Plant material and experimental conditions
Hydroponic experiment was carried out on Henan Agricultural University, Zhengzhou, China. Wheat seeds (cv Zhengmai 379, Triticum aestivum L.) were surface-sterilized in 0.5% Na-hypochlorite for 15 min and seeds were then rinsed carefully with deionized water. The seeds were germinated on a plastic net floating on deionized water and seven-day-old rice seedlings with similar size were transfer to the incubators to growth in a controlled chamber with photoperiod of 16 h/8 h (day/night) at 25℃/20℃, RH 75%, and light intensity of 200 ± 25 µmol m−2 s−1. The full nutrient solution (FNS) contained: 4.0 mM Ca(NO3)2·4H2O,6 mM KNO3,1 mM NH4H2PO4, 2 mM MgSO4·7H2O, 46.2 μM H3BO3, 9.1 μM MnCl2·4H2O, 0.3 μM CuSO4·5H2O, 0.8 μM ZnSO4·7H2O, 100 μM FeNaEDTA, 0.2 μM (NH4)6Mo7O24·4H2O. The solution pH was adjusted to 6.5 using 1 M HCl or NaOH. Half-strength nutrient solution was applied for the first 7 d and changed to full strength solution for another 4 d. Each treatment was replicated four times. Five treatments was set as follow: (1) CK (without B); (2) CK+B (B was added after 7 days of culture); (3) CK+Cd (Cd was added after 7 days of culture); (4) CK+Cd+B (Cd and B were added after 7 days of culture); (5) preB+Cd (B seven-day pretreatment before Cd exposure). Cd was applied as CdCl2 solution with a final Cd concentration of 5 µM. After 23 d Cd treatments, root and leaf samples rinsed in distilled water were harvested and used fresh or kept frozen in liquid N2 for various biochemical assays.
Cd concentration analysis
Plants were divided into root and shoot fractions and oven-dried at 70℃ to constant weight. These fractions were then digested with mixed acid (HNO3:HClO4 (4:1, v:v)) in a Microwave Digestion System. Cd concentration was determined by graphite furnace atomic absorption spectrometry (GFAAS). The translocation factor (TF) was calculated as follows: TF = shoot CCd / root CCd.
Malondialdehyde (MDA) determination
The level of lipid peroxidation is estimated as the amount of MDA determined by the thiobarbituric acid (TBA) reaction as described by Qin et al. (2018). Briefly, 500 mg of fresh root or shoot samples was homogenized with 2 mL 5% v/v trichloroacetic acid (TCA) and centrifuged at 10,000×g for 10 min. Then, 1 mL 0.5% v/v TBA in 20% v/v TCA was added to 1 mL supernatant and the homogenate was boiled for 30 min at 95 ℃. The mixture was followed by an immediate cooling on ice to stop the reaction and centrifuged at 10,000×g for 5 min. The absorbance was determined at 450 nm, 532 nm and 600 nm and MDA concentration was estimated by the formula: MDA (μmol/g FW) = 6.45(OD532-OD600)–0.56 OD450.
Antioxidant enzyme activities
Antioxidant enzyme activities (SOD, CAT and POD) in roots and leaves were determined spectrophotometrically according to previous literatures and make some modifications (Molins et al. 2013; Wu et al. 2017). Fresh tissues (0.5 g) were ground in liquid nitrogen to a fine powder using a mortar and pestle. The powder was transferred to a pre-cooled (4℃) mortar and pestle with 5 mL of 50 mM phosphate buffer (pH 7.8), containing 0.12 mM EDTA and 2% (w/v) polyvinylpolypyrrolidone. The homogenate was centrifuged at 12,000×g at 4°C for 20 min. The supernatant was used for assays of the antioxidant enzyme activities. The assay of SOD was carried out briefly as follows: the assay mixture (total 3 mL) for SOD contained 0.05 M phosphate buffer, containing 12 µM EDTA and 13 mM L-methionine, 75 µM nitroblue tetrazolium (NBT), 2 µM riboflavin in 5 mM KOH and 0.05 mL enzyme extract. Reaction was initiated by placing the glass test tubes in between two fluorescent tubes. By switching the light on and off, the reaction was started and terminated, respectively. The increase in absorbance due to formazan formation was read at 560 nm. The activity was expressed as ΔA560 (U mg-1 protein min-1). CAT activity was assayed. The reaction mixture for CAT comprised of 1.5 mL of 25 mM phosphate buffer (pH 7.0), 0.3 mL of 0.1 M H2O2 and 0.2 mL of enzyme extract. The change of absorbance value within 210s was measured at 240 nm with the blank setting of deionized water. The CAT activity was expressed as U mg-1 protein min-1. Guaiacol peroxidase activity was determined spectrophotometrically by measuring the oxidation of guaiacol to tetraguaiacol at 470 nm. The reaction mixture contained 1 mL of 50 mM phosphate buffer (pH 5.5), 1 mL of 0.3% H2O2, 0.95 mL 0.2% (v/v) guaiacol, and 0.05 mL enzyme extract. The change rate of absorbance value was recorded at 470nm within 210s, and the number of reads was recorded every 30s. A peroxidase activity unit (U) was determined by △A470 changing by 0.01 per minute and the activity was expressed as U mg-1 protein min-1.
