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Plant Growth Regulation 00: 1–10, 2001.
© 2001 Kluwer Academic Publishers. Printed in the Netherlands.
The use of the electrolyte leakage method for assessing cell membrane
stability as a water stress tolerance test in durum wheat
Mohammed Bajji, Jean-Marie Kinet and Stanley Lutts*
Laboratoire de Cytogénétique, Université catholique de Louvain, 5 (bte 13) Place Croix du Sud, B-1348,
Louvain-la-Neuve, Belgium; *Author for correspondence (e-mail: [email protected]; phone:
+32-10-472037; fax: +32-10-473435)
Received 2 January 2001; accepted in revised form 17 April 2001
Key words: Cell membrane injury, Drought tolerance, Electrolyte leakage, Osmotic stress, Polyethylene glycol,
Triticum durum
Abstract
This work was carried out to adapt the electrolyte leakage technique to durum wheat and then to evaluate its
relevance in the assessment of the cell membrane stability as a mechanism of water stress tolerance in this species. The method currently used is based on in vitro desiccation of leaf tissues by a solution of polyethylene
glycol (PEG) and a subsequent measurement of electrolyte leakage into deionised water. It consists of three successive steps: (1) a washing treatment to remove solutes from both leaf surfaces and cells damaged by cutting;
(2) a stress period during which the leaf tissues are plunged in a PEG-solution and (3) a rehydration period
during which after-effects of the stress are evaluated. During the washing period, the major part of electrolytes
was removed within 15 min. Varying the stress conditions influenced both the percent and the kinetics of electrolyte leakage during rehydration. Electrolyte leakage exhibited a characteristic pattern reflecting the condition
of cellular membranes (repair and hardening). In practice, we recommend a 15-minute washing time, a 10-hour
stress period and 4 h of rehydration. The extent of the cell membrane damage not only correlated well with the
growth responses of wheat seedlings belonging to various cultivars to withholding water but also with the recognised field performances of these cultivars. The relative proportion of endogenous ions lost in the effusate
during the rehydration step may vary strongly according to the element analysed and the precise nutritional status of the plant should therefore be considered. However, an increase in inorganic ion leakage does not fully
explain the recorded PEG-induced increase in electrical conductivity (EC) during the subsequent rehydration step
and organic ions are probably also involved in such an increase.
Introduction
Drought resistance is the result of various morphological, physiological and biochemical characteristics. Its
genetic improvement in crop plants requires the identification of appropriate drought resistance mechanisms and particularly the development of suitable
methodologies for their measurement in large breeding populations.
Cell membranes are one of the first targets of many
plant stresses and it is generally accepted that the
maintenance of their integrity and stability under water stress conditions is a major component of drought
tolerance in plants. The degree of cell membrane injury induced by water stress may be easily estimated
through measurements of electrolyte leakage from the
cells. The method is based on an in vitro stress of leaf
tissues by a PEG solution and a subsequent measurement of electrolyte leakage into an aqueous medium
(Sullivan and Ross 1979). It has an enduring appealbecause it requires readily available and inexpensive
equipment, it is not destructive of whole plants, is
easily used on plant material from a variety of cultural systems and it is suitable for analysing large
number of samples. Such a technique has also been
applied to quantify damages to cell membranes in
XPS 53702 (GROW) – product element 352339 – ICPC/Grafikon
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various abiotic stress conditions such as low (Coursolle et al. 2000; Tamura 2000; Vainola and Repo
2000) and high (Ismail and Hall 1999; Maheswary et
al. 1999; Saelim and Zwiazek 2000) temperatures, air
pollution (Garty et al. 2000), salt stress (Chen et al.
1999; Sreenivasulu et al. 2000), acid conditions
(Spencer and Ksander 1999), heavy metals (De and
Mukherjee 1996) and even in response to biotic
stresses (Adam et al. 2000; Sriram et al. 2000).
