Sei sulla pagina 1di 9

Plant Science 170 (2006) 511–519

Comparative study of cadmium effects on membrane lipid

composition of Brassica juncea and
Brassica napus leaves
Issam Nouairi a, Wided Ben Ammar a, Nabil Ben Youssef a,
Douja Ben Miled Daoud a, Mohamed Habib Ghorbal b,
Mokhtar Zarrouk a,*
Laboratoire Caractérisation et Qualité de l’Huile d’Olive, Institut National de Recherche Scientifique et Technique,
B.P. 95, 2050 Hammam-Lif, Tunisia
Unité Nutrition et Métabolisme Azotés et Protéines de Stress, Département des Sciences Biologiques, Faculté des Sciences de Tunis,
Campus Universitaire, 1060 Tunis, Tunisia
Received 26 April 2005; received in revised form 7 October 2005; accepted 7 October 2005
Available online 28 October 2005

The term phytoremediation is used to describe the clean-up of heavy metals from contaminated soils by plants. In this study, we examined
cadmium (Cd) accumulation by Brassica napus in comparison with the known Cd-hyperaccumulator Brassica juncea, in a hydroponic medium. Cd
treatment was applied as a concentration series between 0 and 50 mM for 15 days. Most of the Cd taken up was retained in roots in both species,
however, B. juncea accumulated more Cd in the shoots compared to B. napus. Excess metal supply resulted in an increase in the lipid content of B.
juncea leaves grown under cadmium treatment, but did not affect fatty acids composition. In contrast, an alteration in the lipid composition of B.
napus leaves was observed together with a decrease in the lipid contents. The amounts of chloroplastic lipids: monogalactosyldiacylglycerol
(MGDG), digalactosyldiacylglycerol (DGDG), sulfolipids (SL) and phosphatidylglycerol (PG) decreased drastically under the effect of metallic
treatments. Whereas, amounts of extrachloroplastic lipids: phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were significantly
increased. The latter finding suggested that PC and PE synthesis was enhanced by metal application. Moreover, the levels of polyunsaturated fatty
acids mainly linolenic acid (C18:3) and hexadecatrienoı̈c acid (C16:3) and that of trans palmitoleic acid (C16:1t) from PG declined. So, cadmium
seems to affect preferentially chloroplastic lipids containing higher levels of polyunsaturated fatty acids. Lipid changes in B. juncea, the well
known Cd-hyperaccumulator specie, revealed a more stability of its cellular membranes to cadmium-stress as compared to Cd-sensitive specie, B.
# 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: Cadmium; Brassica juncea; Brassica napus; Lipid composition; Fatty acids; Leaves

1. Introduction ties, and gene expression [2–4]. Many of these effects can be
interrelated through a general action on membrane biogenesis
Heavy metal contamination of soils and waterways is a and integrity, which in turn can occur because lipid metabolism
serious environmental problem with potentially harmful is altered. Plant membrane structure may be regarded as the first
consequences to agriculture and human health [1]. In non- ‘‘living’’ structure that is a target for heavy metal toxicity [5].
tolerant plant species, heavy metals have been shown to affect a Although all plants can be affected by high levels of heavy
wide range of plant cellular activities including photosynthesis, metals, some species are quite tolerant to lower amounts. In this
respiration, mineral nutrition, membrane structure and proper- context, Brassica juncea has been identified as a high biomass-
producing crop with the capacity to take up and accumulate
heavy metals such as Cd, Cu, Ni, Zn, Pb and Se [6]. This
* Corresponding author. Tel.: +216 71 430 855; fax: +216 71 430 934. tolerance has practical application in bioremediation and in
E-mail address: (M. Zarrouk). efforts to colonize polluted sites [7].
0168-9452/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved.
512 I. Nouairi et al. / Plant Science 170 (2006) 511–519

