*2.9. Determination of PAO Enzymatic Activities and H2O2 Content*

Amine oxidase was determined as previously described by Su et al. [34] and Urra et al. [9]. Leaf samples (0.5 g) were ground with liquid nitrogen, homogenized 2:1 (*v/w*) in 100 mM sodium phosphate buffer (pH 6.5), and centrifuged at 12,000× *g* for 20 min at 4 ◦C. One-hundred-microliters of the recovered supernatant was mixed with the 3 mL reaction mix, which contained 2.5 mL sodium phosphate buffer (100 mM, pH 6.5), 200 μL 15 mM 4-aminoantipyrine/0.2% (*v/v*) *N*, *N*-dimethylaniline, 100 μL 250 U ml−<sup>1</sup> peroxidase, and 100 μL 20 mM putrescine as a substrate. The CuAOPut, PAOSpm, and PAOSpd were determined using Putrescine, Spermine, and Spermidine as substrates, respectively. A 0.01 value of the changes in absorbance at 555 nm was assayed to one activity unit of the PAO enzyme after being incubated for 30 min at 22 ◦C. Control samples without polyamines were used to calculate these activities. Leaf H2O2 content was determined by specific detection kits according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China).

#### *2.10. Antioxidant Assay*

To assess the antioxidant enzyme activity, 0.5 g leaf samples were homogenized with 3 mL ice-cold 50 mM phosphate buffer (pH 7.8), which contained of 2 mM L-ascorbic acid, 2% (*w/v*) PVPP, and 0.2 mM EDTA. Then, the homogenates were centrifuged for 20 min at 12,000× *g*, and supernatants were collected to determine the enzyme activity. The activities of SOD, CAT, APX, and POD were assayed as previously described [30]. AsA/DHA and GSH/GSSG were measured as described by Zhong et al. [35]. The GABA content was determined by the Berthelot reaction with some modifications [36]. Leaf samples (0.5 g) were ground with methanol, centrifuged at 6000× *g* for 15 min, and the supernatant was discarded. The sediment was dissolved in 1.5 mL ddH2O and heated in a water bath at 50 ◦C for 2 h, followed by centrifugation for 15 min at 7000× *g*. A volume of 1 mL supernatant was mixed with 100 μL 2 mol L−<sup>1</sup> AlCl3 and oscillated, and then centrifuged for 10 min at 12,000× *g*. The supernatant was added with 300 μL KOH and incubated for 5 min, then centrifuged at 12,000× *g* for 10 min. The GABA content was measured according to the following procedure: 300 μL supernatant was mixed with the reaction mix, composed of 500 μL 0.1 mol L−<sup>1</sup> sodium tetraborate (pH 10.0), 400 μL 6% phenol, and 600 μL 5% sodium hypochlorite. The solution was boiled for 10 min and then placed in an ice bath for 5 min. Finally, the absorbance at 645 nm was measured after shaking the solution with 2 mL 60% ethyl alcohol. The assessment of the total flavonoid concentration was determined by Zhishen et al. [37], with some modifications. Dried leaf samples (0.5 g) were homogenized with 2 mL 80% ethanol, and then added 300 μL 20 mol L−<sup>1</sup> NaNO2; 3 mL 1 mol L−<sup>1</sup> AlCl3 was added after 5 min, and after 6 min, 2 mL 1 mol L−<sup>1</sup> NaOH was added and mixed well. Finally, the absorbance was measured at 510 nm.

#### *2.11. Analysis of Gene Expression*

Total RNA was extracted with the RNAsimple Total RNA Kit (Tiangen, DP419) and reverse transcribed with the HiScriptTM qRT SuperMix for qPCR (+gDNA wiper) kit (Vazyme, Nanjing, China). The qPCR reaction was performed by the ABI VII7 real-time PCR system (Applied Biosystems, Waltham, MA, USA). *Actin* was used as the tomato reference gene. The qRT-PCR primers are listed in Supplemental data S3 (Table S2).

#### *2.12. Statistics*

The data are presented as the means ± SDs and were analyzed using SPSS 20 statistical software. The experimental data were analyzed with Duncan's multiple range test at *p* < 0.05.

