The effect of cadmium on lipid and fatty acid

Biologia 63/1: 86—93, 2008 Section Cellular and Molecular Biology DOI: 10.2478/s11756-008-0002-6

The effect of cadmium on lipid and fatty acid biosynthesis in tomato leaves Wided Ben Ammar1, Issam Nouairi2, Mokhtar Zarrouk2 & Fatma Jemal1 1

Unité de Recherche “Nutrition et Métabolisme Azotés et Protéines de Stress (UR 99/09-20)”, Faculté des Sciences de Tunis, Campus Universitaire, 2092 Tunis El Manar, Tunisie; e-mail: [email protected] 2 Laboratoire Caractérisation et Qualité de l’Huile d’Olive, Centre de Biotechnologie de Borj Cedria, B.P. 901–2050 Hammam-Lif, Tunisie

Abstract: This research aims to examine the effect of cadmium uptake on lipid composition and fatty acid biosynthesis, in young leaves of tomato treated seedlings (Lycopersicon esculentum cv. Ibiza F1). Results in membrane lipids investigations revealed that high cadmium concentrations affect the main lipid classes, leading to strong changes in their composition and fatty acid content. Thus, the exposure of tomato plants to cadmium caused a concentration-related decrease in the unsaturated fatty acid content, resulting in a lower degree of fatty acid unsaturation. The level of lipid peroxides was significantly enhanced at high Cd concentrations. Studies of the lipid metabolism using radioactive labelling with [1-14 C]acetate as a major precursor of lipid biosynthesis, showed that levels of radioactivity incorporation in total lipids as well as in all lipid classes were lowered by Cd doses. In total lipid fatty acids, [1-14 C]acetate incorporation was reduced in tri-unsaturated fatty acids (C16:3 and C18:3); While it was enhanced in the palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0) and linoleic (C18:2) acids. [1-14 C]acetate incorporation into C16:3 and C18:3 of galactolipids [monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG)] and some phospholipids [phosphatidylcholine (PC) and phosphatidylglycerol (PG)] was inhibited by Cd stress. Our results showed that in tomato plants, cadmium stress provoked an inhibition of polar lipid biosynthesis and reduced fatty acid desaturation process. Key words: biosynthesis; cadmium; fatty acid; lipids;Lycopersicon esculentum; radioactivity labelling. Abbreviations: DGDG, digalactosyldiacylglycerol; DW, dry weight; GL, glycolipids; MGDG, monogalactosyldiacylglycerol; NL, neutral lipids; PC, phosphatidylcholine; PG, phosphatidylglycerol; PL, phospholipids; TBARS, thiobarbituric acid-reacting substances; TL, total lipids.

Introduction Heavy metals are recognized as environmental pollutants and are released from both industrial and agricultural sources. The intensive use of high-phosphate fertilizers in agriculture leads to an increased accumulation of metal ions, especially cadmium, in the soil (Taylor 1997). Cadmium is a non-redox-reactive heavy metal, and its toxicity is believed to be due to its action on a wide range of plant cellular activities. In non-tolerant plant species, cadmium has been shown to affect the mineral distribution (Ben Ammar et al. 2005), the nitrogen (Chaffei et al. 2003) and carbon assimilation (Gouia et al. 2003), the photosynthetic processes (Fargašová 2004; Ben Ammar et al. 2005) and the enzymatic activity (Sch¨ utzend¨ ubel et al. 2001; Sobkowiak et al. 2004). Many of these effects can be interrelated through a general action on membrane biogenesis and integrity, which in turn can occur because lipid metabolism is altered. Certainly direct effects of Cd treatment on the lipid composition of membranes have been reported (Ros et al. 1990; Fodor et al. 1995; Hernandez & Cooke 1997; Jemal et al. 2002; Nouairi et al. 2006) which may

