Montecarlo hybrid tomato seeds purchased from a commercial nursery (Seminis, Barásoain, Navarra, Spain) were used as plant material for exogenous Spm experiments. Tomato seeds were cultivated in seedling trays filled with peat moss, perlite and vermiculite (80:10:10). After germination, seedlings were transplanted to 10-cm diameter pots filled with peat moss and maintained under greenhouse conditions (70% relative humidity, 200 µmol photons m^-2 s^-1 light intensity, natural photoperiod day/night cycle with temperatures averaging 25.0°C and 18.0°C, respectively) and watered three times a week with half-strength Hoagland solution. Temperature and relative humidity were recorded regularly with a portable USB datalogger (OM-EL-WIN-USB, Omega, NJ, United States).

To study the effect of exogenous Spm in the tolerance of plants to different stresses and their combination, we treated 4-week-old tomato plants with Spm by watering with 0.5 L of distilled water containing 0.5 mM Spm (Sigma-Aldrich, St. Louis, MO, USA; Xu et al., 2020) once a day during a week, and control (CT) plants were watered in parallel with 0.5 L of distilled water ( Supplementary Figure 1 ). After finishing Spm treatments, Spm-treated and not treated plants were subjected to four different conditions: CT, salinity (S, 150 mM NaCl), herbicide paraquat (PQ, 1.5 μM PQ), and the combination of salinity and paraquat (S+PQ, 150 mM NaCl + 1.5 μM PQ). Plants were watered three times a week for 15 days with each stressor described above (S, PQ and S+PQ) in a half-strength Hoagland solution. Therefore, eight experimental groups were designed (CT, CT+Spm, S, S+Spm, PQ, PQ+Spm, S+PQ, S+PQ+Spm), subjecting 5 plants to each stress treatment, and all experiments were repeated at least three times ( Supplementary Figure 1 ). Once all stress treatments were completed, the number of healthy leaves (leaves with no symptoms of damage; Balfagón et al., 2019) and plant height were scored for all control and stressed plants, followed by sampling leaves in an intermediate position in the canopy in liquid N₂. Samples were stored at -80°C until further use. For each analysis described below, at least three independent technical repeats per biological repeat and experimental group were performed.

Photosynthetic rate, stomatal conductance, transpiration rate and PSII efficiency were measured simultaneously on plants subjected to the different stress treatments between 9:30 and 11:30 A.M. Leaf gas exchange parameters (photosynthetic rate, stomatal conductance and transpiration rate) were measured by using a LCpro⁺ portable infrared gas analyzer (LI-6800, LICOR, Lincoln NE, USA) under ambient CO₂ and moisture. After instrument stabilization, six measurements were taken on three different mature fully expanded leaves in three replicate plants from each treatment. PSII efficiency was analyzed on the same leaves and plants using a portable fluorometer (FluorPen FP-MAX 100, Photon Systems Instruments, Czech Republic).

Approximately 200 mg of ground frozen leaf tissue was homogenized in 2 mL of 80% ethanol (Panreac) by sonication for 30 min. Homogenates were then centrifuged at 12000 g for 10 min and different aliquots of the supernatant were mixed either with 20% trichloroacetic acid or with a mixture of 20% trichloroacetic acid and 0.5% thiobarbituric acid. Both mixtures were incubated in a water bath at 90°C for 1 h and, after cooling down, samples were centrifuged at 5000 g for 5 min at 4°C. The absorbance of the supernatant was read at 440, 534 and 600 nm against a blank, and malondialdehyde (MDA) concentration was calculated as described in Zandalinas et al. (2017).

H₂O₂ accumulation in leaves was measured by using a commercial kit (Amplex Red hydrogen peroxide-peroxidase assay, Molecular Probes/Invitrogen, Eugene, OR, USA) with few modifications. Briefly, 500 μL of 50 mM sodium phosphate buffer at pH 7.4, containing 50 μM of Amplex Red reagent and 0.05 U mL^-1 of horseradish peroxidase, was added to approximately 40 mg of frozen leaf tissue and incubated for 30 min at room temperature in darkness. Then, samples were centrifuged at 12000 g for 12 min at 4°C and 50 μL of supernatants were transferred into new opaque tubes. Absorbance at 560 nm was measured by using a NanoDrop Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). The concentration of H₂O₂ in each sample was determined from a standard curve consisting of 0, 0.5, 1, 3, 6, and 9 μM H₂O₂. After absorbance measurements, H₂O₂ accumulation per mg of fresh weight was calculated.

