Tomato (Solanum lycopersicum cv. ‘184’) seeds were sown on June 21, 2023, and transplanted to a greenhouse on August 3, 2023, when the tomato seedlings developed four true leaves. The tomato plants were cultivated in a trough-based substrate system with drip irrigation using plastic mulch. After the first flower cluster bloomed, the pollination date was recorded. Ten days after pollination, fruit of uniform size at the fruit-setting stage (diameter ≈ 2 cm) were selected. The plants were marked and treated with different concentrations of exogenous ALA using various methods. Treatments were administered every 10 days until maturity. The experimental treatments are presented in Table 1.

For the ALA treatment as root irrigation, 200 mL of the solution was applied to each plant during each treatment. For the ALA treatment as a foliar spray, a solution containing 0.01% Tween-20 was sprayed until the leaf surface was uniformly covered with droplets. During each treatment, the entire upper and lower surfaces of the four leaves surrounding the fruit clusters were sprayed evenly. The ALA solution sprayed on the fruit surface contained 0.01% Tween-20, and the entire fruit surface was evenly sprayed to ensure that it was covered with droplets. Each treatment was replicated thrice (with three randomly arranged cultivation trays in the greenhouse). To ensure consistency in the effects of ALA treatment, boundary plants were excluded, and six uniformly growing tomato plants were randomly marked in each replicate. Exogenous ALA treatments were conducted at 18:00 (when temperatures drop rapidly in winter in the northwestern region of China) after the greenhouse curtains were closed, followed by 12 h of darkness. All other agronomic management practices were consistent across treatments. During sampling, five mature tomato fruit were randomly selected for each treatment, with three replicates. Samples were immediately brought back to the laboratory for determination of appearance and nutritional quality with the aim of identifying the optimal ALA concentration for root, leaf, and fruit applications. At the fruit-setting stage of the fourth truss of tomato plants, the selected concentrations from the previous screening were applied as follows: root treatments (CK, ALA-1), designated as CK1 and T1; leaf treatments (CK, ALA-2), designated as CK2 and T2; and fruit treatments (CK, ALA-1), designated as CK3 and T3. Treatments were applied every 10 days starting from the fruit-setting stage until the fruit reached maturity. Based on the morphological and color changes observed during fruit development, the dynamic sampling process was categorized into three stages. According to the description by Shinozaki et al. (33), the standard for fruit ripening stages is defined as follows: the mature green stage (full-sized green fruit), the breaker stage (less than 10% green transitioning to orange), the maturity stage (entirely pink fruit), and the red ripe stage (fully red fruit). In this experiment, the mature green stage was observed at 116–123 days after planting, breaker stage at 130–137 days, and full maturity at 144 days.

The single fruit weight was measured using an analytical balance. The soluble sugar content was determined using a method adapted from Grandy et al. (34), with minor modifications. The absorbance of the solution was measured at 620 nm using a UV-1800 ultraviolet–visible spectrophotometer (Shimadzu, Japan) The soluble sugar concentration calculated using the standard curve ranges at 0–160 μg·mL⁻¹. Each treatment was replicated thrice, and the average value was calculated. Titratable acidity was determined by grinding 5 g of fresh tomato fruit into a fine paste, which was then transferred to a conical flask. The volume was adjusted to 50 mL using deionized water, and the mixture was filtered. The filtrate was then titrated with 0.1 mol·L⁻¹ sodium hydroxide (containing two drops of 1% phenolphthalein) until a faint pink color persisted. The procedure was repeated thrice and the average value was calculated. The sugar-to-acid ratio of tomato fruit was calculated as the ratio of soluble sugar content to organic acid content. The soluble solid content was measured as follows: a sample was cut along the equator, and the juice was squeezed out and placed on the prism surface of a PAL-1 handheld refractometer (ATAGO, Japan) to avoid any seeds. This process was repeated if seeds were present. The sample was allowed to sit for 1 min to ensure uniformity and the absence of bubbles, after which the reading was recorded and expressed as a percentage. The soluble protein content was quantified by homogenizing fresh tomato fruit (0.5 g) in 5 mL of distilled water on ice, followed by centrifugation at 8479 × g for 10 min. The supernatant (1 mL) was mixed with Coomassie Brilliant Blue G-250 and incubated for 3 min. Absorbance was measured at 595 nm using a UV-1800 spectrophotometer (Shimadzu, Japan), and the soluble protein concentration was determined based on a standard curve with a concentration range of 0–100 μg·mL⁻¹ with each treatment replicated thrice (units: mg·g⁻¹ FW). Vitamin C (ASA) content was determined using the 2,6-dichlorophenolindophenol (DCPIP) method. Fresh tomato samples (0.5 g) were homogenized in 1.5 mL of 2% oxalic acid in an ice bath, followed by the addition of 1% oxalic acid to form a homogeneous paste. The mixture was transferred to a 50 mL volumetric flask, rinsed with 1% oxalic acid, and supplemented with 30% zinc sulfate (0.5 mL) and 15% potassium ferrocyanide (0.5 mL). The solution was diluted to 50 mL with 1% oxalic acid, filtered, and the filtrate was collected for analysis. The extract [2 mL of DCPIP solution and xylene (5 mL) were added to 4 mL of the extract]. The solution was shaken and allowed to separate, and the pink upper layer was measured at 500 nm using a UV-1800 spectrophotometer (Shimadzu, Japan). The absorbance was recorded after blanking with xylene. The ascorbic acid concentration was calculated using a standard curve within the range of 0–100 mg·mL^-1, with each treatment repeated in triplicate.