have a direct effect on membrane permeability. In addition, Cd treatment reduced the ATPase activity of the plasma membrane fraction of wheat and sunflower roots (Fodor et al. 1995) and could account for increased leakage of nutrients (Ros et al. 1990). These modifications have also been found in plants grown under several environmental stresses. Thus, in the literature there are examples of changes in the phospholipids composition due to low temperatures (Lynch & Steponkus 1987) or salinity (Douglas & Walker 1984), for example, modification of sterols content under salinity (Mansour et al. 1994) or changes in the unsaturation ratio of fatty acids of plants subjected to salt (Zenoff et al. 1994) and metallic (Jemal et al. 2002; Nouairi et al. 2006) stress. Changes in the properties of cellular membranes, observed under metallic stress, may be preceded from the inhibition of lipid biosynthesis and/or degradation by metallic stress. It has been demonstrated that cadmium, like copper (Quartacci et al. 2001; Tripathi et al. 2006) and aluminium (Yamamoto et al. 2001), induced membrane lipid peroxidation by generating reactive oxygen species, which initiate peroxidation of

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polyunsaturated fatty acids (Ben Youssef et al. 2005). Membrane peroxidation is a complex process, which is normally initiated following the abstraction of a hydrogen atom from endogenous unsaturated fatty acids, resulting in the production of lipid radicals. Lipid peroxidation can also be initiated enzymatically through a sequential concerted action of lipoxygenase. Lipoxygenase catalyzes the oxygenation of long chain fatty acids containing a cis,cis-1,4–pentadiene structure to hydroperoxides. Linoleic and linolenic acids are the most abundant fatty acids of this structure in plants. Cd stress has been shown to stimulate lipoxygenase activity (Ben Youssef et al. 2005) and consequently may explain the alteration in unsaturated fatty acids content, observed in Cd-treated plants. Cd action on the lipid peroxidation has been well established; however, its effect on the membrane lipid and fatty acids biosynthesis has not been clearly investigated. The aim of the present work was therefore to examine the effects of Cd stress on lipid and fatty acid biosynthesis in tomato leaves. Material and methods Growth conditions Tomato seeds (Lycopersicon esculentum Mill. cv. Ibiza F1) were first sterilized in 10% (v/v) H2 O2 for 20 min, then thoroughly washed with distilled water and germinated on moistened filter paper at 25 ◦C in the dark for 8 days. Uniform seedlings were then transferred to a continuously aerated nutrient solution (pH 6.0) containing: 2.5 mM KH2 PO4 , 0.5 mM Ca(NO3 )2 , 3 mM KNO3 , 0.5 mM MgSO4 , 100 µM Fe-K-EDTA, 5 µM MnSO4 , 1 µM ZnSO4 , 1 µM CuSO4 , 30 µM H3 BO3 , 1 µM (NH4 )6 Mno7 O24 . Cadmium was added to the medium as CdCl2 at the following concentrations: 0, 1, 25 and 50 µM. After 7 days of Cd treatment, young leaves (developed after application of Cd treatment) were harvested and used for chemical analyses. Lipid extraction and analysis The lipids were extracted according to the method of Allen & Good (1971). Measurements were carried out on the young leaves (appeared after Cd treatment). The plant tissues were fixed in boiling water for 5 min to denature phospholipases (Douce 1964) and then homogenized in chloroform/methanol mixture (1/1, v/v). The homogenate was centrifuged at 3,000 g for 15 min. The lower chloroformic phase containing lipids was aspirated and evaporated at 40 ◦C under vacuum using a rotary evaporator or under a nitrogen flow. The residue was immediately re-dissolved in 2 mL of toluene/ethanol mixture (4/1, v/v) for conservation. Total lipids extracted were separated into phospholipids (PL), glycolipids (GL) and neutral lipids (NL) on silica gel TLC plates, by the procedure of Lepage (1967). To visualize the lipid bands, the plates were sprayed with I2 vapour. Lipid classes were identified by comparison with lipid standards and by specific stains for PL and galactolipids. Fatty acids from total lipids were methylated by the method of Metcalfe et al. (1966). Fatty acid methyl esters were separated and quantified with a gas chromatograph Hewlett-Packard model 4890D equipped with a 30 m × 0.25 mm × 0.25 µm film thickness fused Silica capillary column (Innowax) coupled to a flame ionisation detector (Column

