L-Aminoguanidine Induces Imbalance of ROS/RNS Homeostasis and Polyamine Catabolism of Tomato Roots after Short-Term Salt Exposure

Polyamine (PA) catabolism mediated by amine oxidases is an important process involved in fine-tuning PA homeostasis and related mechanisms during salt stress. The significance of these amine oxidases in short-term responses to salt stress is, however, not well understood. In the present study, the effects of L-aminoguanidine (AG) on tomato roots treated with short-term salt stress induced by NaCl were studied. AG is usually used as a copper amine oxidase (CuAO or DAO) inhibitor. In our study, other alterations of PA catabolism, such as reduced polyamine oxidase (PAO), were also observed in AG-treated plants. Salt stress led to an increase in the reactive oxygen and nitrogen species in tomato root apices, evidenced by in situ fluorescent staining and an increase in free PA levels. Such alterations were alleviated by AG treatment, showing the possible antioxidant effect of AG in tomato roots exposed to salt stress. PA catabolic enzyme activities decreased, while the imbalance of hydrogen peroxide (H2O2), nitric oxide (NO), and hydrogen sulfide (H2S) concentrations displayed a dependence on stress intensity. These changes suggest that AG-mediated inhibition could dramatically rearrange PA catabolism and related reactive species backgrounds, especially the NO-related mechanisms. More studies are, however, needed to decipher the precise mode of action of AG in plants exposed to stress treatments.


Introduction
Salt stress is one of the most important abiotic stress factors threatening agriculture and food security globally [1]. Under increasingly challenging climate conditions, more efforts are needed to understand the mechanisms of salt stress and salt tolerance of crop plants [2]. Plants have evolved many different strategies to cope with salinity stress, for example, by synthesizing compounds that alleviate salt stress injury [3].
Polyamines (PAs) are hub molecules in drought and salt stress in plants [4,5]. These essential N-containing molecules occur in different forms in plants, can be free, bound Our knowledge about the involvement of PAs and their catabolism in short-term salt stress is limited [47], and their contribution to triggering other signaling pathways has not been studied. The objective of this research was to study and give an overview of the short-term effect of salt stress and AG treatment on PA catabolism and related pathways to decipher the significance of PA catabolism and related pathways in the regulation of the fast stress responses of tomato roots.

Plant Material and Growth Conditions
The tomato plants used in this study were Solanum lycopersicum Mill. L. cv. Rio Fuego. Seeds were germinated at 26 • C for 3 d in the dark, and the seedlings were subsequently transferred to perlite for 2 weeks. The plants were then placed in a hydroponic culture, as described [8]. The plants were grown for 6 weeks in a controlled environment under 200 µmol m −2 s −1 photon flux density (F36W/GRO lamps, OSRAM SYLVANIA, Danvers, MA, USA), with a 12/12-h light/dark period, day/night temperatures of 24/22 • C, and relative humidity of 55-60%.

NaCl-and AG-Inhibitor Treatments
Salinity treatment was applied to 6-week-old tomato plants by supplementing the hydroponic solution with 100 and 250 mM NaCl for 1 h in the absence or presence of 1 mM L-aminoguanidine (AG). The concentration of AG was defined to inhibit DAO activity as described [37]. Whole root samples were used for biochemical analysis, whereas root apical tips were used for fluorescent staining. The experiments were repeated three times. In order to avoid the diurnal changes of PA homeostasis, sampling was performed at the same time in all experiments.

Polyamine Catabolic Enzyme Activities
DAO (EC 1.4.3.6) and PAO (EC 1.4.3.4) activities were estimated spectrophotometrically, as described by [26] with some modification. Approximately 200 mg of root tissue was homogenized in liquid N 2 , and 0.6 mL extraction buffer was added to each sample. The extraction buffer contained 0.2 M TRIS (hydroxymethyl)aminomethane (pH 8.0); 10% glycerol; 0.25% Triton X-100; 0.5 mM phenylmethanesulfonyl fluoride (PMSF); 0.01 mM leupeptin in 100 mM potassium phosphate buffer (pH 6.6). The homogenates were left on ice for 20 min and centrifuged for 10 min at 7000× g at 4 • C (Eppendorf centrifuge 5424R, Eppendorf GMBH, Hamburg, Germany). A 150 µL aliquot of the supernatant was combined with 600 µL 100 mM potassium phosphate buffer (pH 6.6), and then the reaction was started by adding 22.5 µL of 2-aminobenzaldehyde (from 10 mg/mL stock solution) and 1 M putrescine (Put) for DAO and 1 M spermidine (Spd) for PAO activity measurements. The reaction mixture was incubated for 1.5 h at 37 • C, and the reaction was stopped by adding 50 µL of 20% (w/v) trichloroacetic acid (TCA). The absorbance was determined at 430 nm (KONTRON, Milan, Italy). The enzyme activity was expressed as specific activity (U g −1 FW), where one unit (U) represents the amount of enzyme catalyzing the formation of 1 µmol of ∆ 1 -pyrroline min −1 .

