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REGULAR ARTICLE
Drought Stress Response in Tunisian Populations of Faba Bean
(Vicia faba L.)
Imtiez Ziadi Backouchi*, Marwa Aouida and Moez Jebara
University Tunis El Manar, Centre de Biotechnologie Borj Cedria, Laboratoire des Légumineuses. BP 901, 2050
Hammam Lif, Tunisie.
ABSTRACT
Twelve Tunisian populations of Vicia faba were subjected to moderate (50% FC) and severe (30% FC) drought stress
in order to screen genotypes for drought tolerance. The experiment was conducted to evaluate the effect of drought
severities on physiological traits, gas exchange, lipid peroxidation, chlorophyll content and antioxidative enzymes in
Vicia faba populations. Water stress induced significant variations in agronomic character among studied populations.
Moderate water stress increased the MDA content in leaves of Mateur major population and Mahdia, Eljem, Mateur
and Fahes minor populations. In addition it decreased in Ltaifia, Takelsa leaves. Sever water stress enhanced the
MDA content in Mateur major population, Mahdia, Ltaifia, Fahes and reduced in Takelsa leaves. In addition, leaf
internal CO2 concentration, net photosynthetic rate, transpiration rate, stomatal conductance and water use efficiency
of the populations were affected by the reduced soil water content. Vicia faba populations studied increased their
chlorophyll content independently of applied water stress, they had an arbitrary response. Our results showed the
importance of SOD and GPOX activities in leaves of faba bean against drought stress conditions and a complementary
APX activity to that of SOD while no major function of CAT activity in Vicia faba populations. Results indicated
that Mateur major population as Ltaifia and Takelsa minor populations were the most tolerant to water stress.
However, Mahdia and Fahes minor populations were the most sensitive. Ltaifia was a genetic homogeneous
population and water deficit tolerant performance can be considered as a selected cultivar with very interesting
agronomic character. Takelsa was a heterogeneous genetic population with high water stress tolerance thus may be
very interesting for breeding program. Sensitive Fahes population had a high level of heterogeneity presents an
interesting reservoir of genes for breeding program.
The tolerant populations have initially high MDA values which were an indicator of water stress tolerance.
Keywords: Drought stress, Vicia faba L, Physiological traits, Gas exchange, Lipid peroxidation, Chlorophyll content,
Antioxidative enzymes.
INTRODUCTION
The faba bean (V. faba L.) plant is one of the most
important crops in Tunisia due to its high nutritive
value in terms of both energy and protein contents.
Therefore, increasing faba bean production is one of
the most important targets of agricultural policy in
Tunisia. Recent statistics (2007, 2008, 2009 and
2010) showed important increase in the part devoted
to faba bean among total grain legume (68%). This
increase was due to the important growth in faba
bean small seeded acreage in the last period
becoming in 2010 season 25,000 ha, about three
times more important than the grown area during
the eighties period. It was expected that the increase
in faba bean small seeded will continue to reach in
2016, about 35,000 ha as projected by the Ministry
of Agriculture 1.
In order to define the criteria needed to develop
cultivars suitable for drought-prone areas, it was
Journal of Plant Biology Research 2015, 4(1): 55-72 eISSN: 2233-0275
pISSN: 2233-1980
http://www.inast.org/jpbr.html
*Corresponding author: Imtiez Ziadi Backouchi Corresponding author e-mail: [email protected]
Tel: +21625387353 Fax: +21679325948
J. Plant Bio. Res. 2015, 4(1): 55-72
56
first necessary to identify the traits associated with
drought response and to explore how they were
expressed in this species 2.
Faba bean was more sensitive to drought than some
other grain legumes including common bean, pea
and chickpea 3. Although genotypic differences in
the response of faba bean to drought have been
documented 4, 2, 5. The development of drought-
tolerant cultivars was essential to improve the yield
stability of faba bean. Plant breeders have
considerable capacity to evaluate breeding material
for drought tolerance under field conditions based
on grain yield at drought-prone sites 6, 7. The
genotypes that show better performance under
hostile environments generally possess some
unidentified physiological attributes of tolerance to
environmental stresses in good conditions 8.
Understanding the physiological basis of drought
tolerance in faba bean was important to identify
traits, which can be used as indirect selection
criteria.
Abiotic stresses are considered the most important
factors for yield reductions in agricultural crops. It
is estimated that yield losses in agricultural crops
due to different abiotic stresses include 15% due to
low temperature, 17% due to drought, 20% due to
salinity, 40% due to high temperature, and 8% due
to other environmental factors 9.
Water is an essential factor during the entire life of
plant growth, from seed germination to the final
growth stage 10.
The rate of plant growth depends on several
important events, such as cell division, cell
enlargement and cell differentiation, as well as
genetic, morphological, physiological, and
ecological events and their complex interactions,
which are severely affected by abiotic stress11.
When plants are exposed to harsh conditions (e.g.,
salinity stress), plants exhibit certain adaptive
features, which may be morphological, anatomical,
physiological, or biochemical in nature to minimize
the deleterious effects of unfavorable environmental
conditions 12 and to help plants to sustain and
thrive under stress conditions. In this regard, plants
perceive stress through their roots and send signals
to change their metabolism for the activation of
defense mechanisms in different parts of the plant
13.
The detoxification of ROS in plant cells can be
categorized as enzymatic and non-enzymatic in
almost all plants. The non-enzymatic antioxidants
include ascorbic acid, tocopherols, flavonoids,
phenolics and carotenoids. The important anti-
oxidant enzymes include peroxidase (POD),
superoxide dismutase (SOD), catalase (CAT),
glutathione reductase (GR), and ascorbate
peroxidase (APX) 14.
There were many reports in the literature that
underlined the intimate relationship between
enhanced or constitutive antioxidant enzyme
activities and increased resistance to environmental
stress 15.
