| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online January 11, 2006; 10.1104/pp.105.071910 Plant Physiology 140:444-456 (2006) © 2006 American Society of Plant Biologists Glutamine Synthetase-Glutamate Synthase Pathway and Glutamate Dehydrogenase Play Distinct Roles in the Sink-Source Nitrogen Cycle in Tobacco1Unité de Nutrition Azotée des Plantes (C.M.-D., M.R.-C., K.P., M.L., M.-H.V., M.F., T.J., A.S.) and Laboratoire Commun de Cytologie (O.G., J.K.), Institut National de la Recherche Agronomique, 78026 Versailles cedex, France
Glutamate (Glu) metabolism and amino acid translocation were investigated in the young and old leaves of tobacco (Nicotiana tabacum L. cv Xanthi) using [15N]ammonium and [2-15N]Glu tracers. Regardless of leaf age, [15N]ammonium assimilation occurred via glutamine synthetase (GS; EC 6.1.1.3) and Glu synthase (ferredoxin [Fd]-GOGAT; EC 1.4.7.1; NADH-GOGAT; EC 1.4.1.14), both in the light and darkness, and it did not depend on Glu dehydrogenase (GDH; EC 1.4.1.2). The [15N]ammonium and ammonium accumulation patterns support the role of GDH in the deamination of [2-15N]Glu to provide 2-oxoglutarate and [15N]ammonium. In the dark, excess [15N]ammonium was incorporated into asparagine that served as an additional detoxification molecule. The constant Glu levels in the phloem sap suggested that Glu was continuously synthesized and supplied into the phloem regardless of leaf age. Further study using transgenic tobacco lines, harboring the promoter of the GLU1 gene (encoding Arabidopsis [Arabidopsis thaliana] Fd-GOGAT) fused to a GUS reporter gene, revealed that the expression of Fd-GOGAT remained higher in young leaves compared to old leaves, and higher in the veins compared to the mesophyll. Confocal laser-scanning microscopy localized the Fd-GOGAT protein to the phloem companion cells-sieve element complex in the leaf veins. The results are consistent with a role of Fd-GOGAT in supplying Glu for the synthesis and transport of amino acids. Taken together, the data provide evidence that the GS-GOGAT pathway and GDH play distinct roles in the source-sink nitrogen cycle of tobacco leaves.
Plants utilize nitrate, ammonium, and dinitrogen (N2) molecules as external nitrogen sources. Ammonium is the final form of inorganic nitrogen prior to the synthesis of organic nitrogen compounds. Ammonium is also produced via internal metabolic reactions, including photorespiration, hydrolysis of nitrogen carrying and storage molecules, and amino acid conversion (Ireland and Lea, 1999  ). In nonleguminous C3 plants, such as tobacco (Nicotiana tabacum), the photorespiratory ammonium production by the oxidative decarboxylation of Gly exceeds by about 10-fold the primary nitrate reduction in the vegetative leaves. In the senescing leaves, a large amount of ammonium is produced as a result of protein hydrolysis (Hörteinsteiner and Feller, 2002). Therefore, it is essential that toxic ammonium be immediately reassimilated into organic molecules for nitrogen cycling. Ammonium is assimilated into the Gln amide group, which is then transferred to the position of 2-oxoglutarate, yielding two molecules of Glu by the concerted reaction of Gln synthetase (GS; EC 6.1.1.3) and Glu synthase (ferredoxin [Fd]-GOGAT; EC 1.4.7.1; NADH-GOGAT; EC 1.4.1.14). Nitrogen is then incorporated into Asp, Ala, Asn, and other amides and amino acids. Gln-dependent Asn synthetase (AS; EC 6.3.5.4) provides Asn, which serves as a nitrogen carrier together with Gln and Glu.
Numerous studies have been carried out to define the roles of enzymes in nitrogen assimilation and remobilization, tightly interrelated processes during plant growth and development (Miflin and Habash, 2002 To better understand the role of GDH and GOGAT in Glu metabolism in the coordinated reaction with GS, we studied the kinetics of in vivo turnover of [15N]Glu fed to leaf discs during aging of tobacco plants. The time course of [15N]ammonium assimilation into the amino acids was then determined in vivo in young and old leaves. To understand the cellular compartmentation of Glu synthesis and amino acid translocation, we investigated the tissue-specific expression and the cellular localization of Fd-GOGAT in tobacco plants transformed by a fusion between the promoter of the Arabidopsis (Arabidopsis thaliana) Fd-GOGAT gene (GLU1) and a reporter gene.
