# Diurnal changes in the expression of glutamate dehydrogenase and nitrate reductase are involved in the C/N balance of tobacco source leaves

Céline Masclaux-daubresse, Marie-Hélène Valadier, Elisa Carrayol, Michèle Reisdorf-Cren, Bertrand Hirel

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Céline Masclaux-daubresse, Marie-Hélène Valadier, Elisa Carrayol, Michèle Reisdorf-Cren, Bertrand Hirel. Diurnal changes in the expression of glutamate dehydrogenase and nitrate reductase are involved in the C/N balance of tobacco source leaves. Plant, Cell and Environment, 2002. hal-04495760

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# Diurnal changes in the expression of glutamate dehydrogenase and nitrate reductase are involved in the C/N balance of tobacco source leaves

C. MASCLAUX-DAUBRESSE<fnref n="1" />, M.-H. VALADIER<fnref n="1" />, E. CARRAYOL<fnref n="1" />, M. REISDORF-CREN<fnref n="1" />,<fnref n="2" /> & B. HIREL<fnref n="1" />

## ABSTRACT

A novel cDNA encoding glutamate dehydrogenase (GDH) from tobacco (Nicotiana tabacum), named *gdh1*, was characterized. The *gdh1* mRNA was detected in roots, stems and source/senescent leaves. In order to investigate diurnal regulation of *gdh1* in leaves, the content in *gdh1* mRNA was measured every 3 h over a 48 h period and compared to *nia* and *gs2* mRNA levels, encoding, respectively, nitrate reductase (NR) and chloroplastic glutamine synthetase (GS2). In source leaves, *gdh1* mRNA levels exhibit diurnal fluctuations. A 12 h shift was observed between the day-night rhythms of *gdh1* and *nia* expression. Metabolite contents were also measured and a shift in the day-night fluctuations of both glutamate (GLU) and γ-aminobutyric acid (GABA) was observed between sink and source leaves, whereas the diurnal rhythm of α-ketoglutarate showed no change. A possible role of GDH in the shift of GLU and GABA contents is discussed. Leaf disc experiments showed that *gdh1* expression is enhanced in conditions of continuous darkness. This trend is inhibited by sucrose feeding. The opposite was observed for *nia* expression. An important outcome of this work is the reverse regulation of *gdh1* and *nia* genes. A possible role of sugars and amino acids in the co-regulation of *gdh1* and *nia* genes is suggested.

**Key-words:** C/N balance; diurnal rhythm; glutamate dehydrogenase; nitrate reductase; senescence; sink/source relationships; sugar regulation.

## INTRODUCTION

At first, glutamate dehydrogenase (GDH, EC 1.4.1.2) was considered as the key enzyme for ammonia assimilation in plants (Sims & Folkes 1964). Since the discovery of glutamate synthase (GOGAT), the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway has been shown to be the major process for ammonia assimilation

into amino acids (Miflin & Lea 1980). Therefore, the role of GDH in plant metabolism is still a matter of debate (Ireland & Lea 1999 for review). Because of its low affinity for ammonia, it has been suggested that GDH was a catalytic enzyme involved in the deamination of glutamate (Pahlich 1996). Moreover, GDH has been identified in mitochondria and a high range of ammonia concentrations has been found in this organelle (5–10 mM) (Ireland & Lea 1999). From this it was proposed that GDH could synthesize glutamate in mitochondria (Givan 1979; Wallsgrove et al. 1983). Due to its connection with carbon and nitrogen metabolism, a possible role of GDH as an adaptive enzyme whose action depends on the carbon skeleton or glutamate requirements of the cell, has been proposed (Ireland & Lea 1999; Stitt et al. 2002).

GDH can be a homo- or hetero-hexameric protein complex. The genes that encode the different subunits are differentially expressed according to plant organs and tissues, as well as C/N balance (Cammaerts & Jacobs 1985; Ireland & Lea 1999; Morkunas et al. 2000). Therefore, the content in GDH isoenzymic forms is largely modulated and this might be one integrative aspect of GDH activity regulation (Cammaerts & Jacobs 1985; Loulakakis & Roubelakis-Angelakis 1996).

To date, the regulation of GDH expression has been described mainly through C/N feeding experiments (Tassi et al. 1984; Athwal, Pearson & Laurie 1997). In *Arabidopsis*, GDH activity and *gdh* genes are differentially regulated according to the supply of N or C metabolites (Cammaerts & Jacobs 1985; Melo-Olivera, Olivera & Coruzzi 1996; Turano et al. 1997). In several plant species, sugar feeding appeared to be a signal inhibiting *gdh* expression and GDH activity (Laurière & Daussant 1983; Tassi et al. 1984; Maestri et al. 1991; Melo-Olivera et al. 1996) whereas the addition of ammonia stimulated both. Studies of GDH regulation during plant development have indicated an age-related increase in leaf GDH activity (Thomas 1978; Laurière & Daussant 1983; Kar & Feierabend 1984; Ireland & Lea 1999 for a review). The age-related induction of GDH in a range of plant tissues was explained either by an increase in ammonia content or by the depletion of carbo-

<footnote n="1">¹Unité de Nutrition Azotée des Plantes, INRA, Centre de Versailles, Route de Saint Cyr, 78026 Versailles Cedex, France and</footnote>

<footnote n="2">²Université de Versailles-Saint Quentin-en-Yvelines, Département de Biologie, 45 avenue des Etats Unis, 78035 Versailles Cedex, France</footnote>

<footnote>Correspondence: Céline Masclaux-Daubresse. Fax: +33 1 30 83 30 96; e-mail: masclaux@versailles.inra.fr</footnote>
hydrates that are usually observed during senescence (Srivastava & Singh 1987). Indeed, natural senescence can be mimicked by dark adaptation or sugar starvation. Some studies have shown dark-induced *-gdh* expression and -GDH activities (Peeters & Van Laere 1992; Melo-Olivera *et al.* 1996; Turano *et al.* 1997). Therefore, it is likely that in some plants *gdh* behaves as a dark-induced gene (Fujiki *et al.* 2000a).

Recently, we have shown that in tobacco leaves, *gdh* mRNA can be detected exclusively in source leaves where it accumulated during ageing (Masclaux *et al.* 2000). Therefore, source leaves of tobacco appeared to be a good model to study *gdh* age-related regulation. In this study, we observed that, in source leaves, GDH activity increased and *gdh* mRNA accumulated, whereas sucrose content decreased when ammonia accumulated (Masclaux *et al.* 2000). Our interpretation, with respect to the physiological aspects, was coherent with knowledge from previous works. However, our picture was static and relative to events taking place at 1100 h, the time when the plant material was harvested.

