Role of glutamate dehydrogenase and phosphoenolpyruvate carboxylase activity in ammonium nutrition tolerance in roots

Plant Physiol. Biochem. 40 (2002) 969–976 www.elsevier.com/locate/plaphy

Original article

Role of glutamate dehydrogenase and phosphoenolpyruvate carboxylase activity in ammonium nutrition tolerance in roots Berta Lasa *, Silvia Frechilla, Pedro M. Aparicio-Tejo, Carmen Lamsfus Department Ciencias del Medio Natural, Universidad Pública de Navarra, 31006 Pamplona, Spain Received 29 March 2002; accepted 17 June 2002

Abstract Spinach (Spinacea oleracea L. “Correnta F1”) and pea (Pisum sativum L. “Macrocarpon”) plants were grown in a hydroponic culture with nitrate (5 mM), or ammonium (5 mM) as the nitrogen source. Dry matter accumulation declined dramatically in spinach plants fed with ammonium, whereas there was no change in pea plants when compared with nitrate-fed plants. Data obtained from δ15N, the organic nitrogen content, N-assimilation enzyme activity, glutamine synthetase (L-glutamate:ammonia-ligase; EC 6.3.1.2), glutamate dehydrogenase (L-glutamate:NAD+-oxidoreductase; EC 1.4.1.2) and enzymes from the tricarboxylic acid cycle suggest that ammonium incorporation into organic nitrogen is localized in the roots in pea plants and in the shoots in spinach plants. Distribution of incorporated ammonium (in shoots and roots) may determine ammonium tolerance. Our results show that unlike in spinach plants, in pea plants, an ammonium-tolerant species, GDH enzyme plays an important role in ammonium detoxification by its incorporation into amino acids. Furthermore, phosphoenolpyruvate carboxylase (phosphate:oxaloacetate-carboxy-lyase; EC 4.1.1.31) and pyruvate kinase (ATP:pyruvate-2-Ophosphotransferase; EC 2.7.1.40) activities reflect a major flow of carbon for ammonium assimilation through oxalacetate in pea plants and through pyruvate in spinach plants. The differences in the sensitivity to ammonium between the species are discussed in terms of differences in the site of ammonium assimilation as well as in the nitrogen assimilation ways. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Ammonium; Glutamate dehydrogenase; Phosphoenolpyruvate carboxylase; Pisum sativum; Spinacea oleracea

1. Introduction Most plant species can utilize as nitrogen sources both + − NH4 and NO3 . Although inorganic nitrogen is predominantly available to plants as nitrate in most soils, in certain soil and in hydroponic cultures, ammonium can be the

Abbreviations: Arg, arginine; Asn, asparagine; Asp, aspartic acid; δ13C, carbon isotope abundance; δ15N, nitrogen isotope abundance; Fd, ferredoxin; GDH, glutamate dehydrogenase; Gln, glutamine; Glu, glutamic acid; Gly, glycine; GS, glutamine synthetase; GOGAT, glutamate synthase; HPLC, high performance liquid chromatography; ISDH, isocitrate dehydrogenase; MDH, malate dehydrogenase; ME, malic enzyme; PEPC, phosphoenolpyruvate carboxylase; PDC, pyruvate dehydrogenase complex; PK, pyruvate kinase; RuBP, (ribulose-bisphosphate); TCA cycle, tricarboxylic acid cycle; Thr, threonine; Tyr, tyrosine * Corresponding author. E-mail address: [email protected] (B. Lasa). © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 9 8 1 - 9 4 2 8 ( 0 2 ) 0 1 4 5 1 - 1

majority nitrogen ion. High concentrations of ammonium nitrogen in the soil or in nutrient solution may lead to accumulation of toxic amounts of ammonium ions in the plants [15,22]. Depending on the nitrogen source, the response of plant species or cultivars varies widely. For example, plant growth can be unchanged, inhibited or stimulated when ammonium is added in nutrient solutions as a nitrogen source [23]. Nitrate ions can accumulate in the vacuoles; thus most plant species can tolerate high nitrate concentrations without any sign of toxicity. This has led several authors to postulate that nitrate may play an important role as an osmotic agent. Ammonium absorbed by the plant is rapidly metabolized into organic nitrogen compounds [8]. Free ammonium can be an uncoupler of photophosphorylation in vitro and could potentially cause a decrease in net photosynthesis and therefore in the growth of the plants [15], although direct evidence for this occurring in vivo is lacking [12].

