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Journal of Experimental Botany, Vol. 47, No. 295, pp. 203-210, February 1996
Journal of
Experimental
Botany
Growth and nitrogen assimilation in nodules in response
to nitrate levels in Vicia faba under salt stress
M.P. Cordovilla, F. Ligero and C. Lluch1
Departamento de Biologfa Vegetal, Facultad de Ciencias, Universidad de Granada, E-18071 Granada, Spain
Received 10 June 1995; Accepted 18 October 1995
Abstract
This study analyses the effects of salt on the effective
symbiosis of faba bean (Vicia faba L. var. minor cv.
Alborea) and salt-tolerant Rhizobium leguminosarum
biovar. viciae strain GRA19 grown with two KNO3 levels
(2 and 8 mM). The addition of 8 mM KNO3 to the growth
medium increases plant tolerance to salinity even with
a concentration of 100 mM NaCI. This KN0 3 level in
control plants reduced the N 2 fixation. For 2 and 8 mM
KNO, the plants treated with NaCI reduced N 2 fixation
to identical values. The activity of the enzymes mediating ammonium assimilation in nodules (GS, NADHGOGAT and NADH-GDH) was decreased by high KN0 3
levels. The results show that NADH-GOGAT activity
was more markedly inhibited than was GS activity by
salinity, therefore NADH-GOGAT limits the ammonium
assimilation by nodules in V. faba under salt stress.
The total proline content in the nodule was not related
to salt tolerance and thus does not serve as a salttolerance index for V. faba.
Key words: Glutamate synthase, glutamine synthetase, N2
fixation, nitrate, salinity.
Introduction
Salinity threatens irrigated agriculture in many semi-arid
and arid regions of the world (Epstein, 1980; Norlyn,
1980; Staples and Toenniessen, 1984). The faba bean is
considered moderately sensitive to salinity (Lauchli, 1984)
and is a particularly important crop in many of these
regions (World Resources, 1987).
A plant's response under stress varies depending on
the degree of salt stress, the stage of growth, the amount
of available nutrient elements, and the type and form of
the nutrient elements in the rhizosphere. Two major
effects have been identified as the probable causes of
salinity toxicity in various plants: the ionic effect and the
osmotic effect. The ionic effect includes interference with
nitrogen uptake, dislocation of nitrogen assimilation and
protein assembly, interference with the transport of essential ions within the plant, and a lowering of net photosynthetic rates in the affected plants. The osmotic effect
is associated with lack of cell-wall extension and cell
expansion leading to cessation of growth (Downton,
1977; Huffaker and Rains, 1986).
In legumes, salt stress from 50 to 200 mM NaCI significantly limits productivity by adversely affecting the
growth of the host plant, the root nodule bacteria,
symbiotic development and, finally, the nitrogen fixation
capacity (Rai and Prasad, 1983; Bekki et al., 1987;
Delgado et al., 1993).
Salt stress is a major constraint in the production of
legume crop species, particularly when the nitrogen
needed for the growth of these plants is derived from
symbiotic fixation. Plants dependent on KNO3-nitrogen
are less sensitive to salt stress (Lauter et al., 1981;
Singleton and Bohlool, 1984; Tu, 1981).
It is generally observed that salt stress promotes the
accumulation of ammonium, nitrate, and free amino acids
in plants, while it reduces protein synthesis (Pessarakli
et al., 1989a). Udovenko et al. (1970), investigating bean
and pea plants in sand culture with various inorganic N
and salt sources, showed a decreased incorporation of
ammonium into amino-acid compounds by these plants.
Under salt stress conditions, the non-protein-N fraction
increased in peas and beans, whereas the protein-N fraction changed irregularly in stressed plants (Udovenko
et al., 1970).
The present study compares the effects of NaCI on
' To whom correspondence should be addressed: Fax: +34 58 2432 54.
Abbreviations: ARA, acetylene reduction activity; GS, glutamine synthetase; NADH-GDH, NADH-dependent glutamate dehydrogenase; NADH-GOGAT,
NADH-dependent glutamate synthase.
