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First published online November 24, 2004; 10.1104/pp.104.047548 Plant Physiology 136:4308-4317 (2004) © 2004 American Society of Plant Biologists Glutamate Dehydrogenase of Tobacco Is Mainly Induced in the Cytosol of Phloem Companion Cells When Ammonia Is Provided Either Externally or Released during PhotorespirationUnité de Nutrition Azotée des Plantes, Institut National de la Recherche Agronomique, 78026 Versailles cedex, France (T.T.-L., S.F.-M., M.-A.P.d.C., B.H.); Laboratoire d'Androgénèse et Biotechnologie Végétale, Université de Picardie Jules Verne, 80039 Amiens cedex, France (F.D., R.S.); and Laboratoire de Biochimie et Biologie Moléculaire, Unité Mixte de Recherche Physiologie Moléculaire des Semences, Unité Fédérative de Recherche Sciences, Université d'Angers, 49045 Angers cedex 01, France (M.-A.P.d.C.)
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. Tercé-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.
Ammonium is the ultimate form of inorganic nitrogen available to the plant. It can originate from a wide variety of metabolic processes such as nitrate reduction, photorespiration, phenylpropanoid metabolism, utilization of nitrogen transport compounds, amino acid catabolism, symbiotic nitrogen fixation (Hirel and Lea, 2001  ), and insect digestion in carnivorous plants (Schulze et al., 1997). It is then incorporated into an organic molecule, 2-oxoglutarate, by the combined action of the enzymes Gln synthetase (GS) and Glu synthase (Gln-2-oxoglutarate aminotransferase; GOGAT) to allow the synthesis of Gln and Glu. Both amino acids are further used as amino group donors for the synthesis of virtually all the nitrogen-containing molecules including the other amino acids needed for protein synthesis and nucleotides used as basic molecules for RNA and DNA synthesis (Hirel and Lea, 2001). The GS/GOGAT cycle is the major mechanism of ammonium assimilation in higher plants regardless of the various sources of ammonium listed above. However, it has often been argued that other enzymes have the capacity to assimilate ammonium, leading to the hypothesis that alternative pathways might operate under particular physiological conditions when the GS/GOGAT pathway may not be able to fulfill its function (Harrison et al., 2003).
One of these alternative pathways is the reaction catalyzed by the mitochondrial NAD(H)-dependent Glu dehydrogenase (GDH; EC 1.4.1.2), which possesses the capacity to assimilate ammonium in vitro utilizing the organic molecule 2-oxoglutarate to synthesize Glu. This observation led a number of authors to propose that GDH could operate in the direction of ammonium assimilation (Yamaya and Oaks, 1987
Studies on tobacco (Nicotiana tabacum) leaf source/sink relationships have shown that GDH is induced when plants are grown on ammonium as the sole nitrogen source (Tercé-Laforgue et al., 2004
To further investigate the putative role of externally applied ammonium in the regulation of GDH protein synthesis, the subcellular localization of the enzyme was studied in ammonium fed (NH4+) and nitrogen limited (NL) plants with a particular emphasis on highly vascularized organs where the enzyme is mostly present. In addition, the subcellular localization of GDH was also investigated in transformed tobacco plants expressing a partial ferredoxin-dependent GOGAT (Fd-GOGAT) cDNA in the antisense orientation during the transition from CO2 enrichment (where photorespiration is inhibited or greatly reduced) to air (where photorespiration is a major process of ammonium production in leaves; Ferrario-Méry et al., 2000
Relative Ammonium Concentration in the Phloem Sap
To determine the concentration of ammonium and amino acids in the phloem, samples of sap were collected in NH4+ and NL plants, in Fd-GOGAT antisense plants, and in untransformed control plants using the same young leaf in all experiments. This young leaf was used in both NH4+ and NL plants because older leaves were not able to exude. In Fd-GOGAT antisense plants, photorespiration and thus ammonium production in the mitochondria has been shown to be higher in young photosynthetically active leaves (Leegood et al., 1995
GDH Activity and Protein Levels in Stems and Midribs of Plants Grown on Low Nitrogen and Ammonium and in Fd-GOGAT Antisense Plants
In previous publications we have shown that ammonium is one of the main effectors responsible for the increase or the induction of GDH at the activity, protein, and gene expression levels (Tercé-Laforgue et al., 2004 Both GDH aminating and deaminating activities were therefore measured in stems of NH4+ and NL plants following manual separation of the central cylinder (containing most of the phloem tissues) from the cortical parenchyma cell layers. Using this technique, it was possible to demonstrate that GDH aminating activity was always higher in the central cylinder compared to the cortical cell layers, along the stem length in both NH4+ and NL plants (Table II). In addition, highest activity was measured in the central cylinder in the basal stem part of NH4+ plants, whereas at the top of the stems its activity was 3 times lower regardless of the mode of nitrogen nutrition. Although this technique was not fully accurate in terms of tissue separation, it demonstrated that the enzyme is more active in the zone of the stem containing vascular tissue. A similar pattern was observed for GDH deaminating reaction, but its activity was approximately 10 times lower compared to its aminating counterpart (data not shown). GDH aminating activity was also much higher in the leaf midrib of NH4+ plants (Table II).
