Effect of salinity on root-nodule conductance to the oxygen diffusion in the Cicer arietinum-Mesorhizobium ciceri symbiosis.

Abstract

Nodule conductance to O2 diffusion has been involved as a major factor of the inhibition of N2 fixation by soil salinity that severely reduces the production of grain legumes. In order to determine the effect of this constraint on the nodule conductance, oxygen uptake by the nodulated roots of Cicer arietinum was measured by recording the concentration of O2 as a function of pO2 in a gas-tight incubator. After germination and inoculation with the strain Mesorhizobium ciceri UPMCa7, the varieties Amdoun 1 and INRAT 93-1 were hydroponically grown in a glasshouse on 1L glass bottles filled with nutrient solution containing 25 mM NaCl. Salinity induced a marked decrease in shoot (30% versus 14%), root (43% versus 20%), and nodule biomass (100% versus 43%) for Amdoun 1 relative to INRAT 93-1. Although salinity completely prevented nodule formation in the sensitive variety Amdoun 1, nodule number and biomass were higher in the first than in the second variety in the absence of salt. This effect was associated with a significantly higher O2 uptake by nodulated root (510 versus 255 micromol O2 plant(-1)h(-1)) and nodule conductance (20 versus 5 microm s(-1)) in Amdoun 1 than in INRAT 93-1. Salinity did not significantly change the nodule conductance and nodule permeability for INRAT 93-1. Thus, the salt tolerance of this variety appears to be associated with stability in nodule conductance and the capacity to form nodules under salt constraint.

Full text

Page 1

Journal of Plant Physiology 164 (2007) 1028—1036
Effect of salinity on root-nodule conductance
to the oxygen diffusion in the Cicer
arietinum–Mesorhizobium ciceri symbiosis
Boulbaba L’taiefa,b,c,?, Bouaziz Sifib, Mainassara Zaman-Allahb,
Jean-Jacques Drevonc, Mokhtar Lachaa ˆla
aDe ´partement de Biologie, Faculte ´ de Sciences de Tunis, Campus universitaire 1060, Tunisia
bLaboratoire des grandes cultures, INRAT, Rue He ´di Karray 2080 Ariana, Tunisia
cUMR1220 Rhizosphe `re and symbiotes INRA-AGRO.M, Place Viala, 34060 Montpellier cedex 01, France
Received 20 December 2005; accepted 12 May 2006
KEYWORDS
Cicer arietinum;
Nodule conductance;
Oxygen diffusion;
Rhizobia;
Symbiotic nitrogen
fixation
Summary
Nodule conductance to O2diffusion has been involved as a major factor of the
inhibition of N2fixation by soil salinity that severely reduces the production of grain
legumes. In order to determine the effect of this constraint on the nodule
conductance, oxygen uptake by the nodulated roots of Cicer arietinum was
measured by recording the concentration of O2as a function of pO2in a gas-tight
incubator. After germination and inoculation with the strain Mesorhizobium ciceri
UPMCa7, the varieties Amdoun 1 and INRAT 93-1 were hydroponically grown in a
glasshouse on 1L glass bottles filled with nutrient solution containing 25mM NaCl.
Salinity induced a marked decrease in shoot (30% versus 14%), root (43% versus 20%),
and nodule biomass (100% versus 43%) for Amdoun 1 relative to INRAT 93-1. Although
salinity completely prevented nodule formation in the sensitive variety Amdoun 1,
nodule number and biomass were higher in the first than in the second variety in the
absence of salt. This effect was associated with a significantly higher O2uptake by
nodulated root (510 versus 255mmolO2plant?1h?1) and nodule conductance (20
versus 5mms?1) in Amdoun 1 than in INRAT 93-1. Salinity did not significantly change
the nodule conductance and nodule permeability for INRAT 93-1. Thus, the salt
tolerance of this variety appears to be associated with stability in nodule
conductance and the capacity to form nodules under salt constraint.
& 2006 Elsevier GmbH. All rights reserved.
ARTICLE IN PRESS
www.elsevier.de/jplph
0176-1617/$-see front matter & 2006 Elsevier GmbH. All rights reserved.
doi:10.1016/j.jplph.2006.05.016
Abbreviations: Conr, oxygen consumption by nodulated-root; EURS, efficiency in utilisation of the rhizobial symbiosis; nDW, nodule
dry weight; pO2, partial pressure of O2; sDW, shoot dry weight; SNF, symbiotic nitrogen fixation
?Corresponding author. De ´partement de Biologie, Faculte ´ de Sciences de Tunis, Campus universitaire 1060, Tunisia.
Tel.: +21697408012.
E-mail address: lboulaba@yahoo.com (B. L’taief).

