Botanical Studies (2006) 47: 51-60.
*
Corresponding author: E-mail: mkaoua@fstg-marrakech.
ac.ma; Tel: 044-43-31-63; Fax: 044-43-31-70.
Comparative sensitivity of two Moroccan wheat varieties
to water stress: the relationship between fatty acids and
proline accumulation
Mimoun EL KAOUA
1,
*, Rachid SERRAJ
2
, Mohamed BENICHOU
3
, and Driss HSISSOU
1
1
Laboratoire de Biotechnologie et Phytopathologie Moleculaire, Departement de Biologie, Faculte des Sciences et Tech-
niques Gueliz, Universite Cadi Ayyad B.P. 549, Av. Abd el karim El Khattabi, Marrakech, Maroc
2
Crop Physiology Laboratory, International Crops Research Institute for the Semi-Arid Tropics, Po Patancheru 502 324,
India, CGIAR-ICRISAT
3
Department of Biology, Laboratory of Biochemistry Cadi Ayyad University, BP2390 Marrakech, Morocco
(Received October 12, 2004; Accepted August 2, 2005)
ABSTRACT.
Membrane lipids, peroxidation, β-carotene content, proline accumulation, and photosynthetic
activity were analyzed in two Moroccan wheat (Triticum aestivum L.) varieties, Nasma (adapted to irrigated
zone) and Tigre (adapted to semi-arid zone). Plants were grown in plastic pots under laboratory irrigation and
water stress conditions. One set of plants was subjected to regular water irrigation and another to water stress
conditions created by water deprivation. After 30 days of water shortage (corresponding to 8% of field capaci-
ty), the leaves’ fatty acid contents, especially that of Octadecatrienoic acid (18:3 .
9,12,15
), i.e. the major unsatu-
rated fatty acid, significantly decreased. The decrease was more pronounced in Nasma varieties than in Tigre.
The amount of galactolipids, phospholipids, and β-carotene decreased in droughted plants of both local wheat
varieties while the content of neutral lipids increased. Lipid peroxidation, assessed as the content of malondi-
aldehyde (MDA), was found to be augmented under stress. (The rate of increase was 41% and 19% in Nasma
and Tigre respectively). The amount of proline accumulated in leaf segments of both varieties subjected in
vitro to osmotic stress was suppressed by addition of octadecanoic or octadecadienoic acids. The inhibition of
photosynthetic capacity under osmotic stress was reduced when fatty acids were added in medium. A positive
relationship was observed between proline accumulation and membrane stability. The mechanisms of these
physiological responses to water stress are discussed.
Keywords: Lipids; Photosynthetic capacity; Proline; Triticum aestivum; Water stress.
Abbreviations: FAME, fatty acid methyl esters; MGDG, monogalactosyldiacy-glycerol; DGDG,
digalactosyldiacy-glycerol; PH, phospholipids; NL, neutral lipids; TBARS, 2-thiobarbituric acid-reactive
substances; TBA, 2-thiobarbituric acid; MDA, malondialdehyde; RM, reference medium; PEG, Polyethylene
glycol; 16:0, palmitic acid; 16:1, palmitoleic acid; 18:0, stearic acid; 18:1, oleic acid; 18:2, linolenic acid;
18:3, linolenic acid.
INTRODUCTION
Photosynthetic CO
2
assimilation is known to be
severely inhibited by water stress (Boyer, 1970;
Kriedemann, 1986; Vu et al., 1987). This is partly due
to the closing of the stomata that also reduces leaf
transpiration
and prevents the development of excessive
water stress in plant
tissues. Furthermore, under water
stress conditions the chlorophyll contents as well as the
carotenes and other foliar pigments can be decreased
(Iturbe Ormaetxe et al., 1998). Inhibition of photosynthetic
capacity under water stress conditions is related to a
decrease in PSII activity with changes in specific intrinsic
and extrinsic proteins involved in electron transport. This
inhibition is a consequence of reduced oxygen-evolving
capacity and additional damage within reaction centers
(Eastman et al., 1997). In cotyledons of clusterbean
(Cyamopsis tetragonoloba L.), water stress induced a
quantitative loss in the D1 protein, an increase in thylakoid
lipid peroxidation and a decline in the level of β-carotene
(Deo and Biswal, 2001). On the other hand, the role of
β-carotene in protecting isolated PS II reaction centers
from photoinhibition damage has been demonstrated
(Yang et al., 2002).