Respiratory Ammonium Release from Glu
In this experiment, we hypothesized that Glu is deaminated in both young and old leaves. (1) Glu is involved in the photorespiratory nitrogen cycle during the day and provides Gly and 2-oxoglutarate in the peroxisome through the reaction of Glu:glyoxylate aminotransferase (Fig. 1). Two molecules of Gly are in turn converted to CO2, ammonia, and Ser via the Gly decarboxylase multienzyme complex and Ser hydroxymethyltransferase. (2) Glu deamination through the anaplerotic pathway, involving GDH, could participate in mitochondrial respiration during the day/night cycle (Masclaux-Daubresse et al., 2002
To investigate Glu behavior in young and old leaves, leaf discs were incubated with [2-15N]Glu either in the light or in the dark. Total ammonium levels were higher in young than in old leaves (Fig. 2A). No remarkable difference was detected in ammonium levels between the light and dark conditions. The addition of Met sulfoximine (MSO) dramatically inhibited GS activity, whereas it did not affect the aminating and deaminating activities of GDH (data not shown). Following MSO treatment, ammonium reassimilation through GS was inhibited and the increase in total ammonium content via photorespiration became apparent as the difference between the light and dark treatments both in old and young leaves, except at 30 min in young leaves (Fig. 2B).
Release of [15N]ammonium from [2-15N]Glu was higher in the light compared to the dark both in young and old leaves (Fig. 3, A and B). In the light, the addition of MSO led to similar rates of [15N]ammonium release from [2-15N]Glu in old and young leaves (Fig. 3A). However, the young leaves accumulated higher amounts of total ammonium than the old leaves in the light (Fig. 2, A and B). In the dark, the inhibition of ammonium assimilation by MSO resulted in higher [15N]ammonium accumulation in old than in young leaves after 120 min (Fig. 3B). The results suggest that an extra [15N]ammonium was released from [2-15N]Glu deamination in the dark by the reaction independent per se of photorespiration in the old leaves.
Kinetics of Ammonium Assimilation into Amide and Amino Nitrogen The kinetics of [15N]ammonium assimilation were determined in young and old leaves in the light (Fig. 4) and in the dark (Fig. 5). In the light, the high 15N labeling was first detected as [5-15N]Gln as early as 5 min (Fig. 4, A and B), then as [2-15N]Glu, [2-15N]Gln, and other amino acids, such as Ala, Gly, and Ser, after 10 min (Fig. 4, A–D). These labeling kinetics correlate with a high efficiency of the GS activity that can use Glu as soon as it is synthesized by GOGAT (Fig. 6). The delay of [2,5-15N]Gln labeling relative to [2-15N]Gln can be explained by (1) a differential availability of chloroplastic [2-15N]Glu pool as a substrate for the cytosolic GS1 and chloroplastic GS2; (2) a higher availability of NH4+ than 15NH4+; or (3) a preference of GS toward nonisotopic ammonium as a substrate. The 15N enrichment patterns of these amino acids were similar between the young and old leaves, despite a slightly lower enrichment in the young leaves (Fig. 4, A–D). The addition of MSO resulted in the complete inhibition of the labeling of [5-15N]Gln, [2-15N]Glu, [2-15N]Gln, and [2,5-15N]Gln (data not shown), suggesting that GS catalyzes the sole efficient entry of ammonium in our conditions.
When young and old leaf discs were treated with azaserine (AZA), the [5-15N]Gln labeling was unchanged (Fig. 4, E and F). In contrast, [15N] enrichment in [2-15N]Glu, [2,5-15N]Gln, and [2-15N]Gln were substantially decreased, while the remaining label of these amino acids was slightly higher in young than in old leaves (Fig. 4, E and F). These results suggest that AZA did not completely inhibit the total GOGAT activity and that the difference was due to the higher GOGAT activity in young than in old leaves. Both MSO and AZA did not inhibit the GDH aminating and deaminating activities (data not shown). As the GDH aminating activity was induced in old leaves (Masclaux et al., 2000 ), it was expected that GDH assimilates ammonium in the old leaves. However, the 15N-labeling patterns in the old leaves did not show significant differences from those in the young leaves (compare Fig. 4, A and B). Moreover, AZA inhibited the labeling of [2-15N]Glu, [2,5-15N]Gln, [15N]Ser, [15N]Ala, and [15N]Gly in the young leaves and to a higher extent in the old leaves (Fig. 4, E–H). The labeling patterns correlate with the operation of the GS-GOGAT cycle. The feeding of [15N]ammonium resulted in the recovery of label in Gly, Ser, and Ala in young and old leaves with a constant increase during 120 min (Fig. 4, C and D). The labeling rates of these amino acids were almost as high as those of Glu and Asp (Fig. 4, A–D). In contrast, the labeling of Pro