Kinetic Properties and Ammonium-dependent Regulation of Cytosolic Isoenzymes of Glutamine Synthetase in Arabidopsis
2004; Elsevier BV; Volume: 279; Issue: 16 Linguagem: Inglês
10.1074/jbc.m313710200
ISSN1083-351X
AutoresKeiki Ishiyama, Eri Inoue, Akiko Watanabe‐Takahashi, Mitsuhiro Obara, Tomoyuki Yamaya, Hideki Takahashi,
Tópico(s)Polyamine Metabolism and Applications
ResumoGlutamine 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 (Km = 2450 ± 150 μm) and glutamate (Km = 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 (Km for ammonium < 10 μm; Km 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; 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 (Km = 2450 ± 150 μm) and glutamate (Km = 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 (Km for ammonium < 10 μm; Km 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 1The abbreviations used are: GS, glutamine synthetase; GFP, green fluorescent protein; EGFP, enhanced GFP. 1The abbreviations used are: GS, glutamine synthetase; GFP, green fluorescent protein; EGFP, enhanced GFP.; EC 6.3.1.2) is responsible for the primary assimilation of ammonium in higher plants (1Oaks A. Hirel B. Annu. Rev. Plant Physiol. 1985; 36: 345-365Crossref Google Scholar, 2Lam H.M. Coschigano K.T. Oliveira I.C. Melo-Oliveira R. Coruzzi G.M. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 569-593Crossref PubMed Scopus (670) Google Scholar, 3Ireland R.L. Lea P.J. Singh B.K. Plant Amino Acids: Biochemistry and Metabolism. Marcel Decker, New York1999: 49-109Google Scholar, 4Tobin A.K. Yamaya T. J. Exp. Bot. 2002; 52: 591-604Crossref Google Scholar). 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) (1Oaks A. Hirel B. Annu. Rev. Plant Physiol. 1985; 36: 345-365Crossref Google Scholar, 2Lam H.M. Coschigano K.T. Oliveira I.C. Melo-Oliveira R. Coruzzi G.M. Annu. Rev. Plant Physiol. Plant Mol. 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Biol. 1999; 39: 551-564Crossref PubMed Scopus (19) Google Scholar, 22Sakakibara H. Shimizu H. Hase T. Yamazaki Y. Takao T. Shimonishi Y. Sugiyama T. J. Biol. Chem. 1996; 271: 29561-29568Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 24Oliveira I.C. Coruzzi G.M. Plant Physiol. 1999; 121: 301-309Crossref PubMed Scopus (176) Google Scholar). In the roots of legumes, GS1 is regulated by ammonium supplied from the environment or the symbiotic nitrogen fixation (18Hirel B. Bouet C. King B. Layzell D. Jacobs F. Verma D.P.S. EMBO J. 1987; 6: 1167-1171Crossref PubMed Google Scholar, 19Cock J.M. Mould R.M. Bennett M.J. Cullimore J.V. Plant Mol. Biol. 1990; 14: 549-560Crossref PubMed Scopus (37) Google Scholar, 20Miao G.H. Hirel B. Marsolier M.C. Ridge R.W. Verma D.P.S. Plant Cell. 1991; 3: 11-22PubMed Google Scholar, 21Terce-Laforgue T. Carrayol E. Cren M. Desbrosses G. Hecht V. Hirel B. Plant Mol. Biol. 1999; 39: 551-564Crossref PubMed Scopus (19) Google Scholar). Transgenic studies with the soybean GS1 promoter suggested that ammonium-dependent regulation is specific for nitrogen assimilation in leguminous plants; the soybean-derived GS1 promoter was not able to display the ammonium-induced expression of reporter activity in tobacco (20Miao G.H. Hirel B. Marsolier M.C. Ridge R.W. Verma D.P.S. Plant Cell. 1991; 3: 11-22PubMed Google Scholar). This poses questions of whether the stimulation of GS1 expression by ammonium is genuinely associated with symbiosis and whether dicotyledonous plants are furnished or not furnished with the regulatory mechanisms that may perceive the ammonium signal. The availability of ammonium also caused changes in the expression of some of the GS1 genes in non-leguminous plants. For example, among the five isoenzymes of GS1 (GS1a, GS1b, GS1c, GS1d, and GS1e) identified from maize (9Sakakibara H. Kawabata S. Takahashi H. Hase T. Sugiyama T. Plant Cell Physiol. 