Resolving The Role of Plant Glutamate Dehydrogenase

Resolving the Role of Plant Glutamate Dehydrogenase: II.
Physiological Characterization of Plants Overexpressing the

Two Enzyme Subunits Individually or Simultaneously
1
Adaptation des Plantes a` leur Environnement, Unite Mixte de Recherche 1318, Institut Jean-Pierre Bourgin, Institut National de la
Recherche Agronomique (INRA), Centre de Versailles-Grignon, RD 10, 78026 Versailles cedex, France
2
Equipe 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, France
3
Biologie du Fruit et Pathologie, Unite Mixte Recherche 1332, Institut National de la Recherche Agronomique (INRA), Centre de
Bordeaux-Aquitaine, BP81, 71 avenue Edouard Bourlaux, 33882 Villenave dOrnon cedex, France
4
Dipartimento di Bioscienze, Universita` di Parma, Parco Area delle Scienze 11/A, 43100 Parma, Italy
*Corresponding author: E-mail, hirel@versailles.inra.fr; Fax, + 33-130833096.
(Received April 12, 2013; Accepted July 22, 2013)
Keywords: Carbon metabolism Glutamate Glutamate

dehydrogenase Glutamine Subunits Tobacco.
Downloaded from http://pcp.oxfordjournals.org/ by guest on June 4, 2015
Glutamate dehydrogenase (GDH; EC 1.4.1.2) is able to carry

out the deamination of glutamate in higher plants. In order
to obtain a better understanding of the physiological function of GDH in leaves, transgenic tobacco (Nicotiana tabacum L.) plants were constructed that overexpress two genes
from Nicotiana plumbaginifolia (GDHA and GDHB under the
control of the Cauliflower mosiac virus 35S promoter), which
encode the a- and b-subunits of GDH individually or simultaneously. In the transgenic plants, the GDH protein accumulated in the mitochondria of mesophyll cells and in the
mitochondria of the phloem companion cells (CCs), where
the native enzyme is normally expressed. Such a shift in the
cellular location of the GDH enzyme induced major changes
in carbon and nitrogen metabolite accumulation and a reduction in growth. These changes were mainly characterized
by a decrease in the amount of sucrose, starch and glutamine in the leaves, which was accompanied by an increase in
the amount of nitrate and Chl. In addition, there was an
increase in the content of asparagine and a decrease in
proline. Such changes may explain the lower plant biomass
determined in the GDH-overexpressing lines. Overexpressing
the two genes GDHA and GDHB individually or simultaneously induced a differential accumulation of glutamate
and glutamine and a modification of the glutamate to glutamine ratio. The impact of the metabolic changes occurring
in the different types of GDH-overexpressing plants is discussed in relation to the possible physiological function of
each subunit when present in the form of homohexamers or
heterohexamers.
Regular Paper
There`se Terce-Laforgue1, Magali Bedu1, Celine Dargel-Grafin1, Frederic Dubois2, Yves Gibon3,
Francesco M. Restivo4 and Bertrand Hirel1,*
Abbreviations: AGPase, ADP-glucose pyrophosphorylase; C,

carbon; CaMV, Cauliflower mosaic virus; CC, companion
cell; Fd, ferredoxin; GDH, glutamate dehydrogenase;
GOGAT, glutamate synthase; GS, glutamine sythase; N, nitrogen; NMR, nuclear magnetic resonance; NR, nitrate reductase; WT, wild type.
Introduction
Until recently, the physiological role of the enzyme glutamate
dehydrogenase (GDH; EC 1.4.1.2) in higher plants was still a
matter of intensive debate, mainly because the enzyme catalyzes in vitro the reversible amination of 2-oxoglutarate to form
glutamate. The GDH enzyme is therefore theoretically able to
assimilate ammonium nitrogen (N), or release carbon (C) molecules in vivo. Although there was a considerable amount of
evidence that >95% of the ammonia available to higher plants
is assimilated via the glutamine synthase (GS)/glutamate synthase (GOGAT) pathway (Labboun et al. 2009, Hirel et al. 2011,
Lea and Miflin 2011), it has been regularly argued that GDH
could 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 the
a-subunit of GDH is able to assimilate part of the ammonium
released following salinity stress (Skopelitis et al. 2006). Other
groups have argued that GDH operates in the direction of glutamate deamination (Robinson et al. 1992, Fox et al. 1995,
Stewart et al. 1995, Glevarec et al. 2004, Masclaux-Daubresse
et al. 2006, Miyashita and Good 2008). Despite the controversy
as to the physiological function of GDH, it is now clear that the
enzyme 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: journals.permissions@oup.com
Plant Cell Physiol. 54(10): 16351647 (2013) doi:10.1093/pcp/pct108 ! The Author 2013.
1635
T. Terce-Laforgue et al.
1636
construction of plants transformed with nucleotide sequences

