resolving the role of plant glutamate dehydrogenase

Resolving the Role of Plant Glutamate Dehydrogenase: II.Physiological Characterization of Plants Overexpressing theTwo Enzyme Subunits Individually or SimultaneouslyThere`se Terce-Laforgue1, Magali Bedu1, Celine Dargel-Grafin1, Frederic Dubois2, Yves Gibon3,Francesco M. Restivo4 and Bertrand Hirel1,*1Adaptation des Plantes a` leur Environnement, Unite Mixte de Recherche 1318, Institut Jean-Pierre Bourgin, Institut National de laRecherche Agronomique (INRA), Centre de Versailles-Grignon, RD 10, 78026 Versailles cedex, France2Equipe dAccueil Ecologie et Dynamique des Syste`mes Antropises (EDYSAN), Agroecologie, Ecophysiologie et Biologie integrative (AEB),Faculte des Sciences, 33 rue Saint Leu, 80039 Amiens cedex 1, France3Biologie du Fruit et Pathologie, Unite Mixte Recherche 1332, Institut National de la Recherche Agronomique (INRA), Centre deBordeaux-Aquitaine, BP81, 71 avenue Edouard Bourlaux, 33882 Villenave dOrnon cedex, France4Dipartimento di Bioscienze, Universita` di Parma, Parco Area delle Scienze 11/A, 43100 Parma, Italy*Corresponding author: E-mail, [email protected]; Fax, + 33-130833096.(Received April 12, 2013; Accepted July 22, 2013)

Glutamate dehydrogenase (GDH; EC 1.4.1.2) is able to carryout the deamination of glutamate in higher plants. In orderto obtain a better understanding of the physiological func-tion of GDH in leaves, transgenic tobacco (Nicotiana taba-cum L.) plants were constructed that overexpress two genesfrom Nicotiana plumbaginifolia (GDHA and GDHB under thecontrol of the Cauliflower mosiac virus 35S promoter), whichencode the a- and b-subunits of GDH individually or simul-taneously. In the transgenic plants, the GDH protein accu-mulated in the mitochondria of mesophyll cells and in themitochondria of the phloem companion cells (CCs), wherethe native enzyme is normally expressed. Such a shift in thecellular location of the GDH enzyme induced major changesin carbon and nitrogen metabolite accumulation and a re-duction in growth. These changes were mainly characterizedby a decrease in the amount of sucrose, starch and glutam-ine in the leaves, which was accompanied by an increase inthe amount of nitrate and Chl. In addition, there was anincrease in the content of asparagine and a decrease inproline. Such changes may explain the lower plant biomassdetermined in the GDH-overexpressing lines. Overexpressingthe two genes GDHA and GDHB individually or simultan-eously induced a differential accumulation of glutamateand glutamine and a modification of the glutamate to glu-tamine ratio. The impact of the metabolic changes occurringin the different types of GDH-overexpressing plants is dis-cussed in relation to the possible physiological function ofeach subunit when present in the form of homohexamers orheterohexamers.

Keywords: Carbon metabolism Glutamate Glutamatedehydrogenase Glutamine Subunits Tobacco.

Abbreviations: AGPase, ADP-glucose pyrophosphorylase; C,carbon; CaMV, Cauliflower mosaic virus; CC, companioncell; Fd, ferredoxin; GDH, glutamate dehydrogenase;GOGAT, glutamate synthase; GS, glutamine sythase; N, nitro-gen; NMR, nuclear magnetic resonance; NR, nitrate reduc-tase; WT, wild type.

Introduction

Until recently, the physiological role of the enzyme glutamatedehydrogenase (GDH; EC 1.4.1.2) in higher plants was still amatter of intensive debate, mainly because the enzyme cata-lyzes in vitro the reversible amination of 2-oxoglutarate to formglutamate. The GDH enzyme is therefore theoretically able toassimilate ammonium nitrogen (N), or release carbon (C) mol-ecules in vivo. Although there was a considerable amount ofevidence that >95% of the ammonia available to higher plantsis assimilated via the glutamine synthase (GS)/glutamate syn-thase (GOGAT) pathway (Labboun et al. 2009, Hirel et al. 2011,Lea and Miflin 2011), it has been regularly argued that GDHcould operate in the direction of ammonia assimilation(Yamaya and Oaks 1987, Oaks 1995, Melo-Oliveira et al.1996). For example, it has been shown that in leaves thea-subunit of GDH is able to assimilate part of the ammoniumreleased following salinity stress (Skopelitis et al. 2006). Othergroups have argued that GDH operates in the direction of glu-tamate deamination (Robinson et al. 1992, Fox et al. 1995,Stewart et al. 1995, Glevarec et al. 2004, Masclaux-Daubresseet al. 2006, Miyashita and Good 2008). Despite the controversyas to the physiological function of GDH, it is now clear that theenzyme plays a negligible role in the assimilation of ammonium

Plant Cell Physiol. 54(10): 16351647 (2013) doi:10.1093/pcp/pct108, available online at www.pcp.oxfordjournals.org! The Author 2013. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.All rights reserved. For permissions, please email: [email protected]

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because: (i) photorespiratory mutants lacking the plastidic GSisoenzyme are not able to grow in normal air (Blackwell et al.1987, Leegood et al. 1995); (ii) 15N-nuclear magnetic resonance(NMR) labeling studies have clearly demonstrated that in trans-genic plants containing up to five times the amount of GDHenzyme, there was no direct incorporation of ammonia intoglutamate, even when GS, the main enzyme responsible forammonia assimilation, was inhibited (Labboun et al. 2009).Moreover, in GDH-deficient mutants totally lacking root andleaf GDH activity, which grow normally under most conditions,it has recently been shown that the main function of theenzyme is to provide 2-oxoglutarate to the plant when Cbecomes limiting (Fontaine et al. 2012).