6 Oxford University Press 1996
204 Cordovilla et al.
nitrogen-fixing V. faba plants grown with two KN0 3
levels. The aim was to assess the effect of salt on plant
growth, nodulation and N2 fixation in faba bean plants
grown in a solution culture containing 2 and 8 mM KNO3
and inoculated with salt-tolerant Rhizobium. The effect
of salinity and the two KNO3 levels on the activity of
cytosolic GS, NADH-GOGAT and NADH-GDH from
nodules of salt-stressed plants was also examined.
Materials and methods
glutamate dehydrogenase (EC 1.4.1.2) activities were assayed
spectrophotometrically at 30 °C by monitoring the oxidation of
NADH at 340 nm essentially as indicated by Groat and Vance
(1981) and Singh and Srivastava (1986), always within 2 h of
extraction. Two controls (without a-ketoglutarate and without
glutamine in the case of GOGAT, without NH^ and without
a-ketoglutarate in the case of GDH) were used to correct for
endogenous NADH oxidation. The decrease in absorbance
(linear at least 10 min) was recorded for 8 min in a Beckman
DU-70 spectrophotometer.
Protein determination
The soluble proteins in tissue extracts were determined by
Bradford's method (Bradford, 1976), with bovine serum
albumin
(Merck, fraction V) as the standard.
Commercial cultivar Alborea of Vicict faba L. var. minor was
bought from Semillas Pacifico S.A. (Sevilla, Spain). PreContents of reduced nitrogen determination
germinated seeds were planted in Leonard jars (2 per jar) with
The products of acid digestion from the modified Kjeldahl
vermiculite and a nutrient solution (Rigaud and Puppo, 1975),
inoculated with R. leguminosarum bv. viciae strain GRA19, procedure were steam-distilled, after which N content was
determined by mass spectrometry as described by Bremner
which has been described as salt-tolerant (Cordovilla, 1993),
(1965), and Pessarakli and Tucker (1985).
and cultured in a growth chamber. Procedures and growth
conditions were as described before (Cordovilla et al., 1994).
Plant material and growth conditions
Proline determination
Salt and nitrate treatments
Plants were grown on minus NaCl for 18 d from planting, after
which the jars were separated into four groups. The first group
continued growing on NaCl-free solution as control plants. For
the second, third and fourth groups the NaCl treatrnent began
on day 18 reaching the final salt concentration on day 24 (50,
100 and 200 mM NaCl, respectively).
The previous assay with NaCl included two KNO3 levels, 2
and 8 mM, in each case added to the growth medium
immediately after transplanting.
Harvest
Plants were harvested every 3d for 12 d. Harvesting started
24 d after transplanting, with 6 replicates per harvest. The
plants were removed from the jars, the roots thoroughly rinsed
with water, blotted dry on filter paper, and nodules picked and
kept on ice. Shoot, root and nodule dry weights were recorded
after 24 h at 70 CC. Six plants per treatment were used for
nodule dry weight.
Nitrogen fixation assays
Nitrogenase (EC 1.7.99.2) activity was determined by acetylene
reduction on the entire root systems of 6 plants, as well as in
small nodulated root portions of the remaining plants as
described by Cordovilla et al. (1994). The aliquots were
analysed for ethylene in a Perkin Elmer 8600 gas chromatograph
equipped with a Poropak R column (Ligero et al., 1986).
Preparation of cell-free extracts and enzyme assays
Trie maleic acid-KOH buffer and the extraction of nodule
enzymes followed the procedure of Cordovilla et al. (1994).
The supernatant obtained by 30 000 g centrifugation was
assumed to be plant cell cytoplasm and used to measure enzyme
activities and soluble protein.
Glutamine synthetase (EC 6.3.1.2) was determined by the
hydroxamate synthetase assay, adapted from Farnden and
Robertson (1980) and Kaiser and Lewis (1984). Assays were
optimized for the amount of enzyme to give a linear reaction
within at least 30 min. Two blanks without enzyme and without
L-glutamate were also analysed.
NADH-glutamate synthase (EC 1.4.1.14) and NADH-
Samples (1 g fresh weight) were homogenized with 10 ml of 3%
(w/v) sulphosalicylic acid. The homogenate was centrifuged at
2500 g at 2°C for 10 min. The resulting supernatant was used
to determine the proline content.