temperature 210 ◦C). Both the injector and detector were maintained at 230 ◦C and 250 ◦C, respectively. Nitrogen was used as the carrier gas at the flow of 1 mL/min with split injector system (split ratio 1:100). For measuring the amount of fatty acids, heptadecanoic acid (C17:0) was added as internal standard before methylation. Calculation of fatty acid contents was obtained using an integrator Hewlett-Packard model 3390 A. Lipid peroxide determination Lipid peroxide was determined by measuring the concentration of thiobarbituric acid-reacting substances (TBARS), as described by Alia (1995). A fraction of leaf tissue was homogenized in 5% (w/v) trichloroacetic acid. After centrifugation, a sample of the supernatant was added to 20% trichloroacetic acid containing 0.5% (w/v) thiobarbituric acid. The mixture was incubated at 95 ◦C for 30 min, then quickly cooled. After the tube was centrifuged at 10,000 g for 10 min, the absorbance was measured at 532 nm. Radiolabelling protocol For biosynthesis studies, we have used the [1-14 C]acetate (1.95 GBq/mmol) purchased from Amersham International p.l.c., (Amersham, Bucks, U.K.). Radioactive labelling of lipids was achieved by laying microdroplets of the radioactive precursor on attached leaves of either control or treated plants. After incubation at 1, 2, 6, 12 and 24 h, leaves were gently washed with deionized water in order to remove the precursor remaining on the leaf surface. Total lipids (TL) were extracted according to the method of Allen & Good (1971). PL, GL and NL were separated by TLC on silica gel plates 60 (Merck) with the following solvent mixtures: chloroform:acetone:methanol:acetic acid:water (50:20:10:10:5, v/v) (Lepage 1967). After development, bands were located with iodine vapours or spraying the plates with 0.1% rhodamine 6G in ethanol. Individual lipids were identified by comparison with lipid standards and by specific strains for PL and GL. The TL, GL, PL and NL-associated radioactivity was determined by liquid scintillation counting (Beckman LS 6500 Liquid Scintillation Counter). The radioactive fatty acids, in the form of methyl esters, from the TL and the various lipid classes were separated by TLC from silica gel impregnated of silver nitrate. The methyl esters of the marked fatty acids were deposited in bottom of the plate. The elution solvent was composed of hexane: ethylic ether: formic acid in the proportions 7:3:0.8 (v/v). The spots were scraped and the radioactivity was measured by liquid scintillation. Statistics In all experiments three replicates were performed for each time of exposure to Cd and each tested concentration. Data presented are the means ± SD of three independent experiments.

Results Lipid content and fatty acid composition Changes in the polar lipids amounts extracted from tomato leaves produced in cadmium treated seedling are given in Figure 1. A significant decline in the total lipid content was observed, especially at high Cd concentrations. This decline was associated with a corresponding reduction in the amounts of all lipid classes.

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Table 1. Fatty acid composition in total lipids, galactolipids and phospholipids of tomato leaves from plants exposed to cadmium for 7 days.a Cd (µM)

TL

MGDG

DGDG

PC

PG

0 1 10 50 0 1 10 50 0 1 10 50 0 1 10 50 0 1 10 50

C16:0 19.80 19.60 21.00 26.00 17.72 16.62 21.77 28.57 30.30 32.48 25.30 23.15 38.87 39.65 42.49 46.15 31.63 29.95 36.21 38.63

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.2 0.1 0.3 1.4 0.2 0.4 1.2 0.8 0.6 0.8 1.1 0.1 0.2 0.0 1.5 0.2 0.8 0.3 1.2 0.4

C16:1 02.00 01.90 00.90 00.00 01.78 01.28 01.75 00.94 01.16 01.53 00.96 01.19 01.78 01.47 02.12 02.02 24.21 25.39 20.81 19.75