encoding the a- and b-subunits of GDH isolated from Chlorella
sorokiniana. These plants exhibited properties such as increased
growth and improved stress tolerance. More recently, overexpression of Escherichia coli gdhA in tobacco and maize grown
under controlled or greenhouse conditions led to an increase in
plant biomass production accompanied by enhanced resistance to water deficit (Mungur et al. 2006, Lightfoot et al.
2007). In field experiments, the yield of the E. coli gdhA transgenic plants was also increased (Lightfoot et al. 2007), indicating
that the enzyme may be an important component controlling
plant productivity, a hypothesis that was previously put forward following a quantitative genetic study (Dubois et al. 2003,
Gallais and Hirel 2004). However, whether changes in C and N
assimilation and partitioning following metabolism through the
new 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-subunits
of Nicotiana plumbaginifolia GDH have been constructed.
Phenotypic analysis of the transgenic plants was carried out
to evaluate the impact of increased GDH activity on the
plant growth and on the main metabolites representative of
leaf and root C and N metabolism. Such an investigation was
also conducted to determine if each of the two enzyme subunits had a specific physiological function when overexpressed
individually or simultaneously.
Results
Overexpression of GDH
To produce tobacco plants containing elevated amounts of the
a- and b-subunits of GDH, two N. plumbaginifolia cDNAs that
encode 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, after
selection and regeneration on kanamycin, homozygous T3
transgenic lines overexpressing the two GDH subunits were
obtained. The overexpressors were selected by measuring
both NAD(H)-dependent aminating and NAD-dependent deaminating GDH activity. For each construct, 35S-NpGDHA and
35S-NpGDHB, two independent transformants were selected
(lines A1, A2 and B1, B2 respectively), which exhibited the highest amount of leaf GDH activity. Two transgenic lines (GDHA/B)
expressing both genes (lines A2b1 and B1a2) were obtained by
reciprocal crossing between the GDHA and GDHB overexpressors 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-GDH
deaminating activity were increased by at least 4-fold and up to 7fold compared with the wild type (WT) control plants (Fig. 1). In
roots, the increase in enzyme activity was much lower in the six
different transgenic lines, being approximately 2-fold higher compared with the WT. In a previous study, enzyme activity staining
because: (i) photorespiratory mutants lacking the plastidic GS

isoenzyme 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 transgenic plants containing up to five times the amount of GDH
enzyme, there was no direct incorporation of ammonia into
glutamate, even when GS, the main enzyme responsible for
ammonia assimilation, was inhibited (Labboun et al. 2009).
Moreover, in GDH-deficient mutants totally lacking root and
leaf GDH activity, which grow normally under most conditions,
it has recently been shown that the main function of the
enzyme is to provide 2-oxoglutarate to the plant when C
becomes limiting (Fontaine et al. 2012).
In parallel to the whole-plant physiological and molecular
studies, it has been shown that the GDH enzyme is mainly, if
not 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 the
mitochondria and induced in the cytosol of CCs, when the ammonium concentration increased above a certain threshold
(Terce-Laforgue et al. 2004a). This finding led these authors to
hypothesize that the enzyme may act as a sensor to evaluate the
metabolic status of the plant with respect to ammonium and
sugar concentrations, including the amount of C- and
N-containing molecules circulating via the phloem stream. In
line with this hypothesis, it has been suggested by a number of
authors that each of the two GDH subunits (a and b), which are
encoded by two distinct nuclear genes, may have a specific biological function (Melo-Oliveira et al. 1996, Pavesi et al. 2000,
Restivo 2004, Purnell et al. 2005). The a and b polypeptides
can be assembled as homohexamers or heterohexamers composed of different ratios of the two, thus leading to the formation
of seven active isoenzymes in roots, stems and leaves (Fontaine
et al. 2006). More recently, the occurrence of a third active GDH
subunit (termed g) has been demonstrated in the roots of
Arabidopsis thaliana. The ability of the g-subunit to assemble
with the a- and b-subunits (Fontaine et al. 2012) has rekindled
an interest in investigating more closely the metabolic or regulatory role of the different GDH subunits. However, the physiological significance of the organ- and metabolic-dependent
variability of the ratio between the three GDH isoenzyme subunits and its regulation is still unclear, and may not solely be
explained in terms of metabolic function (Skopelitis et al. 2006,
Purnell et al. 2007, Fontaine et al. 2013). As an example, an analysis of tobacco transgenic plants with increased and decreased
amounts of the b-subunit of GDH demonstrated that N metabolism in general and ammonia assimilation in particular were
practically unchanged (Purnell et al. 2005).
In addition to trying to decipher the physiological function
of the enzyme using transgenic plants and mutants with altered
GDH activity, there have been a number of attempts to produce transgenic plants with increased enzyme activity in order
to examine the impact of such genetic manipulation on model
and crop plant performance. For example, a US patent was
granted to Schmidt and Miller (1999) that described the
Resolving the role of glutamate dehydrogenase
NAD(H)-GDH activity
(nmol min-1 mg-1 DW)
80
60
40
20
0
A1
A2
B1
B2
A2b1 B1a2
OE
WT
A1
A2
B1
B2
A2b1
A2
1 B1a2
1 2
OE
20
15
10
Fig. 1 GDH activity in WT and transgenic tobacco plants overexpressing two GDH genes from Nicotiana plumbaginifolia. (A) NAD(H)GDH aminating activity. (B) NAD-GDH deaminating activity. The
enzyme activity was measured in leaf (black columns) and root
(white columns) samples of the wild type (WT) and in transgenic
lines overexpressing GDHA (two independent lines A1, A2), GDHB
(two independent lines B1, B2) and the two genes simultaneously
GDHA/B (two independent lines A2b1, B1a2). The mean for the six
transgenic lines is indicated by OE (overexpressors). GDH activity was
measured on three individual plants of each line. Values are
the mean SE. Differences between NAD(H)- or NAD-GDH activity
between the WT and the six transgenic lines were significant at a
P-value < 0.05.
following PAGE confirmed that in the leaves of the two GDHA

