*Article* **Ethanol Positively Modulates Photosynthetic Traits, Antioxidant Defense and Osmoprotectant Levels to Enhance Drought Acclimatization in Soybean**

**Md. Mezanur Rahman 1, Mohammad Golam Mostofa 1,2,\*, Ashim Kumar Das 3, Touhidur Rahman Anik 4, Sanjida Sultana Keya 1, S. M. Ahsan 5, Md. Arifur Rahman Khan 6, Minhaz Ahmed 3, Md. Abiar Rahman 3, Md. Motaher Hossain <sup>7</sup> and Lam-Son Phan Tran 1,\***


**Abstract:** Drought is a major environmental threat to agricultural productivity and food security across the world. Therefore, addressing the detrimental effects of drought on vital crops like soybean has a significant impact on sustainable food production. Priming plants with organic compounds is now being considered as a promising technique for alleviating the negative effects of drought on plants. In the current study, we evaluated the protective functions of ethanol in enhancing soybean drought tolerance by examining the phenotype, growth attributes, and several physiological and biochemical mechanisms. Our results showed that foliar application of ethanol (20 mM) to drought-stressed soybean plants increased biomass, leaf area per trifoliate, gas exchange features, water-use-efficiency, photosynthetic pigment contents, and leaf relative water content, all of which contributed to the improved growth performance of soybean under drought circumstances. Drought stress, on the other hand, caused significant accumulation of reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, and malondialdehyde, as well as an increase of electrolyte leakage in the leaves, underpinning the evidence of oxidative stress and membrane damage in soybean plants. By comparison, exogenous ethanol reduced the ROS-induced oxidative burden by boosting the activities of antioxidant enzymes, including peroxidase, catalase, glutathione *S*-transferase, and ascorbate peroxidase, and the content of total flavonoids in soybean leaves exposed to drought stress. Additionally, ethanol supplementation increased the contents of total soluble sugars and free amino acids in the leaves of drought-exposed plants, implying that ethanol likely employed these compounds for osmotic adjustment in soybean under water-shortage conditions. Together, our findings shed light on the ethanol-mediated protective mechanisms by which soybean plants coordinated different morphophysiological and biochemical responses in order to increase their drought tolerance.

**Keywords:** antioxidant enzymes; gas exchange features; osmotic adjustment; oxidative damage; photosynthesis; reactive oxygen species; water deficiency; water-use-efficiency

**Citation:** Rahman, M.M.; Mostofa, M.G.; Das, A.K.; Anik, T.R.; Keya, S.S.; Ahsan, S.M.; Khan, M.A.R.; Ahmed, M.; Rahman, M.A.; Hossain, M.M.; et al. Ethanol Positively Modulates Photosynthetic Traits, Antioxidant Defense and Osmoprotectant Levels to Enhance Drought Acclimatization in Soybean. *Antioxidants* **2022**, *11*, 516. https:// doi.org/10.3390/antiox11030516

Academic Editor: Nafees A. Khan

Received: 5 February 2022 Accepted: 3 March 2022 Published: 8 March 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

Water scarcity is undeniably the most critical environmental constraint limiting agricultural output worldwide. Moreover, growing water demand due to increased population pressure, ongoing climate change-mediated erratic precipitation patterns, and rising temperature will further intensify the drought situation in many regions of the world [1]. Drought stress can trigger a wide array of negative consequences in plants by altering their morphological, physiological, biochemical, cellular, and molecular responses, all of which impede plant growth and development [2,3]. A plethora of studies have reported that water scarcity reduces biomass production and stem elongation, disrupts cellular turgor pressure, restricts water uptake, and interrupts gas exchange performance and nutrient acquisition. Drought stress can also stimulate reactive oxygen species (ROS) accumulation and membrane lipid peroxidation, which ultimately leads to poor growth and even death of plants in severe cases [4–6].

