants) to determine the contribution of carbon metabolism to tiller growth. Results Metabolomic analysis indicated the effects of carbon metabolism disorder such as reductions in the levels of sugar metabolites (e.g., sucrose and glucose 6-phosphate) in the shoot basal portions of the gs1;2 mutant seedlings. Decrease in sucrose caused by the lack of GS1;2 was successfully restored to the wild-type levels by introducing OsGS1;2 cDNA into the mutants. In the basal portions of the shoots, the lack of GS1;2 caused low expression of cytosolic fructose 1,6-bisphosphatase2 (OscFBP2), which is a key cytosolic sucrose synthesis enzyme; it is especially important in the Phloem Companion Cells of the nodal vascular anastomoses. NH4 + supply upregulated OscFBP2 expression in the shoot basal portions of the wild type but not in those of the gs1;2 mutants. Rice mutants lacking cFBPase2 presented with ~ 30% reduction in total cFBPase activity in the basal portions of their shoots. These mutants displayed reductions in sucrose levels of the basal portions of their shoots but not in their leaf blades. They also had relatively lower tiller numbers at the early growth stage. Conclusions Metabolomic analysis revealed that the lack of GS1;2 reduced sucrose metabolism in the basal portions of the shoots. Our results indicated that sucrose reduction was caused by the downregulation of OscFBP2 expression in the basal portions of the gs1;2 mutant shoots. The reduction in sucrose content caused by the lack of cFBPase2 resulted in lower tiller number at the early growth stage. Therefore, adequate sucrose supply via cFBPase2 may be necessary for tiller growth in the basal portions of rice shoots
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lack of cytosolic glutamine synthetase1 2 activity reduces nitrogen dependent biosynthesis of cytokinin required for axillary bud outgrowth in rice seedlings
Plant and Cell Physiology, 2017Co-Authors: Miwa Ohashi, Tomoyuki Yamaya, Keiki Ishiyama, Soichi Kojima, Mikiko Kojima, Hitoshi Sakakibara, Toshihiko HayakawaAbstract:: A mutation abolishing cytosolic glutamine synthetase1;2 (GS1;2) activity impairs assimilation of ammonium into glutamine in both roots and basal portions of shoots, and severely decreases axillary bud outgrowth (tillering) in mutant rice seedlings. Although the gs1;2 mutant phenotype is independent of strigolactone, which inhibits tillering, it also demonstrates glutamine- or related metabolite-responsive biosynthesis of cytokinin (CK), which promotes tillering. Here, we examined the connection between GS1;2 and CK biosynthesis during tillering, focusing on basal portions of the shoots as well as apical and axillary bud meristems in the gs1;2 mutant. Despite a sufficient ammonium supply, decreases in precursor CK contents and a decrease in ammonium assimilation into glutamine were observed in basal portions of mutant shoots. Reintroducing expression of OsGS1;2 cDNA driven by its own promoter restored precursor CK contents and ammonium assimilation to wild-type levels. In basal portions of the shoots, glutamine-responsive adenosine phosphate-isopentenyltransferase4 (OsIPT4), which is also predominant in rice roots, was the predominant isogene for IPT, which synthesizes CK. Cell-specific expression of OsIPT4 in Phloem Companion Cells in nodal vascular anastomoses connected to the axillary bud vasculature also decreased in the gs1;2 mutant. Expression of CK-responsive type-A response regulator genes as local indicators of active CKs was also abolished in the axillary bud meristem of the mutant. These results suggest that the lack of GS1;2 activity decreased levels of glutamine or a related metabolite required for CK biosynthesis, causing a deficiency in active CK in the axillary bud meristem necessary for tillering.