RNA was extracted from frozen leaf tissue using an RNeasy Mini kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Total RNA concentration and purity were determined using a Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE) from the ratio of absorbance readings at 260 and 280 nm. Reverse transcription was performed from 1 μg of total RNA using Primer script RT reagent with oligo(dT) primer (Takara Bio Inc., Kusatsu, Japan). The specific primers used for the amplification of each gene are included in Supplementary Table 1 . Primer pairs used for the tomato amplification were designed using free surface Primer3Plus (https://www.primer3plus.com) and NCBI database. Relative expression analysis by RT-qPCR were performed in a StepOne Real-Time PCR system (Applied Biosystems, Foster City, CA, United States). The reaction mixture contained 1 μL of cDNA, 5 μL of SYBRGreen (Applied Biosystems) and 1 μM of each gene-specific primer pair in a final volume of 10 μL. The thermal profile used to analyze the relative gene expression consisted of 10 min at 95°C for pre-incubation, followed by 40 cycles of 10 s at 95°C for denaturation, 10 s at 60°C for annealing and 20 s at 72°C for the extension. Amplicon specificity of the PCR reaction was evaluated by the presence of a single peak in the dissociation curve after the amplification steps. The expression levels of all genes were normalized against the expression of two endogenous control genes (actin [Solyc03g078400] and GAPDH [Solyc05g014470]) based on previous housekeeping selection for tomato (Mascia et al., 2010) and the relative expression was calculated by using REST (Pfaffl et al., 2002). For all genes studied, the reference for S, PQ and S+PQ samples was the expression value obtained for the CT conditions, whereas the reference for S+Spm, PQ+Spm and S+PQ+Spm was the expression value obtained for the CT+Spm conditions. Reference conditions were set as 1. Three technical replicates were analyzed on each biological replicate.

To determine the superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) activities, about 100 mg of frozen leaf tissue was extracted in 2 mL of phosphate buffer at pH 7 in a ball mill (MillMix20, Domel, Železniki, Slovenija). After centrifugation at 14000 g at 4°C for 10 min, supernatants were recovered. Different buffers were used for each enzyme extraction: for SOD, 50 mM phosphate buffer (pH 6.8) with 1.33 mM diethyl-diamino-pentaacetic acid; for CAT and GR, 50 mM phosphate buffer (pH 6.8 and pH 7.5, respectively). The SOD activity was determined following the O 2    − -induced reduction of nitroblue tetrazolium using the xanthine–xanthine oxidase system. CAT activity was determined using the H₂O₂-dependent reduction of titanium chloride. The GR activity was studied following the increase in the absorbance at 412 nm during 2 min as result of the production of the adduct DTNB-GSH after GSSG reduction. The reaction was initiated by adding 10 µL of enzyme extract and the increment in absorbance was recorded during 2 min at 265 nm. Enzyme activity was expressed as enzyme unit (U) per mg of protein. Total protein amount of each extract was determined following the method of Bradford (1976) using Bovine Serum Albumin (BSA) as a protein standard. Further details on enzyme assays are provided in Hossain et al. (2009).

To determine the total antioxidant enzyme activity inhibition (Lado et al., 2016), 50 mg of frozen leaf tissue was extracted in 1 mL of MeOH 80% in a ball mill (MillMix20, Domel, Železniki, Slovenija). After centrifugation at 14000 g at 4°C for 10 min, supernatants were recovered. A buffer containing 738 µM ABTS (Sigma-Aldrich, St. Louis, MO, USA) and 245 µM K₂S₂O₈ (Sigma-Aldrich, St. Louis, MO, USA) was prepared and incubated at room temperature in darkness. Then, a blank with the previous solution buffer was adjusted to an absorbance of 700 nm, and 10 µL of each extract was added to 1 mL of buffer mix. Measurements at 734 nm were recorded at different times (0, 0.5, 1 and 2 mins). Antioxidant activities were determined using ABTS reduction in potassium persulfate and were calculated as U per mg of protein contained in each extract.

Results are presented as the mean ± SD. Statistical analyses were performed by two-tailed Student’s t-test. * denotes statistical significance at P< 0.05 with respect to CT and * means statistical significance at P< 0.05 between Spm-treated and non-treated plants exposed to the same stress condition.

To study the survival and growth of Spm-treated and non-treated tomato plants in response to S, PQ and their combination (S+PQ), plants were subjected to 150 mM NaCl, 1.5 µM PQ or 150 mM NaCl and 1.5 µM PQ, respectively, for 15 days ( Supplementary Figure 1 ). As shown in Figure 1A , survival of tomato plants not treated with Spm significantly decreased in response to S (around 80%) and more markedly in plants subjected to S+PQ (around 60%), whereas all plants survived when subjected to PQ. Interestingly, all Spm-treated plants subjected to the different stresses survived at the end of the different stress treatments ( Figure 1A ). Similarly, plants treated with Spm and subjected to S, PQ and S+PQ showed more healthy leaves and less visible toxicity symptoms such as chlorosis and necrosis (Balfagón et al., 2019), compared to plants not treated with Spm and subjected to the different stresses ( Figure 1B ). In addition, Spm treatment significantly increased tomato growth under control or stress conditions of PQ and S+PQ ( Figure 1C ).