Determination of Free PA Content Using HPLC
Free PA contents were determined as described by [37]. In brief, 200 mg of root was homogenized in 5% perchloric acid. After centrifugation, 2.5 mL of the supernatant was neutralized with 1 mL of 2 M NaOH, and PAs were then derivatized with 10 µL of benzoyl chloride and separated using HPLC. The applied standards were Put, Spd, and spermine (Spm) in the form of hydrochlorides. The results are the means of three independent biological samples expressed in nmol g −1 fresh weight −1 .

RNA Purification and Gene Expression Analyses with Quantitative Real-Time PCR
Total RNA was extracted from 100 mg of tomato root using GeneJET Plant RNA Purification Kit (Thermo Scientific™, K0801 from Thermo Fisher Scientific, Waltham, MA, USA) as recommended by the manufacturer. The isolated RNA was DNase-treated with a TURBO DNA-free™ Kit (Invitrogen by Thermo Fisher Scientific), and first-strand cDNA synthesis of 1 µg of total RNA was carried out with a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems by Thermo Fisher Scientific), using random hexamers. Real-time PCR was carried out with an ABI 7900 Fast Real-Time System (Applied Biosystems by Thermo Fisher Scientific) with the following protocol: 40 cycles at 95 • C for 15 s, 60 • C for 1 min, using Maxima SYBR Green qPCR Master Mix (Thermo Fisher Scientific). The relative expression levels were normalized to both the SlEF1 and SlUBI3 reference genes. The normalized relative transcript levels were calculated according to [48,49] using the 2 −∆∆Ct method, where the relative gene expression level of the untreated control was 1. The specific primers for each examined gene are described in Table S1 and related references are cited in the Supplementary Materials.

Hydrogen Peroxide Determination
The H 2 O 2 levels of the tomato tissues were measured in six-week-old plants using an Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Thermo Fisher Scientific, A22188) as recommended by the manufacturer. Approximately 100 mg of fresh plant material was harvested, ground in liquid N 2 , and diluted in 20 mM potassium phosphate buffer (pH 6.5). The homogenates were centrifuged, and the supernatant was used to measure the H 2 O 2 content. The accumulation of resorufin was determined spectrophotometrically at 560 nm (Thermo Scientific, Multiscan Go Microplate Spectrophotometer). The amount of H 2 O 2 was calculated using a standard curve.

Superoxide Dismutase Enzyme Activity Measurement
Enzyme extracts were prepared as described by [50]. SOD (EC 1.15.1.1) activity measurement was based on the ability of the enzyme to inhibit the photochemical reduction in p-nitro-blue tetrazolium chloride (Sigma-Aldrich, St. Louis, MO, USA) in the presence of riboflavin in the light. One enzyme unit (U) of SOD represents the amount of enzyme causing a 50% inhibition of p-nitro-blue tetrazolium chloride reduction, and its activity was calculated as U g -1 fresh weight.

Determination of SNOs and Nitrite Content
Approximately 100 mg of plant material was harvested immediately after the treatments. After freezing in liquid N 2 , the samples were homogenized twice for 10 s using a Silamat S6 tissue homogenizer (Ivoclar Vivadent, Schaan, Liechtenstein) and 1.7-2.0 mm glass beads (Roth). The homogenized root material was extracted in 500 µL 1× phosphatebuffered saline (PBS) and incubated on ice for 10 min, followed by centrifugation for 10 min Antioxidants 2023, 12, 1614 5 of 16 at 12,435 rpm. The protein content of the plant extract was determined using the Protein Assay Dye Reagent Concentrate (Bio-Rad, Hercules, CA, USA). The quantification of nitrite and SNOs in tomato root tissues was performed using Sievers' Nitric Oxide Analyzer NOA 280i (GE Water & Process Technologies, Ratingen, Germany) as described by [41]. Endogenous nitrite was reduced to gaseous NO by the injection of root extracts into a reaction vessel containing triiodide solution (28.5 mM I 2 , 66.9 mM KI in 77.7% acetic acid) at 30 • C. For SNO detection, endogenous nitrite was scavenged by adding 5% sulfanilamide (w/v, in 1 M HCl) at a dilution of 1:9 to the sample before injection into the triiodide solution.

GC-MS Analysis of TCA Cycle Metabolites
After sample collection, 0.1 g portions of the samples were ground in liquid N 2 and transferred into 2.0 mL safety Eppendorf tubes, containing 30 µL ribitol (1 mg/mL) as an internal standard (ISTD), in 0.5 mL of 60% (v/v) methanol. The tubes were vortex-mixed for 30 s, then placed in an ultrasound bath for 5 min, vortex-mixed again for 15 s, and centrifuged for 5 min at 10,000 rpm, and the supernatant was collected. The extraction was repeated with 0.5 mL of 60% (v/v) methanol and again with 0.5 mL of 90% (v/v) methanol. The supernatant was collected and mixed well. An aliquot (100 µL) was dried in vacuum. For derivatization, methoxyamine hydrochloride dissolved in pyridine (20 mg/mL) was added, incubated at 37 • C for 90 min., then N-trimethylsilyl-N-methyl trifluoroacetamide was added and incubated for 30 min at the same temperature.
The samples were transferred to vials and injected split-mode into the Shimadzu GCMS-TQ equipped with GC column (Phase: HP-5MS length 30 m; ID 0.25 mm; Film thickness: 0.25 µm). A total of 1 µL was injected into the column at 230 • C, and the transfer line and ion source were 250 • C. The carrier gas was He, and a constant flow rate (1 mL/min) was used. The thermal program was 70 • C for 1 min, which increased to 320 • C at a rate of 7 • C/min, and the high temperature was maintained for 5 min. The Kovats retention index was used to identify the standards, and GCMSSolution 4.16 for Shimadzu was used for data processing. Both the GC analyses and data processing were carried out with GCMSSolution 4.16 for Shimadzu using the Wiley and Nist databases.

