Plant materials, growth conditions and treatments Healthy
The seeds of Se-free rice (Oryza sativa L.) CR727 and its hybrid progeny Se-rich red-grain rice Z2057A/CR727 were obtained from the collection of Demonstration Base for International Science & Technology Cooperation of Sichuan Province, Rice Research Institute, Sichuan Agricultural University, China with permission. Rice seeds were sterilized with 1% (v/v) NaClO for 20 min before rinsing 5 times in sterilized double-distilled water (ddH2O). To stimulate uniform germination, seeds were submerged in the sterilized ddH2O and incubated at 37 °C for 3 d in avoidance of light. Seedlings were hydroponically cultured in half-strength Kimura B nutrient solution (pH 5.5) at 25°C, under the condition of 12h light and 12 h dark . The solution was renewed every 3 d to ensure fresh and nutrient stable during a long-term period . In the exogenous Se stress test, the healthy seedlings were continuously growing in presence of a gradient of concentrations of sodium selenate (Na2SeO4, referring as Se hereafter) and mock control for additional 2 d or 14 d [22, 23]. Three independent biological replicates were included in phenotype observations and a variety of assays, while the fully expanded second leaves were harvested to determine growth-related parameters in the assessment of physiological and biochemical responses.
Measurement water status of leaves
Relative water content (RWC) of leaves was introduced to quantify the water status of leaves. Fresh leaves were detached and weighed immediately to 0.1 g and designated as the fresh weight (FW). The same leaves were then soaked in ddH2O at 4 °C in darkness for 24 hours before weight and designated as turgid weight (TW). After that, leaves were subjected to 80 °C for no less than 3d to get sufficient drying prior to weight and designated as for dry weight (DW). The RWC was calculated as (FW-DW)/(TW-DW) × 100% .
Measurement of physiological parameters
Total leaf chlorophyll (Chl) content analysis, fresh leaves (0.1 g) were immersed in 10 ml of dimethyl sulphoxide (DMSO) in the dark for 48 hours, and then the leaf extract was measured at 663 and 645 nm with a spectrophotometer as described by Arnon et al. [25, 26]. The contents of Chl (mg∙g–1) were calculated by the following formula:
The content of superoxide dismutase (SOD) and methane dicarboxylic aldehyde (MDA) were determined by a spectrophotometer-based method. SOD and MDA were first labelled by using a kit (A001–1 SOD and A003 MDA) from Nanjing Jiancheng Bioengineering Institute and according to the manufacturer’s instructions. After the reaction, the appearances of SOD and MDA were determined by subjecting the products to a UV-visible spectrophotometer equipped with cuvettes of 1 cm path length, respectively (T6S, Puxi, Co., Ltd, Beijing, P. R. China). After harvesting the values, the activity of SOD and content of MDA were calculated according to the following formula
Histochemical analysis of reactive oxygen species
To detect the presence of superoxide in leaves, the leaves were incubated in the staining solution of nitrobluetetrazolium (NBT, 0.1%) as described previously . To detect the accumulation of hydrogen peroxide (H2O2), Rice leavesof seedlings after 14 d exogenous treatment with or without (control) Se were collected and stained in a 3,3’-diaminobenzidine-HCl (DAB, 1.0%) solution as described previously .
Measurements of Se and anthocyanin content
An atomic fluorescence spectrophotometer was applied to determine the Se content as described previously [28, 29]. Briefly, after grinding into fine powder, 0.5 g samples were weighted and filled into a glass vial containing a pre-prepared solution of 9 ml HNO3 and 1 mL HClO4. The sampling solutions were ultrasonicated with a fixed setup of parameters (temperature, duration, and frequency) at 20 °C, 4 h, and 100 Hz, respectively, following by a digestion process in the presence of HNO3 in a 180°C-electric hot plate (EH20A Plus, Labtech, USA). The digested products were then diluted with a suitable amount of 37% hydrochloric acid to reduce Se (VI) into Se (IV) within a consistent temperature of 90 °C, as result, a whitish concentrated solution was generated in a volume of 1 ml due to the heating-induced evaporation. The measurements were carried out in an atomic fluorescence spectrophotometer (RGF–6800, Bohui Co., Ltd., Beijing, China). The values were put into the following formula to calculate the content of Se (mg/kg):
where C is the measured value of Se concentration in the digested solution (ng/mL); C0 is the measured value of Se concentration in the control group (without any samples, ng/mL); m is the mass of sample; V is the total volume of digested solution. The measurements were performed in triplicate.
The content of anthocyanin was determined as described previously . Briefly, extraction of rice anthocyanin was completed by using HCl and ethanol solution 0.1 mol/L (1:15, w/v) at 60 ℃ for four hours inside the ultrasonic cleaners (WD–9415B, LiuYi Co., Ltd, Beijing, China). Recovered supernatants were concentrated by using the rotavapor at 45 ℃ (R–210, BUCHI Co., Ltd, Switzerland). Ultrasonic cleaners used to dissolve the remaining solid residues. Final extract reconstituted with deionized water on ultrasonic cleaner before storage. Then solution samples were stored at ‒20 ℃ for further analysis. The absorbance of the solution at 530, 620, and 650 nm was measured using a UV-visible spectrophotometer, respectively. The content of anthocyanin was calculated by putting the values into the following formula:
where V is the total volume of extraction solution (mL); ε is the anthocyanin molar extinction coefficient (4.62×106); m is the mass of sample (g); M is the molecular weight of anthocyanin. The measurements were performed in triplicate. The measurements were performed in triplicate.
Relative genes expressionanalysis
According to the qRT-PCR method , mRNA of two-week treatment rice seedling roots was extracted by using the TRI pure reagent kit (Aid Lab). Primers used in these assays synthesized by Qingke Company (QingkeZixi Co., Ltd., Chengdu, China) are listed in Table 1, and the expression levels were normalized to those of the Actin1 and EF1α as indicated. TransScript® All-in-One First-Strand cDNA Synthesis Super Mix for qPCR (One-Step gDNA Removal) kit (Transgen, Beijing, China) and Universal SYBR® GREEN qPCR Master Mix (2X) were used to perform qRT-PCR on CFX ConnectTM Flex Real-Time PCR System (BIO-RAD Inc., USA). The conditions of thermos cycling were 40 cycles of 95°C for 15 s, 55°C for 15 s, and 72°C for 20 s. The 2−ΔΔCT method was used to calculate the expression levels of target genes [30, 31].
All the phenotype observations and physiological assays were performed in biological triplicates. Data were presented as mean ± standard error (SEM). Statistical analysis was performed with an SPSS 24.0 statistical package (SPSS Inc., Chicago, IL, USA). One-way ANOVA was carried out with multiple comparisons using Duncan’s test to evaluate significant differences at 0.05 probability level.