In parallel to the whole-plant physiological and molecularstudies, it has been shown that the GDH enzyme is mainly, ifnot exclusively, localized in the phloem companion cells (CCs)(Dubois et al. 2003, Terce-Laforgue et al. 2004a, Fontaine et al.2006, Fontaine et al. 2012), but its synthesis was enhanced in themitochondria and induced in the cytosol of CCs, when the am-monium concentration increased above a certain threshold(Terce-Laforgue et al. 2004a). This finding led these authors tohypothesize that the enzyme may act as a sensor to evaluate themetabolic status of the plant with respect to ammonium andsugar concentrations, including the amount of C- andN-containing molecules circulating via the phloem stream. Inline with this hypothesis, it has been suggested by a number ofauthors that each of the two GDH subunits (a and b), which areencoded by two distinct nuclear genes, may have a specific bio-logical function (Melo-Oliveira et al. 1996, Pavesi et al. 2000,Restivo 2004, Purnell et al. 2005). The a and b polypeptidescan be assembled as homohexamers or heterohexamers com-posed of different ratios of the two, thus leading to the formationof seven active isoenzymes in roots, stems and leaves (Fontaineet al. 2006). More recently, the occurrence of a third active GDHsubunit (termed g) has been demonstrated in the roots ofArabidopsis thaliana. The ability of the g-subunit to assemblewith the a- and b-subunits (Fontaine et al. 2012) has rekindledan interest in investigating more closely the metabolic or regu-latory role of the different GDH subunits. However, the physio-logical significance of the organ- and metabolic-dependentvariability of the ratio between the three GDH isoenzyme sub-units and its regulation is still unclear, and may not solely beexplained in terms of metabolic function (Skopelitis et al. 2006,Purnell et al. 2007, Fontaine et al. 2013). As an example, an ana-lysis of tobacco transgenic plants with increased and decreasedamounts of the b-subunit of GDH demonstrated that N metab-olism in general and ammonia assimilation in particular werepractically unchanged (Purnell et al. 2005).

In addition to trying to decipher the physiological functionof the enzyme using transgenic plants and mutants with alteredGDH activity, there have been a number of attempts to pro-duce transgenic plants with increased enzyme activity in orderto examine the impact of such genetic manipulation on modeland crop plant performance. For example, a US patent wasgranted to Schmidt and Miller (1999) that described the

construction of plants transformed with nucleotide sequencesencoding the a- and b-subunits of GDH isolated from Chlorellasorokiniana. These plants exhibited properties such as increasedgrowth and improved stress tolerance. More recently, overex-pression of Escherichia coli gdhA in tobacco and maize grownunder controlled or greenhouse conditions led to an increase inplant biomass production accompanied by enhanced resist-ance to water deficit (Mungur et al. 2006, Lightfoot et al.2007). In field experiments, the yield of the E. coli gdhA trans-genic plants was also increased (Lightfoot et al. 2007), indicatingthat the enzyme may be an important component controllingplant productivity, a hypothesis that was previously put for-ward following a quantitative genetic study (Dubois et al. 2003,Gallais and Hirel 2004). However, whether changes in C and Nassimilation and partitioning following metabolism through thenew GDH enzyme would have an advantage over the GS/GOGAT ammonia assimilatory pathway remained unclear.

To investigate further the function of the two GDH subunits,transgenic tobacco plants overexpressing the a- and b-subunitsof Nicotiana plumbaginifolia GDH have been constructed.Phenotypic analysis of the transgenic plants was carried outto evaluate the impact of increased GDH activity on theplant growth and on the main metabolites representative ofleaf and root C and N metabolism. Such an investigation wasalso conducted to determine if each of the two enzyme sub-units had a specific physiological function when overexpressedindividually or simultaneously.

Results

Overexpression of GDH

To produce tobacco plants containing elevated amounts of thea- and b-subunits of GDH, two N. plumbaginifolia cDNAs thatencode the distinct subunits (GDHA and GDHB) were isolated.The cDNAs were fused with a Cauliflower mosiac virus (CaMV)35S promoter in order to make them constitutive and, afterselection and regeneration on kanamycin, homozygous T3transgenic lines overexpressing the two GDH subunits wereobtained. The overexpressors were selected by measuringboth NAD(H)-dependent aminating and NAD-dependent dea-minating GDH activity. For each construct, 35S-NpGDHA and35S-NpGDHB, two independent transformants were selected(lines A1, A2 and B1, B2 respectively), which exhibited the high-est amount of leaf GDH activity. Two transgenic lines (GDHA/B)expressing both genes (lines A2b1 and B1a2) were obtained byreciprocal crossing between the GDHA and GDHB overexpres-sors as previously described by Labboun et al. (2009).

In fully expanded leaves of the transgenic lines A1, A2, B1, B2,A2b1 and B1a2, both NAD(H)-GDH aminating and NAD-GDHdeaminating activity were increased by at least 4-fold and up to 7-fold compared with the wild type (WT) control plants (Fig. 1). Inroots, the increase in enzyme activity was much lower in the sixdifferent transgenic lines, being approximately 2-fold higher com-pared with the WT. In a previous study, enzyme activity staining

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following PAGE confirmed that in the leaves of the two GDHAtransformants only the most anodal isoenzyme (a-subunit) wasvisible, whereas in the leaves of the two GDHB transformants onlythe most cathodal isoenzyme (b-subunit) was visible. In the twoGDHA/B double transformant lines A2b1 and B1a2, the GDHisoenzyme pattern was typical of that found in the leaves ofN. plumbaginifolia, indicating that in Nicotiana tabacum thetwo subunits a- and b from N. plumbaginifolia had assembledinto an active heterohexameric enzyme (Labboun et al. 2009).