It has been demonstrated recently that electrolyte
leakage measurements may be correlated with several
physiological and biochemical parameters conditioning the plant responses to environmental conditions
such as spectral reflectance (Garty et al. 2000; Vainola
and Repo 2000), antioxidative enzyme synthesis (Liu
and Huang 2000; Sreenivasulu et al. 2000), membrane acyl lipid concentrations (Lauriano et al. 2000),
water use efficiency (Franca et al. 2000; Saelim and
Zwiazek 2000), transverse relaxation time of leaf water (Maheswary et al. 1999), stomatal resistance, osmotic potential and leaf rolling index (Premachandra
et al. 1989). It is therefore not surprising that electrolyte leakage has been recommended as a valuable criterion for identification of stress resistant cultivars in
several crop species (Leopold et al. 1981; Stevanovic
et al. 1997).
However, despite its many advantages, electrolyte
leakage was found to be markedly influenced by various experimental parameters, especially washing
time of collected samples before PEG exposure
(Blum and Ebercon 1981; Premachandra and Shimada 1987), intensity and duration of the PEG treatment (Blum and Ebercon 1981; Vasquez-Tello et al.
1990) and duration of the rehydration period (Bandurska and Gniazdowska-Skoczek 1995; Bandurska et al.
1997; Zwiazek and Blake 1990). Since the degree of
solute leakage varies with species (Leopold et al.
1981; Vasquez-Tello et al. 1990), a careful examination of the technique has to be performed for each
species studied. Another point is that electrolyte leakage measurements quantify the presence of all
charged solutes in the external medium but it does not
give information concerning the identity of these solutes. Electrolyte conductivity measured in the incubating medium may be due to the leakage of both
charged inorganic or organic molecules (Palta et al.
1977). As far as inorganic compounds are concerned,
data concerning the precise nature of leaked ions in
relation to their initial endogenous concentration in
the leaf segments before PEG treatment are surprisingly scanty. If various ions are not involved in the
same proportional manner in the increase of electrolyte leakage after stress exposure, this would imply
that the results of electrolyte leakage measurements
may be influenced by the nutritional status of the leaf
segments analysed, even if mineral nutrition per se is
not the environmental factor analysed for its impact
on cell membrane stability.
In the present study, we performed three sets of
experiments with durum wheat (Triticum durum
Desf), a plant species which has not yet been examined for its response to electrolyte leakage measurements. Our first aim was to determine the appropriate
time required for an adequate leaf sample washing,
the time course of electrolyte leakage in deionised
water following leaf treatment by different PEG concentrations and the stress duration effect on leaf sample electrolyte leakage during rehydration. The second aim was to analyse the behaviour of various cultivars exhibiting different levels of drought-resistance
in field conditions. The third purpose was to quantify
the relative contributions of the various leaked ions
to the measured electrolyte conductivity in relation to
the internal ion content before the stress application.
Materials and methods
Plant materials and growth conditions
This work was performed using four durum wheat
(Triticum durum Desf.) cultivars: Selbera served to
optimise the procedure of electrolyte leakage measurements in this species. Three other cultivars, exhibiting contrasting levels of drought resistance in
field conditions (Kabir 1 (drought sensitive), Omrabi
5 and Haurani (drought resistant)) were used to test
the reliability of the method as a screening tool for
drought tolerance assessment in durum wheat and to
determine the mineral composition of the leachate.
Seeds of Selbera were obtained from INRA (Rabat, Morocco) and those of Kabir 1, Omrabi 5 and
Haurani from ICARDA (Aleppo, Syria) and ENSAINRA (Montpellier, France). Seeds were allowed to
germinate on filter paper moistened with deionised
water for 2 days in a growth chamber (28/20 °C day/
night). Illumination was provided by Sylvania fluorescent tubes (F36W/133-T8/CW) for 16 h/day at a
photon flux density of 170 ␮mol m −2 s −1. The germinating seeds were then transferred to pots (7 × 7 × 8
cm) filled with compost as substrate and maintained
under greenhouse conditions (25/20 °C day/night; un-
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der a 16 h daylength consisting of natural day light
(spring-summer) supplemented with Philips mercury
lamps (HPLN 400 Watts) to reach a minimum photon flux density of about 150 ␮mol m −2 s −1; 65/70%
day/night relative humidity). Compost analysis
showed that the main characteristics of our substrate
were: pH, 5.8; dry matter, 93.4%; nitrogen, 8.4%; total ash, 16.3%; soluble ash, 4.5% and insoluble ash,
11.8%. The mineral content (in mg/100g): K, 43; P,
66; Na, 55; Mg, 128; and Ca, 1406. All plants were
grown under well watered conditions for about 20
days, at which time they had 3 expanded leaves.