Aliquots of 0.25 to 1 ml of crude extract were used, with the
addition of 1 ml of 2.5% (w/v) ninhydrin prepared in glacial
acetic acid at 60% (v/v) and phosphoric acid at 40% (v/v), 1 ml
of glacial acetic acid and sulphosalicylic acid to a total volume
of 3 ml. This reaction mixture is boiled for 60 min, stopped
with ice for 1 or 2 min and 3 ml of toluene is added, whereupon
the mixture is stirred vigorously and the upper phase is used to
measure absorbance at 520 nm. A control with sulphosalicylic
acid was used.
To calculate the proline concentration a model curve was
prepared with proline (Sigma), following the same procedure,
with quantities of between 10 and 100/xg.
Statistical design and analysis
The experimental layout was a randomized complete block
design. All values are means of 6 replicates per treatment. All
results were subjected to multifactor analysis of variance with
a least significant difference (LSD) test between means. Sources
of variance (treatments with salt, treatments with nitrate or
time) were compared with Duncan's test.
Results
The respective growth response to salinity stress of faba
bean plants given 2 and 8 mM KNO3 can be observed in
the data recorded in Table 1. These data clearly indicate
that control plants (not receiving NaCl) given 8 mM
KN0 3 grow more rapidly than plants given 2 mM KNO3.
However, maximum dry-matter accumulation for both
shoots and roots was not significantly affected by the
KNO3 level.
Plants fed 8 mM KNO3 are far more salt-tolerant than
their counterparts fed 2 mM KN0 3 under the experimental conditions. Plants given 8 mM KN0 3 showed no
reduction by salinity in the dry weight of shoots, and
Nodule nitrogen metabolism
205
1
Table 1. Effect of saline treatments during the vegative growth period on dry weight in shoots, roots (g organ' ) and nodules
(g plant ~l) o/V. faba plants inoculated with the salt-tolerant R. leguminosarum GRA19 and grown with 2 and 8 mM KNO3
Data in parenthesis expressed as a percentage of the control for each harvest and K N 0 3 level The least significant difference (LSD) is given for
each plant organ.
Plant organ
NaCl
(mM)
KNO 3
(mM)
Days after salt treatment
12
Shoot
0
2
O
0
50
0.98
1.22
1.18
1.48
0.99(84)
1.26(85)
0.97(82)
1.27(86)
0.94(80)
1.05(71)
(g organ
1.42
1.68
1.26(89)
1.57(93)
1.20(85)
1.69(100)
1.15(81)
1.09(65)
2.09
2.33
1.41(67)
2.07(89)
1.21(58)
2.03(87)
1.19(57)
1.40(60)
2.22
2.28
1.82(82)
2.27(100)
1.61(73)
2.07(91)
1.43(64)
1.50(66)
0.36
0.56
0.40
0.56
0.46
0.63
0.52
0.62
0.56
0.62
0 58
0.72
0 55
0.75
0.63
0 78
0.69
0 62
0.66
0.83
0.59
1.01
0.78
0.95
0.70
0.76
0.82
0.85
0.85
1.07
0.96
0.97
0.87
0.78
0.05
0.04
0.08
0.05
0.07
0.05
0.08
0.04
0.08
0.05
(g plant" 1 )
0.09
0.07
0.09
0.06
0.09
0.06
0.10
0.06
0.11
0.08
0.11
0.07
0.10
0.07
0.11
0.06
0.13
0.11
0.12
0.09
0.13
0.07
0.12
0.06
2
O
O
100
2
200
2
Q
0
Q
O
LSD (0.05) 0.24
Root
0
2
0
0
50
100
200
2
8
2
8
2
0
0
LSD (0.05)0.14
Nodules
0
2
0
0
50
2
100
2
8
2
Q
0
200
LSD (0.05) 0.01
only a reduction of 34% at the 200 mM salinity level,
whereas, even at a concentration of 50 mM, plants given
2 mM KNO3 showed a reduction of 18% in the dry
weight of shoots, this loss increasing with the salinity
level. While the plants fed 8 mM KNO3 were still growing
vigorously at the 50 and 100 mM salinity levels, plants
fed 2 mM were showing signs of wilting.
In plants fed both KNO3 levels, the salinity effect on
growth was more noticeable in the shoot than in the root.