Subcellular localization of GDH in transgenicplants overexpressing the enzyme byimmuno-gold transmission electron microscopy

In order to determine the localization of the GDHA andGDHB gene products of N. plumbaginifolia, immuno-gold

transmission electron microscopy experiments were conductedon leaf and stem sections of the GDHA/B transgenic tobaccoplants. In the fully expanded leaf mesophyll cells of GDH over-expressors, gold particles were detected in the mitochondria ofthe palisade parenchyma cells (Fig. 2A, C). Quantification ofthe gold particles confirmed the strong induction of GDH pro-tein in the leaf mesophyll, considering that the backgroundlevel was around 34 particles mm2 (Supplementary TableS1). Interestingly, the amount of gold particles present in themitochondria of the leaf parenchyma cells of the GDH over-expressors was similar to that found in the stem CCs(Supplementary Table S1). Comparable results were observedin transgenic plants overexpressing GDHA or GDHB individually(Supplementary Table S1). There was no significant labelingabove the background level (corresponding to the unspecificlabeling detected with pre-immune serum) in the mitochondriaof leaf parenchyma cells of the WT plants (SupplementaryTable S1), indicating the absence of GDH protein (Fig. 2B,D). When stem sections of the tobacco GDHA/B transgenicplants were treated with the GDH antiserum, gold particleswere only detected in the mitochondria of the phloem CCs(Fig. 2E). Compared with the WT, a 50% increase(Supplementary Table S1) in the amount of GDH proteinwas observed in the phloem CCs of the GDHA/B transgenicplants (Fig. 2F). No labeling above the background level wasobserved when similar leaf sections were treated with pre-immune serum (Fig. 2G). In the roots of the GDH overexpres-sors, no significant increase in the amount of gold particles, thatwere mainly confined to the phloem CCs, was detected (datanot shown). Compared with the leaves, the much smaller in-crease in NAD(H)-GDH aminating activity determined in theroots of the GDHA, GDHB and GDHA/B plants could explainwhy the sensitivity of the immuno-gold labeling technique didnot allow accurate quantification of the number of gold par-ticles. Similar results were obtained when GDH was localizedand quantified in leaf and root sections of GDHA and GDHBtransgenic plants overexpressing the a- and b-subunits indi-vidually (data not shown).

Phenotypic and physiological characterization ofGDH-overexpressing plants

Plants from each homozygous T3 transgenic line (GDHA, GDHBand GDHA/B) and untransformed WT plants were grown for 8weeks on a complete nutrient solution containing optimalamounts of N in the form of 10 mM NO3

/2 mM NH4+.

Plant biomass production expressed on a total DW basis ofboth the roots and shoots of the three types of GDH over-expressors was significantly lower than that of the WT. Thebiomass of the GDHB plants was the most strongly reduced(up to 70%) compared with the GDHA and GDHA/B plants(Fig. 3A). Only small differences were observed when compar-ing the DW/FW ratio of the different lines, thus indicating thatthe water content was not significantly modified in the GDHoverexpressors (Fig. 3B). Similarly, the leaf soluble protein

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Fig. 1 GDH activity in WT and transgenic tobacco plants overexpres-sing two GDH genes from Nicotiana plumbaginifolia. (A) NAD(H)-GDH aminating activity. (B) NAD-GDH deaminating activity. Theenzyme activity was measured in leaf (black columns) and root(white columns) samples of the wild type (WT) and in transgeniclines overexpressing GDHA (two independent lines A1, A2), GDHB(two independent lines B1, B2) and the two genes simultaneouslyGDHA/B (two independent lines A2b1, B1a2). The mean for the sixtransgenic lines is indicated by OE (overexpressors). GDH activity wasmeasured on three individual plants of each line. Values arethe mean SE. Differences between NAD(H)- or NAD-GDH activitybetween the WT and the six transgenic lines were significant at aP-value< 0.05.


Resolving the role of glutamate dehydrogenase


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Fig. 2 Immuno-gold localization of GDH in tobacco plants overexpressing GDH. Fully expanded leaf mesophyll cell of a GDHA/B overexpressor(A). Detail of the same mesophyll cell showing the presence of a strong labeling in the mitochondria (C). Mesophyll cell of WT plants showing theabsence of gold particles in the mitochondria (B, D). Detail of a companion cell (CC) in a leaf midrib of transgenic GDHA/B overexpressing plants(E). CC in a leaf of WT plants (F). Control section of a mesophyll cell of WT plants treated with pre-immune serum (G). (m), mitochondria; (P),plastids. Bar = 1 mm.




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content was not significantly modified in the GDH overexpres-sors (data not shown). Compared with the WT, root biomasswas not significantly modified in the three types of GDH over-expressors (data not shown).

In the leaves of three types of GDH overexpressors, thestarch content was considerably reduced, often to 80% of the total soluble carbohydrates under ourexperimental conditions) was also reduced in the different GDHtransgenic lines, ranging from 55% to 70% of that of the WT.There was little difference in the sucrose content of the threetypes of GDH overexpressors (Fig. 3D). For the other two sol-uble carbohydrates, glucose and fructose, no significant differ-ences were observed between the WT and the GDHoverexpressors (data not shown). Interestingly, the Chl contentwas almost 3-fold higher in the GDH overexpressors, without

any obvious preferential accumulation in the GDHA, GDHB orGDHA/B plants (Fig. 3E). A 2-fold increase in the leaf nitratecontent was observed in the different GDH overexpressors. Thisincrease was slightly lower in the GDHA plants (Fig. 3F). Theleaf ammonium content was much lower compared with thatfound for nitrate (around 20 5 nmol mg1 DW) but was simi-lar in the WT and the three different GDH overexpressors. In theroots of the different GDH overexpressors, the content of thevarious metabolites measured in the leaves was not significantlymodified in the three types of GDHA, GDHB and GDHA/B trans-genic plants in comparison with the WT (SupplementaryTable S2). Only ammonium was significantly increased in thethree types of GDH overexpressors. This increase was muchhigher in the GDHB overexpressors.