Optimisation of the electrolyte leakage measurement
for the estimation of cell membrane stability in
durum wheat
In order to determine the time course of electrolyte
leakage during sample washing, the uppermost fully
expanded leaf blade of 10 plants from cultivar Selbera were collected, immediately weighed and cut
into segments (ca. 1 cm). Segments originating from
the same leaf were put into 20 ml of deionised water
in a test tube and washed slowly using a rotary shaker
(100 rpm) at room temperature to remove solutes
from both leaf surfaces and damaged cells due to cutting. Electrical conductivity (EC) of the same sample
was measured at various washing times (0, 15, 30, 45,
60, 75 and 90 min) using a LF 92 conductimeter
(WTW GmbH, Weilheim, Germany). In order to estimate the amount of electrolytes released during each
15-min interval, only the newly-released electrolytes
were taken into account and expressed in relative
terms as, for example after a 15 min washing period,
(EC 15-EC 0)/leaf fresh weight, where EC 0 and EC 15
represent electrical conductivities at the beginning (0)
and after 15 min respectively.
In order to quantify the time course of electrolyte
leakage during rehydration, leaf segments were collected as mentioned above, washed for 15 min and
allowed to stand in 20 ml of PEG (average molecular
weight of 10,000) solutions for 15 h in the dark at
25 °C. The PEG concentrations were 0, 10, 20 and
30% corresponding to osmotic potentials of −0.1,
−0.22, −0.75 and −1.46 MPa, respectively, estimated
with a vapour pressure osmometer (Wescor 5500).
Leaves of six different seedlings were used for each
treatment. After the stress period, the leaf segments
were washed quickly for three times with deionised
water and then placed in 6 ml of deionised water. An
initial electrical conductivity measure (ECi) was
taken at the beginning of this rehydration period.
Then, the tubes containing the segments were returned into the dark at 25 °C and subsequent measurements (ECf) were done at different times of rehydration (0.5, 1.5, 3.5, 7.5 and 22.5 h). Following these
readings, samples were autoclaved, cooled at 25 °C
and the total electrical conductivity (ECt) was measured. Electrolyte leakage was expressed as: (ECfECi)/(ECt-ECi) × 100.
Finally, the effect of the duration of PEG treatment
on electrolyte leakage during rehydration was quantified on samples incubated either in 0% (deionised
water) or 30% PEG during different times (0.5, 1, 2,
4, 10, 16 and 24 h) in the dark at 25 °C (10 different
plants per treatment). Electrical conductivity was
measured before (ECi) and after 4 h of rehydration
(ECf) and ultimately after autoclaving (ECt). Electrolyte leakage was expressed as (ECf-ECi)/(ECtECi) × 100.
Comparison of cultivars differing in drought
resistance using both electrolyte leakage and growth
measurements
Three cultivars varying in their drought resistance
(Kabir 1, sensitive; Omrabi 5 and Haurani, resistant)
under both field and greenhouse conditions (Ali Dib
et al. 1994; Bajji 1999; Bajji et al. 2000b, 2000c; Simane et al. 1993) were used in this experiment. Potted plants (60 plants per cultivar) were grown in controlled greenhouse conditions as described above.
Five leaves per cultivar were collected, cut in 1 cm
segments, washed for 15 min in sterile deionised water and then exposed either to 0% (control) or to 30%
PEG 10,000 for 15 h in the dark. Electrolyte leakage
was then measured before (ECi) and after (ECf) 4 h
of rehydration and ultimately after autoclaving (ECt).
Cell membrane injuries were expressed as an index
of injury (Flint et al. 1967) calculated as I d ⫽ 关
共Rs ⫺ Rc兲/共1 ⫺ Rc兲兴 ⫻ 100, where Rs and Rc represent
(ECf−ECi)/(ECt−ECi) for control or PEG-treated tissues, respectively.