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.1 0.2 0.0 0.0 0.0 0.3 0.1 0.1 0.7 0.3 0.3 0.1 0.1 0.1 0.3 0.0 0.6 0.1 0.1 0.2

C16:3 07.50 ± 07.30 ± 05.60 ± 04.60 ± 19.73 ± 19.68 ± 13.62 ± 11.78 ± 01.22 ± 01.01 ± 00.90 ± 00.76 ± 00.56 ± 00.03 ± 00.50 ± – 01.03 ± 01.62 ± 02.46 ± 01.77 ±

0.5 0.3 0.2 0.2 0.2 0.1 0.1 0.0 0.2 0.0 0.1 0.1 0.0 0.1 0.1 0.4 0.1 0.0 0.2

C18:0 01.50 01.80 02.90 03.00 02.20 02.98 07.27 10.37 03.77 02.73 02.17 01.83 03.88 03.37 06.17 05.78 02.54 02.50 06.32 06.22

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.4 0.2 0.1 0.2 0.1 0.0 0.3 0.3 0.5 0.3 0.2 0.1 0.2 0.3 0.2 0.2 0.2 0.2 0.5 0.5

C18:1 03.20 03.40 04.10 07.10 03.45 03.14 04.40 03.32 01.70 01.98 06.48 07.35 09.00 09.62 14.27 14.85 16.93 16.95 19.41 19.12

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

C18:2

0.7 0.2 0.4 0.2 0.1 0.1 0.1 0.3 0.1 0.1 0.6 0.1 0.5 0.2 0.2 0.0 0.8 0.1 0.0 0.1

22.40 23.40 27.10 24.50 05.28 05.19 06.71 06.91 06.74 06.98 05.17 04.85 22.07 21.71 17.38 15.12 07.88 07.41 05.66 04.33

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.5 1.3 0.2 1.2 0.4 0.2 0.6 0.1 0.2 0.2 0.1 0.1 0.1 0.4 0.3 0.2 0.4 0.2 0.3 0.2

C18:3 43.60 ± 0.3 42.60 ± 0.8 38.40 ± 0.4 34.80 ± 0.2 49.84 ± 1.8 51.11 ± 0.9 44.48 ± 0.4 38.11 ± 0.6 55.11 ± 0.8 53.29 ± 0.6 59.02 ± 1.2 60.87 ± 0.4 23.84 ± 0.8 24.15 ± 1.1 17.07 ± 0.3 16.08 ± 002 15.78 ± 0.2 16.18 ± 0.4 10.13 ± 0.2 10.18 ± 0.1

a Total

lipids (TL), galactolipids (MGDG, monogalactosyldiacylglycerol; DGDG, digalactosyldiacylglycerol) and phospholipids (PC, phosphatidylcholine; PG, phosphatidylglycerol). Data are expressed in percentage of total fatty acid (%). Data are the means ± SD of three independent experiments. C16:0, palmitic acid; C16:1, hexadecenoic acid; C16:3, hexadecatrienoic acid; C18:0, stearic acid; C18:1, oleic acid; C18:2, linoleic acid; C18:3, linolenic acid.

300

-1

TBARS (nmol,g DW)

400

200

100

0 0

1

25

50

Cd (µM)

Fig. 1. Total lipid, phospholipid, galactolipid and neutral lipid composition in leaves of tomato seedling treated for 7 days with different concentration of CdCl2 . Data are the means ± SD of three independent experiments.

