*3.3. Ethanol Improved Photosynthetic Pigment Contents in Soybean Plants Subjected to Drought Stress*

The contents of different photosynthetic pigments (e.g., Chl *a*, *b*, total Chls, and carotenoids) were determined to examine whether ethanol improves these parameters under drought circumstances (Figure 3A–E). We found that drought stress caused a significant reduction of Chl *a*, *b*, total Chls, and carotenoid levels in 'D' plants in comparison with 'WW' plants (Figure 3A–E). Contrarily, a substantial improvement in the amounts of Chl *a*, *b*, total Chls, and carotenoids were observed in 'Eth + D' plants when equated to those in 'D' plants (Figure 3A–E). In comparison with 'WW' plants, exogenous ethanol also significantly augmented the levels of total Chls, Chl *a*, Chl *b*, and carotenoids in 'Eth' plants (Figure 3A–E).

**Figure 3.** Effect of exogenously supplied 20-mM ethanol on (**A**) Chl *a*, (**B**) Chl *b*, (**C**) total Chls, and (**D**) carotenoid contents in the trifoliate leaves of soybean plants subjected to drought stress for a period of 8 days. (**E**) Heatmap of the fold-change values of the aforementioned parameters in soybean plants under different treatments. Bars represent means with standard errors (*n* = 4). Different letters shown above the bars are used to indicate significant differences among the treatments (*p* < 0.05). WW, Eth, D, and Eth + D indicate water-sprayed well-watered plants, ethanol-sprayed well-watered plants, water-sprayed drought-exposed plants, and ethanol-sprayed drought-exposed plants, respectively. Chl, chlorophyll; Cars, carotenoids; FW, fresh weight.

#### *3.4. Ethanol Protected Soybean Plants from Drought-Induced Oxidative Stress*

To explore the role of ethanol in alleviating drought stress-mediated oxidative stress, we examined ROS generation in soybean leaves by executing histochemical staining of O2 •− and H2O2, as well as quantifying the levels of H2O2, MDA, and EL (Figure 4A–F). In comparison with 'WW' plants, drought stress led to a significant accumulation of O2 •− and H2O2 in 'D' plants, as evidenced by scattered but conspicuous dark-blue spots (O2 •−) and deep dark-brown polymerization patches (H2O2) (Figure 4A,B). In line with these results, 'D' plants also displayed significantly higher levels of H2O2, MDA, and EL percentage than 'WW' plants (Figure 4C–F). By comparison, 'Eth + D' plants exhibited substantially lower accumulation of ROS, as well as lower levels of H2O2, MDA, and EL percentage than 'D' plants (Figure 4A–F). 'Eth' plants also displayed reduced accumulation of ROS compared with 'WW' plants (Figure 4A–C,F). Nonetheless, comparable MDA levels and EL percentages were observed between 'Eth' and 'WW' plants (Figure 4D–F).

**Figure 4.** Effect of exogenously supplied 20-mM ethanol on ROS accumulation, and the contents

of hydrogen peroxide (H2O2), malondialdehyde (MDA), and electrolyte leakage in the leaves of soybean plants subjected to drought stress for a period of 8 days. (**A**) Nitroblue tetrazolium (NBT) staining for detection of superoxide (O2 •−) and (**B**) diaminobenzidine (DAB)-staining for detection of H2O2. Estimated levels of (**C**) H2O2, (**D**) MDA, and (**E**) electrolyte leakage in the leaves of soybean plants. (**F**) Heatmap of the fold-change values of the aforementioned parameters in soybean plants under different treatments. Bars represent means with standard errors (*n* = 4). Different letters shown above the bars are used to indicate significant differences among the treatments (*p* < 0.05). WW, Eth, D, and Eth + D indicate water-sprayed well-watered plants, ethanol-sprayed well-watered plants, water-sprayed drought-exposed plants, and ethanol-sprayed drought-exposed plants, respectively. EL, electrolyte leakage; FW, fresh weight; ROS, reactive oxygen species.

#### *3.5. Ethanol Improved Antioxidant Defense in Soybean Plants Subjected to Drought Stress*

Next, we further examined the activities of some important antioxidant enzymes to evaluate the involvement of ethanol in improving antioxidant defense to alleviate oxidative stress (Figure 5A–F). We found that drought significantly reduced the CAT activity while enhancing the activities of GST and POD, and the number of total flavonoids in 'D' plants in comparison with 'WW' plants (Figure 5A,C–F). However, we did not observe any significant differences in the activity of APX between 'D' and 'WW' plants (Figure 5B,F). On the other hand, ethanol supplementation substantially increased the activities of CAT, APX, GST, and POD, and the contents of total flavonoids in 'Eth + D' plants in relation to 'D' plants (Figure 5A–F). Notably, the activities of CAT, APX, GST, and POD were remarkably improved in 'Eth' plants relative to 'WW' plants; however, the amounts of flavonoids did not significantly differ between 'Eth' and 'WW' plants (Figure 5A–F).