PHYSIOLOGY
pg_0002
52
Botanical Studies, Vol. 47, 2006
At the cellular level, cell membranes are one of the
first targets of plant stresses (Levitt, 1972). The ability of
plants to maintain, under drought, membrane integrity is
what determines tolerance towards drought stress (Vieira
da Silva et al., 1974). Membrane stability is a widely used
criterion to assess crop drought tolerance (Premachandra
and Shimada, 1988; Zuily-Fodil et al., 1990). During
severe water stress, a decrease in the membrane lipid
contents has been observed (Monteiro de Paula et al.,
1990). This decrease is mainly due to an activation of
degradation processes (Monteiro de Paula et al., 1993).
The phospholipase and galactolipase activities have been
shown to be stimulated in stressed and senescing plants
(Kanituga and Gemel, 1984; O’Sullivan et al., 1987; EL-
Hafid et al., 1989). It was also reported that in cotyledons
of clusterbean ( Cyamopsis tetragonoloba L.) the
peroxidation of the thylakoid lipids increases under water
stress conditions (Deo and Biswal, 2001).
Water stress increases the production of reduced
reactive oxygen species (ROS) in chloroplasts (Smirnoff,
1993). In plants, the chloroplasts are a major source of
activated O
2
(Asada and Takahashi, 1987; Foyer et al.,
1994). The superoxyde radical of O
2
is produced by
photoreduction of O
2
at PSI and PSII, and singlet O
2
is
formed by energy transfer to O
2
from triplet excited state
chlorophyll (Asada and Takahashi, 1987). The negative
effects of stress may be partly due to the oxidative damage
resulting from the imbalance between production of
activated O
2
and antioxidant defence mechanisms (Foyer
and Harbinson, 1994). Other subcellular compartments
of leaves, such as mitochondria and peroxisomes,
are potential generators of O
2
and H
2
O
2
, mainly as a
consequence of electron transport and enzymatic reactions
(Del Rio et al., 1992). Under water stress, the formation of
ROS is usually exacerbated. Water stress caused a marked
increase in oxidative damage to proteins, particularly
in mitochondria and peroxisomes. Bartoli et al. (2004)
have suggested that mitochondria are the main target for
oxidative damage to proteins under drought conditions.
Since drought is a major constraint to cereal production
and yield stability in most regions in Morocco, the purpose
of this work was to evaluate differences in the response to
water stress of two Moroccan wheat varieties, i.e., Nasma
(adapted to irrigated zones) and Tigre (adapted to rainfed
zones). For that, we report on changes in fatty acids, lipid
peroxidation (commonly taken as indicator for oxidative
stress), and on the relationship between photosynthetic
activity and proline accumulation under osmotic stress.
Proline accumulation under osmotic stress conditions
was previously supposed to be involved in osmotic
adjustment (Delauney and Verma, 1993). In this work, we
have evaluated the effect of saturated or unsaturated fatty
acids on proline accumulation and photosynthetic activity
altered under osmotic stress conditions. Previous studies
have shown that proline osmo-induced in canola leaf was
suppressed by the addition of PUFAs to stress medium
containing PEG (Huguet-Robert et al., 2003).
MATERIALS AND METHODS
Plant materials and growth conditions
Seeds of two bread wheat (Triticum aestivum L.)
varieties that are adapted to irrigated conditions (Nasma)
or rainfed semi-arid conditions (Tigre) were provided by
SONACOS (National Company for Seeds Marketing in
Morocco, Souihla Road, Marrakech City).
Seeds were germinated on moist filter paper placed in
glass Petri dishes (9 cm in diameter) in the dark at 25±1°C.
Seedlings were transferred at stage 1-2 on Zadoks scale,
i.e. two leaves unfolded (Zadoks et al., 1974), into plastic
pots (24 cm in height and 20 cm in diameter) containing a
mixture of peat, garden soil, and sand (2:1:1 v/v/v) with a
water saturation capacity of 75%. Four seedlings per pot
and 15 pots for each variety and condition were used. The
pots were placed under natural day and night temperatures
(20 ± 4°C and 7 ± 3°C, respectively) and under sunlight
(10 ± 2 h). All plants were irrigated every two days with
250 ml of tap water.
