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Role of glutamate availability oncadmium induced changes of nitrogenand glutamate in tomato plants(Solanum lycopersicon)Chiraz Chaffei-Haouari a b , Afef Hajjaji-Nasraoui a , ElisaCarrayol b , Maud Lelendais b , Mohamed Habib Ghorbel a &Houda Gouia aa Unité de recherche Nutrition et métabolisme azotés etprotéines de stress (99UR/09-20), département de biologie,Faculté des sciences de Tunis , Campus Universitaire El ManarI , 1060 , Tunis , Tunisie E-mail:b Unité de Nutrition azotée des plantes , INRA , route de Saint-Cyr, F-78027 , VersaillesPublished online: 26 Apr 2013.

To cite this article: Chiraz Chaffei-Haouari , Afef Hajjaji-Nasraoui , Elisa Carrayol , MaudLelendais , Mohamed Habib Ghorbel & Houda Gouia (2011) Role of glutamate availability oncadmium induced changes of nitrogen and glutamate in tomato plants (Solanum lycopersicon) ,Acta Botanica Gallica: Botany Letters, 158:1, 57-69, DOI: 10.1080/12538078.2011.10516254

To link to this article: http://dx.doi.org/10.1080/12538078.2011.10516254

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Acta Bot. Gallica, 158 (1), 57-69, 2011.

Role of glutamate availability on cadmium induced changes of nitrogenand glutamate in tomato plants (Solanum lycopersicon)

by Chiraz Chaffei-Haouari(1,2), Afef Hajjaji-Nasraoui(1), Elisa Carrayol(2), Maud

Lelendais(2), Mohamed Habib Ghorbel(1) and Houda Gouia(1)

(1)Unité de recherche Nutrition et métabolisme azotés et protéines de stress (99UR/09-20), départe-

ment de biologie, Faculté des sciences de Tunis, Campus Universitaire El Manar I, 1060 Tunis,

Tunisie; chiraz_ch2001@yahoo.fr

(2) Unité de Nutrition azotée des plantes, INRA, route de Saint-Cyr, F-78027 Versailles

Abstract.- Maximal glutamine synthetase (GS, EC 6.3.1.2) activity and freeaminoacid accumulation occur together in the leaves and roots. GS and specificGS activities although declining in the stressed leaves. In the other hand, thedecrease of GS activity in the roots is contrarily accompanied by an increase inspecific GS activity in roots in treated plants. Maximal GS activity is correlatedwith the peak degradation of the free aminoacid and protein in the two organs.Western blots analysis from Cd treated plants shows a decrease in chloroplasticGS (GS2) and an increase in cytosolic GS (GS1) proteins. In the roots, free ami-noacids accumulated as NADH-dependent glutamate synthase (NADH-GOGAT,EC 2.6.1.5.3) and specific activities increase. Maximal leaf NADH-GOGAT acti-vity occurs simultaneously with maximal leaf aminating glutamate dehydrogena-se (NADH-GDH, EC 1.4.1.2) activity.

Key words : cadmium - glutamate metabolism - tomato.

Résumé.- La valeur maximale de l’activité glutamine synthétase (GS, CE6.3.1.2) et l'accumulation des teneurs d'acide aminé sont corrélées dans lesfeuilles et dans les racines. L’activité GS et l’activité spécifique de la GS dans lesfeuilles montrent une diminution en présence du cadmium. Contrairement auxfeuilles, la diminution de l’activité GS dans les racines est accompagnée d’uneaugmentation de l’activité spécifique de la GS au niveau des racines des plantestraitées. Le maximum d’activité GS est correlé avec le pic de dégradation desaminoacides et des protéines dans les deux organes. L’analyse des Westernblot des plantules traitées montre une diminution de la quantité protéique de laGS chloroplastique (GS2) et une augmentation de la quantité protéique de la GScytosolique (GS1). Dans les racines, l’accumulation des acides aminés estparallèle à l’augmentation des activités NADH-GDH-dépendante, NADH-GOGAT et leurs activités spécifiques. La valeur maximale de l’activité NADH-GOGAT est observée simultanément avec le maximum d’activité de la glutamatedéshydrogénase NADH-dépendante.

