Changes in the cellular localization of cytosolic glutamine synthetase protein in vascular bundles of rice leaves at various stages of development

Sakurai, Nozomu; Hayakawa, Toshihiko; Nakamura, Teiji; Yamaya, Tomoyuki

doi:10.1007/bf00200297

Cited by 77 publications

(76 citation statements)

References 22 publications

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“…The amount of proteins applied to the gel is indicated in the figure legends. The membrane was incubated with the anti-GS1 polyclonal antibody and goat anti-rabbit IgG alkaline phosphatase conjugate (Promega, Madison, WI) as described previously (28). Proteins cross-reacting with the antibodies were visualized using 5-bromo-4-chloro-3-indolylphosphate-p-toluidine and nitroblue tetrazolium chloride (Promega).…”

Section: Methodsmentioning

confidence: 99%

“…GS Enzyme Assay-The Gln synthetic activity of GS was determined by quantifying L-Gln synthesized from ammonium and L-Glu (28 Extraction of Proteins from Arabidopsis Plants-Approximately 0.2 g of frozen root or leaf tissues were homogenized in an equal volume of GS extraction buffer (50 mM Tris-HCl pH 7.6, 10 mM MgCl 2 , 10 mM 2-mercaptoethanol, and 5% (w/v) polyvinylpyrrolidone) (28) using a chilled mortar and pestle or with MM-300 mixer mill (Qiagen). The homogenates were centrifuged at 20,000 ϫ g for 10 min under 4°C.…”

Section: Methodsmentioning

confidence: 99%

“…One hundred fifty grams of cultured cells were homogenized in 500 ml of GS extraction buffer using a chilled warring blender (28). The homogenate was centrifuged at 27,000 ϫ g for 15 min.…”

Section: Methodsmentioning

confidence: 99%

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Kinetic Properties and Ammonium-dependent Regulation of Cytosolic Isoenzymes of Glutamine Synthetase in Arabidopsis

Ishiyama

Inoue

Watanabe-Takahashi

et al. 2004

Journal of Biological Chemistry

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177

198

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Glutamine synthetase (GS; EC 6.3.1.2) is a key enzyme of nitrogen assimilation, catalyzing the synthesis of glutamine from ammonium and glutamate. In Arabidopsis, cytosolic GS (GS1) was accumulated in roots when plants were excessively supplied with ammonium; however, the GS activity was controlled at a constant level. The discrepancy between the protein content and enzyme activity of GS1 was attributable to the kinetic properties and expression of four distinct isoenzymes encoded by GLN1;1, GLN1;2, GLN1;3 and GLN1;4, genes that function complementary to each other in Arabidopsis roots. GLN1;2 was the only isoenzyme significantly up-regulated by ammonium, which correlated with the rapid increase in total GS1 protein. GLN1;2 was localized in the vasculature and exhibited low affinities to ammonium (K m ‫؍‬ 2450 ؎ 150 M) and glutamate (K m ‫؍‬ 3.8 ؎ 0.2 mM). The expression of the counterpart vascular tissue-localizing low affinity isoenzyme, GLN1;3, was not stimulated by ammonium; however, the enzyme activity of GLN1;3 was significantly inhibited by a high concentration of glutamate. By contrast, the high affinity isoenzyme, GLN1;1 (K m for ammonium < 10 M; K m for glutamate ‫؍‬ 1.1 ؎ 0.4 mM) was abundantly accumulated in the surface layers of roots during nitrogen limitation and was down-regulated by ammonium excess. GLN1;4 was another high affinity-type GS1 expressed in nitrogen-starved plants but was 10-fold less abundant than GLN1;1. These results suggested that dynamic regulations of high and low affinity GS1 isoenzymes at the levels of mRNA and enzyme activities are dependent on nitrogen availabilities and may contribute to the homeostatic control of glutamine synthesis in Arabidopsis roots.Glutamine synthetase (GS 1 ; EC 6.3.1.2) is responsible for the primary assimilation of ammonium in higher plants (1-4). Ammonium is assimilated into glutamine and glutamate through a consecutive reaction of GS and glutamate synthase (GOGAT), the so-called GS/GOGAT cycle. Plants have two types of GS isoenzymes that localize in different compartments: one located in the cytosol (GS1) and the other in the plastid/chloroplasts (GS2) (1-4). GS1 is the major form of GS in plant roots, and the ammonium taken up from the soil is directly converted to Gln by its reaction. Molecular biological studies have identified a number of genes encoding GS1 from various plant species (5-9). The presence of multiple GS1 isoenzymes complicates the overall understanding of their physiological functions. The isoenzymes of GS1 show organ-and cell-specific patterns of expression and are developmentally regulated (10 -17). In addition, the expression of GS1 is metabolically regulated by the availability of nitrogen and carbon sources (18 -22, 24).In the roots of legumes, GS1 is regulated by ammonium supplied from the environment or the symbiotic nitrogen fixation (18 -21). Transgenic studies with the soybean GS1 promoter suggested that ammonium-dependent regulation is specific for nitrogen assimilation in leguminous plants; the soybean-derived GS1...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Kinetic Properties and Ammonium-dependent Regulation of Cytosolic Isoenzymes of Glutamine Synthetase in Arabidopsis

