*3.3. Responsiveness of Put Gene Expression under Hormone, Polyamine, and PQ Treatment*

The hormones in the plant kingdom play crucial roles in plant growth and development. Therefore, four different hormone-induced Put transcript-level changes were analyzed. After ABA treatment, the expression of all Puts was induced at 30 min, while that of *Put2* and *Put5* was induced after 6 h (Figure 2A). The expression of all *Puts* was induced at 30 min after SA treatment, which were also upregulated in the later time points, except for *Put*4 and *Put6* (Figure 2A). In contrast, the transcripts of *Put6*, *Put7,* and *Put8* were reduced initially following GA3 and ETH treatments, and ETH also reduced the *Put4* and *Put5* expression, a GA3 induced both. *Put2* and *Put3* were reduced after GA3 treatment, and then induced after 6 h of treatment, Put1 was induced after GA3 and ETH treatments (Figure 2A).

To examine the possible roles of *Put* genes in the tomato, transcripts of *Puts* after three treatments with polyamines, including Spermidine, Putrescine, and Spermine, were analyzed. Exogenous Spermidine treatment induced all *Put* genes, and *Put2* was the most obvious, followed by *Put5* (Figure 2B). However, the expression of all *Put* genes was reduced by Putrescine, and the reduction magnitude differed among them. After the seedlings were treated with Spermine, all *Put* genes were upregulated, but reduced after 12 h treatment. Additionally, exogenous PQ treatment caused upregulation of all Put genes similar to that of Spermidine treatment (Figure 2B).

#### *3.4. Differential Expression of Put Genes during Abiotic Stress*

Upon exposure to drought stress, the mRNA transcript of *Put1–5* was rapidly induced, and *Put2* was the most significant, but other members were reduced (Figure 2C). Salinity stress resulted in the downregulation of *Put3*, *Put4*, *Put6*, *Put7,* and *Put8*, and upregulation of *Put1*, *Put2,* and *Put5*. Cold stress resulted in the most pronounced induction of *Put4*. Furthermore, when the seedlings were submitted to heat stress, sustained and stable upregulation of *Put6* was observed, whereas two genes (*Put7* and *Put8*) were reduced post-treatment (Figure 2C).

**Figure 2.** Expression patterns of *Put* genes in tomatoes under hormone, polyamine, paraquat, or abiotic stress. (**A**) Heatmap representation of the responsiveness of *Put* genes after treatment with ABA, SA, GA3, and ET. The scale (0 to 3) represents the expression level (from low to high). (**B**) Heatmap representation of the responsiveness of *Put* genes after treatment with Spd, Put, Spm, and PQ. The scale (0 to 3) represents the expression level (from low to high). (**C**) Heatmap representation of the responsiveness of *Put* genes after drought, salt, cold, and heat treatment. The scale (0 to 3.5) represents the expression level (from low to high). qRT-PCR was conducted after treatment, according to the methods section. The expression of WT at 0 min in the different treatments was set to 1.

#### *3.5. Expression Analysis of Put Genes in Different Tissues*

To research the functions of the Put family of genes in the tomato, the expression patterns of *Put* in various tissues (e.g., root, stem, leaf, bud, flower, and fruit) were performed by qRT-PCR. As shown in Figure 3, the transcripts of *Put2* and *Put5* had high levels in the leaves and roots, *Put3* exhibited high levels of expression in the leaves and flowers, while *Put4* showed a high level of transcript expression in the roots and flowers. In addition, all *Put*, except *Put6*, *Put7* and *Put8*, were highly expressed in flowers. Subsequently, the spatial expression pattern of *Put2* and *Put5* was analyzed by qRT-PCR in vegetative tissues of WT plants with salt stress. *Put2* and *Put5* showed increased expression in organs of plants under salt stress, with the highest level of *Put2* transcripts in the leaves, and the highest level of *Put5* transcripts in the roots. After 7 days of salt treatment, the *Put2* gene is significantly more highly induced than *Put5* in the leaves (Figure 3B,C).