The remaining material (about 40 plants per cultivar) was used for growth estimation. Water stress was
imposed by withholding water at the whole plant
level from one group (stressed) of each cultivar for 9
days. The second group was watered as usual (every
2 days, control). Shoot dry weights (48 h in an oven
at 80 °C) were determined on day 0 (DW 0) and after
9 days (DW 9) for both control and stressed seedlings
(8 plants per cultivar were used in each case). Shoot
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relative growth rate (RGR) was determined on a dry
weight basis as: RGR ⫽ 关共lnDW 9兲 ⫺ 共lnDW 0兲兴/9.
Identification and quantification of leaked ions
Uppermost fully expanded leaves collected on 12 unstressed plants of cultivars Kabir 1, Omrabi 5 and
Haurani were shared out in two lots. The first one was
used for estimation of internal ion content, quantified
after digestion of organic matter by 35% HNO 3 using
an inductively coupled argon plasma emission spectrophotometer (Jobin-Yvon JY 48) as previously described (Bajji et al. 2000a). The other set of leaves
were carefully weighed and washed as described
above and then subjected either to 0 or 30% PEG for
15 h in the dark. The leaves were then rinsed and
transferred into 6 ml of deionised water. Leachates
from these leaves (aliquots of 50 ␮ l) were collected
after 4 h of rehydration and analysed for ion concentration. For each element, the percentages of ions
leaked in the rehydration medium was estimated.
Statistical analysis
At least two independent series of each experiment
were performed and resulted in similar tendencies.
Data presented hereafter are pooled, for each experiment, from the repeated series. All measurements
were made on at least 5 individual seedlings per treatment. Statistical analysis (ANOVA) at the 5% level
was performed for all measured parameters. When the
main effect was significant, differences between
means were evaluated for significance by using the
Scheffe F-test.
Results
Optimisation of the electrolyte leakage measurement
for the estimation of cell membrane stability in
durum wheat
Figure 1 illustrates solute removal from leaf samples
as electrolyte leakage to leaf fresh weight ratio during the washing phase. A large part of the electrolytes
was removed within the first 15 min. Another lower
and almost constant part was washed away during the
successive following 15 min periods up to 90 min
from the start of washing. The lowest amount of electrolytes released during this sample washing was observed following the last 15 min interval (75–90).
Figure 1. Amount of newly-released electrolytes into deionised
water at different 15-min intervals from start of washing leaf segments of the Selbera cultivar. Because of unequal leaf sample size,
electrolyte leakage was expressed in relative terms and estimated
by the electrolyte leakage (EL) to leaf fresh weight (LFW) ratio.
Vertical bars are SE (n = 10).
During the rehydration period, PEG-induced electrolyte leakage (% of total electrolyte leakage) increased markedly and significantly between 0 and 3.5
h and then remained rather constant up to 22.5 h (Figure 2). The initial increase was larger the higher the
PEG concentration during the stress period. Statistical analysis showed that there were significant differences between all the treatments except between 10
and 20% PEG. In controls (0% PEG), electrolyte
leakage did not follow the same pattern as in the
presence of PEG; a slight and linear increase was observed during the rehydration period up to 22.5 h
reaching values less than 10% compared with more
than 40 and 60% after the 20 and 30% PEG treatments, respectively.
In leaves previously treated with 30% PEG, electrolyte leakage (% of total electrolyte leakage) after 4
h of rehydration increased markedly with the increase
of the stress period until 10 h (Figure 3). For longer
durations, there was no further increase as indicated
by the statistical analysis. However, in non-stressed
leaves (0% PEG), a lesser and constant leakage of
electrolytes was recorded whatever the treatment period. Significant differences were obtained between 0
and 30% PEG even for a short time stress period (0.5
h).
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Figure 2. Electrolyte leakage (% of total electrolyte leakage) during rehydration of control and PEG-treated samples. Leaf segments
of the Selbera cultivar were treated with 0 (control), 10, 20 and
30% PEG for 15 h then rehydrated in deionised water in the dark
during different times. Vertical bars are SE (n = 6).
Figure 3. The effect of the stress duration on electrolyte leakage
(% of total electrolyte leakage) of leaf segments of the Selbera cultivar. Samples were treated by PEG solution at 0 or 30% during
different times, then rehydrated in deionised water in the dark for
4 h. Vertical bars are SE (n = 10).