Statistical Analysis
The experiments were carried out in three independent biological repetitions. The data are given as the mean ± standard deviation (SD), calculated from the combination of biological repetitions. Two-way analysis of variance (ANOVA) was carried out using GraphPad Prism version 8.0.1.244 for Windows (GraphPad Software, La Jolla, CA, USA), with significance level 0.05 (p ≤ 0.05) (interaction analysis can be seen in Table S3). Different letters on the bars denote significant differences (p = 0.05) based on Tukey's post hoc test for multiple comparisons.

AG Has a Nonspecific Inhibitory Effect on NaCl-Induced Concentration-Dependent Alterations in DAO and PAO Activities of Tomato Roots
In order to investigate the capacity of AG as a DAO inhibitor, 1 mM AG was added to the nutrient solution, and tomato roots were treated for 1 h. NaCl treatment alone did not affect DAO activity, but a significant reduction in PAO activity was detected by salt stress, which was concentration-dependent ( Figure 1). In the presence of AG, DAO activities in tomato roots were enhanced by increasing the concentration of salt ( Figure 1).

Free PA Levels Are Reduced by AG Treatment Independent of NaCl Concentration
In order to determine the effect of AG on PA metabolism, free PA concentrations were determined in salt-and AG-treated tomato roots. Total PA contents were lower in AGtreated roots, which were not influenced by salt stress. Put levels were considerably reduced by AG with or without salt treatment. Spd concentration was reduced by AG only in the presence of 250 mM NaCl, whereas Spm contents were not influenced by either AG or salt ( Figure 2).

Free PA Levels Are Reduced by AG Treatment Independent of NaCl Concentration
In order to determine the effect of AG on PA metabolism, free PA concentrations were determined in salt-and AG-treated tomato roots. Total PA contents were lower in AG-treated roots, which were not influenced by salt stress. Put levels were considerably reduced by AG with or without salt treatment. Spd concentration was reduced by AG only in the presence of 250 mM NaCl, whereas Spm contents were not influenced by either AG or salt ( Figure 2).

Free PA Levels Are Reduced by AG Treatment Independent of NaCl Concentration
In order to determine the effect of AG on PA metabolism, free PA concentrations were determined in salt-and AG-treated tomato roots. Total PA contents were lower in AGtreated roots, which were not influenced by salt stress. Put levels were considerably reduced by AG with or without salt treatment. Spd concentration was reduced by AG only in the presence of 250 mM NaCl, whereas Spm contents were not influenced by either AG or salt ( Figure 2).  Free polyamine contents of control and AG-treated tomato roots with and without NaCl treatment (100 and 250 mM). Data of PA contents are the mean ± SD of three biological replicates. Different letters denote significant differences, two-way ANOVA, following Tukey's post hoc test p = 0.05.

AG Induces Expression of PA Biosynthetic and Catabolic Genes
In order to study the effect of AG on the activity of genes implicated in the biosynthesis of PAs, transcript levels of ADC1 and ADC2 genes encoding arginine decarboxylase, and ODC1 coding for ornithine decarboxylase were determined. Both ADC1 and ADC2 were induced by salt, to different degrees. ADC1 transcript levels were 2 and 12 times higher in 100 mM and 250 mM NaCl-treated roots, respectively. AG treatment slightly enhanced ADC1 expression in control roots and in the presence of 100 mM NaCl (two and three times, respectively), whereas it reduced ADC1 expression by 60% compared to its 250 mM NaCl-treated control when stronger salt stress was applied. ADC2 expression was increased three-fold in salt-treated roots. AG slightly enhanced ADC2 transcription in control and 100 mM NaCl-treated roots (1.75-and 4.1-fold, respectively), whereas expression of this gene was slightly reduced only by 28% with AG at stronger salt stress ( Figure 3). ODC1 expression was 1.5-fold increased by AG treatment in roots treated with no or 100 mM NaCl, but it was reduced by 250 mM NaCl with or without AG ( Figure S1).