After appearance of the 5th leaf, the pots were divided
into three groups according to water stress treatments:
control plant (continuous irrigation), plants subjected to
water deprivation corresponding to 15% of field capacity
(evaluated by gravimetric measurement) for 15 days,
and plants subjected to water deprivation corresponding
to 8% of field capacity for 30 days. For all subsequent
experiments, leaf material was collected from newly
developed leaves during the stress period.
Measurement of leaf pigments
Leaf pigments were extracted from 0.5 g of leaves
with acetone/distilled water (90:10 v/v); the chlorophyll
contents were determined spectrophotometrically (GBC
UV/VIS 916) using a kinetic method of controlled
pheophytinization (Laval-Martin, 1985). Addition of 5 μl
of HCl (3 N) to 3 ml of the pigment extracts was sufficient
to produce total pheophytinization of both chlorophylls.
For the determination of β-carotene contents, 20 ml of
pigment extract were saponified by 10 ml of 15% (w.v
-1
)
KOH under darkness for 60 min. Carotene pigments
were extracted by addition of petroleum ether, and the
absorbance of β-carotene in the suspension was measured
by a spectrophotometer (GBC UV/VIS 916) at 443
nm. The β-carotene amounts were calculated using the
coefficient ε = 0.2630 ml·μg
-1
·cm
-1
(Laval-Martin and
Mazliak, 1979).
Photosynthetic oxygen evolution
The photosynthetic oxygen evolution by the leaf tis-
sues, exposed to saturating light (420 μmol·m
-2
·s
-1
pro-
vided by lamps HMB-160 W) was measured using a
modified Clark type electrode (YSI Model 5300) sensitive
to a variation of about 3 μM O
2
·min
-1
. The consistency of
photosynthetic response was checked by measuring two
successive dark to light (12 min/condition) transitions
pg_0003
EL KAOUA et al. — Wheat lipid modification under water stress
53
for each sample. The experiments were carried out in a
temperature-controlled cuvette of 3 ml at 25°C in a buffer
(50 mM Tris, pH 7.5; 1 mM MgCl
2
) containing 40 mM
NaHCO
3
(El Kaoua and Laval-Martin, 1995) on 50 mg of
fresh leaf segments (leaf 5) maintained previously in the
same buffer for 15 min. The photosynthetic activity was
expressed in μml of O
2
·μmol chlorophyll
-1
·h
-1
.
In comparison with control, the effect of osmotic stress
on leaf photosynthetic activity in the suspension medium
was evaluated by adding 25% of PEG-10 000 alone or
in combination with 25 μM of fatty acids (18:0 or 18:2).
This concentration of PEG 10 000 provided - 0.95 MPa
osmotic potential. Tap water provided only - 0.05 MPa
as measured with an A0300 KNAUER Automatic Semi-
Micro Osmometer and calculated using the equation of
Michel et al. (1983):
Ψ=8.3141×10
-5
×(273.15+°C)×mosmol/Kg×10
-1
=MPa
Proline assay
Wheat seedlings were grown for 6 weeks in hydroponic
Hoagland’s medium in controlled environment chambers
at 22°C and under constant illumination provided by TSE
standard lamps (Type Preheated F40W/54 daylight) giving
about 80 μmol·m
-2
·s
-1
with a 16-h photoperiod. During the
culture period, the medium was changed eve ry four days.
Developed leaves were cut into segments (1.0 / 0.5 mm),
and 100 mg was transferred to a reference medium (5
mmol·l
-1
HEPES, 10 mmol·l
-1
KCl, 1.5 mmol·l
-1
CaCl
2
, pH
6.0) (Huguet-Robert et al., 2003).
Osmotic stress was created by adding 25% (≡ -0.95
MPa) of PEG-10000 to the reference medium. The Petri
dishes containing segments of leaves were divided into
six groups: (i) stress medium with PEG solution only, (ii)
stress medium supplemented with 25 μM of octadecanoic
acid, (iii) stress medium supplemented with 50 μM of
18:0, (iv) stress medium supplemented with 25 μM of
18:2, (v) stress medium supplemented with 50 μM of
octadecadienoic acid; and (vi) the control group was
represented by leaf segments in the reference medium
(without PEG). The pH was adjusted to 6.0. The Petri
dishes were closed, placed under continuous light, and
shaken continuously at 22°C.