**Figure 3.** The expression profiles of the tomato Put family of genes in different tissues. (**A**) Heatmap representation of the relative expression of *Put* genes in different tomato tissues. The scale (0 to 3) represents the expression level (from low to high). (**B**,**C**) The spatial expression of *Put2* and *Put5* were performed by qRT-PCR in different organs of WT plants without NaCl (Control) and salt stress (NaCl) (7 days salt treatment). The WT expression in the control condition was set to 1. The data in B and C are presented as mean values ± SD; n = 3. Different letters indicate significant differences between treatments (*p* < 0.05, Duncan's multiple range test). Three independent experiments were performed with similar results.

#### *3.6. Functional Analysis of Put Genes in Yeast*

To examine the potential function of these proteins in polyamine transport, cDNA fragments containing the ORF were cloned and introduced into the yeast expression vector pYES2, driving expression under the *GAL1* promoter. High concentrations of polyamines or paraquat are toxic to wild-type yeast strains, whereas mutant *agp2*Δ lacking the polyamine uptake transporter protein impairs the sensitivity to the high concentration of polyamines. The candidate positive polyamine transporter was introduced to the yeast mutant *agp2*Δ. The expression of polyamine transporter proteins (Put1, Put3, Put6, and Put7) did not affect the phenotype of the *agp2*Δ mutant, however, among the eight Put proteins, Put2 and Put5 transformants were more sensitive than other Put transformants under 25 mM Spermidine conditions. The expression of Put2 or Put5 in the *agp2*Δ mutant conferred sensitivity to 25 mM Spermidine (Figure 4A). In addition, transformants appeared sensitive

to the polyamine's analog PQ, showing that these proteins are also involved in the uptake of PQ.

**Figure 4.** Comparison of polyamines, Na+, and K+ uptake among tomato *Puts* in yeast. (**A**) Functional complementation of tomato *Puts* in the yeast mutant *agp2*Δ. *agp2*Δ-*Puts* vector strains were grown overnight on SC medium supplemented with 2% galactose. Cell suspensions (the starting OD600 is 0.5) were serially diluted as indicated and 3 μL of each were spotted onto YP-galactose plates containing 25 mM spermidine or 1.5 mM paraquat. Plates were photographed after 3–4 days of incubation at 30 ◦C. The data are representative of one of three independent experiments. EV, empty vector. (**B**) Time course uptake of Spermidine and Putrescine for 0, 10, 20, 30, 40, 50, and 60 min to determine the intracellular amount of Spermidine or Putrescine. (**C**) Functional analysis of tomato *Puts* in the yeast mutant G19 (Δ*ena1–4*) (does not mediate Na<sup>+</sup> uptake) under NaCl treatment, and in the yeast mutant CY162 (a K+ uptake-deficient mutant strain) under deficiency medium. Cell suspensions (the starting OD600 is 0.5) were serially diluted as indicated and 3 μL of each were spotted onto YP-galactose plates containing 100 mM NaCl or 1.5 mM KCl. Plates were photographed after 3–4 days of incubation at 30 ◦C. The data are representative of one of three independent experiments. EV, empty vector. (**D**) Time course uptake of Na<sup>+</sup> and K+ for 0, 10, 20, 30, 40, 50, and 60 min to determine the external levels of Na+ or K+.

Further analysis of these genes in light of their competence to transport polyamine was done by incubating with the liquid AP medium supplemented with either 15 μm Spermidine or Putrescine. Transformants of these *Put* genes improved the ability to transport Spermidine or Putrescine than in those of the *agp2*Δ mutant. These transformants possessed higher uptake of Spermidine or Putrescine relative to that mediated by the *agp2*Δ mutant. The Put2- and Put5-transformants were effective for Spermidine and Putrescine, respectively. The uptake ability was more pronounced for Put2 than for Put5 (Figure 4B).