1992; 33: 49-58Google Scholar, 13Li M.G. Villemur R. Hussey P.J. Silflow C.D. Gantt J.S. Snustad D.P. Plant Mol. Biol. 1993; 23: 401-407Crossref PubMed Scopus (114) Google Scholar), the mRNA of GS1c and GS1d accumulated following application of ammonium to roots, whereas those of GS1a and GS1b were suppressed under the same condition (22Sakakibara H. Shimizu H. Hase T. Yamazaki Y. Takao T. Shimonishi Y. Sugiyama T. J. Biol. Chem. 1996; 271: 29561-29568Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). In Arabidopsis, five putative genes for GS1, GLN1;1, GLN1;2, GLN1;3, GLN1;4, and GLN1;5, are encoded in the genome (23Arabidopsis Genome Initiative Nature. 2000; 408: 796-815Crossref PubMed Scopus (7006) Google Scholar). Oliveira and Coruzzi (24Oliveira I.C. Coruzzi G.M. Plant Physiol. 1999; 121: 301-309Crossref PubMed Scopus (176) Google Scholar) reported that the application of sucrose to the medium causes induction of GLN1;1, GLN1;2, and GLN1;3 mRNAs and that amino acids attenuated the effect of sucrose, suggesting that a metabolic regulation of GS1 is associated with the relative abundance of carbon skeleton versus amino acids accumulated in the root tissue. These results suggest a negative feedback regulation of GS1 by Gln or the downstream nitrogen metabolites as in the case of ammonium transporters. However, the exact functional roles and physiological diversities of the individual GS1 isoenzymes in Arabidopsis have not been well characterized. In the present study, we determined the kinetic properties and ammonium response of the Arabidopsis GS1 isoenzymes. We demonstrated that GS1 expressed in roots (GLN1;1, GLN1;2, GLN1;3, and GLN1;4) can be classified into two distinct groups, the high affinity and low affinity enzymes localized at the root surface and vasculature, respectively. The characteristics of the GS1 isoenzymes in Arabidopsis were fitted into the negative regulatory pathways of ammonium influx and assimilation, which is apparently different from those operated positively for the active assimilation of externally supplied ammonium in the roots of legumes and maize. The results presented here clearly demonstrated that the multiplicity of cytosolic GS1 in higher plants does not simply provide functional redundancy but, rather, confers specified roles to the individual isoenzymes bearing distinctive kinetic properties and ammonium responsiveness. This is the first report presenting the complete and precise data of enzymatic properties and cell type-specific localization of GS1 isoenzymes in higher plants. Plant Culture Conditions—Arabidopsis thaliana ecotype Columbia (Col-0) was used for all experiments. Plants were cultured in a growth chamber controlled at 22 °C with 60% relative humidity under 16-h light and 8-h dark cycles. The light intensity was 40 μmol m-2 s-1. Plants were grown under sterile conditions on agar medium containing 7 mm nitrate (25Inaba K. Fujiwara T. Hayashi H. Chino M. Komeda Y. Naito S. Plant Physiol. 1994; 104: 881-887Crossref PubMed Scopus (118) Google Scholar). Arabidopsis cell culture (26Mathur J. Szabados L. Schaefer S. Grunenberg B. Lossow A. Jonas-Straube E. Schell J. Koncz C. Koncz-Kalman Z. Plant J. 1998; 13: 707-716Crossref PubMed Scopus (109) Google Scholar) was propagated in Murashige and Skoog liquid medium supplemented with 2.5 mm potassium phosphate and 4.5 μm 2,4-dichlorophenoxyacetic acid by continuous shaking under dark conditions. Cloning of Arabidopsis GLN1 cDNAs—Molecular biological experiments were carried out according to the standard protocols (27Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). The coding sequences of GLN1 cDNAs encoding GS1 were isolated by reverse transcriptase PCR. Primers were designed according to the annotations of the nucleotide sequence of Arabidopsis genome (23Arabidopsis Genome Initiative Nature. 