AG Induces Expression of PA Biosynthetic and Catabolic Genes
In order to study the effect of AG on the activity of genes implicated in th biosynthesis of PAs, transcript levels of ADC1 and ADC2 genes encoding arginin decarboxylase, and ODC1 coding for ornithine decarboxylase were determined. Bot ADC1 and ADC2 were induced by salt, to different degrees. ADC1 transcript levels wer 2 and 12 times higher in 100 mM and 250 mM NaCl-treated roots, respectively. AG treatment slightly enhanced ADC1 expression in control roots and in the presence of 10 mM NaCl (two and three times, respectively), whereas it reduced ADC1 expression b 60% compared to its 250 mM NaCl-treated control when stronger salt stress was applied ADC2 expression was increased three-fold in salt-treated roots. AG slightly enhance ADC2 transcription in control and 100 mM NaCl-treated roots (1.75-and 4.1-fold respectively), whereas expression of this gene was slightly reduced only by 28% with AG at stronger salt stress ( Figure 3). ODC1 expression was 1.5-fold increased by AG treatmen in roots treated with no or 100 mM NaCl, but it was reduced by 250 mM NaCl with o without AG ( Figure S1). To test whether the altered enzyme activities were derived from the changes in gen expression, we determined the transcript levels of the PA catabolism genes. CuAO expression was slightly reduced, only by 30% after AG treatment and only in 100 mM NaCl-treated roots ( Figure 4). Considerable variation was observed when the expressio of PAO genes [21] was tested. Transcript levels of PAO1 were enhanced by salt in concentration-dependent manner, displaying 30-fold induction in 250 mM NaCl-treate roots. AG enhanced PAO1 expression at moderate salt stress but reduced it when high sa stress was applied. Expression of PAO2 was not affected either by 100 mM NaCl or by AG at this salt concentration, while 250 mM NaCl induced PAO2 activity 2.5-fold, which wa reduced in the presence of AG. Transcript levels of PAO4 were reduced to half by sa treatments but were not affected by AG (Figures 4 and S2). Collectively, the PAO gene showed higher expression in the 250 mM NaCl-treated plants, which was reduced by AG except for PAO4 and PAO5. To test whether the altered enzyme activities were derived from the changes in gene expression, we determined the transcript levels of the PA catabolism genes. CuAO expression was slightly reduced, only by 30% after AG treatment and only in 100 mM NaCl-treated roots ( Figure 4). Considerable variation was observed when the expression of PAO genes [21] was tested. Transcript levels of PAO1 were enhanced by salt in a concentration-dependent manner, displaying 30-fold induction in 250 mM NaCl-treated roots. AG enhanced PAO1 expression at moderate salt stress but reduced it when high salt stress was applied. Expression of PAO2 was not affected either by 100 mM NaCl or by AG at this salt concentration, while 250 mM NaCl induced PAO2 activity 2.5-fold, which was reduced in the presence of AG. Transcript levels of PAO4 were reduced to half by salt treatments but were not affected by AG (Figures 4 and S2). Collectively, the PAO genes showed higher expression in the 250 mM NaCl-treated plants, which was reduced by AG, except for PAO4 and PAO5.   Figure 3. Data of each bar are the mean ± SD of three biological replicates. Asterisks denote significant differences from untreated control, Student t test, ** p < 0.01, *** p < 0.001.

ROS Levels Are Reduced by AG
To test whether H2O2 could be altered by reduced PA catabolism, H2O2 levels were determined in AG-and salt-treated roots. Salt stress enhanced H2O2 content in a concentration-dependent manner, which was blocked by AG treatment. To further investigate peroxide-derived H2O2 generation, we subsequently tested the influence of salt and AG on SOD activity. Salt stress slightly reduced SOD, which was inhibited by AG in the absence of salt or when roots were treated by 100 mM NaCl. At higher salt stress, AG had no influence on SOD ( Figure 5). These results suggested that SOD activity can be affected by AG in tomato roots, but its influence on H2O2 content is negligible. To test the influence of AG on superoxide production, the accumulation of superoxide anions was tested using in situ DHE assay. Superoxide levels were enhanced by 100 mM but not by 250 mM NaCl. AG had either no or a slightly negative effect on superoxide levels in the root tips ( Figure 6).  Figure 3. Data of each bar are the mean ± SD of three biological replicates. Asterisks denote significant differences from untreated control, Student t test, ** p < 0.01, *** p < 0.001.

ROS Levels Are Reduced by AG
To test whether H 2 O 2 could be altered by reduced PA catabolism, H 2 O 2 levels were determined in AG-and salt-treated roots. Salt stress enhanced H 2 O 2 content in a concentrationdependent manner, which was blocked by AG treatment. To further investigate peroxidederived H 2 O 2 generation, we subsequently tested the influence of salt and AG on SOD activity. Salt stress slightly reduced SOD, which was inhibited by AG in the absence of salt or when roots were treated by 100 mM NaCl. At higher salt stress, AG had no influence on SOD ( Figure 5). These results suggested that SOD activity can be affected by AG in tomato roots, but its influence on H 2 O 2 content is negligible.   Figure 3. Data of each bar are the mean ± SD of three biological replicates. Asterisks denote significant differences from untreated control, Student t test, ** p < 0.01, *** p < 0.001.