In the leaves of the three types of transgenic lines overex-pressing the two GDH subunits individually or simultaneously,a 4-fold increase in soluble asparagine was accompanied on

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Fig. 3 Phenotypic and physiological characterization of WT and transgenic plants overexpressing GDH. Total plant biomass production in theWT and in the GDHA, GDHB and GDHA/B overexpressors. (A). DW/FW ratio (B). Leaf starch content (C). Leaf soluble sugar content (su-crose+ glucose+ fructose) (D). Chl (E) and nitrate (F) contents. White column, WT; pale gray columns, GDHA overexpressors A1, A2; dark graycolumns, GDHB overexpressors B1, B2; and white and gray columns, GDHA/B overexpressors A2b1, B1a2. The black column (OE) represents themean of the six GDH overexpressing lines. Values are the mean SE. Asterisks indicate a t-test with a P-value< 0.05.




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average by a 50% decrease in glutamine (Table 1). Proline wasalso considerably decreased in the transgenic lines, with thesoluble content being as low as 10% of that of the WT, depend-ing on the line examined. The pattern of soluble glutamateaccumulation was different in the GDHA, GDHB and GDHA/Bplants; the concentration was higher in the GDHA transgenics,lower in the GDHA/B transgenics and practically unchanged inthe GDHB plants. A low but significant accumulation of serine(30%) was detected in most of the six overexpressors (Table 1).For the additional soluble amino acids regrouped as others, nosignificant differences were detected in their concentrationin the leaves of the WT when compared with the GDHA,GDHB and GDHA/B plants. In addition, the concentrations ofthe individual amino acids in the phloem sap and rootswere not significantly modified in the different GDH over-expressors in comparison with the WT (SupplementaryTables S3, S4).

Enzyme activity profiling

The activities of a range of enzymes involved in the main stepsof C and N primary assimilation were determined in the leavesof the WT and transgenic lines. The selective nitrate reductase(NR) activity, which corresponds to the actual regulated NRactivity in vivo (Lea et al. 2004), was reduced by 50% in thethree types of GDH overexpressors (Fig. 4A). In contrast, thepotential maximal NR activity was not modified (Fig. 4B). A3-fold increase in the activity of ADP-glucose pyrophosphory-lase (AGPase) was observed in the GDHA, GDHB and GDHA/Boverexpressors when compared with the WT (Fig. 4C). Aslightly greater increase was also observed for NADH-depend-ent GOGAT (Fig. 4D), whereas the activity of the ferredoxin(Fd)-dependent enzyme was not significantly different in com-parison with the WT (see also Fontaine et al. 2006). The activ-ities of the 17 other enzymes measured using the robot-basedplatform (Gibon et al. 2004a) were not significantly different(data not shown). The positive control displaying the NAD(H)-GDH activity measurements (Fig. 4F) confirmed that under the

changed experimental high-throughput conditions, there wasstill at least a 5-fold increase in leaf GDH activity in agreementwith that shown in Fig. 1.

Discussion

A few reports have demonstrated a positive impact ofNADP(H)-GDH overexpression on the growth and physio-logical traits of a range of higher plants (Schmidt and Miller1999, Ameziane et al. 2000, Mungur et al. 2006, Lightfoot et al.2007, Abiko et al. 2010, Egami et al. 2012). In contrast, in thepresent investigation, the biomass production of the overex-pressing tobacco was reduced by 3050%, depending on theGDH subunit composition used for the genetic manipulation.Whether this was due to the use of the genes encoding thehigher plant NAD(H)-dependent enzyme rather than theNADP(H)-dependent enzyme from lower organisms remainsto be determined. In tomato plants overexpressing a tomatoNAD(H)-dependent GDH enzyme, an accumulation of aminoacids was observed in the fruits; however, no detailed informa-tion on plant phenotype was provided (Kisaka et al. 2007).Although there are strong lines of evidence that anNADP(H)-dependent GDH activity does not occur in higherplants (Fontaine et al. 2013), the presence of sufficient amountsof NADP(H) in the mitochondrial matrix (Mller et al. 2001),compatible with the Km of the prokaryotic enzyme from bac-teria (Sakamoto et al. 1975) or green algae (Schmidt and Miller1999, Lawit et al. 2003), indicates that overexpression of anNADP(H)-dependent enzyme could have an impact on plantmetabolism. The fact that the fungal enzyme is able to assimi-late ammonium (Abiko et al. 2010) may explain why expressingan NAD(H)-dependent plant enzyme, which does not assimi-late ammonium, has a different impact on plant metabolismand growth. This was confirmed in tobacco and tomato plantsoverexpressing an NADP(H)-dependent enzyme (Kisaka andKida 2003, Mungur et al. 2005), where an increase in the leafglutamine content was observed. In contrast, we found that in

Table 1 Concentration and proportion of free amino acids in leaves of WT tobacco plants and the three types of GDH overexpressors

WT A1 A2 B1 B2 A2b1 B1a2

Amino acids

Aspartate 28.3 1.4 (17.5) 23.6 3.5 (15) 31.1 2.6 (21.1) 14.2 0.6* (11.4) 14.3 2.3* (14.1) 13.2 1.8* (12.4) 14.9 1.6* (15.5)

Serine 9 0.3 (5.6) 12.1 1.1* (7.9) 12.7 2.9* (8.2) 12.3 1.5* (9.7) 10.5 1.3 (10.4) 11.1 1.7 (10.3) 6.4 0.6 (6.6)