was very low. The AZA treatment substantially inhibited the transfer of label to these amino acids (Fig. 4, G and H). In the dark, [15N]ammonium was assimilated into [5-15N]Gln by GS at 5 min after the [15N]ammonium addition in both young and old leaves (Fig. 5, A and B). Compared to the light conditions, [5-15N]Gln enrichment was higher in the dark (60%–70% versus 30%–40%). In contrast, the labeling of [2-15N]Glu, [2-15N]Gln, [2,5-15N]Gln, and [2-15N]Asp was delayed and lower than 5% during the first 30 min, presumably because of a lower Fd-GOGAT activity in the dark. It can be noted that, after 30 min, the labeling rates of [2-15N]Glu, [2-15N]Gln, [2,5-15N]Gln, [2-15N]Asp, and [4-15N]Asn were increased more in old than in young leaves (compare Fig. 5, A and B), suggesting that old leaves might contain metabolic pathways that increase Glu labeling in the dark. The dark treatment caused a substantial reduction of labeling in [15N]Gly, [15N]Ser, and [15N]Ala, probably as a consequence of the low labeling of [2-15N]Glu (compare Fig. 5, C and D, and Fig. 4, C and D, respectively). The AZA treatment in the dark completely inhibited 15N enrichment in [2-15N]Glu, [2-15N]Gln, [2,5-15N]Gln, and [2-15N]Asp in the old leaves, whereas residual labeling remained in the young leaves (compare Fig. 5, B and F, and A and E, respectively). These results suggest that if old leaves contain particular metabolic pathways that increase the levels of Glu in the dark, such pathways are AZA sensitive. In addition, the MSO treatment completely inhibited label transfer into [2-15N]Glu, [2,5-15N]Gln, and [2-15N]Gln in the old leaves (data not shown), ruling out a role for GDH. It is interesting to note that the dark treatment favored [4-15N]Asn formation in young and old leaves, even in the presence of AZA (Fig. 5, A, B, E, and F). Because [2-15N]Asp labeling was absent in the AZA-treated leaves in the dark, 15NH4+ and [5-15N]Gln served as the potential amino donor to [4-15N]Asn in the dark (Fig. 6). The labeling of Gly, Ser, Ala, and Pro was inhibited by the addition of AZA (compare Fig. 5, C and G, and D and H, respectively).
Amino acids were analyzed in phloem sap from six leaf ranges, collected along the tobacco main axis, from leaf 9 (old leaf) to leaf 30 (young leaf), according to Masclaux et al. (2000)
Pro continuously decreased in the phloem with developmental age, and a similar reduction was observed in the blades (Table I). The phloem sap also transported high levels of Ser, especially in the mature leaves found at the intermediate position between young and old leaves. In the leaf blades, Ser remained almost constant (8%) along the main axis (Masclaux et al., 2000 ). It can be noted that the most abundant amino acids found in phloem (Gln, Asn, Ser, and Pro) are all derived from Glu, the most abundant amino acid present in the leaf blade. Gly, Ala, Glu, and Asp levels remained constant in exudates from the leaves collected along the main axis (Table I).
Two transgenic tobacco lines (GLU1::GUS*1 and GLU1::GUS*2) harboring the GLU1 promoter of the Arabidopsis Fd-GOGAT fused to a GUS reporter gene (GLU1::GUS) were subjected to a comparative study of the enzymes of nitrogen metabolism in young and old leaves. Nitrate reductase activity in the old leaf discs was as low as 25% to 30% of the activity in the young leaf discs (Table II). A similar reduction was observed for GS (21%–25%). In contrast, the NADH-dependent aminating and NAD-dependent deaminating activities increased about 1.5-fold and 2.4-fold, respectively, in the old leaves. GOGAT activity decreased to about one-third in the old leaves: 27% to 28% for Fd-GOGAT and 32% to 35% for NADH-GOGAT.
Concomitant with the enzyme activity, the level of Fd-GOGAT protein in the mesophyll decreased to 30% to 42% of that in the young leaf mesophyll (Fig. 7A). In contrast, the higher levels of Fd-GOGAT protein were recovered in the veins (72%–75%) and in the petiole (1.1–1.8-fold) from the old leaves (Fig. 7A).
To understand the function of the GS-GOGAT cycle that was active in NH4+ reassimilation regardless of leaf age, the tissue-specific expression of GOGAT was determined in the young and old leaves. The activity of the Fd-GOGAT GLU1 promoter decreased to a higher extent in the mesophyll than in the veins during aging (Fig. 7B). GLU1 promoter expression was quantified by in vitro GUS activity in the mesophyll, veins, and petioles of the young and old leaves and also in the roots of two transgenic tobacco lines (Table III). GLU1 promoter activity in the mesophyll decreased by 42% to 44% in the old leaves compared to the activity in the young leaves, whereas its decrease was only by 7% to 12% in the vascular tissues of veins and petioles from the old leaves. GUS activity remained relatively constant in the roots (Table III).