In order to understand the ways in which tolerance may amount of this powder was dissolved in 3 parts of 1 M HNO3
occur, research into the role of membranes and in the and 1 part of 1 M HClO4, and the metal concentration in
metabolism of lipids (as major membrane components) is solution was analyzed by atomic absorption spectrophotometry
needed for several reasons: (i) alterations in the composition of (Philips PYE Unicam PU 9000).
the plasma membrane may change membrane permeability
and, consequently, net metal ion uptake [8]; (ii) the level of lipid 2.3. Chlorophyll content of young leaves
peroxidation which can alter many metabolic processes in the
cell is determined, to an important extent, by the degree of fatty Small lamina discs were cut from fresh leaves, and
acid unsaturation of membrane lipids [9]. Membrane unsatura- chlorophylls were extracted in dark for 72 h in 80% acetone.
tion has been shown to be closely related to heavy metal Chlorophylls were determined according to Strain et al. [14].
tolerance in a number of higher plants [2]; and (iii) the rapid
turnover of membrane components may represent a strategy for 2.4. Lipid extraction and separation
adaptative modification to metal stress.
Cadmium accumulation in soil and water now poses a major Measurements were carried out on the second pair of leaves
environmental and human health problem, the use of metal grown after Cd treatment of each species. The lipids were
accumulating plants to remove toxic metals, including Cd, from extracted according to the method of Folch et al. [15] modified
soil and aqueous streams has been proposed as a possible by Bligh and Dyer [16]. The plant tissues were fixed in boiling
solution to this problem [10]. The aim of the present study was water for 5 min to denature phospholipases [17] and then
to compare the temporal pattern of Cd accumulation and lipid homogenized in chloroform:methanol mixture (2:1, v/v). The
composition changes in the leaves of B. juncea, identified as a homogenate was centrifuged at 3000  g for 15 min. The lower
metal accumulator and a high biomass crop plant within the chloroformic phase containing lipids was aspirated and
Brassicaceae family [11], with a Cd-sensitive specie from the evaporated at 40 8C under vacuum using a rotary evaporator
same family, Brassica napus [12,13]. or with Nitrogen gas. The residue was immediately redissolved
in 2 ml of Toluene:ethanol mixture (4:1, v/v) for conservation.
2. Materials and methods Phospholipids (PL), glycolipids (GL) and neutral lipids (NL)
were separated by TLC on silica gel plates 60 (Merck) with the
2.1. Plant material following solvent mixtures: chloroform:acetone:methanol:ace-
tic acid:water (50:20:10:10:5, v/v/v/v) [18]. After development,
Seeds of Indian mustard (B. juncea, accession no. 426308) bands were located with iodine vapours or spraying the plates
identified as a metal accumulator [6] were obtained from the with 0.1% Rhodamine 6G in ethanol. Individual lipids were
North Central Regional Plant Introduction Station (Ames, IA), identified by comparison with lipid standards and by specific
and oilseed rape (B. napus L., cv. Drakkar) were germinated on strains for PL and GL.
vermiculite and grown hydroponically for 2 weeks in black
containers with 6 l of continuously aerated nutrient solution 2.5. Fatty acids determination
(pH 5.5) contained 136 mg/l KH2PO4, 492.3 mg/l Ca(NO3)2,
303 mg/l KNO3, 17.4 mg/l K2HPO4, 146 mg/l MgSO4, Fatty acids from total lipids and lipid classes were
1.98 mg/l MnCl2, 0.294 mg/l CuSO4, 0.287 mg/l ZnSO4, methylated by the method of Metcalfe et al. [19]. Methyl
1.85 mg/l H3BO3, 0.37 mg/l (NH4)MoO4 and 20 mM Fe- esters of fatty acids were separated and quantified with a
EDTA. The containers were placed in a greenhouse chamber Hewlett-Packard model 4890D gas chromatograph equipped
with day/night temperature and humidity regime of 25/20 8C with a 30 m  0.25 mm  0.25 mm film thickness fused Silica
and 55/75% relative humidity (RH), respectively. A 16 h (daily) capillary column (Innowax) coupled to a flame ionisation
photoperiod was used. The nutrient solution was changed every detector (Column temperature 210 8C). Both the injector and
3 days until the experiment was started. Cadmium was added to detector were maintained at 230 and 250 8C, respectively.
the medium as CdCl2 in four concentrations: 0, 10, 25 and Nitrogen was used as the carrier gas at 1 ml/min with split
50 mM. during treatment with CdCl2, the hydroponic medium injector system (split ratio 1:100). For measuring the amount of
was changed daily to avoid depletion of Cd in nutrient solution. fatty acids, heptadecanoic acid C17:0 was added as internal
After 15 days of heavy metal treatment, samples were standard before methylation. Calculation of fatty acid contents
harvested and used for chemical analyses. was obtained using an integrator.

2.2. Cadmium accumulation 3. Results and discussion

Total shoot and root accumulation of Cd in B. juncea and 3.1. Cd accumulation in Brassica juncea and Brassica
B. napus were determined after 15 days of treatment in order to napus
estimate the test plants usefulness in phytoremediation of Cd-
contaminated sites. Roots and shoots were harvested, washed in Data from atomic absorption spectrometry showed that the
deionized water for 2 min, air dried at 80 8C for 2 days, and then Cd content in both roots and shoots increased significantly with
ground into a fine powder using a pestle and mortar. A known exposure to increasing cadmium concentrations (Fig. 1). Cd
I. Nouairi et al. / Plant Science 170 (2006) 511–519 513

Fig. 1. Cd accumulation in shoots (A); and roots (B) of B. juncea (&) and
B. napus (&) exposed to various Cd concentrations in the nutrient solution for
Fig. 2. Shoot dry weight after 15 days of treatment with different Cd concentra-
15 days. Data represent the mean  S.D. of three independent experiments.
tions. Error bars represent the mean  S.D. of three independent experiments.

accumulation was higher in roots than in shoots in both species. decrease of shoot dry weight in both species (Fig. 2). After Cd
Very high amounts of Cd were taken up by roots and the treatment, root dry weight was reduced by about 56 and 29% as
accumulation rates in roots of B. juncea and B. napus were compared to control plants for B. juncea and B. napus,
comparable (Cd accumulation were up to 4725  583 and respectively, at high Cd concentration (Fig. 3). Growth
4626  690 mg Cd g 1 on dry weight basis, respectively, when reduction in response to Cd-stress was also reported for
plants were exposed to 100 mM Cd). In parallel, B. juncea Phaseolus vulgaris [25], Pisum sativum [26], B. napus [12], B.
appeared to accumulate three times more Cd in the shoots than juncea [23] and for various Brassica species after exposure to
B. napus (values for 15 days exposure: 1450  245 and excess Zn or Cu [27]. These authors hypothesized that the
555  120 mg Cd g 1 dry wt., respectively, when plants were observed decrease in shoot Fe and Mn contents may
exposed to 100 mM Cd). For this reason, B. juncea may be significantly contribute to the inhibition of plant growth, and
useful in some phytoremediation strategies in regions with to the competition between Cd and essential cations resulting in
excessive cadmium accumulation, and can be defined as an decreased nutrient uptake and transport. However, other data
hyperaccumulator plant as it accumulates at least 1000 ppm indicate a significant decrease of Mn content in roots and shoots
metal in the shoots [20]. of B. juncea and B. napus plants in response to Cd stress
The relatively high concentrations of Cd (10, 25, 50 and [12,23].
100 mM) freely available to the plants were chosen to evaluate Over the range of Cd concentrations used, a significant
the ability of B. juncea and B. napus to withstand severe stress. reduction in the total Chl content of the young leaves of B.
Similar Cd concentrations may occur on field contaminated juncea and B. napus was observed from 10 mM Cd in solution
sites [1,21] especially when the phytoremediation process is (Fig. 4). According to Stobart et al. [28], the decrease in
enforced by increasing Cd availability to the plants with chlorophyll content is due to inhibition of protochlorophyllide
artificial chelators such as EDTA or EGTA [22]. In a direct reduction and inhibition of aminolevulinic acid synthesis.
comparison of B. juncea and B. napus in hydroponic solution
containing Cd, B. juncea accumulate more Cd in the shoots than
B. napus. Several reports suggest that B. juncea is capable to
accumulate high levels of Cd within its shoots from soil or
hydrponic solution [23,24]. In B. juncea, Cd appeared to
accumulate preferentially in the trichomes of the youngest leaf
surface [23]. The storage of Cd in trichomes may represent a
detoxification mechanism, since trichomes represent an
external tissue to the leaf.