Amino acid profile analysis: the preparation of tomato fruit samples and analysis of free amino acid components were conducted according to the method described by Nimbalkar et al. (35), with slight modifications. Twenty amino acid standards were obtained from Merck Millipore (Billerica, MA, United States) and Sigma-Aldrich (St. Louis, MO, United States). For the extraction process, 0.1 g of frozen tomato powder was mixed with 1 mL of 0.5 M hydrochloric acid solution. The mixture was vortexed at 3701 × g for 20 min using a vortex mixer (MX-S, Scilogex, San Diego, CA, United States) and then subjected to ultrasonic extraction at 25°C for 20 min (SB-800 DT, NingBo Scientz Biotechnology Co., Ltd., Ningbo, China). After ultrasonication, the sample was centrifuged at 20,000 × g for 20 min (3-18KS; Sigma, Osterode am Harz, Germany). The supernatant was filtered through a 0.22 μm aqueous filter, and 5 μL of the filtrate was injected into an HPLC–MS system (LC–MS, Agilent, #1290–6,460, Santa Clara, CA, United States) for quantitative analysis. The HPLC conditions were as follows: an Agilent InfinityLab Poroshell 120 HILIC-Z column (2.1 × 100 mm, 2.7 μm) was used; a 200 mM ammonium formate stock solution was prepared with water and the pH was adjusted to 3 with formic acid. Mobile phase A was composed of water and ammonium formate stock solution (9:1), and mobile phase B consisted of acetonitrile and ammonium formate stock solution (9:1); both mobile phases had a final concentration of 20 mM. The flow rate was set at 0.5 mL·min-1 and the column temperature was maintained at 25°C. The MS conditions were as follows: the ionization mode was ESI positive mode; the drying gas temperature was 330°C, nebulizer pressure was 35 psi, gas flow rate was 13.0 L·min⁻¹, sheath gas temperature was 390°C, sheath gas flow rate was 12 L·min⁻¹, and capillary voltage was 1,500 V.

The content of glutamate was derived from the free amino acid content determination data described above.

The GABA content was determined using a γ-aminobutyric acid (GABA) assay kit (Suzhou Keming Biotechnology Co., Ltd., Suzhou, China), following the manufacturer’s instructions. Each treatment was performed in triplicate, and the average value was calculated. The instructions for the assay kit are as follows. Weigh approximately 0.1 g of fresh tomato sample and add 1 mL of extraction buffer. The mixture was thoroughly homogenized and transferred to an EP tube. Incubate the tube in a 95°C water bath for 2 h, ensuring the cap is tightly sealed to prevent water loss. After cooling, centrifuge at 8,000 × g for 10 min at 25°C, and collect the supernatant for analysis. Take 90 μL of the supernatant (for the blank, use 90 μL of extraction buffer) and add 150 μL of Reagent 1 and 120 μL of Reagent 2. The solution was thoroughly mixed and incubated at room temperature for 5 min. Then, add 180 μL of Reagent 3, mix well, and incubate in a 95°C water bath for 10 min. After cooling on ice, add 600 μL of Reagent 4 and mix thoroughly. Then, 1 mL of the mixture was transferred into a 1 mL glass cuvette, and the absorbance was measured at 640 nm. The absorbance values were recorded as samples (A_sample) and blanks (A_blank). The difference in absorbance is calculated as ∆A = A_sample − A_blank. Finally, calculate the GABA content using the regression equation provided by the kit under standard conditions.