Proline extraction and determination
Proline content was determined 5 min after the leaf
segments were cut, and the segments were maintained for
6 h or 20 h in the different mediums. Proline was extract-
ed from 100 mg of fresh matter with 3 ml of methanol:
chloroform: water (12:5:1,
v/v/v). The homogenate was
centrifuged at 2,500 g for 5 min, and the supernatant was
recuperated and used for proline estimation.
Free proline was quantified by spectrophotometer by
means of a colorimetric reaction with ninhydrin according
to the method of Singh et al. (1973) modified by
Paquin
and Lechasseur (1979). 1ml of supernatant was transferred
to test tube and heated in a water bath until methanol
evaporation. 0.33 ml of ninhydrin solution (0.01 g of
ninhydrin, 0.166 ml of sulphuric acid 6 M and 0.25 ml of
glacial acetic acid), 0.33 ml of glacial acetic acid and 0.33
of water were added to 0.33 ml of sample. Then the tube
was cooled to room temperature, and 2 ml of toluene was
added. After 30 s of shaking, two phases were separated,
and the absorbance of the upper phase was read at 520 nm.
Proline concentrations were
determined by comparison to
a 0- to 300 μg proline standard
curve and then expressed
on a (μg g
-1
) DW basis.
Lipids extraction and separation
Leaf tissues (0.5 g FW) were ground in an ice-cold
mortar and fixed in 15 ml of boiling 1.5% NaCl solution.
Lipids were then extracted with 100 ml of a mixture of
chloroform/methanol/water (8:4:1 v/v/v) (El Kaoua et
al., 1991). After a strong manual mixing, the emulsion
was centrifuged at 5,000 rpm (HEMLE - Type Z383 -
Max.Speed 17,000 rpm - Max. RCF 26.810xg - Rotor
220.87 VO1) for 5 min, and the recuperated chloroform
hypophase was evaporated at 35°C and stored under
nitrogen gas at -20°C until separation and analysis of the
different fatty acid classes.
A spot of the chloroform lipid solution was deposited
on silica gel TLC plates (Kieselgel 60). One-dimensional
separation was performed in a saturated atmosphere with
the solvent mixture of acetone, acetic acid, and water
(100:2:1 v/v/v) (Christie, 1982). The spots corresponding
to the different lipid classes were visualized under UV
light after the plate was sprayed with an aqueous solution
of rhodamine 6 G (0.02%, w/v). The bands correspond-
ing to galactolipids (MGDG and DGDG), phospholipids,
and neutral lipids were identified by their different Rought
front of migration (Rf).
Esterification and methylation of fatty acids
Before esterification, an internal standard consisting
of 40 μg of 19:0 (nonadecanoic acid, Sigma) was added,
followed by sulphuric acid/methanol (2.5/97.5 v/v). After
two hours at 65°C, 2 ml of petroleum ether and 1 ml of
distilled water were added, and the fatty acid methyl
esters (FAME) present in the upper phase were collected
and analysed with a gas chromatograph (Varian Star
Chromatography Workstation, CP 3380 CG) equipped
with a capillary column (Carbowax 20M, 0.35 mm ×
25 m) and using nitrogen as carrier gas at a pressure of
5.6 bar. The separation was carried out at 160°C for 50
min, then 210°C for 1 h 30 min; the detector was a flame
ionisation type (FID).
The FAMEs were identified by comparison of the re-
tention times with those of known standards, and determi-
nation of fatty acid composition was based on percentage
of each peak area. The quantitative analysis of the FAME
was performed by using an integrator (CP-Scanview;
Chromatography Application Database, Version 6.0) based
on the known quantity (40 μg) of 19:0.
pg_0004
54
Botanical Studies, Vol. 47, 2006
Determination of malondialdehyde
The peroxidation of leaf lipids was measured by the
TBA method, as described by Iturbe-Ormaetxe et al.