Page 2

Introduction
Root nodules are the sites of a beneficial
symbiotic association between legume plants and
soil bacteria commonly denominated as rhizobia.
The plant supplies the bacteria with an energy
source, malate or succinate. In turn, the bacteria
reduce, i.e., fix, the atmospheric N2gas to NH4
providing the latter to the plant for assimilation
into amino acids, protein and other essential
nitrogenous compounds. Since nitrogenase, the
enzyme responsible for N2 fixation, is O2 labile,
legume nodules have evolved mechanisms to down-
regulate their permeability to O2and maintain the
infected-cell O2 concentration at approximately
5–50nM. This is extremely low compared with the
level of approximately 250mM for cells in equili-
brium with air (Witty et al., 1987; King and Layzell,
1991; Denison et al., 1992; Hunt and Layzell, 1993;
Minchin, 1997). By controlling their permeability to
O2diffusion, nodules are able to reduce or increase
the supply of O2to the infected cells, and thereby
regulate their metabolism (de Lima et al, 1994).
The metabolism of legume nodules is thought to be
O2-limited in ambiantal conditions, as gradual
increases in the external partial pressure of O2
(pO2) result in significant stimulations of nitrogen-
ase activity (Drevon et al., 1988; Hunt et al., 1989)
and nodule respiration (Ribet and Drevon, 1995).
The specific sensitivity of the symbiotic nitrogen
fixation (SNF)-dependent legumes to salinity is well
documented for initiation, development and func-
tion of nodules (Singleton and Bohlool, 1983; Bekki
et al., 1987; Serraj et al., 1998; Ramos et al., 1999;
Garg and Gupta, 2000; Saadallah et al., 2001). An
application of salt or drought decreases the nodule
permeability (Sprent, 1972; Vance and Heichel,
1991; Drevon et al., 1994; Gonza ´lez et al., 2001;
Serraj et al., 2001). This decrease is associated
with a contraction of nodule inner-cortex cells
(Serraj et al., 1995) and an increase in acid
abscissic content of the nodule (Irekti and Drevon,
2003). The subsequent inhibition of nitrogenase
activity and nodule respiration is compensated by
raising pO2 in the nodulated-root rhizosphere
(Serraj et al., 1994). Alternatively, the inhibition
of the sucrose synthase (Arrese-Igor et al., 1999)
and various enzymes of the sucrose hydrolysis
(Anthon and Emerich, 1990) by salinity has been
argued to induce a deficiency of carbon for
bacteroids associated with an accumulation of
sucrose up to 40–70% of the total nodule sugar
content. The subsequent inhibition of nitrogenase
would result in O2 accumulation in the infected
zones, inducing the decrease in nodule permeabil-
ity. Additionally, it has been argued that the
+,
limitations of O2 diffusion imposed by structural
modifications due to salinity are compensated by
the decrease of nodule growth and the formation of
a large number of small nodules facilitating the O2
entry in nodules by increased contact area with
external medium (Hunt and Layzell, 1993).
Cicer arietinum is one of the most frequently
grown grain legumes in semi-arid regions. Cultivars
grown in India are either native- (desi) or medi-
terranean- (kabuli) types. The kabuli types have
been found to be more tolerant to salinity than the
desi types (Dua and Sharma, 1995). However, the
nodulation pattern of the two groups in saline soils
has not been examined, and the role of the
bacterial symbiont overall yield in salt-affected
soil remains controversial. Root nodules are the site
of a beneficial symbiotic association between
legume plants and soil bacteria commonly denomi-
nated as rhizobia. The plant supplies the bacteria
with an energy source, malate or succinate. In turn,
the bacteria fix the atmospheric N2 gas to NH4
providing the later to the plant for assimilation into
amino acids, protein and other essential nitrogen-
ous compounds. Since nitrogenase, the enzyme
responsible for N2 fixation, is O2 labile, legume
nodules have evolved mechanisms to regulate-
down their permeability to O2 and maintain the
infected-cell O2 concentration at circa 5–50nM
(Minchin, 1997). Sprent (1972), Vance and Heichel,
(1991), Drevon et al. (1994); Gonza ´lez et al. (2001)
and Serraj et al. (2001) concluded that exposure to
salt or drought decreases the nodule permeability.
Serraj et al. (1994) showed that the salt-induced
inhibition of nitrogenase activity and nodule re-
spiration is compensated by raising pO2 in the
nodulated-root rhizosphere.
Since most of the above conclusions were
obtained with nodules of Glycine max, Phaseolus
vulgaris and Medicago truncatula, the aim of this
work was to examine the effect of salinity on the
oxygen consumption of C. arietinum nodules, and
compare its values in contrasting lines for their
symbiotic tolerance to salinity.
+,
Materials and methods
Plant material and culture conditions
Growth in pots
The selection of two contrasting varieties and
search of toxicity levels was carried out in experi-
ment under greenhouse conditions with plant
growth in pots. The seeds of four chickpea varieties
Amdoun 1, INRAT 93-1 (Be ´ja 1), INRAT 88 (Bouchra)
ARTICLE IN PRESS
Cicer arietinum nodule conductance under salt stress 1029