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lack of cytosolic glutamine synthetase1 2 in vascular tissues of axillary buds causes severe reduction in their outgrowth and disorder of metabolic balance in rice seedlings
Plant Journal, 2015Co-Authors: Miwa Ohashi, Toshihiko Hayakawa, Keiki Ishiyama, Soichi Kojima, Miyako Kusano, Atsushi Fukushima, Atsushi Hanada, Keiichi Kanno, Yoshiya SetoAbstract:Summary The development and elongation of active tillers in rice was severely reduced by a lack of cytosolic glutamine synthetase1;2 (GS1;2), and, to a lesser extent, lack of NADH-glutamate synthase1 in knockout mutants. In situ hybridization using the basal part of wild-type seedlings clearly showed that expression of OsGS1;2 was detected in the Phloem Companion Cells of the nodal vascular anastomoses and large vascular bundles of axillary buds. Accumulation of lignin, visualized using phloroglucin HCl, was also observed in these tissues. The lack of GS1;2 resulted in reduced accumulation of lignin. Re-introduction into the mutants of OsGS1;2 cDNA under the control of its own promoter successfully restored the outgrowth of tillers and lignin deposition to wild-type levels. Transcriptomic analysis using a 5 mm basal region of rice shoots showed that the GS1;2 mutants accumulated reduced amounts of mRNAs for carbon and nitrogen metabolism, including C1 unit transfer in lignin synthesis. Although a high content of strigolactone in rice roots is known to reduce active tiller number, the reduction of outgrowth of axillary buds observed in the GS1;2 mutants was independent of the level of strigolactone. Thus metabolic disorder caused by the lack of GS1;2 resulted in a severe reduction in the outgrowth of axillary buds and lignin deposition.
Soichi Kojima - One of the best experts on this subject based on the ideXlab platform.
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Additional file 2: of Reduction in sucrose contents by downregulation of fructose-1,6-bisphosphatase 2 causes tiller outgrowth cessation in rice mutants lacking glutamine synthetase1;2
2018Co-Authors: Miwa Ohashi, Toshihiko Hayakawa, Keiki Ishiyama, Soichi Kojima, Miyako Kusano, Atsushi Fukushima, Tomoyuki YamayaAbstract:Figure S2. In situ hybridization of OscFBP1 in the basal portions of the shoots. Longitudinal sections of the basal portions of the shoots were prepared from wild-type (WT) (a-c, g-i) and gs1;2 mutant (gs1;2) (d-f) rice grown hydroponically in the presence of 1 mM NH4Cl until the fourth leaf stage. The antisense probe for the OscFBP1 transcript was hybridized with the longitudinal sections from the shoot basal portions of wild-type (a-c) and gs1;2 mutant (d-f) rice. The sense probe for the OscFBP1 transcript was hybridized with the sections from the shoot basal portions of the wild type (g-i) as a negative control. The Phloem Companion Cells of the nodal vascular anastomoses (a, d, g), the internodal parenchyma Cells (b, e, h), and the leaf sheath (c, f, i) are shown. The red arrowhead in (a, d) indicates the hybridization signal of the OscFBP1 transcript in the Phloem Companion Cells of the nodal vascular anastomoses. Abbreviation: cc, Companion cell. Scale bars: 20 μm (a, b, d, e, g, h) and 50 μm (c, f, i). (TIF 5981 kb
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Outgrowth of Rice Tillers Requires Availability of Glutamine in the Basal Portions of Shoots
'Springer Science and Business Media LLC', 2018Co-Authors: Miwa Ohashi, Toshihiko Hayakawa, Keiki Ishiyama, Soichi Kojima, Noriyuki Konishi, Kazuhiro Sasaki, Mitsue Miyao, Tomoyuki YamayaAbstract:Abstract Background Our previous studies concluded that metabolic disorder in the basal portions of rice shoots caused by a lack of cytosolic glutamine synthetase1;2 (GS1;2) resulted in a severe reduction in the outgrowth of tillers. Rice mutants lacking GS1;2 (gs1;2 mutants) showed a remarkable reduction in the contents of both glutamine and asparagine in the basal portions of shoots. In the current study, we attempted to reveal the mechanisms for this decrease in asparagine content using rice mutants lacking either GS1;2 or asparagine