(1998), in which the reactive substance was quantified as
MDA, an end product of lipid peroxidation. Lipid perox-
ides were extracted from 0.5 g (fw) of leaves with 5 ml of
5% (w/v) metaphosphoric acid and 100 μL of 2% (w/v)
butyl hydroxytoluene (in ethanol). After centrifugation
(15,000 g for 20 min), the chromogen was formed by mix-
ing 0.5 ml of supernatant, 50 μl of 2% (w/v) butyl hydrox-
ytoluene, 0.25 ml of 1% (w/v) TBA (in 50 mM NaOH),
and 0.25 ml of 25% (v/v) HCl. The reaction mixtures were
incubated at 100°C for 30 min and cooled to room tem-
perature. The chromogen formed was extracted by adding
1 ml of n-butanol to the mixture followed by vigorous
shaking, the butanol and aqueous phases were separated
by centrifugation, and the absorbance of the TBARS was
determined as TBA-MDA complex at 532 nm using a
spectrophotometer UV (GBC UV/ Vis 916). The amount
of MDA was calculated: ε = 155.0 mM
-1
·cm
-1
(Shalata and
Tal, 1998).
Statistical analysis
The calculated mean values ± SD are reported in the
tables and figures below. The significance of statistical
differences between samples and each treatment were
evaluated by the t-test.
RESULTS
Lipids analysis
Under control conditions, the fatty acids consistently
identified in the total leaf lipids of the two wheat varieties
were 16:0, 16:1, 18:0, 18:1, 18:2 and 18:3. However, 16:0
and 18:3 represent together about 80% of total fatty acids
(Table 1).
Water stress caused a decrease of fatty acid content
in both Nasma and Tigre varieties. This decrease was
more dramatic for the unsaturated 18:3 octadecatrienoic
acid in Nasma and was paralleled by an increase in the
percentages of saturated fatty acids 16:0 and 18:0 (Table
1). The lipid composition was also affected by water
stress in both varieties. Indeed, there was an important
decrease in the percent of galactolipids and phospholipids,
especially in Nasma, and an increase of neutral lipids
under water stress (Figure 1). Lipid peroxidation increased
significantly more in stressed Nasma varieties compared
to Tigre (Figure 2).
Proline accumulation
When the leaves were cut in segments, the proline
content increased about 16 fold in Nasma and threefold
in Tigre (Figure 3) due to water loss by tissues after 6 h
in reference medium. This new proline content remained
unchanged after 20 h in RM, and we have considered this
new content as the control. When leaf segments were sub-
jected to osmotic stress imposed by PEG-10000 during 20
h, they accumulated more proline. This accumulation was
remarkably higher in Nasma compared to Tigre (Figure 3).
Indeed, the proline level increased about 7 and 9 fold in
Nasma and Tigre, respectively. This accumulation was sig-
nificantly lower in either variety when the stress medium
was supplemented with 25 μg of 18:0 or 18:2. When the
same fatty acid concentrations were increased to 50 μg,
the proline content fell significantly in Nasma varieties.
Pigments content
β-carotene content was about twofold higher in Tigre
than in Nasma under irrigated conditions (Figure 4). This
content was significantly reduced (more than 50%) by wa-
ter stress in both varieties, but remained two times higher
in Tigre than in Nasma (Figure 4).
Table 1. Total fatty acid content (mg·g DW
-1
) and fatty acid composition (%)
of leaves of two wheat (Triticum aestivum)
varieties, Nasma and Tigre. The varieties were subjected (Stressed) or not (Control) to water stress, induced by withhold-
ing irrigation after the fifth leaf appearance for 30 days. Results are means ± SD (n = 3).