No reduction in the dry weight of the root was detected
(Table 1) in any of the NaCl levels assayed.
Nodule mass (Table 1) was significantly affected by
KNO3, but the nodules were similar in appearance. The
plants fed 2 mM KNO3 showed no reduction in drynodule mass, whereas plants fed 8 mM KN0 3 showed
reduced dry-nodule mass at the end of the culture for all
NaCl levels, this reduction increasing with the salinity
level.
The KN0 3 concentration did not significantly affect
the distribution of N between roots and shoots.
Furthermore, KN0 3 treatment had no effect on N content
per gram (Table 2) in either shoots or roots for control
plants. In salt-treated plants, the N content of shoots
responded in a manner similar to that of the dry weight
of shoots: the shoots of plants given 2 mM KN0 3 showed
reductions at all NaCl levels assayed, and the shoots of
plants given 8 mM KN0 3 showed significant reductions
only at 200 mM NaCl. For roots, in both KN0 3 concentrations, all NaCl levels reduced the N content, while the
dry weight remained stable.
The high KNO3 (8mM) treatment in control plants
caused a reduction of approximately 30% in ARA per
gram of nodule and a reduction of approximately 48% in
ARA per plant (Table 3). Both specific and total activity
were severely depressed by salinity. The effect of NaCl
on ARA was more pronounced in plants fed 2 mM
KNO3, in such a way that in the last harvest the nitrogenase activity registered the same values for both KNO3
levels. Nevertheless, the evolution of both specific and
total nitrogenase activity was different for the two KN0 3
concentrations, in that the plants given 8 mM KN0 3
showed the same values in all the harvests for 50 and
100 mM NaCl, while in plants given 2 mM KNO3 the
decrease was progressive at all the NaCl levels. The
depression in total activity was partly due to salt reducing
the activity of pre-formed nodules and partly due to the
reduced differentiation of new pink nodules.
The activity of the enzymes mediating ammonia assim-
206 Cordovilla et al.
Table 2. Effect of saline treatments during the vegetative growth period on nitrogen content of shoots and roots (mg g~l DW)
faba plants inoculated with R. leguminosarum GRA19 and grown with 2 and 8 mM KNO3
of\.
The LSD is given for each plant organ.
Plant organ
Shoot
NaCl
(mM)
0
50
100
200
KNO 3
(mM)
Days after salt treatment
0
3
6
9
12
2
8
2
8
2
8
2
8
45.1
46.6
43.8
45.8
33.1
44.2
37.8
42.8
39.0
36.0
(mgg-'DW)
41.9
43.8
37.3
42.2
32.3
39.4
33.9
35.6
38.1
39.0
36.2
41.7
32.3
40.0
33.5
34.8
35.4
39.0
319
37.5
30.8
40.9
26.7
33.2
2
8
2
8
2
8
2
8
39.9
41.0
41.4
41.9
35.9
37.0
40.1
38.7
33.8
36.0
41.5
41.8
37.9
38.6
39.3
35.2
34.7
37.1
42.4
41.9
39.1
36.0
37.2
34.6
29.2
35.7
43.8
42.8
40.4
34.6
37.0
36.2
30.2
34.8
LSD (0.05) 4.1
Root
0
50
100
200
LSD (0.05) 1.1
Table 3 Effect of saline treatments during the vegative growth period on nodule acetylene-reduction activity (ARA) (jimol C2HA
hh~lg~
'g l ' nodule) and total ARA per plant (junol C2H4 h ' plant l) for V. faba plants inoculated with R. leguminosarum GRA19 and
grown with 2 and 8 mM KN03
Parameter
NaCl
(mM)
KNO3
(mM)
Days after salt treatment
0
ARA per unit weight
of nodule
0
50
100
200
2
8
2
8
2
8
2
8
83.0
59.2
LSD (0.05) 13.1
ARA per plant
0
50
100
200
2
8
2
8
2
8
2
8
4.15
2.37
3
6
9
12
(/imol C 2 H 4 h ~ ' g ~ ' nodule)
66.3
62.6
48.3
42.9
63.7
56.5
43.6
45.9
53.7
46.5
41.5
37.3
46.9
42.7
35.4
19.4
62.4
39.9
36.6
39.3
33.7
30.2
33.4
16.0
56.5
38.2
26.0
33.4
22.7
29.3
12.0
14.4
(,xmol C 2 H 4 h ~l
5.30
2.41
4.46
2.29
4.30
1.66
3.75
1.77
6.86
3.19
4.03
2.75
3.37
2.11
3.68
0.96
7.34
4.20
3.12
3.01
2.95
2.05
1.44
0.86
plant" 1 )
5.63
3.00
5.08
2.61
4.19
2.24
4.27
1.17
LSD (0.05) 0.74
ilation in nodules was affected by different KNO3 levels
(Table 4). When the nitrate supply was increased the
activity of GS, NADH-GOGAT and NADH-GDH
decreased. The activity of these enzymes at the last harvest
was severely depressed by salinity; this reduction increased
with higher levels of salinity and of KNO3, NADHGOGAT being the enzyme which showed the greatest fall
in activity. After 3 d of treatment with 50 mM NaCl GS,
NADH-GOGAT and NADH-GDH increased in activity
at both KNO3 levels. For NADH-GOGAT and
NADH-GDH an increase is also detected at 100 mM
NaCl in plants grown with 2 mM KNO3.