In young leaves of Fd-GOGAT antisense plants in which photorespiratory ammonia accumulates following transfer from high CO2 to ambient atmosphere (Ferrario-Méry et al., 2000 ), an increase in GDH activity was found to occur both in the leaf midrib and in the petiole compared to the untransformed control plants (Table II).
Using grapevine (Vitis vinifera) antibodies, already successfully employed for the immunolocalization of GDH in a number of plant species (Dubois et al., 2003
A slight increase in the amount of GDH protein was observed in both the petiole and the midrib of Fd-GOGAT antisense plants following a 3-d transfer from high CO2 to air compared to untransformed plants (Fig. 1B). The variations in GDH activity and in the amount of the corresponding protein in leaf mesophyll tissue are not presented because it was not possible to remove all the minor veins containing some phloem tissue (see below).
To refine the localization of GDH in phloem-rich tissues such as stems, petioles, and midribs and to determine whether ammonium provided externally or internally released during photorespiration had an impact on the subcellular distribution of the protein, immunocytochemical electron microscopy experiments were conducted. In previous physiological studies experiments were performed on leaves that still contained some minor veins in the mesophyll tissue (Masclaux et al., 2000 Figure 2A shows a partial view of a mature leaf mesophyll cell of NH4+ plants after incubation with the GDH antiserum. The section was devoid of gold particles, indicating the absence of GDH protein. However, it is possible that very low amounts of protein were present in the mesophyll cells and therefore at the limit of detection of the immunocytochemical technique. No gold particles were observed when a similar section was treated with preimmune serum (Fig. 2B). Similar results were obtained in NL plants (data not shown). Quantification of gold particles confirmed the absence of GDH both in the mitochondria and cytosol of leaf mesophyll, considering that the background level is around 4 particles/µm2. For the cytosol, the labeling was close to the background level, and the changes in GDH protein content observed both in the mitochondria and in the cytosol of NH4+ plants were not significant (Table III).
In contrast, in the CCs of the minor veins of NH4+ plants gold particles were mostly present in the mitochondria. However, some labeling was detected in the cytosol and the vacuolar material (Fig. 2C). In the CCs of minor veins of NL plants the labeling was weak and mostly visible in the mitochondria (Fig. 2D). Quantification of gold particles showed that in the minor vein CCs the increase in GDH protein content occurred both in the cytosol and in the vacuole of NH4+ plants (Table III). In the leaf midrib (Fig. 3A ) of NH4+ plants, GDH protein was present both in the cytosol and the mitochondria of the CCs, but the strongest labeling was detected inside the vacuole. In the vacuole, the variations observed between organs and treatments were not only due to the density of labeling in the vacuolar material but also dependent on the proportion of cellular material present inside the vacuole. Therefore, the data presented in Table III correspond to an average density of gold particle present in the whole vacuolar area. Although much lower than in the cytosol and the vacuole, an increase in GDH protein in the mitochondria of midribs CCs (Table III) was also observed. Similar results were obtained with a petiole section (data not shown).
Only a few gold particles were visible when a similar leaf midrib section was treated with preimmune serum (Fig. 3B). Controls for the specificity of the labeling were also performed with GS and isocitrate dehydrogenase (IDH) antiserum. Figure 3D shows a partial view of a mature leaf mesophyll cell of NH4+ plants after incubation with the GS antiserum. Gold particles were only present in the plastids. When minor vein or midrib sections of NH4+ plants were treated with the GS antiserum, gold particles were only detected in the cytosol of the CCs (Fig. 3C). The other control using antibodies against IDH clearly shows that the protein is only present in the mitochondria (Fig. 3E). In the three controls we did not observe any labeling above the background level in the vacuolar material of the CCs (Fig. 3, B, C, and E). Moreover, the GDH antiserum appears to be highly specific since only one spot could be detected following two-dimensional western-blot analysis (Tercé-Laforgue et al., 2004 ). At the base of the stem of NH4+ plants large amounts of GDH protein were present both in the mitochondria and the cytosol of the CCs (Fig. 4, A and B ). Some labeling was also detected in the vacuolar material of these cells (Fig. 4, C and D). However, the density of gold particles was much lower compared to that found in the vacuole of the leaf midrib CCs (see Fig. 3A and Table III for comparison). In an equivalent stem section of NL plants GDH protein was mostly present in the mitochondria of the CCs. However, compared to NH4+ plants the labeling was much weaker (Fig. 4E). Quantification of gold particles in the basal stem part CCs of plants fed with NH4+ confirmed a stronger induction of GDH in the cytosol compared to that seen in the mitochondria (Table III). This quantification also shows that there is more protein in the stems compared to midribs. A weak background of gold particles could be seen in the basal stem part cortical parenchyma cells of NH4+ plants (Fig. 4F). In these cells, the number of gold particles was very low compared to CCs containing tissue and not significantly different in NL and NH4+ plants. Moreover, their density was similar to that measured in leaf mesophyll cells (data not shown).