Mots clés : cadmium - métabolisme du glutamate - tomate.

received October 19, 2009, accepted March 11, 2010

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Abbreviations - Cd = cadmium, GS2 = chloroplastic glutamine synthetase, GS1 = cytoso-lic glutamine synthetase, GS = glutamine synthetase, GDH = glutamate dehydrogenase,NADH-GDH = NADH-dependent GDH, NADH-GOGAT = NADH-dependent glutamatesynthase.

I. INTRODUCTION

The synthesis of reserves protein creates a demand for the necessary aminoacid precursors.This demand is met mainly by aminoacids synthesized de novo by the plant (Gouia et al.,2003). Most of N translocates to the tomato plants in the glutamine and asparagine(Chaffei et al., 2004) and they are the main N donors for in situ synthesis of protein ami-noacids in the plant (Hirel & Lea, 2001).

Glutamine synthetase (GS) as the first enzyme of the GS/GOGAT system depends onATP and appears in several isoforms. The most abundant isoform is GS2, which is locatedin the chloroplast and root plastids and is mainly involved in the photorespiratory pathwayin leaves (Wallsgrove et al., 1987; Gouia et al., 2008). In tomato roots, the cytosolic iso-form of GS (GS1) is only present in etiolated seedlings and replaced by GS2 located inroot plastids (Miflin & Habash, 2002). Within any location, the metabolism will differaccording to the developmental stage and the environmental conditions of plants. Thus, thenature of GS and its regulation has to be approached by taking into account the multidi-mensional nature of nitrogen metabolism and appreciating the large differences that occurin glutamine metabolism between various locations in the organ. There are many studiesthat suggest that the plant can sense its reduced nitrogen status and regulate the uptake andreduction of nitrate (Miflin & Habash, 2002). Several lines of evidence have suggested thatglutamine may play an important role (Glass et al., 2002). However, there are arguments(Stitt et al., 2002) indicating that it may be other aminoacids which act as sensors of plantnitrogen status. However, the discovery of NAD(P)H glutamate synthase and later ferre-doxin-dependent glutamate synthase established a route, the glutamate cycle, for NH3 toenter into organic compounds via its assimilation by GS. It is essential that toxic ammo-nium must be immediately reassimilated into organic molecules for nitrogen cycling.Ammonium is assimilated into Gln amide group, which is then transferred to the positionof 2-oxoglutarate, yielding two molecules of Glu by the concerted reaction of Gln synthe-tase and Glu synthase. Plants have evolved to capture available carbon and nitrogen and tostore and transfer them efficiently. To achieve this, plants use individual aminoacids andproteins that differ widely in their C/N ratio. The proposed role of glutamate dehydroge-nase (GDH) is particularly noteworthy. GDH is one of the few enzymes capable of relea-sing amino nitrogen from amino acids to give a keto-acid and NH3 that can be separatelyrecycled to be used in respiration and amide formation, respectively. GDH may be expec-ted to function in the deaminating direction in tissues that are converting amino acids intotransport compounds with a low C/N ratio. In a previous paper (Gouia et al., 2008), wedemonstrated the presence of proteolytic activity capable of releasing aminoacids from theprotein of stressed leaves and roots. In this work we report the presence of GS, GOGATand GDH activity in stressed leaves and roots of tomato. The respective catalytic poten-tials of these enzymes to incorporate N into translocated glutamine and subsequently uti-lise of this N for assimilatory growth in leaves and roots are crucial to the N economy(John et al., 2004). The purpose of this focus paper is to review key aspects of the place of

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glutamate in plant nitrogen metabolism, focusing on its metabolic roles, the question ofwhether its cellular content is physiologically regulated.

