*2.3. Hydroponic Culture Treatments and Plant Growth Analysis*

Subsequently, the primed seeds were placed into a 96 well black hydroponic box with one seed in each well. Each treatment was repeated six times and each repetition had 80 seeds. The hydroponic nutrient solution consisted of 0.5 μM potassium nitrate, 0.5 μM Ca(NO3)2, 0.5 μM magnesium sulfate, 2.5 μM monopotassium phosphate, 2.5 μM ammonium chloride, 100 μM ferric EDTA, 30 μM boric acid, 5 μM manganese sulfate, 1 μM copper sulfate, 1 μM zinc sulfate, and 1 μM ammonium hepta-molybdate ((NH4)6Mo7O24). The hydroponic boxes were repositioned daily within the growth chamber (30 ◦C with an alternation cycle of 12/12 h light/dark) by completely randomized design (CRD). After two weeks of hydroponics, three repeats of each treatment were exposed with 100 μM Cr for 7 days, as well as another three repeats without Cr, used as control. Sampling was carried out after the 7th day of Cr treatment.

The sampling of 21-day-old seedlings was conducted and they were washed with ddH2O to exterminate the Cr residues. The height of seedlings was calculated by using a ruler and their fresh biomass was measured on a scale. To estimate the dry mass, leaves, and roots were dried out in an oven separately at 75 ◦C for 24 h.

#### *2.4. Determination of Photosynthetic Pigments*

The estimation of chlorophyll contents such as chlorophyll a (*Chl* a), chlorophyll b (*Chl* b), total chlorophyll as *Chl* (a + b) and carotenoids (Car) was carried out following the method of Lichtenthaler and Wellburn [27]. Concisely, fresh leaves tissues (0.2 g) were homogenized with pure water and soaked in 3 mL of 95 percent ethanol (*v*/*v*). The mixture was centrifuged for 10 min at 5000× *g* to separate the supernatant. After that, supernatant at the volume of 1 mL aliquot were mixed with 9 mL ethanol (95 percent, *v*/*v*). Thereafter, using an exhausting spectrophotometer, the mixture was measured using absorbance at 665, 649, and 470 nm wavelengths [28]. The following equation was used to determine the chlorophyll contents:

Chlorophyll a (*Chl* a) = 13.95 A665 − 6.88 A649 (1)

$$\text{Chlorophyll b (Chl b)} = 24.96 \text{ A} \theta 49 \text{ } -7.32 \text{ A} \theta 65 \text{ } \tag{2}$$

$$\text{Total chlorophyll content} = \text{Cbl a + Ch}l \text{ b} \tag{3}$$

$$\text{Caroteroids (C}\_{\text{x+c}}) = (1000 \text{ A470 } - 2.05 \text{ C}\_{\text{a}} - 104 \text{ C}\_{\text{b}}) / 245 \tag{4}$$

The values of photosynthetic attributes were measured as milligrams (mg) per liter (L) of plant extract. Parameters related to gaseous exchange were investigated by the methodology of Zhou and Leul [29]. After 2 h of assimilation inside the growth cabinet at 18 ◦C, 1000 molm−<sup>2</sup> s−<sup>1</sup> light intensity, and 60% comparative moisture, the uppermost wholly extended leaves were used to calculate the rate of transpiration (Tr), net photosynthetic rate (Pn), stomatal conductance (gs), the intercellular level of CO2 (Ci), and photochemical efficacy of PS II (*Fv*/*Fm*). ImagingWin software (IMAGING-PAM, Walz, Effeltrich, Germany) was used to examine the colored images for *Fv*/*Fm* and *Fm* levels.

#### *2.5. Estimation of Cr Contents*

The samples of dried root and shoot (0.2 g) per treatment were mixed with 5 mL concentrated HNO3 and HClO4 (5:1, *v*/*v*) on a stovetop at 70 ◦C for around 5 h. Dilution of the samples (digested) was subjected to dilution to a final amount of 10 mL with 2% HNO3 before being tested, and three replicates per treatment were prepared. To examine the Cr along with microelements Na, Cu, K, P, Fe, Ca, Mn, and Zn, scum was measured by an atomic absorption spectrometer (iCAT-6000-6300, Thermo Scientific, Waltham, WA, USA).

#### *2.6. Determination of Electrolyte Leakage and Total Soluble Sugar*

The rice seedling is utilized to estimate electrolyte leakage (dSm−1). Surface sterilization was carried out for 5 g seeds with HgCl2 (1%) and speckled with ddH2O, with four

replications. Subsequently, seedlings were drenched in 25 mL ddH2O, and were left at room temperature for one-day in an incubator. The sample was moved to a new void beaker, up to the volume of 25 mL by adding ddH2O. Electrolyte leakage was reported in dSm−<sup>1</sup> [30]. To estimate the total soluble sugar, samples of 0.5 g in the form of fresh shoots were crushed with pestle and mortar, in an extraction buffer that prepared from phosphate (50 mM, pH 7), ascorbate (1 mM), KCl (100 mM), glycerol (10%, *v*/*v*) and β-mercapto-ethanol (5 mM). Then, the supernatant was amassed into the micro-centrifuge tube through centrifugation at 12,000× *g* for 15 min. Later, the homogenate of three replicates was used to quantify the total soluble protein content [31] and total soluble sugar, by the procedure as for the phenol-sulfuric acid assay [32].

#### *2.7. Estimation of Endogenous Abscisic Acid, Jasmonic Acid and Salicylic Acid Contents*

The quantification of endogenous ABA contents was accomplished by using frozen samples following the protocol of Kim et al. [33] and Kamboj et al. [34]. The endogenous contents of JA were quantified following the detailed procedure of [35]. Extraction and quantification of free SA was carried out via the protocol of Yalpani et al. [36] and Fang et al. [37]. Three biological replicates were used to estimate the endogenous abscisic acid, jasmonic acid and salicylic acid contents.

#### *2.8. Estimating MDA, H2O2 and O2* •− *Contents*

The determination of MDA content was performed with 2-thiobarbituric acid (TBA). Homogenization of ~2 mL extract in 3 mL of TBA (5%) was performed, before being diluted inside 5% of trichloroacetic acid (TCA). Formerly, the grounded and mixed samples were preheated for 15 min at 95 ◦C, and earlier cooled to ice instantaneously and centrifuged at two different wavelengths, viz., 532 nm and 600 nm, via utilizing a UV–vis spectrophotometer (Hitachi U-2910) [38]. The value of MDA content was donated as nmol mg-1 protein. The hydrogen peroxide (H2O2) was determined by following the detailed protocol of Kwasniewski [39]. The H2O2 contents were estimated as μmol g−<sup>1</sup> FW. Superoxide radical (O2 •−) contents were estimated following the method of Jiang and Zhang [40] with a few amendments. Three replications were used to estimate the MDA, H2O2 and O2 •− contents.
