Physiologia Plantarum 115 (4): 531-540 (2002)
2
Effects of water stress on antioxidant enzymes of leaves and
nodules of transgenic alfalfa overexpressing superoxide
dismutases
Maria C. Rubioa, Esther M. Gonzálezb, Frank R. Minchinc, K. Judith Webbc,
Cesar Arrese-Igorb, Javier Ramosa and Manuel Becanaa
aDepartamento
de Nutrición Vegetal, Estación Experimental de Aula Dei, Consejo Superior de
Investigaciones Científicas, Apdo 202, E-50080 Zaragoza, Spain
bDepartamento
de Ciencias del Medio Natural, Universidad Pública de Navarra, E-31006
Pamplona, Spain
cInstitute
of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, SY23 3EB,
United Kingdom
*Corresponding author, e-mail: becana@eead.csic.es
3
The antioxidant composition and relative water stress tolerance of nodulated alfalfa plants
(Medicago sativa L. x Sinorhizobium meliloti 102F78) of the elite genotype N4 and three
derived transgenic lines have been studied in detail. These transgenic lines overproduced,
respectively, Mn-containing superoxide dismutase (SOD) in the mitochondria of leaves and
nodules, MnSOD in the chloroplasts, and FeSOD in the chloroplasts. In general for all line
s, water stress caused moderate decreases in MnSOD and FeSOD activities in both leaves
and nodules, but had distinct tissue-dependent effects on the activities of the peroxidescavenging enzymes. During water stress, with a few exceptions, ascorbate peroxidase and
catalase activities increased moderately in leaves but decreased in nodules. At mild water
stress, transgenic lines showed, on average, 20% higher photosynthetic activity than the
parental line, which suggests a superior tolerance of transgenic plants under these
conditions. However, the untransformed and the transgenic plants performed similarly
during moderate and severe water stress and recovery with respect to important markers
of metabolic activity and of oxidative stress in leaves and nodules. We conclude that the
base genotype used for transformation and the background SOD isozymic composition
may have a profound effect on the relative tolerance of the transgenic lines to abiotic
stress.
Abbreviations - APX, ascorbate peroxidase; Ci, internal CO2 concentration; GPX, guaiacol
peroxidase; MnSOD, FeSOD, CuZnSOD, superoxide dismutases containing Mn, Fe, or Cu plus
Zn as metal cofactors; Ψw, water potential.
4
Introduction
Water stress has profound effects on crop production. Even plants with an optimum water supply
experience transient water shortage periods, where water absorption cannot compensate for
water loss by transpiration (Kramer and Boyer 1997). In addition, many other environmental
stresses, such as cold, salinity and high temperature, have a water stress component. At the mole
cular and cellular levels, drought and other adverse conditions induce oxidative stress in plant
tissues (Thompson et al. 1987, Smirnoff 1993), which can be diagnosed by the accumulation of
lipid peroxides, oxidized proteins, or modified DNA bases (Moran et al. 1994, Halliwell and
Gutteridge 1999).
Superoxide dismutases (SODs) are ubiquituous metalloenzymes that catalyze the dismutati
on of superoxide radical to H2O2 and O2. The superoxide radical is a potential precursor of the
highly oxidizing hydroxyl radical and, therefore, SODs are a critical defense of plants, other
aerobic organisms, and some anaerobes against oxidative stress (Halliwell and Gutteridge 1999).
Three classes of SODs, differing in their metal cofactor, are known in plants. All three SODs
are nuclear-encoded but localized in different subcellular compartments. Typically, CuZnSODs
are in the cytosol and chloroplasts, MnSODs in the mitochondria and peroxisomes, and FeSODs
in the chloroplasts (Bowler et al. 1994, del R o et al. 1998). Transgenic plants overexpressing
SODs in the chloroplasts, mitochondria, and cytosol have been generated (Bowler et al. 1991,
Van Camp et al. 1996). In some cases, transgenic plants showed superior tolerance to oxidative
stress induced by incubation of leaf disks with methylviologen or by exposure of plants to ozone
(Bowler et al. 1991, Sen Gupta et al. 1993). In other cases, but using the same stress inducers,
no beneficial effects were found (Tepperman and Dunsmuir 1990, Pitcher et al. 1991). These
contradictory results were ascribed to differences in the SOD constructs, in the methodology
used to analyze the transformants, and in the growth conditions of the plants (Slooten et al. 1995,
Allen et al. 1997).
The above-mentioned studies were performed mainly with tobacco, but important crop
legumes are now amenable for transformation (Christou 1994). In these plants, SOD and
ascorbate peroxidase (APX) play critical protective roles in nodule activity (Puppo and Rigaud
1986, Dalton et al. 1998). Conceivably then, overexpression of antioxidant enzymes in legumes
5
could provide additional protection to the process of N2 fixation, especially during senescence
and under stress conditions. In a previous study, we have analyzed the SOD composition of
several transgenic lines of alfalfa and characterized three of them at the molecular level (Rubio
et al. 2001). Using the three selected lines (1-10, 4-6 and 10-7) along with the parental line (N4),
we have carried out the present study with two objectives. First, to determine the effects of water
stress on important physiological and biochemical parameters of leaves and nodules. Second, to
find out whether transgenic plants overexpressing SOD isozymes in different subcellular
compartments outperform untransformed plants during water stress. To fulfil these objectives,
we have studied in detail the antioxidant composition and relative water stress tolerance of
nodulated alfalfa plants of the elite genotype N4 and three derived transgenic lines.