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Ammonium originates in the plant from nitrate reduction, direct absorption, photorespiration, dinitrogen fixation or deamination of nitrogenous compounds such as asparagine; it is assimilated into organic molecules primarily by the combined catalytic action of two enzymes, glutamine synthetase (GS) (L-glutamate:ammonia-ligase; EC 6.3.1.2) and glutamate synthase (GOGAT) (L-glutamate:ferredoxinoxidoreductase; EC 1.4.7.1) [28]. Glutamate dehydrogenase (GDH) (L-glutamate:NAD+-oxidoreductase; EC 1.4.1.2) catalyzes the amination of 2-oxoglutarate and the deamination of glutamate; the direction of the activity depends on specific environmental conditions [25,33]. Increase of GDH activity in ammonium-fed plants under stress conditions has been observed implicating GDH to ammonium detoxification [21]. However, the major argument against GDH for + + the assimilation of NH4 is its high Km for the NH4 [39]. Moreover, physiological, molecular and genetic studies have shown that the enzymes GS and GOGAT function as the routes of ammonia assimilation in plants [30,38]. On the other hand, the function of GDH could be related to the replenishment of TCA cycle intermediates via its oxidation to 2-oxoglutarate. It should be noted that in vivo, aspartate aminotransferase and glutamate decarboxylase could also mediate the supply of carbon to the citric acid cycle. Therefore, controversy still exists on the role of GDH in nitrogen and carbon metabolism. Ammonium assimilation requires a substantial contribution of fixed C in both leaves and roots [1]. In leaves, triose-P enters the glycolytic pathway directly [24]. In roots, C is required directly as α-ketoglutarate (by metabolism of sucrose through glycolysis/Krebs) and indirectly by the generation of reducing power (reduced Fd) in the plastids. Phosphoenolpyruvate derived from glucose reacts with carbon dioxide from the atmosphere to yield oxalacetic acid throughphosphoenolpyruvatecarboxylase(phosphate:oxaloacetate-carboxy-lyase; EC 4.1.1.31). The carboxylation of phosphoenolpyruvate in roots is enhanced when ammonium rather than nitrate is the major source of nitrogen [3]. Therefore, PEPC may be an important interface between carbon and nitrogen metabolism.

In this paper, we study the response of two plant species, pea and spinach, to ammonium and nitrate nutrition. These species were chosen because they are of agronomic interest and differ in their nitrogen nutrition; pea is an N2 fixing species and spinach requires great amounts of nitrogen. Therefore, the aim of this paper is to analyze the differences of these species in nitrogen assimilation which could help to elucidate the mechanism of ammonium nutrition tolerance.

2. Results 2.1. Growth and N content The effect of the nitrogen source (nitrate or ammonium) on plant biomass was highly dependent on the plant species. In spinach plants, ammonium nutrition decreased the plant biomass by 83% compared to plants grown with nitrate, whereas it had no significant effect in pea plants. The effect of ammonium on the growth of spinach plants was mainly due to an inhibition of shoot dry matter production, leading to a decrease in the shoot/root ratio (Table 1). Depending on the nitrogen source and the species, there were differences in organic nitrogen content. Thus, in spinach plants, ammonium nutrition resulted in an increase in the organic nitrogen content in the shoots and a decrease in the roots. On the other hand, in pea plant shoots, organic nitrogen content was similar regardless of the nitrogen source, whereas it increased in the roots (Table 1). Soluble protein was affected by nitrogen nutrition in a different way depending on the plant species. Thus, in spinach plant, it decreased both in roots and shoots, whereas it increased in pea plants when ammonium was the nitrogen source. Regardless of the species, there was an accumulation of nitrate mainly in the roots. This accumulation was significantly higher in roots of pea plants than in spinach plants. Free ammonium content data show that this cation was accumulated mainly in roots of both pea and spinach plants fed with ammonium (Table 1).