Fatty acids
Nasma
Tigre
Control
Stressed
Control
Stressed
16:0
Content
%
2.73 ± 0. 43
17.3 ± 0.3
2.43 ± 0.41
28.2 ± 3.1
2.87 ± 0.27
25.9 ± 1.5
1.90 ± 0.31
24.2 ± 2.5
16:1
Content
%
0.57 ± 0.12
3.9 ± 0.3
0.31 ± 0.10
3.5 ± 1.2
0.50 ± 0.02
4.5 ± 0.2
0.30 ± 0.05
4.2 ± 0.3
18:0
Content
%
0.35 ± 0.05
2.4 ± 0.1
0.29 ± 0.01
3.5 ± 0.7
0.24 ± 0.07
2.2 ± 0.5
0.18 ± 0.03
2.9 ± 0.9
18:1
Content
%
0.74 ± 0.13
4.3 ± 0.5
0.47 ± 0.24
5.6 ± 1.7
0.38 ± 0.02
3.6 ± 0.2
0.28 ± 0.05
4.6 ± 1.5
18:2
Content
%
0.65 ± 0.05
4.2 ± 0.2
0.26 ± 0.15
2.9 ± 0.9
0.21 ± 0.07
2.3 ± 0.6
0.20 ± 0.05
2.5 ± 0.4
18:3
Content
%
9.43 ± 0.18
66.6 ± 2.7
4.80 ± 1.37
54.8 ± 4.7
5.59 ± 0.28
60.3 ± 1.9
4.39 ± 0.73
58.9 ± 2.9
pg_0005
EL KAOUA et al. — Wheat lipid modification under water stress
55
Figure 1. Change in the relative amount of lipid classes (expressed as a percent of control) as related to water stress, induced by with-
holding irrigation during 30 days, in leaves of two wheat (Triticum aestivum L.) varieties: Nasma and Tigre: (A) Result represents the
variation of lipid content (in μg
·
g DW
-1
) under water stress. (B) Result expressed as a percent of control. The result are means ± S.D.
(n=3). C: control; S: stressed.
Figure 3. Proline content in leaf segments of two wheat variet-
ies maintained 5 min under ambient conditions (C) or 6 h and
20 h in reference medium (RM). The osmotic stress was created
by the addition of 25% PEG10 000 in RM during 20 h supple-
mented or not with two different concentrations (25 μg or 50 μg)
of stearic acid (18:0) or linolenic acid (18:2). Results are means
± S.D. (n=3).
Figure 2. Malondialdehyde (the end product of lipid peroxida-
tion) quantified in leaves from two cultivars of Triticum aesti-
vum (Nasma was adapted to an irrigated zone, and Tigre to a
semi-arid zone) subjected to moderate (MS) or to severe (SS)
stress induced by withholding irrigation after the fifth leaf ap-
pearance for 15 and 30 days, res pectively. Res ults represent
means ± S.D. (n = 5 separate measurements). (C) = control.
50
45
40
35
30
25
20
15
10
5
0
Nasma
Tigre
Wheat varieties
200
180
160
140
120
100
80
60
40
20
0
C
5 min
RM
6h
RM PEG 18:0-25μg 18:2-25μg 18:0-50μg 18:2-50μg
Culture conditions
20h
pg_0006
56
Botanical Studies, Vol. 47, 2006
Photosynthetic activity
Photosynthetic activity of leaf fragments, measured
by oxygen evolution was more important in Tigre than
in Nasma varieties (Figure 5). The PEG-induced osmotic
stress resulted in a significant decrease of oxygen evolu-
tion in both varieties. However, photosynthesis inhibition
was almost complete in Nasma (94%) while in Tigre 65%
of activity remained after 15 min of treatment (Figure 5).
When the PEG was added with fatty acids, the photosyn-
thesis activity was restored in Nasma. The C18:2 is more
effective than C18:0 at establishing this photosynthetic
activity.
DISCUSSION
Our results showed that water stress leads to a
significant decrease in polar lipids (MGDG and
phospholipids) and an increase in neutral lipids in both
wheat varieties studied, Nasma and Tigre. Neutral lipid
(diacylglycerol and triacylglycerol) accumulation,
previously observed in other plants (Martin et al., 1986;
Hubac et al., 1989; Dakhma et al., 1995), was considered
a defence mechanism of the plant against drought
stress (Hubac et al., 1989). It has also been suggested
that the free fatty acids, released during water stress by
the action of lipases on polar lipids, could be stored in
triacylglycerols to avoid oxidation by free radicals and
activated oxygen forms (Hubac et al., 1989; Dakhma et
al., 1995).
The results showed that the wheat variety Tigre,
adapted to rainfed semi-arid conditions, showed a more
stable phospholipid content after water stress than did
the variety Nasma, which is mostly adapted to irrigated
conditions. These results are in agreement with previous
studies on Vigna unguiculata leaves (Sahsah et al., 1998).
