*3.3. Changes in MDA and H2O<sup>2</sup> Accumulation*

During the 12 days of drought stress, MDA, a product of lipid peroxidation was detected among two cultivars. Compared to their respective controls, the amount of MDA content was increased in both cultivars under stress. However, the amounts of upsurge were the degree of difference. After 12 days of stress, the content of MDA was considerably increased in CO6 (119%) than in VRM (Gg) 1 (49%), further revealing that the VRM (Gg) 1 plants cope with a smaller amount of membrane damage compared to CO6 (Figure 2). Likewise, the accumulation level of H2O<sup>2</sup> in plants as a response to drought stress imposed on VRM (Gg) 1 and CO6 was also estimated (Figure 2). Six days after drought stress, no major changes in H2O<sup>2</sup> content were obtained in both cultivars under stress and control conditions. However, after 12 days, H2O<sup>2</sup> levels exhibited a significant increase between both types of plants compared with respective controls. Notably, the magnitudes of increase were different in CO6 (139%) and VRM (Gg) 1 (51.75%). Under control conditions, both cultivars showed no major difference in MDA and H2O<sup>2</sup> (Figure 2).

#### *3.4. Activity of Enzymatic and Non-Enzymatic Antioxidants*

The effect of drought on enzymatic antioxidants viz., SOD, POD, and CAT were evaluated on the mungbean cultivars (Figure 3). After 12 days of drought stress, the three antioxidant enzyme activities were higher in both cultivars. No major changes in SOD activity were found 6 days after drought stress in the VRM (Gg) 1 and CO6 compared to their respective control. However, a significant increase was found in 12 days after drought stress in both cultivars. The increase in VRM (Gg) 1 was high compared to CO6. Unlike

SOD, POD and CAT activities in the VRM (Gg) 1 and CO6 significantly increased 6 days after drought stress and maintained a higher level after 12 days of drought stress compared to their respective controls. However, the increase in VRM (Gg) 1 was high compared to CO6. Ascorbic acid is one of the most abundant water-soluble antioxidant compounds in plants. The response of ascorbic acid content for drought stress in VRM (Gg) 1 and CO6 was estimated and is presented in Figure 3. The ascorbic acid content was higher in the VRM (Gg) 1 than the CO6. After 12 days of drought stress, the ascorbic acid content was slightly increased in the VRM (Gg) 1. No significant changes were seen in the CO6 than their respective controls. *Horticulturae* **2022**, *8*, x FOR PEER REVIEW 7 of 13

**Figure 2.** (**A**) Effect of drought stress on proline (µg g−1 FW), (**B**) H2O<sup>2</sup> (µmol g−1 FW)**,** and (**C**) MDA contents (µmol g−1 FW) in two mungbean cultivars VRM (Gg) 1 and CO6. 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 2.** (**A**) Effect of drought stress on proline (µg g−<sup>1</sup> FW), (**B**) H2O<sup>2</sup> (µmol g−<sup>1</sup> FW)**,** and (**C**) MDA contents (µmol g−<sup>1</sup> FW) in two mungbean cultivars VRM (Gg) 1 and CO6. 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).

#### *3.3. Changes in MDA and H2O<sup>2</sup> Accumulation 3.5. Transcriptional Profiling of Photosynthesis, Antioxidant, and Stress-Responsive Genes*

During the 12 days of drought stress, MDA, a product of lipid peroxidation was detected among two cultivars. Compared to their respective controls, the amount of MDA content was increased in both cultivars under stress. However, the amounts of upsurge were the degree of difference. After 12 days of stress, the content of MDA was considerably increased in CO6 (119%) than in VRM (Gg) 1 (49%), further revealing that the VRM (Gg) 1 plants cope with a smaller amount of membrane damage compared to CO6 (Figure 2). Likewise, the accumulation level of H2O<sup>2</sup> in plants as a response to drought stress imposed on VRM (Gg) 1 and CO6 was also estimated (Figure 2). Six days after drought stress, no major changes in H2O<sup>2</sup> content were obtained in both cultivars under stress and control conditions. However, after 12 days, H2O<sup>2</sup> levels exhibited a significant increase between both types of plants compared with respective controls. Notably, the magnitudes of increase were different in CO6 (139%) and VRM (Gg) 1 (51.75%). Under control conditions, both cultivars showed no major difference in MDA and H2O<sup>2</sup> (Figure 2). After 12 days of drought stress, four photosynthesis-related genes (*PS II-PsbP, PS II-LHCB, PS I-PsaG/PsaK*, and *PEPC 3*) transcripts levels were analyzed between VRM (Gg) 1 and CO6 by qRT-PCR analysis (Figure 4). Under control conditions, the transcripts of photosynthesis-related genes in VRM (Gg) 1 and CO6 showed no major differences. However, after 12 days of drought stress, transcripts level increased in both cultivars compared with respective controls. Noticeably, the transcripts level was higher in VRM (Gg) 1 than CO6. Further, we analyzed the three antioxidants (*SOD 2*, *POD*, and *CAT 2*) and four drought stress-responsive genes (*HSP-90, DREB2C, NAC 3*, and *AREB 2*) in both cultivars. Like photosynthesis genes, transcripts of antioxidant and drought stress-responsive genes also did not exhibit major differences in VRM (Gg) 1 and CO6 under controlled conditions. However, the transcripts levels were increased in VRM (Gg) 1 than CO6 after 12 days of drought stress. Together, these results suggest that VRM (Gg) 1 exhibited better performance under drought stress compared to CO6, owing to the transcripts differences of these genes under drought stress.