Table 1 Effect of nitrogen nutrition nitrate (5 mM) and ammonium (5 mM) on plant growth parameters, organic nitrogen, soluble protein, nitrate and ammonium content in spinach and pea plants. nd, not detected. Values represent the mean of six samples. Analysis of variance results are represented as follows: S, species effect; N, nitrogen nutrition effect; I, species/nitrogen nutrition interaction effect on a single parameter. Asterisk, significant differences at 95%; ns, no significant differences; na, not applicable in a two-way (species and N-nutrition) analysis of variance Parameters

Growth plant (g DW) Shoot/root ratio Organic nitrogen (%) Soluble protein (mg g–1 DW) Nitrate (µmol g–1 DW) Ammonium (µmol g–1 DW)

Spinach plants

Shoot Root Shoot Root Shoot Root Shoot Root

Pea plants

ANOVA

Nitrate

Ammonium

Nitrate

Ammonium

S

N

I

0.95 4.92 5.6 4.3 164 48 287 687 6 6

0.16 2.02 9.0 2.7 147 17 nd nd 6 110

1.10 1.94 5.2 3.8 115 57 262 984 12 23

0.94 1.95 5.8 6.1 129 69 Nd Nd 14 302

* * * * * * ns * * *

* * * * ns * na na ns *

* * * * * * na na ns *

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Table 2 Effect of nitrogen nutrition nitrate (5 mM) and ammonium (5 mM) on the percentage distribution of amino acids in spinach and pea shoots. Values represent the mean of six samples and are given as percentage of total amino acids. Analysis of variance results are represented as in legend to Table 1 Amino acids

Alanine Arginine Asparagine Aspartic acid Glutamic acid Glutamine Glycine Isoleucine Leucine Lysine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine

Spinach plants

Pea plants

ANOVA

Nitrate

Ammonium

Nitrate

Ammonium

S

N

I

6.4 0.5 5.0 16.9 35.3 7.3 1.9 1.2 1.4 2.2 2.1 1.2 7.9 2.0 2.7 1.4 1.9

1.1 24.0 2.8 1.7 8.9 52.1 0.3 0.2 0.4 0.5 0.2 0.6 3.8 0.5 0.3 0.8 0.7

1.1 3.7 7.6 9.6 16.0 4.1 1.1 3.2 1.9 4.5 1.1 4.1 9.7 11.3 2.4 3.0 1.9

5.4 0.9 36.8 3.9 3.6 10.6 2.1 1.5 2.0 2.0 1.2 1.7 3.0 3.2 0.5 10.4 2.6

* * * ns * * ns * * * * * * * * * *

* * * * * * ns * * * * ns * * * * *

ns * * ns * * * ns ns * * ns ns ns * * ns

The distribution of amino acids in percentages of both the plant species is given in Table 2. In spinach plants, the main amino acids under nitrate nutrition were Glu and Asp, whereas in pea plants, Asp, Glu and Thr were the predominant amino acids. When ammonium was the nitrogen source, this trend changed, in spinach plants, the main amino acids were Gln (52%) and Arg (24%) and in pea plants Asn (37%), Gln (11%) and Tyr (10%). In Table 3, the effect of nitrogen nutrition on the distribution of the different amino acids in roots of both plant species is shown. In spinach plants fed with nitrate, the main amino acids were Glu (21%), Asp (12%) and Gln (12%), while when ammonium was the nitrogen source, the main amino acid was Gln (47%). In pea plants, the effect of nitrogen nutrition was different since nitrate-fed plants showed a relatively high content in Asn (39%) and Gly

(20%). A higher percentage of Asn (67%) and Gln (10%) was found in the presence of ammonium. 2.2. δ15N and δ13C The differences found in δ15N in root and shoot of pea and spinach plants grown with nitrate or ammonium were more significant than differences found in δ13C (Table 4). Shoot and root δ15N varied significantly with nitrogen source and species. The presence of ammonium nutrition caused shoot and root δ15N to become more negative than in nitrate-fed plants. There was a similar decrease of δ15N in the roots of both plant species (50%), however, the decrease of δ15N in the shoots was higher in spinach plants (66%) than in pea plants (34%). Variations of δ13C were lower (3%) in both spinach and pea plants regardless of organ.