The yeast mutant G19 (Δ*ena1–4*) (does not mediate Na<sup>+</sup> uptake) and CY162 (K<sup>+</sup> deficient strain) strain were used to further disclose whether Put could transport Na<sup>+</sup> or K+. The growth status had a significant difference between G19 transformed with Put2 or Put5 than the G19 empty, Put5 had a better growth status, whilst other Put trans-yeast strains were not significant different to the G19-empty vector under 100 mM NaCl treatment (Figure 4C). Similarly, under 0.1 mM KCl, CY162 yeast transformed with Put2 or Put5 had a better-growing status compared to the CY162-empty vector, while other Put trans-yeast strains grew similarly to the CY162-empty vector, indicating that Put2 could transport K+, and Put5 could transport Na<sup>+</sup> and K<sup>+</sup> (Figure 4C). Furthermore, the ion depletion assay exhibited that K+ ions decreased significantly faster than Na<sup>+</sup> inoculated into the liquid AP medium with 200 μM NaCl + 200 μM KCl, confirming the role of Put2 in Na<sup>+</sup> and K+ transport (Figure 4D).

#### *3.7. Put2 Is a Positive Regulator Protein of Tomato Plant Salt Tolerance*

We focused our present study on Put2, since *Put2* was highly expressed in leaves after 7 days of salt stress. Furthermore, Put2 had the highest uptake ability of polyamines among the eight Puts and overexpression of *Put2* increased salt tolerance in yeast (Figures 3 and 4). To further determine the role of Put2 in salt stress, as shown in Figure 5, we generated five overexpression lines (named OE#1 to OE#5) and selected two lines (OE#1 and OE#2) for further study after examining Put2 mRNA levels. Meanwhile, two lines of *Put2* mutants (*put2*#1 *and put2*#2) were generated by CRISPR/Cas9 technology, which induced frameshift mutations. Next, we compared the salt tolerance of these tomato plants. In comparison to WT plants, the *Put2*-OE#1 and *Put2*-OE#2 plants exhibited decreased sensitivity to salt stress with less wilted leaves, higher *Fv/Fm* and dry weight, and lower REL after a salt treatment for 7 days. Whereas *put2* mutants displayed salt hypersensitivity with lower *Fv/Fm* and higher REL than WT. These results suggest that Put2 positively regulates salinity tolerance.

To explore whether Put2 responds to salinity stress through mediating the Na+ balance, we further analyzed the Na+ and K<sup>+</sup> contents in WT, *put2* mutants, and *Put2-*OE plants subjected to NaCl treatment. K<sup>+</sup> and Na+ content did not differ significantly among the genotypes examined under normal conditions. However, after treatment with 200 mM NaCl for 7 days, in comparison to WT plants, the levels of Na+ in shoots parts of the *put2* mutants were much higher, the K<sup>+</sup> content was significantly lower in mutants, and so the ratios of Na+/K+ were increased. Conversely, the contents of Na+ and the Na+/K+ ratio in the *Put2*-OE plants were lower, and the levels of K+ were higher (Figure 5H).

The transcription levels of encoding salt response genes such as *SOS1-3* and *NHX1-3* were examined in the WT, *put2* mutants, and *Put2*-OE plants using qRT-PCR. The mRNA abundance of *SOS1-3* and *NHX1-3* in the mutants was lower than that in WT. On the contrary, the transcript levels of these genes were significantly increased in *Put2*-OE plants. These results demonstrated that Put2, as a positive regulator, is mediated by Na+/K+ homeostasis (Figure S2).

#### *3.8. Put2 Improves Salt Tolerance by Facilitating Polyamines Synthesis*

Previous studies reported that polyamines uptake proteins can facilitate polyamines synthesis, at least in rosette leaves of *Arabidopsis thaliana* plants (Ahmed et al., 2017), which enlightened us to next investigate the polyamine metabolites. In the polyamine synthesis pathway, key representative molecules (Put, Spd, Spm, and Dap) are derived from the amino acid Arg (Figure 6A). Significantly, there was a considerable increase in the concentrations of Arg (the Put/Spd/Spm precursor), Spd, and Put in the leaves of *Put2*-OE plants, but the content in the *put2* mutants were lower than those of WT plants in the

absence of salt stress. When exposed to the salt treatment, the endogenous content of Arg, Spd, and Put increased significantly in *Put2*-OE plants than that of WT and *put2* mutants, whereas the levels of these polyamines in *Put2*-OE plants were still considerably higher than those in the WT plants (Figure 6B–D). Additionally, the Spm and Dap content showed no significant differences among the WT, *put2* mutants, and *Put2*-OE lines (Figure 6E,F).

