*4.1. Identification of Tomato Put Family*

The tomato Put proteins were divided into four groups, which have a high similarity to those in rice and *Arabidopsis* (Figure 1). Differences among these proteins are probably due to environmental impacts, and analysis of the structure and motif in these genes indicated that *Put* genes disclosed a close exon-intron and motif structure, showing that a closer evolutionary pattern in these genes and diverse functional relationships also exist among the other group members (Figure 1). Interestingly, protein analysis indicated that the PotE (Putrescine-ornithine antiporter) motif is highly conserved, suggesting that it may be involved in amino acid and polyamine transport (Figure 1C) [45,46]. A series of cis elements is a specific sequence at the promoter region of a given gene, which is involved in the expression of protein-coding transcripts and is mediated by transcriptional regulation and small RNAs. Repression and activation of gene expression by binding with these cis elements are a general means of modulating various life processes [47]. Plant miRNAs have a function in regulating related genes that are involved in response to abiotic stresses [48]. miR390 was strongly induced after exposure to salinity during lateral root formation in poplar and positively regulated auxin signaling subjected to salt stress [49]. miR6024 negatively mediates the resistance genes and defense system, facilitates disease by the necrotrophic pathogen *A. solani,* and perturbs immunity in the tomato [50]. The module of miR164a-NAM3 affords cold resistance by increasing ethylene production in the tomato [51]. Tomato Put was targeted by miRNAs including miR390, miR6024, and miR164a, which might be associated with various stress responses (Table 2). We also discovered several types of conserved cis-regulatory elements in the promoter regions of Put; these cis elements are associated with transcriptional regulation of the core gene network, and of plant growth and development [52]. The *Put* genes of the tomato contain various cis elements including

stress, hormone, light, auxin, GA, ABA, and methyl jasmonate (MeJA) responsive elements (Figure 1E). The presence of numerous abiotic stress-specific cis-regulatory motifs and hormonal cis elements implicate these genes stimulating the hormone signaling pathways and providing stress tolerance in the tomato [47]. Of note, the cis elements activate their downstream genes after binding to specific transcription factors, playing an important amplifier role during various abiotic/biotic stresses. Meanwhile, we also speculate that Put is involved in salinity stress since these cis-regulatory elements are also closely implicated in salinity tolerance [53].

#### *4.2. Expression Profiles of the Put Gene Family after Treatment with Various Hormones, Polyamines, and Abiotic Stresses, in Different Tissues*

A gene expression profile can provide critical symbols for its biological functions. Here, we examined the expression pattern of the *Put* genes via qRT-PCR under treatment with exogenous hormones and polyamines, as well as abiotic stress conditions. We observed the *Put* genes appeared to be upregulated in ABA treatment. On the other hand, all Put genes, except for *Put7*, were induced by SA. *Put6*, *Put7,* and *Put8* were inhibited initially after GA3 treatment. The expression of five *Put* (*Put4*, *Put5*, *Put6*, *Put7,* and *Put8*) genes were downregulated, and the other *Put* (*Put1*, *Put2* and *Put3*) genes were induced in leaves at 3 and 6 h of ET treatment. Similarly, the critical role of growth factors and hormones in increasing polyamine transport rates in mammalian cells has been demonstrated [54]. Together with the presence of hormone-responsive cis elements and altered transcripts levels after hormone treatment, this implies that Put may have a crucial role in the hormone regulatory pathway. Furthermore, the differential expression profiling of *Put* in response to polyamine treatment was evaluated. All *Put* genes are involved in exogenous polyamines treatment; the *Puts* exhibited varied patterns in response to the same polyamine. For instance, *Put2* and *Put5* were dramatically upregulated, while the other genes were slightly induced after spermidine treatment. However, putrescine resulted in suppression of these *Put* genes. Additionally, spermine led to the upregulation of *Puts*, especially *Put2* and *Put5*. On the other hand, the qRT-PCR analysis revealed pronounced effects of the spermidine and spermine-induced *Put* gene expression (Figure 2B). Since polyamines are used as substrates required for the polyamine uptake proteins [43], the different polyamine responses may involve the substrate specificity of polyamine transport and homeostasis. In fact, rice Put1 is a specific and high-affinity spermidine uptake transporter involved in polyamines uptake, leading to the accumulation of polyamines in yeast [55]. The lower affinity of Put may be the reason for its higher proportion in the free state. Therefore, spermidine, spermine, and putrescine, may severe as Put substrates. Interestingly, after salt and drought stress, *Put2* and *Put5* were significantly induced compared with the others. However, their expression was significantly inhibited under cold and heat temperature stress (Figure 2C). Altogether, these results indicate that *Put* genes are potentially involved in hormone and polyamine induction, as well as in response to salinity stress.