2000; 408: 796-815Crossref PubMed Scopus (7006) Google Scholar) presented in The Institute for Genomic Research (www.tigr.org/tdb/e2k1/ath1/) and the Munich Information Center for Protein Sequences (mips.gsf.de/proj/thal/db/index.html) databases. The locus numbers for GLN1 genes are At5g37600 (GLN1;1), At1g66200 (GLN1;2), At3g17800 (GLN1;3), At5g16570 (GLN1;4), and At1g48470 (GLN1;5). Total RNA was extracted using the RNeasy plant mini kit (Qiagen, Hilden, Germany). Reverse transcription and PCR were carried out using Omniscript reverse transcriptase (Qiagen) and KOD plus DNA polymerase (Toyobo, Tokyo, Japan). The amplified PCR products were cloned into pCR-Blunt II-TOPO (Invitrogen, Carlsbad, CA) and fully sequenced. Quantitative Real Time PCR Analysis—Extraction of RNA and cDNA synthesis was carried out as described above for cDNA cloning. Gene-specific primers are presented in Table I. Constitutive expression of ubiquitin (UBQ2; GenBank™ accession number J05508) was confirmed in parallel (Table I). The PCR products were detected and quantified as SYBR Green fluorescence (Applied Biosystems, Foster City, CA) using the Gene Amp 5700 sequence detection system (Applied Biosystems). The mRNA content was quantitatively determined by using a purified cDNA clone as a standard for its calibration.Table IGene specific primers used for the real-time PCR analysisNameGeneSequenceGln1;1-RFaThe pairs of forward (RF or F) and reverse (RR or R) primers were used to specifically amplify GLN1 and UBQ2 cDNAs.GLN1;15′-CATCAACCTTAACCTCTCAGACTCCACT-3′Gln1;1-RRGLN1;15′-ACTTCAGCTGCAACATCAGGGTTGCTA-3′Gln1;2-RFGLN1;25′-TGTTAACCTTGACATCTCAGACAACAGT-3′Gln1;2-RRGLN1;25′-ACTTCAGCAATAACATCAGGGTTAGCA-3′Gln1;3-RFGLN1;35′-CGTTAACCTCAACCTCACCGATGCCACC-3′Gln1;3-RRGLN1;35′-TCCTCCTTGGCAACGTCGGGGTGGCTG-3′Gln1;4-RFGLN1;45′-AATCAATCTCGATCTCTCCGATTCCACT-3′Gln1;4-RRGLN1;45′-TTCTTCGGCGACAACACTAGGGTCTTCA-3′Gln1;5-RFGLN1;55′-TCTCCTAAACCTTGATCTATCAGACACC-3′Gln1;5-RRGLN1;55′-CTCTTCAGCCTTCACATTGGGATGATC-3′Gln2-RFGLN25′-TTCTCCAACATGTCAGATGAGAGTGCCT-3′Gln2-RRGLN25′-CCAGGTGCTTGACCGGTACTCGAACCA-3′144FUBQ25′-CCAAGATCCAGGACAAAGAAGGA-3′372RUBQ25′-TGGAGACGAGCATAACACTTGC-3′a The pairs of forward (RF or F) and reverse (RR or R) primers were used to specifically amplify GLN1 and UBQ2 cDNAs. Open table in a new tab Preparation of Anti-GS1 Antibody—The cDNA fragment encoding rice GS1, RGS28 (GenBank™ accession number X14245) (7Sakamoto A. Ogawa M. Masumura T. Shibata D. Takeba G. Tanaka K. Fujii S. Plant Mol. Biol. 1989; 13: 611-614Crossref PubMed Scopus (70) Google Scholar) was inserted into the NcoI site of pTrc99A (Amersham Biosciences). The nucleotide sequence downstream of the translation initiation codon was modified to an AT-rich structure to obtain high expression of the recombinant GS1 protein (22Sakakibara H. Shimizu H. Hase T. Yamazaki Y. Takao T. Shimonishi Y. Sugiyama T. J. Biol. Chem. 1996; 271: 29561-29568Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The first 24-bp portion of the RGS28 coding sequence was deleted by HincII digestion and replaced with the oligonucleotide 5′-TCGAGCCATGGCTTCTTTAACTGATCTCGTC-3′ without changing the encoding amino acid residues. The 3′-end of RGS28 was digested with PflMI and ligated with the oligonucleotide 5′-CTGGAAGCCCCATCATCATCATCATCATTGACCATCAT-3′, adding His6 codons and a stop codon at the C terminus. The modified RGS28 was cloned in pTrc99A and transformed to Escherichia coli JM109. Culture of E. coli and purification of the recombinant protein were performed as described previously (22Sakakibara H. Shimizu H. Hase T. Yamazaki Y. Takao T. Shimonishi Y. Sugiyama T. J. Biol. Chem. 1996; 271: 29561-29568Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). To prepare polyclonal antibody against the rice recombinant GS1 protein, 500 μg of the purified antigen emulsified with adjuvant was injected into a rabbit. Additional injections (500 μg each) were done twice every 2 weeks. Whole blood was obtained from the carotid artery, and the antiserum was collected by centrifugation. Western Blot Analysis—Proteins were separated in 12.5% (w/v) polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Bio-Rad) by electroblotting. 