Comparison of cultivars differing in drought
resistance using both electrolyte leakage and growth
measurements
The data of injury index (Figure 4A) showed that the
drought sensitive cultivar Kabir 1 exhibited the highest values compared with the drought resistant cultivars Omrabi 5 and Haurani. The effects of withholding water on the shoot relative growth rate (RGR,
Figure 4. A. Injury index (%) in leaves of three durum wheat cultivars differing in drought resistance (Kabir 1, sensitive; Omrabi 5
and Haurani, resistant). Samples were treated by PEG solution at 0
or 30 % during 15 h, then rehydrated in deionised water in the dark
for 4 h. Vertical bars are SE (n = 5). B. Shoot relative growth rate
calculated on a dry weight basis (g/gDW/d) of three durum wheat
cultivars differing in drought resistance (Kabir 1, drought sensitive;
Omrabi 5 and Haurani, drought resistant). Seedlings at the 4th leaf
stage were either well watered (control) or water stressed (stressed)
by withholding water for 9 days. Vertical bars are SE (n = 8).
calculated on a dry weight basis) of the three cultivars are shown in Figure 4B. In the control treatment,
shoots of Kabir 1 had significantly higher RGR that
those of Omrabi 5 and Haurani. RGR of Kabir 1 was
significantly reduced (28%) by water stress while that
of Omrabi 5 was not affected. In contrast, Haurani
displayed higher RGR values in the stressed than in
the control treatment, however the differences were
not significant.
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Table 1. Internal ion concentration in leaves of durum wheat and corresponding amounts of ions leaked in deionised water at the end of 4h
of incubation following treatment with 0 or 30% of PEG 10,000 for 15 h. The percentage of ions leaked is given, for each element and each
treatment, in parentheses. Since no significant differences among genotypes were recorded, data were pooled for three durum wheat cultivars
(Kabir 1, Omrabi 5 and Haurani). Values are means of 9 replicates±S.E.
Element
Initial concentration (␮mol g −1 DW)
0%PEG (␮mol g −1 DW)
30%PEG (␮mol g −1 DW)
K
Pi
Mg
Ca
Na
Zn
Cu
Fe
Mn
Ni
TOTAL
818.7 ± 74.4
137.6 ± 14.6
53.4 ± 3.6
102.5 ± 12.0
48.6 ± 5.6
0.77 ± 0.08
0.13 ± 0.01
1.89 ± 0.22
1.65 ± 0.24
0.12 ± 0.01
1165.4
98.1 ± 3.2 (11.9)
1.3 ± 0.2 (0.9)
5.2 ± 0.2 (9.7)
11.3 ± 0.5 (11.0)
46.8 ± 2.1 (96.2)
0.19 ± 0.02 (24.6)
0.07 ± 0.01 (53.8)
1.45 ± 0.23 (76.7)
0.09 ± 0.03 (5.4)
0.08 ± 0.04 (66.7)
164.58 (14.1)
112.2 ± 5.7 (13.7)
2.2 ± 0.1 (1.6)
6.0 ± 0.3 (11.2)
13.2 ± 0.5 (12.9)
47.2 ± 2.9 (97.1)
0.23 ± 0.02 (29.8)
0.09 ± 0.01 (69.2)
1.88 ± 0.17 (99.4)
0.27 ± 0.07 (16.3)
0.11 ± 0.02 (91.6)
183.4 (15.7)
Identification and quantification of leaked ions
Discussion
No significant difference was recorded among cultivars for the initial amount of endogenous ions or the
relative proportion of leaked ions, whatever the element. Consequently, data presented in Table 1 are
pooled for the three genotypes studied. From a quantitative point of view, K, Pi and Ca are the most important ions in leaf segments collected from whole
plants. Our plant material also contained a high value
of Na: although the substrate used in our experiment
should not be considered as “saline” (EC was lower
than 4 mmhos cm −1), it has to be noted that it presents an appreciable sodium adsorption ratio. When
these segments were incubated in deionised water, the
relative amount of ions which leaked out strongly depend on the element considered: for K, Mg and Ca,
the percentage of ions that were lost in the incubating
medium ranged from 9 to 12%. This value, however,
was less than 1% in the case of Pi. In contrast, almost
all sodium leaked out and an important proportion of
microelements (mainly Zn, Cu and Fe) were also lost.
