The Response of Carbon Metabolism and Antioxidant
Defenses of Alfalfa Nodules to Drought Stress and to the
Subsequent Recovery of Plants1,2[W][OA]
Loreto Naya, Ruben Ladrera, Javier Ramos, Esther M. González, Cesar Arrese-Igor,
Frank R. Minchin, and Manuel Becana*
Alfalfa (Medicago sativa) plants were exposed to drought to examine the involvement of carbon metabolism and oxidative stress in
the decline of nitrogenase (N2ase) activity. Exposure of plants to a moderate drought (leaf water potential of 21.3 MPa) had no
effect on sucrose (Suc) synthase (SS) activity, but caused inhibition of N2ase activity (243%), accumulation of succinate (136%)
and Suc (158%), and up-regulation of genes encoding cytosolic CuZn-superoxide dismutase (SOD), plastid FeSOD, cytosolic
glutathione reductase, and bacterial MnSOD and catalases B and C. Intensification of stress (22.1 MPa) decreased N2ase (282%)
and SS (230%) activities and increased malate (140%), succinate (168%), and Suc (1435%). There was also up-regulation
(mRNA) of cytosolic ascorbate peroxidase and down-regulation (mRNA) of SS, homoglutathione synthetase, and bacterial
catalase A. Drought stress did not affect nifH mRNA level or leghemoglobin expression, but decreased MoFe- and Fe-proteins.
Rewatering of plants led to a partial recovery of the activity (75%) and proteins (.64%) of N2ase, a complete recovery of Suc, and a
decrease of malate (248%) relative to control. The increase in O2 diffusion resistance, the decrease in N2ase-linked respiration and
N2ase proteins, the accumulation of respiratory substrates and oxidized lipids and proteins, and the up-regulation of antioxidant
genes reveal that bacteroids have their respiratory activity impaired and that oxidative stress occurs in nodules under drought
conditions prior to any detectable effect on SS or leghemoglobin. We conclude that a limitation in metabolic capacity of bacteroids
and oxidative damage of cellular components are contributing factors to the inhibition of N2ase activity in alfalfa nodules.
Drought is a major factor limiting crop production
and has a particularly negative impact on symbiotic N2
fixation (Sprent, 1972; Zahran, 1999). However, the
causes for the drought-induced inhibition of nitrogenase (N2ase) activity are still uncertain. Studies on
soybean (Glycine max), common bean (Phaseolus vulga-
1
This work was supported by Ministerio de Educación y Ciencia
(MEC)-Fondos Europeos de Desarrollo Regional (grant nos.
AGL2002–02876, AGL2005–01404, and AGL–2005–00274) and by
Gobierno de Aragón (group E33). L.N. and R.L. are the recipients of
predoctoral fellowships (‘‘Formación de Personal Investigador’’
program), and J.R. is the recipient of a postdoctoral contract (‘‘Juan
de la Cierva’’ program) from MEC.
2
Dedicated to Dr. Frank R. Minchin on the occasion of his
retirement.
* Corresponding author; e-mail becana@eead.csic.es; fax 34–976–
716145.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Manuel Becana (becana@eead.csic.es).
[W]
The online version of this article contains Web-only data.
[OA]
Open Access articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.107.099648
1104
ris), and pea (Pisum sativum) have shown that the
inhibitory effects may be mediated by a decrease in
nodule O2 permeability (Durand et al., 1987; Serraj and
Sinclair, 1996; Ramos et al., 1999) and metabolic activity (Dı́az del Castillo and Layzell, 1995). The finding
that the activity of Suc synthase (SS), but not other
carbon and nitrogen metabolism enzymes, rapidly declined in nodules upon imposition of stress provided
strong evidence for a major role of SS in the inhibition
of N2 fixation (González et al., 1995; Gordon et al.,
1997; Ramos et al., 1999). A detailed biochemical analysis of the rug4 mutant of pea, which displays severely
reduced SS activity, further demonstrated an essential
role of SS in symbiosis (Gordon et al., 1999). These and
subsequent results (Gálvez et al., 2005; Marino et al.,
2006) led to the conclusion that SS is a critical regulatory enzyme in nodule carbon metabolism and in the
early response of N2 fixation to drought. The authors
suggested that the inhibition of SS activity restricts the
availability of malate and other dicarboxylic acids for
bacteroid respiration (Fig. 1A), and that this is responsible for the inhibition of N2ase activity (Gordon et al.,
1997; Gálvez et al., 2005).
Another mechanism that could play a role in the
drought-induced inhibition of N2 fixation, but has received much less attention, is oxidative stress. In plant
Plant Physiology, June 2007, Vol. 144, pp. 1104–1114, www.plantphysiol.org Ó 2007 American Society of Plant Biologists
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Departamento de Nutrición Vegetal, Estación Experimental de Aula Dei, Consejo Superior de Investigaciones
Cientı́ficas, 50080 Zaragoza, Spain (L.N., J.R., M.B.); Departamento de Ciencias del Medio Natural,
Universidad Pública de Navarra, Campus de Arrosadı́a, 31006 Pamplona, Spain (R.L., E.M.G., C.A.-I.);
and Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth,
Dyfed SY23 3EB, United Kingdom (F.R.M.)