Table 3 Effect of nitrogen nutrition nitrate (5 mM) and ammonium (5 mM) on the percentage distribution of amino acids in spinach and pea roots. Values represent the mean of six samples and are given as percentage of total amino acids. Analysis of variance results are represented as in legend to Table 1 Amino acids

Alanine Arginine Asparagine Aspartic acid Glutamic acid Glutamine Glycine Isoleucine Leucine Lysine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine

Spinach plants

Pea plants

ANOVA

Nitrate

Ammonium

Nitrate

Ammonium

S

N

I

5.1 3.0 4.5 11.7 21.4 12.5 9.7 2.5 3.5 4.5 2.2 1.3 6.2 2.2 2.8 1.4 3.5

6.6 5.0 4.5 3.1 8.3 47.0 3.0 1.2 2.0 2.9 1.4 1.6 6.2 2.2 0.3 0.4 2.4

0.6 4.2 39.4 7.3 8.4 2.9 20.0 1.7 1.4 3.1 2.4 0.9 0.3 4.3 1.5 5.0 3.1

0.3 3.5 67.0 1.9 2.1 9.8 0.8 0.3 0.3 0.7 0.3 0.3 0.1 1.2 0.4 7.1 1.0

* ns * * * * ns * * * ns * * ns ns * *

ns ns * * * * ns * * * * ns * * * ns *

* ns * * * * ns ns ns ns ns ns * * ns ns ns

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Table 4 Effect of nitrogen nutrition nitrate (5 mM) and ammonium (5 mM) on δ15N (‰) and δ13C (‰) in spinach and pea plants. Values represent the mean of six samples. Analysis of variance results are represented as in legend to Table 1 Isotope abundance (‰)

δ15N

Shoot Root Shoot Root

δ13C

Spinach plants

Pea plants

ANOVA

Nitrate

Ammonium

Nitrate

Ammonium

S

N

I

– 2.08 – 3.32 –33.31 –32.65

–13.90 – 7.32 –34.24 –32.04

– 3.37 – 6.57 –31.73 –29.45

– 9.84 –11.13 –31.60 –30.37

* * * *

* * * ns

* ns * *

2.3. Enzyme activities In Table 5, GS and GDH activities are shown. There were differences in the contribution of both enzymes depending on the species, nitrogen nutrition and plant organ. Thus, GS activity was localized mainly in the shoots, whereas that of GDH was in the roots. Also ammonium stimulated the activity of these enzymes, but in a different way depending on the species. In spinach plants, ammonium increased GS activity both in shoots (twofold) as in roots (threefold) and GDH activity mainly in shoots (threefold). On the other hand, in pea plants, the presence of ammonium stimulated GDH activity mainly in roots (threefold) and slightly in shoots (37%). The supply of carbon skeletons from phosphoenolpyruvate for ammonium assimilation can be met both by phosphoenolpyruvate carboxylase and pyruvate kinase (ATP:pyruvate-2-O-phosphotransferase; EC 2.7.1.40) enzymes. The activity of both the enzymes changed when

ammonium was the nitrogen source when compared to nitrate. However, a different trend was observed depending on the species. Ammonium increased PEPC activity in the shoots (42%) of spinach plants and in the roots (threefold) of pea plants. Furthermore, there was a remarkable increase in PK activity in spinach plants when ammonium was the nitrogen source; this increase was higher in shoots than in roots. In pea plants, the activity of this enzyme did not change in the presence of ammonium (Table 6). The effect of ammonium on the activity of some enzymes of the TCA cycle such as malic enzyme ((S)-malate:NADoxidoreductase; EC 1.1.1.39), malate dehydrogenase (Lmalate:NAD-oxidoreductase; EC 1.1.1.37), isocitrate dehydrogenase (isocitrate:NADP-oxidoreductase; EC 1.1.1.42) and pyruvate dehydrogenase complex (PDC) was also measured and the results are shown in Table 6. The effect of ammonium on the MDH and PDC activity showed a similar trend, since the activity of these enzymes increased in shoots of spinach plants and in roots of pea plants. To the