Steady-state levels of Fd-GOGAT mRNA were measured by relative quantitative reverse transcription (RT)-PCR in mesophyll and veins from young and old leaves of both GLU1::GUS*1 and GLU1::GUS*2 transgenic plants. Equal total amounts of 18S ribosomal RNA were used as the internal standard for RT (data not shown). The level of Fd-GOGAT mRNA in the old mesophyll decreased between 76% and 85% of the mRNA levels in the young mesophyll (Fig. 7C). In the veins, the Fd-GOGAT mRNA level remained higher, ranging between 91% and 110% in the old leaves (Fig. 7C). The different extent of decrease in the mRNA (Fig. 7C) and protein levels of Fd-GOGAT (Fig. 7A) suggests an involvement of posttranscriptional regulation.
Using confocal laser-scanning microscopy, the cellular and subcellular localization of Fd-GOGAT protein was determined in leaf sections of tobacco by the indirect immunofluorescence method. The specific labeling of fluorochrome was detected in the mesophyll cells (Fig. 8A) compared to the control treated with only the first antibody (Fig. 8B). The fluorescence was localized to the chloroplasts of palisade and spongy parenchyma. The intensity of specific labeling and the number of labeled chloroplasts increased in the absence of bovine serum albumin (data not shown). Moreover, the Fd-GOGAT protein was found to be located in the vascular system of minor veins (Fig. 8C). Minor veins revealed a collateral organization with only one layer of phloem below a small number of xylem elements. Higher magnification resolution of the vascular bundles showed that the Fd-GOGAT protein was localized in the phloem companion cells (CC) next to the sieve element (SE). Phloem CCs, which appeared within the focal plane, were found to correlate with the cells that were detected by transmission microscopy (Fig. 8D).
Nitrogen metabolism in old source leaves is characterized by a progressive hydrolysis of stromal proteins and degradation of chloroplasts (Jiang et al., 1993 ; Masclaux et al., 2000; Hörteinsteiner and Feller, 2002). Since the main metabolic process in leaf senescence consists of nutrient remobilization, toxic free ammonium should be rapidly refixed into the amino acids to avoid deteriorating effects and provide nitrogenous forms suitable for source-sink transport. Our 15N-labeling study provides evidence that significant [15N]ammonium was released from [15N]Glu deamination by GDH in the dark to higher extents in old than in young leaves. This is consistent with the higher GDH activity in response to natural senescence (Masclaux et al., 2000; Masclaux-Daubresse et al., 2002). It is estimated that up to one-third of the Glu-dependent respiratory rates in isolated mitochondria can be attributed to the GDH deamination reaction (Aubert et al., 2001). The carbon flow from Glu oxidation becomes important under conditions of carbon limitation in darkness (Scheible et al., 2000) and carbohydrate starvation (Robinson et al., 1992; Turano, 1998). Also, the transition of young leaves to old leaves is characterized in part by a decrease in Suc (Masclaux et al., 2000). Therefore, it is conceivable that GDH supplies 2-oxoglutarate by Glu oxidation for the nitrogen and carbon cycle in old leaves.
Label of [15N]ammonium was rapidly incorporated into [5-15N]Gln by GS in the light and in the dark regardless of leaf age. Kinetics of [15N]ammonium assimilation clearly showed that Fd-GOGAT and/or NADH-GOGAT transferred [5-15N] of Gln to [2-15N]Glu as soon as 10 min after [15N]ammonium feeding in the light, while the [2-15N]Glu labeling by GOGAT was slightly delayed in darkness. Despite the induction of cytosolic GS1 and a partial degradation of chloroplasts in old leaves, the chloroplastic GS2 protein remains predominant over the cytosolic GS1 protein in tobacco leaves (ratio of 75%:25% in the old leaves and 95%:5% in the young leaves; Masclaux et al., 2000
GOGAT occupies the central position of photorespiratory nitrogen metabolism by providing Glu that serves as the amino donor via transaminase reactions. The 15N-labeling patterns of Glu, Gly, Ser, Ala, and Pro in this study correlate with the open flux of nitrogen by import and export of amino acids into and from the photorespiratory nitrogen cycle, respectively (Betsche, 1983