Intriguingly, plants have evolved various adaptive strategies to fight against the detrimental consequences of drought [7]. These adaptive strategies include, but are not limited to, increased leaf succulence, enhanced root growth to acquire more water and essential nutrients from the deeper layer of soils, restricted transpirational water loss, retained optimum photosynthetic rate, and improved water-use-efficiency (WUE) [8,9]. In addition, plants can synthesize many compatible compounds, such as proline (Pro), amino acids, and sugars, to maintain osmotic adjustment under drought circumstances [10]. Moreover, plants have evolved complex oxidative stress protection mechanisms to avoid ROS-induced oxidative damage by increasing the synthesis of non-enzymatic antioxidants, such as carotenoids and flavonoids, and stimulating the activities of enzymatic antioxidants, such as glutathione peroxidase (GPX), peroxidase (POD), glutathione *S*-transferase (GST), ascorbate peroxidase (APX), superoxide dismutase (SOD), and catalase (CAT) [11].

Oilseeds have long been regarded as essential components of human diets and the vital raw materials of many industrial applications for the production of pharmaceutical products, oleochemicals, cosmetics, and biofuels [12]. Soybean (*Glycine max*), in particular, is the world's fourth most important grain crop, accounting for 59% of global oilseed production (www.soystats.com). Soybean acts as a source of 29% edible oil and 70% plant-derived proteins worldwide [6,13]. Importantly, being a legume crop, soybean plays a pivotal role in improving soil fertility through the process known as symbiotic nitrogen-fixation [14,15]. Drought is critical for soybean growth and development and is one of the leading reasons for the soybean yield penalty in arid and semi-arid areas of the world [6,13]. Many strategies, such as gene mining, genetic engineering, and molecular breeding, have been employed to develop soybean varieties with a heightened capacity to survive through water dearth conditions [16]. However, farmers in low-income countries prefer to practice an easy, cost-effective approach that provides immediate agronomic and economic benefits, because biotechnological and breeding research requires more investment and time for developing drought-resilience crops [6,17,18]. Considering these facts, treating plants with cost-effective signaling molecule(s) (SMs) has gained much attention for overcoming drought on numerous agricultural crops, including soybean [6]. Ethanol has emerged as an excellent representative of organic SMs that already showed promising effects in mitigating the adverse impacts of several abiotic stresses, such as chilling stress in rice (*Oryza sativa*) [19] and salt stress in soybean [20], rice, and *Arabidopsis (Arabidopsis thaliana*) [21]. These findings provide a strong rationale for testing the function of ethanol in alleviating the harmful impacts of drought on the economically valuable crop soybean.

In the current research, we intended to investigate whether ethanol could increase the resilience of soybean toward drought stress, as it did in the case of chilling and salt stress tolerance [19–21]. With this objective, we examined various morphophysiological and biochemical parameters, including (i) plant growth features and biomass production, (ii) leaf relative water content and succulence as an indicator of water status, (iii) gas exchange parameters, (iv) contents of different photosynthetic pigments, (v) droughtcaused ROS generation and membrane lipid peroxidation, (vi) activities and/or levels of different enzymatic and non-enzymatic antioxidants, and (vii) accumulation of several osmoprotectants, to deduce ethanol-mediated drought tolerance mechanisms in soybean.

#### **2. Materials and Methods**

## *2.1. Plant Materials, Experimental Design, and Treatments*

Seed germination and pot-culture of soybean (*Glycine max*) variety (BARI soybean #6) were carried out following the procedures described by Rahman et al. [6]. The average minimum and maximum temperatures during the experimental period were 17 and 34 ◦C, respectively, with a relative humidity of about 84%. Ten-day-old healthy soybean seedlings grown in pots (eight plants in each pot) were divided into four treatment groups, including (i) water-sprayed well-watered plants (WW), (ii) ethanol-sprayed well-watered plants (Eth), (iii) water-sprayed drought-exposed plants (D), and (iv) ethanol-sprayed drought-exposed plants (Eth + D). Following the methodology of Rahman et al. [6], drought stress was imposed by withholding water irrigation for 8 days, while the control plants were irrigated regularly during the whole experimental period. Plants from 'Eth' and 'Eth + D' treatment groups were sprayed (8-times in total) with 20-mM ethanol solution (20 mL to each pot), while plants from the 'WW' and 'D' treatment groups were sprayed (8-times in total) with an equal amount of water every day for a period of 8 days. The applied ethanol dose (20 mM) was selected based on the phenotypes obtained from a small-scale experiment (Supplementary Figure S1). After 8-days of drought exposure, the first trifoliate leaves of soybean plants (19-day-old plants) were harvested to determine numerous parameters associated with soybean morphology, physiology, and cellular biochemistry. The experiment was repeated thrice to ensure the accuracy of the experimental outcome.