**Figure 5.** Put2 positively regulates salt tolerance. (**A**) Genotyping of mutations in *put2#1* and *put2#2*. Red letters indicate the target sites, '-' represent sequence deletion, and blue letters represent the protospacer adjacent motif (PAM). (**B**) qRT-PCR analysis of *Put2* transcript levels in WT and Put2 OE (1#, 2#, 3#, 4# and 5#). (**C**) Phenotypes of WT, *put2* mutants, or *Put2* OE lines after exposure with or without salt stress for 7 days. (**D**) *Fv/Fm* in WT, *put2* mutants, or *Put2* OE lines leaves after exposure with or without salt stress for 7 days. The false-color code depicted at the bottom of image range from 0 (black) to 1.0 (purple), showing the level of damage in the leaves. (**E**) Quantitative analysis of *Fv/Fm* as shown in (**D**). (**F**) The relative electrolyte leakage and (**G**) dry weight in WT, *put2* mutants, or *Put2* OE lines after exposure with or without salt stress for 7 days. (**H**) Ion content and Na+/K+ ratio in shoots of WT, *put2* mutants, or *Put2* OE lines after exposure with or without salt stress for 7 days. Data are presented as mean values ± SD; n = 3. Different letters indicate significant differences between treatments (*p* < 0.05, Duncan's multiple range test). At least three independent experiments were performed.

**Figure 6.** The role of Put2 in the regulation of polyamine metabolism and H2O2 content. (**A**) Simplified scheme of polyamine biosynthesis relevant to this study in plants; Arg, arginine, ADC, arginine decarboxylase, Put, putrescine, Spds, spermidine synthase, Spd, spermidine, Spms, spermine synthase, Spm, spermine, PAO, polyamine oxidase, Dap, 1,3-diaminopropane. (**B**) The Arg content in WT, *put2* mutants, or *Put2* OE lines with or without salt stress for 7 days. The content of the polyamines Spd (**C**), Put (**D**), Spm (**E**), and Dap (**F**) in WT, *put2* mutants, or *Put2* OE lines with or without salt stress for 7 days. The Put-dependent CuAO (**G**), Spd-dependent PAO (**H**), and Spm-dependent PAO (**I**) enzymatic activity in WT, *put2* mutants, or *Put2* OE lines with or without salt stress for 7 days. (**J**) The H2O2 content in WT, *put2* mutants, or *Put2* OE lines with or without salt stress for 7 days. Data are presented as mean values ± SD; n = 3. Different letters indicate significant differences between treatments (*p* < 0.05, Duncan's multiple range test). At least three independent experiments were performed.

In leaves, knockout or gain of function of Put2 did not influence the Put-dependent PAO and Spm-dependent PAO activity in the absence or presence of salt treatment (Figure 6G,H). By contrast, *Put2*-OE plants showed an increase in Spd-dependent PAO activity, whereas *put2* mutants exhibited a decrease in this activity, relative to WT plants under normal conditions. During salinity stress, there were no significant changes in the activity of Spd-dependent PAO between WT, *put2* mutants, and *Put2*-OE lines (Figure 6I). Since H2O2 is one of the PAO reaction products, the levels of H2O2 were measured. The leaves of *Put2*-OE plants showed an evident increase in H2O2 content in the absence of salt stress, whereas the largest rise was shown in *put2* mutants in the presence of 200 mM NaCl (Figure 6J), proving that *put2* mutants experience more severe oxidative damage from salt stress than WT and *Put2*-OE plants. Thus, it is possible that Put2-induced H2O2 production is closely associated with PAOs under normal conditions. On the other hand, Put2 contributes to the decrease of H2O2 content under salt conditions, which declined oxidative damage and enhanced salt tolerance. These results demonstrate that Put2 is required for some polyamines metabolism intermediates (including Arg, Spd, and Put), as well as the enzyme (PAO).