3.2. Cd inhibits shoots and roots growth

A detailed growth analysis of shoots and roots of both

species revealed that when B. juncea and B. napus plants were
exposed to various cadmium concentrations in hydroponic
culture, growth rate declined dramatically after 15 days of Cd
treatment. Cd exposure of B. juncea and B. napus also produced
a severe decrease in shoots dry weight, with the highest Cd Fig. 3. Root dry weight after 15 days of treatment with different Cd concentra-
concentration (100 mM) causing, respectively, 65 and 75% tions. Error bars represent the mean  S.D. of three independent experiments.
514 I. Nouairi et al. / Plant Science 170 (2006) 511–519

Fig. 5. Total lipid content of young leaves of B. juncea (&) and B. napus (&)
Fig. 4. Chlorophyll content of young leaves of B. juncea and B. napus exposed exposed to various Cd concentrations in the nutrient solution for 15 days. Lipid
to various Cd concentrations in the nutrient solution for 15 days. Data represent content was estimated from the amounts of fatty acids. Data represent the
the mean  S.D. of three independent experiments. mean  S.D. of three independent experiments.

In vitro studies have demonstrated dramatic effects of Cd on (0 mM Cd) for each species, respectively. The drastic alteration
photosynthesis at various levels such as (i) direct effects on showed in lipid content of B. napus leaves, can be explained by
Photosystem II (PSII) electron transport [29]; (ii) decrease of Cd inducing disturbance of the membrane lipid turnover.
plastoquinone levels in the chloroplast [30]; (iii) inhibition of Cadmium metal enhanced lipoxygenase activity, which respon-
the Calvin cycle [31], or (iv) failure in chlorophyll biosynthesis sible for catalysing lipid peroxidation by using membrane lipid
[32]. And may therefore strongly affect biomass production. components as substrates, particularly unsaturated fatty acids
The recently reported Cd-induced reduction in growth, leaf [36]. Heavy metals are also involved in many ways in the
chlorophyll content and photochemical quantum yield of production of activated oxygen species that actively induce
photosynthesis for 10-day-old seedlings of B. napus grown peroxidation of membrane lipids [4]. A lower total lipid content
under high light intensity [12]. Indicates, that in vivo Cd effects has been demonstrated in plants subjected to metals stress [37,8].
on photosynthesis depend on plant species, plant age, time of The reduction in the total lipid amount (Fig. 5) of B. napus leaves,
Cd exposure. including a severe decrease ( 81%) at 50 mM Cd of glycolipids
However, the absence of Cd effects on photosynthetic (GL) content (Table 1) the main lipids of B. napus leaf, which in
activity during 4 days treatment as observed with adult B. the control plant, accounted for about half of the total lipids. Cd
juncea plants [24] most likely results from an effective excess (50 mM) caused a remarkable decrease by 84 and 81% in
protection of chloroplasts by cellular detoxification mechan- the amounts of the two principal GL molecules in particular the
isms, keeping Cd in the cytosol below a critical threshold level. monogalactosyldiacylglycerol (MGDG) and digalactosyldiacyl-
The absence of Cd effect on photosynthetic performance of glycerol (DGDG), respectively (Table 1). We have found, at the
B. juncea did not exclude the significant decrease of same Cd concentration (50 mM), an increase by 13% in the
chlorophyll content in this study, a similar result was also amount of phospholipids fraction (PL). The contents of
observed by Salt et al. [23] in B. juncea plants exposed to phosphatidylcholine (PC) and phosphatidylethanolamine (PE),
various Cd concentrations in the nutrient solution for 7 days, expressed as mg g 1 dry wt., increased by 118 and 88%,
and may be caused by a growth reduction despite maintenance respectively, in B. napus leaves (Table 1). Furthermore, the
of full photosynthetic activity. MGDG/DGDG ratio was reduced by 20% at 50 mM Cd in
comparison with the control plants (Table 2). Change in the ratio
3.3. Cd effects on fatty acids and lipid composition of these two molecules might alter the proportion of lipids with
different phase structures. In fact, MGDG is considered a lipid
Plant cell membranes are dynamic in behaviour, with a lipid indispensable for PSII activity, because it regulates the
composition changing with variations in the environment, and supramolecular structure of PSII complexes [38]. The decrease
may be considered as the first ‘‘living’’ structure that is target in the MGDG/DGDG ratio can be interpreted as a loss of PSII
for heavy metal toxicity [33]. Certainly direct effects of Cd complex stability [8]. In comparison with the control, plants of B.
treatments on the lipid composition of membranes have been napus grown at 50 mM Cd showed a rearrangement in the lipid
reported [34,35]. In this study, we have found that the amount of membranes composition (Table 2). Whereas the proportions of
total lipids, which evaluated from the amount of total fatty acids chloroplastic lipids such as MGDG, DGDG and phosphatidyl-
and expressed in mg fatty acid g 1 dry wt., was changed in glycerol (PG) decreased, that of PC and PE increased (Table 2).
treated plants, and was remarkably dependent on metal doses Changes in the PL composition due to Cd treatment may affect
and plant species. The results given in Fig. 5 indicate a dramatic the fluidity and also the intrinsic-membrane protein activities as a
decrease by 52% of the total lipid content in the young leaf of B. result of alterations in the lipidic environment in which they are
napus plants grown at 50 mM Cd for 15 days. While it increased embedded [8]. The PC/PE ratio was slightly increased at high
by 19% in B. juncea leaves in comparison with control plants cadmium dose (50 mM).
I. Nouairi et al. / Plant Science 170 (2006) 511–519 515

