*2.5. Damage Index; Determination of MDA and H2O<sup>2</sup> Contents*

MDA content estimation was done following the Stewart and Bewly method [33] to determine lipid peroxidation. Leaf samples (1 g) were homogenized in 0.1% of trichloroacetic acid (TCA) and then centrifuged at 12,000 rpm for 15 min at 4 ◦C. After centrifugation, 0.5 mL of the supernatant was collected and mixed with a 1 mL volume of 20% TCA containing a 0.5% thiobarbituric acid (TBA) solution. The sample was incubated for another 30 min at 95 ◦C and placed in ice bath to stop the reaction followed by centrifuging at 12,000 rpm for 10 min. The resulting solute was measured for absorbance at 532 nm. MDA content was estimated by the 155 mM−<sup>1</sup> cm−<sup>1</sup> extinction coefficient. Results were stated as µmol/g fresh weight. The H2O<sup>2</sup> content was determined following the method previously elucidated by Loreto and Velikova [34]. Leaf samples (1 g) were homogenized in ice-cold 0.1% TCA and centrifuged at 12,000 rpm for 15 min at 4 ◦C. After that, 0.5 mL of 10 mM potassium phosphate buffer and 0.75 mL of 1 M KI were added to 0.5 mL of the supernatant. The absorbance was measured at 390 nm against a blank, and the H2O<sup>2</sup> content was inferred by a standard calibration curve, previously made solutions with known H2O<sup>2</sup> concentration. H2O<sup>2</sup> concentration was expressed as µmol/g fresh weight.

#### *2.6. Assay of Antioxidant Enzymes*

Approximately 1 g of sampled leaves were weighed and finely chopped to powder with liquid nitrogen. About 10 mL of ice-cold 50 mM potassium phosphate buffer with 0.1-mM Na<sup>2</sup> EDTA and 1% (*w*/*v*) polyvinylpyrrolidone (PVP) was used to homogenize the powder. The pH of the buffer should be 7.8 for SOD and POD assays whereas it is 7.0 for CAT assay. After filtering the homogenate with a 4-layered muslin cloth, it was centrifuged at 12,000 rpm at 4 ◦C for 15 min. The aliquot part in the supernatant was gathered for enzyme activity assays. For recording the SOD activity, the protocol of Madamanchi and Alscher [35] was followed. The amount of enzyme that reduces nitroblue tetrazolium (NBT) to half was referred to be one unit of SOD. The solute was read at an absorbance of 560 nm. As guaiacol was the electron donor, the POD activity was recorded at 470 nm as proposed by Chance and Maehly [36] in which one unit POD function was referred to as one unit change in absorbance of 0.01 unit in a minute. As described by Aebi [37], the activity of CAT was found. In an interval of 2 min, a reduction in the absorbance at 240 nm was recorded after the digestion of H2O2. It is found that one unit of CAT produces 1 mM of H2O<sup>2</sup> in a minute for which the results are expressed in units/mg of protein.

#### *2.7. Ascorbic Acid Content*

Ascorbic acid (referred to as vitamin C) was measured following the method previously reported by Arakawa et al. [38] with minor modifications. Leaf samples and 5 mL of 5% trichloroacetic acid (TCA) were homogenized in the mortar and centrifuged at 10,000 rpm for 10 min at 4 ◦C. Then, 1 mL of clear supernatant, 1 mL of 5% TC, 1 mL alcohol, 0.5 mL 0.4% phosphoric acid (H3PO4)-alcohol, 1 mL of 0.5% 4,7-diphenyl-1,10 phenanthroline (BP)-alcohol, and 0.5 mL 0.03% ferric trichloride (FeCl3)-alcohol were added into to a tube and incubated at 40 ◦C for 1 h. The reaction was ended at room temperature, and absorbance was measured at 534 nm with a spectrophotometer (Unicam UV-330, Cambridge, UK). Results were expressed as the unit's µmol/g fresh weight.

#### *2.8. RNA Isolation, cDNA Synthesis, and qRT-PCR Analysis*

According to the manufacturer's guidelines, total RNA was isolated using an RNeasy plant mini kit (Qiagen, Hilden, Germany) and treated with RNase-free DNAseI (Promega, Madison, WI, USA). The RNA quantity was assessed using the bio spectrometer (Eppen-

dorf, Hamburg, Germany) based on the absorbance ratio at 280 nm. Further, the quality of RNA was tested on 1% agarose gel via electrophoresis. The first-strand cDNA was done by transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, Penzberg, Germany) following the manufacturer's guidelines. Sequence information of the following genes, photosystem II oxygen-evolving complex protein (*PS II-PsbP*), photosystem II chlorophyll A/B binding protein (*PS II-LHC*), photosystem I PsaG/PsaK (*PS I-PsaG/PsaK*), phosphoenolpyruvate carboxylase 3 (*PEPC 3*), superoxide dismutase 2 (*SOD 2*), peroxidase (*POD*), catalase-2 (*CAT 2*), heat shock protein-90 (HSP-90), dehydration responsive elementbinding transcription factor (*DREB2C*), NAC transcription factor 3 (*NAC 3*), and abscisic acid-responsive elements-binding factor 2 (*ABF 2*) were obtained from NCBI database [https://www.ncbi.nlm.nih.gov/ (accessed on 29 May 2019); *Vigna radiata* var. radiata (Mungbean)]. Primer 5.0 software was used to design the corresponding primer pairs of the concerned gene sequences for qRT-PCR reaction (Table S1) and were verified to produce a single peak in the melting curve by using a Light Cycler 480® Real-Time PCR System (Roche Applied Science, Penzberg, Germany). Aliquots for qRT-PCR reactions included 10 µL of final volumes containing 1 µL of cDNA, 0.5 µL each primer (10 µM), and 5 µL (2×) of FastStart Essential DNA Green Master mix (Roche Applied Science, Penzberg, Germany) and 3 µL of ddH2O. All reactions were performed in 96-well plates using a Light Cycler 480® Real-Time PCR System with three technical replicates. The thermal conditions are as follows: 95 ◦C for 5 min, followed by 40 cycles of 95 ◦C for 10 s, 60 ◦C for 30 s, and then 72 ◦C for 30 s. Actin gene (internal control) from mungbean was used to normalize, and transcripts change was calculated using the 2−∆∆CT method.