Metabolite extraction and metabolite profiling analysis
The identification of differential metabolites was performed by Shanghai Lu Ming Biological Technology Co. Ltd. 80 mg accurately weighed sample was transferred to a 1.5 mL Eppendorf tube. Two small steel balls were added to the tube. 20 µL of 2-chloro-l-phenylalanine (0.3 mg/mL) dissolved in methanol as internal standard and 1 mL mixture of methanol and water (7/3, v/v) were added to each sample, samples were place at -20℃ for 2 min. Then grinded at 60 HZ for 2 min, and ultrasonicated at ambient temperature for 30 min after vortexed, then placed at -20℃ for 20 min. Samples were centrifuged at 13,000 rpm, 4℃ for 10 min. 0.3 mL of supernatant in a brown and glass vial was dried in a freeze concentration centrifugal dryer. 0.4 mL mixture of mixture of methanol and water (1/4, v/v) were added to each sample, samples vortexed for 30s, then placed at 4℃ for 2 min. Samples were centrifuged at 13,000 rpm, 4℃ for 5 min. The supernatants (150 µL) from each tube were collected using crystal syringes, filtered through 0.22 µm microfilters and transferred to LC vials. The vials were stored at -80℃ until LC-MS analysis. QC samples were prepared by mixing aliquots of the all samples to be a pooled sample.
An ACQUITY UHPLC system (Waters Corporation, Milford, USA) coupled with an AB SCIEX Triple TOF 5600 System (AB SCIEX, Framingham, MA) was used to analyze the metabolic profiling in both ESI positive and ESI negative ion modes. An ACQUITY UPLC BEH C18 column (1.7 µm, 2.1×100 mm) were employed in both positive and negative modes. The binary gradient elution system consisted of (A) water (containing 0.1% formic acid, v/v) and (B) acetonitrile (containing 0.1% formic acid, v/v) and (B) acetonitrile (containing 0.1% formic acid, v/v) and separation was achieved using the following gradient: 0 min, 5% B; 2 min, 20% B; 4 min 25% B; 9 min 60% B; 14 min 100% B; 18 min 100% B; 18.1 min 5% B and 19.5 min 5% B. The flow rate was 0.4 mL/min and column temperature was 45℃. All the samples were kept at 4℃ during the analysis. The injection was 10 µL. Data acquisition was performed in full scan mode (m/z ranges from 70 to 1000) combined with IDA mode. The QCs were injected at regular intervals (every 10 samples) throughout the analytical run to provide a set of data from which repeatability can be assessed.
The acquired LC-MS raw data were analyzed by the progqenesis QI software (Waters Corporation, Milford, USA). The internal standard was used for data QC (reproducibility). Metabolites were identified by progenesis QI (Waters Corporation, Milford, USA) Data Processing Software, based on public databases such as http://www.hmdb.ca/; http://www.lipidmaps.org/ and self-built databases. The positive and negative data were combined to get a combine data which was imported into R ropls package. Principle component analysis (PCA) and (orthogonal) partial least-squares-discriminant analysis (O)PLS-DA were carried out to visualize the metabolic alterations among eperimental groups, after mean centering and Pareto variance (Par) scaling, respectively. The Hotelling’s T2 region, shown as an ellipse in score plots of the models, defines the 95% confidence interval of the modeled variation. Variable importance in the projection (VIP) ranks the overall contribution of each variable to the OPLS-DA model, and those variables with VIP > 1 are considered relevant for group discrimination. The differential metabolites were selected on the basis of the combination of a statistically significant threshold of variable influence on projection (VIP) values obtained from the OPLS-DA model and p values from a two-tailed Student’s t test on the normalized peak peak areas, where metabolites with VIP values less than 0.05 were considered as differential metabolites.
Statistical analysis
Analysis of variance (ANOVA) was computed for statistically significant differences using the statistical package SPSS, version 20.0 (SPSS, Chicago, IL, USA).All data are the means ± SE of four independent replicates; the mean differences were compared utilizing the LSD's multiple range test (P < 0.05).