Carbon Metabolism and Antioxidants in Drought-Stressed Nodules
cells, this occurs when the generation of reactive
oxygen species (ROS) overwhelms the antioxidant
defenses (Fig. 1B). Indeed, drought induces oxidative
stress in pea nodules (Gogorcena et al., 1995), and
other types of abiotic stress also lead to general decreases of antioxidant activities that are associated
with nodule senescence (Swaraj et al., 1994; Gogorcena
et al., 1997; Hernández-Jiménez et al., 2002; Porcel
et al., 2003). In addition, the application of low concentrations of paraquat, a ROS generator, to the rooting medium of pea plants inhibits SS activity of
nodules prior to N2ase activity, suggesting that SS is
an early target of oxidative stress in nodules (Marino
Plant Physiol. Vol. 144, 2007
et al., 2006). However, in all of these studies, the
response of nodule antioxidants was not analyzed at
the molecular level and in most of them N2ase activity
was not monitored, making it difficult to establish a
relationship between the decrease of antioxidant protection and the loss of nodule function.
Alfalfa (Medicago sativa) is a perennial forage legume
of great agronomical interest that produces indeterminate nodules upon infection of roots with Sinorhizobium
meliloti. Previous agronomical, physiological, and biochemical studies strongly suggest that alfalfa is more
drought tolerant than pea (Moran et al., 1994) and ureideproducing grain legumes (Sinclair and Serraj, 1995).
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Figure 1. Simplified representations of some metabolic routes examined in this work. A, Carbon and
nitrogen metabolism in nodules of
an amide-producing legume, such
as alfalfa. Adapted from Arrese-Igor
et al. (1999). B, Production of ROS
and some major antioxidant systems in legume nodules. Adapted
from Matamoros et al. (2003). AAT,
Asp aminotransferase; ASC, ascorbate; ETC, electron transfer chain;
GS, Gln synthetase; OAA, oxaloacetate; ox met, oxidative metabolism; PEP, phosphoenolpyruvate;
PEPC, phosphoenolpyruvate carboxylase; MDH, malate dehydrogenase; TCA, tricarboxylic acid
cycle.
Naya et al.
the antioxidant defenses of nodules are involved in the
drought-induced inhibition of N2ase activity.
RESULTS
Effect of Drought on N2ase and Nodule Respiration
For example, photosynthesis was inhibited by 77% in
pea leaves having a water potential (Cw) of 21.3 MPa
(Moran et al., 1994), but only decreased by 28% in
alfalfa leaves at a Cw of 21.8 MPa (Rubio et al., 2002).
This inhibition was accompanied by consistent decreases in antioxidant activities and soluble protein
in pea leaves, but by either minor or no changes in
alfalfa leaves. On the basis of this relatively high
drought tolerance, we have selected alfalfa as plant
material to test the hypotheses that the SS activity and
1106
Effect of Drought on Organic Acids, Sugars, and
Associated Enzymes
The effects of drought on the major dicarboxylic
acids (succinate, a-ketoglutarate [aKG], and malate)
and sugars (Suc) of nodules were examined (Fig. 3A),
as they are used by the host cells and bacteroids for the
production of the energy and reducing power required
for N2 fixation and other metabolic reactions (Temple
et al., 1998). The application of a moderate drought
caused an accumulation of Suc (158%), no changes
in malate, and a decrease (223%) in aKG, relative to
Plant Physiol. Vol. 144, 2007
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Figure 2. Expression of N2ase in alfalfa plants exposed to drought stress
and following recovery from drought. A, Steady-state mRNA levels of
the nifH gene, encoding the Fe-protein (component 2), were quantified
by qRT-PCR. Values are means 6 SE of six biological replicates, each
corresponding to RNA extracts from different plants. B, Protein levels of
the MoFe-protein (component 1) and Fe-protein (component 2) of
N2ase. Values are means 6 SE of four western blots that were analyzed
densitometrically. C, Apparent N2ase activity of intact plants measured
as H2 evolution with an open flow-through system. Values are means 6 SE
of five or six replicates. For all panels, treatments are designated as C
(control), D1 (moderate drought stress), D2 (severe drought stress), and
R (recovery). For A, means of D1, D2, and R are indicated with an
asterisk when .2 (up-regulation) or ,0.5 (down-regulation). For B and
C, means of D1, D2, and R marked with an asterisk are significantly
different from C, as determined by the Dunnett’s t test (P , 0.05).
The expression of N2ase (mRNA, protein, and activity) was analyzed in nodulated alfalfa plants subjected to drought stress (Fig. 2). The mRNA level of
the nifH gene (encoding the Fe-protein of N2ase) of
S. meliloti was determined by quantitative reverse
transcription (qRT)-PCR, normalized with housekeeping genes, and expressed relative to values of control plants (Fig. 2A). Following the same criteria for
significant gene up-regulation (ratio .2) or downregulation (ratio ,0.5) in the qRT-PCR analysis as those
generally used in cDNA array studies (El Yahyaoui
et al., 2004), our results show that the nifH mRNA level
in alfalfa nodule bacteroids did not appreciably change
under moderate or severe stress but decreased after
the recovery period. Immunoblots revealed a significant decrease in the contents of the two proteins of
N2ase (MoFe-protein and Fe-protein), especially in nodules from severely stressed plants, and a partial (MoFeprotein) or complete (Fe-protein) recovery in nodules
from reirrigated plants (Fig. 2B). The apparent N2ase
activity was measured as H2 evolution in intact alfalfa
plants ‘Aragón’ using an open flow-through system to
minimize plant disturbance (Minchin et al., 1986). In
alfalfa ‘Aragón,’ this activity consistently decreased
upon application of moderate (243%) and severe (282%)
drought stress, and recovered up to 75% of the control
values after rewatering of plants (Fig. 2C).