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 (28Sakurai N. Hayakawa T. Nakamura T. Yamaya T. Planta. 1996; 200: 306-311Crossref Scopus (65) Google Scholar). Proteins cross-reacting with the antibodies were visualized using 5-bromo-4-chloro-3-indolylphosphate-p-toluidine and nitroblue tetrazolium chloride (Promega). GS Enzyme Assay—The Gln synthetic activity of GS was determined by quantifying l-Gln synthesized from ammonium and l-Glu (28Sakurai N. Hayakawa T. Nakamura T. Yamaya T. Planta. 1996; 200: 306-311Crossref Scopus (65) Google Scholar). A 0.1-ml reaction mixture contained 100 mm tricine-HCl (pH 7.8), 80 mml-Glu, 6 mm ammonium, 8 mm ATP, 20 mm MgSO4, and 8 mm 2-mer-captoethanol. The amount of proteins used for the enzyme reaction is indicated in Figs. 2 and 6 and Table II. The reaction mixture was incubated at 30 °C for 15 min, terminated by heating at 96 °C for 2 min, and filtered through Ultra free-MC 5000 NMWL Filter Unit (Millipore, Bedford, MA). Five microliters of filtrate was incubated with AccQ Fluor reagent (Waters, Milford, MA), and the resulting AccQ-derivative of Gln was measured by Waters 2695 high performance liquid chromatography system on an AccQ Tag column (3.9 mm diameter × 150 mm length; Waters) according to the manufacturer's instructions.Fig. 6GLN1;3 activity is inhibited by Glu. Kinetics of Gln synthetic activities of GLN1;1 (A), GLN1;2 (B), GLN1;3 (C), and GLN1;4 (D) were plotted against the concentration of Glu in the reaction mixture and fitted to the Michaelis-Menten equation (solid line). The GLN1;3 activities shifted out from the Michaelis-Menten equation are indicated as dashed lines. The purified GS1 protein was incubated in a 0.1-ml assay mixture as described under “Experimental Procedures.” One microgram of GLN1;1 and GLN1;2 and 0.5 μg of GLN1;3 and GLN1;4 were used for the GS enzyme assays.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IIThe kinetic properties of the Arabidopsis GS1 isoenzymesKmVmaxaPurified GS1 protein was incubated in a 0.1 ml assay mixture as described under “Experimental Procedures.” One microgram of GLN1;1, GLN1;2, and cell culture-derived GS1 and 0.5 μg of GLN1;3 and GLN1;4 were used for the GS enzyme assays. One katal of enzyme activity was defined as 1 mol of Gln synthesized per second at 30 °C.GluNH4+ATPGluNH4+ATPmmμmμmNanokatal/mg proteinGLN1;11.1 ± 0.4bMean ± S.E. (n = 3).<10300 ± 2029.3 ± 1.627.4 ± 0.721.4 ± 0.4GLN1;23.8 ± 0.22450 ± 1501100 ± 14065.7 ± 0.265.7 ± 1.166.6 ± 4.4GLN1;33.9 ± 0.11210 ± 40850 ± 30162 ± 2493.9 ± 10100.0 ± 0.04GLN1;40.6 ± 0.148 ± 6400 ± 5079.2 ± 165.7 ± 1.573.9 ± 4.2Cell culture GS14.458056010895.269.4a Purified GS1 protein was incubated in a 0.1 ml assay mixture as described under “Experimental Procedures.” One microgram of GLN1;1, GLN1;2, and cell culture-derived GS1 and 0.5 μg of GLN1;3 and GLN1;4 were used for the GS enzyme assays. One katal of enzyme activity was defined as 1 mol of Gln synthesized per second at 30 °C.b Mean ± S.E. (n = 3). Open table in a new tab 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 MgCl2, 10 mm 2-mer-captoethanol, and 5% (w/v) polyvinylpyrrolidone) (28Sakurai N. Hayakawa T. Nakamura T. Yamaya T. Planta. 1996; 200: 306-311Crossref Scopus (65) Google Scholar) using a chilled mortar and pestle or with MM-300 mixer mill (Qiagen). The homogenates were centrifuged at 20,000 × g for 10 min under 4 °C. The supernatants were immediately desalted on PD-10 (Amersham Biosciences) or Probe Quant G-50 Micro Columns (Amersham Biosciences) equilibrated with buffer A (25 mm Tris-HCl, pH 7.6, 1 mm MgCl2, and 10 mm 2-mercaptoethanol). The crude enzyme fractions obtained were stored in 50% (v/v) glycerol. Soluble protein content was determined by the Coomassie Blue dye binding method (Bio-Rad) using bovine serum albumin as a standard Purification of Native GS1 Protein from Arabidopsis Cell Culture— All of the steps of enzyme purification were performed in a cold room (4 °C). One hundred fifty grams of cultured cells were homogenized in 500 ml of GS extraction buffer using a chilled warring blender (28Sakurai N. Hayakawa T. Nakamura T. Yamaya T. Planta. 