A similar investigation was conducted using the top stem part of both NH4+ and NL plants. A very weak labeling was only observed in the top stem CCs of NH4+ plants in both the cytosol and in the mitochondria (Fig. 5A ). At the top of the stem of NL plants, the labeling was mostly present in the mitochondria of CCs and was much weaker in comparison to that of NH4+ plants (Fig. 5B; Table III). Compared to NL plants there was a significant increase in the number of gold particles both in the mitochondria and the cytosol of NH4+ plants (Table III). However, the signal was much lower when compared to that observed in the basal part of the stem, and the vacuole was unlabeled (see Fig. 4, A and B; Table III). Immunolocalization of GDH was also conducted using leaves of Fd-GOGAT antisense plants. When Fd-GOGAT antisense plants were transferred from high CO2 to air, some labeling was detected both in the mitochondria and in the cytosol of the CCs present in the midrib (Fig. 5C). When untransformed control plants were subjected to the same treatment, GDH protein was detected only in the mitochondria (Fig. 5D). Since in the mesophyll parenchyma cells of Fd-GOGAT antisense plants no gold particles were visible either in the cytosol or in the mitrochondria (Fig. 5E), it can be concluded that ammonium released from photorespiration is able to induce the synthesis of GDH specifically in the cytosol of the CCs.
Previous investigations showed that high concentrations of ammonium either provided externally (Cammaerts and Jacobs, 1985 ; Srivastava and Singh Rana, 1987; Lea and Ireland, 1999) or released into the sieve tube from protein hydrolysis (Masclaux et al., 2000; Limami et al., 2002) generally lead to an increase in leaf GDH activity. To our knowledge, this is the first demonstration that GDH increase occurs mainly in stems and midribs and is restricted to the CCs. Subcellular localization of the enzyme using immunogold labeling demonstrates that the amount of GDH increases mainly in the cytosol and vacuolar material and in the mitochondria of the CCs. This phenomenon occurs when ammonia is applied externally to the root system or internally released during the photorespiratory process. This conclusion was corroborated by higher amounts of ammonium translocated through the phloem stream, irrespective of its origin. The increase in both NADH and NAD-dependent activity and GDH content was proportional to the increase in ammonium content of the phloem sap and not directly correlated to the concentration of a particular amino acid.
It is well established that cycling of nitrogen molecules from xylem to phloem largely contributes to nitrogen transport via the phloem (Jeschke and Pate, 1992
Under standard plant growth conditions, the basic level of GDH activity in the CCs, which remains confined to the mitochondria, may have a house-keeping role in a tissue that has to cope with a low oxygen concentration (van Dongen et al., 2003
The picture arising from the immunogold labeling studies is that the increase in the cytosolic GDH is not only due to a higher ammonium level but is also dependent of the anatomy of the organ. GDH induction was observed to be higher in basal parts of the stem, which may be explained by the fact that they are the main site for xylem-phloem exchange of nitrogen (Atkins, 2000 Interestingly, we also found some organ specificities in the distribution of GDH protein, particularly in the leaf midrib, in which the vacuolar material was strongly labeled. It is unlikely that this labeling is an artifact since two positive controls performed either with GS antibodies or IDH antibodies and a negative control performed with preimmune serum confirmed the specificity of the labeling in the cytosol (GS) and in the mitochondria (IDH) of CCs but not in the vacuole. In addition, other experiments showed that the vacuolar labeling was specific to the CCs in the leaf midrib because little vacuolar labeling was observed in other cell types and CCs in minor veins, top stem internodes, and mesophyll cells.