transformants only the most anodal isoenzyme (a-subunit) was
visible, whereas in the leaves of the two GDHB transformants only
the most cathodal isoenzyme (b-subunit) was visible. In the two
GDHA/B double transformant lines A2b1 and B1a2, the GDH
isoenzyme pattern was typical of that found in the leaves of
N. plumbaginifolia, indicating that in Nicotiana tabacum the
two subunits a- and b from N. plumbaginifolia had assembled
into an active heterohexameric enzyme (Labboun et al. 2009).
Subcellular localization of GDH in transgenic

plants overexpressing the enzyme by
immuno-gold transmission electron microscopy
In order to determine the localization of the GDHA and
GDHB gene products of N. plumbaginifolia, immuno-gold
NAD-GDH activity
(nmol min-1 mg-1 DW)
WT
transmission electron microscopy experiments were conducted

on leaf and stem sections of the GDHA/B transgenic tobacco
plants. In the fully expanded leaf mesophyll cells of GDH overexpressors, gold particles were detected in the mitochondria of
the palisade parenchyma cells (Fig. 2A, C). Quantification of
the gold particles confirmed the strong induction of GDH protein in the leaf mesophyll, considering that the background
level was around 34 particles mm 2 (Supplementary Table
S1). Interestingly, the amount of gold particles present in the
mitochondria of the leaf parenchyma cells of the GDH overexpressors was similar to that found in the stem CCs
(Supplementary Table S1). Comparable results were observed
in transgenic plants overexpressing GDHA or GDHB individually
(Supplementary Table S1). There was no significant labeling
above the background level (corresponding to the unspecific
labeling detected with pre-immune serum) in the mitochondria
of leaf parenchyma cells of the WT plants (Supplementary
Table S1), indicating the absence of GDH protein (Fig. 2B,
D). When stem sections of the tobacco GDHA/B transgenic
plants were treated with the GDH antiserum, gold particles
were 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 protein
was observed in the phloem CCs of the GDHA/B transgenic
plants (Fig. 2F). No labeling above the background level was
observed when similar leaf sections were treated with preimmune serum (Fig. 2G). In the roots of the GDH overexpressors, no significant increase in the amount of gold particles, that
were mainly confined to the phloem CCs, was detected (data
not shown). Compared with the leaves, the much smaller increase in NAD(H)-GDH aminating activity determined in the
roots of the GDHA, GDHB and GDHA/B plants could explain
why the sensitivity of the immuno-gold labeling technique did
not allow accurate quantification of the number of gold particles. Similar results were obtained when GDH was localized
and quantified in leaf and root sections of GDHA and GDHB
transgenic plants overexpressing the a- and b-subunits individually (data not shown).
Phenotypic and physiological characterization of

GDH-overexpressing plants
Plants from each homozygous T3 transgenic line (GDHA, GDHB
and GDHA/B) and untransformed WT plants were grown for 8
weeks on a complete nutrient solution containing optimal
amounts of N in the form of 10 mM NO3 /2 mM NH4+.
Plant biomass production expressed on a total DW basis of
both the roots and shoots of the three types of GDH overexpressors was significantly lower than that of the WT. The
biomass 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 comparing the DW/FW ratio of the different lines, thus indicating that
the water content was not significantly modified in the GDH
overexpressors (Fig. 3B). Similarly, the leaf soluble protein
1637
A
C
E
P
m
m
m
m
m
m
P
m
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 the
absence 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.
1638
content was not significantly modified in the GDH overexpressors (data not shown). Compared with the WT, root biomass
was not significantly modified in the three types of GDH overexpressors (data not shown).
In the leaves of three types of GDH overexpressors, the
starch content was considerably reduced, often to <30% of
that of the WT, with the reduction being the greatest in the
GDHB overexpressors (Fig. 3C). The leaf sucrose content (which
represents >80% of the total soluble carbohydrates under our
experimental conditions) was also reduced in the different GDH
transgenic lines, ranging from 55% to 70% of that of the WT.
There was little difference in the sucrose content of the three
types of GDH overexpressors (Fig. 3D). For the other two soluble carbohydrates, glucose and fructose, no significant differences were observed between the WT and the GDH
overexpressors (data not shown). Interestingly, the Chl content
was almost 3-fold higher in the GDH overexpressors, without
20
*
*
16
*
12
*
*
8
4
D
800
600
*
*
400
200
50
*
40
30
20
10
0
Soluble sugars ( nmol mg-1DW)
Chlorophyll (g mg-1DW)
DW/FW ( %)
40
*
*
Nitrate (nmol mg-1DW)
Plant biomass (mgDW)

Starch (nmol mg-1DW)
60
any obvious preferential accumulation in the GDHA, GDHB or