The fatty acid composition of total lipids was summarized in Table 1. We noted that tomato membrane lipids are formed by palmitic (C16:0), palmitoleic (C16:1), hexadecatrienoic (C16:3), stearic (C18:0), oleic (C18:1), linoleic (C18:2) and linolenic (C18:3) acids. Cd treatment reduced the C18:3 levels and increased the C16:0, C18:0, C18:1 ones. To refine our investigation, the fatty acid composition of the galactolipids and the principal PL was analysed in Cd-treated leaves. Table 1 shows that Cd affects differently the C18:3 percentages in the monogalactosyldiacylglycerol (MGDG) and (di-

Fig. 2. Thiobarbituric acid reactive substances (TBARS) in leaves of tomato plants treated for 7 days with different concentrations of CdCl2 . Data are the means ± SD of four independent experiments.

galactosyldiacylglycerol) DGDG molecules. In fact, in the DGDG, the level of C18:3 was constant or increased slightly at higher Cd doses, however, it was reduced in the MGDG. The decline in the C18:3 content was paralleled by an increase of C18:0 and C16:0 percentages (Table 1). The fatty acid composition of phosphatidylcholine (PC) and phosphatidylglycerol (PG) was also changed by Cd treatment. As shown in Table 1, a large decrease in the C18:3 percentages was found, often accompanied by an increase in the C16:0, C18:0 and the C18:1 levels. Peroxidation status To determine the susceptibility of tomato plants to Cd-



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Fig. 3. Effect of Cd stress on biosynthesis of total lipid, galactolipid, phospholipids and neutral lipid in tomato leaves as revealed by radiolabelling experiments using sodium[1-14 C]acetate as a radioactive precursor.

Fig. 4. Effect of Cd stress on biosynthesis of different fatty acids of total lipids in tomato leaves as revealed by radiolabelling experiments using sodium[1-14 C]acetate as a radioactive precursor.


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Fig. 5. Effect of Cd stress on biosynthesis of monogalactosyldiacylglycerol (MGDG) in tomato leaves as revealed by radiolabelling experiments using sodium[1-14 C]acetate as a radioactive precursor.

induced lipid peroxidation, leaves of plants, exposed to a range of Cd concentrations, were harvested and analysed for their levels of lipid peroxidation products. Figure 2 shows that Cd concentration greater than 1 µM caused a significant increase of lipid peroxide products. The TBARS content passed from 76 nmol.g−1 dry weight (DW) in the control leaves to 266 nmol · g−1 DW in leaves treated with 50 µM CdCl2 . The TBARS content was found to be dependent on Cd concentration in growth medium. Effect of cadmium on lipid biosynthesis In order to explain the Cd effect on the membrane lipid composition, experiments with sodium [1-14 C]acetate as precursor were carried out. Our results showed that application of 25 and 50 µM CdCl2 depressed the quantity of radioactivity incorporated in total lipids for all incubation times (Fig. 3). For the time 24 h, the level of acetate incorporation passed from 12.6 × 106 cpm/g DW in control leaves to 2.7 × 106 cpm/g DW in leaves treated with 50 µM CdCl2 . Moreover, Cd treatment reduced the radioactivity amount incorporated in to all lipid classes (Fig. 2). Likewise, the total fatty acids biosynthesis was disrupted by the metallic stress. Figure 4 illustrates that

there was a large decline in the levels of the acetate incorporation in the tri-unsaturated fatty acids (C18:3 and C16:3). However the rate of the radioactivity incorporation in palmitic (C16:0) and stearic (C18:0) acids was increased for all incubation times. Similar result seems to be the consequence of Cd effects on the fatty acids composition of galactolipids and the principal phospholipids (PC and PG). In the MGDG molecule, Cd reduced the radioactivity incorporation into the C18:3 and C16:3 and stimulated the C18:1, C18:2 and C16:0/C18:0 biosynthesis (Fig. 5). In the DGDG, Cd treatment affected differently the tri-unsaturated fatty acids biosynthesis. It inhibited the C16:3 biosynthesis and stimulated the C18:3 production. The radioactivity incorporation into C 16:1 and C16:0/C 18:0 was also stimulated (Fig. 6). It seems that in the DGDG molecule, the cadmium inhibits the biosynthesis of C18 fatty acids and stimulates that of the C16. Figures 7 and 8 show the radioactivity variations in the PC and PG fatty acids. It is clear that Cd reduced the radioactivity level in the tri-unsaturated fatty acids and increased that of the C16:0/C18:0, C16:1 and C18:1.