Asparagine 4.5 0.7 (2.9) 13.2 0.9* (8.7) 15.30 7.9* (8.9) 17.8 7.5* (13.3) 12.5 4.1* (11.7) 22.3 1.0* (20.7) 13.1 1.2* (13.7)

Glutamate 26.8 3.2 (16.6) 48.2 11* (29.5) 32.6 6.3* (21.3) 25.2 1.1 (20.5) 19.7 0.9* (20) 15.4 0.5* (14.3) 10.9 1.5* (11.3)

Glutamine 50 5.8 (30.9) 24.1 9* (13.9) 30.3 4.9* (20) 23.9 4* (18.8) 19.7 5.7* (18.6) 21.0 1.2* (19.5) 18.3 0.3* (19.1)

Proline 16 4.2 (9.8) 6.1 3.4* (3.3) 2.7 0.6* (1.8) 2.1 0.1* (1.7) 1.1 0.2* (1.1) 1.5 0.1* (1.4) 4 0.4* (4.1)

Others 27.5 1.3 (16.7) 36.2 13.5 (21.7) 29.9 1.4 (18.7) 29.2 1.8 (24.6) 24.8 1.3 (24.1) 23 1.7 (21.4) 28.2 1.3 (29.4)

Total 162.1 16.1 (100) 163.5 49.1 (100) 154.6 42.5 (100) 124.7 17.7 (100) 115.6 10.4 (100) 107.5 32.1 (100) 115.8 30.7 (100)

Amino acids were separated and quantified in a fully expanded leaf of the wild-type control plants (WT), GDHA (lines A1 and A2), GDHB (lines B1 and B2) andGDHA/B (lines A2b1 and B1a2) overexpressors.The amino acid concentration was expressed in nmol g1 DW. Their relative proportions is indicated in parenthese. Values are the mean SE. For each line, threeindividual plants were analyzed.*Significant difference between the WT and different transgenic lines with a t-test P-value< 0.05.




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tobacco plants overexpressing an NAD(H)-dependent plantenzyme, the leaves accumulated less glutamine (Table 1).Although difficult to interpret, this result suggests that thephysiological impact of GDH overexpression depends on thetype of GDH enzyme used for the genetic manipulation, as wellas the type of reducing equivalent. In one of our previousstudies, we found that in the same NAD(H)-GDH overexpres-sing plants, leaf glutamine decreased even after a relatively shortperiod of 15N labelling (Labboun et al. 2009). It is thus likely thatin the present study, the decrease in the pool of leaf glutamineis not the consequence of a long-term metabolic adjustmentoccurring during plant development. Interestingly, an increasein the leaf asparagine content was observed in all the studiesmentioned above (Table 1). Such findings show that overex-pression of either NAD(H)- or NADP(H)-dependent GDHactivity has an impact on amino acid metabolism and in par-ticular metabolism derived from glutamate and glutamine,which could greatly influence the translocation and use ofamino acids that can act as N transport molecules duringplant growth and development (Lea and Azevedo 2007, Leaet al. 2007).

In the present investigation, we have provided additionalinformation on the cellular and subcellular location of the add-itional GDH protein produced in the leaves of the overexpres-sors. Using immuno-gold localization, we have shown that theoverexpression of the GDH protein mainly occurred in themitochondria of the leaf mesophyll palisade (Fig. 2), a celltype in which the enzyme was not present in WT plants(Terce-Laforgue et al. 2004a). In the mitochondria of the CCs,where the native enzyme is localized, the increase in theamount of protein was small compared with that found inthe mesophyll. This relatively modest increase in GDH proteinoverexpression in the vascular tissue probably explains why wedid not observe any major modifications in the amino acidcontent and proportions of the phloem sap in the GDH trans-genic plants (Supplementary Table S3). This latter finding alsosuggests that the modifications observed in the leaf amino acidcontent had apparently no impact on their translocation fromthe mesophyll cells in which they accumulate. Similarly, theroot amino acid profile was not significantly modified, confirm-ing that as for other metabolites, the 2-fold increase in NAD(H)-GDH activity in roots was not sufficient to induce detectable

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Fig. 4 Enzyme activity profiling of tobacco leaves from WT and transgenic plants overexpressing GDH. Activity of a number of selected enzymesin the WT and in the GDHA, GDHB and GDHA/B overexpressors. (A) Selective NR activity. (B) Maximal NR activity. (C) AGPase, (D) NADH-GOGAT. (E) Fd-GOGAT. (F) NAD(H)-GDH. White column, WT; pale gray columns, GDHA overexpressors A1, A2; dark gray columns, GDHBoverexpressors B1, B2; and white and gray columns, GDHA/B overexpressors A2b1, B1a2. The black column (OE) represents the mean of the sixGDH overexpressing lines. Values are the mean SE. Asterisks indicate a t-test with a P-value< 0.05.




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changes in metabolite accumulation or translocation from thisorgan, except for ammonium (Supplementary Table S2). Thislatter finding suggests that there is a feedback control mech-anism presumably originating from the leaves that may controlspecifically ammonium uptake and translocation from theroots or that perturb ammonium assimilation.