The effect of drought on enzymatic antioxidants viz., SOD, POD, and CAT were evaluated on the mungbean cultivars (Figure 3). After 12 days of drought stress, the three

activity were found 6 days after drought stress in the VRM (Gg) 1 and CO6 compared to their respective control. However, a significant increase was found in 12 days after drought stress in both cultivars. The increase in VRM (Gg) 1 was high compared to CO6. Unlike SOD, POD and CAT activities in the VRM (Gg) 1 and CO6 significantly increased 6 days after drought stress and maintained a higher level after 12 days of drought stress compared to their respective controls. However, the increase in VRM (Gg) 1 was high

*3.4. Activity of Enzymatic and Non-Enzymatic Antioxidants*

CO6 than their respective controls.

**Figure 3.** Activities of enzymatic and non-enzymatic antioxidants in two mungbean cultivars VRM (Gg) 1 and CO6. (**A**) SOD (Umg−1 Protein), (**B**) POD (Umg−1 Protein), (**C**) CAT (Umg−1 Protein), and (**D**) ascorbic acid (µmolg−1 FW). 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 3.** Activities of enzymatic and non-enzymatic antioxidants in two mungbean cultivars VRM (Gg) 1 and CO6. (**A**) SOD (Umg−<sup>1</sup> Protein), (**B**) POD (Umg−<sup>1</sup> Protein), (**C**) CAT (Umg−<sup>1</sup> Protein), and (**D**) ascorbic acid (µmolg−<sup>1</sup> FW). 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). *Horticulturae* **2022**, *8*, x FOR PEER REVIEW 9 of 13

compared to CO6. Ascorbic acid is one of the most abundant water-soluble antioxidant compounds in plants. The response of ascorbic acid content for drought stress in VRM (Gg) 1 and CO6 was estimated and is presented in figure 3. The ascorbic acid content was higher in the VRM (Gg) 1 than the CO6. After 12 days of drought stress, the ascorbic acid content was slightly increased in the VRM (Gg) 1. No significant changes were seen in the

**Figure 4.** Effect of drought stress on the relative expression level of photosynthesis (*PS II-PsbP, PS II-LHCB, PS I-PsaG/PsaK,* and *PEPC 3*), antioxidants (*SOD 2, POD,* and *CAT 2*), and stress (*HSP-90, DREB2C, NAC 3* and *AREB 2*) related genes in two mungbean cultivars VRM (Gg) 1 and CO6. Values investigated the transcripts level differences of photosynthesis-related genes under **Figure 4.** Effect of drought stress on the relative expression level of photosynthesis (*PS II-PsbP, PS II-LHCB, PS I-PsaG/PsaK,* and *PEPC 3*), antioxidants (*SOD 2, POD,* and *CAT 2*), and stress (*HSP-90,*

followed by the same letter are not significantly different (*p* ≤ 0.05) according to duncan's multiple

In the present study, the response of mungbean cultivars to drought stress was investigated in terms of analyzing the physio-biochemical and transcriptional changes. We found that chlorophyll content and plant dry mass were decreased during drought stress, and the cultivar VRM (Gg) 1 showed a lower decrease compared to CO6, indicating improved photosynthesis and plant growth development. Additionally, VRM (Gg) 1 subjected to drought stress did not show any major changes in RWC. However, the RWC had a significant decrease in CO6, suggesting better water maintaining capacity in VRM (Gg) 1. We also found that when VRM (Gg) 1 and CO6 were exposed to drought stress, the photosynthetic gas exchange parameters (leaf net photosynthetic rate and stomatal conductance) were decreased. Notably, the stomatal conductance in VRM (Gg) 1 plants showed lower decreases than that in CO6 under drought stress. This changing pattern in stomatal conductance is comparable to that in leaf net photosynthetic rate among the plants of VRM (Gg) 1 and CO6, showing that the better leaf net photosynthetic rate in VRM (Gg) 1 was related to the regulation of stomatal conductance. In contrast, the intercellular CO2 concentration of VRM (Gg) 1 was lower than CO6 plants. This fact was because of the varied reduction of leaf net photosynthetic rate in VRM (Gg) 1 and CO6. It might be the reason for increased CO<sup>2</sup> assimilation and decreased intercellular CO<sup>2</sup> concentration in the VRM (Gg) 1 plants compared to CO6. Therefore, photosynthesis and growth in the VRM (Gg) 1 were better when imposed the drought stress. Moreover, we

range test. Bars present means ± SE (*n* = 3).