Table 1
Lipid content (mg g dry weight) of B. napus young leaves from plants treated with different Cd concentrations
Cd (mM) Lipid content (mg g dry wt.)
Glycolipids (GL) Phospholipids (PL) Neutral lipids (NL)
a a a a c b a a b
0 11.47 1.87 1.44 16.78 2.70 1.33 3.28 1.64 0.88 9.83c 7.65a
10 4.96b 1.68a 1.26a 7.90b 7.56a 3.56a 2.50b 1.66a 1.35a 16.63a 4.39b
25 4.45b 1.41b 0.91b 6.76b 6.40a 3.50a 2.40b 1.70a 1.26a 15.26a 5.09b
50 1.80c 0.75c 0.55c 3.10c 5.90a 2.50a 1.00c 0.84c 0.84b 11.08b 2.12c
Results are the means of three replicates. For comparisons among means an analysis of variance was used. For each treatment means in rows followed by different
letters are significantly different at P=0.05.

Table 2
Lipid composition (mol%), MGDG to DGDG ratio and PC to PE ratio of B. napus young leaves from plants treated with different Cd concentrations
Cd (mM) Lipid composition (%) MGDG/DGDG ratio PC/PE ratio
Glycolipids (GL) Phospholipids (PL) Neutral lipids (NL)
0 33.47a 11.29a 4.20a 48.96a 7.88c 3.88b 9.57a 4.78b 2.56b 28.67c 22.32a 2.96a 2.03b
10 16.28b 5.80b 4.35a 26.43b 26.14 12.30a
8.64b 5.73ba 4.66a 57.47b 15.17b 2.80a 2.12b
25 16.50b 5.23b 3.34b 25.07b 23.78b 13.00a 8.91ab 6.31a 4.68a 56.68b 18.91c 3.15a 1.83b
50 11.65c 4.90b 3.68b 20.23c 23.51a 15.33a 6.13c 4.90b 4.90a 66.77a 13.00b 2.37b 2.31a
Data are given as % of total lipids. Results are the means of three replicates. For comparisons among means an analysis of variance was used. The significance of the
letters is the same as in Table 1.

For B. juncea plants exposed for 15 days to different of specific PL expressed as mol% of total lipids showed only an
concentrations of Cd in the nutrient solution, our results showed increase in the relative abundance of PG and PE proportions
an increase in lipid membranes amount (Fig. 5). Leaf total (+34% for PG and +17% for PE with the control plants)
lipids of B. juncea treated with 50 mM Cd increased by 19% in (Table 4). However, PC, phosphatidic acid (PA) and
comparison with control plants grown without Cd. This phosphatidylinositol (PI) proportions being unaffected by
increase can cause an augmentation of the total membrane 50 mM Cd (Table 4). With regard to the PL changes, the
area of the cells in particular the vacuolar membrane. Earlier PC/PE ratio lowered slightly from 3.07 to 2.57 by 50 mM Cd
studies showed that the vacuole is the site for the accumulation (Table 4). In this context, membrane phase behaviour is also
of a number of heavy metals including Zn and Cd [39,40], this regulated by PL composition and an appropriate balance
way can be considered as a very important mechanism of heavy between bilayer-forming and non-bilayer-forming lipids [41].
metal tolerance and detoxification with phytochelatins [5]. In The 50 mM Cd supply lowered the PC/PE ratio in B. juncea,
B. juncea, cadmium treatment (50 mM) caused an increase in which may have led to enriched non-lamellar-forming domains
the amounts of all lipid classes in comparison with control and phase separation of non-lamellar-forming lipids [42].
(+8% for GL, +29% for PL and +19% for neutral lipids (NL)) Furthermore, we also observed that the MGDG/DGDG ratio
(Table 3). Analysis of the lipid composition of B. juncea leaf increased by 85% in B. juncea plants at 50 mM Cd treatment.
membranes showed that exposure to 50 mM Cd for 15 days had This higher MGDG/DGDG ratio can be correlated with a stress
no effect on the relative abundance of various lipids. Analysis resistance of B. juncea in our observation. Similar results were

Table 3
Lipid content (mg g dry weight) of B. juncea young leaves from plants treated with different Cd concentrations
Cd (mM) Lipid content (mg g dry wt.)
Glycolipids (GL) Phospholipids (PL) Neutral lipids (NL)
b a c b b b c b b
0 6.01 4.39 0.41 10.81 4.82 1.56 1.93 1.03 1.07 10.43c 4.29b
10 4.19c 3.31b 1.66a 9.17c 5.59a 2.44a 2.35b 0.97b 1.35a 12.72b 3.04c
25 5.83b 4.48a 0.82b 11.14ab 5.60a 2.27a 2.93a 2.27a 1.09b 14.18a 3.28c
50 7.71a 3.05b 0.87b 11.65b 5.62a 2.18a 3.07a 1.21b 1.40a 13.51ab 5.23a
Results are the means of three replicates. For comparisons among means an analysis of variance was used. The significance of the letters is the same as in Table 1.
516 I. Nouairi et al. / Plant Science 170 (2006) 511–519

Table 4
Lipid composition (mol%), MGDG to DGDG ratio and PC to PE ratio of B. juncea young leaves from plants treated with different Cd concentrations
Cd (mM) Lipid composition (%) MGDG/DGDG ratio PC/PE ratio
Glycolipids (GL) Phospholipids (PL) Neutral lipids (NL)
a a a a b c c b b
0 23.48 17.17 1.60 42.26 18.83 6.12 7.55 4.04 4.21 40.77b 16.76a 1.36b 3.07a
10 16.80b 13.29b 6.68b 36.78b 22.44a 9.78a 9.44b 3.88b 5.42a 50.98a 12.18b 1.26b 2.29b
25 20.39a 15.66a 2.87c 38.93b 19.56b 7.96b 10.26a 7.94a 3.82b 49.55a 11.46b 1.30b 2.45b
50 25.38a 10.06c 2.86c 38.31b 18.50b 7.19b 10.12a 3.98b 4.63b 44.44ab 17.21a 2.52a 2.57b
Data are given as % of total lipids. Results are the means of three replicates. For comparisons among means an analysis of variance was used. The significance of the
letters is the same as in Table 1.

