A method for improving the nutritional quality of tomato fruits at high temperature

Abstract

This invention discloses a method for improving the nutritional quality of tomato fruits under high temperatures, belonging to the field of agricultural technology. The method includes: under high-temperature conditions, after the tomatoes have grown to the first inflorescence and are bearing fruit, spraying a serotonin-rhamnolipin mixture diluted with water onto the leaves and / or fruits; wherein the final concentration of serotonin in the diluted serotonin-rhamnolipin solution is 50-100 μM, and the concentration of rhamnolipin is 50-100 mg / L. This invention regulates the homeostasis of reactive oxygen species in fruits and protects the activity of key enzymes in the carotenoid synthesis pathway through serotonin, while simultaneously utilizing rhamnolipin to enhance the penetration and delivery efficiency of serotonin into fruit tissues and improve its stability. The two work synergistically to achieve precise quality control during fruit development. This invention can significantly increase the content of soluble solids, soluble sugars, lycopene, and carotene in tomato fruits under high temperatures without affecting the single fruit weight, fruit shape, or other appearance and commercial characteristics. It has the advantages of simple operation, low cost, and safety with no residue.

Description

Technical Field

[0001] This invention mainly relates to the field of agricultural technology, specifically a method for improving the nutritional quality of tomato fruits under high temperatures. Background Technology

[0002] Tomatoes are extremely sensitive to high temperatures during their growth. When ambient temperatures remain consistently high, photosynthesis in tomatoes is inhibited, pollen viability decreases, pollination and fertilization are hindered, and fruit set rate is significantly reduced. More importantly, high temperatures severely inhibit the synthesis and accumulation of flavor and nutrients such as sugars, organic acids, and lycopene in the fruit, leading to poor color development, insufficient sugar content, and bland flavor, resulting in a significant decrease in the fruit's commercial value and market competitiveness. The problem of high-temperature stress on tomatoes in summer greenhouse cultivation is becoming increasingly prominent. How to ensure the normal development and quality formation of tomato fruits under high-temperature conditions has become an urgent technical challenge to be solved in the field of tomato cultivation and quality control.

[0003] Currently, production methods to cope with high-temperature stress mainly rely on physical measures such as shading netting and misting for cooling. While these measures can alleviate heat damage to some extent, they cannot fundamentally solve the core problems caused by high temperatures, such as pollen abortion, reduced fruit set, and deterioration of fruit quality. In terms of chemical regulation, there are reports of technologies that enhance the high-temperature tolerance of tomatoes through exogenous substances. For example, Chinese invention patent CN115849977B discloses a water-soluble fertilizer for tomatoes, its preparation method and application, and a tomato planting method. This method enhances the tomato's adaptability to high-temperature environments by adding methyl jasmonic acid to the fertilizer, while simultaneously adding lycopene to promote lycopene synthesis and improve fruit coloring. Chinese invention patent application CN119325867A discloses a method to improve tomato fruit yield and quality under high-temperature conditions. Spraying glutathione solution on tomato leaves during hot weather significantly increases the weight of individual fruits, the yield per plant, and the content of soluble solids, soluble sugars, and vitamin C in the fruit. In addition, Chinese invention patent CN121377864 A discloses a water-soluble fertilizer and its preparation method for improving the high temperature resistance and stress resistance of tomatoes. It proposes to enhance the heat resistance of tomatoes from a metabolic level by adding substances related to the polyamine metabolic pathway, such as L-arginine, L-methionine, spermidine and methyl jasmonate.

[0004] However, the existing technologies still have significant shortcomings. First, most existing technologies focus on improving the overall stress resistance of the plant, primarily addressing the improvement of leaf heat tolerance or the maintenance of fruit yield. Their specific regulatory effect on fruit flavor and quality (especially key quality indicators such as soluble sugar content and sugar-acid ratio) under high-temperature stress is very limited. For example, while spraying glutathione or methyl jasmonate during the seedling stage can improve plant heat tolerance, it does not significantly promote the accumulation of soluble sugars and lycopene in mature fruits. Second, the timing of existing technologies is mostly during the seedling or vegetative growth stages, making it difficult to play an effective role during the critical window period of fruit sugar accumulation and flavor compound synthesis. Third, the mechanisms of action of existing solutions are relatively simple, mostly limited to supplementing nutrients or activating a specific stress resistance signaling pathway, lacking systematic regulation of the fruit quality metabolic network.

[0005] Serotonin (5-hydroxytryptamine, 5-HT) is an endogenous neurotransmitter metabolite found in plants, and recent studies have shown its involvement in the regulation of plant stress responses. Rhamnolipids are glycolipid biosurfactants produced by *Pseudomonas aeruginosa*, capable of reducing interfacial tension on leaf surfaces and improving the wetting and spreading ability of spray solutions on the cuticle of leaves and fruits. However, the combined application of serotonin and rhamnolipids for the regulation of tomato fruit quality, especially for specifically enhancing fruit flavor and quality under high temperatures through exogenous spraying during fruit development, has not been reported to date.

