Plant materials and layout
Maize seeds (Sahiwal-2002) were procured from Maize and Millets Research Institute (MMRI), Yousaf Wala Sahiwal, Pakistan. Seeds were immersed in 30 % (v/v) H2O2 for 5 min. for sterilization, washed with deionized water for 24 h and dried. Seeds were placed in Petri dishes lined with double layer of What-man # 02 filter papers. The surface of each filter paper was moistened with 15 mL of H2O and kept in dark condition 25 ± 2 °C for a 48 h. After germinations, six seeds were planted in plastic pots (depth; 40 cm and diameter; 35 cm) in sterilized sand with a particular particle size 0.25 mm. The sand was soaked for 24 h in 30 % (v/v) HCl solution to remove all cations and anions and then thoroughly rinsed with deionized water three time (with 24 h soaking). The pots were treated with 0 and 150 µm Cd2+ applied as CdCl2 and 0, 0.5, 1, 2.5, 5 and 10 mM Ca2+ levels using Ca(NO3)2 as source . The ½-strength Hoagland's (Hoagland 1938) nutrient solution was applied to the seedlings throughout the experimentation. The Hoagland solution was applied to the plants to saturate the sand at 3 days interval with draining any past solution left in sand. All pots were arranged as CRD with 3 replications under controlled glasshouse environment with day/night temperature of 24 ± 4°C/14 ± 2°C and RH 58-60 % (Rady et al. 2019).
Plant sampling and measurements
Plants material was sampled at the seedling stage to determine plant growth attributes, physio-biochemical traits, ROS, and enzymes of antioxidants defense system. Harvested seedlings were washed with distilled water and growth attributes were recoded. Leaf samples of maize seedlings were frozen at –80°C for physio-biochemical traits and antioxidants. Sampled seedlings were dried in oven at 70 °C to achieve a constant dry weight for determination of root (RDW) and shoot (SDW) dry weight.
The shoot length (SL) of plants from each treatment was measured from sand level to the topmost leaf of the plant. The roots of seedlings were carefully removed from the sand for recording root length (RL). Root (RFW) and shoot fresh weight (SFW) of seedlings were measured immediately after excision. The leaf area (LA) was estimated by measuring length × width × 0.68.
Chlorophyll contents were assessed as described by Arnon (1949) and carotenoids following the method of Davis (1976). For the appraisal of chlorophyll contents, 0.1 g of leaf sample was grounded in 5 mL of acetone (80 %). Extract was filtered through a Whatman # 02 filter paper (GE Healthcare, UK) and absorbance was recorded through a spectrophotometer (Hitachi U-2910, Tokyo, Japan) at 645, 663, and 480 nm. The values of photosynthetic pigments were calculated by using the following formula.
Here, V characterizes the volume of acetone and (FW) showed the leaf fresh weight.
Determination of relative membrane permeability
The fresh leaf samples were collected and washed thoroughly with 4 changes of water to eradicate any adhered electrolytes on the surface. The leaves were cut into small discs with a borer and placed in the small glass test tube containing deionized water (10 mL), The ECo was measured by the help of Cond/Salinity meter (TPS AQUA-CPA). The test tubes were incubated for 24 h at 4 °C and EC1 was measured. The tubes were then wrapped with aluminum foil, autoclaved for 10 min. at 100 kPa and (EC2) was recorded. The ratio of % ion leakage was computed as designated by Yang et al. (1996).
Assessment of biochemical traits
Anthocyanin content was appraised according to the method of Giusti and Wrolstad (2001). The 0.1 g of leaf was pulverized in trichloroacetic acid (TCA) by using pestle and mortar. The homogenized material was transferred to test tubes and shifted to water bath at 80 °C for 20 min. Homogenized material was centrifuged at 12,000 xg for 10 min. in the absorbance was noted at 516 and 700 nm using a spectrophotometer (Hitachi U-2910, Tokyo, Japan). Acetone was run as blank and amount of monomeric anthocyanin contents was calculated as follows.
Here, A= (A510-A700) MW= 449.2 and ε= 26900 [ε is the molar absorptivity measured the amount of cyanidin-3-glucoside pigment and DF is dilution factor].
Oxidative stress markers (MDA and H2O2)
Lipids peroxidation (LPX) was quantified by means of malondialdehyde (MDA contents) by following Heath and Packer (1968). LPX content was determined by the reaction of thiobarbituric acid-TCA with trichloroacetic acid-TCA. The 0.25 g leaf sample was grinded in 500 µL of TCA (0.1 %) and then centrifuged at 15,000 xg. An aliquot (1 mL) was taken and mixed with 2 mL of 0.5 % of TBA and 20% TCA. Test tubes containing reactants were incubated at 85 °C for 20 min. and reaction was terminated in an icebox. Absorption was recorded at 532 and 600 nm by spectrophotometer (Hitachi U2910, Tokyo, Japan). All absorption ODs (at 532nm) were subtracted from 600 nm. LPX concentration was calculated by using 155 mM cm-1 as an extinction coefficient.