Materials and methods
Plant material and propagation
Alfalfa (Medicago sativa L.) lines used in this study were provided by B. McKersie (Research
Triangle Park, NC, USA). Line N4 (WT) is the nontransgenic parental line. Lines 1-10 (MnSOD
mit) and 4-6 (MnSODchl) were transformed to overexpress Nicotiana plumbaginifolia MnSOD
in the mitochondria or in the chloroplasts, respectively. Line 10-7 (FeSODchl) was transformed
to overexpress Arabidopsis thaliana FeSOD in the chloroplasts. All three constructs included the
35S promoter. Details of gene constructs, transformation protocols, screening of transformants,
propagation of clones, and inoculation of plants with Sinorhizobium meliloti strain 102F78 were
described earlier (Rubio et al. 2001). Plants were grown on pots containing a 2/1 (v/v) mixture
of perlite and vermiculite in growth cabinets set at the following conditions: 16-h photoperiod,
PPFD of 300 µmol m-2 s-1, 25/20C (day/night), and 70/80% RH (day/night). Plants were
irrigated three days a week alternatively with distilled water and nutrient solution supplemented
with 0.5 mM NH4NO3 (Gogorcena et al. 1997).
Application of water stress
Approximately 40 days after transferring plants to pots, they were separated at random into five
groups (see Figure legends for details of plant numbers). These were labeled as control (unstress
6
ed), mild water stress (S1), moderate water stress (S2), severe water stress (S3), and recovery
(R). Control plants were kept under optimal water conditions throughout the experiment, wherea
s the other four groups were subjected to water stress by withholding irrigation. Water potential
(Ψw) was measured with a pressure bomb (Soil Moisture Equipment, Santa Barbara, CA, USA)
using representative leaves, situated in the middle of the plant, to monitor the progression of
water deficit. Measurements were made on two to four leaves from different plants within each
treatment.
Plants from the S1, S2, and S3 stress treatments were harvested when leaf Ψw values (±SE
of 12-20 replicates) reached -1.29 ± 0.03 MPa, -1.80 ± 0.02 MPa, and -2.83 ± 0.06 MPa,
respectively. The S3 stage was usually reached after 7 days of withholding water. For the
recovery treatment, plants were allowed to attain the S3 stress stage and were then rewatered for
3 days. Control plants were harvested in between S3 and R plants. Average Ψw values (±SE of 819 replicates) for control and R plants were -0.93 ± 0.08 MPa and -1.15 ± 0.05 MPa,
respectively. Samples of leaves (0.25 g) and nodules (25 mg) to be used for biochemical
analyses were flash-frozen in liquid N2 and stored at -80C.
Physiological parameters
Photosynthesis was measured in the same type of leaves using a LI-6200 portable photosynthesis
system equipped with a LI-6250 CO2 analyzer (Li-COR, Lincoln, NE, USA). Gas-exchange
parameters of leaves, including stomatal conductance, transpiration and internal CO2
concentration (Ci), were measured simultaneously with photosynthesis using the same
equipment. Leaf areas were determined with a portable area meter LI-3000A. All measurements
were made on three to five leaves of different plants from each treatment.
In vivo nitrogenase activity and root respiration were determined as H2 and CO2 evolution,
respectively, from sealed roots in a 79% Ar, 21% O2 gas stream generated using an open flowthrough gas system (Witty and Minchin 1998) with electrochemical H2 sensors (City
Technology, Portsmouth, UK) and an IR gas analyzer (ADC, Hoddesdon, UK). Measurements
were made on intact, undisturbed plants housed in a controlled environment cabinet (Gogorcena
et al. 1997).
7
Assay of total SOD activity
All enzymes were extracted at 0-4¡C and activities were measured spectrophotometrically at 25C
within the linear region for both time and enzyme concentration.
Leaves (0.1 g) were thoroughly ground in a plastic centrifuge tube with 2 ml of a medium
containing 50 mM potassium phosphate buffer (pH 7.8), 0.1 mM EDTA, 1% (w/v) PVP-10, and
0.1% (v/v) Triton X-100. The extract was centrifuged at 13000g for 20 min. Nodules (25 mg)
were homogenized in an Eppendorf tube with 200 ml of the same extraction medium. The
extracts were centrifuged at 13Ê000g for 5 min and the supernatants used to assay SOD activity.
Dialysis of the extracts was not necessary when using the method described in detail below.
Total SOD activity was assayed in a medium consisting of 1 ml of superoxide-generating
solution, 20 ml of 0.5 mM KCN, and 25 ml (leaves) or 5 ml (nodules) of enzyme extract. The
superoxide-generating solution contained 50 mM potassium phosphate buffer (pH 7.8), 0.1 mM
EDTA, 1 mM xanthine, and 1 mM ferric cytochrome c. The reaction was initiated by addition of
sufficient xanthine oxidase (approximately 16 ml of diluted enzyme) to produce an increase in
the absorbance at 550 nm of 0.050 per min. Diluted xanthine oxidase was prepared by adding 20
ml of stock solution (Sigma) to 800 ml of 50 mM potassium phosphate (pH 7.8). This
preparation was kept in ice and discarded every day. A low concentration of KCN (10 mM) was
included in the assay medium of total SOD to inhibit mitochondrial cytochrome c oxidase
without affecting CuZnSOD activity. One unit of SOD activity was defined as the amount of
enzyme required to inhibit the reduction of ferric cytochrome c by 50% (McCord and Fridovich
1969). Boiled extracts showed < 4% residual SOD activity.