The aim of this study was to investigate the effects
of drought stress on physiological traits, chlorophyll
content, nitrogen content, photosynthetic
efficiency, lipid peroxydation and changes in
enzyme activities (APX, CAT, GPOX, and SOD) in
Tunisian Vicia faba populations in order to
determine the tolerant and sensitive populations to
water deficit stress.
Tolerant to drought stress and homogenous genetic
populations are recommended for use by farmers.
Heterogeneous genetic population with high water
stress tolerance and sensitive population had a high
level of heterogeneity present an interesting
reservoir of genes for breeding program. In
addition, it is necessary to identify the traits
associated with drought response and to explore
how they are expressed in Vicia faba in order to
identify the criteria needed to develop cultivars
suitable for drought stress.
MATERILS AND METHODS
Plant material and growth conditions
Experiment was conducted under greenhouse in the
Centre of Biotechnology of Borj Cedria (CBBC),
Tunisia. Three Vicia faba major populations
(Eljem, Alia and Mateur) and seven Vicia faba
minor populations (Korba, Eljem, Mahdia, Mateur,
Fahes, Takelsa and Ltaifia) were collected from
different regions in Tunisia during 2010 and two
reference varieties Locale and Saber02 were used
for study. Seeds were surface disinfected by
immersion in ethanol 95% for 30 seconds, followed
by immersion in a 5% sodium hypochlorite solution
for 10 minutes and then rinsed five times with sterile
distilled water.
Seeds were soaked for 24 h in distilled water. The
trial was conducted in plastic pots of 2.5 l volume.
J. Plant Bio. Res. 2015, 4(1): 55-72
57
The pots were filled with a mixture of agricultural
soil of nursery CBBC.
All plants were grown in individual pots. The
studied populations underwent three water
treatments (90, 50 and 30% of field capacity). Five
replicates for each population per treatment were
used. Drought treatment was applied at flowering
stage. For enzyme activity plant were frozen in
liquid N2 and stored at -80°C. Plant morphological
(plant height, number of flowers, aerial fresh
weight, length of roots, number of nodules, root
fresh and dry weight), leaf physiological ( Stomatal
conductance (gs), net photosynthesis (A),
transpiration rate (E) and internal CO2
concentration (Ci), chlorophyll content, nitrogen
content) and biochemical (lipid peroxidation,
protein content and antioxidant enzyme )
measurements were performed.
Determination of chlorophyll content
Leaf chlorophyll content was
spectrophotometrically determined according to
16 from 100 mg fresh leaf tissues extracted in dark
for 72 h in 80 % acetone. Extract absorbance was
measured at 645 and 663 nm.
Nitrogen content assessment
The total nitrogen was determined by the micro-
Kjeldahl method 17 after submitting the plant
material to oxidation by sulphuric digestion
(H2SO4).
Measurements of photosynthetic gas exchange
Stomatal conductance (gs), net photosynthesis (A),
transpiration rate (E) and internal CO2
concentration (Ci) were determined at flowring
using a portable gas exchange measuring system
(LCpro+, UK). Measurements were done between
10:00 and 12:00 h under atmospheric CO2 and full
sunlight. Water use efficiency (WUE) was
calculated as photosynthetic rate divided by
transpiration rate (A /E).
Total soluble proteins and antioxidant activities
Total soluble protein was measured according to
Bradford’s method 18. A sample of 1 g of fresh
leaves and 1 g of fresh root were ground in a mortar
with 10 % (w/w) polyvinyl- pyrrolidone in 1 mL of
50 mM phosphate buffer (pH7.8) containing 0.1
mM EDTA, 0.1 % (v/v) Triton X-100, 1 mM
phenylmethanesulfonyl fluoride (PMSF). The
extract was centrifuged at 13,000 g for 20 min, and
the supernatant was used to determine enzyme
activities. The extraction buffer contained in
addition 5mM ascorbate for analysis of APX 19.
Superoxide dismutase (SOD) activity was
determined spectrophotometrically by measuring its
ability to inhibit the photoreduction of nitroblue
tetrazolium (NBT) at 560 nm20. The reaction
solution contained 50 mM K-phosphate (pH 7.8),
0.1 mM EDTA, 10 mM L-methionine, 2.7 µM
riboflavin and 75 µM NBT. One unit of SOD
activity was defined as the amount of enzyme that
inhibited 50 % of NBT photoreduction at 25 °C.
Guaicol peroxidase (POX) was assayed according
to 21 following the evolution of the kinetics of the
enzyme at 470 nm during 1 min ( = 26.6 mM-1 cm-
1). POX activity was measured as oxidation of
guaiacol (9 mM) in the presence of H2O2 (19 mM).
Lipid peroxidation
Lipid peroxidation in leaves was assayed using the
thiobarbituric acid (TBARS) method modified
according to22. A sample of 500 mg fresh leaves
was homogenized in 3 ml of 0.1 % TCA solution.
The homogenate was centrifuged at 10,000 g for 20
min, and 0.5 ml of the supernatant was added to 1
ml of 0.5 % TBA in 20 % TCA. The absorbance of
the supernatant was determined at 532 nm. The
value for non-specific absorption at 600 nm was
subtracted. The amount of malondialdehyde (MDA)
was calculated using the extinction coefficient =
155 mM-1 cm-1.
Statistical analysis
Data were submitted to analysis of variance
(ANOVA) using the STATISTICA software
(http://www.statsoft.com). Means were compared
by the Fisher’s LSD test (P < 0.05).