PEG treatment slightly increased the proportion of
ions lost during the subsequent rehydration period for
almost all elements: however, such an increase was
unexpectedly limited for the most important ions and
no significant differences were recorded among K, Pi,
Ca and Mg in this respect. If we estimate the mean
proportion of endogenous ions that were lost during
the rehydration period, only an absolute increase of
1.6% (corresponding to a relative increase of 11.3%)
was recorded in PEG – comparatively to non-treated
leaf segments.
What is the most adequate procedure for electrolyte
leakage measurement in durum wheat?
In the present study, we tried to design an appropriate
procedure to evaluate cell membrane injury caused by
an osmotic agent in durum wheat. The procedure is
based on electrolyte leakage measurements in leaf tissues immersed in deionised water after exposure to
an osmotic stress. As electrolyte leakage is greatly influenced by both plant and leaf age and sampling position of the leaf (Bandurska and Gniazdowska-Skoczek 1995; Adam et al. 2000; Premachandra and Shimada 1987), all experiments were conducted at the
same seedling stage, always using the whole uppermost fully expanded leaf blade. Our results will therefore be free from the error caused by these factors.
The basic method consists of three successive
steps, namely washing, dehydration and rehydration.
Different times of washing are reported in the literature from a few minutes (Bandurska and Gniazdowska-Skoczek 1995; Blum and Ebercon 1981) to 60 min
(Zwiazek and Blake 1990) and even 90 min (Premachandra and Shimada 1987; Vasquez-Tello et al.
1990). In our case, the large part of electrolytes,
which was easily accessible, was removed from the
leaf samples during the first 15 min (Figure 1) and
could be attributed to the surface adhering electrolytes and/or those removed from damaged cells or
from apoplast and vessels (Borochov-Neori and Borochov 1991). However, from 30 to 90 min, the lower
and almost regular amount of leakage could however
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arise from intact cells due to the simple effect of immersion and hypo-osmotic shock induced by deionised water. According to Pràsil and Zàmecnìk (1998),
electrolyte leakage from plant tissue in deionised water is a function of time. A rapid leakage would occur
from the intercellular free space regions followed by
slower releases across the plasma membrane and then
tonoplast. Such a pattern would be expected to follow a triple exponential function. In our work, only
two distinct phases could be identified, suggesting
that the tested durations of rinsing were not long
enough to detect the impact of immersion on tonoplast permeability. Since the aim of this step of the
electrolyte leakage test is to remove only solutes from
leaf surfaces, damaged cells due to cutting and intercellular spaces, we can recommend 15 min as a sufficient washing time.
During the rehydration period, electrolyte leakage
increased markedly and significantly between 0 and
3.5 h then remained rather constant up to 22.5 h (Figure 2). In many studies devoted to the assessment of
cell membrane stability in response to an osmotic
stress, a duration of 24 h was usually considered as a
rehydration period despite the use of different species,
molecular weights of PEG and times and intensities
of the stress (Agarie et al. 1995; Bandurska and
Gniazdowska-Skoczek 1995; Blum and Ebercon
1981; Stevanovic et al. 1997; Vasquez-Tello et al.
1990). Our work suggests 4 h as an appropriate time
for the rehydration phase in durum wheat since beyond this time (i) there was no further significant increase in leakage in response to the PEG stress and
(ii) non negligible losses due to the immersion effect
could occur. In this way, the method became less
time-consuming for routine work.
Previous results obtained in our laboratory showed
that the amount of electrolyte lost in the PEG solution during osmotic stress treatment is usually quite
low (Bajji, unpublished results). This implies that
electrolye leakage subsequently quantified in deionised water should be considered as a consequence of
hyperosmotic to hypo-osmotic transition rather than a
consequence of hyperosmotic stress sensu stricto.
Concerning the influence of the stress duration on the
membrane leakiness during rehydration, we found
that electrolyte leakage increased with the increase of
the PEG treatment until 10 h (Figure 3). For longer
duration, there was no further increase. The time
passed in the stressing condition leads to stabilisation
in the amount of leakage during subsequent rehydration which is suggestive of hardening of the stressed
tissue (Leopold et al. 1981). From this result, 10 h as
a stress period seems suitable owing to the cessation
of leakage beyond this period. In the literature, this
period was, as in the case of rehydration mentioned
above, often automatically fixed to 24 h (Agarie et al.