synthetase 1 (AS1). The contributions of the availability of glutamine and asparagine to the outgrowth of rice tillers were investigated. Results Rice has two AS genes, and the enzymes catalyse asparagine synthesis from glutamine. In the basal portions of rice shoots, expression of OsAS1, the major species in this tissue, was reduced in gs1;2 mutants, whereas OsAS2 expression was relatively constant. OsAS1 was expressed in Phloem Companion Cells of the nodal vascular anastomoses connected to the axillary bud vasculatures in the basal portions of wild-type shoots, whereas cell-specific expression was markedly reduced in gs1;2 mutants. OsAS1 was up-regulated significantly by NH4 + supply in the wild type but not in gs1;2 mutants. When GS reactions were inhibited by methionine sulfoximine, OsAS1 was up-regulated by glutamine but not by NH4 +. The rice mutants lacking AS1 (as1 mutants) showed a decrease in asparagine content in the basal portions of shoots. However, glutamine content and tiller number were less affected by the lack of AS1. Conclusion These results indicate that in Phloem Companion Cells of the nodal vascular anastomoses, asparagine synthesis is largely dependent on glutamine or its related metabolite-responsive AS1. Thus, the decrease in glutamine content caused by a lack of GS1;2 is suggested to result in low expression of OsAS1, decreasing asparagine content. However, the availability of asparagine generated from AS1 reactions is apparently less effective for the outgrowth of tillers. With respect to the tiller number and the contents of glutamine and asparagine in gs1;2 and as1 mutants, the availability of glutamine rather than asparagine in basal portions of rice shoots may be required for the outgrowth of rice tillers
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Reduction in sucrose contents by downregulation of fructose-1,6-bisphosphatase 2 causes tiller outgrowth cessation in rice mutants lacking glutamine synthetase1;2
'Springer Science and Business Media LLC', 2018Co-Authors: Miwa Ohashi, Toshihiko Hayakawa, Keiki Ishiyama, Soichi Kojima, Miyako Kusano, Atsushi Fukushima, Tomoyuki YamayaAbstract:Abstract Background Our previous transcriptomic analysis revealed that downregulation of nitrogen and carbon metabolism in the basal portions of the shoots inhibited cytosolic glutamine synthetase1;2 (GS1;2), which severely reduced rice tiller number. In the present study, we used rice mutants lacking GS1;2 (gs1;2 mutants) to determine the contribution of carbon metabolism to tiller growth. Results Metabolomic analysis indicated the effects of carbon metabolism disorder such as reductions in the levels of sugar metabolites (e.g., sucrose and glucose 6-phosphate) in the shoot basal portions of the gs1;2 mutant seedlings. Decrease in sucrose caused by the lack of GS1;2 was successfully restored to the wild-type levels by introducing OsGS1;2 cDNA into the mutants. In the basal portions of the shoots, the lack of GS1;2 caused low expression of cytosolic fructose 1,6-bisphosphatase2 (OscFBP2), which is a key cytosolic sucrose synthesis enzyme; it is especially important in the Phloem Companion Cells of the nodal vascular anastomoses. NH4 + supply upregulated OscFBP2 expression in the shoot basal portions of the wild type but not in those of the gs1;2 mutants. Rice mutants lacking cFBPase2 presented with ~ 30% reduction in total cFBPase activity in the basal portions of their shoots. These mutants displayed reductions in sucrose levels of the basal portions of their shoots but not in their leaf blades. They also had relatively lower tiller numbers at the early growth stage. Conclusions Metabolomic analysis revealed that the lack of GS1;2 reduced sucrose metabolism in the basal portions of the shoots. Our results indicated that sucrose reduction was caused by the downregulation of OscFBP2 expression in the basal portions of the gs1;2 mutant shoots. The reduction in sucrose content caused by the lack of cFBPase2 resulted in lower tiller number at the early growth stage. Therefore, adequate sucrose supply via cFBPase2 may be necessary for tiller growth in the basal portions of rice shoots