Amount of H2O2 was quantified by measuring the oxidation of ferrous ions medicated by peroxidase and ferric ions react with the xylenol (Bellincampi et al. 2000). Leaf sample 0.5 g was grounded in 5 mL of 10 mM sodium phosphate buffer (SPB). Centrifugation of homogenized material was done at 15,000 xg. A 2 mL of aliquot was reacted with the assay reagent containing 200 mM sorbitol, 200 µM xylenol, 50 mm H2SO4, and 500 µM ammonium ferrous sulphate. The reactant material was incubated at 24 °C for a 1 h and absorption was recorded at 560 nm by using a spectrophotometer (Hitachi U-2910, Tokyo, Japan).
Cellular antioxidants (APX and POD)
The maize seedlings’ shoot were grounded in liquid nitrogen and extracted with 1 mM L-1 of 5% polyvinylpyrrolidone, and, sodium phosphate buffer (SPB) having pH 7.8. Extracted material was centrifuged at 15,000 xg. Enzyme crude extract was stored at 4 °C for 36 h till analysis.
Ascorbate peroxidase activity (APX)
Activity of APX was quantified by oxidation of ascorbate (Chen and Asada 1989). Reaction was started by adding 10 µL of crude enzyme extract to 2 mL of assay reagent (30 % H2O2, 0.5 mM C6H8O6, and sodium phosphate buffer (SPB) having pH 7.2,). After 30 s of reaction initiation, a shift in absorption was noted at 290 nm for 4 min. on a spectrophotometer (Hitachi U-2910, Tokyo, Japan). Activity of enzyme was estimated through extinction coefficient (2.8 mM cm-1), while the specific activity of the enzyme was calculated on the basis of protein contents and expressed as an mg-1 min.-1 FW.
Peroxidase (POD) activity
POD activity was appraised spectrophotometrically by using the method of Goliber (1989) based on oxidized of guaiacol in the presence of H2O2 and expressed as a Units mg-1 proteins. A 20 µL of the enzyme extract was added to assay reagent (20 mM guaiacol, 10 mM H2O2, and 0.1 M phosphate buffer) and volume was maintained up to 3 mL. Enzyme activity was measured at 460 nm after 60 s interval through a spectrophotometer (Hitachi U-2910, Tokyo, Japan). Enzyme specific activity was expressed on the base of proteins.
Ascorbic acid was determined as described by Nino and Shah (1986). Plant tissues (100 mg) were pulverized in thiobarbituric acid (TCA) and centrifuged 10,000 xg for 10 min. An aliquot (500 µL) was taken with 500 µL of dthiocarbamate (DTC) in glass tubes. Reactants were left for ½ h at 37 °C. Test tubes containing reactant material was transferred to the ice-bath to terminate the reaction. After that, 2 mL of diluted H2SO4 was mixed slowly and left over for ½ h at 37° C in incubator. Extracted material was centrifuged at 12,000 xg. The shift in absorption was measured at 520 nm with the help of a spectrophotometer (Hitachi U-2910, Tokyo, Japan).
Total amino acids
Free amino acid was quantified followed by Hamilton and Van-Slyke (1943) method. The 0.1 g of the leaf sample was grinded and immersed in a potassium phosphate buffer (SPB) overnight. After incubation, 1 mL of plant extract was transferred to 25 mL test tubes after adding 1 mL each of 10 % ninhydrin and 2% of pyridine solution. The test tubes containing reactants were placed in boiling water bath for 1 h. The final volume of samples was made to 25 mL by using deionized H2O. Absorbance was recorded at 570 nm spectrophotometrically (Hitachi U-2910, Tokyo, Japan) and resulting absorbance were compared with the standard curve plotted for leucine.
Soluble proteins were appraised following Lowry et al (1951). Plant sample (0.1 g) was grounded in 50 mM sodium phosphate buffer (SPB) having pH 6.8. The extracted aliquot (500 µL) was mixed in 0.3 mL of deionized H2O and 3 mL of Bio-Rad protein assay dye and vortexed for 15 s. The absorbance was measured spectrophotometrically at 595 nm (Hitachi U-2910, Tokyo, Japan). Soluble proteins were estimated by comparing absorbance of samples with bovine serum albumin (BSA) using a standard value.
Minitab-19, software e (Minitab, LLC, State College, PA, USA) was used to analyze the data. The means values were compared by using the Tukey pairwise comparison test at P≤0.05 following an analysis of variance (ANOVA). The effect of Ca and Cd treatments was assessed by using a multivariate analysis (PCA by ggbiplot), correlation matrix (ggbiplot2) and heatmaps were plotted by customized code (pheatmap) by using R statistical software (R Core Team, 2019). Response curves under cadmium and calcium stress treatments were constructed by fitting a generalized linear model (GLM) in CONACO version 5 for windows.