1996; 200: 306-311Crossref Scopus (65) Google Scholar). The homogenate was centrifuged at 27,000 × g for 15 min. The supernatant was fractionated by (NH4)2SO4 precipitation between 30 and 60% saturation. The precipitate was suspended in 50 ml of buffer A containing 1.2 m (NH4)2SO4 and then immediately loaded onto a HiTrap butyl-FF column (15 ml bed volume) (Amersham Biosciences) equilibrated with the same buffer. The pooled fraction was saturated to 70% (NH4)2SO4 and centrifuged at 27,000 × g for 15 min. The precipitate was suspended in 2 ml of buffer A and loaded for gel filtration chromatography on a HiLoad 26/60 Superdex 200pg (320 ml bed volume) (Amersham Biosciences) equilibrated with buffer A. The fractions containing GS activities were further purified by anion exchange chromatography using a HiTrap Q HP (1-ml bed volume) (Amersham Biosciences) equilibrated with buffer A. Protein was eluted by a linear gradient of NaCl from 0 to 1 m. Expression of Recombinant GS1 Proteins in E. coli—The KpnI and SalI sites were created on the 5′- and 3′-ends of the coding region of GLN1 cDNAs by PCR using KOD-Plus DNA polymerase (Toyobo). The amplified PCR products were cloned into pCR-Blunt II-TOPO (Invitrogen) and fully sequenced. The cDNA inserts were digested by KpnI and SalI and ligated into the KpnI and SalI sites of pQE2 (Qiagen) to produce the His6 fusion constructs, termed pQE2-GLN1;1, pQE2-GLN1;2, pQE2-GLN1;3, and pQE2-GLN1;4, respectively. E. coli HB101 was transformed with the recombinant construct and grown at 30 °C in 300 ml of LB medium containing 1 m sorbitol and 100 μg/ml ampicillin. When A600 reached 0.5, the culture was cooled down to 20 °C. One millimolar isopropyl-1-thio-β-d-galactopyranoside was supplied to induce the expression of recombinant GS1 proteins, and the culture was further incubated at 20 °C for 12 h by continuous shaking (180 rpm). Purification of Recombinant GS1 Enzymes—One volume of E. coli cell pellets collected by centrifugation was re-suspended in 5 volumes of lysis buffer (50 mm NaH2PO4, pH 8, 300 mm NaCl, and 10 mm imidazole). Cell suspensions were incubated for 30 min at 4 °C with lysozyme (Nacalai Tesque, Kyoto, Japan) at the final concentration of 1 mg ml-1. After ultrasonication, the insoluble fraction was removed by centrifugation. The His-tagged protein in the soluble fraction was affinity-purified using a nickel-nitrilotriacetic acid agarose column (1-ml bed volume) (Qiagen). Washing was carried out with a buffer containing 50 mm NaH2PO4, pH 8, 300 mm NaCl, and 20 mm imidazole. Protein was eluted with the same buffer containing 250 mm imidazole. The eluates were desalted and equilibrated in buffer A using PD-10 (Amersham Biosciences). Protein was further purified by anion exchange chromatography with a HiTrap Q HP (1-ml bed volume) (Amersham Biosciences) using the AKTA Prime fast protein liquid chromatography system (Amersham Biosciences). Protein was eluted by 10 ml of a linear NaCl gradient from 0 to 1 m, and the protein peak fractions were analyzed for the presence of a single band of recombinant GS1 protein (40 kDa) on a Coomassie Blue-stained polyacrylamide gel. The Gln biosynthetic activities of the pooled fractions were assayed to determine the kinetic properties. Construction of GLN1 Promoter-Green Fluorescent Protein (GFP) Plants—The fusion gene constructs of Arabidopsis GLN1 gene promoters and EGFP (Clontech) for plant transformation were constructed as follows. The 2463-bp, 2501-bp, 1213-bp, and 1161-bp 5′-promoter regions