ROS Levels Are Reduced by AG
To test whether H2O2 could be altered by reduced PA catabolism, H2O2 levels were determined in AG-and salt-treated roots. Salt stress enhanced H2O2 content in a concentration-dependent manner, which was blocked by AG treatment. To further investigate peroxide-derived H2O2 generation, we subsequently tested the influence of salt and AG on SOD activity. Salt stress slightly reduced SOD, which was inhibited by AG in the absence of salt or when roots were treated by 100 mM NaCl. At higher salt stress, AG had no influence on SOD ( Figure 5). These results suggested that SOD activity can be affected by AG in tomato roots, but its influence on H2O2 content is negligible. To test the influence of AG on superoxide production, the accumulation of superoxide anions was tested using in situ DHE assay. Superoxide levels were enhanced by 100 mM but not by 250 mM NaCl. AG had either no or a slightly negative effect on superoxide levels in the root tips ( Figure 6). To test the influence of AG on superoxide production, the accumulation of superoxide anions was tested using in situ DHE assay. Superoxide levels were enhanced by 100 mM but not by 250 mM NaCl. AG had either no or a slightly negative effect on superoxide levels in the root tips ( Figure 6).

SNO Increase and Nitrite Reduction Could Be Responsible for Absence of Detectable NO
PAs are known to induce NO production in plants. We therefore determined NO levels in salt-and AG-treated tomato root tips using in situ DAF-FM DA fluorescent reaction. While 100 mM NaCl had no effect, 250 mM NaCl treatment led to significantly higher NO accumulation. AG treatment reduced the NO levels only in 250 mM NaCltreated samples (Figure 7). Quantitative data are expressed as pixel intensity. DAF-FM fluorescence was measured at 500 µm away from the root tips and averaged. Note that AG reduced the NO contents of root tips in all cases. Bars indicate ±SE of measurements performed with at least 10 seedlings per each treatment, different letters denote significant differences (two-way ANOVA, Tukey's post hoc test p = 0.05).
NO can be generated from nitrite and nitrosothiols (SNO). In order to compare NO levels with such precursors, nitrite and SNO levels were determined in salt-and AGtreated tomato roots. Nitrite content was higher in roots treated by 100 mM NaCl but was reduced by high salt. Nitrite concentrations were reduced by AG in all samples. SNO levels were enhanced by the NaCl treatments. SNO contents were similar in AG-treated roots in the absence of salt or in the presence of 100 mM NaCl, whereas AG enhanced SNO accumulation in plants stressed by a high concentration of salt (Figure 8). Figure 6. Detection of superoxide anions in control and AG-treated tomato root apices with and without NaCl treatment (100 and 250 mM). Superoxide anion content was measured using a fluorescence probe DHE (dihydroethidium) (scale bar: 100 µm) and expressed as pixel intensity. DHE fluorescence was measured at 500 µm away from the root tips and averaged. Significant reduction occurred in the 100 mM treatment after AG treatment. Bars indicate ±SE of measurements performed with at least 10 seedlings per each treatment, different letters denote significant differences (two-way ANOVA, Tukey's post hoc test p = 0.05).

SNO Increase and Nitrite Reduction Could Be Responsible for Absence of Detectable NO
PAs are known to induce NO production in plants. We therefore determined NO levels in salt-and AG-treated tomato root tips using in situ DAF-FM DA fluorescent reaction. While 100 mM NaCl had no effect, 250 mM NaCl treatment led to significantly higher NO accumulation. AG treatment reduced the NO levels only in 250 mM NaCl-treated samples (Figure 7). Antioxidants 2023, 12, x FOR PEER REVIEW 9 of 17 Figure 6. Detection of superoxide anions in control and AG-treated tomato root apices with and without NaCl treatment (100 and 250 mM). Superoxide anion content was measured using a fluorescence probe DHE (dihydroethidium) (scale bar: 100 µm) and expressed as pixel intensity. DHE fluorescence was measured at 500 µm away from the root tips and averaged. Significant reduction occurred in the 100 mM treatment after AG treatment. Bars indicate ±SE of measurements performed with at least 10 seedlings per each treatment, different letters denote significant differences (two-way ANOVA, Tukey's post hoc test p = 0.05).

SNO Increase and Nitrite Reduction Could Be Responsible for Absence of Detectable NO
PAs are known to induce NO production in plants. We therefore determined NO levels in salt-and AG-treated tomato root tips using in situ DAF-FM DA fluorescent reaction. While 100 mM NaCl had no effect, 250 mM NaCl treatment led to significantly higher NO accumulation. AG treatment reduced the NO levels only in 250 mM NaCltreated samples (Figure 7). Quantitative data are expressed as pixel intensity. DAF-FM fluorescence was measured at 500 µm away from the root tips and averaged. Note that AG reduced the NO contents of root tips in all cases. Bars indicate ±SE of measurements performed with at least 10 seedlings per each treatment, different letters denote significant differences (two-way ANOVA, Tukey's post hoc test p = 0.05).
NO can be generated from nitrite and nitrosothiols (SNO). In order to compare NO levels with such precursors, nitrite and SNO levels were determined in salt-and AGtreated tomato roots. Nitrite content was higher in roots treated by 100 mM NaCl but was reduced by high salt. Nitrite concentrations were reduced by AG in all samples. SNO levels were enhanced by the NaCl treatments. SNO contents were similar in AG-treated roots in the absence of salt or in the presence of 100 mM NaCl, whereas AG enhanced SNO accumulation in plants stressed by a high concentration of salt (Figure 8). Quantitative data are expressed as pixel intensity. DAF-FM fluorescence was measured at 500 µm away from the root tips and averaged. Note that AG reduced the NO contents of root tips in all cases. Bars indicate ±SE of measurements performed with at least 10 seedlings per each treatment, different letters denote significant differences (two-way ANOVA, Tukey's post hoc test p = 0.05).
NO can be generated from nitrite and nitrosothiols (SNO). In order to compare NO levels with such precursors, nitrite and SNO levels were determined in salt-and AGtreated tomato roots. Nitrite content was higher in roots treated by 100 mM NaCl but was reduced by high salt. Nitrite concentrations were reduced by AG in all samples. SNO levels were enhanced by the NaCl treatments. SNO contents were similar in AG-treated roots in the absence of salt or in the presence of 100 mM NaCl, whereas AG enhanced SNO accumulation in plants stressed by a high concentration of salt (Figure 8).