The activity of glutamate acid decarboxylase (GAD) was measured using a plant GAD assay kit (Suzhou Greys Biotech Co., Ltd., Suzhou, China). This procedure was performed according to the manufacturer’s instructions. Each treatment and plant part were analyzed in triplicate, and enzyme activity was calculated based on standard curves.

Total RNA was extracted from tomato fruit using the AG Steady Pure Plant RNA Extraction Kit (Aikeri Biotechnology), and reverse transcription was performed using the AG Evo M-MLV Reverse Transcription Kit (Aikeri Biotechnology). The qRT-PCR reaction system comprised 10 μL, including 0.5 μL of cDNA template, 5 μL of 2 × SYBR Green qPCR Premix (containing ROX Plus) (Aikeri Biotechnology, Beijing, China), and 0.4 μL of each forward and reverse primer, with ddH₂O added to a final volume of 10 μL. Two-step detection was conducted according to the manufacturer’s instructions and quantification was performed using the comparative CT method (36). The tomato actin gene served as an internal reference, and the primer sequences are listed in Table 2.

Data were analyzed using SPSS 26.0, and GraphPad Prism 10.1.2. Tukey’s HSD test was performed to conduct variance analysis, with a significance level set at p < 0.05. Graphical representations were conducted using GraphPad Prism 10.1.2.

As shown in Figure 1A, ALA application at all three sites increased tomato fruit size. Among them, the largest fruit weight in each treatment was obtained with root application of 10 mg·L⁻¹, foliar application of 100 mg·L⁻¹, and fruit application of 100 mg·L⁻¹. As shown in Figure 1B, the application of ALA solutions in different ways enhanced the single-fruit weight of tomatoes. Root application of 10 mg·L⁻¹ ALA significantly increased the single fruit weight at the mature stage by 67.77% compared to the control. Leaf application of 100 mg·L⁻¹ ALA significantly increased the single fruit weight at the mature stage by 42.37% compared to the control. Fruit application of 100 mg·L⁻¹ ALA significantly increased the single fruit weight at the mature stage by 76.24% compared to the control.

The ALA solutions applied to different sites increased the soluble sugar content of tomatoes (Figure 2A). However, root application of 10 mg·L⁻¹ and fruit application of 100 mg·L⁻¹ ALA significantly increased the soluble sugar content in ripe tomatoes.

Specifically, root application of 10 mg·L⁻¹ ALA significantly increased the soluble sugar content in mature tomatoes by 94.52% compared to the control. Leaf application of 50 mg·L⁻¹ and 100 mg·L⁻¹ ALA also showed higher soluble sugar content than the control, although the differences were not statistically significant. Fruit application of 100 mg·L⁻¹ ALA significantly increased the soluble sugar content in mature tomatoes by 78.51% compared to the control.

The ALA solutions at different concentrations applied to different plant sites decreased the titratable acid content in tomato fruit (Figure 2B). Root application of 10 mg·L⁻¹ ALA significantly reduced the titratable acid content in mature tomatoes by 33.61% compared to the control. Leaf application of 50 mg·L⁻¹ and 100 mg·L⁻¹ ALA significantly reduced the titratable acid content in mature tomato peels by 22.05 and 23.62%, respectively. The application of 100 mg·L⁻¹ and 200 mg·L⁻¹ ALA significantly reduced the titratable acid content in mature tomatoes by 19.42 and 12.81%, respectively.

