Plant material. Cuttings selected for this study were healthy, one-year-old honeysuckle (Lonicera japonica Thunb.) from a representative strain cultivated at Liaoning University which continues to be used in additional research in the university's Department of life sciences. The cuttings were obtained in accordance with all relevant institutional, national, and international guidelines and legislation and all appropriate permissions and licenses were obtained for the specimens and materials. The cuttings were cultivated in sterilized sand for ten weeks and then transplanted to a mixture of soil to sand (3:1). The soil was a brown topsoil (0~20 cm, pH 7.06), and was obtained from Liaoning University. The level of basic nutrients in the soil was 0.21 mg/kg calcium, 97.29 mg/kg nitrogen, 8.84 mg/kg phosphorus, 216.98 mg/kg potassium, and 1.50% organic matter. Air-dried soil samples were filtered through a 4.0 mm sieve for use in the experiment.
Cd exposure. The experiments were conducted in a laboratory in Liaoning University 25/17°C day/night temperature, 50~60% relative humidity, 16 h light/8 h dark) starting in March, 2017. Eighteen plastic pots (20 cm diameter×30 cm height) containing 2.5 kg of air-dried, disinfected soil were prepared. A CdCl2•2.5H2O solution of different concentrations of Cd2+ were randomly added to each pot. Six levels of Cd2+ were administered to the pots, including 0 (control), 10, 30, 80, 150, and 200 Cd2+ mg/kg soil (3 replicates in each level). The soil was regularly mixed and allowed to come to equilibrium over a period of 40 days. During this time, the soil was mixed and sprayed with water every week to maintain an 80% water content. Three honeysuckle cuttings with similar growth and height were planted in each pot after the 40 days equilibrium period. Leaves from the cuttings were harvested every ten days from the 50th through the 90th day after the cuttings were placed in the cadmium-containing soils. Leaf samples were wrapped in tin foil, immediately frozen in liquid nitrogen, and stored at -80°C until subsequent analysis.
Measurement of growth parameters. Height (cm) and leaf area (A, cm2) of plants were measured after 90 days of Cd exposure. At that time, all plants were collected. Roots were washed with distilled water, submerged in EDTA-Na2 for 20 min to balance ion levels, then cleaned with deionized water and immediately dried on filter paper. Lastly, the whole above-ground portion of the plant was placed in a 105°C oven to a constant dry weight for the determination of biomass.
Determination of chlorophyll content. Chlorphyll was extracted from 0.5 g fresh leaves with 80% acetone (centrifuging at 5000×g) and the absorbance of the supernatant was measured at 663 and 645nm to determine the level of chlorophyll a, chlorophyll b, and total chlorophyll, respectively .
Determination of ROS levels. The rate of O2.− generation in leaves was determined by the hydroxylamine hydrochloride method  with minor modifications. Initially, 0.1 g leaves were ground in 3 ml 0.05 mol/L phosphate (K-P) buffer (pH 7.8), followed by centrifugation at 5000×g, 3 min at 4°C. Subsequently, 0.5 ml supernatant was mixed with phosphate buffer (pH 7.8) and 1 mol/L hydroxylamine hydrochloride and incubated for 20 min at 25°C. Then, 17 mmol/L p-aminobenzene sulfonic acid and 7 mmol/L 1-naphthylamine were added to the solution and absorbance was measured at 530 nm.
Hydrogen peroxide (H2O2) levels were determined in an extract prepared from 0.5 g leaves in 2.5 ml propanone, which was then centrifuged at 12000×g for 10 min at 4°C. The resulting supernatant was added to a mixture of 0.1 ml 5% Ti(SO4)2 (titanium sulphate) and 0.2 ml NH3 (ammonia), and then centrifuged at 10000×g for 10 min at 4°C. The resulting precipitate was dissolved in 2 mol/L H2SO4 and then re-centrifuged. The absorbance of the supernatant was measured in a spectrophotometer at 415 nm .
Determination of enzymatic and non-enzymatic antioxidant compounds. A total of 0.5 g of fresh leaves were ground in 3.5 ml 50 mmol/L phosphate buffer (K-P; pH 7.8) containing 1.0 mmol/L EDTA-Na2, 1.0 mmol/L ascorbate and 2% (v/v) polyvinylpyrrolidone (PVP), and 1.5 ml saturated ammonium sulfate. The mixture was then centrifuged at 5000×g for 10 min at 4°C. The resulting supernatant was used to measure enzyme activity.