Page 3

and INRAT 87(Neyer) were provided by M. Kharrat
(Field crops Laboratory, Gain Legumes programme,
INRAT Ariana, Tunisia). They were sterilised in 2%
calcium hypochlorite, washed with sterile distilled
water and germinated at 281C in soft agar contain-
ing 100mL of Bergersen solution (Vincent, 1970).
The rhizobial inoculant was prepared from Mesor-
hizobium ciceri UPMCa7 preserved in tubes at 41C
on YEM media (Vincent, 1970). Rhizobia were
grown in liquid YEM solution into an Erlenmeyer
with agitation during 2 days at 281C, in darkness.
Inoculation was performed by soaking 4-day-old
seedlings for 30min with 100mL of inoculant
containing approximately 108cellsmL?1. The seed-
lings were transplanted into plastic pots containing
2kg of the collected sterilised soil from the
Research Area of Tunisian Agricultural Research
Institute, Ariana. Afterwards, they were separated
into five groups (five plants per treatment) and
irrigated with nutrient solution supplemented with
0 (control), 25, 50, 75 and 100mM NaCl. Plants
were harvested at 45d of culture, coinciding with
the onset of flowering. Fresh and dry matter of
shoots, roots and nodules were then determined.
Growth in hydroaeroponics
For O2uptake and nodule conductance, only the
two contrasting varieties Amdoun 1, INRAT 93-1
were used, with the surface seeds were sterilised
and pre-germinated in agar 0.9%. They were
transferred into 1L glass bottles wrapped with
aluminium foil to maintain darkness in the rooting
environment. The roots of selected uniform seed-
lings were gently passed through the hole of a
rubber stopper on the bottleneck, and cotton wool
was fitted at the hypocotyl level to maintain the
root system suspended in the nutrient solution. The
latter contained 0.7mM K2SO4, 1mM MgSO4?7H2O,
1.65mM CaCl2, 22.5mM Fe for macronutrients and
6.6mM Mn, 4mM Bo, 1.5mM Cu, 1.5mM Zn, 0.1mM for
micronutrients.It
250mmolKH2PO4week?1plants?1. In order to re-
produce natural conditions, salt was added during
the first days after transfer into the glass bottle.
During the first 2 weeks, i.e. before nodule
function, the nutrient solution was complemented
with 2mM urea, and was renewed every 2 weeks
thereafter without urea. It was initially added with
1mL of M. ciceri UPMCa7 inoculum containing
approximately 108cellsmL?1. Its pH was main-
tained to 7.0 by adding 0.2gL?1CaCO3. It was
aerated with a flow of 400mLL?1min?1of filtered
air via a compressor and ‘‘spaghetti tube’’ distribu-
tion system. Plants were grown in a temperature-
controlled glasshouse with night/day temperatures
of circa 20/281C and a 16-h photoperiod with
additional lights of 400mmolPARm?2s?1.
wassupplied with
Oxygen consumption by nodulated root
One day before the measurement with the device
shown in Fig. 1, the level of the nutrient solution
was lowered to one-third of the volume of the
culture bottle for the majority of nodules to be in
ARTICLE IN PRESS
Figure 1. Device for the in situ measurement of oxygen consumption by nodulated-root of Cicer arietinum. Between
each confinement, the circuit was opened and washed by a renewed gaseous mixture monitored by mass-flow metres.
Oin ¼ influx O2; Oex ¼ efflux O2.
B. L’taief et al. 1030