The ALA solutions applied to different sites increased the sugar-acid ratio in tomatoes (Figure 2C). However, root application of 10 mg·L⁻¹, foliar application of 50 mg·L⁻¹ and 100 mg·L⁻¹, and fruit application of 100 mg·L⁻¹ and 200 mg·L⁻¹ ALA significantly increased the sugar-to-acid ratio in ripe tomatoes. In contrast, root application of 50 mg·L⁻¹ ALA reduced the sugar-to-acid ratio, while other ALA treatment concentrations had no effect on this parameter. Root application of 10 mg·L⁻¹ ALA significantly increased the sugar-acid ratio in mature tomatoes by 1.90 times compared to the control. Leaf application of 50 mg·L⁻¹ and 100 mg·L⁻¹ ALA significantly increased the sugar-acid ratio in mature tomatoes by 0.78 times and 0.60 times, respectively. Fruit application of 100 mg·L⁻¹ ALA and 200 mg·L⁻¹ ALA significantly increased the sugar-acid ratio in mature tomatoes by 1.25 times and 0.42 times, respectively, compared to the control.

The ALA solutions applied to different plant sites enhanced the soluble solid content of tomatoes (Figure 3A). Specifically, root application of 10 mg·L⁻¹ ALA significantly increased the soluble solids content in mature tomatoes by 41.71% compared to the control. Leaf application of 50 mg·L⁻¹ and 100 mg·L⁻¹ ALA significantly increased the soluble solids content in mature tomatoes by 13.88 and 9.09%, respectively. Fruit application of 100 mg·L⁻¹ and 200 mg·L⁻¹ ALA significantly increased the soluble solids content in mature tomatoes by 15.50 and16.67%, respectively, compared to the control. Figure 3B shows that ALA solutions applied to different plant sites enhanced the soluble protein content in tomatoes. Application of 10 mg·L⁻¹ ALA irrigated on root significantly increased the soluble protein content in mature tomatoes by 82.71% compared to the control. Leaf application of 50 mg·L⁻¹ and 100 mg·L⁻¹ ALA significantly increased the soluble protein content in mature tomatoes by 114.71 and 241.05%, respectively, compared to the control. Fruit application of 100 mg·L⁻¹ ALA increased the soluble protein content in mature tomatoes by 152.32% compared to the control.

Exogenous ALA solutions applied to different plant sites enhanced ascorbic acid content in tomatoes (Figure 3C). However, root application of 10 mg·L⁻¹, foliar application of 50 mg·L⁻¹ and 100 mg·L⁻¹, and fruit application of 100 mg·L⁻¹ ALA significantly increased the ascorbic acid content in ripe tomatoes, whereas other ALA treatment concentrations had no effect on ascorbic acid content. Root application of 10 mg·L⁻¹ ALA significantly increased the ascorbic acid content in mature tomatoes by 5.06% compared to the control. Leaf application of 50 mg·L⁻¹ and 100 mg·L⁻¹ ALA significantly increased the ascorbic acid content in mature tomatoes by 4.79 and 5.33%, respectively. Fruit application of 100 mg·L⁻¹ ALA significantly increased the ascorbic acid content in mature tomatoes by 1.31% compared to the control.

Figure 4 illustrates the effect of exogenous ALA application to various plant parts on the free amino acid composition of tomato. In the present study, 20 free amino acids, including both essential and non-essential amino acids, were detected during the three ripening stages. The essential amino acids in humans include phenylalanine, methionine, isoleucine, tryptophan, valine, leucine, histidine, and threonine. Non-essential amino acids include glutamic acid, cysteine, arginine, glycine, serine, proline, tyrosine, alanine, cystine, aspartic acid, asparagine, and glutamine. Based on taste properties, these 20 amino acids were categorized into four groups: umami (glutamic and aspartic acids), bitter (arginine, histidine, leucine, tyrosine, valine, isoleucine, and phenylalanine), sweet (proline, alanine, threonine, glycine, serine, and asparagine), and aromatic (phenylalanine, tyrosine, and tryptophan). The levels of these amino acids, particularly umami, increased significantly during fruit maturation.

During the mature green stage of the fruit, the total amino acid content was 21.08% higher with root-applied exogenous ALA than with the other treatments (Figure 4A). The umami amino acid content was also higher (24.33%) than that of the control.