GDHA/B plants (Fig. 3E). A 2-fold increase in the leaf nitrate
content was observed in the different GDH overexpressors. This
increase was slightly lower in the GDHA plants (Fig. 3F). The
leaf ammonium content was much lower compared with that
found for nitrate (around 20 5 nmol mg 1 DW) but was similar in the WT and the three different GDH overexpressors. In the
roots of the different GDH overexpressors, the content of the
various metabolites measured in the leaves was not significantly
modified in the three types of GDHA, GDHB and GDHA/B transgenic plants in comparison with the WT (Supplementary
Table S2). Only ammonium was significantly increased in the
three types of GDH overexpressors. This increase was much
higher in the GDHB overexpressors.
In the leaves of the three types of transgenic lines overexpressing the two GDH subunits individually or simultaneously,
a 4-fold increase in soluble asparagine was accompanied on
100
80
60
*
*
40
20
0
2000
*
1500
*
*
1000
*
*
*
500
0
Fig. 3 Phenotypic and physiological characterization of WT and transgenic plants overexpressing GDH. Total plant biomass production in the
WT and in the GDHA, GDHB and GDHA/B overexpressors. (A). DW/FW ratio (B). Leaf starch content (C). Leaf soluble sugar content (sucrose + glucose + fructose) (D). Chl (E) and nitrate (F) contents. White column, WT; pale gray columns, GDHA overexpressors A1, A2; dark gray
columns, GDHB overexpressors B1, B2; and white and gray columns, GDHA/B overexpressors A2b1, B1a2. The black column (OE) represents the
mean of the six GDH overexpressing lines. Values are the mean SE. Asterisks indicate a t-test with a P-value < 0.05.
1639
changed experimental high-throughput conditions, there was

still at least a 5-fold increase in leaf GDH activity in agreement
with that shown in Fig. 1.
average by a 50% decrease in glutamine (Table 1). Proline was

also considerably decreased in the transgenic lines, with the
soluble content being as low as 10% of that of the WT, depending on the line examined. The pattern of soluble glutamate
accumulation was different in the GDHA, GDHB and GDHA/B
plants; the concentration was higher in the GDHA transgenics,
lower in the GDHA/B transgenics and practically unchanged in
the 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, no
significant differences were detected in their concentration
in the leaves of the WT when compared with the GDHA,
GDHB and GDHA/B plants. In addition, the concentrations of
the individual amino acids in the phloem sap and roots
were not significantly modified in the different GDH overexpressors in comparison with the WT (Supplementary
Tables S3, S4).
Discussion
Enzyme activity profiling

The activities of a range of enzymes involved in the main steps
of C and N primary assimilation were determined in the leaves
of the WT and transgenic lines. The selective nitrate reductase
(NR) activity, which corresponds to the actual regulated NR
activity in vivo (Lea et al. 2004), was reduced by 50% in the
three types of GDH overexpressors (Fig. 4A). In contrast, the
potential maximal NR activity was not modified (Fig. 4B). A
3-fold increase in the activity of ADP-glucose pyrophosphorylase (AGPase) was observed in the GDHA, GDHB and GDHA/B
overexpressors when compared with the WT (Fig. 4C). A
slightly greater increase was also observed for NADH-dependent GOGAT (Fig. 4D), whereas the activity of the ferredoxin
(Fd)-dependent enzyme was not significantly different in comparison with the WT (see also Fontaine et al. 2006). The activities of the 17 other enzymes measured using the robot-based
platform (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
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)
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)
1.5 0.1* (1.4)
Serine
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)
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)
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)
Total
4 0.4* (4.1)
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) and
GDHA/B (lines A2b1 and B1a2) overexpressors.
The amino acid concentration was expressed in nmol g 1 DW. Their relative proportions is indicated in parenthese. Values are the mean SE. For each line, three
individual plants were analyzed.
*Significant difference between the WT and different transgenic lines with a t-test P-value < 0.05.
1640
A few reports have demonstrated a positive impact of

