Arabidopsis (Arabidopsis thaliana) ecotype Columbia was used in this study. The β-glucuronidase (GUS) reporter line CAT2pro::GUS and mutants myc2 (SALK_017005), cat2-1 (SALK_076998), and myc2 cat2-1 were previously reported (Zhang et al., 2020). The mutant jar1 (CS8072) was obtained from the Arabidopsis Biological Resource Center (ABRC). Seeds were surface sterilized for 5 min in 5% commercial bleach, washed three times with sterile water, and plated on 1/2 Murashige and Skoog (MS) medium (pH 5.8) (Sigma-Aldrich, St. Louis, MO, USA) containing 1% sucrose and 1% agar. Plant seeds were stratified at 4°C for 3 days in the dark and then transferred to chambers. The seedlings grown vertically at 22°C and 100 μmol m⁻² s⁻¹ illumination under 16 h light/8 h dark conditions for 5 days were transferred to 1/2 MS medium without or with NaCl or MeJA for another 5 days, and the lengths of newly grown roots and seedling fresh weight were measured and statistically analyzed.

The treated or untreated seedlings were used for DAB to assay H₂O₂ accumulation as we described previously (Zhang et al., 2020a,b). Briefly, the seedlings were incubated in freshly prepared DAB staining solution (1 mg/ml DAB and 0.1% Tween 20 in 10 mM Na₂HPO₄) for about 8 h and then rinsed with 70% ethanol several times to remove the chlorophyll. The images of the leaves were captured using a digital camera.

The salt- or MeJA-treated or untreated seedlings were ground to a fine powder under liquid nitrogen and suspended in freshly prepared cold protein extraction buffer (50 mM potassium phosphate buffer, pH 7.8, 0.2 mM ethylenediaminetetraacetic acid (EDTA)-Na₂, 0.1 mM ascorbic acid, and 1% polyvinyl polypyrrolidone, PVPP). After centrifugation at 12,000 g for 10 min at 4°C, the supernatant was transferred to a new tube for further use. The protein concentration was assayed by the Bradford method, and CAT activity was determined according to the published methods (Aebi, 1984). CAT activity was assayed by monitoring the consumption of H₂O₂ at 240 nm.

Treated or untreated seedlings were collected for total RNA isolation, first-strand cDNA synthesis, and qRT-PCR as we described previously (Liu et al., 2017a; Zhang et al., 2020a). The constitutively expressed ACTIN2/8 gene was used as an internal control. All experiments were repeated at least three times. The primer sequences are listed in Supplementary Table 1.

The GUS histochemical staining experiment was performed according to our previously reported method (Zhang et al., 2020a,b). About 5-day-old CAT2::GUS transgenic seedlings were treated with 0.25 μM MeJA or 125 mM NaCl for 1 day and then incubated at 37°C in GUS staining solution (100 mM sodium phosphate buffer, pH 7.5, 10 mM EDTA, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 1 mM 5-bromochloro-3-indolyl-b-D-glucuronide, and 0.1 % Triton X-100). Then, the seedlings were rinsed with 70% ethanol several times to remove the chlorophyll, and images were captured using a digital camera.

Jasmonic acid levels were determined according to our previously reported methods (Yuan et al., 2017; Zhang et al., 2020a). Briefly, about 200 mg of the treated and untreated seedlings were collected and ground into fine powder under liquid nitrogen. Dihydrojasmonic acid (DHJA) of 20 ng was added as an internal standard for JA. The purified samples were then methylated by diazomethane, re-suspended in 100 μl ethyl acetate, and analyzed by gas chromatography-mass spectrometry-selected ion monitoring (GC/MS-SIM) (Trace GC Ultra/ISQ, Thermo Fisher Scientific, Waltham, MA, USA). The compounds were separated on an Rtx-5MS (30 mm × 0.25 mm × 0.25 mm) column. The chromatographic parameters were as follows: 40°C for 1 min after injection, followed by sequential temperature ramps of 25°C/min to 150°C, 5°C/min to 200°C, 10°C/min ramp to 240°C, and 240°C for 10 min. The monitored ions were m/z 151 and 153 for JA and DHJA, respectively.