The most striking result was the large decrease in the leafstarch concentration, indicating that the capacity of the plantto store carbohydrates was considerably altered. This alterationof C metabolism was also observed following the detection of asignificant decrease in the leaf soluble carbohydrate content,mainly represented by sucrose (Fig. 3). If we consider that inleaves more photosynthates are diverted into starch when su-crose accumulates (Stitt et al. 1984), it is therefore likely that thedecrease in leaf starch content is the consequence of a reduc-tion in sucrose synthesis, indicated by its lower level of accu-mulation. The possibility that other mechanisms such asautophagy (Wang et al. 2013) may be involved in the regulationof starch degradation can also be considered. The increase inmaximal AGPase activity, one of the key enzyme involved instarch synthesis (Fig. 4), is much more difficult to explain, if weconsider that its regulation is complex and not fully character-ized (Martin and Smith 1995, Li et al. 2012, Sonnewald et al.2013, Thormalen et al. 2013). However, it has been shown thatin Arabidopsis AGPase activity decreases at the beginning of theday when starch starts to accumulate, thus indicating thatthere is not necessarily a direct correlation between the totalactivity of the enzyme and the rate of starch synthesis (Gibonet al. 2004b). Whether it is the modified N status resulting fromthe overexpression of GDH or the decrease in leaf starch con-tent that are the regulatory controls that stimulate the increasein AGPase activity remains to be determined. Nevertheless, thisfinding further indicates that in the GDH overexpressors inves-tigated in this work, the regulation of important steps involvedin the synthesis of C- and N-containing molecules has beenaltered. Interestingly, these steps did not involve the accumu-lation of organic acids, since there were no significant changesin the accumulation of 2-oxoglutarate (data not shown), theproduct of the deaminating activity of GDH. At the same time,nitrate uptake and assimilation also appeared to be modified inthe leaves of the GDH overexpressors, since we observed a de-crease in the proportion of selective NR activity (Fig. 4), fittingwell with the observed accumulation of nitrate. Such results arein agreement with previous reports that showed that the levelsof soluble sugars can affect the level of nitrate and vice versa(Scheible et al. 1997). It is therefore likely that introducing GDHactivity into a cell type in which the enzyme is not normallypresent triggers profound modifications of the cross-talk be-tween the C and N assimilatory pathways by modifying theregulation of the transduction pathways involved (Scheibleet al. 1997, Matt et al. 2002, Nunes-Nesi et al. 2010). One canalso hypothesize that in addition to its metabolic role, GDHmay be another important sensor like TOR, SnRk1 and PII thatcould regulate key steps of C, N and associated energy supply(Nunes-Nesi et al. 2010). For example, the alteration of the

glutamine and glutamate content and to some extent theirratio in the GDH overexpressors may also explain the observedperturbation in central metabolism, since the two amino acidsare important signal molecules involved in the regulation of Cand N metabolism.

The observed perturbations in carbohydrate and amino acidmetabolism in the leaves strengthens the idea that GDH may bean important component of the C and N regulatory network,since it has been demonstrated that the enzyme makes a con-siderable contribution to the control of glutamate homeostasis(Labboun et al. 2009), by providing C skeletons when there is ashortage of carbohydrates (Miyashita and Good 2008, Fontaineet al. 2012). The lower plant biomass production by the GDHoverexpressors (Fig. 3) may thus be explained by the fact thatless nitrate is utilized by the transgenic plants to synthesizeglutamine, or that in the WT the accumulation of glutamineinhibits nitrate uptake (Girin et al. 2010). However, these per-turbations in the N assimilatory pathway had no impact on leafprotein accumulation but were rather the origin of a metabolicadjustment, which reduces the accumulation of both solubleand insoluble carbohydrates as proposed in the regulatoryscheme presented in Fig. 5. The finding that on a DW basisthere is more Chl in the leaves of the transgenic lines indicatesthat in addition to central C and N metabolism, the productionof the photosynthetic machinery was also altered even thoughthe plants had less biomass (Fig. 3). The glutamate level wasincreased only in the GDHA overexpressors, whereas the Chlcontent was increased in all the three types of transformants.Such an increase in the leaf Ch content, at least in the GDHAtransformants, may be directly or indirectly related to the factthat glutamate, the substrate of GDH, is also the precursor ofChl biosynthesis (Czarnecki et al. 2011). The possibility that theaccumulation of asparagine could be the result of a metabolicperturbation induced by GDH overexpression such as the de-crease in its precursor glutamine is also an attractive hypothesis(Fig. 5). Such a perturbation could at the same time induce adecrease in the leaf proline content, as glutamate and glutam-ine may also be used as substrates for its synthesis (Brugie`reet al. 1999, Forde and Lea 2007).

When examining the changes in the amino acid content ofthe various overexpressing plants, the most interesting resultwas the finding that the glutamate content was different in thethree types of GDH overexpressors. Although only the steady-state levels of metabolites were measured and not their fluxesand site of accumulation, this finding could suggest that it is theresult of an aminating activity of the a-subunit previously re-ported by Skopelitis et al. (2006). However, when 15NH4

+ wassupplied to the leaves of GDHA lines A1 and A2, in the presenceof a GS inhibitor, no labeling was detected in the amino groupof glutamate, thus indicating that the a-subunit was not able toaminate 2-oxoglutarate in vivo (Labboun et al. 2009). It isknown that a number of enzymes and metabolic pathwaysare involved in maintaining the concentration of glutamatewithin plant cells (Forde and Lea 2007), and these includeboth forms of GOGAT. However, there was little difference in




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the activity of Fd-GOGAT, and the increase in NADH-GOGATactivity in the leaves observed in the overexpressors was notspecific for the GDHA transformants (Fig. 4).