NADP(H)-GDH overexpression on the growth and physiological traits of a range of higher plants (Schmidt and Miller
1999, Ameziane et al. 2000, Mungur et al. 2006, Lightfoot et al.
2007, Abiko et al. 2010, Egami et al. 2012). In contrast, in the
present investigation, the biomass production of the overexpressing tobacco was reduced by 3050%, depending on the
GDH subunit composition used for the genetic manipulation.
Whether this was due to the use of the genes encoding the
higher plant NAD(H)-dependent enzyme rather than the
NADP(H)-dependent enzyme from lower organisms remains
to be determined. In tomato plants overexpressing a tomato
NAD(H)-dependent GDH enzyme, an accumulation of amino
acids was observed in the fruits; however, no detailed information on plant phenotype was provided (Kisaka et al. 2007).
Although there are strong lines of evidence that an
NADP(H)-dependent GDH activity does not occur in higher
plants (Fontaine et al. 2013), the presence of sufficient amounts
of NADP(H) in the mitochondrial matrix (Mller et al. 2001),
compatible with the Km of the prokaryotic enzyme from bacteria (Sakamoto et al. 1975) or green algae (Schmidt and Miller
1999, Lawit et al. 2003), indicates that overexpression of an
NADP(H)-dependent enzyme could have an impact on plant
metabolism. The fact that the fungal enzyme is able to assimilate ammonium (Abiko et al. 2010) may explain why expressing
an NAD(H)-dependent plant enzyme, which does not assimilate ammonium, has a different impact on plant metabolism
and growth. This was confirmed in tobacco and tomato plants
overexpressing an NADP(H)-dependent enzyme (Kisaka and
Kida 2003, Mungur et al. 2005), where an increase in the leaf
glutamine content was observed. In contrast, we found that in
B
*
1.0
0.0
AGPase activity
(nmol min-1 mg-1DW)
A2
B1
B2 A2b1B1a2 OE
WT A1
20
*
*
*
15
10
5
0
A2
B1
40
30
20
10
0
A2
B1
B2 A2b1 B1a2 OE
A2
B1
B2 A2b1 B1a2 OE
1.0
*
0.8
0.6
*
*
0.4
02
0.2
0.0
B2 A2b1B1a2 OE
WT A1
WT A1
4
4
3
3
2
2
1
1
0
A2
B1
60
B2 A2b1 B1a2 OE
Fd-GOGAT activity
(nmol min-1 mg-1DW)
WT A1
0.5
WT A1
NADH-GOGAT activity
(nmol min-1 mg-1DW)
1.5
Maximal NR activity
(nmol min-1 mg-1DW)
2.0
*
NAD(H)-GDH activity
(nmol min-1 mg-1DW)
Selective NR activity
(nmol min-1 mg-1DW)
*
*
*
40
20
0
WT A1
A2
B1
B2 A2b1B1a2 OE
Fig. 4 Enzyme activity profiling of tobacco leaves from WT and transgenic plants overexpressing GDH. Activity of a number of selected enzymes
in the WT and in the GDHA, GDHB and GDHA/B overexpressors. (A) Selective NR activity. (B) Maximal NR activity. (C) AGPase, (D) NADHGOGAT. (E) Fd-GOGAT. (F) NAD(H)-GDH. White column, WT; pale gray columns, GDHA overexpressors A1, A2; dark gray columns, GDHB
overexpressors B1, B2; and white and gray columns, GDHA/B overexpressors A2b1, B1a2. The black column (OE) represents the mean of the six
GDH overexpressing lines. Values are the mean SE. Asterisks indicate a t-test with a P-value < 0.05.
tobacco plants overexpressing an NAD(H)-dependent plant

enzyme, the leaves accumulated less glutamine (Table 1).
Although difficult to interpret, this result suggests that the
physiological impact of GDH overexpression depends on the
type of GDH enzyme used for the genetic manipulation, as well
as the type of reducing equivalent. In one of our previous
studies, we found that in the same NAD(H)-GDH overexpressing plants, leaf glutamine decreased even after a relatively short
period of 15N labelling (Labboun et al. 2009). It is thus likely that
in the present study, the decrease in the pool of leaf glutamine
is not the consequence of a long-term metabolic adjustment
occurring during plant development. Interestingly, an increase
in the leaf asparagine content was observed in all the studies
mentioned above (Table 1). Such findings show that overexpression of either NAD(H)- or NADP(H)-dependent GDH
activity has an impact on amino acid metabolism and in particular metabolism derived from glutamate and glutamine,
which could greatly influence the translocation and use of
amino acids that can act as N transport molecules during
plant growth and development (Lea and Azevedo 2007, Lea
et al. 2007).
In the present investigation, we have provided additional

information on the cellular and subcellular location of the additional GDH protein produced in the leaves of the overexpressors. Using immuno-gold localization, we have shown that the
overexpression of the GDH protein mainly occurred in the
mitochondria of the leaf mesophyll palisade (Fig. 2), a cell
type 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 the
amount of protein was small compared with that found in
the mesophyll. This relatively modest increase in GDH protein
overexpression in the vascular tissue probably explains why we
did not observe any major modifications in the amino acid
content and proportions of the phloem sap in the GDH transgenic plants (Supplementary Table S3). This latter finding also
suggests that the modifications observed in the leaf amino acid
content had apparently no impact on their translocation from
the mesophyll cells in which they accumulate. Similarly, the
root amino acid profile was not significantly modified, confirming that as for other metabolites, the 2-fold increase in NAD(H)GDH activity in roots was not sufficient to induce detectable
1641
1642
glutamine and glutamate content and to some extent their