Analysis of SOD isozyme composition
SOD isozymes were individualized and identified on 15% polyacrylamide native gels by
incubation with specific inhibitors (3 mM KCN or 5 mM H2O2 for 1 h) and subsequent staining
for SOD activity (Rubio et al. 2001). Activity bands resistant to KCN but inhibitable by H2O2
were assigned to FeSOD isozymes and those resistant to both inhibitors to MnSOD isozymes.
Representative gels were also stained for peroxidase activity to rule out the possibility of
artifactual SOD activity for any of the bands. The same leaf and nodule extracts as those loaded
8
on gels were assayed for KCN-insensitive SOD activity (MnSOD+FeSOD) using the ferric
cytochrome c method and including 3 mM KCN in the assay medium. The individual FeSOD
and MnSOD activities were then calculated by applying the relative proportions calculated by gel
densitometry to the KCN-insensitive SOD activity.
Assay of other antioxidant enzymes
APX, catalase (EC 1.11.1.6), and guaiacol peroxidase (GPX, EC 1.11.1.7) were extracted from
0.1 g leaves or 25 mg nodules with 2 ml or 200 ml, respectively, of optimized media. The extract
ion medium for leaf APX contained 50 mM potassium phosphate buffer (pH 7.8), 1% PVP-10,
5 mM ascorbate, and 0.1% Triton X-100. The extraction media for nodule APX and all the other
enzymes from leaves and nodules had identical composition to the medium for leaf APX except
that 0.1 mM EDTA was included.
APX activity was assayed by following the disappearance of ascorbate at 290 nm (Asada
1984) for 3 min (40-s lag period) using 30 ml (leaves) or 5 ml (nodules) of extract. Additional
controls were run to correct for APX-independent ascorbate oxidation. These controls included
boiling of enzyme extracts, incubation with the inhibitors KCN (1 mM) and p-chloromercuriphe
nylsulfonic acid (0.5 mM; pCMPSA), and omission of extract (nonenzymatic activity) or H2O2
(ascorbate oxidase activity). Nonenzymatic activity was virtually zero, but oxidase activity was
especially high in nodules and APX activities in leaves and nodules were corrected accordingly.
As expected, APX activities were lost either by boiling the extracts or in the presence of the
above inhibitors. pCMPSA inactivates cytosolic and chloroplastic APX but has no effect on
GPX (Amako et al. 1994). Therefore, the inhibition of APX activity by pCMPSA indicated that
ascorbate oxidation by the GPX present in the extracts was negligible and that genuine APX
activity was being measured.
Catalase activity was assayed by following the decomposition of H2O2 at 240 nm (Aebi
1984) for 2 min with 30 ml extract (leaves) or for 1 min with 5 ml extract (nodules). As
expected, boiling of extracts and addition of 1 mM KCN or 5 mM aminotriazole in the assay
medium inhibited catalase activity.
GPX activity was measured by following the oxidation of pyrogallol at 430 nm (Amako et
al. 1994) for 3 min with 10 ml extract (leaves) or for 1 min with 2.5 ml extract (nodules).
9
Extracts were preincubated for 5 min with 0.5 mM pCMPSA in 50 mM potassium phosphate
buffer (pH 7.0). Then, 20 mM pyrogallol and 0.1 mM H2O2 were added to initiate the reaction.
Because APX may also catalyze pyrogallol oxidation to some extent, pCMPSA was included in
the assay medium to inactivate APX and thus ensure the accurate measurement of GPX activity.
Additional controls included boiling of extracts and incubation with inhibitors. The small
residual activity (<15%) found after boiling nodule extracts was used to correct GPX activities of
nodules. GPX activity was completely inhibited by 1 mM KCN.
Oxidative damage of lipids and proteins
The content of lipid peroxides in leaves was measured in terms of 2-thiobarbituric acid reactive
substances (TBARS) exactly as previously described (Iturbe-Ormaetxe et al. 1998), except that
the absorbance of the chromogen in the butanol phase at 532 nm was corrected by the
nonspecific absorption at 600 nm (Dhindsa et al. 1981).
Oxidized proteins were quantified by derivatization of carbonyl groups with 2,4-dinitrophenyl
hydrazine to form the corresponding dinitrophenyl-hydrazones, according to the procedure of
Levine et al. (1990), which was adapted for plant tissue as described by Matamoros et al. (1999).
Statistical analyses
Six series of plants grown independently under identical environment conditions and subjected
to the same water stress treatments were required to obtain sufficient leaf and nodule material.
Thus, each series was considered to be a repetition of the whole experiment. Physiological
parameters of control and stressed plants were measured from at least four repeat series and
values did not significantly differ among series based on analysis of variance. The same occurred
with biochemical parameters, although in this case at least two repeat series of plants were used.
To study the effect of water stress for each line, means were subjected to analysis of variance
and compared with the Duncan's multiple range test. The number of independent samples
(replicates) used for the calculation of the means is stated in each table or figure.