Page 4

direct contact with the gaseous phase. The
circulation of the gaseous phase in the circuit from
the nodulated-root environment through the oxy-
meter was driven by a peristaltic pump with a flow
of 400mLmin?1that favoured the homogenisation
of the O2 concentration in the gas and liquid
phases. The incubator at the level of the plant-
stem through the bottle- neck was tightened with a
seal of a non-toxic Rhodorsyl polymer (RTV 385)
that was poured as a liquid and stopped by the
cotton fitted around the stem. Thus, the micro-
spaces, which might have been a source of gas leak
along the plant stem, were filled before polymer-
isation. The measurement consisted in recording,
with an oxymeter (Abiss, La Verpille `re, France), the
pO2decrease during successive confinement period
with varying initial pO2, as previously described in
details by Jebara and Drevon (2001). A steady
decrease in pO2 corresponded to a stationary
nodulated-root respiration and constant nodule
permeability. After 5min registration during steady
state, the devise was flushed with a new N2–O2
gaseous mixture established in less than 10min
with mass-flow metres (Tylan, F38296, La Verpil-
le `re). For each 5min confinement, the Conr could
be calculated as follows:
Conr ¼ DpO2(V/24.2)(60/t) with DpO2¼ (initial?
final)pO2
in% of the
V ¼ volume of gas phase in l; 24.2 ¼ volume of
1mol pure gas in experimental conditions in l;
t ¼ time between initial and final O2measurement
in min, i.e. 5. Conr was expressed in mmol O2
consumed per hour per plant.
atmospheric pressure;
Biomass and nodule surface
The plants were harvested after the gas exchange
measurements and separated into shoots, roots and
nodules. Shoot, root and nodule dry mass (DM) were
determined after drying for 2 days at 701C. Because
chickpea nodules are indeterminate, with unlimited
growth, they are often multilobular, each lobe
representing to an N2-fixing zone.
Consequently, the nodule numbers included lobe
numbers. Each unit nodule N2-fixing zone was
considered as a cylinder of 3mm diameter and
3mm length, for the calculation of whole nodule
surface as follows: S ¼ nPDH, with n the nodule
number per plant, D the fresh nodule diameter and
H the length of the fresh nodule. Nodule surface
was expressed in mm2. The EURS was estimated by
the slope of the regression model of shoot biomass
as a function of nodule biomass. For a linear
adjustment curve, i.e. y ¼ ax þ b, b corresponds
to the shoot biomass production without nodules
(g shoot dry weight (sDW0)), and a to the EURS as
(gsDM–gsDM0) g?1nDM. The Statistic software was
used to perform the statistical analyses of biomass,
correlation analyses and regressions of gas ex-
changes parameters as a function of external pO2.
The analysis of variance and the standard deviation
of the means were used to determine the sig-
nificance (Po0.05) of differences in symbiotic
effectiveness.
Results
Selection of two contrasting varieties and
search of toxicity levels in sand culture.
Data in Fig. 2A show genotypic differences in the
of shoot growth of the four chickpea varieties.
Without salt, Amdoun 1 show the higher production
of shoot biomass (1.2gDMsplant?1), with the INRAT
87varietydisplaying
(0.33gDMsplant?1). With salt, the production was
differentially decreased depending on the variety.
For example, at 25mM NaCl, only the Amdoun 1
variety presented a significant decrease in shoot
biomass production. With concentrations higher
than 25mM NaCl, shoot biomass of all varieties
decreased, but Amdoun 1, the more vigorous
variety in absence of salt, presented the higher
decrease (more than 50%) and INRAT 93-1 displayed
the lower one.
Thechickpea varieties
growth of root in the control treatment (Fig. 2B)
with Amdoun 1 being significantly more productive
than INRAT 93-1, INRAT 88 and INRAT 87. In these
conditions, the root biomass ranged from 0.92 to
0.531gDMsplant?1in Amdoun 1 and INRAT 87,
respectively. The effect of salt on root growth was
similar to that of shoots, so that Amdoun 1
displayed a high salt sensitivity to NaCl treatment
whereasINRAT93-1 presented
behaviour.
Moreover, our results showed that nodules are
the most sensitive organs to the salinity (Fig. 2C),
with 25mM NaCl. Salinity entirely prevented the
formation of symbiosis between the sensitive
variety Amdoun 1 and INRAT 87 and Mesorhizobium
ceceri UPMCa7 rhizobia. In contrast, the variety
INRAT 93-1 and INRAT 88 showed a decrease in
nodule biomass formation only in salinity. With
concentrations higher than 25mM NaCl, salinity
completely prevented the nodulation in all chick-
pea varieties.
In continuation of this study, we worked with the
two contrasting varieties Amdoun 1 and INRAT 93-1
and with 25mM NaCl.
thelower one
expresseddifferent
an opposite
ARTICLE IN PRESS
Cicer arietinum nodule conductance under salt stress1031

Page 5

Shoots, roots and nodule growth of two
contrasting varieties in hydroaeroponics
Data in Fig. 3A show that salinity induced a
significant decrease of 30% and 14% in shoot growth
for Amdoun and INRAT 93/1, respectively. The root
biomass was affected more by salinity with a
significant decrease of 43% and 20% for Amdoun 1
and INRAT 93/1 (Fig. 3B). Nodule biomass and number
without salt was higher in Amdoun than INRAT 93/1
(Fig. 3C). Salinity entirely prevented the nodule
formation between the sensitive line, Amdoun 1, and
M. ciceri UPMCa7, INRAT 93 showed a decrease only in
nodule biomass formation under salinity.
In order to assess whether the efficiency in
utilisation of the M. ceceri UPMCa7rhizobia varied
among the two contrasting varieties, the shoot
mass of each individual plant was plotted as a
function of its nodule mass. The results showed a
significant correlation between both parameters
for the two varieties, regardless of the treatment.
The slope of the linear regression was significantly
increased under salt for INRAT 93-1. A significant
difference in this efficiency parameter was found,
with a negative correlation with INRAT 93-1,
whereas it was positive for Amdoun 1.
O2uptake and nodule conductance
Theresponseofnodulated-rootO2
uptake
on variation of rhizospheric pO2was measured in
order to assess the effect of salt on nodule
permeability, as well as the subsequent inhibition
ARTICLE IN PRESS
0
1
2
3
Root DM, g plant-1
B
0
0.1
0.2
0.3
Nodule DM, g plant-1
C
0
1
2
3
Shoot DM, g plant-1
0 mM NaCl
25 mM NaCl
A
35
56
45
Amdoun 1 INRAT 93-1
Figure 3. Effect of NaCl (25mM) on shoots (A), roots (B)
and nodule (C) biomass of two chickpea varieties:
Amdoun 1 and INRAT 93-1. NaCl 25mM was added to
the medium at the beginning of the culture (at the first
day of seedling transfer on the culture medium). Plants
were harvested 45 DAS (day after sowing). Data are the
means7SD of five replicates. The numbers on the
histogram indicate the nodule number per plant.
0.0
1.2
0.4
0.8
1.2
1.6
0 2550 75100
NaCl, mM
Root DM, g plant-1
Shoot DM, g plant-1
0.0
0.08
0.4
0.8
B
Amdoun1
INRAT 93-1
INRAT 88
INRAT 87
A
0.00
0.02
0.04
0.06
Nodule DM, g plant-1
C
Figure 2. Effect of salinity on shoot (A), root (B) and
nodule (C) biomass of 4 chickpea varieties: Amdoun 1,
INRAT 93-1, INRAT 88 and INRAT 87. The NaCl concentra-
tions of 0, 25, 50, 75 and 100mM were added to the
irrigation solution at the beginning of the culture (at the
first day of seedling transfer on the culture medium).
Plants were harvested 45 day after sowing (DAS). Data
are the means7SD of five replicates.
B. L’taief et al. 1032