The *Put* genes exhibited a divergent expression pattern in different tissues. *Put2* and *Put5* displayed the highest expression in leaves and roots, and all genes had high expression in flowers and fruit (Figure 3A). Similarly, the Put family appears to have a distinct tissue expression profile in *Arabidopsis* and *Citrus sinensis* [43,44], and a divergent pattern of intracellular localization [13], which implied specialization in a spatial manner. Furthermore, *Put2* was more significantly induced by salt stress than *Put5* in leaves and roots, indicating that the functional role of *Put2* related to salt stress may be important. In the tomato, the functional role of *Put2* in abiotic stress tolerance remains largely unknown.

#### *4.3. Put2 Contributes to Polyamins Biosynthesis and Catabolism Associated with Salt Tolerance*

Although our results above have shown that Put is involved in abiotic stress, its function is yet to be understood, especially regarding polyamine transport and salt stress. Previous studies have shown that yeast is an excellent heterologous expression system to study the function of genes in polyamine transport and salt stress [43]. Here, we used the

yeast model to preliminarily investigate their function in polyamine uptake and salt stress tolerance (Figure 4). The results showed that transformants of *agp2*Δ mutants expressing *Put2* and *Put5* had higher sensitivity to spermidine and paraquat, indicating that both function as an importer. Here, we also showed that the yeast *agp2*Δ cells' capacity to transport paraquat may be compensated by Put2 and Put5 (Figure 4A), since paraquat is transported by the polyamine transport system [12]. A time course absorption experiment directly provided evidence that *Put2* and *Put5* encoded a transporter that can regulate polyamines import, with high activity of polyamine uptake for Put2 (Figure 4B). Furthermore, overexpression of *Put2* increased salt tolerance in yeast, hampered the influx of Na+, and enhanced K+ uptake (Figure 4C,D). Indeed, polyamine transporters have recently been linked to the regulation of salt stress through promoting Na<sup>+</sup> efflux and K<sup>+</sup> channels [56]. Thus, combining the previous results in this article (Figures 2–4), we speculate that the induction of *Put2* expression by salt may be regulating the polyamines and Na+/K+ homeostasis to alleviate salt damage.

To further validate the function of Put2, the *put2* mutants and overexpression lines were generated (Figure 5A,B). Our results showed that *put2* mutants were more shriveled than WT plants under salinity stress, whereas overexpression elevated salt tolerance (Figure 5C). Likewise, *Put2*-OE plants displayed increased levels of Fv/Fm and dry weight, and reduced levels of relative electrolyte, which agrees with an increase in salt tolerance and a decrease in Na<sup>+</sup> content and the Na+/K+ ratio (Figure 5D–H). Similarly, Put3 is critical for Na<sup>+</sup> and K<sup>+</sup> homeostasis by physically interacting with SOS1 and SOS2, forming a complex with SOS2 under stress conditions [56]. As such, the induction of *SOS1-3* and *NHX1-3* in *Put2*-OE plants could also synergistically activate the SOS1 and SOS2 (Figure S2). Thus, subsequent increases in the Put2 activity would enhance salt tolerance by activating the Na+/H+ exchange activity.

Polyamines, an important regulator in the plant kingdom, are necessary for plant growth, development, and the stress response. The dynamic balance of polyamines in the plant is stringently regulated by polyamine synthesis, degradation, and transport [57,58]. The latter was previously involved in subcellular polyamine transport through the complementation experiment in yeast [55], and the transport of paraquat in *Arabidopsis* [12]. While the implication of *Put2* in polyamine biosynthesis and catabolism was not noted. Here, under control conditions, overexpression of *Put2* increases the endogenous Arg, Spd, and Put content, which failed to increase in *put2* mutants. Upon salt stress, meanwhile, the polyamine content in WT plants performed much better than *put2* mutants, and *Put2*-OE plants were better than the WT (Figure 6A–D). NaCl supply treatment also increased the activation of polyamine synthesisrelated genes more clearly in *Put2*-OE plants (Figure 8), demonstrating the important function of Put2 in the polyamine biosynthesis process. On the other hand, considering polyamine catabolism, the higher activity of PAOspd confirmed the acceleration of polyamine catabolic reactions in *Put2*-OE plants (Figure 6I), and evidenced that Put2 positively regulates PAO activity. In addition, two main sources of ROS were indicated to exist in plants, including NADPH oxidases and polyamines catabolism by PAO activity [8,59]. In *put2* leaves, H2O2 was lowered with respect to WT plants under normal conditions; however, an upregulation was observed in *Put2*-OE plants, and likewise PAO activity and *PAO* gene expression were altered (Figures 6J and 8), further demonstrating that Put2 attributes to PAO activity. Accordingly, enhancement of PAO activity has been shown to alleviate salinity damage and increase the polyamines and H2O2 content [60,61]. Therefore, Put2 may contribute to governing polyamine biosynthesis and catabolism. However, we cannot completely rule out other possibilities, for example, Put2 may be capable of regulating the long-distance and appropriate tissue distribution of polyamines [62].