10
Results
Effects of water stress on physiological parameters
Three stress levels were applied to plants by withholding water, giving an average leaf Ψw of
-1.29 MPa (S1), -1.80 MPa (S2), and -2.83 MPa (S3). Plants at the S1 and S2 stages were
visually indistinguishable from control (unstressed) plants (leaf Ψw of -0.93 MPa), whereas
thoseat the S3 stage exhibited some wilting. Rewatering of these plants for three days (R)
caused complete rehydration of plant tissue (leaf Ψw of -1.15 MPa). Measurements of
photosynthesis and gas-exchange parameters during stress and recovery indicated that the three
stress levels were physiologically relevant and that their effects were reversible to different
extents.
Photosynthesis was similar in control plants of all four lines (Fig. 1), as were the dry
weights of leaves, stems, roots, and nodules (data not shown). Mild water stress had no effect on
photosynthesis of transgenic lines but inhibited that of WT by 20%. Moderate and severe water
stress inhibited photosynthesis of all lines by 25% and 50%, respectively. After rewatering of
plants, photosynthetic activities recovered >86% of the control values (Fig. 1). Simultaneous
measurements of gas-exchange parameters indicated that there was significant stomatal closure
already at mild stress (83% decrease in stomatal conductance for WT and 45-70% for
transgenic lines) and that this was further aggravated at moderate stress (72-85% decrease for all
lines) and at severe stress (82-90% decrease for all lines). However, the photosynthesis/Ci ratios
are clearly more relevant than the values of stomatal conductance per se to assess the relative co
ntribution of stomatal vs nonstomatal factors to the stress-induced decline of photosynthesis
(Long and Hallgren 1985). For all lines, the photosynthesis/Ci ratios (in percent) of plants at
mild or moderate stress were similar or slightly superior to those of control plants (Fig. 1),
indicating that the inhibition of photosynthesis at mild and moderate stress can be entirely
accounted for by stomatal closure (Long and Hallgren 1985). In contrast, the photosynthesis/Ci
ratio substantially decreased in severely-stressed plants of all lines except perhaps in FeSODchl
(Fig. 1). This indicates that the additional decrease in stomatal conductance at severe stress
11
relative to moderate stress (<10%) cannot explain the additional decrease (20-36%) of
photosynthesis and hence that nonstomatal factors are also limiting photosynthetic activity.
Rates of N2 fixation were measured on intact WT and MnSODmit plants, since the latter
was the only line expressing the transgene in the nodules. Control (unstressed) plants showed
comparable values for nitrogenase activity (14.2±0.4 and 15.9±1.2 mmol H2 min-1 g-1 dry
weight (mean ± SE of 3 replicates), for WT and MnSODmit, respectively. These values
decreased by 81% and 78%, respectively, at mild water stress and became too low to measure at
moderate and severe water stress. In WT and MnSODmit plants, water stress produced a
decrease in nitrogenase-linked respiration and an increase in the O2 diffusion resistance of
nodules and in the carbon cost of nitrogenase activity. However, there were no significant
differences between the lines in relation to the magnitude of these effects.
Effects of water stress on antioxidant activities and oxidative damage
The total SOD activity of leaves remained relatively unchanged during water stress, except for a
25% decrease in the activity of the MnSODchl and FeSODchl plants at severe stress (Fig. 2).
Rewatering of plants produced a complete recovery of total SOD activity, except for FeSODchl
(80% of control). Separate determinations of MnSOD (KCN/H2O2-insensitive) and FeSOD
(KCN-insensitive) using SOD activity gels in the presence of inhibitors revealed significant
differences. The MnSOD activity of WT decreased by approximately 30% during stress,
whereas that of MnSODchl was unaffected. Upon rewatering, the MnSOD activity of WT
recovered to 75% of control and that of transgenic lines recovered completely. The MnSOD
activity of FeSODchl declined by 28% at mild and moderate stress but then increased to nearly
control values at severe stress and recovery; in contrast, its FeSOD activity increased slightly at
mild stress and decreased to approximately 35% of control at severe stress and following
rewatering. The FeSOD activity of MnSODchl, unlike its MnSOD activity, increased slightly at
mild stress but then decreased at severe stress by 30% relative to the control (Fig. 2).
Water stress had distinct effects on the enzyme activities involved in the removal of H2O2
in leaves. GPX activity did not change, whereas total APX activity (cytosolic plus chloroplastic)
varied moderately and catalase activity was markedly affected (Fig. 3). Total APX activity of
WT remained constant at mild and moderate water stress, whereas the activity of MnSODmit
12
and MnSODchl increased between 50 and 80% in the same conditions and that of FeSODchl
increased between 20 and 40% at moderate and severe stress. Comparisons of APX activities
extracted in the absence and presence of ascorbate enabled us to estimate cytosolic APX activity
(Amako et al. 1994). This activity accounted for 70% of total APX in WT and FeSODchl and
50% in MnSODmit and MnSODchl. Likewise, the increase of total APX activity in the latter
two lines during water stress was found to be mainly due to an increase in cytosolic APX
activity (data not shown). The catalase activity of leaves increased by 2-fold in WT at moderate
stress and by 71% in MnSODmit at mild stress, but in both lines the activity declined below
control levels during intensification of stress and after rewatering of plants (Fig. 3). Catalase
activity of MnSODchl showed a 4-fold increase at mild water stress and then progressively
returned to control values. In contrast, the activity of FeSODchl was unaffected by mild water
stress, increased approximately 2-fold at moderate and severe stress, and declined below control
values after recovery of plants. The protein content in leaves of MnSODchl and FeSODchl
showed decreases of only 16 to 24% at severe stress and upon recovery (Fig. 3).