An open flow-through system was also used to
measure simultaneously H2 and CO2 evolution, and
hence to determine N2ase activity and some related
parameters in control and drought-stressed plants of
alfalfa ‘N4’ (Table I). A moderate drought stress induced a sharp decline (281%) in total N2ase activity,
which was accompanied by less pronounced decreases
in total root respiration (254%) and in N2ase-linked
respiration (266%). The discrepancy between the extent of inhibition of N2ase activity and its associated
respiration can be explained by major increases in the
carbon cost of N2ase (1126%) and in the O2 diffusion
resistance of nodules (1158%).
Carbon Metabolism and Antioxidants in Drought-Stressed Nodules
Table I. Effect of drought stress on parameters related to N2ase activity, respiration, and O2 diffusion
resistance in alfalfa nodules
Treatments are designated as C (control) and D1 (moderate drought stress). Values are means 6 SE of six
replicates, and those marked with an asterisk are significantly different based on the Dunnett’s t test (P ,
0.05). TRR, Total root respiration; GMR, nodulated root growth and maintenance respiration; NLR, N2aselinked respiration; R (2Ar), O2 diffusion resistance in 79% N2 1 21% O2; R (1Ar), O2 diffusion resistance in
79% Ar 1 21% O2.
Parameter
Units
C
D1
D
%
mmol H2 min21 plant21
mmol CO2 min21 plant21
mmol CO2 min21 plant21
mmol CO2 min21 plant21
mol CO2 mol21 H2
s m21, 3 1026
s m21, 3 1026
values of control nodules. Intensification of stress had
no further negative effect on the aKG content of nodules, but led to a consistent accumulation of succinate
(168%), malate (142%), and Suc (1435%). Rewatering
of plants caused the return of succinate and Suc to
control values, whereas it sharply decreased (248%)
the nodule contents of aKG and malate.
Drought also affected the activities of some carbon
and nitrogen metabolism enzymes in nodules (Fig.
3B), albeit the effects were less intense than those
observed for carbon metabolites (Fig. 3A). In fact, the
activities of SS, isocitrate dehydrogenase (ICDH), alkaline invertase (AI), and Glu synthase (GOGAT) in
nodules of moderately stressed plants did not significantly differ from those of control plants. Application
of severe drought stress only led to a minor decrease
(212%) in ICDH activity, but caused a significant decline (230%) in SS, AI, and GOGAT activities. Upon
rewatering of plants, SS activity returned to control
values, ICDH and AI activities remained higher than
1.40
6.82
3.45
3.43
1.96
0.26
0.56
0.27*
3.12*
2.03*
1.15*
4.42*
0.67*
1.04*
281
254
241
266
1126
1158
186
the control (120%), and GOGAT activity did not
recover (Fig. 3B).
Effect of Drought on Antioxidant Enzymes of Nodule
Host Cells
The high specificity and sensitivity afforded by qRTPCR was central for the quantification of mRNAs
encoding various isoforms of antioxidant enzymes,
such as the superoxide dismutases (SODs), from the
nodule host cells (Fig. 4A). Gene-specific primers
(Supplemental Table S1) were designed for RT-PCR
amplification of the cDNAs coding for CuZnSODc,
CuZnSODp, plastidic FeSOD, and mitochondrial
MnSOD. According to the criteria mentioned above
for changes in gene expression to be considered significant, there was up-regulation of CuZnSODc and
FeSOD under moderate drought and of CuZnSODp
during recovery, but no changes in the expression of
MnSOD for any of the treatments (Fig. 4A).
Figure 3. Contents of carbon substrates and associated enzyme activities in nodules of alfalfa plants
exposed to drought stress and following recovery
from drought. A, Contents of dicarboxylic acids and
Suc in nodules. B, Specific activities of ICDH, SS, AI,
and GOGAT in nodules. Plant treatments and statistical analysis are as described in Figure 2. Values are
means 6 SE of six to 10 replicates.
Plant Physiol. Vol. 144, 2007
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N2ase
TRR
GMR
NLR
Carbon cost
R (2Ar)
R (1Ar)
Naya et al.
Figure 4. Steady-state levels of mRNAs encoding antioxidant enzymes (A) and other important proteins (B) in the nodule host cells of
alfalfa plants exposed to drought stress and
following recovery from drought. Plant treatments are designated as in Figure 2. Values are
means 6 SE of four to six biological replicates,
each corresponding to RNA extracts from different plants. Means of D1, D2, and R are indicated with an asterisk when .2 (up-regulation)
or ,0.5 (down-regulation).
Effect of Drought on Antioxidant Enzymes of Bacteroids
Free-living and symbiotic S. meliloti contain two
SOD and three catalase isoforms. The MnSOD (sodA) is
located in the cytosol (Santos et al., 1999), whereas the
CuZnSOD (sodC) is located in the periplasm (Ampe
et al., 2003). Catalase A (katA) is mainly expressed in
the bacteroids, catalase C (katC) in the bacteria within
the infection threads, and catalase B (katB) in both the
bacteria and bacteroids (Jamet et al., 2003). Using specific primers (Supplemental Table S1), we examined
the effect of drought stress on the expression of the five
1108
genes in the bacteroids (Fig. 5). Application of a
moderate water deficit to plants led to a modest, yet
significant, increase in the sodA mRNA level, which
returned to control values under severe stress and consistently decreased in the recovery treatment. In contrast, no changes were observed in sodC expression for
any treatment. Interestingly, the kat genes were differentially expressed with drought stress, although the
changes were modest in many cases (Fig. 5). Whereas
katB and katC were up-regulated in nodules of moderately stressed plants, the mRNA level of katA markedly decreased during drought stress and remained
low upon rewatering of plants. Also, in nodules of
severely stressed plants, the katB gene remained upregulated, but the mRNA level of katC did not differ
significantly from the control.