Diurnal changes in metabolite contents such as sugars or ammonia are well-known (Scheible, Krapp & Stitt 2000) and many enzymes involved in carbohydrate and nitrogen metabolism also exhibit diurnal rhythms. The influence of metabolites as signals that can modulate the diurnal fluctuation of the expression of certain genes has been largely debated (Cheng *et al.* 1992; Matt *et al.* 1998; Klein *et al.* 2000; Scheible *et al.* 2000; Matt *et al.* 2001; Lillo, Meyer & Ruoff 2001 for a review). If we assume that GDH plays a role in and is influenced by the C/N balance, then potential *gdh* day-night oscillations could be important. In the present study, we report the isolation and the characterization of a novel cDNA encoding GDH from tobacco (*Nicotiana tabacum*) and named *gdh1*. The *gdh1* mRNA was detected in roots, stems and source leaves. In source leaves, *gdh* expression followed a diurnal rhythm. Moreover, mRNA content was enhanced by continuous darkness and inhibited by sucrose feeding. An important outcome of this work is the opposite regulation of *gdh1* and nitrate reductase (*nia*) genes. Results are discussed in relation of nitrogen- and carbon-management that occur during leaf ageing and day-night transitions.

# MATERIALS AND METHODS

## Plant material and growth

Tobacco (*Nicotiana tabacum*, cv. Xanthi XHFD8; INRA, Versailles, France) was grown on a clay loam soil during 4 months until flowering.

For diurnal rhythm experiments, each emerging leaf was numbered and tagged from the base of the seedling. Nine plants of uniform development and numbering, were divided into three batches (A, B and C) and transferred to a controlled environment growth chamber (16 h photoperiod, 350/400 µmol photons m⁻² s⁻¹, day/night temperature 26/18 °C). Plants were automatically watered with N12

(10 mM NO₃⁻ and 2 mM NH₄⁺) (Coïc & Lesaint 1971) for 1 min (flow rate for each plant: 50 mL min⁻¹) every 2 h. When plants were flowering, they had grown up to about 45 leaves which were numbered. Leaf disc tissues (1 cm²) were randomly collected avoiding the veins, from source leaves (numbered 13, 14 and 15) and sink leaves (numbered 30, 31 and 32) (see Masclaux *et al.* 2000). Leaf discs were pooled on the three-plant batch basis (as described as above) to give three batches of source-leaf tissue and three batches of sink-leaf tissue, respectively. Leaf discs were collected over a 48 h period, at each designated sampling time (i.e. 1200, 1500, 1800, 2200, 2400, 0300, 0600, 0700 and 0900 h). Leaf tissue was frozen in liquid nitrogen and immediately reduced to a homogeneous powder that was stored at -80 °C until use in all further experiments.

For sucrose feeding and dark treatment experiments, leaf discs (1 cm²) were collected, avoiding the veins, from 12 tobacco plants, 3 h after the beginning of the light period. The discs were rinsed in distilled water and floated on 20 mL of buffer (10 mM MES pH 6.5, 40 mM KCl and 10 mM CaCl₂) in Petri dishes. The discs were incubated for 5 h in the growth chamber (23 °C), with or without sucrose (50 mM) in dark or light (150 µmol quanta m⁻² s⁻¹) condition. In each floating solution, molality was maintained at 280 mmol kg⁻¹ by the addition of polyethylene glycol (PEG, 200 AMW). The leaf discs were collected and frozen in liquid nitrogen before storing at -80 °C. The viability of the leaf discs was checked as discussed previously (see Ferrario-Mery *et al.* 2001).

## Chlorophyll, phaeophytin and total protein determinations

The chlorophyll and phaeophytin content was estimated on crude leaf extracts (Arnon 1949; Vernon 1960). Soluble protein was determined using a commercially available kit (Coomassie Protein assay reagent, Bio-Rad, Hercules, CA, USA).

## Metabolite extraction and analysis

NO₃⁻, amino acids and carbohydrates were determined after ethanolic extraction of frozen leaf disc powder (50 mg fresh weight (FW) mL⁻¹ of 80% (v/v) ethanol in water for 2 h at 4 °C). The extract was centrifuged at 12 000 × g for 5 min. The pellet, kept for the determination of starch, was resuspended in water and incubated at 100 °C for 2 h. Starch was determined after release of glucose by incubation with α-amylase and amyloglucosidase (Boehringer Mannheim, Mannheim, Germany) at 50 °C for 3 h in 20 mM acetate buffer (pH 4.6). Ethanol was removed from the supernatant under vacuum and the residue suspended in water to determine the levels of NO₃⁻, amino acids and soluble carbohydrates. The NO₃⁻ was measured according to Cataldo *et al.* (1975). Total amino acids content was assayed by the Rosen colorimetric method using glutamine as a reference (Rosen 1957). Individual amino
acid composition was determined on pooled samples extracted from an equal dry weight of each leaf material by ion-exchange chromatography (Rochat & Boutin 1989). Soluble carbohydrates were determined by an enzymatic method using a commercially available assay kit (Boehringer Mannheim). The α-ketoglutarate was extracted in 1 M HClO₄ (1 mL per 100 mg FW) and determined enzymatically (Bergmeyer 1965; Scheible *et al.* 1997).

Free NH₄⁺ was extracted in 2% solution of 5-sulfosalicylic acid (50 mg FW mL⁻¹) and determined by the phenol hypochlorite colorimetric method (Berthelot reaction) using (NH₄)₂SO₄ as a reference.

## Protein extraction, fractionation and enzymatic assays

Enzymes were extracted from frozen leaf material stored at -80 °C in cold extraction buffer containing 50 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM MgCl₂, PVP 0.5%(w/v), β-mercaptoethanol 0.1% (v/v) and 4 µM leupeptin. All extractions were performed at 4 °C. Glutamate dehydrogenase NAD and NADH activities were measured as described by Turano *et al.* (1996).

For GDH fractionation, 2 g FW of old/source leaf tissue were ground in extraction buffer as described above. The extract was filtered and loaded onto a DEAE-Sephacel (Pharmacia, Uppsala, Sweden) column as described by Brugière *et al.* (1999). After elution, using a NaCl gradient (0–0.4 M), 48 fractions were collected according to Becker, Carrayol & Hirel 2000, and assayed for GDH activity. Protein fractions were concentrated using micro concentrators (Microcon YM-3; Amicon, Bedford, MA, USA) for Western blot experiments in order to contain the same amount of (NADH) GDH activity.

## Gel electrophoresis and Western blot analysis

Proteins (10 µg) were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) (Laemmli 1970). The percentage of polyacrylamide in the running gels was 10%. Denatured proteins were electrophoretically transferred to nitro-cellulose membranes. Polypeptide detection was performed using polyclonal anti-serum raised against grape leaf GDH (Loulakakis & Roubelakis-Angelakis 1990).

## Statistics

Results are presented as mean values for the three plant-material batches (as described as above) with standard errors (n = 3).

## Screening of a Nicotiana tabacum library

A λgt10 cDNA library of tobacco was kindly provided by Dr Klaus Palme (Max-Plank Institut für Züchtungsforschung, Köln, Germany). The library was screened with a

random primed ³²P-labelled 443 bp *gdh* probe (Masclaux *et al.* 2000) and cDNA were cloned as described by Dubois *et al.* (1996). The nucleotide sequencing of the *gdh1* cDNA was carried out at the Genopole E.S.G.S, Evry, France.