Data are means (±SD) of three biological replicates, and the asterisks indicate statistically significant differences (^*p < 0.05, ^**p < 0.01, and ^***p < 0.001, Student's t-test). Bars with different letters indicate significant differences at p < 0.05 by two-way ANOVA with Tukey's multiple comparison test.

Previous reports documented different roles of JA in salt stress tolerance in different plant species (Velitchkova and Fedina, 1998; Qiu et al., 2014; Hazman et al., 2015; Kurotani et al., 2015; Valenzuela et al., 2016). Our recent report showed that JA represses CAT2 expression to stimulate leaf senescence (Zhang et al., 2020a), shedding a light on whether and how JA affect plant salt stress response through the modulation of CAT2 expression. To understand how JA involves plant response to high salinity, we first determined JA accumulation treated with or without salt stress. About 5-day-old wild-type plant seedlings were transferred onto 1/2 MS medium containing different concentrations of additional NaCl and treated for 5 days. Our results showed that JA accumulation in salt-treated plants was significantly higher than that in the untreated control (Figure 1A), indicating that salt stress induces JA accumulation in plants.

To dissect the effect of JA on salt stress response in Arabidopsis, we examined the seed germination of the wild-type plant on 1/2 MS medium with the high salinity in the presence or absence of JA. Our results showed that low concentration of exogenous JA alone did not but high salinity significantly affected seed germination and cotyledon greening process, and exogenous JA further enhanced the salt stress effects (Figures 1B,C), suggesting a negative regulatory role of JA in plant salt stress tolerance. To verify this, 5-day-old wild-type plant seedlings were subjected to high salinity in the presence or absence of JA. Similarly, JA also increased plant sensitivity to salt stress as evidenced by shorter newly grown roots and lower seedling fresh weight in salt plus JA-treated plants compared with those of the salt-treated plants (Figures 1D–F). Consistently, two-way ANOVA analysis showed that the interaction between the two treatments (MeJA and NaCl) in Figure 1F had significant differences (Supplementary Table 2), further supporting that exogenous JA decreases plant seedling salt stress tolerance.

To confirm the role of endogenous JA in plant salt stress tolerance, a loss-of-function mutant of JASMONATE RESISTANT1 (JAR1), jar1, which is defective in catalyzing the formation of bioactive JA, JA-Ile (Staswick et al., 2002), was employed. Compared with the wild-type plant, the jar1 mutant had higher germination and cotyledon greening rates when subjected to salt stress (Figures 2A–C). Consistently, the jar1 mutant exhibited higher salt stress tolerance than the wild-type plant in terms of newly grown root length and seedling fresh weight (Figures 2D–F), further supporting that JA represses plant tolerance to salt stress.

Salt stress severely disturbs normal cellular physiological processes in plants, resulting in extensively higher accumulation of ROS, such as H₂O₂. It has been reported that increased antioxidant ability confers plants an enhanced salt stress tolerance (Hernandez et al., 2000). Therefore, we sought to investigate whether JA reduces plant salt stress tolerance through changing H₂O₂ accumulation by performing a DAB staining experiment. Our results showed that the wild-type plants treated with both salt stress and JA had obviously higher H₂O₂ accumulation than the salt-stressed plants (Figures 3A,B), indicating that exogenous JA promotes salt stress-induced H₂O₂ accumulation.

To test whether the effect of JA on plant salt stress tolerance was primarily due to its promotion of H₂O₂ accumulation, a ROS GSH was used to treat the plant in the presence of high salinity and JA. Our results showed that exogenously applied GSH significantly reduced the H₂O₂ accumulation of the plants treated with JA and salt stress (Figures 3A,B). In addition, seed germination, root length, and fresh weight were higher in both salt- and GSH-treated plants than those in the salt-treated control (Figures 3C–H). We also detected H₂O₂ levels in the jar1 mutant treated with salt stress. We found that, consistent with its increased salt stress tolerance, the jar1 mutant accumulates less H₂O₂ than the wild-type plant when treated with high salinity (Figure 3I). These results indicate that increased JA impairs plant salt stress tolerance at least partially due to promoting ROS accumulation.