The observed decrease in galactolipids localised on
the chloroplast envelope, stroma lamellae, and grana
system (Bahl et al., 1976) indicates that water stress
affects the chloroplast membranes. This could contribute
to the inhibition of photosynthesis, which is more
important in Nasma than in Tigre. In fact, Nasma showed
a larger decrease in MGDG and a higher inhibition of
photosynthesis. A decline in MGDG content under water
stress conditions has been considered by several authors
to be a feature of drought-sensitive cultivars (Hubac et al.,
1989; Pham Thi et al., 1990; Quartacci et al., 1995). It was
also previously shown in cotton that water stress increased
the activity of MGDG-hydrolases (El-Hafid et al., 1989).
Similarly, a more pronounced decrease in lipid contents
in drought-sensitive than in drought-tolerant cultivars has
been observed in cowpea and was explained mainly by
an activation of lipid degradation processes (Monteiro
de Paula et al., 1993). However, tolerance to water stress
may involve different mechanisms, including the capacity
to maintain high levels of antioxidants (Sairam et al.,
1998). Relative to Tigre, the irrigation-dependant Nasma
variety presented a higher degree of peroxidation and
contained less β-carotene pigments. It was reported that
β-carotenes play a role as antioxidants (Sairam et al.,
1998). Carotenoids can directly deactivate singlet oxygen
(
1
O
2
) and can also quench the excited triplet state of
chlorophyll, thus indirectly reducing the formation of
1
O
2
Figure 4. β-carotene (μg
·
g
-1
DW) in leaves of two wheat (Triti-
cum aestivum) cultivars (Nasma and Tigre) subjected to water
stress induced by withholding irrigation for 30 days (8% of field
capacity). Results are means ± S.D. (n=3).
2.5
2
1.5
1
0.5
0
Nasma
Tigre
Wheat varieties
Figure 5. P hotosynthetic capacity (μmole O
2
·
μmole chloro-
phyll
-1
·
h
-1
) in leaf discs of two wheat (Triticum aestivum) culti-
vars, Nasma and Tigre. Effect of osmotic stress created by 25%
PEG (≡ -0.95 MPa) added or not with fatty acid (octadecanoic,
18:0, or octadecadienoic acid, 18:2) for 15 min in leaf disc sus-
pension medium. Values are the means ± S.D. (n = 5 separate
measurements).
140
120
100
80
60
40
20
0
C
PEG PEG+18.0 PEG+18.2
Treatment type
pg_0007
EL KAOUA et al. — Wheat lipid modification under water stress
57
species (Sieferman-Harms, 1987; Foyer and Harbinson,
1994).
A higher proline accumulation was observed in the
irrigation-dependent variety Nasma, in which higher per-
oxidation levels and a higher decrease in unsaturated fatty
acids were also observed. Proline accumulation in stressed
plants is a well documented phenomenon. It has been
suggested that this amino acid acts as an organic solute
directly involved in osmotic adjustment to osmotic stress
and/or in protecting macromolecules under various envi-
ronmental perturbations (for a review, see Delauney and
Verma, 1993). In this work, we noticed that the accumula-
tion of the proline is more important in the most sensitive
stress varieties. This accumulation can be correlated with
the cell damage; therefore, it is probably one of the conse-
quences of stress.
In addition, our results clearly showed that addition of
octadecanoic or octadecadienoic acid decreased osmo-
induced proline accumulation significantly and restored
photosynthetic activity (Figure 5). This effect of 18:0 or
18:2 addition can be explained by their biosynthesis of
octadecatrienoic acid (18:3), an abundant polyunsatu-
rated fatty acid found in the galactolipids of thylakoid
membranes. Such a pathway might be involved in the
synthesis of polar lipids and membrane stabilization. In
addition after its formation, 18:3 might be converted into
methyl-jasmonate (Me-JA) via 12-oxophytodienoic acid
(12-OPDA) in chloroplast (Vick and Zimmerman, 1984).
Me-JA is considered to be a regulator of plant growth and
development (Sembdner and Parthier, 1993). Me-JA and
other cyclopentanone compounds, mainly jasmonic acid
(JA) and its amino acid conjugates, influence a variety of
physiological properties, especially those related to stress
tolerance (Parthier, 1991). Our results suggested a positive
relationship between fatty acids and photosynthetic capac-
ity in the two wheat varieties studied. It is known that a
decrease in fatty acid unsaturation results in a decrease
of membrane fluidity (Shinitzky, 1984). The water stress
induced a decrease in the degree of unsaturation in the
phospholipids (Svenningsson and Liljenberg, 1986) and
in MGDG (Zuily-Fodil, et al., 1992). On the other hand,
drought-induced peroxidative forms of wheat have been
shown to attack polyunsaturated fatty acids in common
bean (Ferrari-Iliou et al., 1993).