RESULTS
J. Plant Bio. Res. 2015, 4(1): 55-72
58
I. Modulation of morphological parameters
under water stress
The Vicia faba major populations Alia and Mateur
had the highest plants (61 and 56 cm); while Eljem
had the shortest (46 cm) and variation under water
stress was not significantly affected. Particularly at
30% of field capacity (FC), plant height decreased
and under 50% FC plant height was also decreased,
excepted Eljem population which not significantly
increased (54 cm). All plant height Vicia faba minor
populations were decreased under 50% FC excepted
Eljem and Saber 02 which showed the increase of
their plant height (1,16 and 11,37%). At 30% of FC,
plant height of minor populations decreased
exceptionally Mateur and Ltaifia populations that
proved a clear recovery however Takelsa and Korba
(9,04 and 10,72 %) were the less affected ones.
The number of flowers was strongly affected by
water stress for all tested populations. The Vicia
faba major Alia was the most affected at 50% of FC
and Eljem was the most sensitive under sever
drought stress, while Mateur population was the
most tolerant to water stress. (Figure1A). The minor
populations Eljem as Saber02 variety were the most
tolerant while Mateur as locale variety were the
most affected under moderate stress. Korba and
Ltaifia populations were the least affected but Eljem
and Mahdia ones were strongly affected by 30% of
FC (Figure 2A).
Only Eljem major population stimulated growth at
50% of FC whereas at sever water stress, all major
populations elucidated important reduction in their
aerial fresh weight. Mateur with higher production
was the most tolerant (Figure 1B). Aerial fresh
weight increase at 50% of FC among Eljem minor
population and Saber 02 variety. Mahdia and Fahes
populations indicated the lowest values. The most
of populations pointed out the decrease of aerial
fresh weight under 30% of FC excepted Mateur and
Takelsa populations that indicated recovery (Figure
2B). In addition to its ability to tolerate water stress,
Takelsa population was heterogeneous as well as
interesting for breeding program.
Under moderate drought stress condition Eljem and
Alia major populations lived an increase in their
aerial dry weight. While during severe drought
stress condition, all major populations evinced a
reduction in their aerial dry weight moreover
Mateur population was the most tolerant at 30%
(Figure 1C). At 50% of FC, minor population
demonstrated a reduction in aerial dry weight
particularly Mahdia population was the most
affected furthermore excepted Eljem and Saber 02
which exhibited increase of aerial dry weight. At
30% of FC, aerial dry weight of all populations was
decreased excepted Mateur population. In general
Takelsa and Ltaifia populations were the least
affected by tested water stress, at two levels of stress
(Figure 2C).
Results proved that water deficit conditions induced
important variations on length of roots. Vicia faba
major populations indicated a reduction on the
length of roots except Mateur population that its
length of roots increased with an increase of water
stress (Figure 1D). Eljem minor population was the
most tolerant although Mahdia was the most
sensitive at 50% of FC. However, Korba, Latifia
and Saber 02 marked an increase of length of roots
at 30% of FC principally Fahes population was the
more sensitive and its roots were most reduced
(Figure 2D).
Mateur population had the more fitness to nodulate.
At 50% of FC, only Alia population number of
nodules increased and sever stress (30%) reduced
significantly number of nodules. It was observed
treatment effect on major populations (Figure 1E).
All minor populations showed decrease of number
of nodules at 50% of FC excepted Eljem population
that illustrated an increase of nodules number.
Number of nodules increased for Korba, Mateur,
Saber 02 and Ltaifia populations with sever tested
drought stress, which is directly related to the
tolerance of these populations. The number of
nodules in Fahes population was more affected by
water stress indicating its sensitivity to water deficit
(Figure 2E). Fahes population with a high level of
heterogeneity presents an interesting reservoir of
genes for breeding program.
Results indicated that water deficit conditions had
significant effect on root dry weight of Vicia faba.
Besides, major population Mateur had the highest
root dry weight at 50% of FC. The root dry weight
decreased significantly for all populations under
sever drought stress (Figure 1F). The Eljem and
Takelsa minor populations were the most resistant
however Mahdia was the most affected with
moderate stress. However, Takelsa population had
the important root dry weight while Fahes had the
lowest root dry weight at 30% of FC. The trend
demonstrated by Fahes population may be
J. Plant Bio. Res. 2015, 4(1): 55-72
59
indicative of sensitivity to water stress conditions
(Figure 2F).
Figure 1. Effect of water stress on number of flowers, aerial fresh weight, aerial dry weight, length of roots,
number of nodules and roots dry weight in Vicia faba major populations. Different letters denote significant
differences (LSD, P<0.05)
Figure 2. Effect of water stress on number of flowers, aerial fresh weight, aerial dry weight, length of roots, number
of nodules and roots dry weight in Vicia faba minor populations. Different letters denote significant differences (LSD,
P<0.05).
II. Effect of water stress on gas exchange
J. Plant Bio. Res. 2015, 4(1): 55-72
60
The leaf internal CO2 concentration (Ci) was
increased for Eljem and Mateur major populations
under moderate stress and Ci was increased with
30% FC for Alia and Mateur populations (Table 1).
All minor populations showed increase in Ci 50% of
FC excepted Ltaifia population that showed slight
decrease. Also, all minor populations showed Ci
increase and Ltaifia and Takelsa populations were
the most affected under severe drought stress (30%)
(Table 2).
Applied moderate stress decreased transpiration rate
(E) of all major populations and only Eljem and
Mateur populations exhibited recovery with sever
water deficiency (Table 1). The (E) decreased with
50%FC for all minor populations excepted Mahdia
and Saber02 that demonstrated increase in their
transpiration rate. Similarly, FC transpiration rate
decreased for all minor populations with 30% FC
and excepted Eljem population as Saber02 and
Locale varieties their transpiration rate increased
(Table 2).