Our above results showed that JA weakens plant salt stress tolerance through increasing H₂O₂ accumulation, prompting us to further study whether CAT is regulated by JA in plant response to salt stress as JA downregulates CAT2 expression during leaf senescence in our previous report (Zhang et al., 2020a). To do this, we first assayed the expression of CAT2 in the wild-type and jar1 mutant seedlings treated with high salinity, and our qRT-PCR results showed that salt stress treatment extensively induced the expression of CAT2 both in the wild-type and jar1 mutant, while the jar1 mutant had significantly higher CAT2 expression than the wild-type (Figure 4A), revealing that JA negatively regulates CAT2 expression in plant under salt stress conditions. To strengthen this note, we also examined CAT2 expression in salt-treated wild-type plant seedlings in the presence or absence of JA. We found that high salinity stress activates CAT2 expression while JA dampened its expression in the wild-type plant (Figure 4B). Such effect of JA on CAT2 expression in plant response to salt stress was confirmed by using the transgenic plant of GUS reporter gene driven by CAT2 promoter (Zhang et al., 2020a), and our results showed that the GUS staining in salt-treated CAT2::GUS plants was darker than that in the untreated control while this staining was extensively repressed by JA (Figure 4C), indicating that JA downregulates CAT2 expression in plant response to high salinity. In line with this, the CAT activity in both JA and salt-treated wild-type plants was significantly lower than that of the salt-treated plants (Figure 4D).

MYC2 is a master transcriptional factor regulating the expression of most JA downstream genes (Kazan and Manners, 2013; Santino et al., 2013). Therefore, we assessed whether MYC2 involves JA-regulated CAT2 expression in plant response to salt stress. We treated the wild-type and myc2 mutant with the high salinity in the presence or absence of JA and assayed their CAT2 expression using qRT-PCR. Our results showed that salt-induced CAT2 expression was largely repressed by JA in the wild-type plant but not in the myc2 mutant (Figure 4B). In addition, the GUS staining in the myc2 CAT2::GUS plant was darker than that in the CAT2::GUS plant when treated with both salt and JA (Figure 4C), revealing that MYC2 is required for JA-mediated suppression of CAT2 expression in plant response to salt stress. Further, we examined the CAT activity in the wild-type and myc2 mutant plants treated with salt and JA, and we found that JA treatment inhibited CAT activity in salt-treated wild-type plant while such effect of JA was largely compromised in the myc2 mutant (Figure 4D), further supporting the role of MYC2 in JA-mediated repression of CAT2 expression in plant response to salt stress.

Increased CAT2 expression and CAT activity of the myc2 mutant suggested higher salt stress tolerance. Thus, we evaluated the tolerance of the myc2 mutant to high salinity. Our results showed that the myc2 mutant seeds had higher germination and cotyledon greening rate than the wild-type plant when germinating on 1/2 MS medium containing high salinity plus or minus JA (Figures 5A–C). In addition, the myc2 mutant exhibited higher salt stress tolerance than the wild-type plant when challenged with salt stress as evidence by longer newly grown root and higher seedling fresh weight of the mutant in the presence or absence of JA (Figures 5D–F). These results clearly indicated that MYC2 participates in JA-mediated negative regulation of plant salt stress tolerance.

Our above results showed that JA suppresses CAT2 expression in an MYC2-dependent manner in plant response to salt stress. To genetically support this note, we further determined the salt stress phenotype of the myc2 cat2-1 double mutant. We found that the cat2-1 mutant was indeed very sensitive to high salinity as previously reported (Bueso et al., 2007; Li et al., 2015), while the increased salt stress tolerance of the myc2 mutant was completely reverted by the mutation of CAT2 in myc2 cat2-1 mutant in terms of seed germination, newly grown root length, and seedling fresh weight (Figures 6A–F). Consistently, lower ROS accumulation in the myc2 mutant compared with that in the wild-type plant was significantly increased by the cat2-1 mutation (Figures 6G,H).

Together with the above results, our study reveals that JA reduces Arabidopsis plant salt stress tolerance by repressing CAT2 expression in an MYC2-dependent manner.