Ⅰ. INTRODUCTION
Alfalfa (Medicago sativa L.), a perennial legume, has high nutritional value and rapid digestion, making it a highly valuable potential forage for livestock (Marten et al., 1988). In Korea, alfalfa is cultivated in rice paddies and reclaimed land to increase self-sufficiency in high-quality forage. However, spring droughts have a significant impact on alfalfa productivity (Jeong et al., 2024). Furthermore, according to the RCP 8.5 scenario, the frequency of droughts in the Korean Peninsula is projected to increase steadily (Nam et al., 2015).
Drought stress causes water deficit in plants and increases intracellular reactive oxygen species (ROS), which damage cell membranes, organelles, proteins, and DNA (Farooq et al., 2009;Gill and Tuteja, 2010). To cope with drought stress, plants activate various defense mechanisms, one of which is defense respiration, which replenishes energy deficits and activates defense mechanisms (Jardine and McDowell, 2023). During this process, the acetate fermentation pathway is activated, leading to the accumulation of acetate and ethanol. These volatile organic compounds (VOCs), including ethanol, are released into the atmosphere via transpiration and function as airborne signaling molecules that induce stress-responsive reactions in neighboring plants (Holopainen and Gershenzon, 2010). The role of ethanol as a stress-defense signaling molecule among plants suggests that exogenous ethanol application may similarly enhance drought tolerance. In plants, ethanol serves as a precursor of acetic acid and is oxidized through the sequential actions of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) (Bashir et al., 2022;Jardine and McDowell, 2023). Ethanol absorbed by plants is not only converted to acetate and used as an energy source, but it has also been reported to enhance drought tolerance by activating ABA signaling in Arabidopsis, inducing stomatal closure, and supplying scarce carbon through gluconeogenesis (Bashir et al., 2022). Furthermore, ethanol treatment has been reported in crops such as soybean (Rahman et al., 2022) and cassava (Vu et al., 2022). However, the effect of ethanol treatment on drought tolerance in alfalfa, a major forage crop, has not yet been reported. Therefore, in this experiment, different concentrations of ethanol were applied to growing alfalfa plants, and then plant growth and physiological and biochemical responses were investigated.
Ⅱ. MATERIALS AND METHODS
1. Plant growth and ethanol treatment
Alfalfa (Medicago sativa L.), cv. Vernal, was used as the plant material. Plants were grown in pots filled with horticultural soil:sand (2:1, v/v) under regular irrigation for 21 days. Ethanol solutions at concentrations of 0, 50, 100, 250, 500 and 1000 mM were prepared by diluting 99.9% ethanol with distilled water. The experiment was arranged with three pots for each ethanol concentration. Each pot containing 10 plants was treated with the prepared ethanol solutions by bottom watering for 5 min. Treatments were applied 3 times every 2 days over a period of 4 days. After ethanol pretreatment, water supply was withheld for 9 days to impose drought stress, whereas control plants were irrigated normally. All alfalfa plants were maintained in a controlled growth chamber at 23°C, 55% relative humidity, and under a light regime of 220 μmol m⁻²·s⁻¹ with a 16 h light/8 h dark photoperiod.
2. Measurement of relative water content
The relative water content (RWC) of alfalfa shoots was measured according to the method of Zhang et al. (2019). Briefly, the fresh weight (FW) of sampled alfalfa shoots was measured, and the turgid weight (TW) was measured after overnight incubation in distilled water. After drying in a 70°C dry oven for 72 h, the dry weight (DW) was measured. The RWC was then calculated using the measured FW, TW, and DW. For RWC measurement, four biological replicates were used for each treatment. RWC (%) = [(FW – DW) / (TW – DW)] × 100
3. Measurement of chlorophyll a, b and carotenoid content
The chlorophyll a (Chl-a), chlorophyll b (Chl-b), and carotenoid content of alfalfa leaves were analyzed using the method of Yoon et al. (2021). For Chl-a, Chl-b measurement, three biological replicates were used for each treatment.
