2.1. Plant materials, growth conditions and treatments Healthy
Healthy selenium-free rice (Oryza sativa L.) CR727 and selenium-rich red hybrid rice Z2057A/CR727 seeds were collected with the consent of the Demonstration Base for International Science & Technology Cooperation of Sichuan Province, Rice Research Institute, Sichuan Agricultural University, Chengdu, Sichuan, China. Rice seeds were surface sterilized with NaOCl [1% (v/v)] for 20 min and washed 5 times with double distilled water (ddH2O) followed by imbibition for 48 hours. For germination, the seeds were placed on plastic nets, floating on distilled water (dH2O) and kept in an incubator (37 ± 1°C) under dark for 72 hours. Uniformly germinated seeds were sorted and cultivated in a growth 1000ml plastic chamber with half-strength Kimura B nutrient solution and housed under a light-dark cycle of 12 h:12 h at 25 ± 2 °C, using a hydroponic solution [17]. The nutrient solution was adjusted to a pH of 5.5 using 1 M NaOH and renewed at 3 days interval [16]. After 12 days, healthy seedlings were exposed to environmentally toxic Se stress by supplying 10, 20, 40 and 80μM sodium selenate [Na2SeO4, referred to as Se hereafter] in the nutrient solution [10]. The control and Se-treated seedlings were grown for 14 additional days in the above-stated conditions [22]. The fully expanded second leaves of rice seedlings were harvested to determine growth-related parameters associated with physiological and biochemical responses. Three independent replications of each treatment were used in determining each parameter.
2.2. Measurements of leaf water status
Leaf relative water content (RWC) was calculated based on fresh weight (FW), turgid weight (TW) and dry weight of leaves samples. Approximately 0.1 g fresh leaves were detached and immediately weighed as the FW, then soaked in de-ionized water for 24 hours at 4 °C in the dark and weighted as TW. Leaf samples were then dried in an 80 °C oven for at least 72 hours prior to being weighted for DW. The RWC was calculated as (FW-DW)/(TW-DW) × 100% [23].
2.3 Measurements of physiological parameters
Total leaf chlorophyll (Chl) content analysis, fresh leaves (0.1 g) were immersed in 10 ml of dimethyl sulphoxide in the dark for 48 hours, and then the leaf extract was measured at 663 and 645 nm with a spectrophotometer [24]. According to the manufacturer’s instructions of the kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), superoxide dismutase (SOD) and methane dicarboxylic aldehyde (MDA) were determined by the Shimadzu UV-visible spectrophotometer on path length cuvettes of 1 cm (T6S, Puxi, Co., Ltd, Beijing, P. R. China).
2.4 Histochemical Analysis
After 14 days of treatment, rice leaves were collected for histochemical analysis. 3,3’-diaminobenzidine (DAB, 1.0%) staining was performed to detect hydrogen peroxide (H2O2) accumulation [25]. To observe the accumulation of reactive oxygen species (ROS) in leaves, nitroblue tetrazolium (NBT, 0.1%) was performed as previously described [25].
2.5 Measurements of selenium and anthocyanins content
To determine the Se content, we used the atomic fluorescence spectrophotometer (RGF–6800, Bo Hui Co., Ltd, Beijing, P. R. China) as previously described [26, 27]. To determine the anthocyanins content, we used the Shimadzu UV-visible spectrophotometer according to the previously described [11].
2.6 Relative genes expression analysis
According to the qRT-PCR method [3], 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 (Qingke Zixi 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 ofthermos 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[28].
2.7Statistical Analysis
All analyses were performed in triplicate. Data expressed as mean ± standard error (SEM). One-way ANOVA was carried out with multiple comparisons using Duncan’s test to detect significant differences among the five groups with different concentration sodium selenate treatments at p ≤ 0.05. All statistical analysis was performed with the SPSS 24.0 statistical package (SPSS Inc., Chicago, IL, USA).