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lack of cytosolic glutamine synthetase1 2 activity reduces nitrogen dependent biosynthesis of cytokinin required for axillary bud outgrowth in rice seedlings
Plant and Cell Physiology, 2017Co-Authors: Miwa Ohashi, Tomoyuki Yamaya, Keiki Ishiyama, Soichi Kojima, Mikiko Kojima, Hitoshi Sakakibara, Toshihiko HayakawaAbstract:: A mutation abolishing cytosolic glutamine synthetase1;2 (GS1;2) activity impairs assimilation of ammonium into glutamine in both roots and basal portions of shoots, and severely decreases axillary bud outgrowth (tillering) in mutant rice seedlings. Although the gs1;2 mutant phenotype is independent of strigolactone, which inhibits tillering, it also demonstrates glutamine- or related metabolite-responsive biosynthesis of cytokinin (CK), which promotes tillering. Here, we examined the connection between GS1;2 and CK biosynthesis during tillering, focusing on basal portions of the shoots as well as apical and axillary bud meristems in the gs1;2 mutant. Despite a sufficient ammonium supply, decreases in precursor CK contents and a decrease in ammonium assimilation into glutamine were observed in basal portions of mutant shoots. Reintroducing expression of OsGS1;2 cDNA driven by its own promoter restored precursor CK contents and ammonium assimilation to wild-type levels. In basal portions of the shoots, glutamine-responsive adenosine phosphate-isopentenyltransferase4 (OsIPT4), which is also predominant in rice roots, was the predominant isogene for IPT, which synthesizes CK. Cell-specific expression of OsIPT4 in Phloem Companion Cells in nodal vascular anastomoses connected to the axillary bud vasculature also decreased in the gs1;2 mutant. Expression of CK-responsive type-A response regulator genes as local indicators of active CKs was also abolished in the axillary bud meristem of the mutant. These results suggest that the lack of GS1;2 activity decreased levels of glutamine or a related metabolite required for CK biosynthesis, causing a deficiency in active CK in the axillary bud meristem necessary for tillering.
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lack of cytosolic glutamine synthetase1 2 in vascular tissues of axillary buds causes severe reduction in their outgrowth and disorder of metabolic balance in rice seedlings
Plant Journal, 2015Co-Authors: Miwa Ohashi, Toshihiko Hayakawa, Keiki Ishiyama, Soichi Kojima, Miyako Kusano, Atsushi Fukushima, Atsushi Hanada, Keiichi Kanno, Yoshiya SetoAbstract:Summary The development and elongation of active tillers in rice was severely reduced by a lack of cytosolic glutamine synthetase1;2 (GS1;2), and, to a lesser extent, lack of NADH-glutamate synthase1 in knockout mutants. In situ hybridization using the basal part of wild-type seedlings clearly showed that expression of OsGS1;2 was detected in the Phloem Companion Cells of the nodal vascular anastomoses and large vascular bundles of axillary buds. Accumulation of lignin, visualized using phloroglucin HCl, was also observed in these tissues. The lack of GS1;2 resulted in reduced accumulation of lignin. Re-introduction into the mutants of OsGS1;2 cDNA under the control of its own promoter successfully restored the outgrowth of tillers and lignin deposition to wild-type levels. Transcriptomic analysis using a 5 mm basal region of rice shoots showed that the GS1;2 mutants accumulated reduced amounts of mRNAs for carbon and nitrogen metabolism, including C1 unit transfer in lignin synthesis. Although a high content of strigolactone in rice roots is known to reduce active tiller number, the reduction of outgrowth of axillary buds observed in the GS1;2 mutants was independent of the level of strigolactone. Thus metabolic disorder caused by the lack of GS1;2 resulted in a severe reduction in the outgrowth of axillary buds and lignin deposition.
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Resolving the Role of Plant Glutamate Dehydrogenase: II. Physiological Characterization of Plants Overexpressing the Two Enzyme Subunits Individually or Simultaneously.