The accumulation of proline would be merely a conse-
quence of membrane disorganization brought on by water
stress. This is further supported by the addition of fatty
acids, which probably regenerates the oxidized lipids of
the membrane. Very recent findings (Huguet-Robert et al.,
2003) have shown that osmo-induced proline accumula-
tion in canola leaf discs is inhibited by polyunsaturated
fatty acids. The authors suggest that PUFAs were convert-
ed to methyl-jasmonate, which could actually behave as a
more potent suppressor of the proline response.
In conclusion, our results showed that water stress
induced a decrease in polar lipid content and an increase
in neutral lipids in both local varieties studied. A strong
decrease in phospholipids, galactolipids, unsaturated
fatty acids and a higher degree of peroxidation were
observed in the irrigation-dependant variety, suggesting
more disorganized and unstable membranes. This could
be associated with higher metabolic damage as they can
be ascertained through proline accumulation. However,
further studies are required for the physiological analysis
of the interactions between membrane lipids and proline
metabolism, and their respective roles in response to
drought. Also, we need in the future work to analyse phos-
phatidylglycerol fatty acids from chloroplasts, especially
hexadecenoic acid (16:1 cis and trans) which play
important roles in environmental stress.
Acknowledgements. Part of this work was supported by
the Biology Department of the Faculty of Science and
Technology, Marrakech and by the Project No. p5t1/03
(Centre National de Coordination et de la Planification
de la Recherche Scientifique et Technique du Maroc). We
thank the Director of SO.NA.CO.S (National Company
for Seeds Marketing in Morocco) for a generous donation
of the wheat seeds used in this study.
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兩種摩洛哥小麥對水逆境之不同敏感度：脂肪酸和脯氨酸累
積量之相關性
Mimoun EL KAOUA
1
, Rachid SERRAJ
2
, Mohamed BENICHOU
3
, and Driss
HSISSOU
1
1
Laboratoire de Biotechnologie et Phytopathologie Moleculaire, Departement de Biologie, Faculte des
Sciences et Techniques Gueliz, Universite Cadi Ayyad B.P. 549 - Av. Abd el karim El Khattabi, Marrakech, Maroc
2
Crop Physiology Laboratory, International Crops Research Institute for the Semi-Arid Tropics, Po
Patancheru 502 324, India, CGIAR-ICRISAT
3
Department of Biology, Laboratory of Biochemistry Cadi Ayyad University, BP2390 Marrakech, Morocco
細胞½脂，過氧化，乙型-胡蘿½素，脯氨酸累積，及光合作用都分別於兩種摩洛哥小麥 (Triticum
aestivum L.) 品種，Nasma （適應於灌溉區）及 Tigre（適應於半乾旱區），測定。供試植物在實驗室分
別於供水及乾旱條件下生長於塑½盆內。缺水 30 天後（相當於產量潛能之 8%），葉中之脂肪酸含量
（尤其是 18C3 雙鍵，即18:3 .9,12,15；此乃主要的不飽和脂肪酸）顯著地減少。減少程度 Nasma 比
Tigre明顯。半乳糖脂，磷脂，及乙型-胡蘿½素含量在乾旱條件下於兩種小麥品種中都減少，但中性脂
肪含量.增加。脂½之過氧化（以 malondialdehyde 量評估）於乾旱時增加（增率於 Nasma 及 Tigre 分
別為 41% 及 19%）。兩種小麥品種葉子於離體滲壓逆境時脯氨酸之累積量可添加 18C 脂肪酸及 18C2
雙鍵脂肪酸予以抑制。當培養基內添加上述兩種脂肪酸時，光合作用之受滲透逆境造成之抑制程度可
減½。脯氨酸之累積和½安定度兩者間具正相關。本文討½這些生理反應和水逆境之關係。
關鍵詞：Triticum aestivum；水逆境；光合作用能力；脂½；脯氨酸。