Ascorbate peroxidase (APX; EC 22.214.171.124) activity: A 1 ml reaction mixture containing phosphate buffer (pH 7.0), 0.83 ml ascorbate, 0.13 ml H2O2, 0.04 ml crude enzyme was utilized. Ascorbate consumption was measured by the reduction in absorbance at 290 nm over 1 min. APX activity was calculated using an extinction coefficient of 2.8 L/(mmol cm) .
Dehydroascorbate reductase (DHAR; EC 126.96.36.199) activity: A 1 ml reaction mixture containing 0.7 ml phosphate buffer, 0.1 ml reduced glutathione (GSH), 0.1 ml dehydroascorbate (DHA), and 0.1 ml crude enzyme extract was utilized. DHAR activity was calculated from the changes of absorbance at 265 nm over 1 min, using an extinction coefficient of 14 L/(mmol cm) .
Monodehydroascorbate reductase (MDAR; EC 188.8.131.52) activity: A 1 ml reaction mixture containing phosphate buffer (pH 7.6), 0.9 ml ascorbate, 0.04 ml ascorbate oxidase, 0.03 ml NADPH, and 0.03 ml of crude enzyme extract was utilized. MDAR activity was determined by measuring the consumption of NADPH as indicated by the change in absorbance at 340 nm over 1 min, using an extinction coefficient of 6.2 L/(mmol cm) .
Glutathione reductase (GR; EC 184.108.40.206) activity: A 1 ml reaction mixture containing 0.86 ml oxidized glutathione (GSSG), 0.1ml NADPH, and 0.04 ml of crude enzyme extract was utilized. GR activity was calculated from the change in absorbance at 340 nm over 1 min, using an extinction coefficient of 2.8 L/(mmol cm) .
GSH was determined according to the method of Yu et al.  with a few modifications. Fresh leaves (0.15 g) were extracted with 1.75 ml 5% (w/v) sulfosalicylic acid, followed by centrifugation at 12000×g for 4 min at 4°C. The supernatant was used to determine the reduced and total glutathione content. Initialy, a 0.6 ml 0.1 mol/L phosphate buffer (K-P; pH 7.0; containing 0.5 mol/L EDTA) and 50 µl 3 mmol/L DTNB (5,5'-dithiobis-(2-nitrobenzoic acid)) was added to the supernatant and the content of reduced glutathione was determined by measuring the absorbance of the solution at 412 nm for 5 min. Then, 0.5ml phosphate buffer, 50 µl DTNB, 0.1 ml NADPH (0.4 mmol/L), and 2 µl GR were added to the supernatant and the solution was incubated for 20 min. Total glutathione was determined by measuring absorbance at 412 nm. Oxidized glutathione (GSSG) content was determined based on the difference between the values of total glutathione content and reduced glutathione content (GSSG). The obtained values were used to determine the GSH/GSSG ratio.
Determination of non-protein thiols (NPTs) and proline content. Non-protein thiols (NPTs) were measured as previously described by Sharma et al. . Initially, 0.1 g of sample was ground in 5 ml 1 mol/L HCl and 1 mol/L EDTA, and centrifuged at 10000×g for 3 min at 4°C. The supernatant was then added to 0.5 ml phosphate buffer (pH 7.8), and 0.5 ml 6 mmol/L DTNB, N levels were determined by measuring the change in absorbance at 412 nm.
Proline content was determined using the acid ninhydrin assay . Initially, 0.1 g fresh leaves were ground in 5 mL 3% sulfosalicylic acid. The homogenate was centrifuged at 10000×g for 3 min at 4°C. The supernatant was mixed in a 1:1:1 ratio with glacial acetic acid, and 2.5% acid ninhydrin, boiled at 100°C for 30 min and finally cooled. Then 6 mL of toluene was added, after thorough mixing, the chromophore-containing toluene was separated, absorbance was measured at 520 nm taking blank toluene as a control.
Statistical analysis. One-way ANOVA followed by a Duncan’s multiple range test at a 5% level was used to statistically analyze the effect of cadmium on plant growth, chlorophyll content, O2·− production rate, H2O2 content, proline content, GSH levels, NPTs content, and antioxidant enzyme activity (APX, DHAR, MDAR and GR). A Pearson correlation coefficient was calculated for each treatment to explore the relationship between O2·− production rate, H2O2 content, proline content, GSH, NPTs content, APX, DHAR, MDAR, and GR. A redundancy analysis (RDA) and a permutation test were performed for each treatment to determine the key variables explaining changes in the O2·− production rate and H2O2 content. The predictor variables included proline content, GSH, NPTs content, APX, DHAR, MDAR, and GR. A significance level of P<0.05 was used in the Pearson correlation analysis, RDA, and Permutation tests. The Pearson correlation analysis, RDA, and permutation tests were carried out in R 3.5.2 . The ANOVA was carried out using SPSS software.