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Fig. 6. Effect of Cd stress on biosynthesis of digalactosyldiacylglycerol (DGDG) in tomato leaves as revealed by radiolabelling experiments using sodium[1-14 C]acetate as a radioactive precursor.

Fig. 7. Effect of Cd stress on biosynthesis of phosphatidylcholine (PC) in tomato leaves as revealed by radiolabelling experiments using sodium[1-14 C]acetate as a radioactive precursor.


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Fig. 8. Effect of Cd stress on biosynthesis of phosphatidylglycerol (PG) in tomato leaves as revealed by radiolabelling experiments using sodium[1-14 C]acetate as a radioactive precursor.

Discussion Plant cell membranes are dynamic in behaviour, with a lipid composition changing according to variations in the environment and may be considered as the first “living” structure that is a target for heavy metal toxicity (Quartacci et al. 2001). In this study, we have found that the membrane lipid composition was changed in treated leaves, and was dependent on metal doses (Fig. 1). The supply of Cd induces a decrease in the TL content. Similar reduction was due to a sharp decrease in GL, PL and NL. The same tendency was reported as a result of heavy metal toxicity (Quartacci et al. 2001; Ben Yousef et al. 2005; Nouairi et al. 2005), salinity (Ben Hamed et al. 2005) and drought stress (Gigon et al. 2004). Changes in the PL level due to metallic treatment may affect the fluidity. The intrinsic-membrane protein activities may also be modified by the alterations in the lipid environment in which they are embedded (Quartacci et al. 2000). The lipid degradation was generally interpreted as causing a decrease in the total membrane area of the cells, which would indicate a decrease in the number and size of cellular organelles, such as chloroplasts (Djebali et al. 2002). The drastic alteration in lipid content could be due to a slowing down of the lipids biosynthetic processes as well as to an acceleration of the degradative phenomena. In order to confirm this hypothesis and to monitor rapidly any disturbances to lipid metabolism, in tomato treated with Cd, experiments with sodium (1-14 C) acetate were performed. Our results, represented in Fig-

ure 3, showed a decrease in 14 C incorporation into total lipids. Similar result was a consequence of Cd action in the GL and PL biosynthesis. On the other hand, the analysis of the total fatty acids composition showed a lower unsaturation level in the leaves of plants grown with Cd supply (Table 1). In TL, the percentage of C18:3, the abundant fatty acid, decreased and that of C16:0 and C18:0 increased. The same variation has been found in the MGDG and phospholipids tested (Table 1). An exception was the fatty acid composition of the DGDG, where a distinct increase of the C18:3 percentage and a decrease in that of C16:0 was observed (Table 1). This response has been found in thylakoid membranes of Cu-treated spinach (Maksymiec et al. 1992). It has been suggested that the high level of thylakoid lipid unsaturation may be required to maintain the fluidity degree needed for the diffusion of lipophilic compounds and/or may confer a suitable geometry to the lipid molecules. Furthermore, phospholipid fatty acids affect the bilayer properties regulating its fluidity and permeability and the activities of membrane-bound enzymes as well (Quartacci et al. 2001). The decrease in the unsaturation level might be related to direct reaction of oxygen free radicals with unsaturated lipids and/or to an inhibition of the fatty acids biosynthesis. It has been shown that cadmium also enhanced lipoxygenase activity, which is responsible for catalysing lipid peroxidation by using membrane lipid components as substrates, particularly unsaturated fatty acids (Howlett & Avery 1997; Ben Youssef et al. 2005) which can explain the high level of