H2S Levels Displayed Different Changes during Salt Stress
Recent studies have suggested that H2S could interact with NO and H2O2 metabolic pathways [55]. Therefore, H2S concentrations were measured in salt-and AG-treated root tips, using a specific H2S staining method, WSP-1. H2S content was not affected by 100 mM NaCl but was increased nearly two-fold by 250 mM NaCl. AG treatment had no or only a slight influence on H2S concentrations (Figure 9). Figure 9. Detection of hydrogen sulfide in control and AG-treated tomato root apices with and without NaCl treatment (100 and 250 mM). Hydrogen sulfide content was measured using a fluorescence probe WSP-1 (Washington State Probe-1) (scale bar: 100 µm) and expressed as pixel intensity. WSP-1 fluorescence was measured at 500 µm away from the root tips and averaged. Bars indicate ±SE of measurements performed with at least 10 seedlings per each treatment, different letters denote significant differences (two-way ANOVA, following Tukey's post hoc test p = 0.05).

GABA and the TCA Cycle as a Possible Explanation of Decreased PA Levels
Put degradation could result in enhanced GABA production in plants [56]. As reduced PA levels could occur during AG application, we determined the concentrations of GABA and several TCA cycle metabolites in salt-and AG-treated roots ( Figure S3). GABA contents slightly increased after 1 h of salt stress, but the difference was not significant. AG reduced the GABA levels in control roots but had no effect on salt-treated roots.
To test possible changes in TCA cycle metabolites, concentrations of citrate, succinate, fumarate, and malate were measured in salt-and AG-treated tomato roots. Moderate salt stress (100 mM NaCl) had only a slight effect on these metabolites, with the exception of fumaric acid, whose concentration was elevated two-fold. Higher salt stress reduced concentrations of succinic, fumaric, and citric acids. AG treatment alone led to

H 2 S Levels Displayed Different Changes during Salt Stress
Recent studies have suggested that H 2 S could interact with NO and H 2 O 2 metabolic pathways [55]. Therefore, H 2 S concentrations were measured in salt-and AG-treated root tips, using a specific H 2 S staining method, WSP-1. H 2 S content was not affected by 100 mM NaCl but was increased nearly two-fold by 250 mM NaCl. AG treatment had no or only a slight influence on H 2 S concentrations ( Figure 9).

H2S Levels Displayed Different Changes during Salt Stress
Recent studies have suggested that H2S could interact with NO and H2O2 metabolic pathways [55]. Therefore, H2S concentrations were measured in salt-and AG-treated root tips, using a specific H2S staining method, WSP-1. H2S content was not affected by 100 mM NaCl but was increased nearly two-fold by 250 mM NaCl. AG treatment had no or only a slight influence on H2S concentrations (Figure 9). Figure 9. Detection of hydrogen sulfide in control and AG-treated tomato root apices with and without NaCl treatment (100 and 250 mM). Hydrogen sulfide content was measured using a fluorescence probe WSP-1 (Washington State Probe-1) (scale bar: 100 µm) and expressed as pixel intensity. WSP-1 fluorescence was measured at 500 µm away from the root tips and averaged. Bars indicate ±SE of measurements performed with at least 10 seedlings per each treatment, different letters denote significant differences (two-way ANOVA, following Tukey's post hoc test p = 0.05).

GABA and the TCA Cycle as a Possible Explanation of Decreased PA Levels
Put degradation could result in enhanced GABA production in plants [56]. As reduced PA levels could occur during AG application, we determined the concentrations of GABA and several TCA cycle metabolites in salt-and AG-treated roots ( Figure S3). GABA contents slightly increased after 1 h of salt stress, but the difference was not significant. AG reduced the GABA levels in control roots but had no effect on salt-treated roots.
To test possible changes in TCA cycle metabolites, concentrations of citrate, succinate, fumarate, and malate were measured in salt-and AG-treated tomato roots. Moderate salt stress (100 mM NaCl) had only a slight effect on these metabolites, with the exception of fumaric acid, whose concentration was elevated two-fold. Higher salt stress reduced concentrations of succinic, fumaric, and citric acids. AG treatment alone led to Figure 9. Detection of hydrogen sulfide in control and AG-treated tomato root apices with and without NaCl treatment (100 and 250 mM). Hydrogen sulfide content was measured using a fluorescence probe WSP-1 (Washington State Probe-1) (scale bar: 100 µm) and expressed as pixel intensity. WSP-1 fluorescence was measured at 500 µm away from the root tips and averaged. Bars indicate ±SE of measurements performed with at least 10 seedlings per each treatment, different letters denote significant differences (two-way ANOVA, following Tukey's post hoc test p = 0.05).