Effect of Drought on Antioxidant Metabolites
The major redox metabolites of nodules were quantified using enzymatic methods and high-performance
capillary electrophoresis (CE). This enabled us to determine not only the total amounts of ascorbate and
thiol tripeptides, but also the proportions of their oxidized forms (Fig. 6). The content of total ascorbate
(ascorbate 1 dehydroascorbate) in control alfalfa nodules was 5.9 mmol g21 dry weight (0.9 mmol g21 fresh
weight), which is in the range reported for other legume nodules (Dalton et al., 1986; Gogorcena et al.,
1995, 1997). Ascorbate decreased with moderate (230%)
and severe (258%) drought stress and partially recovered (79% of control) upon rewatering of plants,
whereas dehydroascorbate did not significantly vary
with any of the treatments (Fig. 6). Dehydroascorbate
accounted for approximately 19% of total ascorbate in
nodules of control and moderately stressed plants and
increased to 33% during severe stress and recovery,
proportions below the range of 40% to 80% observed
by Groten et al. (2006) and Marino et al. (2007) in pea
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The expression of other antioxidant enzymes (Fig.
4A), as well as of other important proteins (Fig. 4B),
was investigated in nodules. Cytosolic ascorbate peroxidase (APXc) and catalase are involved in the direct
scavenging of H2O2; cytosolic glutathione reductase
(GRc) in the detoxification of H2O2 and maintenance of
glutathione in the reduced form; ferritin in the binding
of free iron in a nontoxic form; and g-glutamylcysteine
synthetase, glutathione synthetase, and homoglutathione synthetase (hGSHS) in the synthesis of thiol
compounds (Matamoros et al., 2003; Puppo et al., 2005).
The only relevant findings were the up-regulation of
GRc under moderate drought and of APXc under severe drought (Fig. 4A), and the down-regulation of
hGSHS and SS under severe drought (Fig. 4B). Also,
the imposition of stress or the subsequent recovery
of plants from drought had no significant effect on
the content of leghemoglobin (Lb), which was approximately 0.12 mg mg21 protein for all treatments, and
on the activities of CuZnSODc, CuZnSODp, MnSOD,
FeSOD, APXc, GR, and catalase (data not shown).
However, the protein level of the CuZnSODc isoform
increased slightly in nodules of plants exposed to a
moderate or severe (125%) drought stress and more
markedly in nodules of plants recovered from drought
(1148%).
Carbon Metabolism and Antioxidants in Drought-Stressed Nodules
nodules of a comparable developmental stage. In this
respect, we should note that, in our hands, the proportion of dehydroascorbate increased up to 50% to
60% if alfalfa or common bean nodules were not
harvested directly into liquid nitrogen but left instead
to stand on ice for less than 1 h (J. Loscos, M.A.
Matamoros, and M. Becana, unpublished data).
Mature alfalfa nodules have been reported to contain approximately two-thirds of GSH (gGlu-Cys-Gly)
and one-third of hGSH (gGlu-Cys-bAla), a structurally related homolog that is present exclusively in
some legume species and tissues (Matamoros et al.,
2003). This was confirmed by our determinations in
control nodules of 4 mmol g21 dry weight (0.7 mmol g21
fresh weight) of GSH and 2.1 mmol g21 dry weight
(0.3 mmol g21 fresh weight) of hGSH (Fig. 6). Moderate
or severe drought stress had no effect on the contents
of GSH or hGSH, whereas, during the recovery of
plants, GSH significantly decreased (230%) but hGSH
remained constant. As a result, the proportion of GSH
declined from 67% in control plants to 52% in droughtrecovered plants, and the proportion of hGSH increased
concomitantly from 33% to 48% (Fig. 6). The oxidized
forms of the two thiol tripeptides remained fairly
constant and at a low level (,4%) during drought and
subsequent recovery.
DISCUSSION
The results of this study with alfalfa reveal that the
decline of N2ase activity with drought does not involve an inhibition of SS activity but a metabolic
limitation of bacteroids. This may be caused by alterations in the O2 availability and respiratory capacity of
bacteroids and by the oxidative damage of nodule cell
components. Two major differences in the response of
Effect of Drought on Lipid Peroxidation and
Protein Oxidation
The accumulation of oxidatively damaged lipids
and proteins is a marker of oxidative stress in plant
and animal tissues. These products arise, among other
mechanisms, by the oxidative attack of lipids and proteins by ROS (Stadtman, 1992; Halliwell and Gutteridge,
1999) and can be conveniently detected and quantified
by HPLC and immunological methods. To determine
if alfalfa nodules were experiencing oxidative stress
under drought conditions, malondialdehyde (MDA),
a product of membrane lipid peroxidation, was quanPlant Physiol. Vol. 144, 2007
Figure 6. Contents of antioxidant metabolites in nodules of alfalfa
plants exposed to drought stress and following recovery from drought.
Plant treatments and statistical analysis are as described in Figure 2.
Values are means 6 SE of five to seven replicates.