II. MATERIAL AND METHODS

A. Plant material and growth conditions

After imbibition, seeds of tomato (Solanum lycopersicon Mill. cv 63/5F1) were germi-nated on moistened filter paper at 25 °C in the dark. After 7 days, uniform seedlings weretransferred to 6 litres plastic beakers (8 plants per beaker) filled with continuously aerated,basal nutrient solutions of an initial pH 5.8-6, containing 3 mM KNO3, 0.5 mM Ca(NO3)2,2.4 mM KH2PO4, 0.5 mM MgSO4, 100 µM Fe-K2-EDTA, 30 µM H3BO3, 5 µM MnSO4,1 µM CuSO4, 1 µM ZnSO4, and 1 µM (NH4)6Mo7O24. Plants were grown in a growthchamber (26 °C/70% relative humidity during the day, 20 °C/90% relative humidity duringthe night). The photoperiod was 16 h daily with a light irradiance of 150 µmol.m-2.s-1 atthe canopy level. At the age of 10 days after transplant, cadmium was added to the mediumas CdCl2 at 0 to 50 µM. After one week of Cd treatment, plants were separated into shootsand roots. Roots were rapidly washed three times with distilled water, then samples werestored in liquid nitrogen for subsequent analysis or dried at 70 °C for at least three days inorder to determine both dry material and ionic contents.

B. Analytical methods

Cd content in leaves and roots was analyzed by digestion of plant material in concen-tral HNO3/HClO4 mixture (3/1, v/v). The metal concentrations were determined by atomicabsorption spectrophotometry (Perkin-Elmer Analyst 300). Chlorophyll and pheophytincontent were determined on crude leaf extracts (Torres Netto et al., 2002). Soluble proteinwas determined using a commercially available kit (Coomassie Protein assay reagent,BioRad, California, USA; Bradford, 1976). Total aminoacid content was assayed by theRosen colorimetric method using glutamine as a reference. Individual aminoacid compo-sition was determined on pooled samples extracted from an equal dry weight of three plantrepeats using ion-exchange chromatography (Oliveira et al., 1997).

C. Enzymatic assays

Enzymes were extracted from frozen leaf and root material stored at -80 °C. All extra-ctions were performed at 4 °C.

Glutamine synthetase activity was determined by the semi-synthetic reaction withhydroxylamine and ATP as co-substrates using the method of O’Neal & Joy (1973). Afterthe reaction, mixture was incubated at 37 °C for 15 min, the reaction was terminated byadding acidic FeCl3 solution (2% (W/V) TCA and 3.5% (W/V) FeCl3 in 2% HCl). Theproduction of γ-glutamylhydroxamate was measured with a spectrophotometer at 540 nm.One unit of GS activity was the amount of enzyme catalysing the formation of 1 µmolγ-glutamylhydroxamate.min-1 at 37 °C. GS activity was expressed on the basis of gramfresh weight.

The activity NAD(H)-GDH was measured at 340 nm at 30 °C. Composition of NADH-GDH assays was that of Loulakakis and Roubelakis-Angelakis (1990). The reaction mix-ture contained 150 mM Tris-HCl pH 8, 100mM NH4Cl, 10 Mm α-ketoglutaric acid pH 7.4,0.3 mM NADH, 100 µl enzyme extract. A control was carried out without NADH and a

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blank test in which the α-ketoglutaric acid and NH4Cl were omitted was always perfor-med. Measurements were carried out on two independent assays each time. NAD+-GDHactivity measurements were performed in the presence of 100 mM Tris-HCl pH 9,20.4 mM CaCl2, 0.4 mM L-glutamate pH 7.4, 28.4 mM NAD+ and 50 µl enzyme extract.The GDH activity was expressed as µmol NAD(H) per min and per g FW.

Fd-GOGAT activity was determined using reduced methyl viologen as the electrondonor (Lea & Miflin, 2003). The reaction mixture consisted of 200 mM KH2PO4-KOH pH7.5, 10 mM glutamine (Gln), 10 mM 2-ketoglutarate, 15 mM methyl viologen,1 mM amino-oxyacetic acid (transaminase inhibitor), and extract. After 5 min of pre-incu-bation at 30 °C, the reaction was started by the addition of reductant solution (47 mgNa2S2O4, 50 mg NaHCO3 in 1 ml of water). After 30 min of incubation at 30 °C, the reac-tion was terminated by adding 1 ml of ethanol and then shaking vigorously. Fd-GOGATactivity was determined by the quantitative measurement of glutamate using HighPerformance Liquid Chromatography (HPLC) as described previously (Esposito et al.,2003). The activity was expressed as nmol glutamate formed min-1.mg-1 protein.

