4.3. Measurement Index

Six seedlings exhibiting similar growth patterns were selected from different treatments. After wiping away the surface water, the corn was divided into above-ground and root sections and the biomass was weighed. Firstly, the maximum height of the whole plant was manually measured using a ruler and this was repeated for each of the eight plant treatments. Secondly, the length and width of all fully expanded leaves were measured using a ruler, and the leaf area was calculated (single leaf area of fully expanded leaves = leaf length × maximum leaf width × 0.75). Furthermore, the overall root morphology was studied using an Epson Perfection V850 Pro Photo scanner (Epson America lnc., Long Beach, CA, USA). Subsequently, the total root length, the total root surface area, the total root volume, the average root diameter, and other parameters were analyzed using WinRHIZO 2007 (Regent Instruments Inc., Québec, QC, Canada) and the fresh weight was obtained after scanning the root system. The base of each stem was cut at 3 cm, scanned using an Epson scanner (placed on a flat plane), and the average diameter was analyzed using WinRHIZO 2007 software, with six repetitions for each treatment. The number of lateral roots 10 cm from the base of the main root was determined. Finally, a fresh sample was placed inside a 105 °C oven for 30 min, dried at 80 °C to measure the constant weight, and the above-ground and root system dry weights were obtained.

The net photosynthetic rate (P_n) was measured using a portable photosynthetic instrument (Li6800; LI-COR Inc., Lincoln, NE, USA) for five full-spread leaves of the selected plant between 9:00 and 11:00 a.m. The chlorophyll content in five full-spread leaves of the selected plant was measured by using a chlorophyll meter (SPAD-502, Minolta, Tokyo, Japan).

Plant cell membranes play an important role in maintaining the cellular microenvironment and normal metabolism. Under stress conditions, the cell membrane is damaged by electrolyte extravasation, which increases the conductivity of the extract. The third leaf was selected for each treatment. Leaves were rinsed with distilled water, dried, and cut into small pieces. Then, a 0.1 g sample was weighed and placed in a glass test tube. A DDSJ-308A conductivity meter was used to measure the relative conductivity at room temperature between 20 and 25 °C. Each sample was measured three times, and the average was recorded as R1. After measuring the relative conductivity at room temperature, the test tubes were placed in a boiling water bath for 30 min. After cooling to room temperature, the conductivity was measured again. Each sample was measured three times, and the average was recorded as R2. Relative conductivity = (R1/R2) × 100%.

On day 18 after cadmium treatment, about 0.2 g of fresh corn leaves were ground and homogenized with 1 mmol EDTA and 1%PVPP of phosphate buffer extract. A microplate reader (Spark Multi-functional microplate reader) was used to determine the leaf physiology. MDA content was tested as described by Xia et al. [49]. Activities of SOD (EC 1.15.1.1) and POD (EC EC1.11.1.7) were determined by the method of Qiu et al. [50]. The content of soluble protein was tested as reported by Bradford [51].

The MDA content in plant leaves was determined via the thiobarbituric acid colorimetric method. The extract was reacted with a hot water bath (60 °C,15 min) of 0.75% TBA solution containing 10% TCA and then centrifuged. Then, the absorbance of the extract was measured at 450 nm, 532 nm, and 600 nm. The MDA content in the sample was (μmol·g⁻¹·Fw) = [MDA concentration in the extract (μmol/mL) × total amount of the extract (mL)/sample fresh weight (g)]. The enzyme activity was calculated as U/mg protein based on the measured soluble protein.

Superoxide dismutase (SOD; EC1.15.1.1): Riboflavin is prone to oxidation under light and aerobic conditions to produce superoxide ions, thus reducing NBT and resulting in a blue color with a maximum absorption peak at 560 nm. The SOD enzyme is an oxygen-radical scavenger, and the enzyme activity in plants is indicated by the degree of reaction. The SOD enzyme activity unit (U) was measured via the nitroblue tetrazolium (NBT) photochemical reduction method and represented as the NBT photoreduction inhibition by 50%.

Peroxidase (POD; EC1.11.1.7): The peroxidase extract reacted with guaiacol and hydrogen peroxide to produce a brown solution with a maximum absorption luminosity at 475 nm. The guaiacol colorimetric method was used to measure the POD activity in the plant shoots, with an increase of 0.01 in the absorbance value considered the enzyme activity unit. It was measured rapidly under the catalytic reaction of hydrogen peroxide and once every 15 s for a total of 5 min. The POD enzyme activity (U/mg·FW·min) was calculated based on the reaction curve.

The soluble protein content was determined with the Coomassie brilliant blue G-250 method; 30 μL of extract and 170 μL of Coomassie bright blue G-250 solution were mixed and then left for 5 min, with the measured absorbance value at 595 nm (minus the control) used in the standard curve equation to calculate the protein content in the reaction system.

All parts of the plant were ground, crushed, and weighed to obtain the dry weight. Then, 0.05 g of sample was added into a tetrafluoro crucible, 20 mL of nitric acid: perchloric acid (3:1) was added for nitration for 12 h, and the tetrafluoro crucible was placed onto an electric heating plate (at about 170 °C) in a fume hood to remove the acid [52]. The reaction was stopped when the volume of the solution became the size of a soybean, and the solution was subsequently cooled, fixed at a 25 mL volume with 3% nitric acid, shaken well, and stored for further use. Finally, the Cd content was determined using an Optima 2100 DV inductively coupled plasma emission spectrometer from Perkin Elmer (PE).

The health risk index [2] was calculated as follows: health risk index = C (CD) × coefficient (C) × daily food intake (DFI)/average body weight (ABW) × oral reference dose of cadmium (Ordc). Here, C (CD) is the Cd content (mg/kg); C is the correction coefficient, with a value of 0.085; and DFI is 0.4 kg/person/d according to the provisional standard proposed by Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO). The adult ABW was set to 70 kg and Ordc was 0.001 mg/kg/day, according to the United States Environmental Protection Agency (US EPA) (1985).