## Southern blot analysis

Genomic DNA was extracted according to Paszkowski *et al.* (1984). Genomic DNA (100 µg) was digested and resolved by electrophoresis using a 0.8% agarose gel, transferred to a nylon membrane (Genescreen biotechnology systems; NEN Life Sciences Products, Boston, MA, USA) and hybridized with the random primed ³²P-labelled 443 bp *gdh* probe (Masclaux *et al.* 2000). Hybridization was performed at 65 °C according to Church & Gilbert (1984). Filters were washed with 2 × SET (0·06 M Tris-HCl pH 8, 0·3 M NaCl, 4 mM EDTA) at room temperature for 5 min and at 65 °C for 10 min Additional washings were performed successively using 1 × SET and 0.5 × SET at 65 °C for 20 min before drying and exposure to X-ray film.

## Extraction of total RNA and Northern blot analysis

Total RNA was extracted as described by Verwoerd, Dekker & Hoekema (1989) from plant material stored at -80 °C. Northern blot analysis was performed as described previously (Masclaux *et al.* 2000). The following ³²P-labelled probes were used for mRNA detection: *nia* from *Nicotiana sylvestris* (Vaucheret *et al.* 1989), *gs2* from *N. tabacum* (Becker *et al.* 1992) and a 1121 bp *Eco*RI fragment of the *gdh1* cDNA isolated in this work. Hybridization was performed under high stringency conditions as described for Southern blot analyses. The relative amounts of mRNA were determined by densitometric scanning of Northern blot autoradiograms (Power Look II scanner; UMAX Data systems, Taiwan) and quantified by an advanced quantifier (J-D Match; BioImage System Corp., Ann Arbor, MI, USA).

# RESULTS

## Cloning and sequence analysis of the tobacco glutamate dehydrogenase *gdh1* cDNA

A DNA fragment of 443 bp containing part of the coding region of a *N. tabacum* *gdh* gene (Masclaux *et al.* 2000) was used as a probe to screen a tobacco seedling cDNA library (provided by Dr Klaus Palme at the Max-Planck Institut für Züchtungsforschung, Köln, Germany). A single clone containing an open reading frame of 1630 nucleotides and designated *gdh1* was isolated (Accession number AJ420266). The corresponding translation product gave a polypeptide of 411 amino acids with a predicted molecular mass of 44·5 kDa and a pHi of 7·33. The deduced amino acid sequence showed an homology of 86 and 95% with *Nicotiana plumbaginifolia* *gdhA* and *gdhB*, respectively (Ficarelli, Tassi & Restivo 1999) and 92% with
*Legdh1* of *Solanum lycopersicum* (Purnell, Stewart & Botella 1997). The amino acid sequence deduced from *gdh1* showed all of the structural characteristics of known plant *gdh* protein sequences (Turano *et al.* 1997; Purnell *et al.* 1997).

A genomic Southern blot analysis was performed under high stringency conditions using as a probe the ³²P-labelled 443 bp fragment originally used to screen the cDNA library. Since this sequence did not contain any *Eco*RI, *Hinc*II or *Ssp*I restriction sites, tobacco genomic DNA was digested with these three enzymes. Following autoradiography, a single major band and three minor bands were visible (Fig. 1). Using low stringency hybridization, the four hybridization signals were of similar intensity (data not shown). This result suggests that GDH is encoded by more than one gene in *N. tabaccum* and that the *gdh1* sequence corresponds to one of them.

## Organ-specific expression of *gdh1* mRNA in *N. tabaccum*

Northern blot analysis was performed using the same RNA extracts previously isolated from anthers, pistils, petals, stems, midribs, young leaves and roots (Dubois *et al.* 1996) and the ³²P-labelled 1121 bp *gdh1 Eco*RI fragment as a probe. Compared to floral organs and midribs, higher amounts of *gdh1* mRNA were detected in stem and root tissues (Fig. 2). As previously observed (Masclaux *et al.*

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Genomic Southern blot analysis of *gdh1* in *N. tabaccum*. Genomic DNA was digested with *Eco*RI (lane 1), *Ssp*I (lane 2) and *Hinc*II (lane 3). The size of the DNA markers is indicated in kilobases and the unspecific hybridization of the 1·6, 1·0 and 0·5 kbp fragments of the λ 1 kb ladder (Boehringer Mannheim) is shown in lane L.</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>**Figure 2.** Northern blot analysis of *gdh1* expression in various organs from *N. tabaccum*. Total RNA were extracted from anthers, pistils, corollas, stems, midribs, young leaf blades and roots. Ten micrograms of total RNA were loaded in each track. mRNA was detected in stems and roots after hybridization with the ³²P-labelled *gdh1* probe.</figcaption></figure>

2000), the gene was not expressed in young leaf mesophyll tissue (Fig. 2).

## Characterization of the GDH protein in source leaves

Two main sections along the plant axis of a mature tobacco plant (35 leaves) have been defined previously (Masclaux *et al.* 2000). The upper section contains the youngest leaves (45–15) which behave as sink organs. In the lower section, older/senescent leaves (9–15) behave as source organs and they have been characterized by the expression of specific marker genes and proteins such as *gdh*. Therefore, leaf mesophyll tissues from source leaves (13–15) were used for GDH protein characterization.

Figure 3a shows the elution profile of GDH activity following DEAE-Sephacel ion exchange chromatography of a protein extract prepared from source leaves. A single peak of GDH activity was detected whether the enzyme activity was measured in the aminating or the deaminating direction (Fig. 3a). Protein fractions containing GDH activity were subjected to Western blot analysis using GDH-specific antiserum (Loulakakis & Roubelakis-Angelakis 1990). A single protein exhibiting a molecular mass of about 42 kDa was detected in each fraction, the highest GDH content corresponding to the fractions containing the highest enzyme activity (Fig. 3b).

These results suggest that, in tobacco source leaves at least, GDH activity is associated with a single type of subunit. However, it cannot be excluded that the enzyme contains several polypeptides of identical molecular mass that could differ in charge (Loulakakis & Roubelakis-Angelakis 1990).

## Day-night fluctuation in the expression of *gdh1* mRNA

All previous studies of *gdh1* expression in tobacco leaves were performed on samples collected in the middle of the light period (Masclaux *et al.* 2000). In order to determine
<figure><img src="image_4.png" /></figure>

**Figure 3.** DEAE-Sephacel chromatography of GDH activity in source leaves of *N. tabacum*. (a) Relative GDH aminating and deaminating activities were measured in eluted fractions and expressed as a percentage of the maximum activity. A single major peak for both activities was observed (fractions 14–22). The results are the mean ± Sn (Sn = stan- dard error with *n* = 3). (b) West- ern blot analysis of GDH protein content in fractions 14– 22 shows a single band.

whether the *gdh1* gene expression was subject to day–night
fluctuations, total RNA were extracted from leaf disc sam-
ples collected during a 48 h period from both young/sink
(*n* = 30–32) and old/source (*n* = 13–15) leaves. Northern
blot analysis was performed using the *EcoRI* fragment of
*gdh1* cDNA as a probe.