#### *4.4. Put2-Mediated Antioxidant Capacity Establishes Suitable ROS Levels under Salt Stress*

PAO activity has been reported to contribute to an increase in salt tolerance through the production of H2O2 [63]. However, *Put2*-OE plants treated with NaCl showed that the increase in H2O2 content was lower than in WT plants. Furthermore, the H2O2 content increased considerably in *put2* mutants accompanied by serious salt stress injury, which correlated inversely with the activities of PAO (Figure 6J). Thus, PAO-induced H2O2 production was stunted under salt conditions in *Put2*-OE plants; therefore, another mechanism to eliminate H2O2 must exist. Indeed, plants exposed to salt stress generate a super-excess of ROS, which is highly toxic and can overwhelm the PAO-induced H2O2, ultimately damaging cellular activity and leading to plant death [8]. In the current study, *Put2*-OE plants exhibited obviously increased activities of antioxidant enzymes after salt treatment. Additionally, the ASA-GSH cycle was also activated by overexpression of *Put2*, which could be another exploration for Put2 enhanced salt tolerance, as the enzymatic system and scavenging procedure could be activated in *Put2*-OE plants (Figures 7 and 8). These findings indicate that Put2 functions as an important regulator linking polyamines and ROS, and affects both the production and elimination of ROS. The results coincided with a previous study showing that Put2 promoted phyA-mediated germination by sensing seed oxidation and protecting the decaying seed from oxidative damage [64]. Therefore, there is strong evidence that Put2 increased salt tolerance probably by promoting antioxidants in plants. A similar report has been conducted showing that CsPUT4 can protect against cold stress by modulating polyamine homeostasis and turning on the antioxidant enzyme defense system in the sweet orange [44]. Meanwhile, GABA and flavonoids, as free radical scavengers, have been exhibited to alleviate salinity damage and heat damage by inducing polyamine enhancement [65,66], which are increased in *Put2*-OE plants under salinity stress; this further demonstrates positive feedback regulation by Put2.

#### **5. Conclusions**

In this work, eight Put family proteins were found in the tomato, and their chromosomal location, structure, phylogenetic tree, and physiochemical properties were investigated. Furthermore, molecular characterization was performed in yeast to understand their involvement in polyamine uptake and salt stress tolerance. Additionally, the cis elements in the promoter, miRNAs targeting Put, and the expression profiles of *Put* genes in different tissues and their responses to exogenous hormones and polyamines, as well as abiotic stress, were analyzed, proving they may play a vital function in abiotic stress, growth, and development. Furthermore, we show the role of Put2, which, to our knowledge, is the first polyamine uptake protein characterized in the tomato shown to play a role in salinity tolerance. Firstly, in yeast, Put2 was highly tolerant to salt stress, as indicated by less Na+ invasion and K+ efflux, which also could be attributable to an enhancement in the absorption of polyamines. Importantly, overexpression of *Put2* in the tomato decreased salinity sensitivity, evidenced by enhanced polyamine biosynthesis and catabolism and maintained Na+/K+ homeostasis, in addition to activated ROS-scavenging enzyme activities and nonenzymatic antioxidant process. These findings shed light on Put2-regulated salinity tolerance in the tomato. Here, we provide comprehensive deciphering of the mechanisms of Put2 for enhancing salt tolerance and some valuable evidence for interpreting the potential functions of tomato Put genes in abiotic stress tolerance.

Clearly, further studies are required to understand the precise function of Put and the upstream and downstream targets of the individual Puts. Generating loss- and gainof-function mutations and characterizing their roles will provide useful tools, generating new evidence and new findings.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antiox12020228/s1, Figure S1. Cartoon of the predicted topology of tomato Put proteins. Transmembrane domains are indicated as blue rectangle. Figure S2. Relative transcript levels of SOS1, SOS2 and SOS3 (A), NHX1, NHX2 and NHX3 (B) gene expression in 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 S3. The MDA content in 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. Supplemental data S1. The protein sequences of Put in Arabidopsis, rice and tomato, respectively. Supplemental data S2. The Put genes Cis-element distribution. Supplemental data S3. Primers used in this study.

**Author Contributions:** M.Z.: Writing—original draft. L.Y., W.L. and H.Q.: Methodology. B.L. and R.H.: Review and editing. X.Y. and Y.K.: Supervision. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was financially supported by Guangdong Provincial Special Fund for Modern Agriculture Industry Technology Innovation Teams: No. 2023KJ122, Guangzhou basic and applied basic research foundation (SL2022A04J00131), GuangDong Basic and Applied Basic Research Foundation (2214050009633).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are contained within the article and Supplementary Material.

**Conflicts of Interest:** The authors declare no conflict of interest.