4. Measurement of total soluble carbohydrate content
The total soluble carbohydrate content of alfalfa shoots was determined according to the method of Maria del Rosario M. et al. (2020). A 20 mg plant sample was homogenized in liquid nitrogen using a mortar and pestle, 2 mL of 80% (v/v) ethanol was added, and the mixture was incubated at 80°C for 10 min in a water bath. The supernatant was centrifuged at 3,500 rpm for 15 min, and the supernatant was recovered. The supernatant was diluted 1:1 (v/v) with 80% (v/v) ethanol, mixed with 5% (w/w) phenol and sulfuric acid, and incubated at 25°C for 25 min. The absorbance of the extract after the reaction was measured at 490 nm using a spectrophotometer. A standard curve was then constructed using D-glucose, and the total soluble carbohydrate content was calculated. For total soluble carbohydrate content measurement, three biological replicates were used for each treatment.
5. Measurement of hydrogen peroxide (H2O2)
The H2O2 content of alfalfa shoots was determined according to the method of Sharova et al. (2023). Briefly, 50 mg of leaf tissue was homogenized in liquid nitrogen and extracted with 0.9 mL of 50 mM phosphate buffer (pH 7.0). The homogenate was centrifuged at 6,000 × g for 25 min, and 0.9 mL of the resulting supernatant was mixed with 0.3 mL of 0.1% (v/v) titanium tetrachloride dissolved in 20% (v/v) sulfuric acid. The absorbance of the reaction mixture was measured at 410 nm using a spectrophotometer, and H2O2 concentration was calculated using an extinction coefficient of 0.28 μM-1·cm-1. For H2O2 content measurement, three biological replicates were used for each treatment.
6. Statistical analysis
All statistical analyses were performed using IBM SPSS (IBM SPSS Statistics for Windows, Version 27.0. Armonk, NY, USA) using one-way ANOVA (Tukey’s HSD, p<0.05).
Ⅲ. RESULTS AND DISCUSSION
1. Effects of ethanol pretreatment on growth of alfalfa plants
To evaluate the effects of ethanol pretreatment on alfalfa growth, 21-day-old plants were exposed to different concentrations of ethanol (0–1000 mM). Following the pretreatment, irrigation was completely withheld, and drought stress was imposed for 9 days. Plant growth parameters, including survival rate, fresh weight, and dry weight, were subsequently assessed. As shown in Fig. 1, the majority of plants pretreated with 250 mM ethanol survived prolonged drought stress, whereas all plants in the control and those exposed to higher ethanol concentrations failed to survive (Fig. 1). These results indicate that ethanol pretreatment at 250 mM is optimal for inducing drought tolerance in alfalfa.
Under well-watered conditions, fresh weight was significantly increased in plants pretreated with 100 and 250 mM ethanol compared with the control. However, no significant differences were observed among treatments under drought conditions (Fig. 2A). Similarly, dry weight did not differ significantly between ethanol-pretreated and non-treated control plants, regardless of water availability (Fig. 2B). Previous studies have reported that ethanol treatment promotes growth in soybean under wellwatered conditions (Rahman et al., 2022), whereas growth inhibition was observed in Arabidopsis thaliana following ethanol application under similar conditions (Bashir et al., 2022). Consistent with these findings, ethanol pretreatment in alfalfa enhanced plant growth under non-stress conditions, while under drought stress, it did not appear to directly promote biomass accumulation.
2. Effects of ethanol pretreatment on relative water content (RWC)
To examine the effects of ethanol pretreatment on the relative water content (RWC) of alfalfa, fresh weight (FW), turgid weight (TW), and dry weight (DW) of alfalfa shoots were measured, and RWC was calculated based on these parameters (Fig. 3).
Under well-watered conditions, no significant differences in RWC were observed among the treatments. In contrast, under drought stress, RWC increased with increasing ethanol concentration and was significantly higher in plants pretreated with 250 mM ethanol compared with the control (Fig. 3). Previous studies have reported that ethanol treatment induces abscisic acid (ABA) signaling in Arabidopsis, thereby suppressing transpiration and contributing to the maintenance of plant water status (Bashir et al., 2022). Based on the present results, ethanol pretreatment in alfalfa is thought to similarly enhance water retention under drought stress through a mechanism comparable to that reported in Arabidopsis.