#### *3.9. Put2 Decreases ROS Levels under Salinity Stress*

The production of ROS is known to be increased under stress conditions, and H2O2 is the most stable ROS. To determine whether Put2 reduces ROS accumulation through antioxidant enzyme and non-enzymatic compound regulation under salinity stress, the following relevant indicators were detected. During salt stress, the level of H2O2 and MDA, two indicators of oxidative damage during salt stress, were significantly lower in *Put2*-OE plants than in WT; increases in H2O2 and MDA content were detected in *put2* mutants (Figures 6J and 7A). Subsequently, antioxidant enzymes, including SOD, CAT, APX, and POD, are in the midst of the primary enzyme defense against ROS. The activities of SOD, CAT, APX, and POD were more pronounced in *Put2*-OE than in WT. In contrast, *put2* mutants showed lower antioxidant enzyme activity than WT (Figure 7), which is consistent with lower levels of H2O2 and MDA in *Put2*-OE than in WT and *put2* mutants. These results indicate that Put2 improves salinity tolerance by reducing oxidative damage.

To confirm whether the Put2-mediated ROS decrease is regulated by non-enzyme compounds, we determined the effects of glutathione redox homeostasis, GABA, and flavonoid contents in *put2* mutants and *Put2*-OE plants, which however, revealed no difference from the WT under normal conditions, except for GABA. The GABA content in *put2* mutants was significantly lower than that of WT and *Put2*-OE plants before salt treatment. Overexpression of *Put2* also considerably elevated the AsA/DHA and GSH/GSSG ratios, as well as the content of GABA and flavonoid, while knockout of *put2* compromised the increase in these parameters compared to that in the WT under salinity conditions (Figure 8).

#### *3.10. Put2 Triggers Upregulation of Polyamine Synthesis and Is Related to Detoxification Gene Expression*

To investigate the transcriptional regulation of polyamine synthesis and detoxification by Put2, we examined the transcript levels of 27 genes involved in polyamine synthesis, ROS detoxification, and GABA synthesis by qRT-PCR in leaves under normal or salinity conditions. qRT-PCR analysis of polyamine synthesis genes in *put2*#1 mutant and *Put2*-OE#1 plants revealed that the set genes of encoding polyamine synthesis (including *ADC1*, *ADC2*, *SPDS1*, *SPDS2*, *SPMS1*, *SPMS2*, *PAO1*, *PAO3,* and *PAO5*) were upregulated in *Put2*-OE#1 plants; in contrast, they were downregulated in *put2*#1 mutants Furthermore, salinity treatment caused relative increases in the expression of polyamine synthesis genes only in WT and *Put2*-OE#1 plants (column 3 vs. column 1, and column 5 vs. column 3), while they decreased in *put2#*1 mutants (column 4 vs. column 3). Collectively, the analysis of differentially expressed genes also suggests that the modifications in gene expression in response to the stress conditions are more intense in the *Put2*-OE#1 plant. Additionally, we noticed an interesting finding with the greatest upregulation of the *PAO5* gene in the five *PAOs* genes in the *Put2*-OE#1 plant

under normal conditions, which may be a mechanism for the relatively higher PAO activity and levels of H2O2 in *Put2* OE plants (Figures 6J and 8).

**Figure 7.** Put2 enhances salt tolerance via positively regulating ROS-scavenging enzyme activity and nonenzymatic antioxidant process. The MDA content (**A**), SOD (**B**), CAT (**C**), APX (**D**), and POD (**E**) activity in WT, *put2* mutants, or *Put2* OE lines with or without salt stress for 7 days. (**F**) The ratio of ascorbic acid (AsA) to dehydroascorbate (DHA), (**G**) the ratio of glutathione (GSH) and glutathione disulfide (GSSG), (**H**) the gamma-aminobutyric acid (GABA), and (**I**) the total flavonoids in WT, *put2* mutants, or *Put2* OE lines with or without salt stress for 7 days. Data are presented as mean values ± SD; n = 3. Different letters indicate significant differences between treatments (*p* < 0.05, Duncan's multiple range test). At least three independent experiments were performed.