Because of the trauma caused to the leaves every 3 h due
to leaf disc sampling, control samples of unwounded leaves
were collected at 36 and at 57 h. Nitrate reductase gene
(nia) expression, which is known to be subjected to large
day-night fluctuations (Galangau *et al.* 1988; Scheible *et al.*
2000), was used as a positive control. Although different
results concerning the day-night fluctuations of plasticid
GS gene (gs2) expression have been reported in the litera-
ture (Becker *et al.* 1992; Matt *et al.* 2001), gs2 mRNA con-
tent was used as a negative control in the Northern blot
experiments (Fig. 4). A 18S rRNA probe was used as a
loading control. A diurnal rhythm for nia mRNA was
detected whether sink or source leaves were examined
(Fig. 4). Although the steady state level of nia mRNA was
lower in old/source leaves (Masclaux *et al.* 2000) it reached
a maximum of expression during the night, increasing
between 1800 and 0600 h, and a minimum during the day,
decreasing from 0700 to 1500–1800 h (Fig. 4).

In sink leaves, no *gdh1* transcripts were detected what-
ever the time of extraction (data not shown). In old/source
leaves, *gdh1* mRNA levels exhibited diurnal fluctuations,
rising between 0700 and 1500–1800 h and then decreasing
dramatically from 1800 h to the end of the dark period until
0600 h (Fig. 4). This result shows that, in source leaf meso-
phyll cells, a shift of 12 h occurs between the day–night

rhythm of *gdh1* and *nia* RNA levels. In contrast, we did not
detect any diurnal oscillations in either the aminating or
deaminating activities of the GDH enzyme (data not
shown).

**Metabolite diurnal fluctuations**

Levels of carbohydrates and nitrogen metabolites were
measured in both young/sink and old/source leaves of
tobacco. Regardless of the age of the leaf, characteristic
diurnal oscillations were detected for starch and sucrose
(Matt *et al.* 1998; Fig. 5A–D. No oscillations were detected
for nitrate (Fig. 5E & F). Although the sink leaf free ammo-
nia content tended to accumulate during the day, probably
as a result of photorespiratory ammonia accumulation,
these minor fluctuations were not observed in source leaves
(Fig. 5G & H). No fluctuations were observed in the amino
acid content of sink leaves, whereas the total amino acids
accumulated in source leaves during the first part of the
light period (Fig. 5I & J).

A day–night fluctuation was observed in α-ketoglutarate
levels that were similar to those described by Scheible *et al.*
(2000) (Fig. 5K & L). It is interesting to note that, in sink
leaves, α-ketoglutarate accumulated when ammonia
reached its lowest concentration during the day–night
cycles.

In both sink and source leaves, diurnal rhythms were
detected for glutamine (GLN), glycine (GLY), aspartate
(ASP) and asparagine (ASN) (Fig. 6). ASP and ASN accu-
mulated during the first part of the night (Fig. 6E–H)
whereas GLN and GLY accumulated during the light
<figure><img src="image_5.png" /><figcaption>**Figure 4.** Diurnal changes in *gdh1* and *nia* mRNA accumulation during 48 h of a 18 h light : 6 h dark photoperiod. The dark and light periods are indicated by closed and open bars, respectively. Old/source and young/sink leaf sampling began at 1200 h and samples were numbered from 12 to 57, corresponding to the time in hours after the beginning of the experiment. Total RNA was extracted from each sample and *gdh1*, *nia* and *gs2* mRNA contents were analysed by Northern blotting and specific ³²P-labelled probe hybridization. A 18S ribosomal probe was used as a loading control. (A) The result of the Northern blot analysis. (B) Quantification of the accumulation of the different transcripts by the values of the densitometric scanning of the bands corresponding to each transcript. Each value was corrected according to the one found for 18S control.</figcaption></figure>

period (Fig. 6A–D). Amounts of GLY were higher in source leaves in comparison with sink leaves suggesting that glycine decarboxylase could be the main limiting factor in the photorespiratory pathway in ageing leaves.

Serine (SER), glutamate (GLU) and γ-aminobutyric acid (GABA) also showed a day-night fluctuation but the rhythm patterns were different between sink and source leaves (Fig. 6I–N). In sink leaves, GLU and GABA accumulation was observed at the beginning of the night and in the middle of the day, respectively (Fig. 6K & M). In source leaves, the accumulation of both of these amino acids was shifted by about 6 h in comparison with sink leaves (see Fig. 6L & N and 6K & M, for comparison). Moreover, in both types of leaf, a 5-6 h delay was observed between the accumulation of GABA preceding that of GLU (see Fig. 6K & L and 6M & N, for comparison).

Accumulation of proline (PRO) occurred only in sink leaves at the end of the light period–beginning of the night period (Fig. 6O & P) just as GLN. Thus, GLN and PRO might be the two main nitrogen storage compounds at the end of the light period in sink leaves. PRO accumulation was attenuated after a 24 h day-night; this could be as a

consequence of leaf disc sampling. Such a hypothesis was reinforced by measuring the PRO content of samples collected from unwounded controls after 36 h. The 36 h unwounded leaf tissues contained the same amount of PRO (814 nmol (mg Pheo)⁻¹) as the early-collected leaf discs (see Fig. 6O).

## Influence of sucrose and light on *gdh1*, *nia* and *gs2* expressions

To better understand the putative regulatory control mechanisms controlling the day-night fluctuation of *gdh1* expression in tobacco source leaves, we have investigated both the effect of a dark treatment and sucrose feeding on *gdh1* transcript accumulation using leaf discs (Ferrario-Mery *et al.* 2001). The expression of both *nia* and *gs2* mRNA was monitored in parallel and used as controls.

In leaf discs isolated from source leaves and placed under light, *gdh1* transcripts were barely detectable (Fig. 7, lane 2). Sucrose feeding resulted in a decrease of *gdh1* transcript content (Fig. 7, lane 1). In contrast, when leaf discs were
incubated in the dark, the *gdh1* mRNA content was strongly enhanced (Fig. 7, lane 4). The dark-induced accumulation of *gdh1* transcripts was inhibited when sucrose was added to the incubation medium (Fig. 7, lane 3).

On the other hand, in dark-treated leaf discs, *nia* gene expression was strongly induced by sucrose (Fig. 7 lane 3) in comparison with samples placed in the light with or without sucrose (Fig. 7, lanes 1 and 2). In light-treated leaf discs, *nia* transcript accumulation under sucrose feeding (Fig. 7, lane 1) was lower than in dark-treated discs (Fig. 7, lane 3), suggesting that dark-treated tissues were sugar starved and then more sensitive to sucrose feeding.

<figure><img src="image_6.png" /></figure>

**Figure 5.** Diurnal changes of starch (A; B); sucrose (C; D); nitrate (E; F); ammonium (G; H), amino acids (I; J) and α-ketoglutarate (K; L) contents in both young/sink (A; C; E; G; I; K) and old/source (B; D; F; H; J; L) tobacco leaf mesophyll tissues. The shaded blocks represent the dark period. The numbers show the time in hours after the beginning of the experiment. Phaeophytin (Pheo) or chlorophyll (Chl) contents in young and old leaves were, respectively, 2·2 (±0·2) and 0·9 (±0·1) µg (mg FW)⁻¹. Quantities of starch, sucrose and amino acids were expressed in µmol (mg Chl)⁻¹. Quantities of ammonium and α-ketoglutarate were expressed in µmol (mg Pheo)⁻¹. Quantities of nitrate were expressed in mmol (mg Chl)⁻¹. The results are the mean ± Sn (Sn = standard error with n = 3).

