A. thaliana (ecotype Columbia-0) seeds were sterilized and sown in half-strength Murashige and Skoog (MS) liquid medium supplemented with 1% sucrose and 0.1% agar. The plants were grown under previously described conditions (Sako et al., 2016). Four-day-old plants were treated with ethanol (Wako, Japan), acetone (Wako, Japan), methanol (Wako, Japan), N'N-dimethylformamide (DMF) (Wako, Japan), dimethyl sulfoxide (DMSO) (Wako, Japan), or sterilized deionized water for 24 h, with or without a subsequent treatment with 100 mM NaCl (Wako, Japan). The NaCl solution was added into the medium containing the solvents (Figure S1A). The survival rate of 20 plants was calculated 4 days after the NaCl treatment. The experiment was conducted using three biological replicates.

For rice experiments, Oryza sativa L. cv. Nipponbare seeds were germinated in water at 30°C for 2 days, and then transferred to plastic pots containing granular soil (Bonsoru No. 2; Sumitomo Chemical, Tokyo). The plants were grown in a vat filled with water at 30°C for 2 weeks under a 14-h light:10-h dark photoperiod. For the salinity stress test, soil moisture was removed by leaving the pots on Kimtowel (Nippon Paper Crecia) for 20 min. The pots were then incubated in a 0, 0.3, or 0.6% (corresponding to 0, 51 or 103 mM, respectively) ethanol solution for 4 days. After the ethanol treatment, the pots were left on Kim towel for 20 min to remove soil moisture. The pots were subsequently transferred to a 200 mM NaCl solution and incubated for 5 days. The data was analyzed from 12 plants for each treatment. The experiment was conducted using two independent biological replicates.

Four-day-old A. thaliana plants were treated with 0.3% (51 mM) ethanol for 24 h and then exposed to 100 mM NaCl for 72 h. We then measured the chlorophyll content of 30–50 mg seedlings for each treatment as previously described (Kim et al., 2003). The experiment was conducted with three biological replicates. Statistical significance was determined by ANOVA, followed by post-hoc Tukey's tests. Means that differed significantly (P < 0.05) are indicated by different letters.

Total RNA was extracted from 5-day-old A. thaliana seedlings that were treated with 0.3% (51 mM) ethanol for 24 h, with or without a subsequent treatment with 100 mM NaCl for 2 h. Sterilized deionized water was used as a negative control. The RNA was extracted using the Plant RNA reagent (Thermo Fisher Scientific) as previously described (Nguyen et al., 2015). The quality of the extracted total RNA was evaluated using a Bioanalyzer system (Agilent). The RNA was extracted from 30 plants. The experiment was conducted using three biological replicates.

A microarray analysis was completed as previously described (Nguyen et al., 2015). The microarray data underwent a one-way ANOVA method and were deposited in the GEO database (GEO ID: GSE95202). Each treatment was analyzed using four biological replicates. A total of 30 plants were used for each treatment. Genes with an expression log₂ ratio ≥ 0.7 [t-test analysis, Benjamini–Hochberg correction (FDR) ≤ 0.05] were identified as upregulated genes.

We synthesized cDNA using the QuantiTect Reverse Transcription Kit (QIAGEN) for a quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The qRT-PCR was conducted as previously described (Sako et al., 2016). We used AtACT2 as a reference gene. The experiment was conducted using three biological replicates. A total of 30 plants were used for each treatment. The qRT-PCR primer sequences were as follows: AtZAT10: 5′-ACATCCCTCCGATCCCTGAA-3′ and 5′-ACCGGAAAGTCAAACCGAGG-3′; AtZAT12: 5′-TCCGATGGGACAAGCTTTGG-3′ and 5′-AAGCCACTCTCTTCCCACTG-3′; AtAPX1: 5′-GCACTATTGGACGACCCTGT-3′ and 5′-AGCAAACCCAAGCTCAGAAA-3′; AtAPX2: 5′-AAGTTGAGCCACCTCCTGAA-3′ and 5′-GTGTGTCCACCAGACAATGC-3′; AtACT2: 5′-GATCTCCAAGGCCGAGTATGAT-3′ and 5′-aCCCATTCATAAAACCCCAGC-3′.

Five-day-old A. thaliana plants treated with 0.3% (51 mM) ethanol for 24 h, with or without a subsequent treatment of 100 mM NaCl for 12 h were used for an APX assay. The experiment was conducted using three biological replicates. Proteins were extracted from 30 plants and the APX assay was conducted as previously described (Bradford, 1976; Mostofa et al., 2015). The protein content was determined using bovine serum albumin as a standard.