Table 5 Effect of nitrogen nutrition nitrate (5 mM) and ammonium (5 mM) on GS and GDH activity in spinach and pea plants. Values represent the mean of six samples. Analysis of variance results are represented as in legend to Table 1 Enzymatic activities

–1

GS (nkat g

FW)

GDH (nkat g–1 FW)

Spinach plants

Shoot Root Shoot Root

Pea plants

ANOVA

Nitrate

Ammonium

Nitrate

Ammonium

S

N

I

42.9 7.8 1.5 4.2

89.0 27.7 4.8 6.7

91.8 12.9 2.7 3.3

108.4 5.7 3.7 10.0

* * ns *

* * * *

* * * *

Table 6 Effect of nitrogen nutrition nitrate (5 mM) and ammonium (5 mM) on PEPC (phosphoenolpyruvate carboxylase), PK (pyruvate kinase), ME (malic enzyme), MDH (malate dehydrogenase), PDC (pyruvate dehydrogenase complex) and ISDH (isocitrate dehydrogenase) activity in spinach and pea plants. Values represent the mean of six samples. Analysis of variance results are represented as in legend to Table 1 Enzymatic activities

PEPC (nkat g

–1

FW)

PK (nkat g–1 FW) ME (nkat g–1 FW) MDH (µkat g–1 FW) PDC (nkat g–1 FW) ISDH (nkat g–1 FW)

Spinach plants

Shoot Root Shoot Root Shoot Root Shoot Root Shoot Root Shoot Root

Pea plants

ANOVA

Nitrate

Ammonium

Nitrate

Ammonium

S

N

I

7.5 4.2 7.5 9.3 1.4 2.1 1.00 0.65 0.75 0.55 14.9 11.2

10.7 4.5 160.1 94.2 1.2 3.5 2.85 0.52 2.7 0.45 28.1 14.9

9.8 2.7 20.9 9.5 1.3 3.7 2.14 0.43 0.84 0.78 18.0 28.1

5.8 8.5 22.9 8.5 1.4 4.8 2.49 0.82 0.92 1.69 25.7 28.1

ns * * * ns * * ns * * ns *

ns * * * ns * * * * * * *

* * * * ns ns * * * * * ns

B. Lasa et al. / Plant Physiol. Biochem. 40 (2002) 969–976

contrary, in the presence of ammonium, ME activity increased in roots of both the species, similarly isocitrate dehydrogenase activity increased in roots of both species (twofold) and in shoots mainly in spinach plants.

3. Discussion Spinach plants are highly sensitive to ammonium nutrition, which is mainly assimilated in the shoots, whereas pea plants tend to assimilate ammonium in the roots and show a tolerance to this nitrogen source. These results suggest that the ammonium-assimilating capacity of different plant organs plays a crucial role in ammonium tolerance. Although the shoot is considered to be more sensitive to ammonium than the root [4], the toxicity caused by the accumulation of ammonium does not seem to cause a different response between species, since our results show that there are no differences in shoot ammonium accumulation. The nitrogen source and patterns of assimilation can strongly influence intra-plant patterns in δ15N. Significant intra-plant variation can be observed when nitrate is the primary nitrogen source, but little variation is observed when ammonium is the source. It appears to be the case that ammonium is assimilated immediately in the root, with organic nitrogen in shoots and roots being the product of a single assimilation event. Nitrate assimilation can occur in both roots and shoots. Fractionation during assimilation by nitrate reductase causes the δ15N of unassimilated nitrate to become enriched in relation to organic nitrogen. The δ15N of leaves can be greater than in the roots because the nitrate available for assimilation is enriched in relation to root nitrate because it originates from a pool that has already been exposed to assimilation [11]. This is consistent with our results with respect to nitrate-fed plants; however, ammonium-fed plants showed different intra-plant variation of δ15N depending on the species. Pea plants showed fewer differences in intra-plant variation of δ15N, which could be a result of an assimilation of ammonium carried out principally in roots. On the other hand, spinach plant showed greater intra-plant variation of δ15N, which could be explained in terms of a distribution of ammonium assimilation between roots and shoots or of different ways of ammonium assimilation with respect to pea plants. Both the concentration and composition of the pool of free amino acids can be distinctly affected by the nitrogen source [5]. Thus, maize seedlings grown in the presence of ammonium, accumulate mainly amides, whereas when nitrate is supplied, the main organic N-compound is glutamate [19]. Our results show that ammonium nutrition decreases aspartate and glutamate both in spinach and pea plants, whilst increasing amide content, but in a different way depending on the species. Thus, in pea plants, the main amide is asparagine and in spinach plants glutamine. This fact can be related to the site of ammonium assimilation, since the ability of legume roots to export asparagine