1995; Bandurska and Gniazdowska-Skoczek 1995;
Blum and Ebercon 1981; Premachandra and Shimada
1987; Stevanovic et al. 1997).
As a conclusion of this part of the present work,
we recommend 15 min as the appropriate time required for leaf segment washing, 10 h as an adequate
stress duration and about 4 h as a rehydration period.
These findings show that a widely used technique
such as electrolyte leakage measurement can be adjusted to a particular species and, in this manner, it
becomes less time-consuming.
Is the electrolyte leakage measurement related to
the drought resistance level of durum wheat
cultivars?
The possibility of using electrolyte leakage measurement to evaluate water stress tolerance in durum
wheat has been studied here jointly with seedling
growth of three contrasting cultivars following a period of water shortage. The drought sensitive cultivar
Kabir 1 showed greater relative membrane injury (expressed as injury index) than the two drought resistant ones Omrabi 5 and Haurani (Figure 4A). As far
as seedling relative growth rate (calculated on a dry
weight basis) is concerned, Kabir 1 was, once again,
the only cultivar affected by water stress at the shoot
level (Figure 4B). Hence, our results indicate that the
responses of leaf electrolyte leakage to PEG-induced
osmotic stress not only correlate well with the effect
of withholding water on seedling growth but also with
field drought resistance. Indeed, observations made
with field-grown plants have given the following results: Kabir 1 was found to be characterised by a poor
yield stability and is highly sensitive to water stress
(Ali Dib et al. 1994). Omrabi 5 is an early-maturing
cultivar selected under dry and hot conditions (Simane et al. 1993); it is characterised by its high yield
potential and stability (Ali Dib et al. 1994; Simane et
al. 1993). Haurani is a Syrian landrace adapted to
drought (Simane et al. 1993). It should however be
underlined that in our study, the impact of drought
imposed at the whole plant level on cell membrane
stability quantified by the electrolyte method was not
considered: electrolyte leakage was quantified after
an osmotic shock imposed by PEG on leaf segments
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collected on non-stressed plants. Different results
would have been expected if whole plants were submitted to water stress for days or even weeks before
harvesting leaf segments for electrolyte leakage estimation, because water stress may modify the chemical composition and physical structure of biological
membranes (Lauriano et al. 2000; Senaratna et al.
1987), which, in turns, has a direct impact on the rate
of electrolyte leakage (Knowles et al. 2001).
By using the electrolyte leakage method, we have
demonstrated that maintenance of membrane integrity
in leaf segments under osmotic stress correlates with
the drought tolerance in durum wheat estimated on
the basis of the growth of whole plants. The way we
estimated electrolyte leakage suggest that it may be
used as a “predictive” criterion of putative water
stress resistance in whole plants. The varietal differences reported here and in our previous studies (Bajji
1999; Bajji et al. 2000a, 2000c) between the three
cultivars may offer partial explanations for the differential tolerance to drought stress observed in these
cultivars. Although it is not clear how much membrane competence may contribute to drought tolerance in durum wheat, this evidence suggests that it
may be an important component. Less membrane
damage in the present work was correlated with an
increased capacity to accumulate sugars at the leaf
level during water stress (Bajji 1999; Bajji et al.
2000c). In fact, it was hypothesised that sugars, particularly nonreducing disaccharides such as sucrose
and trehalose (in few species) interact with cellular
membranes to increase the stability of the lipid layers
(Nilsen and Orcutt 1996). The protective mechanism
of these solutes is still uncertain, but one hypothesis
suggests that under desiccation, molecules normally
associated with the phospholipid head-groups are replaced with sugars (Leopold and Vertucci 1986). This
may prevent lateral phase transition and the formation
of lipid domains which have the potential of forming
inverted micelles and thus increasing membrane leakage. Increased accumulation of such compatible solutes in leaf tissues of the drought resistant cultivars
would reduce dehydration damage and promote
growth during and after water stress (Bajji et al.
2000c).