also reported for aluminium-resistant cultivar of Triticum extent of unsaturation did not change (Table 5). Among the
aestivum [43]. Concerning fatty acid composition, we have different fatty acids, it is noteworthy that excess cadmium
found that the unsaturation of lipids was altered in young leaves decreased dramatically the PG-16:1 trans percentage (Table 5).
of B. napus plants grown under different doses of Cd (Table 5). The lower unsaturation of MGDG and PG lipids, considered
Membrane unsaturation has been shown to be closely related to indispensable for PSII activity [38], might have altered the
heavy metal tolerance in a number of higher plants [2]. The intimate lateral contact between the membrane-spanning
metal induced a decrease in the MGDG, DGDG, NL and PG domains of integral proteins and the lipid bilayer [44]. In fact,
unsaturation and increased that of PC and PE, but the total it has been suggested that the high level of unsaturation of

Table 5
Fatty acid composition (mol%) of lipids of B. napus young leaves from plants treated with different Cd concentrations
Lipid categories Cd (mM) C16:0 C16:1c C16:1t C16:3 C18:0 C18:1 C18:2 C18:3 Unsaturation
b b b a c c c b
MGDG 0 2.01 0.70 0.44 38.72 0.20 0.60 2.41 55.02 97.89a
10 4.70a 0.76b 0.50b 29.21b 0.41c 1.22b 3.61b 59.73a 95.03b
25 5.10a 1.25a 0.90a 28.90b 1.22b 3.10a 3.91b 55.72b 93.78c
50 4.92a 1.10a 0.80a 28.73b 2.00a 2.85a 4.76a 55.00b 93.24c
DGDG 0 23.60a 0.43c 2.01a 3.38a 2.50c 3.00c 5.21b 60.01a 74.04a
10 24.21a 0.71b 2.17a 3.04ab 6.70b 10.73b 15.02a 37.60b 69.27b
25 24.13a 2.30a 0.42b 2.80b 8.51a 10.26b 14.32a 37.48b 67.58b
50 22.56b 2.11a 0.31b 2.50c 9.05a 11.01a 14.30a 38.30b 68.53b
SL 0 44.70b 1.10c 0.22 Tr 4.30c 5.20c 14.61a 29.30a 50.43a
10 46.81b 1.30c Tr 0.83b 5.24b 8.36b 10.13b 27.50a 48.12a
25 50.12a 1.90b Tr 0.60b 7.42a 11.00a 12.47ab 16.61c 42.58b
50 40.26c 3.05a Tr 1.10a 8.31a 11.10a 14.71a 21.67b 51.63a
PC 0 22.70a 0.94a 0.20 0.90b 5.63a 7.91b 37.72a 24.11b 71.78c
10 15.40b 0.71b Tr 1.01b 5.02a 8.68a 36.00b 33.30a 79.70b
25 15.01b 0.90a Tr 0.90b 5.70a 8.11b 36.04b 33.40a 79.35b
50 13.60c 0.82ab Tr 1.41a 6.26a 8.89a 35.41b 33.83a 80.36a
PE 0 28.21a 0.71b Tr 0.91a 6.03b 7.10b 38.02b 19.17b 66.45c
10 25.20b 0.86b 0.32a 0.91a 5.53c 8.10a 36.21b 23.00a 69.43b
25 20.70c 1.91a 0.20a 1.10a 6.20b 8.43a 37.13b 24.48a 73.25a
50 21.01c 2.12a Tr 0.85a 7.10a 8.12a 39.91a 21.05b 72.05ba
PG 0 25.48b 0.40b 29.61a 5.22a 1.91c 5.30b 12.01a 20.20b 72.76a
10 39.20a 1.02a 12.30b 4.70a 4.53b 5.11b 9.12b 24.17a 56.42b
25 40.15a 1.17a 13.91b 2.20b 10.20a 7.94a 11.62a 13.00c 49.84c
50 38.70a 1.01a 13.80b 1.30c 9.61a 8.12a 11.91a 15.60c 51.74c
NL 0 24.91a 1.31c 3.20b 16.32a 2.83c 3.40c 8.10c 40.11a 72.44a
10 23.50ab 3.62b 4.60a 11.03b 4.52b 3.60c 10.22b 39.00a 72.07a
25 21.20c 7.68a 2.00c 6.72c 8.80a 7.52b 11.70b 34.50b 70.12b
50 22.70bc 7.10a Tr 7.44c 10.10a 9.86a 13.01a 29.90c 67.31c
Total lipids 0 17.30b 0.90a 1.30a 13.60a 2.11c 5.11c 13.32c 44.90a 79.13a
10 16.75b 0.73ab 1.12a 5.19c 4.90b 9.09a 20.12b 36.86b 73.11a
25 20.91a 0.63b 1.00a 9.01b 4.10b 6.25b 17.99b 38.37b 73.25a
50 18.21b 0.31c 0.66c 5.80c 7.02a 7.09ba 28.15a 32.91c 74.92a
Results are the means of three replicates. For comparisons among means an analysis of variance was used. The significance of the letters is the same as in Table 1 (Tr, trace).
I. Nouairi et al. / Plant Science 170 (2006) 511–519 517