Interestingly, a low [15N]Glu labeling by the GS-GOGAT cycle was detected in old leaves after 30 min of [15N]ammonium feeding in the dark. This indicates that the nitrogen cycle takes place in the dark, albeit at lower rates. Given that Ala partially replaces Glu in the transaminase reaction with higher efficiency in the dark, [15N]Glu would become apparent. Consistently, peroxisomal Glu:glyoxylate aminotransferase can use both Glu and Ala as the amino donor at equal rates (Igarashi et al., 2003
The spatial distribution of the enzymes of amino acid synthesis correlates with the pathway of intra- and intercellular transport of amino acids. Fd-GOGAT found in the CC-SE complex corresponds to the expression of the GLU1 promoter-GUS transgene within phloem cells (Feraud et al., 2005
The role of amino acid transport was first assigned for NADH-GOGAT in rice (Oryza sativa) because it was found in the vascular tissues, particularly in the metaphloem, metaxylem-parenchyma, and mestome sheath cells of the vascular bundles, whereas it was not detected in the mesophyll, CCs, and SEs (Tobin and Yamaya, 2001
Plant Materials and Growth Conditions
Tobacco (Nicotiana tabacum cv Xanthi XHFD8; Institut National de la Recherche Agronomique, Versailles, France) plants were grown on a clay loam soil in a greenhouse under natural lighting (temperature ranging between 20°C and 30°C) until flowering. Twelve plants were watered every 2 d with a 10 mM nitrate and 2 mM ammonium nutrient solution (Coïc and Lesaint, 1971
Transgenic tobacco plants harboring the promoter of the Fd-GOGAT gene (GLU1) of Arabidopsis (Arabidopsis thaliana ecotype Columbia) were prepared by transforming tobacco cv Xanthi (Ziegler et al., 2003
15N-labeling experiments were performed using leaves of 10-week-old wild-type tobacco plants. Discs of 1 cm in diameter were prepared from leaves numbered 10 to 11 (old) and 22 to 23 (young) from bottom to top. Discs were floated on 10 mM MES buffer, pH 6.5, containing 10 mM CaCl2, 40 mM KCl, and 2% (w/v) polyethylene glycol, either in the light (photosynthetic photon flux density, 250 µmol photons m–2 s–1) or in the dark, with or without 1 mM MSO or 1 mM AZA for 1 h. Afterward, [15N]ammonium (99% enrichment) or [2-15N]Glu (99% enrichment; Euriso-top S.A.) was added to the medium. Leaf discs were dipped into the labeling solutions, quickly transferred, and rinsed with a large volume of water before freezing in liquid nitrogen (time 0). Leaf discs were further incubated in the light or in the dark and collected at 5, 10, 20, 30, 45, 60, 120, and 240 min. Samples were frozen in liquid nitrogen prior to analysis.
Total amino acids and ammonia were extracted with 2% (w/v) sulfosalicylic acid. Extracts were centrifuged at 17,500g for 20 min to eliminate cellular debris. Amino acids were applied to a column (AG 50W-X8 resin, 100–200 mesh, H+-form, 5 x 0.5 cm; Bio-Rad Laboratories), washed with 4 mL water, and eluted with 2.5 mL of 6 M NH4OH, then with 1 mL water. Total amino acids were determined by the method of Rosen (1957)
[15N]Ammonium released from [2-15N]Glu was determined after purification and derivatization steps as described by Fujihara et al. (1986)
Phloem exudates were collected from 10-week-old wild-type tobacco plants in the light period as described previously (Chaffei et al., 2004
Nitrate reductase was extracted, and activity and activation state, expressed as the ratio of 10 mM MgCl2-dependent activity to 5 mM EDTA-dependent activity, were measured as described by Ferrario-Méry et al. (2000)
Protein samples (20–50 µg) were subjected to SDS-PAGE electrophoresis (Laemmli, 1970
Total RNA was extracted from the GLU1::GUS transgenic tobacco plants using a kit according to the manufacturer's instruction (Qiagen). To carry out relative quantitative PCR, ribosomal RNA was used as an endogenous standard, and first-strand cDNA was synthesized from 2 µg RNA using an Omniscript RT kit (Qiagen). An abundance of initial cDNA strands between samples was corrected using Quantum RNA 18S internal standards (Ambion). PCR was performed on a LightCycler instrument (Roche). The following specific primer sets were used. GLU was used for Fd-GOGAT: forward primer, 5'-GGACCTGAAAATGCATCTCA-3'; reverse primer, 5'-GCCAAATGCTGCTGATAAAC-3'. The reaction was carried out using 1:20 and 1:40 dilutions of cDNA. PCR was hot started at 95°C and carried out for 32 cycles composed of 94°C for 30 s, 46°C for 90 s, and 72°C for 90 s. PCR products were visualized by ethidium bromide in agarose gel and bands were quantified by scanning of samples with a FLA-5000 imaging system (FujiFilm France S.A.S.).