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reflects a capacity to assimilate nitrogen not possessed by roots of other species [29]. This high potential of legume roots to process nitrogen may be related to their predisposition to infection by the Rhizobium species and to the development of the symbiotic relationship [29]. Ammonium assimilation is mainly carried out by the concerted action of GS–GOGAT [18]. Our results show that GS activity is located mainly in the shoots. In spinach plants, the presence of ammonium increased GS activity compared to nitrate-fed plants. This suggests that there is no relationship between GS activity and ammonium tolerance. Even though the GS/GOGAT pathway is the major route in higher plants [27], they are able to use other alternative routes such as those catalyzed by GDH, which catalyzes the reversible amination of 2-oxoglutarate to yield glutamate. Previous experiments have shown that GDH activity increased in pea plants when switched from nitrate to ammonium and this increase was correlated with an increase in the amino acid content, showing that the aminating activity of GDH may have a role in the ammonium detoxification (data not shown). In this case, GDH activity was higher in ammonium-fed plants compared to nitrate-fed plants in both the species. However, in spinach plants, which assimilate ammonium mainly in shoots, although GDH activity increased in shoots (threefold in ammonium-fed plants), ammonium assimilation is carried out mainly by the action of GS. On the other hand, in pea plants, ammonium assimilation is located in roots, and in this organ, GDH is the predominant enzyme, which increased threefold compared to nitrate-fed plants. This suggests a possible relationship between GDH activity and ammonium tolerance. The metabolic pathways of nitrogen and carbon are linked since nitrogen assimilation requires carbohydrates for the production of energy and the provision of carbon skeletons. Besides, while nitrate can be accumulated in vacuoles, ammonium ions are toxic and need to be rapidly assimilated. Thus, ammonium assimilation requires availability of carbon skeletons that increase the TCA cycle carbon flow [40]. In this experiment, we verified that the activity of the enzymes implicated in the TCA cycle, such as MDH, PDC and isocitrate dehydrogenase, is stimulated under ammonium nutrition in both species, and this result highly correlates to organic acid content [20]. The stimulation of these enzymes is mostly found in the shoots in spinach plants and in the roots in pea plants, indicating once again that these species assimilate ammonium in different organs. The stimulation of respiration that occurs in vascular plants during nitrogen uptake has been ascribed to an initial activation of PK and PEPC leading to an increased production of citric acid cycle carbon skeletons needed for ammonium assimilation [17]. Our results show that ammonium nutrition stimulates PK and PEPC activity. However, there was a difference in behavior depending on the species: in spinach plants, the main way which provides carbon skeletons is PK, while in pea plants, PEPC is the preferential way. Taking into account the different sensitivity to

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ammonium nutrition between these species, it appears that PEPC is more efficient than PK for providing carbon skeletons. PEPC is believed to play a variety of physiological roles in plants, including the anaplerotic fixation of CO2 for the synthesis of dicarboxylic acids used as respiratory substrates, and the supplement of carbon skeletons for ammonium assimilation [36]. The capacity of the plant to provide carbon skeletons through carbon dark fixation by roots may determine the capacity of the plant to assimilate ammonium, especially under conditions in which the supply of 2-oxoglutarate is limited. It may also constitute an alternative way to supply carbon skeletons, in addition to respiratory 2-oxoglutarate. The incorporation of carbon fixed by PEPC may also change the δ13C values; these values are expected to be less negative than the values of CO2 fixed by RuBP carboxylase [31]. However, in our experiment, the possible supply of carbon by PEPC in roots of pea plants does not reflect changes in δ13C that corroborate this process. In summary, the differences found in ammonium nutrition response between spinach (ammonium-sensitive species) and pea (ammonium-tolerant species) plants seem to be related to differences in the site of ammonium assimilation as well as to the assimilation route. Thus, the present results provide a relationship between ammonium root assimilation and ammonium tolerance. Moreover, our results highlight the importance of the GDH and PEPC as enzymes more efficient for ammonium detoxification.