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Figure 5. Steady-state levels of mRNAs encoding antioxidant enzymes
in the bacteroids of alfalfa plants exposed to drought stress and
following recovery from drought. Plant treatments are designated as
in Figure 2 and statistical analysis is as described in Figure 4. Values are
means 6 SE of four to six biological replicates, each corresponding to
RNA extracts from different plants.
tified by reaction with thiobarbituric acid (TBA) and
subsequent HPLC analysis of the corresponding colored adduct (Fig. 7A). Moderate and severe drought
stress caused similar consistent increases (60%–73%)
in the nodule content of MDA, which remained at that
high level upon rewatering of plants. The pattern of
oxidized proteins in nodules was also examined in
gels upon derivatization of the protein carbonyl groups
with 2,4-dinitrophenylhydrazine (DNP) using a commercial antibody raised against the hydrazone derivatives (Fig. 7B). Such immunoblots revealed that,
relative to controls, the levels of oxidatively modified
proteins were higher in nodule extracts from droughtstressed plants and similar in nodule extracts from
plants recovered from drought. Taken together, these
results provide conclusive evidence that drought induces oxidative stress in alfalfa nodules, a situation
previously reported for pea nodules (Moran et al.,
1994) and leaves (Iturbe-Ormaetxe et al., 1998).
Naya et al.
carbon metabolism to drought were found with respect to previous results with other legumes. First, in
alfalfa nodules, a moderate drought stress inhibited
N2ase activity by 43% (Fig. 2) but had no effect on SS
mRNA (Fig. 4B) and activity (Fig. 3), whereas concomitant decreases in SS and N2ase activities were
observed in common bean (Ramos et al., 1999), pea
(Gálvez et al., 2005), and soybean (González et al.,
1995). Second, in alfalfa nodules subjected to severe
drought stress, when N2ase was inhibited by 82%, the
concentrations of malate and succinate increased by
40% and 68%, respectively (Fig. 3), whereas they declined progressively with drought in pea nodules
(Gálvez et al., 2005). Consequently, in alfalfa nodules
under stress conditions, SS activity was sufficient to
sustain the production of both organic acids from Suc.
The accumulation of Suc under drought stress was
also reported for other legume nodules and was mainly
attributed to the inhibition of SS activity (González
et al., 1995; Ramos et al., 1999; Gálvez et al., 2005). Our
results, especially with moderately stressed nodules,
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Figure 7. Oxidative damage of lipids and proteins in nodules of alfalfa
plants exposed to drought stress and following recovery from drought.
For both sections, treatments are designated as C (control), D1 (moderate drought stress), D2 (severe drought stress), and R (recovery). A,
Content of MDA in nodules. Values are means 6 SE of four replicates
and are statistically compared as described in Figure 2. B, Immunoblot
analysis of carbonyl derivatives of proteins from nodules. Lanes labeled
C#, D1#, D2#, and R# were loaded with aliquots of the corresponding C,
D1, D2, and R extracts, in which the derivatization step was omitted
(negative controls). All lanes of the 10% SDS gel contain 15 mg of
nodule protein. Approximate molecular masses in kilodaltons are given
on the right and bars mark immunoreactive proteins with an enhanced
signal in D1, D2, or R as compared to C. The immunoblot shown is
representative of four of them, each loaded with nodule extracts from
plants grown in different series.
are in sharp contrast with this explanation and suggest
that the accumulation of Suc in alfalfa nodules is
caused by a still active import of Suc from the shoot,
together with a limitation of Suc consumption in the
nodules due to impairment of respiratory activity. Furthermore, the accumulation of dicarboxylic acids and
Suc with drought stress (Fig. 3) indicates that nodule
metabolism and, in particular, N2ase activity and
respiration are not limited by the provision of reduced
carbon.
The existence of metabolic limitations underpinning
the drought-induced decline of N2ase activity was
initially proposed on the basis that it was only partially
restored by raising external O2 concentration (Dı́az del
Castillo and Layzell, 1995; Serraj and Sinclair, 1996). A
potential limiting factor is Lb degradation (Guerin
et al., 1990; Irigoyen et al., 1992), but there was no effect
of drought on the Lb content of alfalfa nodules, in
agreement with earlier studies on pea (González et al.,
1998) and soybean (Gordon et al., 1999). Another metabolic constraint for nodule activity under drought
stress conditions could be a decrease in the respiratory capacity of the bacteroids (Dı́az del Castillo and
Layzell, 1995). This conclusion is supported by the
accumulation of malate and succinate (Fig. 3), the
main respiratory substrates of bacteroids (Lodwig and
Poole, 2003), and by the reduction of N2ase-linked
respiration (Table I). In accordance with this hypothesis, the increase in resistance to O2 diffusion would be
in response to an increase in O2 concentration in the
infected zone, due to reduced bacteroid respiration.
The decline of organic acids in nodules of droughtrecovered plants would then be explained by a reactivation of both N2ase activity and respiration in the
bacteroids. Although our results cannot discern whether
an alteration of bacteroid respiration is a cause or an
effect of the inhibition of N2ase activity, the molecular
analysis of N2ase expression provides some insight
(Fig. 2). Moderate and severe drought stress did not
significantly affect the mRNA levels of nifH, but did
decrease, to different extents, the contents of MoFeprotein and Fe-protein in bacteroids. These observations suggest that the loss of N2ase activity is, at least
in part, caused by protein degradation, in addition to
other probable factors such as a decrease in ATP and
reducing power. Interestingly, rewatering of plants
decreased the nifH mRNA level but allowed the partial
(MoFe-protein) or total (Fe-protein) recovery of the
N2ase proteins, which may be indicative of an increased translation of the nifH transcript or of a higher
stability of the Fe-protein in the drought-recovered
plants. In any case, N2ase activity was not completely
restored, which suggests that nodule metabolism remains affected even after 2 d of plant rewatering. This
metabolic limitation is evident by the significantly
lower content of MoFe-protein in bacteroids and by
the declines in malate content and GOGAT activity in
nodules of drought-recovered plants (Fig. 3). In fact,
GOGAT, which is a primary enzyme for nitrogen
assimilation in alfalfa nodules (Temple et al., 1998),
Carbon Metabolism and Antioxidants in Drought-Stressed Nodules
Plant Physiol. Vol. 144, 2007
determine if oxidative stress and effects on N2ase
protein content occur prior to, and therefore may be
responsible for, the observed reduction in bacteroid
respiration.