The high levels of GDH in the vacuole remain enigmatic, although GDH has been found previously localized inside multivesicular bodies associated with vacuolar autophagic activity. This lytic activity of the vacuole already reported in the senescing flowers of grapevine (Paczek et al., 2002
In conclusion, our results suggest that GDH plays a dual role in CCs, either in the mitochondria when mineral nitrogen availability is low or in the cytosol when ammonium concentration increases above a certain threshold. An attractive hypothesis is that GDH, when induced by ammonium in the cytosol, may act as a sensor to evaluate the carbon/nitrogen status of the plant particularly with respect to ammonium and sugar concentration and/or fluxes through the phloem stream. A putative sensing role for GDH is in keeping with the current view that the continuity between CCs and sieve tubes is one of the key elements for metabolite translocation and signaling during plant growth and development (van Bel, 2003
Plant Material and Growth
Tobacco (Nicotiana tabacum) cv xanthi XHFD8 (Institut National de la Recherche Agronomique [INRA], Versailles, France) was grown on coarse sand. From the bottom of the seedlings, each emerging leaf was numbered and tagged. From a batch of 6-week-old plants, 12 plants of uniform development and numbering 7 leaves were selected. These were transferred to a controlled environment growth chamber (16 h light, 350–400 µmol photons m–2 s–1, 25°C; 8 h dark, 18°C) and watered with either a NH4+ solution (5 mM NH4+) or an NL solution (0.1 mM NO3–). For both NH4+ and NL solutions the basic mixture contained 1.25 mM K+, 0.25 mM Ca2+, 0.25 mM Mg2+, 1.25 mM H2PO4–, 0.75 mM SO42–, 21.5 µM Fe2+ (Sequestrene; Syngenta Agro S.A.S, Rueil Malmaison, France), 23 µM B3+, 9 µM Mn2+, 0.3 µM Mo2+, 0.95 µM Cu2+, and 3.5 µM Zn2+. For the NH4+ solution ammonium was supplied as 1 mM (NH4)2SO4 plus 3 mM NH4Cl, and for the NL solution NO3– was supplied as 0.1 mM KNO3. Plants were automatically watered for 1 min (flow rate for each plant, 50 mL min–1) every 2 h. Four plants were used for each nitrogen feeding condition. Four weeks after sowing, leaves were numbered 8, 9, 11, 13, 15, and 20 (from bottom to top) for both NH4+ and NL plants as described by Tercé-Laforgue et al. (2004)
The growth and culture of transformed and untransformed tobacco plants were similar to those described in Ferrario-Méry et al. (2000)
Lyophilized plant material was used for metabolite extraction. Free ammonium was extracted with 2% (w/v) 5-sulfosalicylic acid (1 mL/10 mg dry weight [DW]) as described by Ferrario-Méry et al., (1998)
Phloem exudates were collected from leaf 18 of NH4+, NL, and Fd-GOGAT antisense plants grown as described above. Phloem exudates were obtained using the technique described by King and Zeevaart (1974)
Enzymes were extracted from frozen leaf material stored at –80°C. All extractions were performed at 4°C. GDH was measured as described by Turano et al. (1996)
For metabolite analyses and measurement of enzyme activities, results are presented as mean values for four plants with SEs (SE = SD/
Proteins were extracted from frozen leaf material in cold extraction buffer containing 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM MgCl2, 0.5% (w/v) polyvinylpyrrolidone, 0.1% (v/v) Proteins were scanned on the western-blot membranes with a Power Look II scanner (UMAX Data Systems, Taipei, Taiwan) and quantified with the software the public domain NIH Image program (developed at the United States National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image).
Leaf fragments (2–3 mm2), mesophyll, midribs, or stems were fixed in freshly prepared 1.5% (w/v) paraformaldehyde in phosphate buffer 0.1 M, pH 7.4, for 4 h at 4°C. For immunolocalization, material was dehydrated in an ethanol series (final concentration 90% [v/v] ethanol) then embedded in London Resin white resin (Polysciences, Warrington, PA). Polymerization was carried out in gelatin capsules during 10 h at 54°C. For immunotransmission electron microscopy studies, ultra thin sections were mounted on 400-µm mesh nickel grids and allowed to dry at 37°C. Sections were first incubated with 5% (v/v) normal goat serum in T1 buffer (0.05 M Tris-HCl buffer containing 2.5% [w/v] NaCl, 0.1% [w/v] BSA, and 0.05% [v/v] Tween 20, pH 7.4) for 1 h at room temperature then with anti-GDH rabbit serum (Loulakakis and Roubelakis-Angelakis, 1990
Polyclonal antiserum raised against tobacco GS2 (Hirel et al., 1984
We gratefully acknowledge Dr. Judith Harrison for critical reading of the manuscript and François Gosse for technical assistance. The antibodies to grapevine GDH were a generous gift from Professor Kaliopi Roubelakis-Angelakis. Received June 3, 2004; returned for revision October 12, 2004; accepted October 14, 2004.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.047548. * Corresponding author; e-mail hirel{at}versailles.inra.fr; fax 33–1–30–83–30–96.
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ABSTRACT
RESULTS
, where SD is the standard deviation and n the number of replicates). 
-mercaptoethanol and 4 µM leupeptine, and separated by SDS-PAGE (Laemmli, 1970
-ketoglutarate are metabolite signals involved in nitrate reductase gene transcription in untransformed and transformed tobacco plants deficient in ferredoxin-glutamine-