4. Methods 4.1. Plant material and growth conditions Spinach (Spinacea oleracea L. ‘Correnta F1’) and pea (Pisum sativum L. ‘Macrocarpon’) seeds were sown in vermiculite/perlite (2/1) and irrigated with distilled water. After 1 week, seedlings were transferred to a continuously aerated (flowing rate = 12 ml s–1) hydroponic culture with six seedlings per tank of 7 l and three tanks per treatment for each species. The nutrient solution was replaced every 5 d and the macroelement solution composition for the different treatments is shown in Table 7. The microelement composition was as described by Rigaud and Puppo [35]; plants were fed with nitrate (5 mM) as the sole nitrogen source applied as Ca(NO3)2 or with ammonium (5 mM) as the sole nitrogen source applied as (NH4)2SO4. The pH of the solutions was controlled daily and maintained near neutral (6.6 ± 0.4) by adding 5 mM CaCO3. Plants were grown under controlled conditions, at 30/20 °C (day/night) temperature, 60/80% relative humidity, 16/8 h photoperiod and 400 µmol m–2 s–1 photosynthetic photon flux (fluorescent: GTE Sylvania F96T12/CV/VHO; incandescent 40 W Phillips). Growth period was 28 and 21 d for spinach and pea, respectively.

Table 7 Macroelement content in nutrient solution nitrate (5 mM) and ammonium (5 mM). Values are expressed in g l–1 N-source

K2HPO4 KCl CaSO4 MgSO4 Na2FeEDTA CaCO3 Ca(NO3)2 (NH4)2SO4 CaSO4

Nitrate

Ammonium

0.2 0.2 0.09 0.1 0.025 0.5 0.41 – –

0.2 0.2 0.09 0.1 0.025 0.5 – 0.33 0.34

4.2. Growth and nitrogen analysis At the end of the growth period, six plants were separated into roots and shoots and dry weights were determined by drying in an oven at 80 °C for 48 h. For nitrogen analyses, dried samples were ground and sieved through a filter with 0.8 mm pores. Organic nitrogen content was determined by the Kjeldahl method [2]. 4.3. Enzyme analysis Phosphoenolpyruvate carboxylase (phosphate:oxaloacetate-carboxy-lyase; EC 4.1.1.31) and GDH (Lglutamate:NAD+-oxidoreductase; EC 1.4.1.2) were extracted from frozen plant material (0.5 g) which was ground in a mortar with liquid nitrogen and 5 ml of extraction buffer containing 100 mM maleic acid-KOH buffer (pH 6.8), 100 mM sucrose, 2% 2-mercaptoethanol and 15% etylenglycol. Extracts were filtered through one layer Miracloth (Calbiochem), centrifuged first at 3500 × g for 8 min at 4 °C and at 30,000 × g for 20 min at 4 °C and the supernatant used for the assay. PEPC activity was determined by coupling the reaction to NADH-oxidation mediated by malate dehydrogenase (L-malate:NAD-oxidoreductase; EC 1.1.1.37) [13]. GDH activity was determined in the aminating direction by following the absorption change at 340 nm by NADH-oxidation [16]. GS (L-glutamate:ammonialigase; EC 6.3.1.2) was extracted from frozen plant material (0.5 g), using mortar and pestle with liquid nitrogen and 5 ml of the following medium: 50 mM Tris–HCl buffer (pH 8), 1 mM EDTA, 10 mM 2-mercaptoethanol, 5 mM dithiothreitol, 10 mM MgSO4, 1 mM cysteine and 0.6% polyvinylpolypyrrolidone. Extracts were filtered through one layer Miracloth (Calbiochem), centrifuged at 35,000 × g for 20 min at 4 °C and the supernatants used for the assay. GS activity was determined by a biosynthetic assay based on glutamyl hydroxamate synthesis [32]. Malate dehydrogenase (L-malate:NAD-oxidoreductase; EC 1.1.1.37), malic enzyme ((S)-malate:NAD-oxidoreductase; EC 1.1.1.39), pyruvate kinase (ATP:pyruvate-2-O-phosphotransferase;