**4. Discussion**

*DREB2C, NAC 3* and *AREB 2*) related genes in two mungbean cultivars VRM (Gg) 1 and CO6. 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).

#### **4. Discussion**

In the present study, the response of mungbean cultivars to drought stress was investigated in terms of analyzing the physio-biochemical and transcriptional changes. We found that chlorophyll content and plant dry mass were decreased during drought stress, and the cultivar VRM (Gg) 1 showed a lower decrease compared to CO6, indicating improved photosynthesis and plant growth development. Additionally, VRM (Gg) 1 subjected to drought stress did not show any major changes in RWC. However, the RWC had a significant decrease in CO6, suggesting better water maintaining capacity in VRM (Gg) 1. We also found that when VRM (Gg) 1 and CO6 were exposed to drought stress, the photosynthetic gas exchange parameters (leaf net photosynthetic rate and stomatal conductance) were decreased. Notably, the stomatal conductance in VRM (Gg) 1 plants showed lower decreases than that in CO6 under drought stress. This changing pattern in stomatal conductance is comparable to that in leaf net photosynthetic rate among the plants of VRM (Gg) 1 and CO6, showing that the better leaf net photosynthetic rate in VRM (Gg) 1 was related to the regulation of stomatal conductance. In contrast, the intercellular CO2 concentration of VRM (Gg) 1 was lower than CO6 plants. This fact was because of the varied reduction of leaf net photosynthetic rate in VRM (Gg) 1 and CO6. It might be the reason for increased CO<sup>2</sup> assimilation and decreased intercellular CO<sup>2</sup> concentration in the VRM (Gg) 1 plants compared to CO6. Therefore, photosynthesis and growth in the VRM (Gg) 1 were better when imposed the drought stress. Moreover, we investigated the transcripts level differences of photosynthesis-related genes under drought stress. *PS II-PsbP, PS II-LHCB, PS I-PsaG/PsaK*, and *PEPC 3* are major genes related to photosynthesis. In our study, following 12 days of drought stress, the transcripts levels of all the genes excluding *PS I-PsaG/PsaK* considerably increased in both cultivars compared to their control. Notably, the transcripts level in VRM (Gg) 1 was high compared to CO6, suggesting that VRM (Gg) 1 had better photosynthetic capacity than CO6 under drought stress. Proline accumulation is an important metabolic response to drought in plants and it is also employed as an indicator to regulate the drought tolerance. After 6 and 12 days of drought stress, VRM (Gg) 1 had a much higher level of proline than CO6. Collectively, these results are in line with the reports of Li et al. [39]. Ansari et al. [40] Favero Peixoto-Junior et al. [41], who described that the genotype is referred to as tolerant to drought stress if it keeps better photosynthetic performance, chlorophyll content, RWC, plant dry mass, and proline under stress conditions.

Many studies showed that drought stress causes oxidative damage, characterized as an accumulation of H2O<sup>2</sup> and MDA [42,43]. Our results showed that, after the drought stress, the accumulation of H2O<sup>2</sup> and MDA was low in VRM (Gg) 1 whereas high in CO6. A lower H2O<sup>2</sup> and MDA content in VRM (Gg) 1 specified that it has stable ROS scavenging and better protective mechanism. In several crops, including mungbean, wheat, and muskmelon [28,40,44] under drought stress, the genotypes with contrasting drought tolerance showed differences in H2O<sup>2</sup> and MDA content. Additionally, a higher accumulation of proline in VRM (Gg) 1 was vital, and it acts as a compatible solute that prevents the protein and membrane structure while also scavenging ROS to maintain the cellular redox level under drought stress and agrees with the statement of Yamada et al. [45]. Plants with tolerance to abiotic stress possess a robust antioxidant system to defend them from oxidative stress by keeping increased antioxidant enzymes and antioxidant molecule activity and contents under stress conditions [46]. SOD, POD, and CAT are major enzymes protecting the plants against ROS-induced oxidative damage [14,47]. Many research reports detailed that the up-regulated expression of SOD, POD, and CAT leads to decreased ROS production under stress conditions [44,48]. In our study, SOD, POD, and CAT activities

were heightened over time in VRM (Gg) 1 compared with the CO6 under drought stress and corroborate with the low ROS production observed in VRM (Gg) 1. Abid et al. [44] and Ali et al. [28] showed SOD, POD, and CAT activities were higher in VRM (Gg) 1 than CO6 under drought stress which was in concurrence with our findings. Likewise, the accumulation of non-enzymatic antioxidant ascorbic acid was higher in VRM (Gg) 1 than in CO6. However, the increase was non-significant, and only a marginal increase was observed. Taking together, we conclude that VRM (Gg) 1 has a stronger antioxidant system than CO6.