Preliminary quantification of oxidized lipids and proteins in leaves showed that they
accumulated significantly at severe water stress but not at mild and moderate stress. Likewise,
no detailed measurements of oxidative damage were made in nodules because initial data showe
d no differences in the accumulation of lipid peroxides between WT and MnSODmit (the only
line expressing the transgene in nodules). We therefore measured oxidative damage only in
leaves of control (unstressed) plants and in those subjected to severe stress and recovery (Fig. 4).
Severe water stress caused a general, although modest (23 to 36%), increase in TBARS in the
leaves of all four lines. It also led to a moderate accumulation of oxidized proteins (carbonyl
groups) in leaves of MnSODmit (44%) and MnSODchl (27%), but had no effect on the other
two lines. Upon rewatering of plants, TBARS and total carbonyls returned to control levels in all
four lines (Fig. 4).
Antioxidant enzyme activities were measured in nodules of the same plants. In all four
lines, the total SOD activity of nodules was not affected by mild stress, decreased by 20% at
13
moderate stress and by 30% at severe stress, then remained at this level after recovery of plants
(Fig. 5). There was a progressive decline of MnSOD activity of all lines, with decreases in the
range of <25% at mild stress, 20 to 40% at moderate stress, and 40 to 60% at severe stress.
Recoveries were to >70% of the control values. However, there were only minor changes in the
FeSOD activity of FeSODchl and moderate decreases in that of WT, whereas the decline in
MnSODmit and MnSODchl was in the range of 40 to 60%. Upon rewatering of plants, the
FeSOD activity of MnSODmit and MnSODchl returned to the mild stress levels, but that of WT
and FeSODchl remained 20% lower (Fig. 5).
Water stress also had a differential effect on the activities involved in H2O2 scavenging in
nodules, although the responses were rather similar for all four lines (Fig. 6). The APX activity
of FeSODchl was only slightly (<20% decrease) affected by any stress treatment, whereas that
of the other lines declined by 40% at mild stress and recovered partially or completely to the
control values with progression of water stress and rewatering of plants. The GPX activity of
nodules was slightly affected by mild or moderate stress, and declined by 35 to 45% at severe
stress for all lines. Catalase activity, unlike its leaf counterpart, was inhibited by moderate and
severe water stress, albeit at levels always <30%. This activity remained at values 20% lower
than the controls during rewatering. The soluble protein content of nodules decreased by only 20
to 30% at severe stress and remained at this level in the transgenic lines during recovery, but
returned to control values in WT (Fig. 6).
Discussion
In plants of all four lines subjected to severe water stress (leaf Ψw of -2.8 MPa), photosynthesis
was inhibited by 50% (Fig. 1) and the leaf content of oxidized lipids and proteins increased by
23 to 44% (Fig. 4) relative to control (unstressed) plants. This reflects a general superior
tolerance of alfalfa to water stress with respect to other legumes such as pea, where severe water
stress (reached with a leaf Ψw of -1.9 MPa) caused the virtual suppresion of photosynthesis and
an increase of 45 to 67% in the oxidative damage of the leaves (Iturbe-Ormaetxe et al. 1998).
At mild stress, transgenic lines showed, on average, 20% higher photosynthetic activity
14
than the WT (Fig. 1). This relatively worse photosynthetic performance of WT plants should be
mainly ascribed to a larger decrease in stomatal conductance (83% in WT vs 45-70% in
transgenics), which suggests variations in stomatal density or size. The photosynthesis/Ci ratios
(Fig. 1) indicate that in most, if not all, lines the inhibition of photosynthesis at mild and
moderate stress is due to stomatal closure, whereas at severe stress both stomatal and
nonstomatal factors are involved. One such nonstomatal factor is probably damage of the
photosynthetic machinery, as there is accumulation of oxidized lipids and proteins in the leaves
at severe stress (Fig. 4).
The response of SODs and associated antioxidant enzymes to water stress was compared in
WT and transgenic alfalfa. This was deemed of interest because previous work had showed that
in water-stressed pea leaves there is a correlative increase between cytosolic CuZnSOD and
APX activities (and transcripts), suggesting the coordinated expression of both enzymes (Mittler
and Zilinskas 1994). In alfalfa leaves, however, the cytosolic CuZnSOD activity was low and in
general there was no correlation between H2O2-producing (MnSOD or FeSOD) and H2O2scavenging (APX, catalase, GPX) enzyme activities. The possible exception would be the MnSO
Dchl plants, where MnSOD and APX activities clearly exceeded the control values during water
stress (compare Figs. 2 and 3). The maintenance of higher MnSOD and APX activities during
stress in MnSODchl plants may be related to the fact that a fraction of both enzymes is located
to the chloroplasts, because it did not occur in MnSODmit plants, which bear the same construc
but targeted to the mitochondria.