<figure><img src="image_7.png" /></figure>

**Figure 6.** Diurnal changes of glutamine (A; B); glycine (C; D); aspartate (E; F); asparagine (G; H); serine (I; J); glutamate (K; L); GABA (M; N) and proline (O; P) contents in both sink (A; C; E; G; I; K; M; O) and source (B; D; F; H; J; L; N; P) tobacco leaf mesophyll tissues. The shaded lanes represent the dark period. The numbers show the time in hours after the beginning of the experiment. Standard errors were not shown because of sampling (see Materials and methods). Note that scales are different for GLY amount in source and sink leaves.

In light-treated leaf discs, the accumulation of gs2 mRNA was higher than in dark-treated leaf discs (Fig. 7, lanes 1 and 2 compared with lanes 3 and 4). The addition of sucrose to leaf discs incubated either in dark or light condition, led to a decrease in the amount of gs2 transcript. Sucrose did not then reverse the negative effect of darkness (Fig. 7, lanes 1 and 3 compared with lanes 2 and 4), suggesting that gs2 behaves like other genes encoding enzymes involved in photosynthesis (Krapp, Quick & Stitt 1991; Wingler *et al.* 1998).

We did not observe any modifications in either GDH protein content or GDH activity using the same leaf disc samples incubated either in the light or in the dark, treated or not with sucrose (data not shown).
<figure><img src="image_8.png" /></figure>

**Figure 7.** Effect of sucrose feeding and dark treatment on mRNA accumulation. Northern blot experiments, using *gdh1*, *nia* or *gs2* ³²P-labelled probes and 10 µg of total RNA extracted from leaf discs, incubated in the presence of light with sucrose (lane 1, + S) or without sucrose (lane 2, -S), in the presence of dark with sucrose (lane 3, + S) or without sucrose (lane 4, -S). A 18S ribosomal probe was used as a loading control.

DISCUSSION

In a previous work, we have shown that ageing induced
gradual changes in metabolite and enzymatic contents in
leaves (Masclaux *et al.* 2000). Two main sections along the
tobacco main axis could be roughly defined and character-
ized in terms of importance of the occurring metabolic
pathways (Masclaux *et al.* 2000). Upper leaves are defined
as a sink section mainly involved in primary carbon and
nitrogen assimilation. The lower leaves were part of a
source section involved in nitrogen remobilization and
characterized by the induction of GDH and cytosolic
glutamine synthetase (GS1). However, the comparison of
sink and source leaves metabolism was based on data from
leaf samples taken at 1100 h. Since numerous enzymes
involved in C and N metabolism and either their substrate
or products are known to exhibit day–night fluctuations
(Scheible *et al.* 2000), *gdh1* gene expression was monitored
in both source and sink leaves along a 48 h day–night
period.

Northern blots revealed a rhythm for *gdh1* mRNA accu-
mulation in source leaves whereas no *gdh1* transcripts
could be detected in young leaves. The *gdh1* mRNA oscil-
lations showed a minimum at the beginning of the light
period (0600 h) and a peak in the afternoon (1800 h). The
*gdh1* mRNA level oscillated in an opposite way compared
with that of *nia* transcripts along the day–night period. This

could reflect the fact that the expression of the gene encod-
ing NR is turned on when that of the gene encoding GDH
is turn off and reciprocally.

The NR oscillating system is well documented. The diurnal rhythm of NR is not likely to be governed by a clock receiving input through phytochrome receptors but rather dependent on photosynthetically active light absorbed by chlorophylls (Becker *et al.* 1992; Lillo 1994). It is based on a transcription–translation feedback loop in which the positive component that increases *nia* gene transcription is related to light and the component exerting a negative effect is a product of nitrogen assimilation. GLN was suggested to be a good candidate to exert the negative feedback on *nia* day–night fluctuation (Klein *et al.* 2000; Lillo *et al.* 2001 for review), even though some reports have shown that *nia* expression and glutamine accumulation are not so clearly related (Ferrario-Mery *et al.* 2001). Contrary to *nia* regulation, both ammonia and amino acids (GLN for example) are able to induce *gdh* gene expression (Melolivera *et al.* 1996; Turano *et al.* 1997; Masclaux, unpublished; Fig. 8). Moreover, in the present work, we show that glutamine oscillations in source leaves are in phase with *gdh1* mRNA fluctuations (Figs 4, 6E & F). Therefore, it is attractive to suggest that, as for *nia* gene regulation, GLN is a possible effector for the diurnal modulation of *gdh1* (Fig. 8). However, if the biological clock oscillator models are true (Lillo *et al.* 2001), a negative regulation should also be linked to a feedback from a product supplied from the *in vivo* GDH activity (Fig. 8). In the present study, we show that *gdh1* expression is negatively regulated by sucrose, a C metabolite. However a role for sugar as an effector in the feedback regulation of *gdh1* is not obvious, since there is no evidence that GDH activity can modulate sugar content. Another possibility is that the sugar-signalling pathway controlling *gdh1* expression is mediated *via α*-KG replen-

<figure><img src="image_9.png" /></figure>

**Figure 8.** Schematic representation of the antiparallel regulation of the *gdh* and *nia* genes. Arrows indicate positive effects and bars negative effects on gene regulation. *nia*: nitrate reductase gene; *gdh* glutamate dehydrogenase gene, GLN: glutamine.
<figure><img src="image_10.png" /><figcaption>**Figure 9.** Schematic representation of the hypothetical evolution of the role of GDH in the C/N balance during source leaf ageing. PK: pyruvate kinase (Scheible et al. 2000); CS: citrate synthase (Scheible et al. 2000); NADP-ICDH: NADP-isocitrate dehydrogenase (Scheible et al. 2000); NR: nitrate reductase; GS1: cytosolic glutamine synthetase; AMT1;1: ammonium transporter (Gazzarrini et al. 1999); NTCP-23: cysteine protease (Ueda et al. 2000); AA: amino acids; α-KG: α-ketoglutarate.</figcaption></figure>

ishment, i.e. via the product of the GDH deaminating activity (Morcuende *et al.* 1998; Koch *et al.* 2000). Interestingly, Ferrario-Mery *et al.* (2001) have shown recently that α-KG feeding can promote the accumulation of *nia* transcripts in tobacco leaves. From this, it will be useful to test whether α-KG can induce *gdh1*.

Opposite changes in *gdh1* and *nia* expression observed through reversed day–night oscillations are also illustrated by sucrose feeding and dark-induction experiments. The transcript level of *gdh1* was increased by a dark treatment and decreased by the addition of sucrose. Sucrose feeding was able to abolish the dark induction of *gdh1* as well as the dark inhibition of *nia*. Moreover, leaf developmental changes such as ageing emphasized the opposition between *nia* and *gdh1* regulation. Since sugars produced via photosynthesis became limiting in old/source leaves (Masclaux *et al.* 2000), sugar availability could once more be a potential signal in age-related *gdh1* and *nia* expression.