Leaf sections were incubated in 50 mM sodium phosphate, pH 7.0, 0.1 mM K3[Fe(CN)6], 0.1 mM K4[Fe(CN)6], and 1.9 mM 5-bromo-4-chloro-3-indolyl-
Leaf sections were hand cut using razor blades. Sections were fixed in 3.7% (w/v) formaldehyde for 1 h, in 50 mM PIPES buffer, pH 6.9, 5 mM MgSO4, and 5 mM EGTA (MTSB) for 30 min, then in 6.5 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.3, 14 mM NaCl, and KCl (PBS) for 15 min. Tissues were dehydrated in a graded ethanol series (30%, 50%, 70%, 90%, and 97% [v/v] in PBS). Samples were incubated in a mixture of 100% wax and 97% ethanol (1:1, v/v) at 40°C overnight, and embedded in 100% wax at 40°C for 2 h. Sections (10 µm) were prepared using a microtome and air dried on polylysine-coated glass slides. Samples on slides were dewaxed and rehydrated through a degraded ethanol series (97%, 90%, and 50% [v/v] in PBS). Samples were blocked with 1% (w/v) bovine serum albumin in PBS (blocking solution), and incubated with rabbit antibody against tobacco Fd-GOGAT diluted in blocking solution. After three washes of 5 min each in PBS, slides were incubated with goat anti-rabbit IgG labeled with Alexa 405 (Molecular Probes) in blocking solution. After washing, slides were sealed with PBS. As controls, sections were incubated with preimmune sera as the primary antibodies or without the primary antibodies, and with Alexa 405-labeled secondary antibodies. Immunofluorescence was observed using a spectral confocal laser-scanning microscope (Leica TCS SP2 AOBS). Microscopic observations were carried out with a laser diode (25 mW, 405 nm), and generated using a Leica HC PL APO 63x/1.20 Water Corr/0.17 Lbd.BL objective. Low-speed scan (200 lines/s) images (512 x 512 pixels) were generated. Alexa 405 fluorescence was collected with a specific bandwidth (407–427 nm) after a spectral adjustment to eliminate the background noise. The red autofluorescence of tissues was observed between 509 and 628 nm. In some experiments, another photomultiplier was used to collect simultaneously the transmitted light with the same configuration as above. Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: for GLU1, Y09667 and U39287; and for GLU2, U39288.
We thank Dr. David Tepfer for proofreading the manuscript. We are grateful to François Gosse for the culture and maintenance of plant materials. Received September 23, 2005; returned for revision October 31, 2005; accepted December 5, 2005.
1 This work was supported by the Université de Versailles-Saint Quentin-en-Yvelines (grants to M.R.-C. and K.P.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Akira Suzuki (suzuki{at}versailles.inra.fr). Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.071910. * Corresponding author; e-mail suzuki{at}versailles.inra.fr; fax 33–1–30–83–30–96.
Atkins C (2000) Biochemical aspects of assimilate transfers along the phloem path: N solutes in lupin. Aust J Plant Physiol 27: 531–537 Aubert S, Bligny R, Douce R, Gout E, Ratcliffe RG, Roberts JK (2001) Contribution of glutamate dehydrogenase to mitochondrial glutamate metabolism studied by 13C and 31P nuclear magnetic resonance. J Exp Bot 52: 37–45 Betsche T (1983) Aminotransferase from alanine and glutamate to glycine and serine during photorespiration in oat leaves. Plant Physiol 71: 961–965 Blackwell RD, Murray AJS, Lea PJ, Joy KW (1988) Photorespiratory amino donors, sucrose synthesis and the induction of CO2 fixation in barley deficient in glutamine synthetase and/or glutamate synthase. J Exp Bot 39: 845–858 Bradford MM (1976) A rapid and sensitive method for utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254[CrossRef][ISI][Medline] Carvalho H, Pereira S, Sunkel C, Salema R (1992) Detection of a cytosolic glutamine synthetase in leaves of Nicotiana tabacum L. by immunocytochemical methods. Plant Physiol 100: 1591–1594 Chaffei C, Pageau K, Suzuki A, Gouia H, Ghorbel MH, Masclaux-Daubresse C (2004) Cadmium toxicity induced changes in nitrogen management in Lycopersicon esculentum leading to a metabolic safeguard through an amino acid storage strategy. Plant Cell Physiol 45: 1681–1693 Chaves Das Neves HJ, Vasconcelos AMP (1987) Capillary gas chromatography of amino acids, including asparagine and glutamine: sensitive gas chromatographic-mass spectrometric and selected ion monitoring gas chromatographic-mass spectrometric detection of the N,O(S)-tert.