*2.3. Determination of Biomass and Root Morphology, Root Vigor and Root Fe3+ Reductase Activity*

After 15 days of treatment, five seedlings were randomly picked from each treatment, and the selected plants were cut from the same part, divided into above-ground and belowground parts, any water on the plant surface was dried with absorbent paper and the fresh weight was measured. The samples were then placed in an electric thermostatic drying oven (Heratherm™ General Protocol Ovens, 51028148, Thermo Scientific™, Waltham, MA, USA) set to 105 ◦C for 30 min. After adjusting the temperature to 80 ◦C, the material was dried to a constant weight before measuring the dry weight. For root morphology measurements, the whole root system of a plant was scanned with a root system scanner (Epson Perfection V800 Photo, B11B223201, Epson America, Inc., Los Alamitos, CA, USA). The analysis was done using a root scanner (WinRhizo PRO, version 2017, Regent Instruments Inc., Quebec City, QC, Canada), and parameters such as the total root length, total surface area, total volume and average diameter were read [25]. Root vigor was determined by the triphenyl tetrazolium chloride (TTC) method [26]. Fe3+ reductase activity was determined according

to the method of Ekmekcioglu C [27]. Three biological replicates for each treatment were set in treatments.

#### *2.4. Determination of Photosynthetic Pigment Content and Photosynthetic Index*

On the 10th day, chlorophylls (Chl) such as Chla, Chlb and carotenoids were measured in the third fully-expanded leaf [28]. About 0.1 g of leaf tissue was placed in a tube containing 96% ethanol in the dark for about 24 h until the leaves turned completely white. The absorbance values of chlorophyll extracts at 470 nm, 649 nm and 665 nm were measured with a spectrophotometer (UV-2450, Shimadzu, Kyoto Prefecture, Japan), and chlorophyll a, chlorophyll b and carotenoid contents were calculated.

The photosynthetic indexes such as the net photosynthetic rate (Pn), transpiration rate (Tr), intercellular CO2 concentration (Ci) and stomatal conductance (Gs) were measured using a portable photosynthetic apparatus (LI-6800, Li-COR Inc., Lincoln, NE, USA) on a clear day at around 10 a.m. The parameters were set to a flow rate of 500 <sup>μ</sup>mol·s−1, leaf temperature of 28 ◦C and CO2 concentration of 400 <sup>μ</sup>mol·mol<sup>−</sup>1; a CO2 cylinder was used to stabilize the CO2 environment [22].

Following 24 h of darkness, the seedling leaves were sampled to test the maximum photochemical efficiency, i.e., Fv/Fm [29]. In addition, the actual photochemical efficiency of PSII (ΦPSII), photosynthetic electron transfer rate (ETR), photochemical quenching coefficient (qP) and non-photochemical quenching coefficient (NPQ) were measured after 30 min of plant exposure to natural light conditions [30].

#### *2.5. Determination of Antioxidant Properties and Osmoregulatory Substances*

A fresh-leaf or root sample (0.3 g) was placed in a pre-cooled pestle and mortar and ground to a fine frozen powder under liquid nitrogen, followed by homogenization in 3 mL 50 mM phosphate buffer (pH 7.8) in an ice bath. Then, homogenate centrifugation was done at 12,000× *g* for 15 min at 4 ◦C. The supernatant was used to determine the peroxidase (POD) [31], catalase (CAT) [32] and superoxide dismutase (SOD) [33] activity. Activity analyses of POD, CAT and SOD were performed as described previously [34]. Three biological replicates for each treatment were performed. The lipid peroxidation level was measured by estimating the malondialdehyde (MDA) content in roots using thiobarbituric acid (TBA) [35]. Electrolyte leakage (%) was estimated by measuring ion leakage from roots according to the method of Shou [36]. The roots (which weighed 0.1 g) were placed in centrifuge tubes, then each tube was filled with 20 mL of distilled water. The conductivity (A1) was first measured after shaking the tube well, then the conductivity (A2) was again measured after shaking the tube in the shaker for 2 h. Finally, the sample was boiled and cooled to room temperature to measure the conductivity (A3). Relative electrolyte leakage was measured as follows: Relative conductivity = (A2−A1)/(A3−A1). The content of H2O2 in leaves and roots was determined by the method of Willekens [37]. The content of O2•− in leaves and roots was analyzed by the method previously described by Li et al. [38]. Proline and soluble protein contents were determined by the methods of Bates [39] and Bradford [40], respectively. Meanwhile, the free amino acids and soluble sugar contents were determined by the method of Zhang et al. [41]. Each treatment was repeated three times to ensure the reliability of the results. The organic acid content was determined by high-performance liquid chromatography [42]. Parameter settings were as follows: a ZORBAX Eclipse XDB-C18 column (4.6 × 250 mm, 5 mm) was used; the mobile phase was set at 0.04 mol·mL<sup>−</sup>1, pH 2.4, KH2PO4-H3PO4 buffer solution; the flow rate was 0.8 mL·min−1; the column temperature was 30 ◦C, the detection wavelength was 210 nm and the injection volume was 10 μL.

#### *2.6. Determination of Sucrose Content and Metabolism-Related Enzyme Activities*

The sucrose content was determined by the hydrochloric acid-resorcinol method previously described by Zhang et al. [43]. We accurately weighed 0.1 g of leaves and roots and took 0.2 and 0.4 mL of supernatants, respectively. After adding 200 μL NaOH, the solution was boiled for 5 min at 100 ◦C, then cooled, before 2.8 mL 30% HCL and 0.8 mL 0.1% resorcinol were added, with the contents shaken well. Then, they were placed in a water bath at 80 ◦C for 10 min for the reaction to occur, and after cooling, the OD value was measured at 480 nm. Three replicates of each treatment were performed. Standard curves with different concentration gradients of sucrose were prepared with the standard solution and used to calculate the actual sucrose content in leaves and roots. To analyze the activities of sugar metabolism-related enzymes, frozen samples of leaves were weighed to 0.1 g. Sucrose synthase (SS), sucrose phosphate synthase (SPS), acid convertase (AI) and neutral convertase (NI) activities were determined using the corresponding enzyme activity assay kits (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China).

#### *2.7. Determination of Polyamine Content*

Polyamines extraction from tomato seedlings was performed using the methods described by Flores and Galston [44]. The content of polyamines was determined by HPLC (high-performance liquid chromatograph UltiMate3000, ThermoFisher Scientific™, Waltham, MA, USA). The instrumentation and settings for endogenous polyamine analyses were as follows: a ZORBAX Eclipse XDB-C18 column (4.6 × 250 mm, 5 mm) and mobile phase (methanol: acetonitrile: water = 58:2.5:39.5) were used with a detection wavelength of 230 nm, flow rate of 1 mL·min−1, column temperature of 30 ◦C and injection volume of 10 μL. The organic solvents used above were of chromatographic-grade purity and the water was ultrapure. The mobile phase was configured for use after ultrasonic sonication beforehand.