GABA and the TCA Cycle as a Possible Explanation of Decreased PA Levels
Put degradation could result in enhanced GABA production in plants [56]. As reduced PA levels could occur during AG application, we determined the concentrations of GABA and several TCA cycle metabolites in salt-and AG-treated roots ( Figure S3). GABA contents slightly increased after 1 h of salt stress, but the difference was not significant. AG reduced the GABA levels in control roots but had no effect on salt-treated roots.
To test possible changes in TCA cycle metabolites, concentrations of citrate, succinate, fumarate, and malate were measured in salt-and AG-treated tomato roots. Moderate salt stress (100 mM NaCl) had only a slight effect on these metabolites, with the exception of fumaric acid, whose concentration was elevated two-fold. Higher salt stress reduced concentrations of succinic, fumaric, and citric acids. AG treatment alone led to reduced amounts of all TCA metabolites tested. In combination with salt stress, AG treatment had no or only a minor influence on the concentrations of these metabolites ( Figure S3 and Table S2).

Discussion
The function of PA catabolism and the enzymes implicated in biosynthesis and catabolism are often studied in relation to specific inhibitors. AG is one of the exogenously applied inhibitor compounds, which are known to block the activity of copper amine oxidase or diamine oxidase. This inhibitor has been demonstrated to decrease DAO enzyme activity, but the effects were found to be different depending on the tested plant species, organ, or the applied dose [36,37]. Short-term salt stress altered PA catabolic responses, similarly to results obtained with tobacco or cucumber, demonstrating a feed-forward ROS amplification loop between ROS-producing NADPH oxidase and the apoplastic PAO [57]. In this study, we tested the specificity of AG when used as a DAO inhibitor in salt-stressed tomato plants. Interestingly, in our experimental conditions, AG was unspecific for DAO and suppressed PAO activity as well (Figure 1). Similar results were reported by Kabala et al. [57]: a 1-h salt treatment did not significantly affect DAO enzyme activity in cucumber, and lower H 2 O 2 contents were maintained. Although inhibition of DAO enzyme activity was expected to increase PA concentrations, especially Put levels, in our study, reduction in PAO activity by AG treatment correlated with reduced PA levels under both saline and non-saline conditions ( Figure 2 and Table S3). To decipher the possible mechanisms of this reduction, the expression of genes which encode PA biosynthesis enzymes such as ADCs and ODC1 and genes which encode DAO and PAO enzymes was studied. We found that salt stress induced ADC expression, especially that of ADC2. Interestingly, ODC1 expression was reduced by 250 mM NaCl and further decreased after AG treatment ( Figure S1). Additionally, 100 mM NaCl increased ADC and ODC expression, which was further enhanced by AG treatment, suggesting that PA levels were increased in these plants ( Figure 3). ADC and ODC could compensate for each other's activity, as revealed by [58]. Meanwhile, 250 mM NaCl was shown to reduce PA content. The expression of genes implicated in PA catabolism in response to salt stress [21] was also tested. PAO1 of tomato is similar to the Arabidopsis AtPAO1, which belongs to Clade 1, and is localized in the cytoplasm. PAO2, PAO4, and PAO5 are in Clade 4, showing peroxisome localization. PAO2 was reported to contribute to ABA-mediated plant developmental processes in Arabidopsis [59]. Transcript levels of PAO4 were reduced to half in salt-treated plants, whereas PAO5 expression was reduced by 100 mM NaCl but increased in higher salt-stress conditions ( Figure S2). PAO5 expression was previously shown to be unchanged by salt stress, showing a role in xylem differentiation [60]. To explain the differences between these genes, further studies are needed.
Salinity strongly affects ROS signaling in plants [61] by involving the catabolic processes of PAs. PA catabolism could induce H 2 O 2 production in the roots of tomato plants exposed to salt stress [35]. AG did not change H 2 O 2 levels compared to the control conditions, providing evidence that AG alone did not induce any additional stress in these plants ( Figure 5). AG could alleviate salt-induced H 2 O 2 accumulation, suggesting that AG might have antioxidant features similar to those reported in soybean [34,36]. To decipher the contribution of other potential biosynthetic processes to H 2 O 2 production, SOD enzyme activity was measured in salt-and AG-treated roots. SOD activity was reduced by AG in control and 100 mM NaCl-treated roots, but not in the 250 mM NaCl-treated ones. To examine the possible contribution of stress-dependent superoxide production to H 2 O 2 accumulation, superoxide levels were also measured. AG could reduce superoxide levels in roots subjected to mild salt stress, but it had no significant effect on superoxide production in control or 250 mM NaCl-treated roots ( Figure 6). These results are consistent with [57], where short-term salt stress induced slight changes in H 2 O 2 and SOD in cucumber. It may arise that other antioxidant defense mechanisms could play a role in alleviating oxidative stress during short-term conditions.