**Figure 5.** Effect of exogenously supplied 20-mM ethanol on antioxidant defense responses in the leaves of soybean plants subjected to drought stress for a period of 8 days. Activities of (**A**) CAT (catalase), (**B**) APX (ascorbate peroxidase), (**C**) GST (glutathione *S*-transferase), and (**D**) POD (peroxidase) and the content of (**E**) total flavonoids in soybean leaves under different treatment conditions. (**F**) Heatmap of the fold-change values of the aforementioned parameters in soybean plants under different treatments. Bars represent means with standard errors (*n* = 4). Different letters shown above the bars are used to indicate significant differences among the treatments (*p* < 0.05). WW, Eth, D, and Eth + D indicate water-sprayed well-watered plants, ethanol-sprayed well-watered plants, water-sprayed drought-exposed plants, and ethanol-sprayed drought-exposed plants, respectively. FW, fresh weight; QE, quercetin equivalent.

#### *3.6. Ethanol Improved Water Status, Osmoprotectant Levels, and Water-Soluble Protein Contents in Soybean Plants Subjected to Drought Stress*

To confirm whether ethanol assists in restraining water loss under drought stress, the levels of relative water content (RWC), osmoprotectants, water-soluble proteins, and total carbohydrates were determined in soybean plant leaves (Figure 6A–G). Upon drought exposure, 'D' plants had a significantly lower level of leaf RWC than 'WW' plants (Figure 6A,G). Interestingly, 'D' plants displayed higher levels of Pro than 'WW' plants (Figure 6B,G). By comparison, ethanol supplementation substantially improved the RWC without further enhancement of Pro in 'Eth + D' plants when compared with 'D' plants (Figure 6A,B,G). 'D' plants also had significantly higher levels of water-soluble proteins, total free amino acids, total soluble sugars, and total carbohydrates than 'WW' plants (Figure 6C–G). Notably, the levels of total free amino acids, total soluble sugars, and total carbohydrates were further escalated by exogenous application of ethanol in 'Eth + D' plants relative to 'D' plants (Figure 6C,E–G). 'Eth' plants displayed a significantly higher level of total soluble sugars and lower amount of water-soluble proteins than 'WW' plants; however, both 'Eth' and 'WW' plants showed comparable data for RWC, Pro, total free amino acids, and total carbohydrates (Figure 6A–G).

**Figure 6.** Effect of exogenously supplied 20-mM ethanol on the levels of (**A**) leaf relative water content, (**B**) proline, (**C**) total free amino acids, (**D**) water-soluble proteins, (**E**) total soluble sugars, and (**F**) total carbohydrates of soybean plants subjected to drought stress for a period of 8 days. (**G**) Heatmap of the fold-change values of the aforementioned parameters in soybean plants under different treatments. Bars represent means with standard errors (*n* = 4). Different letters shown above the bars are used to indicate significant differences among the treatments (*p* < 0.05). WW, Eth, D, and Eth + D indicate water-sprayed well-watered plants, ethanol-sprayed well-watered plants, water-sprayed drought-exposed plants, and ethanol-sprayed drought-exposed plants, respectively. FW, fresh weight; Pro, proline; RWC, relative water content; TFAA, total free amino acids; TSS, total soluble sugars; TCHO, total carbohydrates; WSP, water-soluble-proteins.

#### **4. Discussion**

Soybean growth and productivity are severely affected by drought episodes in many parts of the world [5]. Ethanol, an inexpensive chemical (e.g., \$1.0 for 7.0 gallons of 20-mM ethanol; \$290 for 4 L, Sigma-Aldrich), is known to protect soybean plants from the negative consequences of salinity and chilling stress [19–21]. In this study, we also provided evidence that ethanol supplementation enhanced drought tolerance in soybean plants by reducing drought-induced phenotypic aberrations, such as leaf yellowing and senescence, which corresponded with their better growth and biomass production (Figure 1A–G). Under a water-shortage scenario, plants need to forage water and nutrients from deeper layers of soil [34]. Thus, robust root growth can benefit plants by increasing their water absorption spheres under drought conditions. Our study demonstrated that drought stress attenuated total root biomass, whereas ethanol supplementation improved root biomass significantly (Figure 1F). The greater biomass of roots in ethanol-supplemented soybean plants might allow them to absorb more water from the soils [35], thereby contributing to improved soybean growth under water-deficit situations (Figure 7).