Under stress conditions populations studied showed
different stomatal conductance (gs) values. For
major populations gs decreased under 50% FC
however Eljem and Mateur showed a recovery of gs
values with severe drought stress (Table 1). The
applied moderate stress decrease minor populations
(gs) expected Mahdia population and Saber02
variety, which indicated gs increase. Conversely,
Takelsa and Eljem populations as Locale and
Saber02 varieties showed a gs recovery with 30%
FC (Table 2).
The (A) three major populations decreased with
50% FC however we noted spectacular increase of
Eljem and Mateur (A) under sever water
deficiency reach higher level than control (Table 1).
Moderate drought stress caused significant (A)
reductions in all minor populations excepted
Mahdia population. Also, all minor populations
showed decrease of net photosynthesis rate at 30%
FC excepted Eljem population as Locale and
Saber02 varieties which demonstrated a slight
increase (Table 2).
The major population WUE decreased with 50% FC
besides Eljem and Mateur populations indicated a
recovery with 30% (Table 1). Drought stress (50%)
had a negative effect on minor populations WUE
excepted Mateur and Ltaifia populations that
showed enhanced WUE whereas Mateur and Korba
populations as Locale and Saber02 varieties
exhibited a WUE recovery under sever water
deficiency (Table 2).
J. Plant Bio. Res. 2015, 4(1): 55-72
61
Table 1. Effect of water stress (T1=90% FC, T2= 50% FC, T3= 30% FC) on internal CO2 concentration (Ci, µl l-1), transpiration rate (E, mmol H2O m-2 s-1),
Stomatal conductance (gs, mol m-2 s-1) net photosynthesis (A, μmol CO2 m-2 s-1), and (WUE, µmolCO2 mol-1 H2O) in Vicia faba major populations. Different
letters denote significant differences (LSD, P<0.05).
Table 2. Effect of water stress (T1=90% FC, T2= 50% FC, T3= 30% FC) on internal CO2 concentration (Ci, µl l-1), transpiration rate (E, mmol H2O m-2 s-1),
Stomatal conductance (gs, mol m-2 s-1) net photosynthesis (A, μmol CO2 m-2 s-1), and (WUE, µmol CO2 mmol-1H2O) in Vicia faba minor populations. Different
letters denote significant differences (LSD, P<0.05).
Population Ci E gs A WUE
T1 T2 T3 T1 T2 T3 T1 T2 T3 T1 T2 T3 T1 T2 T3
ELJEM 154b 257a 72 c 1.58a 0.53c 1.37b 0.08a 0.02b 0.07a 9.91b 1.65c 11.56a 6.27b 3.11c 8.44a
ALIA 192a 186a 296a 3.06a 0.69b 0.23b 0.17a 0.03b 0.01b 17.46a 3.67b 0.48b 5.75a 5.29ab 2.1b
MATEUR 76b 238a 121.5ab 1.17ab 0.61b 1.73a 0.05ab 0.03b 0.08a 9.27b 2.18c 11.72a 8.17a 3.57b 6.82ab
Population Ci E gs A WUE
T1 T2 T3 T1 T2 T3 T1 T2 T3 T1 T2 T3 T1 T2 T3
KORBA 126 b 165.5a 161a 2.38a 1.83b 1.57c 0.12a 0.09 a 0.08 a 16.43a 10.46b 9.08c 6.90a 5.71b
5.78b
ELJEM 126.5a 129a 163a 2.3 a 1.35a 1.55a 0.12a 0.06 a 0.07a 15.69a 8.82b 8.95b 6.94a 6.52a 5.77a
MAHDIA 160.5a 174 a 184.5a 1.19a 1.36a 1.01a 0.05a 0.06a 0.04a 6.69a 7.07a 5.02a 5.61a 5.20a
4.94a
MATEUR 257 a 289 a 258.5a 0.23a 0.13b 0.08b 0.01a 0.005a 0a 0.48a 0.53a 0.29b 2.08b 3.92a 3.42a
FAHES 102.5b 208a 232.5a 0.72a 0.42b 0.40b 0.03a 0.015a 0.015a 4.33a 1.49b 1.17b 5.94a 3.54b 2.92b
SABER02 199a 261a 213.5a 0.53c 0.76b 0.98a 0.02a 0.03a 0.04a 2.01a 1.76a 3.5a 3.81a 2.31a 3.46a
LOCALE 160.5a 269 a 201a 0.50a 0.36b 0.57a 0.02a 0.01a 0.02a 2.24a 0.73a 1.97a 4.46a 2.03a 3.52a
TAKELSA 91a 216.5a 321a 0.53a 0.15b 0.17b 0.02a 0.005b 0.01ab 3a 0.34b 0.12b 5.82a 3a 0.71a
LTAIFIA 172.5b 152.5b 334 a 0.70a 0.54a 0.20b 0.02a 0.02a 0.01a 3.08a 2.6b 0.08c 4.39a 4.83a 0.53b
J. Plant Bio. Res. 2015, 4(1): 55-72
62
Table 2. Effect of water stress (T1=90% FC, T2= 50% FC, T3= 30% FC) on internal CO2 concentration (Ci, µl l-1), transpiration rate (E, mmol H2O m-2 s-1),
Stomatal conductance (gs, mol m-2 s-1) net photosynthesis (A, μmol CO2 m-2 s-1), and (WUE, µmol CO2 mmol-1H2O) in Vicia faba minor populations. Different
letters denote significant differences (LSD, P<0.05).