## *2.9. Statistical Analysis*

The experimental data are presented as the mean and standard error of the mean. All statistical analyses were performed using SPSS statistical package (SPSS Inc., Chicago, IL, USA). In order to find out the differences among the groups, Duncan's multiple range test for one-way ANOVA was performed at a *p*-value < 0.05 statistical significance.

#### **3. Results**

#### *3.1. The Effect of Drought Stress on Physiological Traits*

Chlorophyll content, plant dry mass, and RWC were observed in two mungbean cultivars following the 12 days of drought stress and compared with the control (Figure 1). After 12 days under drought stress, CO6 plants showed severe wilting, whereas a few leaves of the VRM (Gg) 1 plants had slowly begun to curl. Notably, considerable reductions in chlorophyll content and plant dry mass were observed in the CO6 compared to the respective control. Next, we determined the RWC at the control and drought stress conditions. Mungbean cultivar VRM (Gg) 1 did not show any considerable changes in RWC when subjected to drought stress. In contrast, RWC had a considerable decrease in the CO6 after 12 days of drought stress.

During the 12 days of drought stress, photosynthetic gas exchange parameters (leaf net photosynthesis rate, stomatal conductance, and intercellular CO<sup>2</sup> concentration) were determined in both cultivars (Figure 1). After 6 days of the drought stress, no major difference in the leaf net photosynthetic rate among VRM (Gg) 1 and CO6 was observed compared to their respective control. However, after 12 days of drought stress, the reduction in CO6 was higher than observed in VRM (Gg) 1. The differences observed in stomatal conductance after drought stress were also similar to the differences seen in leaf net photosynthetic rate, with the same trend as in leaf net photosynthetic rate but to a smaller extent. However, after drought stress, CO6 plants showed an increased intercellular CO2 concentration than in VRM (Gg) 1 revealing the opposite relationship between stomatal conductance and leaf net photosynthetic rate.

conductance and leaf net photosynthetic rate.

**Figure 1.** (**A**) Chlorophyll content (mg g−1 FW), (**B**) relative water content (%), (**C**) stomatal conductance (mol H2O m−2 s −1), (**D**) photosynthesis rate (µmol CO<sup>2</sup> m−2 s −1), and (**E**) intercellular CO<sup>2</sup> concentration (ppm) of two mungbean cultivars (VRM (Gg) 1 and CO6) grown under control and droughtstressed conditions. Values followed by the same letter are not significantly different (*p* ≤ 0.05) according to duncan's multiple range test. Bars present means ± SE (*n* = 3). **Figure 1.** (**A**) Chlorophyll content (mg g−<sup>1</sup> FW), (**B**) relative water content (%), (**C**) stomatal conductance (mol H2O m−<sup>2</sup> s −1 ), (**D**) photosynthesis rate (µmol CO<sup>2</sup> m−<sup>2</sup> s −1 ), and (**E**) intercellular CO<sup>2</sup> concentration (ppm) of two mungbean cultivars (VRM (Gg) 1 and CO6) grown under control and drought-stressed conditions. Values followed by the same letter are not significantly different (*p* ≤ 0.05) according to duncan's multiple range test. Bars present means ± SE (*n* = 3).

compared to their respective control. However, after 12 days of drought stress, the reduction in CO6 was higher than observed in VRM (Gg) 1. The differences observed in stomatal conductance after drought stress were also similar to the differences seen in leaf net photosynthetic rate, with the same trend as in leaf net photosynthetic rate but to a smaller extent. However, after drought stress, CO6 plants showed an increased intercellular CO2 concentration than in VRM (Gg) 1 revealing the opposite relationship between stomatal

#### *3.2. Proline Content 3.2. Proline Content*

Proline accumulation is an eminent metabolic response of plants to drought and is also used as an indicator to determine drought tolerance. The difference in proline content during the 12 days of drought stress was estimated in both cultivars. Under the control conditions, a slight difference was seen in the proline content of both cultivars. VRM (Gg) 1 showed a considerably high amount of proline than CO6 after 6 and 12 days of drought stress. The proline content of VRM (Gg) 1 and CO6 under drought stress are presented in Figure 2. Proline accumulation is an eminent metabolic response of plants to drought and is also used as an indicator to determine drought tolerance. The difference in proline content during the 12 days of drought stress was estimated in both cultivars. Under the control conditions, a slight difference was seen in the proline content of both cultivars. VRM (Gg) 1 showed a considerably high amount of proline than CO6 after 6 and 12 days of drought stress. The proline content of VRM (Gg) 1 and CO6 under drought stress are presented in Figure 2.