-butyldimethylsilyl derivatives. J Chromatogr 392: 249–258[CrossRef][ISI][Medline] Coïc Y, Lesaint C (1971) Comment assurer une bonne nutrition en eau et en ions minéraux en horticulture. Horticulture Française 8: 11–14 DeWitt ND, Sussman MR (1995) Immunological localization of an epitope-tagged plasma membrane pump (H+-ATPase) in phloem companion cells. Plant Cell 7: 2053–2067[Abstract] Ek H, Finlay RD, Söderström B, Odham G (1990) Determination of 15N-labelled ammonium and total nitrogen in plant and fungal systems using mass spectrophotometry. J Microbiol Methods 11: 169–176 Feraud M, Masclaux-Daubresse C, Ferrario-Méry S, Pageau K, Lelandais M, Ziegler C, Leboeuf E, Jouglet T, Viret L, Spampinato A, et al (2005) Expression of a ferredoxin-dependent glutamate synthase gene in mesophyll and vascular cells and functions of the enzyme in ammonium assimilation in Nicotiana tabacum (L.). Planta 222: 667–677[Medline] Ferrario-Méry S, Suzuki A, Valadier M-H, Roux Y, Hirel B, Foyer CH (2000) Modulation of amino acid metabolism in transformed tobacco plants deficient in Fd-GOGAT. Plant Soil 221: 67–79[CrossRef] Ferrario-Méry S, Valadier MH, Godfroy N, Miallier D, Hirel B, Foyer CH, Suzuki A (2002) Diurnal changes in ammonia assimilation in transformed tobacco plants expressing ferredoxin-dependent glutamate synthase mRNA in the antisense orientation. Plant Sci 112: 524–530 Fischer W-N, André B, Rentsch D, Krolkiewicz S, Tegeder M, Breitkreuz K, Frommer WB (1998) Amino acid transport in plants. Trends Plant Sci 3: 188–195[CrossRef][ISI] Fujihara S, Nakashima K, Kurogochi Y (1986) Determination of 15NH3 by gas chromatography-mass spectrometry. Application to the measurement of putrecine oxidation by human plasma. J Chromatogr 383: 271–280[Medline] Hanke GT, Kimata-Ariga Y, Taniguchi I, Hase T (2004) A post genomic characterization of Arabidopsis ferredoxin. Plant Physiol 134: 255–264 Hörteinsteiner S, Feller U (2002) Nitrogen metabolism and remobilization during senescence. J Exp Bot 53: 927–937 Igarashi D, Miwa T, Seki M, Kobayashi M, Kato T, Tabata S, Shiniozaki K, Ohsumi C (2003) Identification of photorespiratory glutamate:glyoxylate aminotransferase (GGAT) gene in Arabidopsis. Plant J 33: 975–987[CrossRef][ISI][Medline] Ireland RJ, Lea PJ (1999) The enzymes of glutamine, glutamate, asparagine, and aspartate metabolism. In BK Singh, ed, Plant Amino Acids. Biochemistry and Biotechnology. Marcel Dekker, New York, pp 49–109 Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusion: Jeschke WD, Pate JS (1991) Modelling of the partitioning, assimilation and storage of nitrate within root and shoot organs of castor bean (Ricinus communis L.). J Exp Bot 42: 1091–1103 Jiang C-Z, Rodermel SR, Shibles RM (1993) Photosynthesis, Rubisco activity and amount, and their regulation by transcription in senescing soybean leaves. Plant Physiol 101: 105–112[Abstract] Kamachi K, Yamaya T, Hayakawa T, Mae T, Ojima K (1992) Vascular bundle-specific localization of cytosolic glutamine synthetase in rice leaves. Plant Physiol 99: 1481–1486 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685[CrossRef][Medline] Lam HM, Hsieh MH, Coruzzi G (1998) Reciprocal regulation of distinct asparagine synthetase genes by light and metabolites in Arabidopsis thaliana. Plant J 16: 345–353[CrossRef][ISI][Medline] Liepman AH, Olsen LJ (2001) Peroxisomal alanine:glyoxylate aminotransferase (AGT1) is a photorespiratory enzyme with multiple substrates in Arabidopsis thaliana. Plant J 25: 487–498[CrossRef][ISI][Medline] Masclaux C, Valadier M-H, Brugière N, Morot-Gaudry J-F, Hirel B (2000) Characterization of the sink/source transition in tobacco (Nicotiana tabacum L.) shoots in relation to nitrogen management and leaf senescence. Planta 211: 510–518[CrossRef][ISI][Medline] Masclaux-Daubresse C, Carrayol E, Valadier M-H (2005) The two nitrogen mobilisation- and senescence-associated GS1 and GDH genes are controlled by C and N metabolites. Planta 221: 580–588[CrossRef][ISI][Medline] Masclaux-Daubresse C, Valadier M-H, Carrayol E, Reisdorf-Cren M, Hirel B (2002) Diurnal changes in the expression of glutamate dehydrogenase and nitrate reductase