Which compounds may be involved in the
stress-induced increase of electrical conductivity?
Our results demonstrate that the contribution of the
different quantified ions to the electrical conductivity
measured during the rehydration step may be extremely variable. Some rare studies which analysed
this aspect in other species concluded that an important part of this electrical conductivity may be attributed to potassium and its unidentified counteranions
(Palliotti and Bongi 1996; Palta et al. 1977; Shcherbakova and Kacperska 1983). In our work, potassium
undoubtedly is the major inorganic ion recorded in
the effusate (Table 1). In contrast, Vasquez-Tello et al.
(1990) reported an important leakage of Pi in bean:
this, obviously, did not occur in our material. According to these authors, such an increase in Pi exudation
may be related to a stimulation of chloroplastic acid
phosphatase but it was recorded after 24 h rather than
4 hours of rehydration. Sodium and calcium were also
reported to afford an appreciable contribution to measured electrical conductivity (Palliotti and Bongi
1996; Premachandra et al. 1989). In our experiment,
almost all the sodium leaked in the external medium,
even in the case of leaf segments which were not
treated by PEG. Martinoia et al. (1986) consider that
most sodium present in the mesophyll is sequestered
in the vacuoles. Thus, the major contribution of Na to
the electrical conductivity suggests that the tonoplast,
and not only the plasmalemma, may be affected by
the rehydration treatment. On the other hand, if some
part of the sodium is present in the apoplast, it may
be present in the effusate without resulting from a loss
of membrane semipermeability, although, in principle, the washing step has been optimised for the elimination of ions from intercellular spaces (see above).
The fact that the proportion of leaked ions was different for the various elements analysed suggests that
PEG stress followed by rehydration had a different
impact on specific transporters involved in the translocation of ions across membranes rather than a mechanical effect of disruption of membrane continuity.
Moreover, we should keep in mind that the total ion
concentration of the tissues was estimated after digestion of organic matter by nitric acid and would thus
consider some ions which were bound to macromolecules and therefore not susceptible to leak out during the rehydration steps. These considerations lead
us to conclude that the nutritional status of tested tissues should be carefully considered, especially if the
method is applied to durum wheat plants exposed to
salt stress, since salinity usually induces an increase
in the endogenous Na as well as a decrease in K.
One of the most important, although unexpected,
observations, results from the fact that the PEG-induced increase in inorganic ions (from 14.1 to 15.7%
9
of endogenous ions) does not explain the recorded
increase in electrical conductivity of the effusate (Figure 3). Indeed, the recorded increase in the leakage
of most ions was only a few percent and only minor
microelements, available in small amounts, exhibited
a large increase in exudation rates (Table 1). Moreover, no differences were recorded among cultivars
for the relative importance of mineral contribution to
recorded increase in electrical conductivity (Table 1)
while differences were recorded for the injury percentages (Figure 4). Obviously, other compounds
should be involved in such an increase of electrical
conductivity. Several authors reported a stress-induced leakage of organic compounds such as UV-absorbing substances (De and Mukherjee 1996), sugars
(Palta et al. 1977) and amino acids (Shcherbakova
and Kacperska 1983). Sugars are often considered as
the major non-electrolyte compounds present in the
effusate (Palta et al. 1977). We suggest that organic
acids should be considered in durum wheat since a
decrease in the pH of effusate may be recorded in the
specific case of PEG-treated leaf segments (Lutts, unpublished results).
The aim of the present work was to analyse the
validity of the quick and inexpensive method of electrolyte leakage measurement as a predictive test for
screening drought resistance in durum wheat. Although the method may indeed be recommended for
this purpose, we conclude that the mineral status of
the plant should be considered since the relative contribution of various ions may be quite different. Other
experiments are also needed in order to identify the
major contributors to the PEG-induced increase in EC
and to test the effect of water stress applied in vivo,
at the whole plant level, on electrolyte leakage assessment.
Acknowledgements
The authors are very grateful to AUPELF-UREF
(ARC “Biotechnologies et tolérance à l’aridité chez
les céréales) for financial support and to ICARDA
(Aleppo, Syria), ENSA-INRA (Montpellier, France)
and INRA (Rabat, Morocco) for kindly providing the
seeds.
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