It would appear that the regulation of glutamate accumula-tion is complex, due to the presence of two different GDHsubunits (a or b) which can assemble into homohexamersand heterohexamers. In line with this hypothesis, the concen-tration of glutamate was increased in the GDHA overexpressors,was not significantly modified in the GDHB overexpressors, butwas significantly reduced in the GDHA/B transgenic plants. Thissuggests that the concentration of glutamate in planta overseveral weeks is differentially controlled and dependent uponthe enzyme subunit composition. The short-term 15N labelingexperiments performed over 18 h using [15N]glutamate did notdemonstrate any difference in the flux of N metabolites be-tween the three types of GDH overexpressors (Labboun et al.2009). This again suggests that there can be both a short-term(15N labeling experiment) and a long-term (present study)metabolic adaptation of the plant to the overexpression ofeach of two GDH subunits or to the heterohexameric enzyme.Whether the different subunits are able to play a specific meta-bolic regulatory role probably depends on their relative abun-dance when the enzyme is in the form of homohexamers orheterohexamers. This suggestion remains to be further investi-gated, using, for example, heterologous protein expression sys-tems. Although less marked, we observed a reduction in theamount of aspartate only in the GDHB and GDHA/B overexpres-sors, which again fits with the hypothesis that a single GDHsubunit or a heteromeric assembly is able specifically to regulatepart of a metabolic pathway involved in N assimilation, or atthe interface between C and N metabolism. Such varied

mechanisms that could regulate amino acid biosynthesis, accu-mulation and further metabolism illustrate the complexity of anumber of metabolic pathways in terms of both topology andstoichiometry (Szecowka et al. 2013). This level of complexitymay be even greater when an enzyme such as GDH is expressedin a different cell type compared with its native environment.As illustrated in Fig. 5, the changes in the glutamate contentalone, or the glutamate to glutamine ratio resulting from theoverexpression of the two GDH subunits individually or simul-taneously, could also influence the regulation of N metabolism(nitrate assimilation, serine, aspartate, asparagine and prolinesynthesis) and thus plant biomass production.

It is therefore important to take into account the occurrenceof the possible regulatory mechanisms shown in Fig. 5, whenNAD(H)-GDH alone, or in combination with other enzymes, isused to manipulate N assimilation for improving N use effi-ciency in plants. This study also highlights the fact that it isimportant to consider the native site of expression of anenzyme before any attempt is made to increase or decreaseits activity in a different cellular compartment or cell type.

Materials and Methods

Plant material and growth

Tobacco (N. tabacum cv. Xanthi XHFD8; INRA, Versailles,France) was grown on coarse (diameter = 12.5 mm) sand(Bellanger-Sopromat) throughout plant development. Fromthe bottom of the seedlings, each emerging leaf was numberedand tagged. From a batch of 6-week-old plants, 12 plants ofuniform development and with seven leaves each were

GLU GLN GLU/GLN PRO ASP ASN SUC STARCH

GDHA ++ - ++ -- 0 ++ - --GDHB 0 - + -- - ++ - --GDHA/B - - 0 -- - ++ - --

Accumulaon of

NO3- Biomass

Fig. 5 Schematic representation summarizing putative regulatory mechanisms occurring in the GDH overexpressors. The black lines with arrowsindicate the effect on plant biomass production. The semi-solid lines with arrows indicate a possible reciprocal regulation. The dotted lines witharrows represent the effect on metabolite accumulation. Positive effect:+ in red and negative effect in blue. In the table, a large accumulationof a metabolite is indicated by (++) in red, a moderate accumulation by (+) in red. A moderate decrease in metabolite content is indicated by () in blue and a strong decrease by ( ) in blue. No change in metabolite accumulation is shown as (0). The blue boxes indicate when theaccumulation of a metabolite (glutamate, glutamate/glutamine ratio and asparagine) is specific to GDHA, GDHB or GDHA/B overexpressors.




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selected. Plants were grown in a controlled environmentgrowth chamber (16 h light, 350400 mmol photons m2 s1,26C; 8 h dark, 18C) and watered with a complete solution(10 mM NO3

and 2 mM NH4+; Coc and Lesaint 1971). Plants

were automatically watered for 1 min (flow rate for each plant:50 ml min1) every 2 h. Three plants were used for each experi-ment. Four weeks later, leaves were numbered 8, 9, 11, 13, 15, 20and 30 (from the bottom to the top) as previously described byTerce-Laforgue et al. (2004b). These plants were separated intoshoots and roots. The entire root system was frozen in liquidnitrogen and immediately reduced to a homogenous powderwhich was stored at 80C and used for all further experi-ments. Leaf 20, corresponding to a fully expanded leaf (photo-synthetic activity of 5.8 mmol m2 s1), was used forphysiological analysis. From this leaf, 1 cm2 sections of leafblade tissue without the main midrib were randomly collectedand pooled in two groups. One was weighed and then lyophi-lized to determine the fresh and dry weights. The other wasweighed, frozen and used to determine the quantity of Chl perFW. The remaining leaf tissue was frozen in liquid N and im-mediately reduced to a homogenous powder which was storedat 80C and used for all further experiments. All the harvest-ing of fresh material was carried out between 13:00 and 17:00 h.

Production of tobacco plants overexpressing GDH

DNA fragments containing the GDHA (NpGDHA) cDNA andthe GDHB (NpGDHB) cDNA from N. plumbaginifolia (Restivo2004) were subcloned in the sense orientation into the binaryvector pBI121 to obtain 35S-NpGDHA and 35S-NpGDHB con-structs using the procedure previously described by Fontaineet al. (2006). Seeds from GDHA and GDHB plants were collectedand the resulting transgenic plants were screened for kanamy-cin resistance. The transgenic plants identified in this gener-ation were classified as T1 plants. Homozygous T3 progeny werethen selected for further biochemical and cytoimmunochem-ical studies. Double transformants were obtained by crossing aGDHA-overexpressing line with a GDHB-overexpressing plantusing each transgenic line either as a female receptor (A2 andB1) or as a male for pollen donor (a2 and b1) to obtain finallytwo independent homozygous double transformants (GDHA/B) called lines A2b1 and B1a2.