An important finding of this work was the contrasting behavior of APX and catalase
activity in leaves with respect to nodules during water stress (compare Figs. 3 and 6). In general,
both enzyme activities (especially catalase) increased in leaves but declined in nodules relative
to control values. The increase in APX and catalase activities in leaves is probably a response to
the enhanced production of H2O2 under water stress.This H2O2 excess would be generated by
photorespiration in the peroxisomes rather than by overexpression of SODs because water stress
is known to enhance photorespiratory rates, the increase in catalase (peroxisomal) was
considerably greater than in APX (extra-peroxisomal), and there was no obvious correlation
15
between overexpressed SODs (especially FeSOD) and APX or catalase. Reports of the effects of
water stress on catalase activity have been so far contradictory. While several authors found only
minor changes (reviewed by Smirnoff 1993), others observed similar increases in catalase
activity to those reported here in water-stressed leaves of young pea plants (Mittler and Zilinskas
1994). However, we found a decline of activity in older plants from different pea cultivars
(Moran et al. 1994, Iturbe-Ormaetxe et al. 1998), suggesting that the response of catalase
activity to water stress is age- and cultivar-dependent. In alfalfa nodules, contrary to the leaves,
water stress caused a decrease in the enzyme activities that produce or scavenge H2O2 (Figs. 5
and 6). The decline in APX, and especially in catalase, may be the result of a general but
reversible slowing down of nodule metabolism, as similar decreasing trends were observed for
SODs, GPX, and soluble protein.
We conclude that in general there were no major differences between WT and transgenic
alfalfa for most parameters used in this study as markers of water stress tolerance. Dry weight
production, photosynthetic activity, leaf soluble protein content, and MnSOD, FeSOD and GPX
activities were similar for all four lines at moderate and severe water stress and following
rewatering of plants. Furthermore, transgenic plants suffered from oxidative stress (judged by
the accumulation of damaged lipids and proteins) and recovered from it similarly to the WT
(Fig. 4). These conclusions are important because previous work on transgenic tobacco
overexpressing MnSOD (Slooten et al. 1995) or FeSOD (Van Camp et al. 1996) in the
chloroplasts has shown enhanced tolerance to oxidative stress induced by methyl viologen in
leaf discs. While convenient for many purposes, this assay is unlikely to reflect tolerance of
plants to oxidative stress generated in natural conditions. In fact, plants overproducing FeSOD
were no more tolerant to salt stress (Van Camp et al. 1996).
The oxidative damage to cell components under water stress conditions is mediated by
superoxide radicals (Smirnoff 1993) and probably hydroxyl radicals because there was oxidation
of amino acid residues of proteins to carbonyl groups (Moran et al. 1994; Fig. 4). Transgenic
alfalfa plants of the RA3 genotype overexpressing MnSOD in the chloroplasts showed lower
membrane injury than the WT as judged by electrolyte leakage from leaf discs (McKersie et al.
1996). Our data show similar membrane damage, estimated as peroxidation of membrane lipids,
and similar overall cellular damage, estimated as oxidation of membrane and soluble proteins.
16
Assuming equal reliability of the markers used to assess stress tolerance, several reasons may
explain the different results. (a) The use of different base genotypes. Thus, McKersie et al.
(1996) used the RA3 genotype (poor agronomic interest) for water stress experiments, whereas
we have used the N4 genotype (selected for its field performance), and it is well-known that
important variations exist in the tolerance to abiotic stress among cultivars of most crops,
including legumes (Moran et al. 1994, Iturbe-Ormaetxe et al. 1998). (b) The different nitrogen
source of plants. The plants used by McKersie et al. (1996, 1999) were grown under
nonnodulating conditions and may differ in stress tolerance from the N2-fixing plants used in
this study. This was shown in pea and alfalfa, which are more tolerant to water stress in
symbiosis than when using combined nitrogen (Antolín et al. 1995, Frechilla et al. 2000). (c) The
different background SOD isozyme composition of the leaves. McKersie et al. (1999) found high
CuZnSOD activities and no FeSOD activity, whereas we observed low CuZnSOD activity and
high MnSOD and FeSOD activities. We conclude that all these factors, along with the use of
tissue-specific promoters, may have a major impact on the performance of the derived transgenic
lines and hence should be considered in any attempt to improve crop tolerance to abiotic stress
by genetic engineering.
Acknowledgments - We thank Gloria Rodríguez for help with harvesting of nodules. M.C.R. was
the recipient of a predoctoral fellowship from the Ministry of Education and Culture. This work
was supported by grant 2FD97-1101 from the Comisión Interministerial de Ciencia y
Tecnología and the European Union and by grants PB98-0522 and PB98-0545 from the
Dirección General de Enseñanza Superior e Investigación Científica. The Institute of Grassland
and Environmental Research is grant aided by the Biotechnology and Biological Sciences
Research Council.
References
Aebi H (1984) Catalase in vitro. Methods Enzymol 105: 121-126
Allen RD, Webb RP, Schake SA (1997) Use of transgenic plants to study antioxidant defenses.