NADH-GOGAT activity was assayed consistently within 2 h of extraction, monitoringNADH oxidation at 340 nm and using two control mixtures (minus 2-ketoglutarate andminus glutamine) to correct for endogenous NADH oxidation. The assay mixture contai-ned 50 mM KH2PO4-KOH, pH 7.5, 10 mM KGA, 10 mM Gln, 1 mM NADH, 1 mMamino-oxyacetate, and extract. The reaction was started by adding glutamine immediatelyfollowing the enzyme preparation. The activity was expressed as µmol oxidized NADHmin-1.mg-1 protein.

D. Electrophoresis and Western blotting

Protein gel blot analysis and gel staining procedure : proteins (10 µg) were separated bySDS-PAGE (Verword et al., 1989). The percentage of polyacrylamide in the running gelswas 10% GS. Denaturated proteins were electrophoretically transferred to nitro-cellulosemembranes. Polypeptide detection was performed using polyclonal antiserum raisedagainst tobacco GS1 and GS2 (Masclaux et al., 2002). Relative GS2 and GS1 proteinamounts were determined by densitometric scanning of Western blot membranes andquantifier using image software (NIH image 1.63, public domain).

The data presented in this work are the average of at least five replicates per treatment;means ± standard error (SE) are given in the figures. Each experiment was carried out induplicate. According to the Tukey test, values ± 0.05 were considered significantly diffe-rent.

III. RESULTS

A. Growth

Treatment of plants during seven days with Cd is accompanied by its progressive upta-ke and accumulation in the plant (Fig. 1A, 1B and 1C) and decreasing of root and leavegrowth and photosynthetic pigment contents (Fig. 1D). The decrease in chlorophyllcontent is parallel to the decrease in pheophytin content at 20 and 50 µM treatments.However, for every concentration used, Cd accumulation is more important in roots thanin leaves (Fig. 1B). Total aminoacids were quantified and we observe that free

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Fig. 1.- Effects of Cd treatment (0, 5, 10, 20 and 50 µM) on (A) Cd accumulation in leaves(■) and roots (□), (B) Cd repartition between leaves and roots, (C) dry weight of leavesand roots, (D) total chlorophyll and pheophytin contents, (E) free aminoacids content inleaves (■) and roots (□). Values are means ± SE of three individual plants. Standard errorsare not shown when they are smaller than the symbol.

Fig. 1.- Effets de la dose du Cd (0, 5, 10, 20 et 50 µM) sur l’accumulation de Cd (A) dansles feuilles (■) et les racines (□), (B) répartition du Cd entre les feuilles et les racines, (C)production de la matière sèche des feuilles et des racines, (D) teneurs en chlorophylletotale et en phéophytine, (E) teneurs en acides aminés totaux dans les feuilles (■) et dansles racines (□).

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aminoacid content increases progressively in leaves and roots of plants when treated bycadmium (Fig. 1E).

B. Glutamine and glutamate contents

Individual aminoacid (glutamate and glutamine) content was also determined and theresults are presented in Fig. 2. Studying the qualitative and quantitative changes in indivi-dual aminoacid contents can thus be informative. In control plants the main aminoacidsencountered in leaves (Fig. 2A) are glutamate (Glu) and glutamine (Gln). Glu as well asGln are the major aminoacids represented in roots (Fig. 2B). This amide (high N/C ratio)is known to be highly reactive and to serve as the major N transport form in plants. In Cd-treated plants, we observed that Gln content increases in leaf tissues (Fig. 2A). In parallel,the low decrease of Glu content may be a consequence of buffering effects through modu-lations in protein content. Indeed, these aminoacids are direct products of the protein cata-bolism and aminoacids anabolism. However, the more drastic changes in aminoacidsproportions are observed in roots. In contrast to leaves, Glu in roots decreases dramatical-ly with Cd increase in the culture medium. Gln content increases clearly at 20 and 50 µMof Cd concentrations, whereas total protein proportions are decreased.