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TBARS (Fig. 2). On the other hand, radioactivity was accumulated in the C16:0/C18:0, the C16:1, the C18:1 and the C18:2 molecules of different lipid classes, extracted from Cd-treated plants (Fig. 4), and decreased in the tri-unsaturted fatty acids (C16:3 and C18:3). The inhibition of some enzymatic activities, such as oleoylPC transferase, oleate and linoleate desaturases, under metallic treatment (Stefanov et al. 1992), may explain the decrease in the fatty acids unsaturatin level. Moreover, similar data indicated that cadmium affected the two lipid biosynthesis pathways (chloroplastic and extrachloroplastic). It can be concluded that this work reports some explanation concerning the alteration on membrane lipids of tomato plants caused by metallic stress. Cadmium reduced lipid and fatty acids biosynthesis and stimulated the peroxidation process. Consequently, Cd stress affected the membrane fluidity and permeability and it can produce irreversible damage to cell functioning. References Alia K.V.S.K., Prasad P. & Pardha Saradhi P. 1995. Effect of zinc on free radicals and proline in Brassica and Cajanus. Phytochem. 42: 45–47. Allen C. & Good P. 1971. Acyl lipid in photosynthetic systems. Methods Enzymol. 23: 523–547. Ben Ammar W., Nouairi I., Tray B., Zarrouk M., Jemal F. & Ghorbel M.H. 2005. Effets du cadmium sur l’accumulation ionique et les teneurs en lipides dans les feuilles de tomate (Lycopersicon esculentum). J. Soc. Biol. 199: 157–163. Ben Hamed K., Ben Youssef N., Ranier A., Zarrouk M. & Abdelly C. 2005. Changes in content and fatty acid profiles of total lipids and sulfolipids in the halophyte Crithmum maritimum under salt stress. J. Plant Physiol. 162: 599–602. Ben Youssef N., Nouairi I., Ben Temime S., Taamalli W., Zarrouk M., Ghorbal M.H. & Ben Miled Daoud D. 2005. Effets du cadmium sur le métabolisme des lipides de plantules de colza (Brassica napus L.) C. R. Biol. 328: 745–757. Chaffei C., Gouia H., Masclaux C. & Ghorbel M. H. 2003. Réversibilité des effets du cadmium sur la croissance et l’assimilation de l’azote chez la tomate (Lycopersicon esculentum). C. R. Biol. 326: 401–412. Djebali W., Chaibi W. & Ghorbel M. H. 2002. Croissance, activité peroxydasique et modifications ultrastructurales induites par le cadmium dans la racine de tomate. Can. J. Bot. 80: 942– 953. Douce R. 1964. Identification et dosage de quelques glycérophosphatides dans des souches normales et tumorales de scorsončres cultivés in vitro. C. R. Acad. Sci. 259: 3066–3068. Douglas T.J. & Walker R.R. 1984. Phospholipids, free sterols and adenosine triphosphatase of plasma membrane-enriched preparations from roots of citrus genotypes differing in chloride exclusion ability. Physiol. Plant. 62: 51–58. Fargašová A. 2004. Toxicity comparison of some possible toxic metals (Cd, Cu, Pb, Se, Zn) on young seedlings of Sinapis alba L. Plant Soil Environ. 50: 33–38. Fodor E., Szabé-Nagy A. & Erdei L. 1995. The effects of cadmium on the fluidity and H+ -ATPase activity of plasma membrane from sunflower and wheat roots. J. Plant Physiol. 147: 87–92. Gigon A., Matos A.R., Laffray D., Zuily-Fodil Y. & Pham-Thi A.T. 2004. Effect of drought stress on lipid metabolism in the leaves of Arabidopsis thaliana (Ecotype Columbia). Ann. Bot. 94: 345–351. Gouia H., Suzuki A., Brultfert J. & Ghorbal M.H. 2003. Effects of cadmium on the coordination of nitrogen and carbon metabolism in bean seedlings. J. Plant Physiol. 160: 367–376. Hernandez L.E. & Cooke D.T. 1997. Modification of the root plasma membrane lipid composition of cadmium-treated Fisuni sativum. J. Exp. Bot. 48: 1375–1381.

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Received February 7, 2007 Accepted October 31, 2007