#### *2.2. Assessment of Growth Parameters*

From each treatment, three randomly selected soybean plants were taken to evaluate the growth performance by measuring shoot height, shoot dry weight (DW), root DW, and total DW following the procedures described by Rahman et al. [22].

#### *2.3. Estimation of Leaf Area, Succulence, Electrolyte Leakage, and Relative Water Content*

Total leaf area per trifoliate was estimated according to the following formula reported by Carleton and Foote [23]:

Leaf area (cm2) = maximum length <sup>×</sup> maximum width <sup>×</sup> 0.75 (correction factor).

Leaf succulence was measured following the comprehensive procedure of Rahman et al. [17]. Leaf electrolyte leakage (EL) percentage was quantified following the protocol of Yang et al. [24] with slight modification. Briefly, 0.2 g of first trifoliate leaves were collected in a 50-mL Falcon tube containing 20 mL of tap water. Initial electrical conductivity (EC1) was taken after incubating the samples at 32 ◦C for 2 h. The samples were heated at 100 ◦C for 30 min followed by cooling down at room temperature to record final EC (EC2). EC of tap water was also measured and referred to as EC0. Finally, the EL (%) was calculated using the following equation:

$$\text{EL (\%)} = (\text{EC}\_1 - \text{EC}\_0) / (\text{EC}\_2 - \text{EC}\_0) \times 100. \text{J}$$

Leaf relative water content (RWC) was estimated following the procedure outlined by Das et al. [20].

#### *2.4. Assessment of Gas Exchange Parameters*

An infrared gas analyzer (LI-6400XT, LI-COR Inc., Lincoln, NE, USA) was utilized to estimate the net photosynthetic rate (*Pn*), the stomatal conductance to H2O (*gs*), the leaf temperature (LT), and the transpiration rate (*E*) as previously described by Rahman et al. [17]. Assessment of photosynthetic parameters was carried out under full sunlight between

11:00 AM and 12:30 PM. WUE parameters, including intrinsic WUE (WUEint) and instantaneous WUE (WUEins), were estimated using *Pn*, *gs*, and *E* following the formulae reported in Rahman et al. [17].

#### *2.5. Determination of Photosynthetic Pigment Contents*

Freshly collected leaves were used to quantify the contents of different photosynthetic pigments, such as chlorophylls (Chls) (e.g., Chl *a*, Chl *b*, and total Chls) and carotenoids, following the protocol outlined by Arnon [25] and Lichtenthaler and Wellbura [26], respectively.

#### *2.6. Quantification of the Content of Total Flavonoids*

The method proposed by Das et al. [20] was followed to quantify the levels of total flavonoids in the leaf tissues of soybean plants.

#### *2.7. Histochemical Analyses of ROS and the Estimation of Hydrogen Peroxide and Malondialdehyde Contents*

Freshly harvested leaves were stained using the solutions of nitroblue tetrazolium (NBT) and 3, 3'-diaminobenzidine (DAB) to visualize the accumulations of superoxide (O2 •−) and hydrogen peroxide (H2O2), respectively, following previously described protocol [20]. The contents of H2O2 and malondialdehyde (MDA) in the leaf tissues were estimated using a spectrophotometer as outlined by Yu et al. [27] and Kim et al. [28], respectively.

#### *2.8. Antioxidant Enzyme Extraction and Assessment of Enzyme Activities*

Enzyme extracts were prepared from soybean leaf samples, and the activities of antioxidant enzymes, including CAT (EC: 1.11.1.6), GST (EC: 2.5.1.18), APX (EC: 1.11.1.11), and POD (EC: 1.11.1.7), were determined following the protocol described by Rahman et al. [17].