Nevertheless, the role of diurnal changes in *gdh1* transcript levels remains to be addressed, since we did not detect any day-night fluctuations in the *in vitro* aminating and deaminating activities of the enzyme. However, it has been shown in a number of studies that it is difficult to measure a reliable enzyme activity *in vitro* that reflects the

actual *in vivo* enzyme activity. Indeed, GDH aminating activity measured *in vitro* is always higher in comparison with its deaminating activity. Moreover, *in vivo*, deaminating activity can be revealed accurately only using ¹⁵N labeling techniques as described previously (Berger *et al.* 1985; Robinson *et al.* 1991; Stewart *et al.* 1995). Therefore, we cannot exclude that there is a day-night fluctuation for GDH activity *in vivo*.

Interestingly, we observed that following the induction of *gdh1* mRNA, a rhythm for GLU, different to that of sink leaves, appeared in source leaves. Indeed, in sink leaves, GLU accumulated at the end of the day as a result of GOGAT activity and was used during the night. In source leaves, GLU accumulated in the first half of the night and was probably used in the second half of the night and in the morning. The behaviour of GLU in source leaves can then be questioned. As no change in GLN day-night oscillation was observed between sink and source leaves and since GABA oscillations were shifted by 6 h just like GLU, we hypothesize that GABA is buffering GLU production (Satya Narayan & Nair 1990; Shelp *et al.* 1995). Moreover, we observe here that the expression of *gdh1* in source leaves oscillated in an opposite way compared with glutamate, while α-KG rhythm remained unchanged
between source and sink leaves. A role for *gdhl* in GLU oscillation shift can therefore be considered.

Interestingly, it has been shown recently that, in tobacco source leaves, the levels of the transcripts and activities of organic acid-producing enzymes (pyruvate kinase (PK), citrate synthase (CS), NADP-isocitrate dehydrogenase (NADP-ICDH)) also show diurnal changes (Scheible *et al.* 2000). As for *gdhl* mRNA, the transcript oscillations of these genes, were out of phase compared with that of *nia* mRNA. We observed in source leaves the same fluctuation patterns as the one described by Scheible *et al.* (2000) for individual amino acids, sugars and *α*-KG. Since GDH activity can work in both the aminating and deaminating directions (as shown only *in vitro*), two hypotheses can be considered. GDH deaminating activity could be used to produce *α*-KG just as PK, CS and NADP-ICDH activities (Scheible *et al.* 2000) in order to supply carbon skeletons usable as an acceptor for the reduced nitrogen products of NR activity. However, because deaminating GDH activity would also release mineral nitrogen, such a hypothesis lacks coherence. In such a context, an aminating pathway that is able to compensate the age-related decrease in Fd-GOGAT activity for GLU synthesis appeared to be more logical as long as *α*-KG is available and NR active (Fig. 9). Nevertheless, this hypothesis is based on *gdhl* mRNA accumulation and not on GDH activity.

In any case, we should bear in mind that *gdhl* and *nia* expressions behave in opposition with regard to senescence. Moreover, we have shown previously (Masclaux *et al.* 2000) that *gs1* transcripts accumulated with ageing before those of *gdh* (Fig. 9). It is assumed that GS1 participates in nitrogen mobilization and compensates for a deficit in the GS2 isoform due to age-related chloroplast collapse. The delay between *gdh* and *gs1* age-related accumulation suggests that the roles of GDH and GS1 are not co-operative. Interestingly, the senescence-induced tobacco cysteine protease gene (NTCP-23) presents a diurnally modulated expression that is very similar to that observed for *gdhl* (Ueda *et al.* 2000). Moreover, the ammonium transporter (AtAMT1; 1) which is induced in source compared to sink leaves (Gazzarrini *et al.* 1999), exhibits in roots an almost identical diurnal rhythm to that of *gdhl* and NTCP-23 in leaves. Rhythmic changes in AtAMT1;1 expression was unfortunately not investigated in leaves, but its homologous LeAMT1;1 was seen to be induced in tomato leaves at the end of the day and during the dark period (von Wiren *et al.* 2000).

We have shown previously that the process of leaf senescence is accompanied by an increase in ammonium content, proteolysis and *gdhl* expression and a decrease in sugar contents and NR activity (Masclaux *et al.* 2000). These observations indicate that proteins and amino acids were certainly catabolized to provide ammonia (Fujiki *et al.* 2000b). As some ammonium transporters are senescence-induced in source leaves (von Wiren *et al.* 2000), we can suppose that a part of the ammonium is mobilized from old senescing mesophyll cells to vascular tissues and further to sink tissues (Fig. 9). In such a context, GDH appeared a

good candidate for glutamate catabolism. GDH could then participate in protein burning, supplying *α*-KG in order to sustain respiration and cell viability for nitrogen mobilization efficiency (Fig. 9; Laurière 1983; Srivastava & Singh 1987).

## ACKNOWLEDGMENTS

The authors thank Dr K. A. Roubelakis-Angelakis (University of Crete, Heraklion, Greece) for providing antibodies raised against the grape GDH protein. We are also grateful to Dr J. P. Boutin and K. Pageau for their help in the amino acid analysis, to Dr J. F. Morot-Gaudry for helpful discussions and to Dr M. Hodges and Dr H. Vanacker for critically reading the manuscript.

## REFERENCES

* Arnon D.I. (1949) Copper enzymes in isolated chloroplasts. Polyphenol oxidase in *Beta vulgaris* L. *Plant Physiology* **24**, 1-15.

* Athwal G.S., Pearson J. & Laurie S. (1997) Regulation of glutamate dehydrogenase activity by manipulation of nucleotide supply in *Daucus carota* suspension cultures. *Physiologia Plantarum* **101**, 503-509.

* Becker T.W., Caboche M., Carrayol E. & Hirel B. (1992) Nucleotide sequence of a tobacco cDNA encoding plasticid glutamine synthetase and light inducibility, organ specificity and diurnal rhythmicity in the expression of the corresponding genes of tobacco and tomato. *Plant Molecular Biology* **19**, 367-379.

* Becker T.W., Carrayol E. & Hirel B. (2000) Glutamine synthetase and glutamate dehydrogenase isoforms in maize leaves: localization, relative proportion and their role in ammonium assimilation or nitrogen transport. *Planta* **211**, 800-806.

* Berger M.G., Woo K.C., Wong S.-C. & Fock H.P. (1985) Nitrogen metabolism in senescent flag leaves of wheat (*Triticum aestivum* L.) in the light. *Plant Physiology* **78**, 779-783.

* Bergmeyer H.U. (1965) Citrate, malate, *α*-ketoglutarate. In: *Methods of Enzymatic Analysis* (ed. H.U. Bergmeyer), pp. 318-334. Academic Press, New York and London; Verlag Chemie, Weinheim, Germany.