Page 6

of nitrogenase-linked respiration. In the first
experiment the measurement was performed with
initial pO2 of 15, 21, 30 and 40kPa O2. Nodule
respiration was stimulated steadily by raising
rhizospheric pO2(Fig. 4). However, this stimulation
was higher for Amdoun than INRAT 93/1. In the
presence of salt, this effect was more marked in
latter variety, but it was not significant at pO2more
than 20Kpa (Fig. 4).
Data in Fig. 5A show that salt did not affect the
nodule permeability, i.e. the slope of the regression
models of O2uptake by nodulated root as a function
of pO2, for INRAT 93-1. Without salt, a significant
difference was found in both the varieties: nodule
permeability for Amdoun 1 in the range of
7mm3h?1compared to 3 for INRAT 93-1.
The nodule conductance was calculated as the
ratio of the slope of the regression of nodulated-
root O2uptake as a function of pO2in Fig. 4 upon
the total nodule surface in Fig. 3C. Without salt, a
significant difference in the regression slope was
found between Amdoun 1 (7mm3h?1) and INRAT
93/1 (3mm3h?1) (Fig. 5B). Salt did not change this
value for INRAT 93/1 significantly. Without salt, a
significant difference in nodule conductance was
found between Amdoun (20mmh?1) and INRAT 93/1
(5mmh?1). The nodule conductance was not sig-
nificantly affected by salt in INRAT 93/1.
The nodulated-root O2uptake at the ambiantal pO2
of 20kPa was calculated by applying the Fick law of
diffusion with the values of nodule conductance and
surface. Without salt, Amdoun showed a value of
510mmolO2pl?1h?1than was significantly higher
than 255mmolO2pl?1h?1for INRAT 93/1. This nodu-
lated-root O2uptake was significantly increased by
salt to 520mmolO2pl?1h?1for INRAT 93/1.
Discussion
The present work shows that the salinity did not
alter the nodule conductance in chickpea nodules
in the INRAT 93-1 variety. This tolerant variety
showed no significant differences in nodule con-
ductance under salinity (Fig. 5B). Consequently,
salt tolerance appears to be associated with
stability in nodule conductance of the tolerant
variety under salinity. This supports their lower
respiration linked to their nitrogenase activity by
comparison to the sensitive variety Amdoun 1 in the
control treatment, as shown by their lower de-
crease of permeability, i.e. the product of nodule
ARTICLE IN PRESS
0
400
800
1200
0 mM NaCl
Amdoun1
0
400
800
1200
1520 30 40
0 mM NaCl
25 mM NaCl
INRAT 93-1
Oxygen uptake, µmol O2.h-1 .plant-1
pO2, KPa
Figure 4. Effect of rhizosphere pO2and NaCl (25mM) on
in situ measurements of the oxygen uptake by chickpea
nodulated-root of Amdoun 1 (Top panel) and INRAT 93-1
(Bottom panel). NaCl 25mM was added to the medium at
the beginning of the culture. Plants were harvested 45
DAS (day after sowing). Data are the means7SD of five
replicates.
0
2
4
6
8
10
Nodule permeability, mm3.h-1
0 mM NaCl
25 mM NaCl
A
0
10
20
30
40
Amdoun1 INRAT 93-1
Conductance to O2 diffusion, µm.s-1
B
Figure 5. Effect of salinity (25 mM NaCl) on: (A) nodule
permeability; and (B) conductance to the oxygen diffu-
sion, in two varieties of chickpea, Amdoun 1 and INRAT
93-1. NaCl 25mM was added to the medium at the
beginning of the culture. Plants were harvested 45 DAS
(day after sowing). Data are the means7SD of five
replicates.
Cicer arietinum nodule conductance under salt stress 1033