C. Glutamine metabolism

The molecular masses of the GS isoenzymes are different. Three bands correspondingto either GS1 (40 Kda), GS2a (44 Kda) or GS2b (42 Kda) proteins can be unambiguouslyvisualized on western blots using antibodies raised against the GS2 of tobacco (Chaffei et

Fig. 2.- Effects of Cd treatment (0, 5, 10,20 and 50 µM) on glutamate, glutamineand protein contents in (A) leaves and(B) roots. Results are expressed in % oftotal amino-acide contents. Values aremeans ± SE of three individual plants.Standard errors are not shown whenthey are smaller than the symbol.

Fig. 2.- Effets de la dose du Cd (0, 5, 10,20 et 50 µM) sur la teneur en glutamate,glutamine et protéine totale soluble dansles feuilles (A) et dans les racines (B).

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al., 2004). In the leaves, the level of GS1 protein is clearly increasing up to approximate-ly 6-fold in the 50 µM treated plants (Fig. 3B). Immunoblot observation and signal quan-tification show that a large amount of the two GS2 isoforms (GS2a and GS2b) is detectedin leaf protein extracts, but could not be clearly detected in root extracts (Fig. 3D).However, their amount continuously decreases with increasing cadmium stress. Incontrast, cadmium treatment induces the appearance of the GS1 isoform that can not bedetected in untreated leaf tissues. In roots, the major isoform was GS1, and its amount isalmost unchanged whatever cadmium treatment applied, especially at 20 µM (Fig. 3D).

Fig. 3.- Effects of Cd treatment (0, 5, 10, 20 and 50 µM) on (A) GS activity and specific GSactivity in leaves, (B) protein content for GS enzymes in leaves (in % of control), (C) GSactivity and specific GS activity in roots, (D) protein content for GS enzymes in roots (in% of control). An equal amount of soluble proteins (10 µg) was added per lane. Values aremeans ± SE of three individual plants. Standard errors are not shown when they are smal-ler than the symbol.

Fig. 3.- Effets de la dose du Cd (0, 5, 10, 20 et 50 µM) sur (A) l’activité de la GS et l’activi-té spécifique de la GS dans les feuilles, (B) la teneur de la protéine GS dans les feuilles(en % de témoin), (C) l’activité de la GS et l’activité spécifique de la GS dans les racines,(D) la teneur de la protéine GS dans les racines (en % du témoin). La même quantité enprotéine totale soluble (10 µg) est mise par puit.

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GS2 signals are low and the quantification threshold allows by the analyser. The sharp

decrease on GS activity in leaves and roots may then be due to the decrease of GS2

(Fig. 3A, Fig. 3C). The increase of specific GS activity in leaves and roots after 10 µM of

Cd results to peak of protein degradation in the highest dose of Cd (20 and 50 µM).

D. Glutamate metabolism

NADH-GOGAT activity was assayed and an increase in NADH-GOGAT activity is

clearly observed in treated plants (Fig. 4A and 4B). However, the NADH-GOGAT speci-

fic activity is only 16% of the NADH-GOGAT activity in roots of tomato plants. The

potential increase of specific NADH-GOGAT activity in leaves and roots proves the high

protein degradation in stressed plants.

Fig. 4.- A: effects of Cd treat-ment (0, 5, 10, 20 and50 µM) on NADH-GOGATactivity; B: specific NADH-GOGAT activity in leavesand roots. Values aremeans ± SE of three indivi-dual plants. Standard errorsare not shown when they aresmaller than the symbol.

Fig. 4.- A : effets de la dose duCd (0, 5, 10, 20 et 50 µM)sur l’activité NADH-GOGAT ;B : activité spécifique NADH-GOGAT dans les feuilles etdans les racines.