For control plants the soluble protein content (Table 5)
was identical to that of plants given 2 and 8 mM KNO3.
Nodule nitrogen metabolism
207
Table 4. Effect of saline treatments during the vegetative growth period on glutamine synthetase (GS) (fimol y-glutamate dehydrogenase
h'1 g'1 FW), glutamate synthase (NADH-GOGAT) and glutamate dehydrogenase (NADH-GDH) (yjnol NADHOX h'^g'xFW)
in
nodules of V. faba plants inoculated by R. leguminosarum GRA19 and grown with 2 and 8 mN KNO3
Parameter
NaCl
(mM)
KNO3
(mM)
Days £ifter salt treatment
0
3
6
9
12
297
276
295
267
267
262
266
245
310
281
296
263
267
258
254
241
309
277
266
247
245
226
228
206
lTl
120
156
127
142
117
111
107
136
120
135
96
116
94
91
79
130
112
129
89
108
88
69
67
112
97
111
91
86
64
49
46
31.5
29.2
34.7
32.8
34.3
28.5
30.1
28.2
32.9
31.2
30.5
26.9
29.7
26.2
27.5
25.0
30.0
27.4
29.6
26.4
29.4
23.5
24.1
22.8
30.4
25.3
29.4
23.3
29.0
23.1
22.3
17.6
(,dnol y-glutamyl-hydroxamate h"'g" 1 FW)
GS
0
50
100
200
2
8
2
8
2
8
2
8
302
283
296
284
307
301
299
270
300
277
LSD (0.05) 7
NADH-GOGAT
0
50
100
200
LSD (0.05) 2
NADH-GDH
0
50
100
200
2
8
2
8
2
8
2
8
2
8
2
(/imol NADH^h-V1 FW)
130
127
32.8
26.5
8
2
8
2
8
LSD (0.05) 1.3
(Hafeez et al., 1988). Similarly in this work, V. faba cv.
Alborea plants given 2 mM KNO3 and exposed to 50 mM
NaCl decrease the shoot dry weight per plant (Table 1).
However, plants fed 8 mM KNO3 showed no reduction
in shoot dry weight when exposed to 50 mM and 100 mM
NaCl, although this parameter was reduced by 200 mM
NaCl. Therefore, in these experiments, plants given 8 mM
KNO3 were more salt tolerant than were plants fed
2 mM KNO3. These responses to salinity are generally
consistent with conclusions that N-fixing plants are more
sensitive to salinity than N-fertilized plants (Lauter et al.,
1981; Alston and Graham, 1982; Yousef and Sprent,
1983).
Torres and Bingham (1973) suggest that NOf deficiency induced by Cl" as a result of antagonism between
Discussion
ions, retards growth in plants exposed to high NaCl
levels. It is conceivable that the addition of NO3" decreases
Growth and dry-matter accumulation of legumes are
the Cl~ level in plant tissues (Feigin et al., 1984).
reportedly reduced by low salinity levels (about 50 mM
Silberbush and Lips (1988) as well as Martinez and Cerda
NaCl) in Vicia faba and Phaseolus vulgaris (Abdel(1989) demonstrate that NOf in solution decreases the
Ghaffar et ai, 1982), Glycine wightii (Wilson, 1970),
Glycine max (Grattan and Maas, 1988), and Vigna radiata accumulation of Cl~, but does not affect the Na + content.