ratio in the GDH overexpressors may also explain the observed
perturbation in central metabolism, since the two amino acids
are important signal molecules involved in the regulation of C
and N metabolism.
The observed perturbations in carbohydrate and amino acid
metabolism in the leaves strengthens the idea that GDH may be
an important component of the C and N regulatory network,
since it has been demonstrated that the enzyme makes a considerable contribution to the control of glutamate homeostasis
(Labboun et al. 2009), by providing C skeletons when there is a
shortage of carbohydrates (Miyashita and Good 2008, Fontaine
et al. 2012). The lower plant biomass production by the GDH
overexpressors (Fig. 3) may thus be explained by the fact that
less nitrate is utilized by the transgenic plants to synthesize
glutamine, or that in the WT the accumulation of glutamine
inhibits nitrate uptake (Girin et al. 2010). However, these perturbations in the N assimilatory pathway had no impact on leaf
protein accumulation but were rather the origin of a metabolic
adjustment, which reduces the accumulation of both soluble
and insoluble carbohydrates as proposed in the regulatory
scheme presented in Fig. 5. The finding that on a DW basis
there is more Chl in the leaves of the transgenic lines indicates
that in addition to central C and N metabolism, the production
of the photosynthetic machinery was also altered even though
the plants had less biomass (Fig. 3). The glutamate level was
increased only in the GDHA overexpressors, whereas the Chl
content was increased in all the three types of transformants.
Such an increase in the leaf Ch content, at least in the GDHA
transformants, may be directly or indirectly related to the fact
that glutamate, the substrate of GDH, is also the precursor of
Chl biosynthesis (Czarnecki et al. 2011). The possibility that the
accumulation of asparagine could be the result of a metabolic
perturbation induced by GDH overexpression such as the decrease in its precursor glutamine is also an attractive hypothesis
(Fig. 5). Such a perturbation could at the same time induce a
decrease in the leaf proline content, as glutamate and glutamine may also be used as substrates for its synthesis (Brugie`re
et al. 1999, Forde and Lea 2007).
When examining the changes in the amino acid content of
the various overexpressing plants, the most interesting result
was the finding that the glutamate content was different in the
three types of GDH overexpressors. Although only the steadystate levels of metabolites were measured and not their fluxes
and site of accumulation, this finding could suggest that it is the
result of an aminating activity of the a-subunit previously reported by Skopelitis et al. (2006). However, when 15NH4+ was
supplied to the leaves of GDHA lines A1 and A2, in the presence
of a GS inhibitor, no labeling was detected in the amino group
of glutamate, thus indicating that the a-subunit was not able to
aminate 2-oxoglutarate in vivo (Labboun et al. 2009). It is
known that a number of enzymes and metabolic pathways
are involved in maintaining the concentration of glutamate
within plant cells (Forde and Lea 2007), and these include
both forms of GOGAT. However, there was little difference in
changes in metabolite accumulation or translocation from this

organ, except for ammonium (Supplementary Table S2). This
latter finding suggests that there is a feedback control mechanism presumably originating from the leaves that may control
specifically ammonium uptake and translocation from the
roots or that perturb ammonium assimilation.
The most striking result was the large decrease in the leaf
starch concentration, indicating that the capacity of the plant
to store carbohydrates was considerably altered. This alteration
of C metabolism was also observed following the detection of a
significant decrease in the leaf soluble carbohydrate content,
mainly represented by sucrose (Fig. 3). If we consider that in
leaves more photosynthates are diverted into starch when sucrose accumulates (Stitt et al. 1984), it is therefore likely that the
decrease in leaf starch content is the consequence of a reduction in sucrose synthesis, indicated by its lower level of accumulation. The possibility that other mechanisms such as
autophagy (Wang et al. 2013) may be involved in the regulation
of starch degradation can also be considered. The increase in
maximal AGPase activity, one of the key enzyme involved in
starch synthesis (Fig. 4), is much more difficult to explain, if we
consider that its regulation is complex and not fully characterized (Martin and Smith 1995, Li et al. 2012, Sonnewald et al.
2013, Thormalen et al. 2013). However, it has been shown that
in Arabidopsis AGPase activity decreases at the beginning of the
day when starch starts to accumulate, thus indicating that
there is not necessarily a direct correlation between the total
activity of the enzyme and the rate of starch synthesis (Gibon
et al. 2004b). Whether it is the modified N status resulting from
the overexpression of GDH or the decrease in leaf starch content that are the regulatory controls that stimulate the increase
in AGPase activity remains to be determined. Nevertheless, this
finding further indicates that in the GDH overexpressors investigated in this work, the regulation of important steps involved
in the synthesis of C- and N-containing molecules has been
altered. Interestingly, these steps did not involve the accumulation of organic acids, since there were no significant changes
in the accumulation of 2-oxoglutarate (data not shown), the
product of the deaminating activity of GDH. At the same time,
nitrate uptake and assimilation also appeared to be modified in
the leaves of the GDH overexpressors, since we observed a decrease in the proportion of selective NR activity (Fig. 4), fitting
well with the observed accumulation of nitrate. Such results are
in agreement with previous reports that showed that the levels
of soluble sugars can affect the level of nitrate and vice versa
(Scheible et al. 1997). It is therefore likely that introducing GDH
activity into a cell type in which the enzyme is not normally
present triggers profound modifications of the cross-talk between the C and N assimilatory pathways by modifying the
regulation of the transduction pathways involved (Scheible
et al. 1997, Matt et al. 2002, Nunes-Nesi et al. 2010). One can
also hypothesize that in addition to its metabolic role, GDH
may be another important sensor like TOR, SnRk1 and PII that
could regulate key steps of C, N and associated energy supply
(Nunes-Nesi et al. 2010). For example, the alteration of the
GLU
GLN
GLU/GLN
PRO
ASP
ASN
SUC
STARCH
GDHA
++
++
--
++
--
GDHB
---
++
GDHA/B
---
++
Accumulaon of
NO3-
Biomass
the activity of Fd-GOGAT, and the increase in NADH-GOGAT