MATERIALS AND METHODS
Biological Material and Plant Treatments
Nodulated plants of alfalfa (Medicago sativa ‘Aragón’ or ‘N4’ 3 Sinorhizobium meliloti 102F78) were grown in 4-L pots (six to eight plants per pot),
containing 2:1 (v/v) perlite:vermiculite, under controlled-environment conditions (16-h photoperiod, 350 mmol m22 s21, 25°C/18°C day/night regime,
70% relative humidity). Plants were watered three times a week, alternatively
with distilled water and with a mineral nutrient solution containing 0.5 mM
NH4NO3 (Gogorcena et al., 1997). Plants of alfalfa ‘Aragón’ were grown for 50
to 55 d and were then separated at random into four groups. One of them
(control) was kept under optimal water conditions and two other groups were
subjected to drought stress by withholding irrigation until the plants reached
a leaf Cw (mean 6 SE) of 21.3 6 0.1 MPa (moderate drought; 5–7 d) and 22.1 6
0.2 MPa (severe drought; 8–10 d). The fourth group of plants was also
subjected to severe drought and then allowed to recover by reirrigation for 2 d
(recovery). Control and recovery plants had a similar leaf Cw of 20.6 6 0.2
MPa. Leaf Cw was measured 1 h after the beginning of the photoperiod in
representative leaves, situated in the upper third of the shoot, with a pressure
bomb (Soil Moisture Equipment). Nodule Cw was measured in the same
plants as leaf Cw using C52 sample chambers coupled to a HR-33T microvoltmeter (Wescor). Values of nodule Cw (mean 6 SE) were 20.8 6 0.1, 21.5 6
0.1, 22.5 6 0.1, and 20.7 6 0.1 MPa for the control, moderate stress, severe
stress, and recovery treatments, respectively. Plants of alfalfa ‘N4’ were
subjected to only a moderate drought stress (Cw of 21.5 6 0.1 MPa) and used
to assess the respiration and carbon cost associated with N2ase activity.
Nodules were harvested into liquid nitrogen and stored at 280°C, except for
samples to be used for dry weight determination, which were dried for 48 h at
80°C.
N2ase Activity and Root Respiration
Total N2ase activity and nodulated root respiration were measured on
intact plants using a flow-through gas system that incorporated H2 detectors
(Witty and Minchin, 1998). Root systems of alfalfa ‘N4’ were sealed in their
growth pots, allowed to stabilize for 18 h in a stream of air enriched with
500 mL CO2 L21, and then exposed to a gas stream of 79% (v/v) argon (Ar) and
21% (v/v) O2. Respiratory CO2 production was measured using an IR gas
analyzer, and N2ase activity was measured as H2 production using an
electrochemical hydrogen sensor (City Technology Ltd.). After steady-state
conditions had been reached following exposure to Ar/O2 (within 65 min), the
O2 concentration in the gas stream was increased over the range 21% to 50%
(8.55–20.45 mmol O2 L21). N2ase-linked respiration was calculated from the
linear relationship between changes in total root respiration and H2 production during the O2 stepping period (Witty et al., 1983).
For alfalfa ‘Aragón,’ the apparent N2ase activity was measured in intact
plants, as indicated above, by measuring H2 evolution with an electrochemical
sensor (Qubit Systems) in an open flow-through gas system under a stream of
79% (v/v) N2 and 21% (v/v) O2 (Witty and Minchin, 1998). The H2 sensor was
calibrated with high-purity gases flowing at the same rate as the sampling
system (500 mL min21).
Enzyme Activities, Soluble Protein, and Lb
All enzymes were extracted from nodules at 0°C to 4°C, and activities were
measured at 30°C within the linear range. The enzymes involved in carbon
and nitrogen metabolism were extracted in an optimized medium (Marino
et al., 2006). Desalted extracts were used to assay SS (EC 2.4.1.13), AI
(EC 3.2.1.26), NADP-dependent ICDH (EC 1.1.1.42), and NADH-dependent
GOGAT (EC 1.4.1.14) by following the reduction of NAD1 (SS, AI), the
reduction of NADP1 (ICDH), or the oxidation of NADH (GOGAT) at 340 nm.
The protocols used for determination of SS and AI (González et al., 1995),
ICDH (Gálvez et al., 2005), and GOGAT (Groat and Vance, 1981) activities
have been described in detail in the corresponding references.
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may be limited by a lower availability of aKG. This
enzyme activity is associated with nodule development and N2 fixation (Groat and Vance, 1981) and
appears to be particularly sensitive to drought stress
(Ramos et al., 1999).
Another potential, and frequently overlooked, metabolic constraint of drought-stressed nodules is the
oxidative damage of nodule components. Our results
reveal that this occurs in alfalfa nodules. The upregulation during drought of a number of genes involved in antioxidant protection, namely, CuZnSODc,
FeSOD, APXc, GRc, sodA, katB, and katC (Figs. 4A and
5), indicates that both the host cells and bacteroids are
probably experiencing oxidative stress. This was confirmed by the accumulation of peroxidized lipids
(estimated as MDA) and oxidatively modified proteins (estimated as carbonyl groups) in nodules during
drought stress and subsequent recovery (Fig. 7). At
least two factors may contribute to ensuing oxidative
stress in alfalfa nodules. First, although the activities
of APX and associated enzymes, as assayed in vitro,
remained fairly constant with drought, a large decrease in the concentrations of ascorbate (Fig. 6) and
probably of NAD(P)H (Gogorcena et al., 1995) may
compromise H2O2 detoxification through the ascorbateGSH cycle in vivo. Second, nonenzymatic formation of
cytotoxic aldehydes and protein carbonyl derivatives
(Fig. 7) requires the generation of highly oxidizing
ROS, such as the hydroxyl radical, which in turn depend on trace amounts of metal ions (Stadtman, 1992;
Halliwell and Gutteridge, 1999). Indeed, catalytic iron
(Gogorcena et al., 1995; Evans et al., 1999) and hydroxyl radical production (Becana and Klucas, 1992)
have been found to increase in senescing nodules.