Plant and Cell Physiology, 2013Co-Authors: Therese Tercé-laforgue, Frederic Dubois, Magali Bedu, Yves Gibon, Francesco M. Restivo, Céline Dargel-graffin, Bertrand HirelAbstract:Glutamate dehydrogenase (GDH; EC 1.4.1.2) is able to carry out the deamination of glutamate in higher plants. In order to obtain a better understanding of the physiological function of GDH in leaves, transgenic tobacco (Nicotiana tabacum L.) plants were constructed that overexpress two genes from Nicotiana plumbaginifolia (GDHA and GDHB under the control of the Cauliflower mosiac virus 35S promoter), which encode the α- and β-subunits of GDH individually or simultaneously. In the transgenic plants, the GDH protein accumulated in the mitochondria of mesophyll Cells and in the mitochondria of the Phloem Companion Cells (CCs), where the native enzyme is normally expressed. Such a shift in the cellular location of the GDH enzyme induced major changes in carbon and nitrogen metabolite accumulation and a reduction in growth. These changes were mainly characterized by a decrease in the amount of sucrose, starch and glutamine in the leaves, which was accompanied by an increase in the amount of nitrate and Chl. In addition, there was an increase in the content of asparagine and a decrease in proline. Such changes may explain the lower plant biomass determined in the GDH-overexpressing lines. Overexpressing the two genes GDHA and GDHB individually or simultaneously induced a differential accumulation of glutamate and glutamine and a modification of the glutamate to glutamine ratio. The impact of the metabolic changes occurring in the different types of GDH-overexpressing plants is discussed in relation to the possible physiological function of each subunit when present in the form of homohexamers or heterohexamers.
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control of the synthesis and subcellular targeting of the two gdh genes products in leaves and stems of nicotiana plumbaginifolia and arabidopsis thaliana
Plant and Cell Physiology, 2006Co-Authors: Jeanxavier Fontaine, Bertrand Hirel, Therese Tercelaforgue, Magali Bedu, Francesca Saladino, Caterina Agrimonti, Thierry Tetu, Francesco Maria Restivo, Frederic DuboisAbstract:;Although the physiological role of the enzyme glutamate dehydrogenase which catalyses in vitro the reversible amination of 2-oxoglutarate to glutamate remains to be elucidated, it is now well established that in higher plants the enzyme preferentially occurs in the mitochondria of Phloem Companion Cells. The Nicotiana plumbaginifolia and Arabidopis thaliana enzyme is encoded by two distinct genes encoding either an α- or a β-subunit. Using antisense plants and mutants impaired in the expression of either of the two genes, we showed that in leaves and stems both the α- and β-subunits are targeted to the mitochondria of the Companion Cells. In addition, we found in both species that there is a compensatory mechanism up-regulating the expression of the α-subunit in the stems when the expression of the β-subunit is impaired in the leaves, and of the β-subunit in the leaves when the expression of the α-subunit is impaired in the stems. When one of the two genes encoding glutamate dehydrogenase is ectopically expressed, the corresponding protein is targeted to the mitochondria of both leaf and stem parenchyma Cells and its production is increased in the Companion Cells. These results are discussed in relation to the possible signalling and/or physiological function of the enzyme which appears to be coordinated in leaves and stems.
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changes in the cellular and subcellular localization of glutamine synthetase and glutamate dehydrogenase during flag leaf senescence in wheat triticum aestivum l
Plant and Cell Physiology, 2005Co-Authors: Thomas Kichey, Jacques Le Gouis, Brigitte Sangwan, Bertrand Hirel, Frederic DuboisAbstract:;In order to improve our understanding of the regulation of nitrogen assimilation and recycling in wheat (Triticum aestivum L.), we studied the localization of plastidic (GS2) and cytosolic (GS1) glutamine synthetase isoenzymes and of glutamate dehydrogenase (GDH) during natural senescence of the flag leaf and in the stem. In mature flag leaves, large amounts of GS1 were detected in the connections between the mestome sheath Cells and the vascular Cells, suggesting an active transfer of nitrogen organic molecules within the vascular system in the mature flag leaf. Parallel to leaf senescence, an increase of a GS1 polypeptide (GS1b) was detected in the mesophyll cytosol of senescing leaves, while the GS protein content represented by another polypetide (GS1a) in the Phloem Companion Cells remained practically constant in both leaves and stems. Both GDH aminating activity and protein content were strongly induced in senescing flag leaves. The induction occurred both in the mitochondria and in the cytosol of Phloem Companion Cells, suggesting that the shift in GDH cellular compartmentation is important during leaf nitrogen remobilization although the metabolic or sensing role of the enzyme remains to be elucidated. Taken together, our results suggest that in wheat, nitrogen assimilation and recycling are compartmentalized between the mesophyll and the vasculature, and are shifted in different cellular compartments within these two tissues during the transition of sink leaves to source leaves.
