Plant material
Wheat seeds were provided by Wheat Research Institute, Henan Academy of Agricultural Sciences.
Plant growth
Hydroponic culture was used for seed germination and seedling growth. Plump wheat seeds were surface-sterilized using 0.5% (w/v) NaClO for 10 min and then rinsed completely with pure water. After being allowed to germinate in a gauze for 5 d, uniform seedlings were transplanted into black plastic containers with Hoagland and Arnon [57] solution. The full-strength solution contained 5.0 mM KNO3, 5.0 mM Ca(NO3)2, 2.0 mM MgSO4·7H2O, 1.0 mM KH2PO4, 50 µM EDTA-Fe, 46 µM H3BO3, 9.0 µM MnCl2,·4H2O, 0.80 µM ZnSO4·7H2O, 0.37 µM Na2MoO4·2H2O, and 0.30 µM CuSO4·5H2O. The nutrient solution was replaced every 3 d. The wheat seedlings were first grown in one-quarter-strength, one-half-strength, and eventually full-strength solutions. The plants were grown in a climate chamber using a temperature setting of 24/22 °C (day/night), a photoperiod of 14/10 h (day/night), and light intensity of 300–320 μmol m-2 s-1.
Experimental design
1. Cd concentration gradient treatments
5-d-old wheat genotype seedlings were treated with CdCl2 at six concentrations: 0 μM, 5 μM, 10 μM, 20 μM, 50 μM, and 100 μM for 12 d. Shoot height and biomass, root length and biomass, and leaf number were recorded.
2. Selection of tolerant and sensitive cultivars
A total of 126 wheat genotypes were cultured in a nutrient solution containing 5 μM Cd2+. Root biomass and length were measured after 12 d.
3. Physiological and transcriptional responses toCd2+ in both cultivars
Wheat seedlings of 12-d were divided into two experimental groups based on Cd-free (normal culture) and Cd (5 μM Cd2+) treatments. After 3 d, the samples were harvested to determine ROS concentrations, antioxidative enzyme activities, antioxidant concentrations, and transcriptome sequencing.
4. Addition of exogenous antioxidants
Wheat seedlings of 12-d-old were separated into 17 experimental groups based on the following treatments: Cd-free (normal culture), Cd (5 μM Cd2+), T1 (20 μM GSH + 5 μM Cd2+), T2 (50 μM GSH + 5 μM Cd2+), T3 (100 μM GSH + 5 μM Cd2+), T4 (0.1 mM AsA + 5 μM Cd2+), T5 (0.4 mM AsA + 5 μM Cd2+), T6 (0.8 mM AsA + 5 μM Cd2+), T7 (20 μM GSH + 0.1 mM AsA + 5 μM Cd2+), T8 (50 μM GSH + 0.1 mM AsA + 5 μM Cd2+), T9 (100 μM GSH + 0.1 mM AsA + 5 μM Cd2+), T10 (20 μM GSH + 0.4 mM AsA + 5 μM Cd2+), T11 (50 μM GSH + 0.4 mM AsA + 5 μM Cd2+), T12 (100 μM GSH + 0.4 mM AsA + 5 μM Cd2+), T13 (20 μM GSH + 0.8 mM AsA + 5 μM Cd2+), T14 (50 μM GSH + 0.8 mM AsA + 5 μM Cd2+), and T15 (100 μM GSH + 0.8 mM AsA + 5 μM Cd2+). After 7 d, the root system architecture (RSA) parameters were analyzed and root microscopy was performed.
Determination of ROS concentrations
The O2− level was measured by the hydroxylamine oxidation method according to the method of Ma et al. [58]. H2O2 reacts with titanium oxysulfate to produce pertitanic acid, which is yellow, and at 410 nm is proportional to the H2O2 concentration.
Determination of lipid peroxidation
The level of lipid peroxidation is usually represented by the MDA content [59]. MDA was determined by condensation with thiobarbituric acid (TBA) to form a red product [59].
Antioxidative enzyme activities and antioxidants assays
Root samples were homogenized with a 50-mM phosphate buffer (PBS, pH = 7.0) containing 1 μM EDTA. The homogenates were centrifuged at 14000 × g for 20 min at 4 °C. Subsequently, the supernatant was used for the enzyme assays. O2- reduces nitroblue tetrazolium chloride (NBT) to produce formazan. SOD can clean up O2-, thus formazan content could represent SOD activity [60]. One unit (U) of SOD activity represents 50% inhibition of formazan production. POD catalyzes H2O2 and forms guaiacol dehydrogenation. Through monitoring the formation of guaiacol dehydrogenation in 470 nm, POD activity was determined. The CAT activity was determined by monitoring the decomposition of H2O2 at 240 nm. APX activity was determined by observing the decrease of AsA in absorbance at 290 nm over 2 min. AsA was determined by reacting the supernatant with Fast Blue B salt and assayed spectrophotometrically at 420 nm. GSH was determined by reacting with 5,5'-Dithiobis-2-nitrobenzoic acid and assayed spectrophotometrically at 412 nm. These substances were determined using an assay kit (Coming Medical Technology Co., Ltd, Suzhou, China).
Microscopic analysis
H2O2 was revealed in root samples by immersion in 0.5% (w/v) 3,3-N-diaminobenzidine tetrahydrochloride (DAB), and then incubation at room temperature until the root turned brown [35]. O2− was detected by reacting with NBT to produce a blue formazan precipitate in the root [61]. The loss of cell viability was observed after staining with 0.25% (w/v) Evans blue solution for 5 min and washing three times with 100 μM CaCl2 solution (pH = 5.6) [62]. A fluorescent microscope (LSM800, Carl Zeiss, Oberkochem, Germany) was used for observation.
High-throughput RNA-seq
Wheat roots were harvested from CK and Cd-treated plants for transcriptome analysis, with three biological replicates each. Using an Illumina Hiseq X Ten platform (Illumina Inc., San Diego, CA, USA), a total of 12 RNA samples were analyzed, which generated 6.0-Gb of sequencing data with 150-bp paired-end (PE) reads per sample. Transcript abundances (FPKM values) were calculated from RNA-seq data using the method described by Zhou et al. [63].
Determination of root architecture
WinRHIZO (Pro 2013a, Regent Instrument Inc.), a root analysis software, was used to measure the RSA parameters, including total root length, average root diameter, number of root tips, total root surface area, and total root volume.
Statistical analysis
Using GraphPad Prism 5.01, the differences among Cd concentration gradient treatments were evaluated with Tukey test. The differences between control and treatment (Cd-free and Cd treatment) or between genotypes (T207 and S276) were evaluated with the Student’s t-test. Differences were considered statistically significant at p < 0.05.