When roots perceive a reduction in soil water availability, they convey this environmental constraint as a stress signal toward the shoots [36]. Accordingly, shoots respond to the signal by producing stress hormones like abscisic acid (ABA) to induce stomatal closure for reducing drought-mediated transpirational water loss [37]. It is well known that the complete closure of stomata causes a sharp decline in the photosynthetic rate, which ultimately leads to growth and yield penalty in crops [38]. On the other hand, a partial stomatal closure might help maintain stomatal conductance and transpiration rates in favor of a properly reprogrammed photosynthesis under a water-shortage condition, allowing plants to maximize their WUE [37,39–41]. Our results revealed that exogenous ethanol treatment improved photosynthetic rate, stomatal conductance, and transpiration rate, resulting in enhanced WUE (carbon gain to water loss ratio), which might contribute to improving phenotypes and biomass production in drought-stressed soybean plants (Figure 1A–C,E–G and Figure 2A–C,E,F). Furthermore, an improvement in transpiration rate resulted in a decrease in LT, which helped keep the leaves cool, as evidenced by minimal leaf wilting symptoms (Figure 1C and 2D). Together, these results indicated that ethanol played a putative role in modulating gas exchange features to improve soybean drought acclimatization performance under water-limited conditions (Figure 7).

In support of these findings, ethanol-sprayed plants also displayed an improved level of photosynthetic pigments (e.g., Chl *a*, Chl *b*, total Chls, and carotenoids) under both well-watered and water-shortage conditions (Figure 3A–D). These findings suggest that ethanol might be involved in either promoting the synthesis or slowing down the degradation rate of photosynthetic pigments, or both, resulting in an improvement in the net photosynthetic rate and biomass production (Figure 1E–G, Figures 2A and 3A–D). In line with our findings, ethanol-mediated protection of photosynthetic pigments has also been reported in strawberry (*Fragaria ananasa*), soybean, and *Arabidopsis* plants [20,21,42]. It is also worth noting that the greater leaf area per trifoliate in ethanol-supplemented plants might play a positive role in increasing photosynthetic rate (Figures 1H and 2A). Leaf area directly influences plants' light interception capacity, and consequently, the overall photosynthetic rate and carbon assimilation process [43,44]. Our results highlighted that ethanol spraying significantly increased leaf area compared with water-sprayed plants, supporting the premise of a positive association between increased leaf area and increased photosynthetic rate (Figures 1H and 2A), which coincides with the previous findings of Rahman et al. [6] and Talbi et al. [45].

**Figure 7.** The regulatory roles of ethanol in alleviating drought-induced adverse effects in soybean plants. Foliar application of exogenous ethanol to drought-stressed soybean plants considerably rescued growth phenotypes, partly through the protection of photosynthetic pigments and improvement of gas exchange parameters, which ultimately increased the overall photosynthetic rate to improve growth performance. Exogenous ethanol also triggered the action of the antioxidant defense system by enhancing the activities of enzymatic antioxidants (e.g., CAT, APX, GST, and POD) and the levels of non-enzymatic antioxidants (e.g., total flavonoids), which together contributed to the protection of soybean plants from reactive oxygen species-induced oxidative stress and membrane damage. Additionally, the external application of ethanol enhanced the levels of osmoprotectants (e.g., free amino acids and soluble sugars) to maintain leaf water status for osmotic adjustment under drought circumstances. Upward (blue) and downward (red) arrows indicate increase and decrease, respectively. APX, ascorbate peroxidase; CAT, catalase; Chls, Chlorophylls; *E*, transpiration rate; GST, glutathione *S*-transferase; *gs*, stomatal conductance to H2O; H2O2, hydrogen peroxide; O2 •−, superoxide; POD, peroxidase; *Pn*, net photosynthetic rate.

A number of studies reported that drought-mediated biomass reduction was correlated with the induction of oxidative damage as a result of increased production of ROS, such as O2 •− and H2O2 [46–48]. In the current study, 'D' plants displayed substantial levels of O2 •−, H2O2, MDA, and EL percentage in the leaves (Figure 4A–E), indicating that drought stress provoked serious oxidative stress and membrane damage in soybean plants. On the other hand, 'Eth + D' plants accumulated less O2 •− and H2O2, as well as a reduced MDA level and EL percentage (Figure 4A–E), demonstrating that the application of external ethanol mitigated ROS-mediated cell membrane damage in drought-exposed leaves. In support of our results, previous reports also revealed that ethanol was involved in the reduction of salt stress-induced oxidative damage in the leaves of soybean, rice, and *Arabidoposis* [20,21]. In this study, we found a positive correlation of induction of the antioxidant defense with the reduced levels of ROS in 'Eth + D' soybean plants (Figure 5). We observed that 'Eth + D' soybean leaves maintained an increase in activities of CAT, APX, and POD, which likely contributed to the detoxification of drought-induced H2O2 [11] (Figure 5A,B,D). Additionally, the greater activity of GST in the leaves of 'Eth + D' plants further confirmed the activation of glutathione-dependent H2O2 removal [49] (Figure 5C). Non-enzymatic antioxidants, such as total flavonoids, were also found to be accumulated in 'Eth + D' plants (Figure 5E). Flavonoids are well-recognized for safeguarding cell membrane integrity from oxidative damage by quenching ROS during water-deficit conditions [6,34,50]. These results support that ethanol addition helped soybean plants to

