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: ziadiimtiez@yahoo.fr

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

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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

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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).

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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

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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

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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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