3. Changes in chlorophyll a, b and carotenoid content
To determine the effects of ethanol treatment on chlorophyll and carotenoid biosynthesis in alfalfa, chlorophyll a, b, and carotenoid content in leaves were measured after drought treatment (Fig. 4). As a result, chlorophyll content significantly decreased in 250 and 500 mM ethanol treatments compared to 0 and 100 mM ethanol treatments. However, carotenoid content did not show a significant difference according to ethanol treatment concentration. According to the research results of Shin et al. (2023), chlorophyll and carotenoid content decreased in Korean mint and sweet wormwood when treated with high concentrations of ethanol, indicating that high concentrations of ethanol treatment induced oxidative stress in plants. In this experiment, chlorophyll a and b decreased to a level that was insignificant compared to the control group in the drought stress treatment group when treated with 100 mM ethanol, but decreased in 250 and 500 mM ethanol treatments. These results suggest that high concentration ethanol treatments causes damage to chlorophyll in alfalfa and does not activate photosynthetic reactions.
4. Changes in total soluble carbohydrate content
To determine the effect of ethanol treatment on the total soluble carbohydrate content of alfalfa, the total soluble carbohydrate content of alfalfa leaves after drought treatment was measured (Fig. 5). As a result, total soluble carbohydrate content significantly decreased in all ethanol treatments compared to the control (0 mM ethanol). Furthermore, the 250 mM ethanol treatment significantly decreased the total soluble carbohydrate content compared to the 100 and 500 mM ethanol treatments. According to Rahman et al. (2022), plants can maintain osmoregulation under drought conditions by synthesizing proline, amino acids, and sugars. Furthermore, Bashir et al. (2022) reported that ethanol treatment leads to the accumulation of ethanol as sugars through gluconeogenesis. However, in this experiment, the total soluble carbohydrate content significantly decreased in all ethanol treatment concentrations. This indicates that in alfalfa, ethanol pretreatment resulted in a different physiological response regarding total soluble carbohydrate content compared to the accumulation pattern often reported in other species.
5. Changes in hydrogen peroxide (H2O2) content
To investigate the effects of ethanol pretreatment on reactive oxygen species (ROS) accumulation in alfalfa, plants were treated with ethanol for 4 days, followed by the imposition of drought stress, and the hydrogen peroxide (H2O2) content in alfalfa shoots was subsequently measured (Fig. 6). Compared with the non-treated control, H2O2 levels were significantly reduced in plants pretreated with 100 and 500 mM ethanol, whereas no significant difference was observed in the 250 mM ethanol treatment. In soybean, Rahman et al. (2022) reported that ethanol pretreatment under drought stress led to a reduction in ROS accumulation. Similarly, in the present study, ethanol pretreatment at 100 and 500 mM decreased H₂O₂ content under drought conditions. However, unlike these treatments, 250 mM ethanol pretreatment did not result in a significant reduction in H2O2 levels. This result suggests that pretreatment with 250 mM ethanol may maintain ROS homeostasis at an optimal level, preventing both excessive ROS accumulation and over-scavenging during drought stress.
As a result of this experiment, ethanol pretreatment in alfalfa did not significantly change growth under drought stress, but it is believed that drought tolerance was improved by changing the stress response mechanism. However, the 250 mM ethanol treatment significantly extended plant survival, which is a critical indicator of drought tolerance. In this treatment group, relative water content (RWC) increased significantly compared to the control group, whereas chlorophyll and total soluble carbohydrate content decreased significantly. In addition, there was no change in carotenoid content and hydrogen peroxide (H2O2) content. Furthermore, the decrease in total soluble carbohydrate content contrasts with the typical accumulation response observed in drought-tolerant plants. This suggests that the survival of the 250 mM group was primarily driven by the maintenance of RWC rather than by ROS scavenging or soluble carbohydrate accumulation. Consequently, the maintenance of RWC, despite the absence of significant biomass gain or ROS reduction, likely played a crucial role in enabling survival. Given that physiological responses varied across concentrations, future studies should explore a broader range of lower ethanol concentrations to fully validate the efficacy of ethanol priming and elucidate the precise mechanisms underlying this survivalfocused tolerance.
Ⅳ. CONCLUSIONS
Ethanol pretreatment improved the drought stress tolerance of alfalfa plants. Pretreatment with 250 mM ethanol increased relative water content (RWC) and plant survival under drought stress. The results of this study will be helpful for the development of drought-tolerant alfalfa in the future.
