*2.9. Determination of Reactive Oxygen Species and Antioxidant Enzyme Activities*

At 2 months after seed sowing, fresh leaves of plants were sampled for the estimation of reactive oxygen species (ROS) through the assessment of electrolyte leakage (EL) and the contents of malondialdehyde (MDA) and hydrogen peroxide (H2O2). Additionally, the activities of enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) were assessed. For the EL estimation, distilled water tubes were used to place leaf samples. Samples were autoclaved at 32 ◦C for period of 2 h, and the observed EC of the solution was termed as EC1. Afterwards, this solution was autoclaved at 121 ◦C for 20 min to measure EC2, and finally EL was calculated using the following equation as described by Dionisio-Sese and Tobita [42]:

$$\text{EL} = (\text{EC}\_1 / \text{EC}\_2) \times 100$$

The concentration of MDA was measured using the method of Heath and Packer (1968) as modified by Dhindsa et al. [43] and Zhang and Kirham [44]. Hydrogen peroxide was recorded through homogenization of samples in phosphate buffer 50 mM (pH 6.5) and centrifugation followed by addition of 20% H2SO4 (*v*/*v*). Samples were centrifuged once more for 15 min, and readings were taken by spectrophotometer at 410 nm absorbance [45]. A spectrophotometer was utilized to record the activities of antioxidant enzymes such as SOD, POD, CAT and APX. Fresh leaf samples were crushed in liquid nitrogen (N2), and 0.05 M phosphate buffer (pH 7.8) was utilized for the purpose of standardization. This was followed by centrifugation at 4 ◦C on 12,000× *g* for a period of 10 min. Supernatant was collected for the sake of antioxidant enzyme activity measurements. The method of Zhang [46] was employed to measure SOD and POD activities, while the Aebi method [47] was used for CAT activity. APX contents were estimated using the method of Nakano and Asada [48].

#### *2.10. Estimation of Cr Contents in Plants*

Digestion of dry shoot and root samples was performed for 1 g of each sample in 4:1 (*v*/*v*) ratio of HNO3:HClO4 as described by Rehman et al. [49]. Finally, the digested samples were run on an atomic absorption spectrophotometer for the estimation of Cr concentrations in the processed samples.

#### *2.11. Statistical Analysis*

The IBM SPSS Statistics for Windows, Version 21.0, was used for the data analyses, using the analysis of variance (ANOVA) tool at a 5% probability level. Tukey's HSD post hoc test was performed for multiple comparison of triplicates.

#### **3. Results**

The current study was envisaged to assess the capability of metal-resistant *Staphylococcus aureus* strain K1 to ameliorate the Cr stress in wheat plants.

#### *3.1. Growth Characteristics of Isolate K1*

The bacterial strain K1, capable of tolerating a Cr concentration of up to 22 mM, was selected for further studies. Morphologically, it is characterized by Gram-positive cocci (≈1 μm) with yellowish golden color. Chemically, it is oxidase- and coagulase-negative and catalase-positive (Table 2). The BLASTn investigation showed that it has a close resemblance (99%) to *Staphylococcus aureus* strain ATCC 12600 (NR\_115606.1) and *Staphylococcus aureus* strain NBRC 100910 (MG971399.1). The similar 16S rDNA gene sequences from GenBank were used to carry out phylogenetic analysis, which also confirmed that the isolate K1 belongs to *Staphylococcus aureus*; therefore, it was named *Staphylococcus aureus* strain K1 (KX685332). This was done in order to remain confident that the bacterial strain used in this study was *Staphylococcus aureus* strain K1, as culture media can sometimes be contaminated with other bacteria.

**Table 2.** Biochemical and morphological characteristics of *S. aureus* strain K1.