Ishiyama

Inoue

Watanabe-Takahashi

et al. 2004

Journal of Biological Chemistry

Self Cite

177

198

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“…Root GS 1 may also have a role in the assimilation of the ammonia derived from nitrate reduction in the roots (Lancien et al 1999). GS 1 is also present in the shoots where it is localized in the vascular tissue Kamachi et al 1992;Pereira et al 1992;Dubois et al 1996;Sakurai et al 1996;Pérez-García et al 1998). The role of GS 1 in the vasculature, however, is not well defined but it has been suggested that it may be involved in the synthesis of glutamine for nitrogen transport (Edwards et al 1990;Dubois et al 1996).…”

Section: Introductionmentioning

confidence: 99%

Biochemical and molecular characterization of transgenic Lotus japonicus plants constitutively over-expressing a cytosolic glutamine synthetase gene

Ortega

Temple

Bagga

et al. 2004

Planta

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Higher plants assimilate nitrogen in the form of ammonia through the concerted activity of glutamine synthetase (GS) and glutamate synthase (GOGAT). The GS enzyme is either located in the cytoplasm (GS 1 ) or in the chloroplast (GS 2 ). To understand how modulation of GS activity affects plant performance, Lotus japonicus L. plants were transformed with an alfalfa GS 1 gene driven by the CaMV 35S promoter. The transformants showed increased GS activity and an increase in GS 1 polypeptide level in all the organs tested. GS was analyzed by non-denaturing gel electrophoresis and ion-exchange chromatography. The results showed the presence of multiple GS isoenzymes in the different organs and the presence of a novel isoform in the transgenic plants. The distribution of GS in the different organs was analyzed by immunohistochemical localization. GS was localized in the mesophyll cells of the leaves and in the vasculature of the stem and roots of the transformants. Our results consistently showed higher soluble protein concentration, higher chlorophyll content and a higher biomass accumulation in the transgenic plants. The total amino acid content in the leaves and stems of the transgenic plants was 22-24% more than in the tissues of the non-transformed plants. The relative abundance of individual amino acid was similar except for aspartate/asparagine and proline, which were higher in the transformants.

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“…The major GS isoform in the leaves is GS 2 and it is located in the mesophyll cells and its major role is to assimilate NH 3 resulting from the reduction of nitrate and to re-assimilate NH 3 released during photorespiration (Lam et al, 1995). GS 1 in leaves and stem is localized primarily in the phloem elements (Brears et al, 1991;Kamachi et al, 1992;Dubois et al, 1996;Sakurai et al, 1996) and it is postulated that it functions to generate Gln for transport.…”

mentioning

confidence: 99%

Constitutive Overexpression of Cytosolic Glutamine Synthetase (GS1) Gene in Transgenic Alfalfa Demonstrates That GS1 May Be Regulated at the Level of RNA Stability and Protein Turnover

2001

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Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of NH 4 ϩ with glutanate to yield glutamine. Gene constructs consisting of the cauliflower mosaic virus (CaMV) 35S promoter driving a cytosolic isoform of GS (GS 1 ) gene have been introduced into alfalfa (Medicago sativa). Although transcripts for the transgene were shown to accumulate to high levels in the leaves, they were undetectable in the nodules. However, significant amounts of ␤-glucuronidase activity could be detected in nodules of plants containing the CaMV 35S promoter-␤-glucuronidase gene construct, suggesting that the transcript for the GS 1 transgene is not stable in the root nodules. Leaves of alfalfa plants with the CaMV 35S promoter-GS 1 gene showed high levels of accumulation of the transcript for the transgene when grown under low-nitrogen conditions and showed a significant drop in the level of GS 1 transcripts when fed with high levels of NO 3 Ϫ . However, no increase in GS activity or polypeptide level was detected in the leaves of transgenic plants. The results suggest that GS 1 is regulated at the level of RNA stability and protein turnover.

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Changes in the cellular localization of cytosolic glutamine synthetase protein in vascular bundles of rice leaves at various stages of development

Cited by 77 publications

References 22 publications

Kinetic Properties and Ammonium-dependent Regulation of Cytosolic Isoenzymes of Glutamine Synthetase in Arabidopsis

Kinetic Properties and Ammonium-dependent Regulation of Cytosolic Isoenzymes of Glutamine Synthetase in Arabidopsis

Biochemical and molecular characterization of transgenic Lotus japonicus plants constitutively over-expressing a cytosolic glutamine synthetase gene

Constitutive Overexpression of Cytosolic Glutamine Synthetase (GS1) Gene in Transgenic Alfalfa Demonstrates That GS1 May Be Regulated at the Level of RNA Stability and Protein Turnover

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