Five-day-old A. thaliana plants treated with 0.3% (51 mM) ethanol for 24 h, with or without a subsequent treatment with 100 mM NaCl for 12 h were stained using a modified version of a published method (Kumar et al., 2014; Mostofa et al., 2015). To detect O2•-, plants were stained for 30 min with 0.05% NBT (w/v) in 50 mM potassium phosphate, pH 7.0. To detect H₂O₂, plants were stained for 5 h with 0.1% DAB in 10 mM potassium phosphate, pH 7.0. Samples were stained under light at room temperature, after which they were cleared with an ethanol:acetic acid (96:4) solution until photographed by a digital microscope (VHX-5000, Keyence). The experiment was conducted using three biological replicates. A total of 30 plants were used for each treatment.

For the rice experiments, 14-day-old O. sativa L. cv. Nipponbare plants treated with or without 0.3% (51 mM) ethanol for 24 h were exposed to 100 mM NaCl for 24 h. The second leaf was stained with NBT or DAB as previously described (Mostofa et al., 2015) and then photographed using the M165 FC fluorescent stereo microscope (Leica).

Four-day-old A. thaliana plants were treated with 0.3% (51 mM) ethanol for 24 h and then exposed to 100 mM NaCl for 72 h. H₂O₂ content was then measured as described previously (Ivanchenko et al., 2013). The experiment was conducted with three biological replicates.

For the rice experiments, 14-day-old O. sativa L. cv. Nipponbare plants were treated with or without 0.3% (51 mM) ethanol for 24 h and then exposed to 100 mM NaCl for 24 h. The second leaf was sampled for H₂O₂ content as described previously (Ivanchenko et al., 2013). The experiment was conducted with three biological replicates.

We examined the effects of five organic solvents on A. thaliana high-salinity stress tolerance (Figures 1A,B, Figure S1A). Wild-type plants grown in liquid culture medium were treated with an organic solvent or water for 24 h, with or without a subsequent treatment with 100 mM NaCl for 4 days (Figure S1A). We observed that the ethanol treatment enhanced A. thaliana high-salinity stress tolerance (Figures 1A,B), although the plants appeared slightly yellow under the non-stress condition. In contrast, plants treated with acetone, methanol, DMF, or DMSO did not exhibit any significant morphological differences compared with the control plants under the non-stress condition. Additionally, plants were unable to survive under the high-salinity stress condition (Figures 1A,B). The effects of various concentrations of organic solvents were also tested as shown in Figure S1. The data showed that the other organic solvents except for ethanol could not rescue plants from high-salinity stress condition. Consistent with these results, we observed that the chlorophyll content was higher in ethanol-treated plants than in the untreated plants under the high-salinity stress condition (Figure 1C).

Because the ethanol-treated plants became slightly yellow, we also evaluated the survival rate of salinity-stressed plants after a 7-day recovery period, during which the plants were transferred to MS liquid medium (Figure S2). A higher recovery rate was observed for ethanol-treated plants (93%) than for untreated seedlings (50%; Figure S2). Our data confirmed that ethanol enhances A. thaliana tolerance to high-salinity stress.

We analyzed the ethanol-induced gene expression levels associated with A. thaliana high-salinity stress tolerance using a microarray. Four-day-old plants treated with 0.3% (51 mM) ethanol or water for 24 h, with or without a subsequent treatment with 100 mM NaCl for 2 h were examined (Figure 2A). We observed that 1,293 genes were more highly expressed in ethanol-treated plants than in the untreated control plants in the absence of high-salinity stress (Figure 2A, Table S1). Among these genes, 240 exhibited upregulated expression following a 2 h NaCl treatment in the absence of ethanol (Figure 2A, Table S2). We observed that 1,323 genes were more highly expressed in plants treated with NaCl in the presence of ethanol than in plants treated with NaCl in the absence of ethanol (Figure 2A, Table S3). Of these genes, 169 overlapped with salinity stress-upregulated genes (Figure 2A, Table S4), while 888 genes overlapped with ethanol-upregulated genes in the absence of NaCl (Figure 2A, Table S5). Furthermore, we detected 134 NaCl-inducible genes that were more highly expressed in ethanol-treated plants than in untreated controls, with or without NaCl treatment (Figure 2A, Table S6).

For a more detailed analysis, we focused on the 994 overlapping genes highlighted in red in the Venn diagram presented in Figure 2A. Of these genes, 35 were related to ROS signaling (Table 1), including genes encoding ROS-scavengers [e.g., APX, CAT, glutathione peroxidase (GPX), peroxiredoxin (PrxR), glutathione S-transferase (GST), and alternative oxidase (AOX)], ROS-scavenging signaling molecules (e.g., ferritin and blue copper proteins, which inhibit the production of HO^•, glutathione reductase, dehydroascorbate reductase, and glutaredoxin). In contrast, the expression of SOD genes encoding O2•- scavengers was unaffected by ethanol under high-salinity condition.

