Plant materials, growth condition, and treatments
The glasshouse experiment was conducted using cotton parental lines, namely, the female 9053 (general upland cotton), the male parent sGK9708 (insect resistant), and their hybrids variety Zhongmian 63. Their seeds were provided by Dr. Daigang Yang (Institute of Cotton Research, Chinese Academy of Agricultural Sciences, Anyang, China). Uniformly sized seeds were surface sterilized with 1%(v/v) sodium hypochlorite solution for 20 minutes, washed with distilled water for 3 times and allowed for water imbibition for 24 h before growing in the sand culture medium. Seed germination was allowed for 5 days at 28℃ in dark. On the 6th day, uniform seedlings were transferred to hydroponic media and allowed to acclimatize for 10 days. The nutrient solution composition is 20 µM H3BO3, 1 µM ZnSO4·7H2O, 0.2 µM CuSO4·5H20, 1 µM MnSO4·H2O, 0.005 µM (NH4)6MO7O24·4H2O, 2498.41 µM Ca(NO3)2·4H2O, 499.7 µM KH2PO4, 1000.12 µM MgSO4·7H2O, 2504.70 µM KNO3,100 µM Na Fe·EDTA, at 22℃/27℃ during 8/16 h dark/light periods respectively, with a light intensity of 180 μmol·m-2·s-1 and 60% relative humidity. Fifteen days of equally sized cotton seedlings were transferred into the nutrient solution with different Cu concentrations. The treatments concentrations were composed of 0 (without addition of Cu), 0.2 (control group), 50, 100, 200 μM, and Cu is supplied as CuSO4·5H2O. The nutrient solutions were continuously renewed every 4 days, and the pH value was adjusted to 5.6-5.7 with 0.1M HCl or 0.1M NaOH. Plants were harvested after 10 days with Cu treatments for future analysis.
Determination of growth parameters
Growth parameters of the cotton seedlings were evaluated by determining leaf area, root, and shoot lengths. The leaf area of seedlings was determined by area-weighing method; the lengths of roots and shoots were recorded using the meter ruler.
Measurement of photosynthetic pigments contents
Chlorophyll and carotenoid concentrations were determined according to Lichtenthaler and Wellburn [27]. Briefly, the top-most expanded leaves were randomly cut and soaked in 80% acetone in the ratio of 1:10 w/v until the pigments were completely extracted when leaf became colorless. The process was performed in darkness. The extracts were centrifuged for 15 min at 4000 g to remove any adhering residues. The supernatant was measured at 663, 646 and 470 nm for chlorophyll a, chlorophyll b and carotenoids, respectively. 80% acetone was used as a blank control.
Determination of copper content
For analysis Cu content, the root and leaf samples were harvested separately, and were rinsed with tap water, and immersed in 20 mM Na2-EDTA for 15 min to remove any trace elements adhering to the tissue. The root and leaf samples were oven dried at 75℃ for 48 h. Dried samples (0.1 g) were ground and acid digested with HNO3 mixture for 24 h at 80℃, followed by Cu estimation using an atomic absorption spectrophotometer.
Detection of H2O2 and MDA level
The accumulation of H2O2 in leaves was measured by monitoring the A415 of the titanium-peroxide complex following the method described by Liu et al. [28]. Absorbance values were calibrated to a standard curve generated with known concentrations of H2O2. Recovery was checked by adding various amounts of H2O2 to the leaf extracts as an internal standard. The level of lipid peroxidation was determined according to Thounaojam et al. [15]. Fresh leaves (0.2 g) were homogenized with 5 ml 0.25% TBA. The homogenate was boiled for 30 min at 95 ℃ and centrifuged at 10,000 g for 10 min. The absorbance of the supernatant was recorded at 532 nm and 600 nm using an extinction coefficient of 155 mM-1 ·cm-1.
Measurement of POD activity and GSH content
Frozen leaf segments were homogenized, centrifuged, and then supernatant was immediately used for the antioxidant enzyme assays. The POD activity was measured by guaiacol oxidation according to the method of Li et al. [29]. To determine the GSH content, samples (0.5 g) were crashed with 5 ml of 10% (w/v) TCA, and the homogenate was centrifuged at 15,000×g for 15 min at 4℃. The GSH content was determined according to the method described previously [30].
Determination of soluble sugar content
Soluble sugar content was determined according to the procedure reported by Muhammad et al [31]. Fresh leaves (0.5g) were homogenized in 80% ethanol, and then incubated at 75℃ for 10 minutes. Forty (40) microliters of the supernatant were mixed with 80% of carbolic acid and 4 mL of concentrated sulfuric acid and then the absorbance was recorded at 490nm. The concentration of soluble sugar was determined by a calibration curve prepared from a sucrose solution and was expressed as mg·g-1 FW.
Statistical analysis
All statistical analysis was performed by SPSS 21.0 computer software package. Data were expressed as mean values ± S.D. with three replicates for each treatment. Differences among the groups were examined by one-way ANOVA followed by LSD. P<0.05 was considered as statistically significant.