#### **3. Results**

#### *3.1. Identification of Tomato Put Family Genes*

To analyze Put proteins, a query search against the tomato genome database was accomplished using *Arabidopsis* and rice Put protein as the control search (Table 1). Eight potential *Put* proteins with high sequence similarity to *AtPut* and *OsPut* were identified and called Put1-8. They had CDs sizes ranging from 1077 bp (Put6) to 1605 bp (Put3), with polypeptides of 359–535 amino acids. The theoretical isoelectric points (pI) of *Put* varied from 5.41 (*Put3*) to 9.37 (*Put8*). The molecular weights (MW) of *Put* ranged from 40.28 (*Put6*) to 58.8 (*Put3*) (Table 1). Additionally, all Puts were predicted to contain transmembrane domains (Figure S1).

**Table 1.** Put gene identification and characterization in the tomato.


*3.2. Analysis of the Phylogenetic, Chromosomal Distribution, Gene Structure, and Promoter Sequences of Tomato Family Members*

To better study the evolutionary relationships among the tomato Put protein sequences and those of other plants, a phylogenetic tree was performed through the Neighbour-Joining method. By comparing the protein sequences of the Put genes, three main groupings could be distinguished. Two *Put* genes, *Put4* and *Put6,* were included in Group I; Group II only included *Put5*, and the remaining five genes (*Put1*, *Put2*, *Put3*, *Put7,* and *Put8*) were classified into Group III (Figure 1A). According to their location, the chromosomal positions of the Put genes were analyzed. Among these *Put* genes, three (*Put4*, *Put6* and *Put7*) were mapped to chromosome 1, and three (*Put1*, *Put2* and *Put3*) were located on chromosome 8, while *Put5* and *Put8* were located in chromosomes 9 and 10, respectively. These results suggest an uneven distribution of these genes on chromosomes (Figure 1B).

To better analyze the evolutionary relationships in *Put* genes, the phylogenetic tree was also constructed. *Put1*, *Put2,* and *Put3* were observed to have a close evolutionary relationship. A close evolutionary relationship was also found in *Put7* and *Put8*, and *Put4* and *Put6*, respectively. Interestingly, *Put5* has a separate evolutionary branch. In addition, their structural domains are highly conserved and contain the polyamine transport protein PotE structural domain (Figure 1C). The Put family gene structure analysis was carried out to learn more about their intron/exon structures. One intron was present in *Put2* and *Put5*, two in *Put3*, while the other five (*Put1*, *Put7*, *Put8*, *Put4,* and *Put6*) were intron deletion genes (Figure 1D). To investigate the feasible roles of Put family genes in different

abiotic stress and developmental steps, we used the PlantCARE database to estimate the presence of *cis*-acting elements in the promoter regions of the Put family. We obtained *cis*-acting elements that are associated with stress, hormone, and light responsiveness. Surprisingly, we only found five typical ethylene responsive motifs (ERE, GCC-box motifs) in the promoter of Put2, but not in that of other Puts (Figure 1E).

**Figure 1.** The characteristics of Put genes in the tomato. (**A**) Phylogenetic relationships of Put genes in Arabidopsis, rice, and tomato. (**B**) Gene distribution within the tomato chromosomes. The chromosome numbers are indicated on the left, and the position marked in the chromosome indicates the location of Put genes. (**C**) The protein motifs and phylogenetic trees in the Put famous members. (**D**) Gene structure of the Put family members in tomatoes. (**E**) Cis element distribution of the Put genes.

miRNAs are an important kind of non-coding signal strand RNAs of around 22 nucleotides that are encoded by genes in the organism and are closely involved in the regulation of genes in answer to various life processes and stresses. A total of 29 miR-NAs targeting the polyamine transporter genes in the tomato are listed in Table 2, including three sly-miRNAs targeting Put1 (sly-miR390a-5p, sly-miR390b-5p, and sly-miR6022); slymiR6024 and sly-miR6026 targeting Put2; sly-miR6023 and sly-miR1916 targeting Put3; six sly-miRNAs targeting Put4 (sly-miR9479-3p, sly-miR6024, sly-miR171c, sly-miR171a, sly-miR9472-5p, and sly-miR9478-3p); sly-miR164a-5p and sly-miR164b-5p targeting Put5; Put6 was targeted by sly-miR1917; six sly-miRNAs targeting Put7 (sly-miR156a, slymiR156b, sly-miR156c, sly-miR390a-5p, sly-miR396a-3p, and sly-miR6023); and three sly-miRNAs targeting Put8 (sly-miR156a, sly-miR156b, sly-miR156c, sly-miR319b, slymiR319c-3p, sly-miR396a-3p, and sly-miR6023) (Table 2).