Table 6
Fatty acid composition (mol%) of lipids of B. juncea young leaves from plants treated with different Cd concentrations
Lipid categories Cd (mM) C16:0 C16:1c C16:1t C16:3 C18:0 C18:1 C18:2 C18:3 Unsaturation
a b a a b b b b
MGDG 0 7.80 1.30 0.70 28.80 1.21 4.70 4.90 50.20 90.81a
10 6.70a 1.61a 0.22b 29.05a 2.60a 6.40a 5.30ab 48.02b 90.70a
25 7.62a 1.23b 0.10b 26.51b 2.01a 2.00c 4.82b 55.40a 90.29a
50 6.80a 0.80c 0.21b 23.90c 2.02a 1.71c 5.40a 59.51a 91.75a
DGDG 0 30.60c 2.01a Tr 2.23a 10.51b 8.00a 6.01a 40.41a 58.77a
10 34.51b 0.70b Tr 1.65b 8.20c 7.71a 6.32a 40.60a 57.19a
25 38.50a 0.11c Tr 1.61b 7.62c 2.70b 6.15a 42.90ab 53.69b
50 39.72a Tr Tr 1.40b 12.80a 2.90b 4.31b 38.70b 47.43c
SL 0 36.80c Tr Tr Tr 14.71b 21.08b 16.50a 8.60a 46.29a
10 45.11b Tr Tr Tr 7.39b 24.50a 15.50a 7.41a 47.61a
25 49.52a Tr Tr Tr 11.30b 5.71d 17.01a 15.00b 37.84b
50 49.40a Tr Tr Tr 22.19a 7.80c 12.20b 8.20a 28.39c
PC 0 43.35a Tr Tr Tr 5.61b 8.20a 22.01b 20.80a 51.07b
10 37.70b Tr Tr Tr 7.90a 9.82a 25.86a 18.51b 54.29a
25 40.61b 0.50a Tr Tr 8.10a 7.82a 24.51a 18.22b 51.26b
50 40.70b 0.52a Tr Tr 9.02a 8.64a 22.20b 18.70b 50.18b
PE 0 45.10b Tr Tr Tr 8.20c 12.60a 23.11b 10.82b 46.63a
10 47.89b 1.71a Tr Tr 9.13b 9.02b 23.21b 8.82b 43.08a
25 42.01b 0.42b Tr Tr 9.01b 4.40c 30.10a 13.80a 48.84a
50 52.00a Tr Tr Tr 12.50a 3.61c 22.32b 9.42b 35.45b
PG 0 31.52a Tr 30.53a 1.80a 4.30a 12.24a 11.41b 8.10b 64.20a
10 31.70a Tr 29.91a 0.50c 6.80a 14.41a 9.80b 6.62c 61.34a
25 29.91a Tr 27.60b 0.91b 5.92a 11.90a 13.90a 9.70a 64.04a
50 32.43a 0.35 31.20a 0.31c 6.41a 6.20b 13.25a 9.70a 61.25a
NL 0 28.60a 4.10a 12.71a 12.02a 0.70a 8.11a 6.00a 17.30a 60.64a
10 40.50b 5.91b 12.02a 13.60a 0.71a 8.31a 6.90a 10.31b 57.36b
25 37.70b 4.20a 6.01b 15.02a 7.20b 6.40b 7.21a 16.04a 55.08b
50 38.50b 2.63c 3.42c 11.01a 7.45b 6.06b 9.10b 21.82c 54.19b
Total lipids 0 16.70b 0.51a 2.77a 14.60a 1.61b 1.90b 12.70b 49.00a 81.59a
10 18.45a 0.50a 2.31a 10.81b 2.80a 2.80a 17.32a 44.50a 78.59a
25 18.90a 0.50a 2.20a 11.40b 2.70a 2.20ab 16.70a 45.90a 79.10a
50 17.71ba 0.42b 2.30a 11.81b 2.84a 1.90b 15.91a 46.50a 79.19a
Results are the means of three replicates. For comparisons among means an analysis of variance was used. The significance of the letters is the same as in Table 1 (Tr, trace).

thylakoid lipids may be required to maintain the degree of required to maintain an optimal PSII activity under metal stress.
fluidity needed for the diffusion of lipophilic compounds and/or The 50 mM Cd supply lowered the others lipids unsaturation
may confer a suitable geometry to the lipid molecules. (Table 6), and can be related to a maintained antioxidative
Furthermore, phospholipid fatty acids affect bilayer properties defence system by a decrease in the cell membrane fluidity of B.
regulating its fluidity and permeability and the activities of juncea leaves. Such as B. napus, the total amounts of lipid
membrane-bound enzymes as well [45]. Thus, the increased of unsaturation of B. juncea young leaves, did not change with
PC and PE unsaturation in B. napus plants grown at different increase of Cd concentrations in the nutrient solution.
concentrations of Cd, may indicate an increase in the fluidity of
the membrane surface which could facilitate the deep penetration 4. Conclusion
of harmful activated oxygen species into the cell membranes,
where susceptible fatty acid double bonds are located [46]. On In conclusion, great differences occurred between B. napus
the other hand, alterations in the unsaturation level of the and B. juncea in Cd accumulation and their membrane lipid
individual lipids did not change the total amount of lipid changes under Cd stressed conditions. We have investigated the
unsaturation of membrane leaves of B. napus plants treated with effect of Cd on the lipid membrane composition. Marked
increased Cd concentrations. Thus, it is reasonable to assume that changes in membrane composition were observed after 15 days
only the fluidity of local microenvironments was affected and not treatment. Some of these changes were specific for each
the fluidity of the bulk of the lipids [47]. In contrast to B. napus, species. It is also debatable whether these changes represent a
cadmium treatment did not significantly affect the MGDG, PG phytotoxic lesion arising from the toxic effects of Cd, or from
and PC unsaturation (Table 6), and did not induce any change in actions initiated to restore optimal physical properties.
the PG-16:1 trans of B. juncea treated plants. This stability of Summing up, we propose that the higher loss of membrane
associated thylakoid lipids (MGDG and PG) unsaturation may be lipids in B. napus plants treated with Cd may be related to
518 I. Nouairi et al. / Plant Science 170 (2006) 511–519