Population Ci E gs A WUE
T1 T2 T3 T1 T2 T3 T1 T2 T3 T1 T2 T3 T1 T2 T3
KORBA 126 b 165.5a 161a 2.38a 1.83b 1.57c 0.12a 0.09 a 0.08 a 16.43a 10.46b 9.08c 6.90a 5.71b
5.78b
ELJEM 126.5a 129a 163a 2.3 a 1.35a 1.55a 0.12a 0.06 a 0.07a 15.69a 8.82b 8.95b 6.94a 6.52a 5.77a
MAHDIA 160.5a 174 a 184.5a 1.19a 1.36a 1.01a 0.05a 0.06a 0.04a 6.69a 7.07a 5.02a 5.61a 5.20a
4.94a
MATEUR 257 a 289 a 258.5a 0.23a 0.13b 0.08b 0.01a 0.005a 0a 0.48a 0.53a 0.29b 2.08b 3.92a 3.42a
FAHES 102.5b 208a 232.5a 0.72a 0.42b 0.40b 0.03a 0.015a 0.015a 4.33a 1.49b 1.17b 5.94a 3.54b 2.92b
SABER02 199a 261a 213.5a 0.53c 0.76b 0.98a 0.02a 0.03a 0.04a 2.01a 1.76a 3.5a 3.81a 2.31a 3.46a
LOCALE 160.5a 269 a 201a 0.50a 0.36b 0.57a 0.02a 0.01a 0.02a 2.24a 0.73a 1.97a 4.46a 2.03a 3.52a
TAKELSA 91a 216.5a 321a 0.53a 0.15b 0.17b 0.02a 0.005b 0.01ab 3a 0.34b 0.12b 5.82a 3a 0.71a
LTAIFIA 172.5b 152.5b 334 a 0.70a 0.54a 0.20b 0.02a 0.02a 0.01a 3.08a 2.6b 0.08c 4.39a 4.83a 0.53b
J. Plant Bio. Res. 2015, 4(1): 55-72
63
III. Effect of water stress on chlorophyll content
III.1. Chlorophyll a content
The chlorophyll a content increased in leaves of
Eljem major population; however it decreased in
leaves of Alia and Mateur populations at moderate
water stress. Sever water stress enhanced
chlorophyll a content in Alia and Mateur leaves and
reduced chlorophyll a content in Eljem leaves
(Figure 3A).
Moderate drought stress induced increase of
chlorophyll a content in leaves of Mahdia, Korba
and Eljem minor populations and decrease in Fahes,
Locale, Takelsa, Ltaifia, Mateur and Saber 02
leaves (Figure 4A).Under sever drought condition
chlorophyll a content increased in leaves of Fahes,
Mahdia, Locale, Korba, Ltaifia, Mateur and
Saber02 and it decreased in leaves of Eljem and
Takelsa populations (Figure 4A)
III.2. Chlorophyll b content
Chlorophyll b content increased in leaves of Eljem
and Mateur major populations and decreased in Alia
leaves at 50% of FC. Under sever water stress
chlorophyll b content increased in leaves of three
major populations (Figure 3B).
Moderate water stress enhanced chlorophyll b
content in Korba and Ltaifia leaves and reduced
chlorophyll b content in leaves of Fahes, Mahdia,
Locale, Eljem, Takelsa, Mateur and Saber02
(Figure 4B). Chlorophyll b content increased in
leaves of Fahes, Mahdia, Locale, Korba, Mateur and
Saber02 and decreased in Eljem leaves at 30% of
FC (Figure 4B).
III.3. Total Chlorophyll content
The total chlorophyll content was enhanced in
Eljem leaves and reduced in leaves of Alia and
Mateur major populations under moderate water
stress (Figure 3C). At sever water stress total
chlorophyll content increased in Alia and Mateur
leaves and decreased in Eljem leaves (Figure 3C).
At 50% of FC total chlorophyll content increased in
leaves of Korba and Eljem minor populations,
however it decreased in Fahes, Mahdia, Locale,
Takelsa, Ltaifia, Mateur and Saber02 leaves (Figure
4C). Sever drougth stress improved total
chlorophyll content in Fahes, Mahdia, Locale,
Korba, and Ltaifia populations and it reduced total
chlorophyll content in leaves of Eljem, Takelsa,
Mateur and Saber02 populations (Figure 4C).
Figure 3. Effect of water stress on chlorophyll content
in leaves of Vicia faba major populations (µg/g FW).
Different letters denote significant differences (Fisher's
LSD, P<0.05)
Figure 4. Effect of water stress on chlorophyll content
(µg/g FW) in leaves of Vicia faba minor populations.
Different letters denote significant differences (Fisher's
LSD, P<0.05)
J. Plant Bio. Res. 2015, 4(1): 55-72
64
IV. Effect of water stress on nitrogen content (N)
Under moderate stress condition nitrogen content
was enhanced in leaves and roots of Eljem major
population and reduced in leaves and roots of
Mateur and Alia populations (Figure 5A). Sever
water stress increased nitrogen content in leaves and
roots of the three major populations studied
(Figure5 B).
The leaf nitrogen content increased in Ltaifia,
Locale, Fahes and Takelsa. However, it decreased
significantly in Mateur population at 50% of FC
(Figure 5C). Takelsa, Korba, Mahdia, and Saber02
showed increase in their leaf nitrogen content and
decrease in Ltaifia, Mateur and Locale at 30% of FC
(Figure 5C). Root nitrogen content augmented for
Fahes, Takelsa, Korba and Mahdia populations and
reduced for Ltaifia, Eljem, Mateur, Saber02 and
Locale at moderate water stress (Figure 5D). Sever
stress condition enhanced root nitrogen content for
Fahes, Takelsa, Korba and Locale and reduced for
Mahdia, Ltaifia, Eljem, Mateur and Saber02 (Figure
5D).
Figure 5. Effect of water stress on nitrogen content in
leaves and roots of Vicia faba populations (N, %).
Different letters denote significant differences (Fisher's
LSD, P<0.05)
V. Effect of water stress on lipid peroxidation
Under moderate drought condition, the MDA
content increased in the leaves of Alia and Mateur
populations and decreased in Eljem population
(Figure 6A). Sever stress enhanced MDA content in
Mateur leaves and reduced MDA content in Alia
leaves (Figure 6A).