AG was suggested as functioning as an irreversible NOS (iNOS) inhibitor widely used for reducing NO production in plants [62]. PAs could be produced from L-arginine, affecting oxidative NO production in plants [63]. In our experimental system, AG reduced NO levels independently of salt stress (Figure 7). A similar effect of AG has been reported in [34], which agrees with other studies. Treatment with 250 mM NaCl could provoke nitrosative stress by inducing reductive or oxidative pathways for NO production, potentially generating reactive nitrogen species for tomatoes, which could be alleviated by AG. To confirm the source of these increased NO levels, we investigated the reductive pathway of NO production. Nitrite could be a source of NO [41,64] in plants. PAs were shown to modulate NR activity in wheat leaves, affecting NO production [65]. Moderate salt stress could enhance nitrite levels, whereas high salinity reduced them, and they were further reduced by AG. SNO is like a stored form of NO. Salinity induced SNO accumulation, which was further increased in 250 mM NaCl-treated tomato roots (Figure 8). There is some evidence for the involvement of SNOs and S-nitrosylation in salinity stress, but these need to be further investigated [66].
Recent results support the role of H 2 S as a signal molecule in salt-stress responses in plants [14,67,68]. Its connection to other signal molecules, such as NO or H 2 O 2 , is almost unknown [69,70]. An early study suggested that NO and H 2 S could interact with each other and regulate each other's level; however, some other evidence indicates an inhibiting effect of H 2 S on NO production [70]. Additionally, it is known that H 2 S treatment could induce an increase in the expression of genes involved in PA biosynthesis during salt stress [70]. Our results revealed that 250 mM NaCl was able to increase the H 2 S levels, but AG had only a minor influence on H 2 S accumulation in tomato roots ( Figure 9). These results confirm that H 2 S production depends on the stress intensity in short-term salt treatment.
To provide further evidence about the decreased PA levels, we also checked the other degradation routes from Put to GABA, which could connect with the TCA cycle as an alternative GABA shunt, contributing to the N balance in plants [71]. Our experiments, however, did not demonstrate any GABA increase or other shift in TCA cycle metabolites by metabolomic analysis ( Figure S3).
As a conclusion, our results confirm AG as a nonspecific inhibitor of PA catabolism in short-term conditions, with implications in ROS and NO signaling (summarized in Figures 10 and S4). AG was suggested as functioning as an irreversible NOS (iNOS) inhibitor widely used for reducing NO production in plants [62]. PAs could be produced from L-arginine, affecting oxidative NO production in plants [63]. In our experimental system, AG reduced NO levels independently of salt stress (Figure 7). A similar effect of AG has been reported in [34], which agrees with other studies. Treatment with 250 mM NaCl could provoke nitrosative stress by inducing reductive or oxidative pathways for NO production, potentially generating reactive nitrogen species for tomatoes, which could be alleviated by AG. To confirm the source of these increased NO levels, we investigated the reductive pathway of NO production. Nitrite could be a source of NO [41,64] in plants. PAs were shown to modulate NR activity in wheat leaves, affecting NO production [65]. Moderate salt stress could enhance nitrite levels, whereas high salinity reduced them, and they were further reduced by AG. SNO is like a stored form of NO. Salinity induced SNO accumulation, which was further increased in 250 mM NaCl-treated tomato roots ( Figure  8). There is some evidence for the involvement of SNOs and S-nitrosylation in salinity stress, but these need to be further investigated [66].
Recent results support the role of H2S as a signal molecule in salt-stress responses in plants [14,67,68]. Its connection to other signal molecules, such as NO or H2O2, is almost unknown [69,70]. An early study suggested that NO and H2S could interact with each other and regulate each other's level; however, some other evidence indicates an inhibiting effect of H2S on NO production [70]. Additionally, it is known that H2S treatment could induce an increase in the expression of genes involved in PA biosynthesis during salt stress [70]. Our results revealed that 250 mM NaCl was able to increase the H2S levels, but AG had only a minor influence on H2S accumulation in tomato roots ( Figure 9). These results confirm that H2S production depends on the stress intensity in short-term salt treatment.
To provide further evidence about the decreased PA levels, we also checked the other degradation routes from Put to GABA, which could connect with the TCA cycle as an alternative GABA shunt, contributing to the N balance in plants [71]. Our experiments, however, did not demonstrate any GABA increase or other shift in TCA cycle metabolites by metabolomic analysis ( Figure S3).
As a conclusion, our results confirm AG as a nonspecific inhibitor of PA catabolism in short-term conditions, with implications in ROS and NO signaling (summarized in Figures 10 and S4). Figure 10. Schematic presentation of potential mechanisms of the effect of AG on PA metabolism and related pathways during short-term salt-stress response to different NaCl concentrations (100 and 250 mM). Question marks indicate the questionable functions of AG during stress, as it can act as an inhibitor of NO synthesis or could induce the expression of PAO genes; however, these functions need to be studied.
Understanding the role of PA catabolism during the early responses of plants to salinity stress could fill the gap in our knowledge about the connection between salt sensing and PA homeostasis, focusing on the interplay between other signal pathways [72]. Precise modes of action of PA catabolism and the related signal pathways need to be deciphered by further studies.