Plant material, growth conditions, and Cd treatments.
Seeds of SY33 and FY9 were obtained from the Shenyang Academy of Agricultural Sciences and the Dongya Seed Company, Shenyang, Liaoning Province, China, respectively. The seeds were sterilized with 1% sodium hypochlorite solution (v/v) for 10 min, rinsed with sterile water at least three times, and then soaked in distilled water for 12 h. One part of soaked seeds was used for germination analysis. The same number seeds were transferred into a petri-dished lined with three filter papers and covered with gauze that were moistened with distilled water or 20 mg L− 1 CdCl2 and placed in an incubator at 28°C in the dark to germinate for 9 days. Germination rates were then calculated, respectively.
The other part of soaked seeds was used for sampled for other assays. The soaked seeds were transferred in to petri-dishes lined with three filter papers and covered with gauze that was moistened with distilled water or CdCl2 at 20 mg L− 1, and germinated for 3 days, then were transferred into plastic containers and cultivated in a 1/4 Hoagland solution (pH 6.0) with 20 mg L− 1 CdCl2 treatment at 28°C and a 16/8 h light/dark photoperiod for 3 or 6 days. A control was also set up, without cadmium. Tissues were collected and stored directly in liquid nitrogen or dried at 80°C until the weight was constant before storage. Each treatment was repeated at least four times, with three seedlings in each replicate.
Calculation of the relative growth rate.
The seed was considered germinated when the radicle or coleoptile was at least 2 mm. The germination rates (%) were calculated as the number of seeds out of the total number of seeds that germinated in 3, 6, and 9 days in either water or Cd solution (20 mg L− 1). The length of the plumules and radicles or shoots and roots of between 10 and 15 of the Cd-treated plants for each time period were measured with a ruler. The biomass was determined by measuring the dry weights of the plumules and radicles or shoots and roots.
Cd estimation.
The dry plant tissues were ground to powder, and then 0.2 g of the dry powder was digested with HNO3 and HClO4 (v/v, 83/17) for 24 h. The Cd concentration was measured using an atomic absorption spectrophotometer.
Determination of the O 2 •− , MDA, and Pro concentrations and the relative electrolyte leakage rates.
To determine the concentration of O2•−, a portion of the sample (2.0 g) was mixed with 3 mL 65 nmol L− 1 of a phosphate buffer (pH 7.8) and centrifuged for 10 min at 10,000 rpm. The supernatant (2.0 mL) was mixed with a phosphate buffer (1.5 mL) and hydroxylamine hydrochloride (0.5 mL) at 25°C for 20 min. Then 2.0 mL of the reaction mixture was mixed with 17 mmol L− 1 sulfanilic acid (2.0 mL) and 27 mmol L− 1 of α-naphthylamine (2.0 mL) at 30°C for 30 min. The absorbance was measured at 530 nm using a UV-T6 spectrophotometer.
The concentrations of MDA were measured using the thiobarbituric acid chromogenic method as described by Aravind and Prasad (2003). The free L-proline concentrations and relative electrolyte leakage rates were measured as described by Bates et al. (1973)
Determination of the plant hormones.
Fresh tissue (1 g) was ground to powder and mixed with 10 mL of 80% methanol and 0.2 g of Crosslinked Polyvinylpyrrolidone at 4°C for 12 h and centrifuged at 15,000 rpm for 10 min. The supernatant was extracted by ethyl acetate three times and then dissolved in methanol and stored at − 20°C. The levels of GA3 and ABA were measured using a slightly modified version of the method described by Jia et al. (2020).
Determination of sugar concentrations.
For the sugar assays, the dried samples were powdered and homogenized in 80% ethanol, boiled at 70°C for 30 min, and centrifuged at 8000 g at 4°C for 10 min. The total soluble sugars in the supernatants were measured as described by McCready et al. (1950). The fructose, glucose, and sucrose concentrations were measured by high performance liquid chromatography (HPLC, Waters 600 HPLC) using the method of Sanchez-Linares et al. (2012).
Enzyme activity assay.
The activity of LOX was determined as described by Surrey (1964). 1 g fresh tissue was powdered and homogenized with 50 mM Hepes (pH 7.0), 5 mM cysteine, and 10 mM EDTA. The mixture was centrifuged at 10,000 g at 4°C for 20 min, and the supernatant was examined for enzymes.
To determine the activities of the antioxidant enzymes, samples of fresh plant tissue (500 mg) were homogenized in 5 mL of 100 mM pre-cooled phosphate buffer (pH 7.5) containing 1 mM EDTA. The homogenate was centrifuged at 12,000 g for 15 min at 4°C and the activities of antioxidant enzymes of the supernatant were determined. The SOD activity was examined spectrophotometrically at 560 nm, as described by Tewari et al. (2008). The POD activity was measured as the guaiacol oxidation at 470 nm by H202, as described by Lacan and Baccou. (1998). The CAT activity was determined by measuring the decrease in H202 at 240 nm, as described by Ishibashi et al. (2008).
To measure the activities of the sucrose metabolism enzymes, fresh plant tissues (1 g) were ground to powder with quartz sand and homogenized with a 50 mM phosphate buffer (pH 7.5) at 4°C. After centrifugation at 12,000 g for 20 min at 4°C, the supernatant was divided into two portions, one of which was analyzed for the activities of SPS, SS, and NI, as described by Saher et al. (2005), and the other was stored at − 80°C. Meanwhile, the precipitate was resuspended in the same extraction buffer with 1 M KCl and agitated continuously for 60 min at 4°C. The homogenate was centrifuged at 12,000 g for 20 min, and then the supernatant was mixed with the stored supernatant solution. The AI activities of the mixture were determined using the method of Saher et al. (2005).
RNA extraction and analysis.
RNA was isolated from tissues using an RNA pure Plant Kit (Qiagen). Total RNA (about 2 µg) was reverse transcribed to synthetic cDNA using a MMLV reverse transcriptase (Promega). Quantitative reverse polymerase chain reaction (qRT-PCR) assays were performed with a real-time PCR detection system (ABI 7500) using SuperReal PreMix Plus Kit (Qiagen). The reaction conditions were 40 cycles of 95°C for 10 s, 60°C for 20 s, and 72°C for 30 s. The ΔΔCT method was used to analyze the transcript levels of the relevant genes (Livak et al. 2001). The primers were used to amplificated SOD gene were 5′-CGGTGCACCAGAAGACGAAG-3′ and 5′-GCCAGTCTTCCACCAGCATT-3′. The CAT gene primers were 5′-TCCCAACTACCTGATGCTGC-3′ and 5′-GTTGGGCTTGCGTATGGTTG-3′. The POD gene primers were 5′-TGGAACACAAGCACGAACCC-3′ and 5′-CCTTCCACAGCGTCTCGTT-3′. The ZmTubulin1 (ID: Zm00001d013367) gene was used as an internal control. The primers of ZmTubulin1 were 5′-GTGTCCTGTCCACCCACTCTCT-3′ and 5′-GGAACTCGTTCACATCAACGTTC-3′.
Statistical analysis.
All data are shown as means and the standard deviation (SD). One-way analysis of variance (ANOVA) and Duncan’s multiple range test were carried out using SPSS version 26. The significance level was P < 0.05. Each experiment had four replicates.