The MDA content increased in Mahdia, Eljem,
Mateur, Fahes and Locale leaves and decreased in
Ltaifia, Korba, Takelsa and Saber02 populations at
50% of FC (Figure 6B).
Mahdia, Ltaifia, Eljem, Mateur, Fahes and Locale
leaves showed increase in MDA content however
Korba and Takelsa leaves exhibited decrease in
their MDA content under severe stress condition
(Figure 6B).
Figure 6. Effect of water stress on lipid peroxidation in
leaves of Vicia faba. Different letters denote significant
differences (Fisher's LSD, P<0.05)
VI. Effect of water stress on protein content
Protein content increased in leaves and roots of Alia
and Eljem major populations with moderate applied
stress while it decreased in leaves and roots of
Mateur. The severe drought stress enhanced protein
content in leaves and decreased it in roots of Alia
and Mateur populations. Contrarily Eljem
population protein content decreased in leaves
while it increased in roots with 30% (Table 3).
The Takelsa, Korba, Mateur and Mahdia minor
populations’ leaves protein content increased with
50% FC and it decreased in all other tested
populations. However roots protein content
J. Plant Bio. Res. 2015, 4(1): 55-72
65
increased only of Ltaifia and Korba populations
(Table 4).
The sever applied drought stress induce recovery of
leaves protein content in Fahes, Korba, Mahdia
populations as Saber02 and Locale varieties and
roots protein content in Takelsa, Fahes, Mahdia,
Mateur populations and Locale variety (Table 4).
VII. Effect of water stress on antioxidative
enzymes
Under moderate drought stress SOD activity
decreased in leaves of Alia and Eljem major
populations and it increased Mateur leaves.
Conversely with applied sever water stress SOD
activity increased in leaves of Alia and Eljem and it
decreased in Mateur leaves (Table 3).
SOD activity increased in Alia and Mateur
populations roots under water moderate drought
condition and it decreased in Eljem roots. The sever
water stress enhanced SOD activity in all major
populations roots (Table 3).
Takelsa, Korba and Mateur minor populations had
initially high SOD activity in leaves (Table 4).
Moderate water stress induced increase of SOD
activity in leaves of Ltaifia, Saber02 and Locale and
it decreased in Takelsa, Fahes, Korba, Mahdia,
Mateur and Eljem leaves. In the same sever water
stress enhanced of SOD activity in leaves of Ltaifia,
Takelsa, Mateur and Eljem populations and it
reduced SOD activity in Fhaes and Mahdia leaves
(Table 4). Roots of Ltaifia and Mateur populations
showed important SOD activity at control
condition. Moderate drought stress increased SOD
activity in roots of Takelsa, Fahes, Korba, Mahdia,
Eljem populations and Locale variety and it
decreased in Ltaifia and Mateur roots (Table 4).
At sever water stress SOD activity enhanced in
roots of Fahes and Eljem populations and it
decreased in Mahdia roots (Table 4).
Leaves of Alia population showed important GPOX
activity at control and moderate water stress.
GPOX activity was stimulated in leaves of Mateur
population and it decreased in leaves of Eljem
population at 50% FC. Sever stress enhanced
GPOX activity in Eljem leaves (Table 3).
GPOX activity increased in roots of Alia and
Mateur major populations and it decreased in Eljem
under moderate drought stress. Sever stress
application augment GPOX activity in roots of
major populations (Table 3).
Moderate water stress enhanced GPOX activity in
leaves of Latifia, Takelsa, Fahes populations and
Saber02 variety. However, it decreased in leaves of
Korba, Mahdia, Mateur and Eljem. GPOX activity
increased in leaves of Ltaifia, Takelsa, Saber02,
Fahes, Mahdia, Mateur and Locale and it was
reduced in Korba and Eljem leaves at 30% FC
(Table 4).
Ltaifia and Mahdia showed initially important
GPOX activity in roots (Table 4).
Moderate water stress increased GPOX activity in
Mateur population roots and it decreased in Ltaifia,
Fahes, Takelsa, Korba and Mahdia roots. Sever
stress enhanced GPOX activity in roots of Korba
and Eljem populations and it reduced in Ltaifia,
Takelsa, Fahes, and Mahdia roots (Table 4).
The present study revealed that application of water
stress was able to enhance the activities of SOD and
GPOX; but reduced activity of CAT and APX in
leaves and roots of Mateur major population.
At 50% of FC there is no relationship between the
behaviors of Eljem major population and its
enzymatic activity.
Water stress induced an increase in both SOD and
GPOX activities in leaves of Ltaifia and Takelsa
minor populations. The higher SOD and GPOX
activities in Ltaifia and Takelsa populations under
drought stress indicated their relative tolerance to
drought conditions.
For leaves of Fahes population SOD activity was
decreased while GPOX activity increased.
CAT and APX had no major antioxidative function
in Vicia faba under water stress conditions.
DISCUSION
This study was conducted to assess physiological
and molecular differences between faba bean
Tunisian populations in response to drought stress.
In this regard, differences among faba bean
cultivars in growth parameters were noticed by El
23, 24, 25, 26, 27. Physiological traits revealed
significant genotypic variation for drought tolerance
in faba bean among the 12 populations studied.
Mateur major population as Ltaifia and Takelsa
minor populations were the most tolerant to water
stress. However, Mahdia and Fahes minor
populations were the most sensitive. Our results
J. Plant Bio. Res. 2015, 4(1): 55-72
66
Table 3. Effect of water stress on the protein content (mg/g FW) and specific activity of antioxidant enzymes (SOD, USOD/μg P), (APX, μM H2O2/min/μg P),
(GPOX, μM H2O2/min/μg P) and (CAT, μM H2O2/min/μg P) in shoots and roots of Vicia faba major populations. Different letters denote significant differences
(Fisher's LSD, P<0.05).