After 12 d of saline treatment at all the NaCl levels, the
soluble protein content of the nodule decreased more in
plants with 2 mM KNO3. This reduction increased with
higher NaCl concentrations in the medium.
The soluble proline content in the nodule (Table 5)
was higher in plants given 8 mM KNO3. The response to
NaCl varied with the NaCl level and KN0 3 level. In the
plants given 2 mM KNO3 the proline content did not
change with 50 mM NaCl, whereas with 100 and 200 mM
NaCl the proline content rose 20 and 10 times, respectively, with regard to the first harvest. Similarly, plants
given 8 mM KN0 3 showed 10-fold proline increases at
all the NaCl concentrations assayed.
208
Cordovilla et al.
Table 5. Effect of saline treatments during the vegetative growth period on soluble protein concentration (mg g~l FW) and proline
content (pmol g~yFW) in nodules 0/V. faba plants inoculated by R. leguminosarum GRA19 and grown with 2 and 8 mN KNO3
Parameter
Protein
NaCl
(mM)
0
50
100
200
KNO 3
(mM)
2
8
2
8
2
8
2
8
Days after salt treatment
0
3
6
9
12
13.3
13.1
13.3
13.6
13.2
13.4
12.4
12.4
12.7
12.9
(mgg-'FW)
13.2
13.3
13.0
12.7
12.4
12.1
12.1
12.3
13.7
13.0
11.8
12.8
11.7
12.4
11.1
12.4
13.1
13.2
11.6
12.5
11.3
12.5
10.7
11.9
0.16
0.40
0.17
0.48
0.17
0.63
0.31
0.84
(mgg-'FW)
0.16
0.52
0.19
0.96
0.66
1.49
1.44
2.17
0.18
0.56
0.23
1.03
2.33
2.31
3.09
5.12
0.18
0.55
0.25
4.31
2.69
6.30
3.09
8.03
Macroptilium,
Neonotonia,
LSD (0.05) 0.5
Proline
0
50
100
200
2
8
2
8
2
8
2
8
0.14
0.30
LSD (0.05) 0.25
This may explain the slight inhibition of growth for
concentrations of 100 mM NaCl in V. faba plants grown
with high KNO3 levels.
Salinity affected shoot growth more than root growth,
as was also reported for beans (Wignarajah, 1990).
In these experiments, shoot N content responded in a
manner comparable to that of growth. However, in the
root, N content was decreased by salinity contrary to
growth, as noted in other legumes (Hafeez et al., 1988;
PessarakJi et al., 19896). Other authors observed no
reduction in N content (Singleton, 1983; Weil and Khalil,
1986). In this research, with greater KN0 3 dosages,
salinity had a less inhibitory effect on the N content in
V. faba cv. Alborea—results which agree with those of
Yousef and Sprent (1983) with other V. faba cvs administered NH4NO3.
The 8 mM KNO3 treatment affected the faba bean
nodule by depressing nodule mass, as opposed to the
findings of Caba et al. (1990). Salt stress, together with
the high KN0 3 level decreases nodule mass, undetected
in plants grown with 2 mM KNO3. In fact, nodulation
and nitrogenase activity correlate negatively with the
inorganic nitrogen concentration in the soil (Alston and
Graham, 1982). In control plants, the ARA was affected
by high KNO3 concentrations (30%). In field experiments,
other authors have reported that V. faba plants can prefer
N2, with an apparent nitrate tolerance (Hardanson et al.,
1991). A notable decline in ARA occurred with low-level
salt stress in plants given 2mM KN0 3 . This finding
corroborates earlier observations concerning Glycine,
Medicago, and
Phaseolus
(Berstein and Ogata, 1966; Lakshmi et al., 1974; AbdelGhaffar et al., 1982; Wilson, 1985), and more recent
findings in Vigna radiata (Hafeez et al., 1988), Cicer
arietinum (Elsheikh and Wood, 1990) and Arachis hypogea (Leidi et al., 1992). Low level NaCl plant growth did
not change; this decline in ARA may be attributable
to a direct effect of salt on nitrogenase. This conclusion agrees with Burns et al. (1985), who reported that
NaCl directly affected nitrogenase purified from
Azotobacter.