are involved in the C/N balance of tobacco source leaves. Plant Cell Environ 25: 1451–1462[CrossRef] Miflin BJ, Habash DZ (2002) The role of glutamine synthetase and glutamate dehydrogenase in nitrogen assimilation and possibilities for improvement in the nitrogen utilization of crops. J Exp Bot 53: 979–987 Nakano K, Suzuki T, Hayakawa T, Yamaya T (2000) Organ and cellular localization of asparagine synthetase in rice plants. Plant Cell Physiol 41: 874–880 Ngai N, Tosai FY, Coruzzi G (1997) Light-induced transcriptional repression of the pea AS1 gene: identification of cis-elements and transfactors. Plant J 12: 1021–1034[CrossRef][ISI][Medline] O'Neal D, Joy KD (1973) Glutamine synthetase of pea leaves. I. Purification, stabilisation and pH optima. Arch Biochem Biophys 159: 113–122[CrossRef][ISI][Medline] Pereira S, Carvalho H, Sunkel C, Saleman HM (1992) Immunocytolocalisation of glutamine synthetase in mesophyll and phloem of leaves of Solanum tuberosum L. Protoplasma 167: 66–73[CrossRef] Robinson SA, Stewart GR, Phillips R (1992) Regulation of glutamate dehydrogenase activity in relation to carbon limitation and protein catabolism in carrot cell suspension cultures. Plant Physiol 98: 1190–1195 Rochat C, Boutin JP (1989) Carbohydrates and nitrogenous compounds change in the hull and in the seed during the pod development of pea. Plant Physiol Biochem 27: 881–887 Rosen H (1957) A modified ninhydrin colorimetric analysis for amino acids. Arch Biochem Biophys 67: 10–15[CrossRef][ISI][Medline] Scheible W-R, Krapp A, Stitt M (2000) Reciprocal diurnal changes of phosphoenolpyruvate carboxylase expression and cytosolic pyruvate kinase, citrate synthase and NADP-fisocitrate dehydrogenase expression regulate organic acid metabolism during nitrate assimilation in tobacco leaves. Plant Cell Environ 23: 1155–1167[CrossRef] Somerville CR, Ogren WL (1980) Inhibition of photosynthesis in Arabidopsis mutants lacking leaf glutamate synthase activity. Nature 286: 257–259[CrossRef] Srivastava HS, Singh RP (1987) Role and regulation of L-glutamate dehydrogenase activity in higher plants. Phytochemistry 26: 597–610[CrossRef] Suzuki A, Burkhart W, Rothstein S (1996) Nitrogen effects on the induction of ferredoxin-dependent glutamate synthase and its mRNA in maize leaves under the light. Plant Sci 114: 83–91 Suzuki A, Rioual S, Godfroy N, Roux Y, Boutin JP, Rothstein S (2001) Regulation by light and metabolites of ferredoxin-dependent glutamate synthase in maize. Physiol Plant 112: 524–530[Medline] Tercé-Laforgue T, Dubois F, Ferrario-Méry S, Pou de Crecenzo M-A, Sangwan R, Hirel B (2004) 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 Physiol 136: 4308–4317 Thomson NW, Whatley JM (1980) Development of non-green plastids. Annu Rev Plant Physiol 31: 375–394[ISI] Tobin AK, Yamaya T (2001) Cellular compartmentation of ammonia assimilation in rice and barley. J Exp Bot 52: 591–604 Tourgeon R (1996) Phloem loading and plasmodesmata. Trends Plant Sci 1: 418–423[CrossRef] Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 71: 4607–4611 Turano FJ (1998) Characterization of mitochondrial glutamate dehydrogenase from dark-grown soybean seedlings. Physiol Plant 104: 337–344 van Bel AJE (2003) The phloem, a miracle of ingenuity. Plant Cell Environ 25: 125–149 Williams BD, Wolf RR (1994) Determination of amino- and amide-15N glutamine enrichment with tertiary butyldimethylsilyl derivatives. Biol Mass Spectrom 23: 682–688[CrossRef][ISI][Medline] Yamaya T, Hayakawa T, Tanasawa K, Kamachi K, Mae T, Ojima K (1992) Tissue distribution of glutamate synthetase in rice leaves. Occurrence of NADH-dependent glutamate synthase protein in the unexpanded non-green leaf blades. Plant Physiol 100: 1427–1432 Yamaya T, Oaks A, Rhodes D, Matsumoto H (1986) Synthesis of [15N]glutamate from [2-15N]glutamate and [15N]glycine by mitochondria isolated from pea and corn shoots. Plant Physiol 81: 754–757 Ziegler C, Feraud M, Jouglet T, Viret L, Spampinato A, Paganelli V, Ben Hammouda M, Suzuki A (2003) Regulation of promoter activity of ferredoxin-dependent glutamate synthase. Plant Physiol Biochem 41: 649–655[CrossRef] This article has been cited by other articles:
|
TOP
ABSTRACT
RESULTS
-D-GlcUA (X-Gluc) at 37°C for 2 to 18 h (Jefferson et al., 1987