The expression of several transcription factor family genes involved in ROS signaling was also induced by ethanol in salt-stressed plants. These genes encoded C₂H₂ zinc finger proteins (AtZAT6_AT5G04340, AtZAT10_AT1G27730, and AtZAT12_AT5G59820), WRKY proteins (AtWRKY6_AT1G62300, AtWRKY25_AT2G30250, and AtWRKY33_AT2G38470), a DREB protein (AtDREB19_AT2G38340), a heat shock factor protein (AtHsfA4A_AT4G18880), and NAC proteins (ANAC019_AT1G52890, ANAC102_ AT5G63790, and ANAC032_AT1G77450) (Tables S2, S5, S6). The AtZAT10 and AtZAT12 transcription factor genes were further analyzed by qRT-PCR. The expression levels of these two genes increased following ethanol and NaCl treatments (Figure 2B). These observations suggest that the salt tolerance conferred by ethanol might be due to the increased production of ROS-related proteins and ROS signaling-related transcription factors, such as ZAT10 and ZAT12.

To characterize the molecular functions of ethanol treatments, the expression of the ZAT10/12-related ROS scavenger genes was analyzed by qRT-PCR. We confirmed that AtAPX1 and AtAPX2 expression levels increased in response to ethanol and NaCl treatments (Figure 3A). To clarify whether ethanol regulates cytosolic APX activity, an APX enzyme assay was performed. At 12 h after the NaCl treatment, total APX activity was higher in ethanol-treated plants than in untreated controls under the high-salinity stress condition (Figure 3B). In contrast, no significant differences were observed between ethanol-treated and untreated control plants under the non-stress condition. Our data indicated that ethanol induces the transcription of APX2 and APX1, and enhances APX activity.

The accumulation of O2•- and H₂O₂, which are the two main ROS components induced by salt stress, normally results in oxidative damage. We investigated the accumulation of O2•- and H₂O₂ in ethanol-treated A. thaliana plants under salt stress conditions using NBT and DAB staining, respectively. The cotyledons of NaCl-treated plants were extensively stained by DAB, indicating H₂O₂ was highly accumulated under high-salinity stress condition. The ethanol treatment resulted in a lower accumulation of H₂O₂ (Figure 4A). The NBT staining results revealed slight differences among treatments, and the plants treated with ethanol accumulated less O2•- than the control plants under the salt stress condition (Figure 4A).

The staining data were further confirmed by H₂O₂ content in shoots. The results were consistent with the DAB staining data. Under the control condition, there were no significant differences between ethanol-treated and non-treated plants (Figure 4B). However, after 12 h NaCl stress condition, the ethanol-treated plants could maintain the H₂O₂ level as stable as those in control condition, while the ethanol-nontreated plants showed higher concentration of H₂O₂ compared with the plants in the control condition and ethanol-treated plants under high-salinity stress condition (Figure 4B). These data indicate that ethanol enhances salinity stress tolerance by ROS detoxification in A. thaliana.

To confirm whether ethanol enhances the tolerance of monocots to salt stress condition, 14-day-old rice seedlings were treated with several ethanol concentrations. We observed that the leaves of untreated plants turned slightly yellow on 5 days after the NaCl treatment (Figure 5A). However, the leaves of salinity-stressed plants treated with 0.3 and 0.6% (51 and 103 mM, respectively) ethanol remained green, suggesting that ethanol enhances salinity stress tolerance in rice as well as A. thaliana (Figures 1, 5A).

Ethanol treatments enhanced ROS detoxification and improved the salt stress tolerance of A. thaliana plants. We used DAB and NBT staining to verify that salinity stress tolerance in rice is due to the detoxification of ROS. Rice leaves were more extensively stained by DAB under salinity stress condition than under control condition (Figure 5B). However, the intensity of the DAB staining decreased in ethanol-treated plants under salt stress condition, suggesting that ethanol inhibited ROS accumulation (Figure 5B). Additionally, there was no clear difference in the NBT staining of ethanol-treated and control plants under high-salinity condition (Figure 5B). The H₂O₂ content of rice leaves was measured and the data showed the highest concentration was detected in plants treated with NaCl for 24 h. In contrast, the plants treated with both NaCl and ethanol showed lower concentration of H₂O₂, which is similar with that of control condition (Figure 5C). These results confirmed our DAB staining data, implying that ethanol increases salinity tolerance in rice by inhibiting ROS accumulation, similar to its effects in A. thaliana plants.

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