To determine whether Spm could affect the photosynthesis and different gas exchange parameters of control plants, and plants subjected to S, PQ and S+PQ, photosynthetic rate, PSII efficiency, transpiration rate and stomatal conductance were monitored in plants treated and non-treated with Spm ( Figure 2 ). Photosynthetic rate levels significantly decreased in response to all individual and combined stresses in non-treated plants compared to CT, reaching the lowest value when plants were subjected to S+PQ ( Figure 2A ). Interestingly, exogenous Spm application enhanced the photosynthetic rate under each individual and combined stress. In this sense, Spm-treated plants subjected to S, PQ and S+PQ increased by 6.78%, 7.67% and 33.3%, respectively, their photosynthetic rate. PSII efficiency was negatively affected by S and S+PQ, and Spm treatment increased these values by 13.21% and 8.29%, respectively ( Figure 2B ). Not surprisingly, stomatal conductance of plants treated and non-treated with Spm from the different stresses similarly corresponded to the transpiration rates measured in these plants ( Figures 2C, D ). Therefore, whereas plants subjected to S and S+PQ, and non-treated with Spm reduced transpiration rate and stomatal conductance levels, PQ did not change or slightly increased these values. Interestingly, Spm treatment increased both gas exchange parameters in CT plants as well as in plants subjected to PQ and S+PQ with respect to the corresponding non-treated stressed plants ( Figures 2C, D ). Taking together, Spm could be involved in promoting stomata aperture in response to PQ and S+PQ, as well as under control conditions.

To gain a better understanding of the impact of each stress condition and the Spm-associated changes on the oxidative stress, H₂O₂ levels, MDA content, RT-qPCR analyses of transcripts involved in detoxification pathways, as well as different antioxidant enzyme (SOD, GR and CAT) activities were studied ( Figures 3 , 4 ; Supplementary Figure 2 ). Plants not treated with Spm and subjected to the different stresses significantly increased their leaf H₂O₂ levels compared to CT, whereas Spm treatment reduced the amount of H₂O₂ accumulated in plants subjected to S, PQ and S+PQ with respect to the corresponding stressed plants not treated with Spm ( Figure 3A ). In addition, the degree of lipid peroxidation in tomato plants was studied by monitoring changes in MDA levels (Taulavuori et al., 2001). As shown in Figure 3B , the level of lipid peroxidation decreased by about 56% and 12% in plants subjected to PQ+Spm and S+PQ+Spm, with respect to PQ and S+PQ, respectively.

The activation of detoxification pathways in Spm-treated and non-treated tomato plants subjected to the different stress conditions was evaluated by assessing the expression of FeSOD, Cu/ZnSOD1, Cu/ZnSOD2, MnSOD, CAT1, GPX, cAPX and GR transcripts as well as enzymatic activities of SOD, GR and CAT ( Figure 4 ; Supplementary Figure 2 ). In general, Spm treatment modified the expression of all the transcripts tested involved in detoxification processes compared to that observed in stressed plants not treated with Spm ( Figure 4A ; Supplementary Figure 2 ). The expression of Cu/ZnSOD1, Cu/ZnSOD2, CAT1 and GPX was, in general, attenuated due to the effect of Spm treatment in plants subjected to the different stresses, whereas the expression of cAPX and GR1 was enhanced as a result of exogenous Spm application compared to that shown in stressed plants not treated by Spm ( Figure 4A ; Supplementary Figure 2 ). To further analyze the role of Spm in ROS scavenging processes, the activities of SOD, GR and CAT were evaluated ( Figures 4B–D ). S or S+Spm did not alter any enzymatic activity, and PQ increased GR activity. Under S+PQ conditions, a significant decrease of SOD and GR enzymatic activities in Spm-treated plants by 21.21% and 66.67%, respectively, was observed compared to non-treated plants subjected to S+PQ. On the contrary, CAT activities did not show any alteration in response to stress in both Spm-treated and non-treated plants ( Figure 4D ).

Additionally, to determine the total antioxidant capacity of plants, analysis of the inhibition of total antioxidant activities was performed ( Table 1 ). Spm treatment enhanced the total antioxidant capacity of plants under CT conditions. Moreover, PQ and S+PQ resulted in increased levels of total antioxidant activity compared to CT values. By contrast, values of total inhibition of antioxidant activities decreased in plants subjected to S+PQ+Spm, compared to S+PQ ( Table 1 ).