Population Pro/µl SOD APX GPOX CAT
T1 T2 T3 T1 T2 T3 T1 T2 T3 T1 T2 T3 T1 T2 T3
ELJEM
Shoots 1.94b 2.12a 1.36c 59.30ab 44.60b 73.37a 1.14a 0.65b 1.59a 1.90b 0.94b 6.02a 41.71b 47.45b 119.14a
Roots 0.47b 0.5b 0.70a 80.68b 18.30c 102.94a 3.58a 2.74a 2.05a 24.62b 17.34b 42.53a 76.29b 86.11b 1937.73a
ALIA
Shoots 1.53b 1.61b 1.73a 33.48b 17.45b 63.25a 0.39a 0.25a 0.40a 2.04b 7.11a 1.23c 306.76b 551.52a 37.46c
Roots 0.53a 0.58a 0.37b 30.56b 64.75b 215.89a 2.95b 5.09a 1.24b 43.00a 18.79b 45.08a 107.74b 95.78b 595.23a
MATEUR
Shoots 1.25b 0.67c 2.06a 55.44b 101.34a 22.966c 1.46a 0.90b 0.34c 1.58b 5.53a 2.45b 289.62a 80.56b 23.82c
Roots 1.02a 0.76b 0.74b 35.28b 91.20a 102.61a 1.39a 1.10a 0.91a 15.97b 23.63a 24.89a 354.03a 72.15b 56.30b
J. Plant Bio. Res. 2015, 4(1): 55-72
67
Table 4. Effect of water stress on the protein content (mg/g FW) and specific activity of antioxidant enzymes (SOD, USOD/μg P), (APX, μM H2O2/min/μg P),
(GPOX, μM H2O2/min/μg P) and (CAT, μM H2O2/min/μg P) in shoots and roots of Vicia faba minor populations. Different letters denote significant differences
(Fisher's LSD, P<0.05).
Population Pro/µl SOD APX GPOX CAT
T1 T2 T3 T1 T2 T3 T1 T2 T3 T1 T2 T3 T1 T2 T3
KORBA
Shoots 0.22c 0.68b 0.86a 308.62a 107.50b 107.56b 3.24a 0.98b 1.12b 14.24a 5.86b 5.53b 973.00a 825.16a 344.53a
Roots 0.31b 0.49a 0.23c 84.53a 157.67a 104.56a 1.38b 2.48ab 3.62a 30.38b 20.21b 110.05a 9053.49a 197.13b 599.83b
ELJEM
Shoots 0.63a 0.63a 0.62a 138.38ab 125.90b 166.41a 2.26b 1.82b 4.39a 9.11a 6.24b 4.56b 157.26b 67.60b 807,94a
Roots 0.67a 0.45b 0.13c 15.74b 152.834b 320.15a 2.91b 2.93b 12.36a 18.57b 23.16b 81.16a 27.77b 54.73b 168.03a
MAHDIA
Shoots 0.72b 1.17a 1.34a 154.95a 85.09b 31.89c 2.53a 0.72b 0.56b 5.08a 1.94b 5.48a 504.11a 37.19b 268.79ab
Roots 0.35a 0.10b 0.32a 123.63b 234.04a 93.92b 1.80a 3.31a 35.71a 120.15a 10.77b 6.89b 2024.57b 8638.88a 152.77b
MATEUR
Shoots 0.33b 0.95a 0.33b 361.41a 65.40b 294.89a 3.17b 0.57c 7.34a 5.84b 4.22b 26.96a 460.15a 52.63b 420.87a
Roots 0.11b 0.05c 0.47a 701.64a 75.12c 191.23b 83.76b 151.33a 5.31c 6.15c 253.75a 33.75b 7676.76a 788.19b 197.00b
FAHES
Shoots 0.79a 0.60b 0.66b 156.45a 136.99a 19.80b 2.87a 1.81b 1.26c 3.36b 17.08a 15.41a 1034.92b 152.93c 2838.10a
Roots 0.31a 0.07b 0.27a 70.38b 471.69a 444.30a 3.40b 7.14a 5.60ab 71.38a 29.71b 72.87a 379.33b 992.06a 427.35b
SABER02
Shoots 1.58a 0.77c 1.04b 28.25c 161.77a 78.63b 0.51b 1.59a 0.85b 3.20c 17.49a 10.86b 31.64b 74.59a 56.98ab
Roots 1.00a 0.87a 0.84a 16.08b 13.08b 66.30a 1.19b 1.43b 2.19a 20.72a 23.79a 21.98a 314.81a 65.98b 65.14b
LOCALE
Shoots 1.58a 0.90b 1.48a 46.93a 76.19a 42.63a 0.94a 1.03a 0.63a 2.28b 2.47b 4.03a 59.18b 397.11a 54.80b
Roots 0.16b 0.17b 0.38a 99.83b 283.55a 76.68b 10.78a 10.85a 2.54b 105.73a 31.14b 30.91b 989.58b 3976.03a 255.84b
TAKELSA
Shoots 0.33c 0.57a 0.41b 315.97a 175.08b 231.96b 4.21a 1.91b 3.66a 8.73b 17.45a 10.45b 2275.13a 105.14b 767.84b
Roots 0.22a 0.16b 0.21a 298.09b 729.16a 327.24b 17.80a 10.84b 5.10c 12.64a 6.26b 1.31c 2146.46a 3206.01a 542.32b
LTAIFIA
Shoots 0.59a 0.54a 0.46b 63.65b 83.26b 221.11a 1.60a 1.83a 2.14a 5.39b 6.24b 11.87a 86.62b 1020.20a 124.79b
Roots 0.07c 0.28a 0.11b 986.32a 236.97b 793.12a 19.37a 2.72c 7.76b 124.23a 89.59ab 75.86b 24140.21a 1851.85b 3188.40b
J. Plant Bio. Res. 2015, 4(1): 55-72
68
Our results showed that water stress was able to
enhance the activities of SOD in leaves of tolerant
populations (Mateur major, Ltaifia and Takelsa) in
agreement with previous study reported higher
constitutive and induced levels of SOD activity in
drought-tolerant cultivar of bean under water stress
28; while sensitive Fahes leaves SOD activity was
decreased, which agree with results reported by 29
indicating that SOD activity decreased in drought-
sensitive wheat cultivars. Increased GPOX activity
in Mateur major population, Ltaifia and Takelsa
minor populations indicated that it played a positive
role in controlling the cellular level of H2O2 under
drought stress conditions in agreement with similar
reported results 30, 31, 32.