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Another putative source of glutamate is the reaction catalysed by the mitochondrial

NADH-dependent GDH. The changes in the NADH-GDH activities were investigated

(Fig. 5). The aminating NADH-GDH activity increases by 3 and 4 fold under Cd treatment

in roots and leaves, respectively (Fig. 5A and 5C). In the 50 µM treated plants, the amina-

ting to deamination GDH activities ratio is equal to 25 and 34 relative to control in the

roots and leaves respectively (data not shown), suggesting that changes in the GDH ami-

nating and deaminating activities are in favour of glutamate synthesis. GDH protein

content is lower in leaves compared to roots (Fig. 5B, D). With increasing Cd stress, the

amount of GDH protein increases in the leaves by approximately 4-fold at 50 µM Cd treat-

Fig. 5.- A: effects of Cd treatment (0, 5, 10, 20 and 50 µM) on NADH-GDH activity and spe-cific NADH-GDH activity in leaves; B: protein content of GDH enzyme in leaves (in % ofcontrol); C: NADH-GDH activity and specific NADH-GDH activity in roots; D: proteincontent of GDH enzyme in roots (in % of control). An equal amount of soluble proteins(10 µg) was added per lane.

Fig. 5.- A : effets de la dose du Cd (0, 5, 10, 20 et 50 µM) sur l’activité NADH-GDH et l’ac-tivité spécifique NADH-GDH dans les feuilles ; B : teneur de la protéine GDH dans lesfeuilles (en % du témoin) ; C : activité NADH-GDH et activité spécifique NADH-GDH dansles racines ; D : teneur de la protéine enzymatique GDH dans les racines (en % dutémoin). La même quantité en protéine totale soluble (10 µg) est mise par puit.

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ment (Fig. 5B). No significant change in GDH protein content is observed in the roots(Fig. 5D). The NADH-GDH activity and specific NADH-GDH activity are potentially cor-related, there is probably a strong role played by this enzymatic protein to regeneratingglutamate from aminoacids and protein degradation under Cd stress conditions.

IV. DISCUSSION

In this study, tomato seedlings were exposed to Cd toxicity by introducing increasingconcentrations of this pollutant into the culture medium. Plant morphology is dramatical-ly affected by Cd stress. The emergence of new organs is altered and that newly emergedleaves are chlorotic. This is paralleled with a decline in plant growth (Fig. 1C). We obser-ve that roots from Cd stressed plants are shorter and less hairy than those from controls.However, treated roots are thicker and stronger, which may have compensated in part theloss of biomass. Indeed, it appears that root biomass is affected to a lesser extent compa-red to leaves (Fig. 1C). Besides, changes in total chlorophyll, pheophytin and free ami-noacids content are good biomarkers for describing the senescence like phenomena relatedto the stress triggered by heavy metal (Chaffei et al., 2004; Küpper et al., 2007).

We find that Cd treated plants accumulate free aminoacids in both leaves and roots, andthey progressively lost protein relative to controls. We suppose that the accumulation ofaminoacids in Cd-treated plants is rather a consequence of protein proteolysis (Chaffei etal., 2006).

A concomitant decrease in total soluble protein and the activities of enzymes involvedin primary nitrogen assimilation (GS and GOGAT) is observed in both leaves and roots(Akira et al., 2003). Inhibition of GS activity by Cd stress has been described in maize, peaand barley seedlings (Tercé-Laforgue et al., 2004). We show that, in tomato, such inhibi-tion is closely linked to a decrease in the cognate proteins.

The loss of Rubisco during senescence is well documented. It was shown in previouswork that nitrogen remobilisation is a senescence associated process that takes place insource organs such as senescing leaves (Akira et al., 2003; Tercé-Laforgue et al., 2004).Glutamine metabolism is of a major importance in plant nitrogen economy since it pro-vides nitrogen to young developing organs. Glutamine metabolism is usually associatedwith the induction of the expression of enzymes such as specific proteases, the cytosolicGS1 isoform and GDH (Chaffei et al., 2006). We have thus investigated whether theseenzymes are expressed in leaves and roots of tomato, when subjected to Cd stress. Weshow that total GS activity is decreased in both roots and leaves of Cd-treated plants, withthe higher inhibition in the leaves. Indeed, protein analysis and transcript steady state levelstudy show that GS2 proteins decrease in Cd treated plants. The GS1 protein remainsconstant in roots, except at 20 µM Cd, and the GS1 isoform increases in leaves of Cd trea-ted-plants. This suggests that, when Cd affects the chloroplastic GS activity, plants inducecytosolic isoform to compensate and continue glutamine biosynthesis.