enhanced rate of catabolism and/or the suppression of lipid [15] J. Folch, M. Lee, G.H. Sloane-Stanley, A simple method for the isolation
of total lipids from animal tissues, J. Biol. Chem. 226 (1957) 497–509.
biosynthesis. Although our data do not exclude the directly
[16] E.G. Bligh, W.J. Dyer, A rapid method of total lipid extraction and
oxidative breakdown of polyunsaturated lipids. The results purification, Can. J. Biochem. Physiol. 37 (1959) 911–917.
observed in B. juncea plants, showed that besides changes in [17] R. Douce, Identification et dosage de quelques glycérophosphatides dans
lipids composition, this specie may have an efficient defence des souches normales et tumorales de scosonères cultivés in vitro, C.R.
strategies which can be related on antioxidative production Acad. Sci. 259 (1964) 3066–3068.
against oxidative stress. Our future goal involves examining the [18] M. Lepage, Identification and composition of turnip root lipids, Lipids 2
(1967) 244–250.
responses of the antioxidative enzymes, and to study the lipid [19] D. Metcalfe, A. Schmitz, J.R. Pelka, Rapid preparation of fatty acid esters
biosynthesis by assessing metabolism through radiolabelling, from lipids for gas chromatographic analysis, Anal. Chem. 38 (1966) 524–
to further elucidate their function in the metal tolerance 535.
mechanisms of hyperaccumulator plants. [20] W.H.O. Ernst, Geobotanical and biogeochemical prospecting for heavy
metal deposits in Europe and Africa, in: B. Markert (Ed.), Plants as
Biomonitors—Indicators for Heavy Metals in the Terrestrial Environment,
Acknowledgment V.C.H., Weinheim, 1993, pp. 107–126.
[21] R. Chaney, Health risks associated with toxic metals in municipal sludge,
Seeds of Brassica juncea cv. 426308 were kindly provided in: G. Bitton, B. Damron, G. Edds, M. Davidson (Eds.), Sludge—Health
by the North Central Regional Plant Introduction Station Risks of Land Application, Ann Arbor Science, Ann Arbor, M.I., 1980, pp.
(NCRPIS), Iowa State University, USA. 59–83.
[22] M.J. Blaylock, D.E. Salt, S. Dushenkov, O. Zakharova, C. Gussman, Y.
Kapulink, B.D. Ensley, I. Raskin, Enhanced accumulation of Pb in Indian
References mustard by soil-applied chelating agents, Environ. Sci. Technol. 31 (1997)
[1] L.S. Di Toppi, R. Gabbrielli, Response to cadmium in higher plants, [23] D.E. Salt, R.C. Prince, J.L. Pickering, I. Raskin, Mechanisms of cadmium
Environ. Exp. Bot. 41 (1999) 105–130. mobility and accumulation in Indian mustard, Plant Physiol. 109 (1995)
[2] W. Maksymiec, Effect of copper on cellular processes in higher plants, 1427–1433.
Photosynthetica 34 (1997) 321–342. [24] A. Haag-Kerwer, H.J. Schäfer, S. Heiss, C. Walter, T. Rausch, Cadmium
[3] S. Rama Deli, M.N.V. Prasad, Membrane lipid alterations in heavy metal exposure in Brassica juncea causes a decline in transpiration rate and leaf
exposed plants, in: M.N.V. Prasad, J. Hagemeyer (Eds.), Heavy Metal expansion without effect on photosynthesis, J. Exp. Bot. 50 (1999) 1827–
Stress in Plants—From Molecules to Ecosystems, Springer, Berlin, 1999, 1835.
pp. 99–117. [25] C. Poschenrieder, B. Gunsé, J. Barceló, Influence of cadmium on water
[4] P.H.P. Peixoto, J. Cambraia, R. Sant Anna, P.R. Mosquim, M.A. Moreira, relations, stomatal resistance, and abscisic acid content in expanding bean
Aluminium effects on fatty acid composition and lipid peroxidation of a leaves, Plant Physiol. 90 (1989) 1365–1371.
purified plasma membrane fraction of root apices of two sorghum [26] M. Ashraf, I.S. Vera, A.B. Andrei, D. Karl-Josef, Genotypic variation of
cultivars, J. Plant Nutr. 24 (2001) 1061–1070. the response to cadmium toxicity in Pisum sativum L, J. Exp. Bot. 56
[5] J.L. Hall, Cellular mechanisms for heavy metal detoxification and toler- (2005) 167–178.
ance, J. Exp. Bot. 53 (2002) 1–11. [27] S.D. Ebbs, L.V. Kochian, Toxicity of zinc and copper to Brassica species:
[6] P.B.A.N. Kumar, V. Dushenkov, H. Motto, I. Raskin, Phytoextraction: the implications for phytoremediation, J. Environ. Qual. 26 (1997) 776–781.
use of plants to remove heavy metals from soils, Environ. Sci. Technol. 29 [28] A.K. Stobart, W.T. Griffiths, I. Ameen-Bukhari, R.P. Sherwood, The effect
(1995) 1232–1238. of Cd2+ on the biosynthesis of chlorophyll in leaves of barley, Physiol.
[7] B.H. Robinson, R.R. Brooks, A.W. Howden, J.H. Kirkman, P.E.H. Gregg, Plant. 63 (1985) 293–298.
The potential of the high biomass nickel hyperaccumulator Berkheya codii [29] T. Baszynski, Interference of Cd2+ in functioning of the photosynthetic
for phytoremediation and phytomining, J. Geochem. Explor. 60 (1997) apparatus in higher plants, Acta Soc. Bot. Poland 55 (1986) 291–304.
115–126. [30] Z. Krupa, G. Öquist, N.P.A. Huner, The influence of cadmium on primary
[8] M.F. Quartacci, C. Pinzino, C.L.M. Sgherri, F. Dalla Vecchia, F. Navari- photosystem II photochemistry in bean as revealed by chlorophyll a
Izzo, Growth in excess copper induces changes in the lipid composition fluorescence—a preliminary study, Acta Physiol. Plant. 14 (1992) 71–76.
and fluidity of PSII-enriched membranes in wheat, Physiol. Plant. 108 [31] H.J. Weigel, Inhibition of photosynthetic reactions of isolated intact
(2000) 87–93. chloroplast by cadmium, J. Plant Physiol. 119 (1985) 179–189.
[9] J.L. Harwood, Environmental effects on plant lipid biochemistry, in: J.L. [32] K. Padmaja, D.D.K. Prasad, A.R.K. Prasad, Inhibition of chlorophyll
Harwood (Ed.), Plant Lipid Biosynthesis—Fundamentals and Agricul- synthesis in Phaseolus vulgaris L. Seedlings by cadmium acetate, Photo-
tural Applications, Cambridge University Press, Cambridge, 1998 , pp. synthetica 24 (1990) 399–405.
305–363. [33] J.L. Harwood, Recent environmental concerns and lipid metabolism, in:
[10] D.E. Salt, I.J. Pickering, R.C. Prince, D. Gleba, S. Dushenkov, R.D. Smith, J.C. Kader, P. Mazliak (Eds.), Plant Lipid Metabolism, Kluwer Academic
I. Raskin, Metal accumulation by aquacultured seedlings of Indian Publishers, Dordrecht, 1995, pp. 361–368.
mustard, Environ. Sci. Technol. 3 (1997) 1636–1644. [34] R.O.C. Ros, D.T. Cooke, R.S. Burden, C.S. James, Effects of the herbicide
[11] G.S. Banuelos, D.W. Meek, Accumulation of selenium in plants grown on MCPA and the heavy metals, cadmium and nickel on the lipid composi-
selenium-treated soil, J. Environ. Qual. 19 (1990) 772–777. tion, Mg2+-ATPase activity and fluidity of plasma membranes from rice,
[12] E.H. Larsson, J.F. Bornman, H. Asp, Influence of UV-B radiation and Cd2+ Oryza sativa (cv. Bahia) shoots, J. Exp. Bot. 41 (1990) 457–462.
on chlorophyll fluorescence, growth and nutrient content in Brassica [35] L.E. Hernández, D.T. Cooke, Modification of the root plasma membrane
napus, J. Exp. Bot. 49 (1998) 1031–1039. lipid composition of cadmium-treated Pisum sativum, J. Exp. Bot. 48
[13] N. Ben Youssef, M. Zarrouk, D.D. Ben Miled, A. Cherif, M.H. Ghorbal, (1997) 1375–1381.
Effets du cadmium sur la composition en lipides foliaires de plantules de [36] J.E. Thompson, C.D. Froese, E. Madey, M.D. Smith, Y. Hong, Lipid
colza (Brassica napus L.), Rivista Italiana delle Sostanze Grasse LXXX metabolism during plant senescence, Prog. Lipid Res. 37 (1998) 119–
(2003) 165–170. 141.
[14] H.H. Strain, B.T. Cope, W.A. Svec, Analytical procedures for the isola- [37] O. Ouariti, N. Boussama, M. Zarrouk, A. Cherif, M.H. Ghorbal, Cad-
tion, identification, estimation and investigation of the chlorophylls, mium- and copper-induced changes in tomato membrane lipids, Phyto-
Methods Enzymol. 23 (1971) 452–476. chemistry 45 (1997) 1343–1350.
I. Nouairi et al. / Plant Science 170 (2006) 511–519 519