Drought stress induced a complementary APX
activity to that of SOD for Vicia faba populations.
Our data agree with 33 results indicating that SOD
activity occurred in coordination with APX activity
in sugarcane plants under water stress.
Results confirmed that CAT activity in Vicia faba
populations under water stress conditions had no
major function which was further supported by the
findings of 32 showing that this enzyme had no
major antioxidative function in fig (Ficus carica L.)
under drought conditions.
Moderate water stress increased the MDA content
in leaves of Mateur major population and Mahdia,
Eljem, Mateur and Fahes minor populations. In
addition, it decreased in Ltaifia, Takelsa leaves.
Sever water stress enhanced the MDA content in
Mateur major population, Mahdia, Ltaifia, Fahes
and reduced in Takelsa leaves. 34 suggested that
MDA content significantly increased in the leaves
of V. faba cultivars in response to drought stress.
The tolerant populations had initially high MDA
values which were an indicator of water stress
tolerance. Which agree with results found by 35 in
drought-tolerant cultivars in tomato under water
stress.
In general, drought stress increased leaf internal
CO2 concentration (Ci) for studied populations
excepted Ltaifia population that showed slight
decrease under moderate water stress. 36 In
studying gas exchange of Jatropha curcas L.
subjected to water stress suggested that an increase
in internal CO2 concentration due to stress could be
happed in response to stomatal closing, but it is not
a universal response, as there have been reports of
both increases 37 and decreases 38 of the
internal CO2 concentration in J. curcas when the
plants have been subjected to water deficit.
According to 39, the response of the stomata to
changes in the internal CO2 concentration are also
strongly dependent on other variables, such as light
intensity, plant water status, temperature and vapor
pressure deficit. These abiotic factors probably do
alter the internal CO2 concentrations.
Transpiration rate (E) decreased in Vicia faba leaves
under water stress conditions. 40 pointed that
transpiration rate in Vicia faba leaves was reduced
to moisture stress.
Transpiration rate increased in Mahdia leaves,
which was sensitive to water stress. At moderate
drought condition 41 noted a larger reduction in
transpiration rate (E) upon moderate drought
detected in the susceptible common bean cultivar
compared to the resistant cultivar.
Moderate water stress decreased the stomatal
conductance (gs) in Vicia faba leaves. Our results
agree with data about decline in stomatal
conductance in plants subjected to drought stress
42, 43. Mateur and Takelsa drought tolerant
populations showed a recovery of gs values with
severe drought stress.
Net photosynthesis rate (A) decreased under water
stress conditions in Vicia faba leaves. 44 reported
that net photosynthetic rate of drought-tolerant
cultivars of chrysanthemum decreased with the
reduced soil water content and the drought sensitive
cultivar showed the largest reduction under drought
treatments.
Drought stress (50%) had a negative effect on Vicia
faba populations WUE excepted Mateur and Ltaifia
minor populations, whereas we noted spectacular
increase of Eljem and Mateur major populations
(WUE) under sever water deficiency reach higher
level than control. Also Mateur and Korba minor
populations as Locale and Saber02 varieties
exhibited a WUE recovery under sever water
deficiency. 45 pointed out that drought-tolerant
wheat cultivars showed an increase in WUE, while
the drought-sensitive cultivars showed a decrease in
WUE.
Vicia faba populations studied increased their
chlorophyll content independently of applied water
J. Plant Bio. Res. 2015, 4(1): 55-72
69
stress, they had an arbitrary response. In studying
tolerance to drought of Jatropha curcas accessions
46 found no reduction in Chla or Chlb contents. In
fact, some increase was observed.
Previous study showed Ltaifia was a genetic
homogeneous population47 and water deficit
tolerant performance so can be considered as a
selected cultivar with very interesting agronomic
character. 48 showed that ‘Greek gardenia’ or
‘Pelion’ can be considered as a cultivar with
homogenous genetic composition and low amount
of variation.
In the same Takelsa was a heterogeneous genetic
population47 with high water stress tolerance thus
may be very interesting for breeding program. As
reported for Olea europaea L. by49 .
Sensitive Fahes population had a high level of
heterogeneity 40 presents an interesting reservoir
of genes for breeding program.
CONCLUSIONS In conclusion, the tested physiological traits showed
that Ltaifia was a genetic homogeneous population
and water deficit tolerant performance could be
considered as a selected cultivar with very
interesting agronomic character. Takelsa was a
heterogeneous genetic population with high water
stress tolerance, thus it may be very interesting for
breeding program. Sensitive Fahes population had a
high level of heterogeneity presents an interesting
reservoir of genes for breeding program. In
addition, water stress enhanced activities of SOD
and GPOX in leaves of tolerant populations (Mateur
major, Ltaifia and Takelsa); while it decreased SOD
activity in leaves of sensitive population (Fahes).
Drought stress induced a complementary APX
activity to that of SOD and no major function of
CAT activity in Vicia faba populations.
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