Moreover, concomitantly to the increase in GS1 protein in leaf crude extracts, we alsoobserve a sharp increase in GDH protein. Cd triggers an increase in GDH protein in bothroots and leaves. The analysis of GDH protein levels shows that the increase in GDH pro-tein is parallel to the increase in GDH aminating activity. Cd thus induces GDH geneexpression in both roots and leaves, but induction in leaves appears to be higher than inroots. This suggests that tomato plants can express several GDH isoenzymes with diffe-rential aminating activities and that Cd treats plants preferentially expressed isoenzymes

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Fig. 6.- Scheme of glutamate pathways synthesis and recycling in tomato plants under Cdstress conditions showing the futile cycle implying GS/GOGAT cycle and the amination-deamination of Glu by glutamate dehydrogenase (GDH).

Fig. 6.- Schéma de la voie de synthèse et recyclage du glutamate chez la tomate en condi-tions de stress cadmique montrant l’intérêt de l’implication du cycle GS/GOGAT et l’ami-nation et la désamination du Glu par la glutamate déshydrogénase (GDH).

with higher in vitro aminating capacity, involved in glutamate synthesis. However, untilnow, there is no evidence that the functionality of GDH during stress as well as duringsenescence still remains and it is attractive to suppose that both GDH and NADH-GOGATare induced in order to compensate the lack of glutamate (Dominguez et al., 2003). Weobserve that NADH-GOGAT activity is increasing in the Cd treated roots. Theses resultstaken together confirm that, when stressed by Cd, plant nitrogen metabolism shifts fromprimary assimilation to recycling of ammonia to remobilize the organic nitrogen mainlyprovided by protein degradation (Feller & Fisher, 1994). Interestingly, we observe that thenitrogen mobilisation process is favoured in Cd treated plants in which the growth and theemergence of new organs are considerably compromised, compared to untreated plants.This then suggests that GS1, GDH and proteolysis are involved here for the building ofnew organs.

In roots, unlike leaves, Glu is highly affected and decreased in treated plants, whereasthe Gln remains relatively stable. Gln, which is also an amide providing a high N/C ratio,is however reactive, can be used by the plant as N storage compound (Raven et al., 2004).The N storage status of roots in plants treated with high Cd dose (50 µM) is also confir-med by the increase in free aminoacids (Fig. 1E). This suggests that the synthesis of 2-oxo-glutarate dedicated to aminoacid synthesis and particularly to Glu maintenance continuesand that the availability of reducing power remains however sufficient (Brian & Peter,2007; Limami et al., 2008).

The major outcome of this work is that almost Cd stress studied shows similar effectsas natural senescence and results in i) a decrease in the expression of the primary N-assi-milation protein GS2, a decrease in total GS activity, ii) an increase in GDH and GS1 pro-

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teins depending, however, on the individual stress, thus suggesting that GDH behaves likea non-specific stress-related gene whereas GS1 is more selective (Fig. 6). As can be seenfrom Fig. 6, there is little doubt that glutamate is a central molecule in aminoacid metabo-lism in higher plants. The -amino group of glutamate is directly involved in both the assi-milation and dissimilation of ammonia and is transferred to all other aminoacids. Inaddition, both the carbon skeleton and -amino group form the basis for the synthesis of -aminobutyric acid (GABA), arginine, and proline. It should also be noted that glutamate isthe precursor for chlorophyll synthesis in developing leaves (Yaronskaya et al., 2006). Thebiochemistry and molecular biology of glutamate metabolism have been reviewed pre-viously (Lea & Miflin, 2003; Suzuki & Knaff, 2005) and this paper provides an overviewof the enzymes involved in the metabolism of glutamate as shown in figure 6, focusing onadvances that have been made in the past few years.

Akira C., I. Hiroyuki, KN. Naoko, M. Amane & M.Tadahoko, 2003.- Exclusion of ribulose-1,5-biphos-phate carboxylase/oxygenase from chloroplasts byspecific bodies in naturally senescing leaves of wheat.Plant Cell Physiol., 44, 914-921.