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glutamate dehydrogenase of tobacco is mainly induced in the cytosol of Phloem Companion Cells when ammonia is provided either externally or released during photorespiration
Plant Physiology, 2004Co-Authors: Therese Tercelaforgue, Frederic Dubois, Rajbir S Sangwan, Sylvie Ferrariomery, Marieanne Pou De Crecenzo, Bertrand HirelAbstract:Glutamate (Glu) dehydrogenase (GDH) catalyses the reversible amination of 2-oxoglutarate for the synthesis of Glu using ammonium as a substrate. This enzyme preferentially occurs in the mitochondria of Companion Cells of a number of plant species grown on nitrate as the sole nitrogen source. For a better understanding of the controversial role of GDH either in ammonium assimilation or in the supply of 2-oxoglutarate (F. Dubois, T. Terce-Laforgue, M.B. Gonzalez-Moro, M.B. Estavillo, R. Sangwan, A. Gallais, B. Hirel [2003] Plant Physiol Biochem 41: 565–576), we studied the localization of GDH in untransformed tobacco (Nicotiana tabacum) plants grown either on low nitrate or on ammonium and in ferredoxin-dependent Glu synthase antisense plants. Production of GDH and its activity were strongly induced when plants were grown on ammonium as the sole nitrogen source. The induction mainly occurred in highly vascularized organs such as stems and midribs and was likely to be due to accumulation of Phloem-translocated ammonium in the sap. GDH induction occurred when ammonia was applied externally to untransformed control plants or resulted from photorespiratory activity in transgenic plants down-regulated for ferredoxin-dependent Glu synthase. GDH was increased in the mitochondria and appeared in the cytosol of Companion Cells. Taken together, our results suggest that the enzyme plays a dual role in Companion Cells, either in the mitochondria when mineral nitrogen availability is low or in the cytosol when ammonium concentration increases above a certain threshold.
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glutamate dehydrogenase in plants is there a new story for an old enzyme
Plant Physiology and Biochemistry, 2003Co-Authors: Frederic Dubois, Therese Tercelaforgue, Mariabegona Gonzalezmoro, J M Estavillo, Rajbir S Sangwan, A Gallais, Bertrand HirelAbstract:Abstract Although good progress has been made to dissect and better understand both the main steps and the regulation of inorganic nitrogen assimilation in higher plants, the role of alternative metabolic pathways which are potentially able to incorporate ammonium into organic molecules is still not fully understood. One of them is the reaction catalysed by the mitochondrial enzyme glutamate dehydrogenase (NAD(H)-GDH, EC 1.4.1.2) which is either able to incorporate ammonium into 2-oxoglutarate to form glutamate or to function in the opposite direction to oxidise glutamate. Although it has been clearly demonstrated by the means of 15 N- or 13 C-labelling experiments that the later reaction occurs in the cell, it has been argued that under certain physiological conditions, when the ammonium concentration reaches a certain threshold, the enzyme is able to function in the aminating direction. More recently, it has been found that in grapes, a high proportion of the protein is located in the mitochondria of the Phloem Companion Cells and that a significant amount of enzyme is present in the cytosolic fraction of senescing flowers. Using cytoimmunochemistry, we confirmed in the present study that, in other higher plant species, GDH protein is localised in the mitochondria of the Phloem Companion Cells and in the cytosol of senescing organs or tissues. These findings open, therefore, new perspectives toward a better understanding of the function of GDH, particularly in relation to stress and plant development. Both transgenic studies performed in the past and the quantitative genetic approach presented in this paper strongly suggest that the reaction catalysed by NAD(H)-GDH is of major importance in the control of plant growth and productivity.