activity in the leaves observed in the overexpressors was not
specific for the GDHA transformants (Fig. 4).
It would appear that the regulation of glutamate accumulation is complex, due to the presence of two different GDH
subunits (a or b) which can assemble into homohexamers
and heterohexamers. In line with this hypothesis, the concentration of glutamate was increased in the GDHA overexpressors,
was not significantly modified in the GDHB overexpressors, but
was significantly reduced in the GDHA/B transgenic plants. This
suggests that the concentration of glutamate in planta over
several weeks is differentially controlled and dependent upon
the enzyme subunit composition. The short-term 15N labeling
experiments performed over 18 h using [15N]glutamate did not
demonstrate any difference in the flux of N metabolites between 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 of
each of two GDH subunits or to the heterohexameric enzyme.
Whether the different subunits are able to play a specific metabolic regulatory role probably depends on their relative abundance when the enzyme is in the form of homohexamers or
heterohexamers. This suggestion remains to be further investigated, using, for example, heterologous protein expression systems. Although less marked, we observed a reduction in the
amount of aspartate only in the GDHB and GDHA/B overexpressors, which again fits with the hypothesis that a single GDH
subunit or a heteromeric assembly is able specifically to regulate
part of a metabolic pathway involved in N assimilation, or at
the interface between C and N metabolism. Such varied
Fig. 5 Schematic representation summarizing putative regulatory mechanisms occurring in the GDH overexpressors. The black lines with arrows
indicate the effect on plant biomass production. The semi-solid lines with arrows indicate a possible reciprocal regulation. The dotted lines with
arrows represent the effect on metabolite accumulation. Positive effect: + in red and negative effect in blue. In the table, a large accumulation
of 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 the
accumulation of a metabolite (glutamate, glutamate/glutamine ratio and asparagine) is specific to GDHA, GDHB or GDHA/B overexpressors.
mechanisms that could regulate amino acid biosynthesis, accumulation and further metabolism illustrate the complexity of a
number of metabolic pathways in terms of both topology and
stoichiometry (Szecowka et al. 2013). This level of complexity
may be even greater when an enzyme such as GDH is expressed
in a different cell type compared with its native environment.
As illustrated in Fig. 5, the changes in the glutamate content
alone, or the glutamate to glutamine ratio resulting from the
overexpression of the two GDH subunits individually or simultaneously, could also influence the regulation of N metabolism
(nitrate assimilation, serine, aspartate, asparagine and proline
synthesis) and thus plant biomass production.
It is therefore important to take into account the occurrence
of the possible regulatory mechanisms shown in Fig. 5, when
NAD(H)-GDH alone, or in combination with other enzymes, is
used to manipulate N assimilation for improving N use efficiency in plants. This study also highlights the fact that it is
important to consider the native site of expression of an
enzyme before any attempt is made to increase or decrease
its 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. From
the bottom of the seedlings, each emerging leaf was numbered
and tagged. From a batch of 6-week-old plants, 12 plants of
uniform development and with seven leaves each were
1643
Production of tobacco plants overexpressing GDH

DNA fragments containing the GDHA (NpGDHA) cDNA and
the GDHB (NpGDHB) cDNA from N. plumbaginifolia (Restivo
2004) were subcloned in the sense orientation into the binary
vector pBI121 to obtain 35S-NpGDHA and 35S-NpGDHB constructs using the procedure previously described by Fontaine
et al. (2006). Seeds from GDHA and GDHB plants were collected
and the resulting transgenic plants were screened for kanamycin resistance. The transgenic plants identified in this generation were classified as T1 plants. Homozygous T3 progeny were
then selected for further biochemical and cytoimmunochemical studies. Double transformants were obtained by crossing a
GDHA-overexpressing line with a GDHB-overexpressing plant
using each transgenic line either as a female receptor (A2 and
B1) or as a male for pollen donor (a2 and b1) to obtain finally
two independent homozygous double transformants (GDHA/
B) called lines A2b1 and B1a2.
Enzyme activity measurements

Proteins were extracted from frozen leaf material stored at
80 C. All extractions were performed at 4 C. Glutamate dehydrogenase [NAD(H)-GDH] was measured as described by
Turano et al. (1996) except that the extraction buffer was
that used by Terce-Laforgue et al. (2004b). A robot-based platform to measure the activity of 21 enzymes that are involved in
central C and N metabolism was carried out using the protocol
described by Gibon et al. (2004a). Each sample contained the
equivalent of 500 mg FW of leaf disks harvested from a fully
expanded tobacco leaves (leaf 5 from the bottom) as described
above. The entire sample was powdered under liquid N and
1644
stored at 80 C until it was used for enzyme activity measurements. The enzymes analysed were: NAD(H)-GDH, NADP(H)GDH and NR measured both as the maximal (without Mg2+ in
the reaction mixture) and selective (with excess Mg2+ in the
reaction mixture) types of enzyme activity; GS, Fd-GOGAT,
aspartate aminotransferase (AspAT), alanine aminotranferase
(AlaAT), phosphoenolpyruvate carboxylase (PEPC), fumarase
(Fum), shikimate dehydrogenase (SDH), total phosphoglucose
isomerase (PGI), phosphoglucomutase (PGM), NAD(H)-malate
dehydrogenase [NAD(H)-MDH], triosephosphate isomerase
(TPI), AGPase, pyruvate kinase (PK), sucrose-phosphate synthase (SPS), transketolase (TK), NADP(H)-glyceraldehyde3-phosphate dehydrogenase [NADP(H)-GAPDH] and acid
invertase (AI). NADH-dependent GOGAT was measured as
described previously (Labboun et al. 2009).
Metabolite extraction and analyses

