Measurements

Germinated seeds with 1 mm radicle length were recorded at every 24 h, and radicle elongation was measured 9 days after incubation. Final germination percentage (TG) at the end of the experiment period was calculated according to the following formula:

Germination index was calculated as arithmetical sum of the total seeds germinated every day up to a period of 9 days.

Germ length was measured using ruler. Five seeds were selected and the average was used for analysis.

Vertical shoot growth rate (VSGR) was calculated according to the method described by Huang and Liu [26] with slight modifications. At the initial and ultimate of the treatment, turf canopy heights were measured four times with a ruler in each flask, and the average height was divided by 9 (days) to calculate VSGR [21]. Fresh weights for shoots and roots were also weighed separately at the end of the experiment (9d).

Plant transpiration was assessed following the procedure described by Yu et al. [27]. Water loss was determined by weighing the plant-flask system at every 24 h. To compare the effect of Cd²⁺ or Pb²⁺ on plants with different initial Tr, the normalized relative transpiration (NRT) was calculated according to the following equation described by Yu et al. [28]:

where C is concentration (mM), t is time period (days), T is absolute Tr (grams per day), i is replicate 1, 2, …, n, and j is control 1, 2, …, m. The relative Tr of controls is always set at 100%.

To determine leaf chlorophyll content, fresh leaves (0.1 g FW, cut into small pieces) was soaked in 15 mL dimethyl sulfoxide and kept in the dark for 72 h according to the method described by Yu et al. [29]. The absorption of leaf extracts at 663 and 645 nm was measured with a spectrophotometer [ultraviolet-2600; UNICO (Shanghai) Instruments, Shanghai, China].

Electrolyte leakage (EL) was used as a reliable and rapid method to assess membrane permeability [10]. Fully expanded leaves (0.1 g FW) were excised and washed three times with deionized water, then cut into ≈0.5-cm long segments and placed into 50 mL vials containing 20 mL distilled water. All vials were shaken for 24 h at room temperature. The initial conductivity (C_i) of the incubation solution was measured with a conductance meter (JENCO-3173; Jeno Instruments, San Diego, CA). Subsequently, the leaves were transferred to and killed in an autoclave at 120°C for 30 min. After 24 h incubation on a shaker at room temperature, the conductance of the incubation solution with killed tissues (C_max) was also determined. The relative EL was calculated using the following equation:

About 0.3 g of fully expanded leaves were homogenized in a pre-chilled mortar and pestle with 4 mL of 50 mM ice-cold phosphate buffer (pH 7.0). Then the homogenate was centrifuged at 15,000 xg for 15 min at 4°C. The supernatant was collected for MDA content, enzyme activities, and soluble protein content determination.

The content of MDA was determined by thiobarbituric acid reaction as described by Heath and Packer [30] with slight modification. Briefly, a reaction solution was made containing 20% (v/v) trichloroacetic acid, 0.5% (v/v) thiobarbituric acid, and 1 mL of enzyme extract in 2 mL volume. The mixture solution was heated at 95°C for 30 min using water bath, and then quickly cooled in an ice-water bath. Eventually, the solution was centrifuged at 10,000 rpm for 10 min. The absorbance of the supernatant at 532 nm was recorded and corrected for unspecific turbidity by substracting the value at 600 nm. The content of MDA was calculated based on this adjusted absorbance and the extinction coefficient of 155 mM^-1cm^-1 [30].

To determine the soluble protein content, a reaction solution containing 70 μL of 150 mM PBS (pH 7.0), 30 μL soluble protein extract and 3 mL color reagent according to the protocol described by Bradford [31]. The absorbance of the reaction solution was measured at 595 nm after 2 min and before 1 h using a spectrophotometer (ultraviolet-2006).

The activity of superoxide distumase (SOD) was determined by monitoring its ability to inhibit the photochemical reduction of nitroblue tetrazolium chloride (NBT) [32]. The absorbance of the irradiated solution at 560 nm was recorded with a spectrophotometer (ultraviolet-2006), and one unit SOD activity was defined as the amount of enzyme required to cause 50% inhibition of the rate of NBT reduction at 560 nm.

Catalase activity (CAT) was determined using the method of Gill et al. [33] by monitoring the disappearance of H₂O₂ at 240 nm. One unit of CAT activity was defined as the absorbance change of 0.01 units per minute.

Peroxidase activity (POD) was determined following the method described by Polle et al. [34] with minor modifications. Absorbance change at 460 nm was recorded every 1 min within the first 3 min for calculating POD activity. One unit POD activity was defined as the absorbance change of 0.01 units per minute.

To determine the Cd or Pb content, the plant materials (shoots and roots) were killed at 105°C for 30 min, and then dried to constant weight at 80°C. Dry samples were ground with a mortar and pestle. About 0.5 g dry samples were ashed, and then subjected to wet digestion with a mixture of concentrated HNO₃ and concentrated HClO₄ at 5:1 (v/v) [3]. Finally, the digested solution was redissolved with nitric acid. The atomic absorption spectroscopy (Shimadzu, Model AA-7000, Japan) was employed to determine the Cd or Pb content [35], and the concentration of Cd or Pb was defined as the Cd or Pb content (mg) per unit plant (kg). Meanwhile, the translocation factor was calculated using the following equation: Translocation factor = metal concentration in shoots/metal concentration in roots [36].

Gene expression was assessed according to the method described by Yu et al. [37]. Leaf samples were collected at 24 h after the initiation of the treatment and flash frozen in liquid nitrogen and then stored at -70°C.

About 0.1 g expanded leaves was homogenized with liquid nitrogen, and total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA). The RNA concentration and purity were determined, and the DNA contamination was removed. RNA (2 μg) was reversely transcribed with oligo (dT) primer using first strand cDNA synthesis kit according to the user manual (Fermentas Canada, Burlington, ON, Canada). Primers of different genes were synthesized according to the previous reports for use in Q-PCR (Table 1). The PCR amplification data were analyzed with Option Monitor version 2.03 (MJ Research).

^yF and R represent forward and reverse.