B. Lasa et al. / Plant Physiol. Biochem. 40 (2002) 969–976

EC 2.7.1.40), isocitrate dehydrogenase (isocitrate:NADPoxidoreductase; EC 1.1.1.42) and overall PDC activity were extracted from frozen plant material (0.5 g), which was ground to a powder in a chilled mortar under liquid nitrogen and extraction buffer (2.5 ml) containing 50 mM 3-(Nmorpholino) propanesulfonic acid buffer (pH 7.0), 5 mM MgCl2, 1 mM EDTA Na2, 20 mM KCl, 10 mM 2-mercaptoethanol and 10 mM dithiothreitol. Extracts were filtered through one layer Miracloth (Calbiochem), centrifuged at 20,000 × g for 30 min at 4 °C. A 1 ml aliquot of the resulting supernatant was desalted at 4 °C on a Bio Gel P6DG column pre-equilibrated with 0.4 ml of 250 mM 3-(N-morpholino) propanesulfonic acid buffer (pH 7.0), 25 mM MgCl2 and 100 mM KCl. MDH activity was determined after Gordon and Kessler [14]. ME activity was assayed at 340 nm as previously described [26]. PK activity was measured at 340 nm by coupling the reaction to NADH-oxidation mediated by lactate dehydrogenase ((S)lactate:NAD-oxidoreductase; EC 1.1.1.27) [10]. Isocitrate dehydrogenase activity was determined as NADPH production at 340 nm [9]. Overall PDC activity was assayed by the coupled-enzyme method of Randall and Miernyk [34] as acetyl coenzyme A-dependent NADH reduction. The protein concentration in the extracts was quantified by the dye-binding microassay based on Bradford [6] using a commercial Bio-Rad kit (Watford, UK) and bovine serumalbumine as a standard. 4.4. Chemical analysis Cell sap was extracted by centrifugation from frozen tissue treated for 5 min at 80 °C. Ammonium was determined in sap by isocratic ion chromatography and nitrate by gradient ion chromatography using a DIONEX 500 system equipped with an electrochemical detector ED40. Amino acid extraction was accomplished as described by Royuela et al. [37] and amino acid composition was determined by HPLC according to Cohen et al. [7]. At the end of the growth period, plants were separated into roots and shoots. Plant material was oven-dried at 80 °C for 48 h and milled. The δ15N and δ13C were determined on subsamples (c. 1 mg dry wt) of shoots and roots using isotope ratio mass spectrometry in continuous flow. Samples were weighed, sealed into tin capsules (5 × 8 mm, Lüdi AG) and loaded into the autosampler of an NC elemental analyser NC 2500 (CE instruments, Milan, Italy). The capsule was dropped into the combustion tube of Cr2O3 and Co3O4Ag at 1020 °C with a pulse of oxygen. The resulting oxidation products, CO2, NxOy and H2O, were swept into the reduction tube Cu wire at 600 °C where oxides of nitrogen were reduced to N2 and excess oxygen was removed. A magnesium perchlorate trap removed the water. N2 and CO2 were separated by GC column (Fused Silica, 0.32 mm × 0.45 mm × 27.5 m, Chrompak) at 32 °C and later, it was introduced into the mass spectrometer (TermoQuest Finnigan model Delta plus,

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Bremen, Germany) via a Finnigan Mat Conflo II. Values of δ (‰) were calculated as: d=

Rsample − Rstandard

where R is the ratio of

Rstandard 15

N/14N or

× 1000,

13

C/12C.

4.5. Statistical analysis Results were examined by two-way (species and N-nutrition) analysis of variance with the statistical software STATVIEW.

Acknowledgements This work was supported by research grant AGL 20000934-CO2-01 CICYT and Gobierno of Navarra OF 96/2000.

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