*2.7. Salt Treatment and Salt Tolerance Assays*

The tomato seedlings (WT, *put2* mutants, and *Put2* overexpression lines) were used for salt tolerance experiments. After seed germination and two cotyledons full expansion, the seedlings were cultured in 250 cm3 plastic pots filled with a peat-vermiculite combination (2:1, *v:v*). The seedlings were placed in a greenhouse at 28 ± 2 ◦C/20 ± 2 ◦C (day/night) under a maximum photosynthetic photon flux density (PPFD) of approximately 1200 μmol m−<sup>2</sup> s−<sup>1</sup> and a relative humidity of 70–80%. They were watered daily using Hoagland nutrient solution. Three-week-old WT and transgenic tomatoes of uniform size and health growth status were selected and subjected to salt stress treatment. The seedlings were treated by watering the plants with 200 mL of 200 mM NaCl every other day for salt stress. The control treatment was replaced with an equal amount of water. The salt stress treatment lasted for 7 days, and pictures were captured. The maximum quantum yield of PSII (Fv/Fm) was examined with the Imaging-PAM system (IMAG-MAXI; Heinz Walz, Effeltrich, Germany), as previously described by Zhong et al. [30]. The relative electrolyte leakage (REL%), Na+, and K+ analysis was performed as described previously by Zhong et al. [30]. The plants were enclosed in envelopes and placed in an oven at 105 ◦C for 30 min, and then the oven temperature was adjusted to 75 ◦C to obtain a permanent dry weight (DW).

### *2.8. Determination of Polyamine Content*

The free polyamines content was analyzed by Agilent 1200 High-performance Liquid Chromatography (HPLC, Agilent Technologies, Santa Clara, CA, USA), as previously described in Zhong et al. [33] with slight modifications. Frozen plant tissue (leaves sample, 0.5 mg) was ground with liquid nitrogen, used 10:1 (*v/w*) of extraction buffer (5% cold aqueous perchloric acid (PCA), *w/w*). Samples were incubated for 1 h at 4 ◦C, then centrifuged at 15,000× *g* for 10 min at 4 ◦C. Volumes of 200 μL of collected supernatant were mixed with 15 μL benzoyl chloride and incubated for 1 h at 60 ◦C in darkness conditions. Four milliliters of saturation NaCl solution was used to quench the reaction and diethyl ether was added; 5 mL cold ethyl acetate was then added to extract polyamines. Then, organic layers were evaporated to dryness, redissolved in 100 μL methanol, and filtered with a 0.45 μm pore nylon filter. A volume of 25 μL extraction solution was used to determine the endogenous polyamines levels. The mobile phase was with 64% (*v/v*) methanol and had a flow rate of 0.8 mL min<sup>−</sup>1. Putrescine, spermidine, spermine, and cadaverine (Sigma, St. Louis, MO 63178, USA) were chosen as standard samples and treated similarly.