Fresh plant material was used for metabolite extraction. Free
ammonium was extracted from leaves with 2% (w/v) 5sulfosalicylic acid (1 ml per 10 mg DW) as described by
Ferrario-Mery et al. (1998) and the content determined by
the phenol/hypochlorite assay (Berthelot reaction). The free
amino acid composition of leaves, roots and phloem exudates
was determined by ion exchange chromatography (Rochat and
Boutin 1989) following pH adjustment to 2.1 with 0.1 N HCl.
Sucrose, glucose, fructose and starch were extracted with 1 M
HClO4 (1 ml per 510 mg DW of plant material) as described by
Ferrario-Mery et al. (1998). The soluble sugars (glucose, fructose
and sucrose) were measured enzymatically using a commercially available kit assay (Boehringer Mannheim). The starch
content of the leaves was determined as described by
Ferrario-Mery et al. (1998).
Phloem sap collection

Phloem exudates were collected from a fully expanded leaf from
plants grown as described above. Phloem exudates were obtained using the technique described by King and Zeevaart
(1974). The leaves were cut off and petioles were re-cut under
water before rapid immersion in the collection buffer. For each
experiment, petioles of fully expanded leaves were placed separately in a solution of 10 mM HEPES, 10 mM EDTA (adjusted to
pH 7.5 with NaOH), in a humid chamber (relative humidity
>90%) in the dark. Exudates were collected for 6 h from
10:00 h to 16:00 h and stored at 80 C. Phloem exudates (in
the EDTA solution) were adjusted to pH 2.1 and centrifuged
to 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 the
number of replicates). Statistical analyses were performed using
the Student t-test and principal component analysis (PCA)
functions of the XLStat-Pro 7.5 (Addinsoft) software.
selected. Plants were grown in a controlled environment

growth chamber (16 h light, 350400 mmol photons m 2 s 1,
26 C; 8 h dark, 18 C) 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 min 1) every 2 h. Three plants were used for each experiment. Four weeks later, leaves were numbered 8, 9, 11, 13, 15, 20
and 30 (from the bottom to the top) as previously described by
Terce-Laforgue et al. (2004b). These plants were separated into
shoots and roots. The entire root system was frozen in liquid
nitrogen and immediately reduced to a homogenous powder
which was stored at 80 C and used for all further experiments. Leaf 20, corresponding to a fully expanded leaf (photosynthetic activity of 5.8 mmol m 2 s 1), was used for
physiological analysis. From this leaf, 1 cm2 sections of leaf
blade tissue without the main midrib were randomly collected
and pooled in two groups. One was weighed and then lyophilized to determine the fresh and dry weights. The other was
weighed, frozen and used to determine the quantity of Chl per
FW. The remaining leaf tissue was frozen in liquid N and immediately reduced to a homogenous powder which was stored
at 80 C and used for all further experiments. All the harvesting of fresh material was carried out between 13:00 and 17:00 h.
Immuno-gold transmission electron microscopy

experiments
Supplementary data
Supplementary data are available at PCP online.
Funding
This work was supported by the Institut National de la
Recherche Agronomique (INRA).
Acknowledgments
We gratefully acknowledge Professor Peter. J. Lea for critical
reading of the manuscript and Francois Gosse for technical
assistance. The antibodies to grapevine GDH were a generous
gift 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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Resolving the Role of Plant Glutamate Dehydrogenase: II.

Physiological Characterization of Plants Overexpressing the

Keywords: Carbon metabolism  Glutamate  Glutamate

Downloaded from http://pcp.oxfordjournals.org/ by guest on June 4, 2015

Glutamate dehydrogenase (GDH; EC 1.4.1.2) is able to carry

Abbreviations: AGPase, ADP-glucose pyrophosphorylase; C,

construction of plants transformed with nucleotide sequences

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because: (i) photorespiratory mutants lacking the plastidic GS

Resolving the role of glutamate dehydrogenase

following PAGE confirmed that in the leaves of the two GDHA

Subcellular localization of GDH in transgenic

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transmission electron microscopy experiments were conducted

Phenotypic and physiological characterization of

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Resolving the role of glutamate dehydrogenase

Soluble sugars ( nmol mg-1DW)

Nitrate (nmol mg-1DW)

Plant biomass (mgDW)

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any obvious preferential accumulation in the GDHA, GDHB or

changed experimental high-throughput conditions, there was

average by a 50% decrease in glutamine (Table 1). Proline was

Enzyme activity profiling

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)

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)

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)

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)

30.3 4.9* (20)

19.7 5.7* (18.6)

21.0 1.2* (19.5)

18.3 0.3* (19.1)

1.5 0.1* (1.4)

6.1 3.4* (3.3)

2.7 0.6* (1.8)

2.1 0.1* (1.7)

1.1 0.2* (1.1)

27.5 1.3 (16.7)

36.2 13.5 (21.7)

Keywords: Carbon metabolism Glutamate Glutamate