Free Rad Biol Med 23: 473-479
17
Amako K, Chen GX, Asada K (1994) Separate assays specific for ascorbate peroxidase and
guaiacol peroxidase and for the chloroplastic and cytosolic isozymes of ascorbate peroxidase
in plants. Plant Cell Physiol 35: 497-504
Antolín MC, Yoller J, Sánchez-Díaz M (1995) Effects of temporary drought on nitrate-fed and
nitrogen-fixing alfalfa plants. Plant Sci 107: 159-165
Asada K (1984) Chloroplasts: formation of active oxygen and its scavenging. Methods Enzymol
105: 422-429
Bowler C, Slooten L, Vandenbranden S, De Rycke R, Botterman J, Sybesma C, Van Montagu
M, Inzé D (1991) Manganese superoxide dismutase can reduce cellular damage mediated by
oxygen radicals in transgenic plants. EMBO J 10: 1723-1732
Bowler C, Van Camp W, Van Montagu M, Inzé D (1994) Superoxide dismutase in plants. Crit
Rev Plant Sci 13: 199-218
Christou P (1994) The biotechnology of crop legumes. Euphytica 74: 165-185
Dalton DA, Joyner SL, Becana M, Iturbe-Ormaetxe I, Chatfield JM (1998) Antioxidant defenses
in the peripheral cell layers of legume root nodules. Plant Physiol 116: 37-43
del Río LA, Pastori GM, Palma JM, Sandalio LM, Sevilla F, Corpas FJ, Jiménez A, LópezHuertas E, Hernández JA (1998) The activated oxygen role of peroxisomes in senescence.
Plant Physiol 116: 1195-1200
Dhindsa RS, Plumb-Dhindsa P, Thorpe TA (1981) Leaf senescence: correlated with increased
levels of membrane permeability and lipid peroxidation, and decreased levels of superoxide
dismutase and catalase. J Exp Bot 32: 93-101
Frechilla S, González EM, Royuela M, Minchin FR, Aparicio-Tejo PM, Arrese-Igor C (2000)
Source of nitrogen nutrition (nitrogen fixation or nitrate assimilation) is a major factor
involved in pea response to moderate water stress. J Plant Physiol 157: 609-617
Gogorcena Y, Gordon AJ, Escuredo PR, Minchin FR, Witty JF, Moran JF, Becana M (1997) N2
fixation, carbon metabolism, and oxidative damage in nodules of dark-stressed common bean
plants. Plant Physiol 113: 1193-1201
Halliwell B, Gutteridge JMC (1999) Free Radicals in Biology and Medicine. 3rd Ed. Oxford
University Press, New York, 936 pp
18
Iturbe-Ormaetxe I, Escuredo PR, Arrese-Igor C, Becana M (1998) Oxidative damage in pea
plants exposed to water deficit or paraquat. Plant Physiol 116: 173-181
Kramer PJ, Boyer JS (1997) Water Relations of Plants and Soils. Academic Press, San Diego,
495 pp
Levine RL, Garland D, Oliver CN, Amici A, Climent I, Lenz A, Ahn B, Shaltiel S, Stadtman ER
(1990) Determination of carbonyl content in oxidatively modified proteins. Methods
Enzymol 186: 464-478
Long SP, Hallgren JE (1985) Measurement of CO2 assimilation by plants in the field and the
laboratory. In: Techniques in Bioproductivity and Photosynthesis, 2nd ed (Coombs J, Hall D
O, Long SP, Scurlock JMO, eds). Pergamon Press, Oxford, pp 62-94
Matamoros MA, Baird LM, Escuredo PR, Dalton DA, Minchin FR, Iturbe-Ormaetxe I, Rubio M
C, Moran JF, Gordon AJ, Becana M (1999) Stress-induced legume root nodule senescence.
Physiological, biochemical, and structural alterations. Plant Physiol 121: 97-111
McCord JM, Fridovich I (1969) Superoxide dismutase. An enzymic function for erythrocuprein
(hemocuprein). J Biol Chem 244: 6049-6055
McKersie BD, Bowley SR, Harjanto E, Leprince O (1996) Water-deficit tolerance and field
performance of transgenic alfalfa overexpressing superoxide dismutase. Plant Physiol 111:
1177-1181
McKersie BD, Bowley SR, Jones KS (1999) Winter survival of transgenic alfalfa
overexpressing superoxide dismutase. Plant Physiol 119: 839-847
Mittler R, Zilinskas BA (1994) Regulation of pea cytosolic ascorbate peroxidase and other
antioxidant enzymes during the progression of drought stress and following recovery from
drought. Plant J 5: 397-405
Moran JF, Becana M, Iturbe-Ormaetxe I, Frechilla S, Klucas RV, Aparicio-Tejo P (1994)
Drought induces oxidative stress in pea plants. Planta 194: 346-352
Pitcher LH, Brennan E, Hurley A, Dunsmuir P, Tepperman JM, Zilinskas BA (1991)
Overproduction of petunia copper/zinc superoxide dismutase does not confer ozone tolerance
in transgenic tobacco. Plant Physiol 97: 452-455
Puppo A, Rigaud J (1986) Superoxide dismutase: an essential role in the protection of the
nitrogen fixation process? FEBS Lett 201: 187-189
19
Rubio MC, Ramos J, Webb KJ, Minchin FR, Gonz lez EM, Arrese-Igor C, Becana M (2001)
Expression studies of superoxide dismutases in nodules and leaves of transgenic alfalfa
reveal abundance of iron-containing isozymes, posttranslational regulation, and
compensation of isozyme activities. Mol Plant Microb Interact 14: 1178-1188
Sen Gupta A, Heinen JL, Scott Holaday A, Burke JJ, Allen RD (1993) Increased resistance to
oxidative stress in transgenic plants that overexpress chloroplastic Cu/Zn superoxide
dismutase. Proc Natl Acad Sci USA 90: 1629-1633
Slooten L, Capiau K, Van Camp W, Van Montagu M, Sybesma C, Inzé D (1995) Factors
affecting the enhancement of oxidative stress tolerance in transgenic tobacco overexpressing
manganese superoxide dismutase in the chloroplasts. Plant Physiol 107: 737-750
Smirnoff N (1993) The role of active oxygen in the response of plants to water deficit and
desiccation. New Phytol 125: 27-58
Tepperman JM, Dunsmuir P (1990) Transformed plants with elevated levels of chloroplastic
SOD are not more resistant to superoxide toxicity. Plant Mol Biol 14: 501-511
Thompson JE, Legge RL, Barber RF (1987) The role of free radicals in senescence and
wounding. New Phytol 105: 317-344
Van Camp W, Capiau K, Van Montagu M, Inz
D, Slooten L (1996) Enhancement of oxidative
stress tolerance in transgenic tobacco plants overproducing Fe-superoxide dismutase in
chloroplasts. Plant Physiol 112: 1703-1714
Witty JF, Minchin FR (1998) Methods for the continuous measurement of O2 consumption and
H2 production by nodulated legume root systems. J Exp Bot 49: 1041-1047
Edited by John G. Scandalios
20
Legends to Figures
Fig. 1. Effect of water stress on photosynthesis and on the photosynthesis/Ci ratio of alfalfa WT
and the derived transgenic lines MnSODmit, MnSODchl, and FeSODchl. For each line, bars
represent control (well-watered plants, o), mild water stress (S1), moderate water stress (S2),
severe water stress (S3), and recovery (R). Data are means ± SE of 10-21 replicates.