Drought stress regulates the expression of genes in plants at both transcriptional and post-transcriptional levels. Drought tolerance in plants is thought to be mediated by many genes and biological pathways. Heat-shock proteins serve as molecular chaperones for various client proteins in abiotic stress response and play a significant role in preventing the plants against abiotic stresses. The plant's *HSP90* genes had a major role in response to abiotic stresses, including drought [49,50]. Song et al. [51] reported that the overexpression of *Hsp90* in *Arabidopsis thaliana* improved the plant's sensitivity to drought stresses. VRM (Gg) 1 exhibited higher expression of *Hsp90* in leaves than CO6 during drought stress, suggesting the possible role of preventing the cells from oxidative damage in VRM (Gg) 1 plants. *DREB* is the key transcription factor playing a pivotal role in drought stress response and tolerance to drought [52–55]. In the present study, the *DREB2C* transcription factor was examined in VRM (Gg) 1 and CO6 under drought stress. After ten days of drought stress, the expression level of *DREB2C* in leaves was expressed considerably higher in VRM (Gg) 1 than in CO6. Similarly, *NAC 3* and *AREB 2*, which play critical roles during various abiotic stresses, also showed higher expression in VRM (Gg) 1 compared to CO6. Previously, several studies demonstrated the possible involvement of *NAC 3* and *AREB 2* in drought tolerance [56,57]. From these outcomes, we inferred that it might be possible that the higher expression of *Hsp90, DREB1, NAC 3,* and *AREB 2* is likely to contribute to the better performance of VRM (Gg) 1 during drought stress.

VRM (Gg) 1 had better photosynthetic activity during drought stress, resulting in fewer losses in chlorophyll, relative water content, and plant dry mass. Furthermore, enhanced antioxidative enzyme activities resulted in decreased H2O<sup>2</sup> and MDA levels in VRM (Gg) 1, limiting oxidative damage. These physio-biochemical alterations positively correlated with increased transcripts of photosynthesis and antioxidant-related genes in VRM (Gg) 1 and were consistent with the earlier studies on, mungbean, faba bean, and alfalfa responses to drought stress [58–60]. The increased transcripts of drought-responsive genes suggest that VRM (Gg) 1 has a stronger genetic base for drought tolerance than CO6. However, supplement research is needed to understand the exact genetic and molecular mechanism underlying drought tolerance.

#### **5. Conclusions**

In this study, we found that the mungbean cultivar VRM (Gg) 1 performed well and exhibited tolerance to drought stress compared to CO6, as supported by the physiobiochemical and gene transcriptional changes. In the future, VRM (Gg) 1 will need to be tested under field conditions before being employed in mungbean drought-tolerant breeding programs. VRM (Gg) 1 is a potential source to detect the quantitative trait locus (QTL)/gene (s) associated with drought tolerance. Collectively, the obtained results from our study could be used in the future search for drought-tolerant genotypes or in breeding programs with an aim to obtain tolerant mungbean genotypes.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/horticulturae8050424/s1. Table S1: Details of primers used for quantitative real-time PCR (qRT-PCR) analysis. Reference [61] is cited in the supplementary materials.

**Author Contributions:** Conceptualization, A.K., N.S. and M.P.; methodology, A.K., V.G.R. and G.A.; formal analysis, S.P., V.M. and M.D.; investigation, G.A., A.K., A.A. and I.M.; resources, M.P. and N.S.; data curation, G.A., M.A. and A.K.; writing—review and editing, G.A. and A.K.; supervision, M.P. and N.S.; funding acquisition, A.K. and N.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** A.K. and N.S. acknowledged the support of the Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Government of India for the DST-SERB NPDF fellowship program (PDF/2016/003676).

**Acknowledgments:** All the authors wish to acknowledge NationalAgricultural Development Programme (NADP)/Rashtriya KrishiVikas Yojana (RKVY)—Government of Tamil Nadu, and Centre of Innovation (CI), Agricultural Collegeand Research Institute, Tamil Nadu Agricultural University, Madurai, for providing instrumentation facilities.

**Conflicts of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflicts of interest.

#### **References**