Page 7

conductance and nodule surface. The latter is
proportional to the nodule respiration supporting
N2 fixation. This shares similarities with the
stability in nodule conductance that has been
found with legumes grown under P deficiency in
previous work with soybean (Ribet and Drevon,
1995) and common-bean (Vadez et al., 1996).
The differences in conductance might be due to
differences among genotypes in response of nodu-
lated-root respiration to raising external pO2.
Indeed, nodule conductance depends upon the
overall permeability of the whole population of
nodules, i.e. the slope of the response curve of
nodulated-root respiration as a function of external
pO2. Consistently, higher permeability was ob-
served for sensitive (Amdoun 1) than for tolerant
variety (INRAT 93-1) (Fig. 5A). The stimulatory
effect of pO2on nodulated root respiration for both
chickpea varieties (Fig. 4) demonstrates that below
25kPa O2, and particularly at ambiantal pO2, the
nodule energetic metabolism supporting N2fixation
is oxygen limited. It confirms the conclusion that
nodule N2-fixing metabolism is not limited by the
shoot supply of photosynthates.
The differences in capacity to form nodules
under salinity can explain the differences in nodule
conductance. Without salt, nodule biomass in the
Amdoun 1 variety was higher than INRAT 93-1,
whereas salinity completely prevented the forma-
tion of symbiosis between the sensitive variety
Amdoun 1 and M. ceceri UPMCa7 rhizobia. In
contrast,variety INRAT
decrease in nodule biomass in salinity. These results
confirm the salt sensitivity of Amdoun 1. Generally,
nodular activity is less affected by salt than
nodulation (Bekki et al, 1987, Hafeez et al, 1988;
Singleton and Bohlool, 1983). However, the infec-
tion process appears to be the most sensitive to salt
(Velagaleti and Marsh, 1989; Zahran and Sprent,
1989). Bhardwaj (1975) showed that the salinity
tolerance limits of Rhizobium and Bradyrhizobium
spp. are much higher than those of the legume
host, which suggested that it is predominantly the
tolerance of the host plant that dictates the
possibility of a successful symbiosis.
The salt-induced lower decrease in nodule con-
ductance in the tolerant variety INRAT 93-1 may be
a systemic response for the tolerant variety to
maintain the same N demand for plant growth per
individual nodule under salinity as under the
control treatment. The increase in efficiency in
utilisation of the rhizobial symbiosis (EURS) under
salinity for the tolerant variety INRAT 93-1 sub-
stantiates the conclusion that the respiratory cost
of N2fixation, and its subsequent contribution to
plant growth, is altered by salinity. The latter could
93-1only showeda
be further addressed by simultaneous measure-
ments of nitrogenase activity and nodule respira-
tion for the contrasting variety with and without
salinity. The stability in nodule conductance in the
salt-tolerant variety INRAT 93-1 could be linked to a
specific use of fixed nitrogen in the osmotic
adjustment. Indeed, chickpea accumulates free
proline in response to hyperosmotic stress, as do
many other organisms (Armengaud et al., 2004)
including P. vulgaris (Raggi, 1994) and Medicago
sativa (Irigoyen et al., 1992) in both leaves and
nodules. However, accumulation of free proline is
not always associated with tolerance to osmotic
constraints, since a large increase in proline was
found by Andrade et al. (1995) in leaves of four
bean cultivars subjected to drought stress. The
greatest increases in proline occurred in the
drought-susceptible cultivars. A negative relation-
ship between salt tolerance and proline accumula-
tion has been reported by other authors (Petrusa
and Winicov, 1997). Thus, it would be interesting to
determine salt effects on free proline accumulation
inthenodulesof contrasting
C. arietinum. Alternatively, the stability in nodule
conductance in the tolerant variety INRAT 93-1 may
result from stimulation of respiratory activity in
nodule tissues in order to produce sufficient malate
for osmotic adjustment, like guard-cells using K+-
malate for the osmoregulation (Tablott and Zeiger,
1998). Malate, mannitol and well-known K+, con-
tribute to the decrease in osmotic potential in
several species (Peltier et al., 1997). Part of the
malate might also be used to offset anion deficit,
Na+
concentration (1–1.5mmolg?1DW) that is
twice higher than that of Cl?in some line of M.
truncatula under salinity (Aydi et al., 2002). This
might compete with malate bacteroid supply for N2
fixation, and result in reduction of N2-dependent
growth in spite of the increase in nodule O2
consumption. Nodule malate is synthesised by
carboxylation of phosphoenolpyruvate (PEP) from
the glycolysis of sucrose coming from photosynth-
esis. The phosphoenolpyruvate carboxylase (PEPC;
EC 4.1.1.31), key enzyme in this malate synthesis
(Ga ´lvez et al., 2000), is stimulated by salt in ILC
1919, a relatively salt tolerant variety of chickpea,
resulting in root-nodule higher malate concentra-
tions (Soussi et al., 1999).
In conclusion, this work is the first report of
nodule stimulation by raising oxygen concentration
in its rhizospheric environment in the C. arieti-
num–M. ciceri symbiosis. It shows that nodule
conductance is not changed by salinity resulting in
lower nodule permeability under salinity for toler-
ant symbioses. Since a larger proportion of respira-
tory energy is associated with maintenance under
varieties of
ARTICLE IN PRESS
B. L’taief et al.1034

