*2.3. Vegetative Growth*

After two weeks of DR-stress, seedlings were washed with tap water. Roots and shoots were separated for measuring both fresh and dry weight. After measuring fresh weight, root and shoot were immediately placed in an individual paper bag and put in an oven for drying. The dry weight of root and shoot were recorded after drying of samples at 105 ◦C for 15 min, and 75 ◦C for three days [34]. Plant height was estimated using a measuring tape.

#### *2.4. Leaf Gas Exchange and Pigments*

Photosynthetic parameters including Pn (net photosynthetic rate), Ci (intercellular CO2 concentration), Gs (stomatal conductance) and Tr (transpiration rate) were assayed (9 to 11 a.m.) by operating a portable photosynthesis system, namely CIRAS-3, bought from Hansatech Co., Amesbury, MA, USA, with controlled measuring chamber conditions (25 ◦C leaf temperature, 360 μM/mol CO2 concentration, and 800 μM/m2/s photosynthetic photon-flux density).

The method of Arnon [35] was followed to measure chlorophyll and carotenoid content. Concisely, slicing of leaf samples (0.1 g) was done, and sliced samples were stored in a dark place after placement in glass test tubes and addition of acetone (80%) per tube. The samples were stored in the dark until they were completely discolored, followed by the centrifugation of sample extract. Finally, Lambda 25 UV/VIS Spectrophotometer was employed to calculate chlorophyll a (Chl a), Chlorophyll b (Chl b), and carotenoid (Caro) via measuring absorbance at 645, 663, and 440 nm, respectively.

#### *2.5. Root Morphology and Root Activity*

After two-weeks of DR-stress, roots of three plants were taken and washed using tap water, to study root morphological traits. Scanning of roots was done using a root scanner, followed by evaluating the scanned images using a root image analysis software (Epson Expression 11000XL, Regent Instruments, Québec, QC, Canada), to obtain the root morphological parameters [36].

The TTC (triphenyl tetrazolium chloride) method, as explained by Comas et al. [37], was followed to estimate root activity (capacity of root deoxidization measured in mg g−<sup>1</sup> h−<sup>1</sup> FW). Briefly, root samples (500 mg) were dipped in 10 mL of a mixed solution of phosphate buffer and TTC (0.4%), which is mixed in a 1:1 ratio. After that, the sample was placed in the dark for one to three hours (37 ◦C), followed by the addition of 2 mL of H2SO4 (1 mol L−1) to avoid the reaction of the solution. Finally, dipped roots were blended and transferred into 10 mL of ethyl acetate cleaning solution in a tube, and the spectrophotometer was used to record solution' absorption at 485 nm.

#### *2.6. Leaf Relative Water Content and Soluble Sugar*

According to the method of Barrs and Weatherley [38], the following formula was used to calculate leaves' *RWC* (relative water content) (Equation (1)):

$$R\text{WC } (\%) = \left[ \frac{FW - DW}{SW - DW} \right] \times 100 \tag{1}$$

where *FW* = fresh weight; *DW* = dry weight; *SW* = saturated weight in water.

The anthrone method of Shi et al. [39] was employed to perform soluble sugar content. In brief, 80% (*V*/*V*) ethanol (2 mL) was added to 0.1 g sample at 80 ◦C, and kept for 30 min. Then, anthrone (2 mL) was mixed to extract (100 μL), followed by boiling for 10 min. Finally, after recording the absorption (at 630 nm), the calibration curve of the sucrose standard was used to calculate the soluble sugar content.

#### *2.7. Oxidative Stress Markers, and Proline*

For calculating H2O2, O2 •−, MDA, and proline contents, liquid nitrogen was used to crush frozen sample (0.1 g) into powder form, followed by isolation using 100 mM phosphate buffer (900 μL, pH 7.4) and guidelines labelled in kits (Nanjing Jiancheng Bioengineering Institute, located in Nanjing, China) of H2O2 (A064), O2 •− (A052), MDA (A003-3), and proline (A107-1), at 405, 550, 530, and 520 nm, respectively. Leaves' EL was measured by following the method of Zhang et al. [40].

#### *2.8. Antioxidant Enzymes Activity*

To examine the antioxidant enzyme activities, powdered leaf sample (0.5 g) was homogenized using 100 mM phosphate buffer (900 μL, pH 7.4) by following kits' description. After that, centrifugation (at 4 ◦C temperature, 12,000× *g* revolutions for 15 min) of homogenized samples was done, and to analyze enzymatic activities, the supernatant was shifted to new falcon tube. The guidelines mentioned in kits (A001-1, A007-1, A123-1, A062-1, A004, A084-3-1, BC0660, and BC0650) were followed to note the activities of SOD, CAT, APX, GR, GST, POD, DHAR, and MDHAR enzymes, at 550, 405, 290, 340, 412, 420, 412, and 340 nm wavelengths, respectively.

The method of Logan et al. [41] was followed to assess the levels of ascorbate (AsA) and oxidized ascorbate (DHA). The procedures of Griffith [42] were followed to measure reduced glutathione (GSH) and oxidized glutathione (GSSG) content.

#### *2.9. Quantitative Real-Time PCR*

Following the manufacturer's instructions, Trizole reagent was used to extract total RNA from individual treatment leaf samples. With the help of agarose gel electrophoresis, a NanoPhotometer® spectrophotometer (Implen, Westlake Village, CA, USA), the extracted RNA' purity and quality were examined. The manufacturer protocol of Vazyme HiScript II Q RT SuperMix for qPCR (+gDNA wiper) 1st strand cDNA synthesis kit (Vazyme, Nanjing, China) was followed to reverse-transcribe the extracted RNA, for complementary DNA (cDNA) synthesis. For qRT-PCR (quantitative real-time PCR) analysis, cDNA was used as templates. The Roche FastStart Essential DNA Green Master kit (Roche, Pleasanton, CA, USA) was employed in an Mx3000 P qPCR system (Agilent Technologies, Santa Clara, CA, USA), and 96-well plates were used to perform qRT-PCR. Supplementary Table S1 provides the detail of primers followed in this study Jahan et al. [43], and Actin was used as a reference gene. The formula of Livak and Schmittgen [44], i.e., 2−ΔΔCt was used to calculate the changes in relative gene expression. Three biological replications were performed for each treatment, and three technical replicates were carried out for each biological replicate.

#### *2.10. Statistical Analysis*

The statistical package SPSS version 22.0 (IBM Corporation, Armonk, NY, USA) was used for the statistical analysis of data. One-way analysis of variance (ANOVA) was completed, and the treatment means were compared using the LSD (least significant difference) test (at *p* ≤ 0.05).