* Brugière N., Dubois F., Limami A., Lelandais M., Roux Y., Sangwan R.S. & Hirel B. (1999) Glutamine synthetase in the phloem plays a major role in controlling proline production. *Plant Cell* **11**, 1995-2011.

* Cammaerts D. & Jacobs M. (1985) A study of the role of glutamate dehydrogenase in the nitrogen metabolism of *Arabidopsis thaliana*. *Planta* **163**, 517-526.

* Cataldo D.A., Haroon M., Schrader T.E. & Youngs V.L. (1975) Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid. *Communications in Soil Science and Plant Analysis* **6**, 71-80.

* Cheng C.-L., Acedo N.G., Cristinsin M. & Conkling M.A. (1992) Sucrose mimics the light induction of *Arabidopsis* nitrate reductase gene transcription. *Proceedings of the National Academy of Sciences USA* **89**, 1861-1864.

* Church G.M. & Gilbert W. (1984) Genomic sequencing. *Proceedings of the National Academy of Sciences USA* **81**, 1991-1995.

* Coïc Y. & Lesaint C. (1971) Comment assurer une bonne nutrition en eau et en ions minéraux en horticulture. *Horticulture Française* **8**, 11-14.

* Dubois F., Brugière N., Sangwan R.S. & Hirel B. (1996) Localization of tobacco cytosolic glutamine synthetase enzymes and the corresponding transcripts shows organ- and cell-specific patterns
of protein synthesis and gene expression. *Plant Molecular Biology* **31**, 803–817.

Ferrario-Mery S., Masclaux C., Suzuki A., Valadier M.H., Hirel B. & Foyer C.H. (2001) Glutamine and α-ketoglutarate are metabolite signals involved in nitrate reductase gene transcription in untransformed and transformed tobacco plants deficient in ferredoxin-glutamine-α-ketoglutarate aminotransferase. *Planta* **213**, 265–271.

Ficarelli A., Tassi F. & Restivo F.M. (1999) Isolation and characterization of two cDNA clones encoding for glutamate dehydrogenase in *Nicotiana plumbaginifolia*. *Plant, Cell and Environment* **40**, 339–342.

Fujiki Y., Ito M., Nishida I. & Watanabe A. (2000a) Multiple signaling pathways in gene expression during sugar starvation. Pharmacological analysis of *din* gene expression in suspension-cultured cells of *Arabidopsis*. *Plant Physiology* **124**, 1139–1147.

Fujiki Y., Sato T., Ito M. & Watanabe A. (2000b) Isolation and characterisation of cDNA clones for the E1β and E2 of branched-chain α-ketoacid dehydrogenase complex in *Arabidopsis*. *Journal of Biological Chemistry* **275**, 6007–6013.

Galangau F., Daniel-Vedele F., Moureaux T., Dorbe M.-F., Leydecker M.-T. & Caboche M. (1988) Expression of leaf nitrate reductase genes from tomato and tobacco in relation to light-dark regimes and nitrate supply. *Plant Physiology* **88**, 0383–0388.

Gazzarrini S., Lejay L., Gojon A., Ninnemann O., Frommer W.B. & von Wiren N. (1999) Three functional transporters for constitutive, diurnally regulated, and starvation induced uptake of ammonium into *Arabidopsis* roots. *Plant Cell* **11**, 937–947.

Givan C.V. (1979) Metabolic detoxification of ammonia in tissues of higher plants. *Phytochemistry* **18**, 375–382.

Ireland R.J. & Lea P.J. (1999) The enzymes of glutamine, glutamate, asparagine and aspartate metabolism. In *Plant Amino Acids, Biochemistry and Biotechnology* (ed. B.K. Singh), pp. 49–109. Marcel Dekker Inc., New York, Basel, Hong Kong.

Kar M. & Feierabend J. (1984) Changes in the activities of enzymes involved in amino acid metabolism during the senescence of detached wheat leaves. *Physiologia Plantarum* **62**, 39–44.

Klein D., Morcuende R., Stitt M. & Krapp A. (2000) Regulation of nitrate reductase expression in leaves by nitrate and nitrogen metabolism is completely overridden when sugars fall below a critical level. *Plant, Cell and Environment* **2000**, 863–871.

Koch K.E., Ying Z., Wu Y. & Avigne W.T. (2000) Multiple paths of sugar-sensing and sugar/oxygen overlap for genes of sucrose and ethanol metabolism. *Journal of Experimental Botany* **51**, 417–427.

Krapp A., Quick W.P. & Stitt M. (1991) Ribulose-1,5-biphosphate carboxylase-oxygenase, other Calvin-cycle enzymes, and chlorophyll decrease when glucose is supplied to mature spinach leaves via transpiration stream. *Planta* **186**, 58–69.

Laemmli U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. *Nature* **227**, 680–685.

Laurière C. (1983) Enzymes and leaf senescence. *Physiologie Végétale* **21**, 1159–1177.

Laurière C. & Daussant J. (1983) Identification of the ammonium-dependent isoenzyme of glutamate dehydrogenase as the form induced by senescence or darkness stress in the first leaf of wheat. *Physiologia Plantarum* **58**, 89–92.

Lillo C. (1994) Light regulation of nitrate reductase in green leaves of higher plants. *Physiologia Plantarum* **90**, 616–620.

Lillo C., Meyer C. & Ruoff P. (2001) The nitrate reductase circadian system. The central clock dogma contra multiple oscillatory feedback loops. *Plant Physiology* **125**, 1554–1557.

Loulakakis K.A. & Roubelakis-Angelakis K.A. (1990) Immuno-

characterization of NADH-glutamate dehydrogenase from *Vitis vinifera* L. *Plant Physiology* **94**, 109–113.

Loulakakis K.A. & Roubelakis-Angelakis K.A. (1996) The seven NAD (H) -glutamate dehydrogenase isoenzymes exhibit similar anabolic and catabolic activities. *Physiologia Plantarum* **96**, 29–35.

Maestri E., Restivo F.M., Gulli M. & Tassi F. (1991) Glutamate dehydrogenase regulation in callus cultures of *Nicotiana plumbaginifolia*: effect of glucose feeding and carbon source starvation on isoenzymatic pattern. *Plant, Cell and Environment* **14**, 613–618.

Masclaux C., Valadier M., 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.

Matt P., Geiger M., Walch-Liu P., Engels C., Krapp A. & Stitt M. (2001) The immediate cause of the diurnal changes of nitrogen metabolism in leaves of nitrate-replete tobacco: a major imbalance between the rate of nitrate reduction and the rates of nitrate uptake and ammonium metabolism during the first part of the light period. *Plant, Cell and Environment* **24**, 177–190.

Matt P., Schurr U., Klein D., Krapp A. & Stitt M. (1998) Growth of tobacco in short-day conditions leads to high starch, low sugars, altered diurnal changes in the Nia transcript and low nitrate reductase activity, and inhibition of amino acid synthesis. *Planta* **207**, 27–41.