In summary, we conclude that a decrease in SS
expression (mRNA and activity) is not the cause of
the drought-induced loss of N2ase activity in alfalfa.
Interestingly, a similar response of nodule metabolism to drought was found in Medicago truncatula (R.
Ladrera, E.M. González, and C. Arrese-Igor, unpublished data). This raises the possibility that the decrease in SS activity observed in other legumes is not
directly responsible for the inhibition of N2ase, but
rather that the negative effects of drought on both
activities are concomitant and mediated through another factor, possibly oxidative damage. Alternatively,
it is possible that, depending on legume species, two
models exist concerning the role of carbon metabolism
in the inhibition of N2ase activity by drought. In soybean, pea, and common bean, the inhibition would be
mediated by SS, leading to a decrease in the organic
acid levels of nodules. In alfalfa and M. truncatula, the
inhibition would not involve SS activity and organic
acids would accumulate in nodules. In any case, the
accumulation of respiratory substrates, lipid peroxides, and oxidized proteins and the decrease of N2ase
proteins and N2ase-linked respiration reveal that both
impairment of bacteroid function and oxidative stress
take place in alfalfa nodules before any detectable
effect on SS expression. Further studies are required to
Naya et al.
spectra, with a maximum of A532 and a shoulder at 495 nm (Iturbe-Ormaetxe
et al., 1998).
The oxidative damage of proteins was measured by derivatization of
carbonyl groups with DNP and subsequent separation of proteins on SDS
gels, using the OxyBlot protein oxidation kit (Chemicon) according to the
supplier’s recommendations. The DNP-hydrazone derivatives of proteins
were detected on membranes using rabbit specific anti-DNP as the primary
antibody (1:150 dilution), a peroxidase conjugate of goat anti-rabbit IgG as the
secondary antibody (1:3,000 dilution), and the SuperSignal West Pico chemiluminescence kit (Pierce).
Immunoblot Analysis
Total proteins of nodules were denatured and prepared for electrophoresis
as described by Gordon et al. (1999). Proteins were resolved in 10% (N2ase) or
12.5% (CuZnSODc) SDS gels, and transferred to polyvinylidene difluoride
membranes (Bio Trace PVDF; PALL Life Sciences) using a Mini Trans-Blot
transfer cell (Bio-Rad) following standard protocols. The primary antibodies
against the Klebsiella pneumoniae Kp1 and Kp2 N2ase proteins (1:5,000) and
spinach (Spinacia oleracea) CuZnSODc (1:3,000) were generously provided by
P.W. Ludden (Madison, WI) and S. Kanematsu (Miyazaki, Japan), respectively.
The secondary antibody (1:10,000 for Kp1 and Kp2; 1:20,000 for CuZnSODc)
was goat anti-rabbit IgG peroxidase conjugate (Sigma). Proteins were detected
on blots with the SuperSignal West Pico chemiluminescent substrate (Pierce).
Densitometric semiquantitative analysis was performed with the Quantity
One software (Bio-Rad).
Carbon and Antioxidant Metabolites
Organic acids and sugars were extracted from nodules in 5% (w/v)
trichloroacetic acid, and samples were processed as described by Wilson and
Harris (1966) with minor modifications (Gálvez et al., 2005). Succinate, malate,
and aKG were quantified by ion chromatography in a DX-500 system (Dionex)
by gradient separation with an IonPac AS11 column (Dionex) according to the
method recommended by the supplier. Suc was analyzed by CE in a Coulter
PACE system 5500 (Beckman) coupled to a diode array detector (Marino et al.,
2006).
Ascorbate and dehydroascorbate were measured as described by Bartoli
et al. (2000) with some modifications (Matamoros et al., 2006) using nodules
harvested directly into liquid nitrogen to avoid artifactual oxidation of
ascorbate. Extracts were made with 1 M HClO4, cleared by centrifugation,
neutralized with 1 M K2CO3 to pH 5.6, and centrifuged again. Two aliquots
were made and one of them was treated with 0.4 mM dithioerythritol for
15 min at room temperature. The aliquots were incubated with 0.05 units of
ascorbate oxidase (Sigma), and the decrease in A265 was monitored until stable
and used to calculate ascorbate concentration based on an extinction coefficient of 14.3 mM21 cm21. Ascorbate was quantified by direct analysis of the
aliquots not treated with dithioerythritol, and dehydroascorbate as the difference in ascorbate concentration between the treated and untreated aliquots.
Thiol compounds were extracted from nodules with 2% (w/v) metaphosphoric acid and 1 mM EDTA. The extracts were cleared by centrifugation and
treated with 65 mM dithiothreitol for 15 min at room temperature. The
concentrations of the thiol tripeptides (reduced plus oxidized forms) were
measured by CE (Marino et al., 2006) using GSH (Sigma) and hGSH (Bachem)
for calibration. The proportion of oxidized thiols was determined in extracts
prepared in the same way, prior to dithiothreitol treatment, by an enzymatic
recycling procedure using yeast GR to reduce the disulfide forms and
vinylpyridine as thiol blocking agent (Law et al., 1983).