Page 8

abiotic stresses (Serraj et al., 1995), more inves-
tigations are necessary to assess the diversion of
photoassimilates and/or fixed nitrogen from growth
processes towards maintenance processes among
contrasting C. arietinum symbioses under salinity.
Acknowledgements
This work was supported by the Aquarhiz Project
no. INCO-CT-2004-509115, including a fellowship
allocated to Boulbaba L’taief for his work in INRA-
ENSAM, Montpellier.
References
Andrade JL, Saavedra AL, Trejo CL. Proline accumulation
in leaves of four cultivars of Phaseolus vulgaris L. with
different drought resistance. J Exp Bot 1995;57:
149–57.
Anthon GE, Emerich DW. Developmental regulation of
enzymes of sucrose and hexose metabolism in effec-
tive and ineffective soybean nodules. Plant Physiol
1990;92:346–51.
Armengaud P, Thiery L, Buhot N, Grenier-de March G.
Savoure ´, Transcriptional regulation of proline bio-
synthesis in Medicago truncatula reveals developmen-
tal specific features. Physiol Plant 2004;120:442–50.
Arrese-Igor C, Gonzalez EM, Gordon AJ, Minchin FR,
Galvez L, Royuela M. Sucrose synthase and nodule
nitrogen fixation under drought and other environ-
mental stresses. Symbiosis 1999;27:189–212.
Aydi S, Jebara M, Abdelly C. Etude comparative de
l’action du sel sur le comportement de deux ligne ´es de
Medicago truncatula en condition de fixation symbio-
tique de l’azote. 1e `res journe ´es maghre ´bines de
biotechnologieet symposium
d’azote. Hammamet, 2002. p. 15–8.
Bekki A, Trinchant JC, Rigaut J. Nitrogen fixation (C2H2
reduction) by Medicago nodules and bacteroids under
sodium chloride stress. Physiol Plant 1987;71:61–7.
Bhardwaj KKR. Survival and symbiotic characteristics of
Rhizobium
in saline-alkali
1975;43:377–85.
Dua RP, Sharma PC. Salinity tolerance of kabuli and Desi
chickpea genotypes. International Chickpea and Pi-
geonpea Newsletter 1995;2:19–22.
De Lima M, Oresnik IJ, Fernando SM, Hunt S, Smith R,
Turpin DH. The relationship between nodule adeny-
lates and the regulation of nitrogenase activity by
oxygen in soybean. Physiol Plant 1994;91:687–95.
Denison RF, Hunt S, Layzell DB. Nitrogenase activity,
nodule respiration and O2 permeability following
detopping of alfalfa and birdsfoot trefoil. Plant Physiol
1992;98:894–900.
Drevon JJ, Deransart C, Irekti H, Payer H, Roy G, Serraj R.
La salinite ´ (NaCl) abaisse la conductance des nodosite ´s
de le ´gumineuse a ` la diffusion de l’oxyge `ne, in: J.J.
satellite:Fixation
soils. PlantSoil
Drevon (Ed.), Facteurs Limitant la Fixation Symbio-
tique de l’Azote dans le Bassin Me ´diterrane ´en, INRA
Editions, les colloques, vol. 77. 1994. p. 73–84.
Drevon JJ, Kalia VC, Heckmann MO, Pe ´delahore P. In situ
openflow assay of acetylene reduction activity by
soybean root nodules: influence of acetylene and
oxygen. Plant Physiol Biochem 1988;26:73–8.
Ga ´lvez S, Hirsch AM, Wycoff KL, Hunt S, Layzell DB,
Kondorosi A. Oxygen regulation of a nodule-located
carbonic anhydrase in alfalfa. Plant Physiol 2000;124:
1059–68.
Garg BK, Gupta IC. Nodulation and symbiotic nitrogen
fixation under salt stress. Curr Agric 2000;24:23–35.
Gonza ´lez EM, Ga ´lvez L, Arrese-Igor C. Abscisic acid
induces a decline in nitrogen fixation that involves
leghaemoglobin, but is
synthase activity. J Exp Bot 2001;52:285–93.
Hafeez FY, Aslam Z, Malik KA. Effect of salinity and
inoculation on growth, nitrogen fixation and nutrient
uptake of (Vigna radiata (L.) Wilczek). Plant Soil 1988;
106:3–8.
Hunt S, King BJ, Layzell DB. Effects of gradual increases
in oxygen on nodule activity in soybean. Plant Physiol
1989;91:315–21.
Hunt S, Layzell DB. Gas exchange of legume nodules and
the reduction of nitrogenase activity. Ann Rev Plant
Physiol Mol Biol 1993;44:483–511.
Irekti H, Drevon JJ. Acide abcissique et conductance a ` la
diffusion de l’oxyge `ne dans les nodosite ´s de haricot
soumises a ` un choc salin. In: J.J. Drevon, B. Sifi,
editors, Fixation Symbiotique de l’Azote et De ´vel-
oppement Durable dans le Bassin Me ´diterrane ´en, INRA
Editions, les colloques, vol. 100. 2003. p. 371–80.
Irigoyen JJ, Emirich DW, Sanchez-Diaz M. Water stress
induced changes in concentrations of proline and total
soluble sugars in nodulated alfalfa (Medicago sativa)
plants. Physiol Plant 1992;84:55–60.
Jebara M, Drevon JJ. Genotypic variation in nodule
conductance to the oxygen diffusion in common bean
(Phaseolus vulgaris). Agronomie 2001;21:667–74.
King BJ, Layzell DB. Effects of increases in oxygen
concentration during the argon induced decline in
nitrogenase activity in root nodule of soybean. Plant
Physiol 1991;96:376–81.
Minchin FR. Regulation of oxygen diffusion in legume
nodules. Soil Biol Biochem 1997;29:881–8.
Peltier JP, Marigo D, Marigo G. Involvement of malate and
mannitol in the diurnal regulation of the water status
in members of Oleaceae. Trees Struct Funct 1997;
12:27–34.
Petrusa LM, Winicov L. Proline status in salt-tolerant
and saltsensitive alfalfa cell lines and plants in
response to NaCl. Plant Physiol Biochem 1997;35:
303–10.
Raggi V. Changes in free amino acids and osmotic
adjustment in leaves of water-stressed bean. Physiol
Plant 1994;91:890–903.
Ramos MLG, Gordon AJ, Minchin FR, Sprent JI, Parsons R.
Effect of water stress on nodule physiology and
independent ofsucrose
ARTICLE IN PRESS
Cicer arietinum nodule conductance under salt stress 1035