Melo-Olivera R., Olivera I.C. & Coruzzi G.M. (1996) *Arabidopsis* mutant analysis and gene regulation define a non redundant role for glutamate dehydrogenase in nitrogen assimilation. *Proceedings of the National Academy of Sciences USA* **93**, 4718–4723.

Miflin B.J. & Lea P.J. (1980) Ammonium assimilation. In *The Biochemistry of Plants Amino Acids and Derivatives* (ed. B.J. Miflin), pp. 169–202. Academic Press, New York, London, Toronto, Sydney, San Francisco.

Morcuende R., Krapp A., Hurry V. & Stitt M. (1998) Sucrose-feeding leads to increased rates of nitrate assimilation, increased rates of α-oxoglutarate synthesis, and increased synthesis of a wide spectrum of amino acids in tobacco leaves. *Planta* **206**, 394–409.

Morkunas I., Lehmann T., Ratajczak W., Ratajczak L. & Tomaszewska B. (2000) The involvement of glutamate dehydrogenase in adaptation of mitochondria to oxidize glutamate in sucrose starved pea embryos. *Acta Physiologia Plantarum* **22**, 389–394.

Pahlich E. (1996) Remarks concerning the dispute related to the function of plant glutamate dehydrogenase: commentary. *Canadian Journal of Botany* **74**, 512–515.

Paszkowski J., Shillito R.D., Saul M., Nandak V., Hohn T., Hohn B. & Potrykus I. (1984) Direct gene transfer to plants. *EMBO Journal* **3**, 2717–2722.

Peeters K.M.U. & Van Laere A.J. (1992) Ammonium and amino acid metabolism in excised leaves of wheat (*Triticum aestivum*) senescing in the dark. *Physiologia Plantarum* **84**, 243–249.

Purnell M.P., Stewart G.R. & Botella J.R. (1997) Cloning and characterisation of a glutamate dehydrogenase cDNA from tomato (*Lycopersicon esculentum* L.). *Gene* **186**, 249–254.

Robinson S.A., Slade A.P., Fox G.G., Phillips R., Ratcliffe R.G. & Stewart G.R. (1991) The role of glutamate dehydrogenase in plant nitrogen metabolism. *Plant Physiology* **95**, 509–516.

Rochat C. & Boutin J.-P. (1989) Carbohydrates and nitrogenous compounds changes in the hull and in the seed during the pod development of pea. *Plant Physiology and Biochemistry* **27**, 881–887.

Rosen H. (1957) A modified ninhydrin colorimetric analysis for amino acids. *Archive of Biochemistry and Biophysics* **67**, 10–15.
Satya Narayan V.S. & Nair P.M. (1990) Metabolism, enzymology and possible roles of 4-aminobutyrate in higher plants. *Phytochemistry* **29**, 367–375.

Scheible W.-R., Gonzalez-Fontes A., Lauerer M., Müller-Röber B., Caboche M. & Stitt M. (1997) Nitrate acts as a signal to induce organic acid metabolism and repress starch metabolism in tobacco. *Plant Cell* **9**, 783–798.

Scheible W.-R., Krapp A. & Stitt M. (2000) Reciprocal diurnal changes of phosphoenolpyruvate carboxydase expression and cytosolic pyruvate kinase, citrate synthase and NADP-isocitrate dehydrogenase expression regulate organic acid metabolism during nitrate assimilation in tobacco leaves. *Plant, Cell and Environment* **23**, 1155–1167.

Shelp B.J., Walton C.S., Snedden W.A., Tuin L.G., Oresnik I.J. & Layzell D.B. (1995) Gaba shunt in developing soybean seeds is associated with hypoxia. *Physiologia Plantarum* **94**, 219–228.

Sims A.P. & Folkes B.F. (1964) A kinetic study of the assimilation of ¹⁵N-ammonia and the synthesis of amino acids in an exponentially growing culture of *Candida utilis*. *Proceedings of the Royal Society of London B Biological Science* **159**, 479–502.

Srivastava H.S. & Singh R.P. (1987) Role and regulation of 1-glutamate dehydrogenase activity in higher plants. *Phytochemistry* **26**, 597–610.

Stewart G.R., Shatilov V.R., Turnbull M.H., Robinson S.A. & Goodall R. (1995) Evidence that glutamate dehydrogenase plays a role in the oxidative deamination of glutamate in seedlings of Zea mays. *Australian Journal of Plant Physiology* **22**, 805–809.

Stitt M., Müller C., Matt P., Gibon Y., Carillo P., Morcuende R., Scheible W.-R. & Krapp A. (2002) Steps towards an integrative view of nitrogen metabolism. *Journal of Experimental Botany* **53**, 959–970.

Tassi F., Restivo F.M., Puglisi P.P. & Cacco G. (1984) Effect of glucose on glutamate dehydrogenase and acid phosphatase and its reversal by cyclic adenosine 3': 5'-monophosphate in single cell cultures of *Asparagus officinalis*. *Physiologia Plantarum* **60**, 61–64.

Thomas H. (1978) Enzymes of nitrogen mobilization in detached leaves of *Lolium temulentum* during senescence. *Planta* **142**, 161–169.

Turano F.J., Dashner R., Upadhayaya A. & Caldwell C.R. (1996) Purification of mitochondrial glutamate dehydrogenase from dark-grown soybean seedlings. *Plant Physiology* **112**, 1357–1364.

Turano F.J., Thakkar S.S., Fang T. & Weisemann J.M. (1997) Characterization and expression of NAD (H)-dependent glutamate dehydrogenase genes in *Arabidopsis*. *Plant Physiology* **113**, 1329–1341.

Ueda T., Seo S., Ohashi Y. & Hashimoto J. (2000) Circadian and senescence-enhanced expression of a tobacco cysteine protease gene. *Plant Molecular Biology* **44**, 649–657.

Vaucheret H., Vincentz M., Kronenberger J., Caboche M. & Rouze P. (1989) Molecular cloning and characterization of the two homologous genes encoding for nitrate reductase in tobacco. *Molecular and General Genetics* **216**, 10–15.

Vernon O.P. (1960) Spectrometric determination of chlorophylls and phaeophytins in plant extracts. *Analytical Chemistry* **32**, 1144–1150.

Verwoerd T.C., Dekker B.M.M. & Hoekema A. (1989) A small-scale procedure for the rapid isolation of plant RNAs. *Nucleic Acids Research* **17**, 2362.

Wallsgrove R.M., Keys A.J., Lea P.J. & Miflin B.J. (1983) Photosynthesis, photorespiration and nitrogen metabolism. *Plant, Cell and Environment* **6**, 301–309.

Wingler A., von Schaewen A., Leegood R.C., Lea P.J. & Quick W.P. (1998) Regulation of leaf senescence by cytokinin, sugars and light. *Plant Physiology* **116**, 329–335.

von Wiren N., Lauter F.R., Ninnemann O., Gillissen B., Walch-Liu P., Engels C., Jost W. & Frommer W.B. (2000) Differential regulation of three functional ammonium transporter genes by nitrogen in root hairs and by light in leaves of tomato. *Plant Journal* **21**, 167–175.

*Received 13 March 2002; received in revised form 22 May 2002; accepted for publication 23 May 2002*