Plant Materiel and growth conditions
Seeds of L. Sativum were collected from the region of Djerba (33° 48' 27.353" N 10° 50' 42.529" E). Seeds were disinfected and germinated in Petri dishes containing two sheets of filter paper soaked with distilled water. Five days after germination, seedlings were transplanted into plastic pots containing 1.3 L aerated and renewed quarter-strength Hoagland nutrient solution (Hoagland and Arnon1940).Plant culture was maintained in a growth chamber under 22°C, 8 h of photoperiod per day and a relative humidity of 86 %.Three weeks after, individual plants were exposed to 0.05 (control), 0.5, 1, 1.5 and 2 mM of zinc, in the form of ZnSO4, added or not with two concentrations (9 or 15 ch: Hahnemannian centesimal) of Zincum Metallicum(ZM) for 7 days prior to the final harvest. At the harvest, fresh weight of roots and leaves were separately recorded for six plants randomly selected from each treatment and used for physiological analysis. Roots and leaves were also harvested from three plants of each treatment and frozen at-80°C or air dried for the biochemical analysis.
Plant growth parameters and water content
The weight of leaf and root samples were measured before (fresh weight: FW) and after (dry weight: DW) dryness at 60 °C for 3 days. Water content (WC) was determined as:
WC = (FW - DW)/FW x 100
Tissue zinc content
Zinc was extracted from 25 mg dried material transferred to 125 mL conical digestion flasks. Twelve (12) mL of triacid mixture of nitric acid, sulfuric acid and perchloric acid (9:2:1 (v/v)) were added to the flasks. Plant materials were digested in cold for 3 h followed by digestion for 2–3 h on a hot plate, until the digest was clear or colorless. The flasks were allowed to cool and the contents were diluted to an appropriate volume (Sahraw at and al, 2002).then assayed by atomic absorption spectrophotometer (Perkin Elmer Analyst 300), using standard with known concentrations.
Chlorophyll and carotenoids content
Chlorophyll (Chl) and carotenoids was extracted from 5 mg of fresh leaf tissue using 25mMTris-HCL (pH 7.6), 1 mM EDTA, 1mM MgCl2and 14 mM β-mercaptoethanol.50 μl of the extract were taken and then homogenized in 1450 μl of 80% acetone. The mixture is stored at 4°C over night. The samples were centrifuged for 15 min at 1500 gand the supernatant was used to identify absorbance of chlorophyll a and b, and carotenoids at 645, 663 and 440.5nm, respectively(Marker and al 1980). Total chlorophyll and carotenoids content (mg g-1 FW) were calculated according the following formula:
Chl a = (12.7×DO663)-(2.69×DO645) ×v/(w×1000)
Chl b = (22.9×DO645)-(4.68×DO663)×v/(w×1000)
Chl tot=(20.2×DO645)-(8.02×DO633) ×v/(w×1000)
Carotenoids =46.95×(DO440.5-0.268×Chl a+b)
Malondialdehyde determination
Oxidative damage in L. Sativum was estimated based on malondialdehyde (MDA) content, which was determined from fresh leaf and root tissue, following the method of Draper et al. 1990. One hundred milligrams were ground in 1 mL of 0.1% trichloroaceticacid at 4°C. After centrifugation at 15000 g for 15 min, a250µl aliquot of supernatant was added to 1ml thiobarbituric acid (prepared in 20% trichloroacetic acid) and heated for 30 min in a water bath at 95°C. Samples were again centrifuged at 10000g for 10 min and the absorbance of the supernatant was measured at 532 nm. After subtracting the nonspecific absorbance at 600 nm, MDA concentration (mol g−1 FW) was determined using a molar extinction coefficient of 155 mM−1 cm−1.
Proline content
Proline content was measured by the method of Bates and al. (1973). Fresh leaves and roots (25mg) were mixed with 1 ml of sulfosalicylic acid (3%). The samples were centrifuguated at 12.000 g for 20 min. A 500 μl aliquot of supernatant were added into 500 μl of sulphosalicylic acid, 1 ml of concentrated acetic acid and 1 ml of ninhydrin. After incubation at 100°C for 1 h, the reaction was stopped by placing the test tube in an ice bath. Two milliliters of toluene were added to the solutions. The organic toluene phase was separated and used to determine the content of proline at 520 nm.