Bradford M.M., 1976.- A rapid and sensitive method forquantitation of microgram quantities of protein utilizingthe principal of protein-dye binding. Anal. Biochem.,72, 248-254.

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Chaffei C., C. Masclaux-Daubresse, H. Gouia & M.H.Ghorbel, 2006.- Purification of glutamate dehydroge-nase isoenzymes from control and cadmium treatedtomato leaf. In: Cadmium toxicity and tolerance in

plants. A.K. Nafees & N. Samiullah, PublishingHousse, New Delhi, India, 137-157.

Chaffei C., K. Pageau, A. Suzuki, H. Gouia, M.H. Ghorbel& C. Masclaux-Daubresse, 2004.- Cadmium toxicityinduced changes in nitrogen management inLycopersicon esculentum leading to a mertabolicsafeguard through an aminoacid storage strategy.Plant Cell Physiol., 45 (11), 1681-1693.

Dominguez M.J., F. Gutiérrez, R. Léon, C. Vilchez, J.M.Vega & J. Vigara, 2003.- Cadmium increases the acti-vity levels of glutamate dehydrogenase and cysteinesynthase in Chlamydomonas reinhardtii. Plant

Physiol. Biochem., 41, 828-832.Esposito S., G. Guerriero, V. Vona, V.D.M. Rigano, S.

Carfagna & C. Rigano, 2005.- Glutamate synthaseactivities and proteins changes in relation to nitrogennutrition in barley; the dependence on different plasti-dic glucose-6P dehydrogenase isoforms. J. Exp. Bot.,56 (409), 55-64.

Feller U & A. Fisher, 1994.- Nitrogen metabolism insenescing leaves. CRC Crist Rev. Plant Sci., 13, 241-273.

Glass A.D.M., D.T. Britto, B.N. Kaiser, J.R. Kinghorn, H.J.Kronzucker, A. Kumar, M. Okamoto, S. Rawat, M.Y.Siddiqi, S.E. Unkles & J.J. Vidmar, 2002.- The regula-

tion of nitrate and ammonium transport systems inplants. J. Exp. Bot., 53, 855-864.

Gouia H., C. Chaffei, M. Debouba & M.H. Ghorbel,2008.- Differential toxicological response to cadmiumstress of bean seedlings grown with NO3

- or NH4+ as

nitrogen source. Intern. J. Bot., 4 (1) 14-23.Gouia H., A. Suzuki, J. Brulfert & M.H. Ghorbal, 2003.-

Effects of cadmium on the co-ordination of nitrogenand carbon metabolism in bean seedlings. J Plant

Physiol., 160, 367-376.Hirel B & P.J Lea, 2001.- Ammonium assimilation. In:

Plant nitrogen. P. Lea & J.F. Morot-Gaudry (eds), 79-99.

John A., O. Raven, L. Handley & A. Mitchell, 2004.-Global aspects of C/N interactions determining plant-environment interactions. J. Exp. Bot., 55 (394), 11-25.

Küpper H., A. Parameswaran, B. Leitenmaier, M. Trtílek& I. Šetlík, 2007.- Cadmium-induced inhibition of pho-synthesis and long-term acclimation to cadmiumstress in the hyperaccumulator Thlaspi caerulescens.

New Phytol., 175 (4), 655-674.Lea P.J & B.J. Miflin, 2003.- Glutamate synthase and the

synthesis of glutamate in plants. Plant Physiol.

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Loulakakis K.A & K.A. Roubelakis-Angelakis, 1990.-Immunocharacterization of NADH-glutamate dehy-drogenase from Vitis vinifera. Plant Physiol., 94, 109-113.

Masclaux-Daubresse C., M.H. Valadier, E. Carrayol, M.Reisdorf-Cren & B. Hirel, 2002.- Diurnal changes inthe expression of glutamate dehydrogenase and nitra-te reductase are involved in the C/N balance of tobac-co source leaves. Plant Cell Envir., 25, 1451-1462.

Miflin B.J & D.Z. Habash, 2002.- The role of glutaminesynthetase and glutamate dehydrogenase in nitrogen

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