maintain a better status of flavonoids, conferring protection against drought-caused oxidative damage. Collectively, ethanol application boosted both enzymatic and non-enzymatic defense to trigger efficient ROS detoxification, thereby diminishing cellular damage for better soybean growth performance under drought stress (Figure 7).

Apart from a vibrant antioxidant defense mechanism, the biosynthesis of low-molecularweight osmotic compounds, such as Pro, appears to be an important adaptive mechanism for conserving water status under water-shortage situations in plants [10,51]. Our results showed that 'D' plants accumulated more Pro but retained less RWC in their leaves than control plants (Figure 6A,B). These observations suggest that Pro accumulation in droughtstressed plants was not sufficient to retain water under severe water-deficient environments, which also corroborated with the findings of Dien et al. [52] and Rahman et al. [6]. Alternatively, 'Eth + D' plants replenished water loss in the leaves without a substantial increase of Pro contents, implying that Pro accumulation might be an indicator of soybean cellular dehydration (Figure 6A,B), as also observed in other plants under drought stress [53,54]. Interestingly, we also observed rising levels of total free amino acids, total soluble sugars, and total carbohydrates in 'Eth + D' plants, in contrast to 'D' plants, suggesting that ethanol might compensate water loss independently of Pro but dependently on free amino acids and total soluble sugars (Figure 6A–C,E,F). A number of previous studies have also reported that free amino acids and soluble sugars also played critical roles in maintaining the water status of plants in responses to abiotic stresses, including drought [6,17,22]. Moreover, increased amounts of amino acids and soluble sugars were also known to assure an adequate supply of nitrogen and carbon for sustaining the better metabolism of plants under stressful conditions [55–57]. Increased accumulations of total free amino acids and total soluble sugars, as well as their protective roles in counteracting drought-caused adverse effects, have also been reported by Du et al. [58] and Rahman et al. [6] in soybean and Zahoor et al. [59] in cotton (*Gossypium hirsutum*) plants.

### **5. Conclusions**

Our study revealed that ethanol, in addition to its growth-promoting effects under normal conditions (Figure 1), improved soybean tolerance to water-deficit stress. We presented the first-ever evidence that supplementation of ethanol improved drought tolerance in soybean by improving root biomass, photosynthetic capacity, and WUE, protecting photosynthetic pigments, reducing ROS-triggered oxidative burst by strengthening antioxidant defense, and uplifting osmoprotectant levels (Figure 7). Nonetheless, it will be interesting to identify the major regulatory pathways that are targeted and modulated by ethanol for developing drought tolerance traits in crop plants. Furthermore, field trials and economic evaluations of ethanol application should also be taken into consideration to verify this cost-effective solution of minimizing drought-induced negative effects and reducing crop yield losses in water-limited adverse conditions.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/antiox11030516/s1, Figure S1. Effect of different concentrations of ethanol on soybean plants subjected to drought stress for a period of 8 days. D, drought; Eth, ethanol.

**Author Contributions:** Conceptualization, M.M.R., M.G.M. and L.-S.P.T.; methodology, M.M.R.; experimental work, M.M.R., A.K.D. and T.R.A.; software, M.M.R. and S.M.A.; formal analysis, M.M.R.; validation, M.M.R., M.G.M. and S.S.K.; investigation, M.M.R.; resources, M.A.R.K., M.A., M.A.R. and M.M.H.; data curation, M.M.R.; writing—original draft preparation, M.G.M., M.M.R., S.S.K. and S.M.A.; writing—review and editing, M.M.R., M.G.M. and L.-S.P.T.; supervision, M.G.M. and L.-S.P.T.; project administration, L.-S.P.T.; funding acquisition, L.-S.P.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors would like to extend their sincere appreciation to the United Soybean Board (USB project #2220-172-0148) for providing financial support to carry out the project works.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article and Supplementary Materials.

**Acknowledgments:** The authors would like to thank the Department of Crop Botany, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, Bangladesh for allowing us to use the spectrophotometer.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