Protein and antioxidants enzyme assays
Fresh plant material (200 mg) was extracted in a buffer containing Potassium hydrogen phosphate, EDTA, PVP and glycerol. The homogenate of each sample was centrifuged at 12.000 rpm for 10min. The supernatant fraction was then assayed for proteins and various antioxidant enzymes.
Proteins contents were analyzed according to the method of Bradford (1976), using the principle of Coomassie G250 blue binding with proteins. The concentration of protein was determined from a standard range of BSA (Bovine Serum Albumin) between 0 and 10 μgmL-1.
The Superoxide dismutase (SOD) activity was determined by using the tetrazolium nitroblue (NBT) / riboflavin according to the method of Beauchamp and Fridovich (1971).Briefly, the samples mixture containing phosphate buffer 50 mM, (pH 7.8), EDTA 0.1 mM, L-methionine 13 mM, riboflavin 2 μM and NBT 75 μM. The reaction was initiated by exposing the reaction mixture for 15 min to a 50 μmol.m-2.s-1 fluorescent light source. The absorbance was spectrophotometry measured at 560 nm.
The Catalase (CAT) activity was determined spectometrically according to the method of Chaparro-Giraldo et al (2000), by measuring the disappearance rate of hydrogen peroxide (H2O2) at 240 nm. The reaction mixture contained potassium phosphate buffer (50nM, pH7), 100µl of enzyme extract and H2O2 at 30%.
The Glutathione peroxidase (GPx) converts H2O2into water (H2O) by the transformation of Glutathion (GSH) to Glutathione disulfide (GSSG).Activity of GPx was assayed by measuring the disappearance rate of H2O2according to the method of Flohe and Gunzler, (1984).This activity was expressed in µmol of GSH/min/mg of protein.
The Glutathione reductase (GR) activity was determined by monitoring the oxidation rate of NADPH at 340 nm (Rao et al. 1996). The reaction mixture (1 ml) contained 100 mM phosphate buffer (pH 7.8), 2 mM EDTA, 0.5 mM oxidised glutathione, 0.5 mM NADPH, 0.2 mM NADPH and the reaction was initiated by adding 100µl of the enzyme extract.
Extraction and determination of total polyphenols compounds
Roots and leaves were air-dried at room temperature for 2 weeks. Samples extracts were obtained by magnetic stirring of 1 g of dry powder per sample in methanol 80% (10 ml)) for 30 min and kept at 4 °Cfor 24 h. The methanolic extracts obtained were filtered through a Whatman filter paper (N°.4) and stored at 4 °C.Colorimetric quantification of the total phenolic compound was conducted using the Foline-Ciocalteu reagent, as described by Dewanto et al. (2002). Briefly, an aliquot of 125 µl of 1/10 diluted sample from each methanolic extract was dissolved in 500 µl distilled water and 125 µl Folin–Ciocalteu reagent. After shaking and resting the mixture for 3min, 1250 µl of 7% Na2CO3 was added to the mixture and adjusted with distilled water to a final volume of 3 ml. The mixture was then incubated for 90 min at room temperature in the dark. The absorbance was read at 760 nm and the total phenolic compound was expressed as milligram gallic acid equivalent per gram of dry weight (mg GAE g-1 DW) through the calibration curve of gallic acid (0-500 mg l-1).
Determination of total flavonoids content
The total flavonoid content was determined by using a colorimetric method described previously (Zhihenet al.1999). An aliquot (75 µl) of 7% sodium nitrite (NaNO2)solution was added to each extract (250 µl). The mixture was shaken for 6 min before adding 0.15 µl of 10% aluminum chloride (AlCl3). After 5 min, 0.5 ml of 1M sodium hydroxide (NaOH) was added. The final volume was adjusted to 2.5 ml with distilled water and thoroughly mixed. Absorbance of the mixture was determined at 510 nm. Total flavonoid was expressed as milligram catechin equivalent per gram dry weight (mg CE g-1 DW) using a calibration curve developed for catechin (0-500 mg l-1).
Statistical analysis
Data were subjected to a one-way ANOVA test using ANOVA, CoStat software, version 6.4, CoHortSoftware, Monterey, CAand means were compared according to Duncan’s multiple-range test at 5 % level of significance.