**Figure 8.** Put2 positively mediates the expression of polyamine synthesis and antioxidant enzymeencoding genes. Heatmap of polyamine synthesis and antioxidant enzyme-encoding genes. Levels were differentially modified in *put2* vs. WT plants, and *Put2* OE vs. WT plants under normal conditions or salinity stress. Log2 fold changes (relative to expression levels in sample of WT plants under control treatment) are shown with a color scale. A chart on the right side of the heatmap shows log2 fold change of expression levels in *put2* (mutant) compared to those in WT plants before salt treatment (column 1), log2 fold change of expression levels in *Put2* OE (overexpression) compared to those in WT plants before salt treatment (column 2), log2 fold change caused by salinity treatment in WT plants (column 3), and log2 fold change caused by salinity treatment in *put2* (column 4) or *Put2* OE (column 5). Statistically significant differences are indicated in bold font (*p* < 0.05).

Similarly, the transcriptional levels of ROS detoxification-related genes, including *Cu/Zn-SOD*, *MDAR*, *DHAR*, *APX*, *GR*, *CAT1*, *POD,* and *GPX*, were further upregulated in the *Put2*-OE#1 plant compared with WT plants after the salinity treatment. By contrast, the induction of ROS detoxification-related genes in response to salt stress was compromised in *put2* mutants; increased ROS-scavenging capacity failed in the *put2*#1 mutant (Figure 8). In addition, the expression of *GAD1*, *GAD2,* and *GAD3*, encoding glutamate decarboxylase, likely the key enzyme for GABA biosynthesis in the tomato, was induced in the *Put2*- OE#1 plant before salt treatment. Accordingly, the transcript levels of *GADs* were further enhanced after salt treatment in the *Put2*-OE#1 plant. Notably, increases in the expression of *GADs* in response to salt stress were suppressed in the *put2*#1 mutant. The enhanced

*GADs* transcripts and GABA content was observed in the WT and *Put2*-OE plants but not in the *put2* mutants after salt stress (Figures 7 and 8). Thus, the results indicate that Put2 participates in enhanced salt tolerance by mediating the ROS-scavenging capacity.

#### **4. Discussion**

Polyamines, which are small polycationic molecular regulators and signaling molecules, not only orchestrate fundamental growth and development in plants, but also induce a series of stress cascades [38]. Polyamines are involved in various pathways in plants and lessen the lethal effects of abiotic stresses by regulating transcription factors, hormonal responses, antioxidant enzymes, and the activation of signaling cascades [39,40]. Polyamine biosynthesis and degradation play important roles in various abiotic stress tolerance pathways and are closely associated with coping with ROS production [41]. The polyamine transporter belongs to the mammalian L-type amino acid transporter family, like polyamine synthesis and metabolism protein, which plays a critical role in plant growth, development, and the stress responses [42]. Though Put has been analyzed in *Arabidopsis*, rice, and the sweet orange, the effects of Put on abiotic stresses in the tomato have not been reported [43,44]. Here, we looked at the physiological and molecular function mechanisms of Put in the resistance to abiotic stress in the tomato. Eight Put proteins in the tomato were identified and characterized at the complete genome level through alignment with *Arabidopsis* Put proteins. Then, extensive analyses on the tomato Put proteins, including phylogenetic development, gene and protein structure, physiochemical characteristics, motifs, miRNAs, and cis elements, and their substrates were performed; and the effects of Puts on abiotic stress tolerance was evaluated in yeast and tomatoes. Furthermore, we demonstrated the importance of the Put2 mediation of polyamine metabolism and antioxidant capacity, and that overexpression of *Put2* increased polyamines to influence the homeostasis of antioxidant capacity, showing that Put2 was a positive regulator for salinity stress in tomatoes. These findings enlarge the comprehension of Put family members, which may be employed in breeding for genetic modification and the development of abiotic stress tolerant crops.