Enzyme activity measurements

Proteins were extracted from frozen leaf material stored at80C. All extractions were performed at 4C. Glutamate de-hydrogenase [NAD(H)-GDH] was measured as described byTurano et al. (1996) except that the extraction buffer wasthat used by Terce-Laforgue et al. (2004b). A robot-based plat-form to measure the activity of 21 enzymes that are involved incentral C and N metabolism was carried out using the protocoldescribed by Gibon et al. (2004a). Each sample contained theequivalent of 500 mg FW of leaf disks harvested from a fullyexpanded tobacco leaves (leaf 5 from the bottom) as describedabove. The entire sample was powdered under liquid N and

stored at 80C until it was used for enzyme activity measure-ments. The enzymes analysed were: NAD(H)-GDH, NADP(H)-GDH and NR measured both as the maximal (without Mg2+ inthe reaction mixture) and selective (with excess Mg2+ in thereaction mixture) types of enzyme activity; GS, Fd-GOGAT,aspartate aminotransferase (AspAT), alanine aminotranferase(AlaAT), phosphoenolpyruvate carboxylase (PEPC), fumarase(Fum), shikimate dehydrogenase (SDH), total phosphoglucoseisomerase (PGI), phosphoglucomutase (PGM), NAD(H)-malatedehydrogenase [NAD(H)-MDH], triosephosphate isomerase(TPI), AGPase, pyruvate kinase (PK), sucrose-phosphate syn-thase (SPS), transketolase (TK), NADP(H)-glyceraldehyde-3-phosphate dehydrogenase [NADP(H)-GAPDH] and acidinvertase (AI). NADH-dependent GOGAT was measured asdescribed previously (Labboun et al. 2009).

Metabolite extraction and analyses

Fresh plant material was used for metabolite extraction. Freeammonium was extracted from leaves with 2% (w/v) 5-sulfosalicylic acid (1 ml per 10 mg DW) as described byFerrario-Mery et al. (1998) and the content determined bythe phenol/hypochlorite assay (Berthelot reaction). The freeamino acid composition of leaves, roots and phloem exudateswas determined by ion exchange chromatography (Rochat andBoutin 1989) following pH adjustment to 2.1 with 0.1 N HCl.Sucrose, glucose, fructose and starch were extracted with 1 MHClO4 (1 ml per 510 mg DW of plant material) as described byFerrario-Mery et al. (1998). The soluble sugars (glucose, fructoseand sucrose) were measured enzymatically using a commer-cially available kit assay (Boehringer Mannheim). The starchcontent of the leaves was determined as described byFerrario-Mery et al. (1998).

Phloem sap collection

Phloem exudates were collected from a fully expanded leaf fromplants grown as described above. Phloem exudates were ob-tained using the technique described by King and Zeevaart(1974). The leaves were cut off and petioles were re-cut underwater before rapid immersion in the collection buffer. For eachexperiment, petioles of fully expanded leaves were placed separ-ately in a solution of 10 mM HEPES, 10 mM EDTA (adjusted topH 7.5 with NaOH), in a humid chamber (relative humidity>90%) in the dark. Exudates were collected for 6 h from10:00 h to 16:00 h and stored at 80C. Phloem exudates (inthe EDTA solution) were adjusted to pH 2.1 and centrifugedto remove debris and EDTA which precipitated at the low pH.

Statistics

For measurement of enzyme activities and metabolite analyses,results are presented as mean values for three plants with SEs(SE = SD/n 1, where SD is the standard deviation and n thenumber of replicates). Statistical analyses were performed usingthe Student t-test and principal component analysis (PCA)functions of the XLStat-Pro 7.5 (Addinsoft) software.




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Immuno-gold transmission electron microscopyexperiments

Leaf fragments (23 mm2), midribs or stems were fixed in freshlyprepared 1.5% (w/v) paraformaldehyde in phosphate buffer0.1 M, pH 7.4 for 4 h at 4C. For immuno-gold transmission elec-tron microscopy experiments, material was dehydrated in anethanol series [final concentration 90% (v/v) ethanol] thenembedded in London Resin white resin (Polysciences).Polymerization was carried out in gelatin capsules during 10 hat 54C. For immunotransmission electron microscopy studies,ultra thin sections were mounted on 400 mesh nickel grids andallowed to dry at 37C. Sections were first incubated with 5% (v/v) normal goat serum in T1 buffer [0.05 M TrisHCl buffer con-taining 2.5% (w/v) NaCl, 0.1% (w/v) bovine serum albumin (BSA)and 0.05% (v/v) Tween-20, pH 7.4] for 1 h at room temperatureand then with anti-GDH rabbit serum (Loulakakis andRoubelakis-Angelakis 1990) diluted 70 times in T1 buffer for 6 hat room temperature. Sections were then washed five times withT1 buffer, twice with T2 buffer [0.02 M TrisHCl buffer containing2% (w/v) NaCl, 0.1% (w/v) BSA and 0.05% (v/v) Tween-20, pH 8]and incubated for 2 h at room temperature with 10 nm colloidalgoldgoat anti-rabbit immunoglobulin complex (Sigma) diluted50 times in T2 buffer. After several washes, grids were treated with5% (w/v) uranyl acetate in water and observed with a PhilipsCM12 electron microscopeat 80 kV. Controls were conductedeither by omitting the primary antibody or by substituting itwith normal rabbit pre-immune serum.

Supplementary data

Supplementary data are available at PCP online.

Funding

This work was supported by the Institut National de laRecherche Agronomique (INRA).

Acknowledgments

We gratefully acknowledge Professor Peter. J. Lea for criticalreading of the manuscript and Francois Gosse for technicalassistance. The antibodies to grapevine GDH were a generousgift from Professor Kaliopi Roubelakis-Angelakis.

Disclosures

The authors have no conflicts of interest to declare.

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resolving the role of plant glutamate dehydrogenase

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