The present study shows that in nodules of V. faba cv.
Alborea nitrogen nutrition interferes with the activity of
the enzymes mediating assimilation of nitrogen. In control
plants, GS activity was approximately 2.4 times higher
than NADH-GOGAT activity, for the two levels of
KN0 3 assayed. The GDH activity was roughly 10 times
and 4 times less than GS and GOGAT values, respectively,
for the two KNO3 levels. Therefore, NH3 is assimilated
via the GS/GOGAT system in the nodule cytosol of
V. faba cv. Alborea. These results correspond with those
for V. faba (Caba et al, 1993) and for alfalfa (Ta et al.,
1986). Groat and Vance (1981) observed that in Medicago
sativa the GDH activity is not associated with nitrogen
fixation either, but rather is related with nodule
senescense.
Salt stress inhibited GS and NADH-GOGAT activities,
a finding in agreement with Bourgeais-Chaillou et al.
(1992), who reported reduced GS and NADH-GOGAT
in the soybean. Treatment with NaCl in V. faba cv.
Nodule nitrogen metabolism
Alborea affected NADH-GOGAT more than GS, as
reported by Billard and Boucaud (1980) for Phaseolus
vulgaris. In the first 3 d salt treatment stimulated (at
certain NaCl concentrations only) the activity of enzymes
involved in ammomium metabolism. This stimulation
may be due to the NH^ and amide accumulation induced
by stress (Hatata, 1982).
The decrease in soluble protein content of the nodules
is a common response to salt stress reported in other
legumes (Bourgeais-Chaillou et al., 1992). The response
may be due to a protein break-down (Mothes, 1956), or
to an alteration in the incorporation of amino acids into
proteins (Stewart and Lee, 1979). Udovenko et al. (1970)
states that salt stress reduces amino acid incorporation
into proteins in V. faba and P. sativum. The effect of salt
on soluble protein in the nodule is less when plants are
grown with high KN0 3 concentrations.
The proline content within the cytosol of the nodule of
V. faba cv. Alborea increased under salt stress, as also
described for other legumes (Kohl et al., 1991). At the
end of culture, the increase in proline, with respect to
plant growth without salt, for 100 mM (15- and 12-fold
for 2 and 8 mM KNO3, respectively) and 200 mM NaCl
(17- and 15-fold for 2 and 8 mM KNO3, respectively)
(Table 5) was similar to that described by Fougere et al.
(1991) for nodules of soybean plants grown in the presence of 150 mM NaCl. Marked increase (10-fold or more)
in free proline occurs in many plants during moderate or
severe water or salt stress; this accumulation, mainly as
a result of increased proline biosynthesis, is usually the
most outstanding change among the free amino acids
(Hanson and Hitz, 1982). Therefore, as reported in roots,
stems, and leaves of other plants, proline accumulation
in nodules may represent an osmoregulatory mechanism.
There is great controversy over proline accumulation,
which appears to be more a symptom of susceptibility to
stress (Hanson and Hitz, 1982) than an adaptive response.
Plants grown with 8 mM KN0 3 reach higher proline
levels than do plants with 2 mM KNO3. However, the
proline level did not always correlate with the ability to
withstand salinity stress. Plants with 8 mM KNO3 and
200 mM NaCl, and plants with 2 mM KN0 3 for all levels
of NaCl showed strong inhibition of growth and significant increases in proline (Tables 1, 2). In these cases, part
of the proline could result from catabolic processes that
accompany a decrease in the growth rate. Thus, in V. faba
cv. Alborea, the proline content of the nodule is not a
reliable index of salt tolerance, as shown for proline
accumulation in leaves of Vigna (Ashraf, 1989).
In conclusion, V. faba cv. Alborea plants given 8 mM
KNO3 tolerate 100 mM NaCl, and an increase in proline
content was noted, correlating with the rise in salt, but
this increase might not be sufficient to confer resistance
in the cultivar used.
209
Acknowledgements
Financial support was obtained through the Andalusian
Research Program and the DGICYT.
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