Page 9

biochemistry of a drought tolerant cultivar of common
bean (Phaseolus vulgaris L.). Ann Bot 1999;83:57–63.
Ribet J, Drevon JJ. Increase in permeability to oxygen
and in oxygen uptake of soybean nodules under
limiting phosphorus nutrition. Physiol Plant 1995;94:
298–304.
Saadallah K, Drevon JJ, Abdelly C. Nodulation et croissance
nodulaire chez le Haricot (Phaseolus vulgaris L.) sous
contrainte saline. Agronomie 2001;21:627–34.
Serraj R, Roy G, Drevon JJ. Salt stress induces a decrease
in the oxygen uptake of soybean nodules and their
permeability to oxygen
1994;91:161–8.
Serraj R, Fleurat-Lessard P, Jaillard B, Drevon JJ.
Structural changes in the inner-cortex cells of soybean
root-nodules are induced by short-term exposure to
high salt or oxygen concentrations. Plant Cell Environ
1995;18:455–62.
Serraj R, Vasquez-Diaz H, Drevon JJ. Effects on nitrogen
fixation, oxygen diffusion and ion distribution in
soybean, common bean and alfalfa. J Plant Nutr 1998;
21:475–88.
Serraj R, Vasquez-Diaz H, Hernandez G, Drevon JJ.
Genotypic difference in the short-term response of
nitrogenase activity (C2H2reduction) to salinity and
oxygen in the common bean. Agronomie 2001;21:
645–51.
Singleton PW, Bohlool BB. Effect of salinity on the
functional components of soybean–Rhizobium japoni-
cum symbiosis. Crop Sci 1983;23:815–8.
diffusion.PhysiolPlant
Soussi M, Lluch C, Ocan ˜a A. Comparative study of
nitrogen fixation and carbon metabolism in two
chick-pea (Cicer arietinum L.) cultivars under salt
stress. J Exp Bot 1999;50:1701–8.
Sprent JI. The effects of water stress on nitrogen fixing
root nodules. New Phytol 1972;71:451–60.
Tablott LD, Zeiger M. The role of sucrose in guard cell
osmoregulation. J Exp Bot 1998;49:329–37.
Vadez V, Rodier F, Payre H, Drevon JJ. Nodule perme-
ability and nitrogenase-linked respiration in bean
genotypes varying in the tolerance to P deficiency.
Plant Physiol Biochem 1998;34:871–8.
Vance CP, Heichel GH. Carbon in N2fixation: limitation or
exquisite adaptation. Ann Rev Plant Physiol Plant Mol
Biol 1991;42:373–92.
Velagaleti RR, Marsh S. Influence of host cultivars and
Bradyrhizobium strains on the growth and symbiotic
performance of soybean under salt stress. Plant Soil
1989;119:133–8.
Vincent JM. Manual for the practical study of root nodule
bacteria. IBP handbook 15. Oxford: Blackwell; 1970.
p. 164.
Witty JF, Skotl L, Revsbech NP. Direct evidence for
changes in the resistance of legume root nodule toO2
diffusion. J Exp Bot 1987;38:1120–40.
Zahran HH, Sprent JL. Effects of sodium chloride and
polyethylene glycol on root-hair infection and nodula-
tion of Vicia faba L. plants by Rhizobium leguminosar-
um. Planta 1989;167:303–9.
ARTICLE IN PRESS
B. L’taief et al. 1036