Fig. 2. Effect of water stress on SOD activities of leaves from alfalfa WT and the derived
transgenic lines MnSODmit, MnSODchl, and FeSODchl. Designation of water stress and
recovery treatments is as described in the legend to Fig. 1. Data are means ± SE of 4-5
replicates.
Fig. 3. Effect of water stress on H2O2-scavenging enzyme activities and protein content of
leaves from alfalfa WT and the derived transgenic lines MnSODmit, MnSODchl, and
FeSODchl. Designation of water stress and recovery treatments is as described in the legend to
Fig. 1.Data are means ± SE of 4-5 replicates.
Fig. 4. Effect of water stress on the oxidative damage of lipids and proteins of leaves of alfalfa
WT and the derived transgenic lines MnSODmit, MnSODchl, and FeSODchl. Designation of
water stress and recovery treatments is as described in the legend to Fig. 1. Data are means ± SE
of 3-4 replicates.
Fig. 5. Effect of water stress on SOD activities in nodules of alfalfa WT and the derived
transgenic lines MnSODmit, MnSODchl, and FeSODchl. Designation of water stress and
recovery treatments is as described in the legend to Fig. 1. Data are means ± SE of 4-5 replicates.
21
Fig. 6. Effect of water stress on H2O2-scavenging enzyme activities and protein content in
nodules of alfalfa WT and the derived transgenic lines Designation of water stress and recovery
treatments is as described in the legend to Fig. 1. MnSODmit, MnSODchl, and FeSODchl. Data
are means ± SE of 4-5.
(mmol m
-2
s -1 )
Photosynthesis / Ci
(µmol m
-2
s -1 )
Photosynthesis
Fig. 1
10
8
6
4
2
0
25
20
15
10
5
0
WT
MnSODmit
MnSODchl
FeSODchl
Fig. 2
LEAVES
DW)
-1
(units mg
Total SOD
4
3
2
1
0
DW)
1.5
-1
(units mg
MnSOD
2.0
1.0
0.5
0
3.0
DW)
-1
(units mg
FeSOD
2.5
2.0
1.5
1.0
0.5
0
WT
MnSODmit
MnSODchl
FeSODchl
Fig. 3
g -1 DW)
100
2.5
80
2.0
60
1.5
40
1.0
20
0.5
-1
0
0
g -1 DW)
-1
(µmol min
150
100
100
50
50
0
0
WT
MnSODmit
MnSODchl
FeSODchl
WT
MnSODmit
MnSODchl
FeSODchl
Protein
150
200
(mg g -1 DW)
GPX
250
200
g -1 DW)
(µmol min
-1
3.0
Catalase
120
(mmol min
APX
LEAVES
(nmol carbonyl groups mg
-1
Oxidized proteins
protein)
(nmol TBARS g
-1
Lipid peroxides
DW)
Fig. 4
LEAVES
150
100
50
0
30
25
20
15
10
5
0
WT
MnSODmit
MnSODchl
FeSODchl
Fig. 5
NODULES
14
DW)
10
-1
(units mg
TotalSOD
12
8
6
4
2
0
-1
(units mg
MnSOD
DW)
3.0
2.5
2.0
1.5
1.0
0.5
0
-1
(units mg
FeSOD
DW)
1.5
1.0
0.5
0
WT
MnSODmit
MnSODchl
FeSODchl
Fig. 6
NODULES
8
4
40
20
2
0
0
600
150
400
100
200
50
(µmol min
-1
200
0
0
WT
MnSODmit
MnSODchl
FeSODchl
WT
MnSODmit
MnSODchl
FeSODchl
Protein
800
(mg g -1 DW)
g -1 DW)
6
60
g -1 DW)
(µmol min
-1
80
Catalase
g -1 DW)
100
-1
GPX
10
(mmol min
APX
120