[38] N. Murata, S.I. Higashi, Y. Fujimura, Glycerolipids in various preparations aluminum-resistant and aluminum-sensitive cultivar of Triticum aestivum,
of photosystem II from spinach chloroplasts, Biochem. Biophys. Acta Physiol. Plant. 96 (1996) 683–691.
1019 (1990) 261–268. [44] D.J. Murphy, The molecular organization of the photosynthetic mem-
[39] W.H.O. Ernst, J.A.C. Verkleij, H. Schat, Metal tolerance in plants, Acta branes of higher plants, Biochim. Biophys. Acta 864 (1986) 33–94.
Bot. Neerl. 41 (1992) 229–248. [45] M.F. Quartacci, E. Cosi, F. Navari-Izzo, Lipids and NADPH-dependent
[40] D.N. De, Plant Cell Vacuoles, CSIRO Publishing, Collingwood, Aus- superoxide production in plasma membrane vesicles from roots of wheat
tralia, 2000. grown under copper deficiency or excess, J. Exp. Bot. 52 (2001)
[41] Y.Y. Leshem, Plant membrane biophysics in development and senescence, 77–84.
in: Y.Y. Leshem (Ed.), Plant Membranes—A Biophysical Approach to [46] W.I. Gruszecki, K. Strzalka, Does xanthophyll cycle take part in the
Structure, Development and Senescence, Kluwer Academic Publishers, regulation of fluidity of the thylakoid membrane? Biochim. Biophys. Acta
Boston, 1992, pp. 111–154. 1060 (1991) 310–314.
[42] P.J. Quinn, W.P. Williams, Structure and dynamic of plant membranes, in: [47] M. Droppa, J. Masojidek, Z. Rozsa, A. Wolak, L.I. Horváth, T. Farkas, G.
J.L. Harwood, J.R. Bowyer (Eds.), Methods in Plant Biochemistry, Horváth, Characteristics of Cu deficiency-induced inhibition of photo-
Academic Press, London, 1984, pp. 297–340. synthetic electron transport in spinach chloroplasts, Biochim. Biophys.
[43] G. Zhang, J.J. Slaski, D.J. Archambault, G.J. Taylor, Aluminum-induced Acta 891 (1987) 75–84.
alterations in lipid composition of microsomal membranes from an