During the breaker stage of the fruit, the total amino acid content was higher with root and leaf ALA applications than in the control groups, with increases of 8.39 and 18.08%, respectively (Figure 4B). Umami amino acids were higher in the root, leaf, and fruit treated with exogenous ALA than in their respective control groups, with increases of 35.06, 16.22, and 21.07%, respectively.

In addition, during the mature stage, the total amino acid content with root and leaf applications was higher than that in the control, with increases of 11.22 and 16.5%, respectively (Figure 4C). The umami amino acid content in fruit treated with roots, leaves, and fruit was higher than that in their respective controls, with increases of 10.03, 20.13, and 7.26%, respectively.

As shown in Figure 5A, the GABA content in tomato fruit was lower at the mature stage than at other stages, indicating that the GABA content in the fruit decreased during the ripening process. However, exogenous ALA application to different parts of the plant increased GABA content in the fruit. During the mature green stage, exogenous ALA applied to the roots, leaves, or fruit significantly increased the GABA content in tomato fruit, with increases of 45.16, 41.83, and 32.54%, respectively, compared with the control groups. In the breaker stage, fruit-applied exogenous ALA significantly increased the GABA content in tomato fruit by 98.41% compared to the control. Although root and leaf applications of ALA showed numerical increases compared to the controls during the maturity stage, these increases were not statistically significant. During the mature stage, the GABA content in tomato fruit decreases rapidly, but exogenous ALA application to the roots, leaves, or fruit effectively slows this decline. Compared to the control, ALA applied to different parts of the plant significantly increased the GABA content in tomato fruit by 433.32, 214.58, and 373.98%, respectively.

The glutamate content increased as the tomatoes matured (Figure 5B). During the mature-green stage, the application of exogenous ALA to plant roots significantly increased the glutamate content in tomato fruit, showing an increase of 32.50% compared with that of the control. However, foliar and fruit applications only showed numerical increases without significant differences compared with the control. During the breaker stage, exogenous ALA applied to different parts significantly increased glutamate content in tomatoes compared to that in the control groups, in which the root treatment improved glutamate content by 33.32%, leaf treatment by 24.25%, and fruit treatment by 28.57%. At the maturity stage, exogenous ALA applied to different parts significantly increased the glutamate content in tomatoes by 15.60, 28.73, and 9.96% in the root, leaf, and fruit treatments, respectively.

In the control and leaf ALA groups, GAD activity decreased as the tomatoes matured (Figure 5C). However, root- and fruit-applied exogenous ALA increased GAD activity as the tomatoes matured. Exogenous ALA applied to different plant parts can enhance GAD activity in tomato plants. In the mature green stage, root and leaf application of exogenous ALA significantly increased GAD activity in tomato fruit by 90.30 and 94.31%, respectively, compared with the control, whereas fruit application showed a numerical increase without a significant difference. In the breaker stage, root and fruit applications of exogenous ALA significantly increased GAD activity by 93.00 and 63.88%, respectively, compared to the control, whereas leaf application showed a numerical increase without a significant difference. At the maturity stage, root and fruit application of exogenous ALA significantly increased GAD activity in tomato fruit by 241.17 and 154.19%, respectively, compared with the control, whereas leaf application showed a numerical increase without a significant difference. As shown in Figures 5D–F, root application of ALA during the mature-green stage significantly increased the relative expression of GAD3 in tomatoes by 167.45% compared to the control. Leaf application of ALA also significantly enhanced the relative expression of GAD3, increasing it by 156.22% compared with the control. Furthermore, fruit application of ALA significantly elevated the relative expression of GAD1 by 60.15% compared with the control. During the breaker stage, root application of ALA significantly increased the relative expression of GAD3 by 64.89% compared with the control. Leaf application of ALA notably enhanced the relative expression of GAD1, increasing it by 63.75% compared with the control. Additionally, fruit application of ALA significantly increased the relative expression of GAD1 by 146.24% compared with the control. In the mature stage, fruit application of ALA significantly boosted the relative expression of GAD1 and GAD2, resulting in increases of 219.37 and 214.26%, respectively, compared to the control.