of GLN1;1, GLN1;2, GLN1;3, and GLN1;4, respectively, were amplified from Col-0 Arabidopsis genomic DNA by PCR using KOD plus DNA polymerase (Toyobo). The BamHI site was created on the 5′-end of the forward primer. The reverse primer containing the NcoI restriction site (CCATGG) was created at the translation initiation site of GLN1 gene to make the promoter-GFP fusion. The amplified PCR products were cloned into pCR-Blunt II-TOPO (Invitrogen) and fully sequenced. The GLN1 promoter regions were cut out as BamHI-NcoI fragments and ligated with the NcoI-NotI fragment of EGFP (Clontech) and the NotI-EcoRI fragment of the nopaline synthase terminator (29Chiu W.L. Niwa Y. Zeng W. Hirano T. Kobayashi H. Sheen J. Curr. Biol. 1996; 6: 325-330Abstract Full Text Full Text PDF PubMed Scopus (1200) Google Scholar). The resultant GLN1 promoter:EGFP:nopaline synthase terminator cassette was placed in the position of the β-glucuronidase and nopaline synthase terminator in the binary plasmid, pBI101 (Clontech). The binary plasmids were transferred to Agrobacterium tumefaciens GV3101 (pMP90) (30Koncz C. Schell J Mol. Gen. Genet. 1986; 204: 383-396Crossref Scopus (1545) Google Scholar) by the freeze-thaw method (31Höfgen R. Willmitzer L. Nucleic Acids Res. 1988; 16: 9877Crossref PubMed Scopus (838) Google Scholar). Arabidopsis plants were transformed according to the floral dip method (32Clough S.J. Bent A.F. Plant J. 1998; 16: 735-743Crossref PubMed Google Scholar). Transgenic plants were selected on GM medium (33Valvekens D. van Montagu M. van Lijsebettens M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5536-5540Crossref PubMed Scopus (1139) Google Scholar) containing 50 mg liter-1 kanamycin sulfate. Fluorescence of GFP in transgenic plants was observed under a BX61 microscope equipped with a FV500 confocal laser scanning system and a 505-525-nm band pass filter (Olympus, Tokyo, Japan), as descried previously (34Yoshimoto N. Inoue E. Saito K. Yamaya T. Takahashi H. Plant Physiol. 2003; 131: 1511-1517Crossref PubMed Scopus (163) Google Scholar). Cytosolic GS1 Is the Major Form in Arabidopsis Roots—The mRNA and protein contents of GS1 and GS2 in Arabidopsis were determined by real time PCR and Western blot analyses (Fig. 1). Arabidopsis plants were grown on agar medium containing 7 mm nitrate as a sole nitrogen source for 14 days. The total GS1 mRNA was calculated as a sum of GLN1;1, GLN1;2, GLN1;3, GLN1;4, and GLN1;5 mRNAs. The gene-specific primer pairs that specifically amplify the transcripts of individual GS isoenzymes are described in Table I. The GS1 mRNA was ∼20-fold more abundant than GS2 mRNA in Arabidopsis roots (Fig. 1A). Western blot analysis indicated accumulation of a 40-kDa protein that corresponds to the molecular size of GS1 in roots (Fig. 1B). GS2 (44 kDa) was hardly detectable in roots (Fig. 1B). The results indicate that GS2 mRNA is weakly expressed in roots as a minor GS isoenzyme; however, judging from the Western blots, the contribution of GS2 to the Gln synthesis is not appreciable as compared with GS1. Thus, we concluded that GS1 is the major isoenzyme that carries out the ammonium assimilation in Arabidopsis roots. In leaves, the mRNAs and proteins for both isoenzymes were expressed. On real time PCR, GS2 mRNA was slightly more abundant than GS1 mRNA in leaves (Fig. 1A). Western blot showed two major bands corresponding to the molecular sizes of GS2 and GS1 subunit proteins, respectively (Fig. 1B). Increased Accumulation of GS1 Protein by Ammonium Treatment Is Not Correlated with the GS Activity—The enzyme activity and the abundance of GS1 polypeptides were simultaneously monitored during a time course of ammonium treatment. Two week-old plants were deprived of nitrogen for 3 days and treated with 10 mm ammonium chloride. Root tissues were harvested for Western blotting and GS
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