Oxidative Damage of Lipids and Proteins
The nodule content of MDA, a major product formed from decomposition
of lipid peroxides, was quantified by HPLC essentially as described elsewhere
(Iturbe-Ormaetxe et al., 1998). The (TBA)2-MDA adduct was resolved on a C18
column (4.6 3 250 mm, 5 mm; Baker), eluted at 1 mL min21 with 5 mM
potassium phosphate, pH 7.0, containing 15% acetonitrile and 0.6% tetrahydrofurane, and was detected at 532 nm. Calibration curves were made with
1,1,3,3-tetraethoxypropane (Sigma) as the standard, which is stoichiometrically converted into MDA during the acid-heating step of the TBA reaction.
The purity of the peak corresponding to the (TBA)2-MDA adduct was routinely monitored by scanning between 400 and 600 nm with a photodiodearray detector. The peaks in the samples and standard showed identical
1112
Transcript Levels
Total RNA was isolated from nodules using the RNAqueous kit (Ambion),
treated with DNaseI (Roche) at 37°C for 30 min, and reverse transcribed using
Moloney murine leukemia virus reverse transcriptase (Promega). qRT-PCR
analysis was carried out with the iCycler iQ system (Bio-Rad) using iQ SYBRGreen Supermix reagents (Bio-Rad) and specific primers for genes expressed
in the nodule host cells and bacteroids (Supplemental Table S1). The PCR
program consisted of an initial denaturation and Taq activation step of 5 min at
95°C, followed by 50 cycles of 15 s at 95°C and 1 min at 60°C. A melting curve
analysis was performed after every PCR reaction to confirm the accuracy of
each amplified product. All reactions were set up in duplicate. The mRNA
levels of alfalfa genes were normalized against EF1-a (El Yahyaoui et al., 2004)
and those of S. meliloti genes against smc00324 and smc02641 (Becker et al.,
2004). Values of treated plants were expressed relative to those of control
plants using the 22DDCt method (Livak and Schmittgen, 2001). The absence of
contamination with genomic DNA was tested by qRT-PCR in all RNA
samples, after the DNase treatment but prior to reverse transcription, using
the primers of the housekeeping genes.
Statistical Analysis
Each measurement of N2ase activity and associated parameters was made
with a pool of three to five intact plants. Each biochemical assay was made with
nodules harvested from plants growing in different pots. Data were obtained
from two (N2ase activity, carbon metabolism, and antioxidant enzymes and
metabolites) or four to six (mRNAs of antioxidant enzymes) series of plants that
were grown independently. The total numbers of replicates are stated in each
figure. For each treatment, the measurements from the different series of plants
were pooled for statistical analysis. For each parameter, the mean values of the
drought and recovery treatments were compared with the controls by ANOVA
and Dunnett’s t test.
Supplemental Data
The following material is available in the online version of this article.
Supplemental Table S1. Primers used for qRT-PCR.
ACKNOWLEDGMENTS
We thank Carmen Pérez-Rontomé (Estación Experimental de Aula Dei,
Zaragoza), Arantza Ederra, and Olga Marqués (Universidad Pública de
Plant Physiol. Vol. 144, 2007
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The SODs (EC 1.15.1.1) were extracted in a medium consisting of 50 mM
potassium phosphate, pH 7.8, 0.1 mM EDTA, 1% (w/v) soluble polyvinylpyrrolidone, and 0.1% (v/v) Triton X-100. Total SOD activity was determined by
the ferric cytochrome c method with modifications (Rubio et al., 2002). The
CuZnSODc, CuZnSODp, plastidic FeSOD, and mitochondrial MnSOD isoforms were individualized in native gels and identified by differential
inhibition with KCN or H2O2 (Rubio et al., 2002). APX (EC 1.11.1.11) and
catalase (EC 1.11.1.6) were extracted in a medium containing 50 mM potassium
phosphate, pH 7.0, and 0.5% soluble polyvinylpyrrolidone, and the activities
were assayed, respectively, following the disappearance of ascorbate at 290
nm (Asada, 1984) and the decomposition of H2O2 at 240 nm (Aebi, 1984). GR
(EC 1.6.4.2) was extracted in a medium consisting of 50 mM Tricine, pH 7.8,
0.2 mM EDTA, 10 mM b-mercaptoethanol, and 1% soluble polyvinylpyrrolidone, and was assayed by following the oxidation of NADPH at 340 nm
(Dalton et al., 1986). Because organelle APXs are inactivated in extraction
media lacking ascorbate, whereas the APXc is stable (Dalton et al., 1986;
Amako et al., 1994), the activity assayed in nodule extracts was only due to
APXc. However, the GR activity assayed corresponded to the sum of the
cytosolic and plastidic isoforms.
Soluble protein was quantified by the dye-binding microassay (Bio-Rad)
using bovine serum albumin as the standard. Lb concentration was determined by the pyridine-hemochrome method, using an extinction coefficient
(556 nm minus 539 nm) of 23.4 mM21 cm21 for the difference spectrum between the reduced (1dithionite) and the oxidized (1ferricyanide) hemochromes (Appleby and Bergersen, 1980).
Carbon Metabolism and Antioxidants in Drought-Stressed Nodules
Navarra, Pamplona) for their valuable technical assistance. This work is part
of the Ph.D. theses of L.N. (supervised by J.R. and M.B.) and R.L. (supervised
by E.M.G. and C.A.-I.).
Received March 15, 2007; accepted April 13, 2007; published April 27, 2007.
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