[0006] Therefore, there is an urgent need in this field for a method that can specifically enhance the flavor quality of tomato fruits (especially the content of soluble sugars and lycopene) under high temperature conditions. By precisely targeting the key period of fruit quality formation, the method can effectively alleviate the inhibitory effect of high temperature on fruit sugar accumulation and flavor substance synthesis from a metabolic perspective, so as to ensure the commercial quality of tomatoes during the hot summer season. Summary of the Invention

[0007] This invention addresses the problem of overly simplistic solutions in existing technologies by providing a significantly different approach. It offers a method to improve the nutritional quality of tomato fruits under high temperatures, addressing the shortcomings of existing technologies that primarily focus on enhancing overall plant resistance or maintaining yield. These technologies lack effective means for specifically regulating the flavor and quality of tomato fruits under high temperatures during fruit development, making it difficult to precisely control sugar accumulation and flavor compound synthesis during critical periods. Consequently, they fail to effectively alleviate the technical problems of decreased sugar content, flavor deterioration, and reduced nutritional quality in tomato fruits caused by high temperatures.

[0008] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows: A method for improving the nutritional quality of tomato fruit under high temperature includes the following steps: (1) Preparation of serotonin-rhamnolipin mixture; (2) After tomatoes grown at high temperatures (referring to temperature conditions above 32°C that affect the normal coloring and sugar accumulation of tomatoes) have grown to the point of fruiting the first inflorescence, spray the leaves and / or fruits with a serotonin-rhamnolipin mixture; the serotonin-rhamnolipin mixture is obtained by diluting the serotonin-rhamnolipin mixture with water by 100-200 times.

[0009] Furthermore, in step (2), the amount of spraying per plant is 5-15 mL, and the amount per hectare is 150 L-450 L.

[0010] Furthermore, in step (2), after the first spray, spray once every 7 days, and spray a total of 3 times during the high-temperature season.

[0011] Further, in step (1), the serotonin-rhamnolipin mixture has a serotonin concentration of 10 mM and a rhamnolipin solution concentration of 10 mg / L; in step (2), the serotonin-rhamnolipin mixture has a serotonin concentration of 50-100 μM and a rhamnolipin solution concentration of 50-100 mg / L.

[0012] Furthermore, step (1) specifically includes: (1-1) First, dissolve serotonin in ultrapure water and add antioxidant; then add ultrapure water to make up the volume to prepare a stock solution with a serotonin concentration of 20 mM. The final concentration of antioxidant in the serotonin stock solution is 0.1 mM. It is required to protect from light and keep at low temperature during preparation. (1-2) Weigh rhamnolipin, add ultrapure water and stir to dissolve, then make up to a final volume to prepare a rhamnolipin stock solution with a final concentration of 20 g / L; (1-3) Finally, the serotonin mother liquor and rhamnolipin mother liquor are mixed in equal volume ratio to obtain the serotonin-rhamnolipin mixture.

[0013] This invention is based on the synergistic effect of serotonin and rhamnolipin. By intervening exogenously during the critical window of tomato fruit quality formation, it achieves a specific improvement in the nutritional quality of fruits under high temperature stress. Its mechanism of action mainly includes the following three aspects:

[0014] (1) Although existing technologies mention the relationship between serotonin and rice heat resistance, they all focus on stress resistance studies during the seedling / vegetative growth stage, and have never addressed quality regulation during fruit development, let alone flavor quality indicators such as soluble sugars and lycopene. In this invention, serotonin, as an important indoleamine endogenous metabolite in plants, can effectively scavenge excessive reactive oxygen species (ROS) accumulated in fruit cells under high temperature stress, alleviate the damage of oxidative stress to chloroplast and mitochondrial function, thereby maintaining the activity of the photosynthetic electron transport chain and ensuring the normal synthesis of photosynthetic products. More importantly, serotonin can protect key enzymes in the carotenoid biosynthesis pathway in fruits (such as phytoene synthase PSY1) from oxidative inactivation, thereby relieving the inhibition of lycopene synthesis by high temperature. At the same time, serotonin promotes the conversion and accumulation of sucrose to fructose and glucose by regulating the activity of enzymes related to sugar metabolism, fundamentally alleviating the problem of decreased soluble sugar content caused by high temperature.

[0015] (2) Rhamnolipids, as a glycolipid biosurfactant, can significantly reduce the interfacial tension between the sprayed solution and the fruit surface, and improve the wetting and spreading ability of droplets on the waxy layer of tomato leaves and the cuticle of fruit. More importantly, rhamnolipids can instantly increase the permeability of the cuticle, and as an efficient delivery carrier, significantly enhance the penetration and delivery efficiency of serotonin into the internal tissues of the fruit (especially the pulp cells), ensuring that serotonin reaches an effective concentration in the target organs, thereby achieving the ideal quality control effect with a lower spraying concentration.

[0016] (3) The present invention adds a certain proportion of rhamnolipin to the mixed reagent to provide an acidic and stable solution system for serotonin, avoiding the disadvantage of serotonin being easily oxidized and degraded when used in high temperature and alkaline environment, and giving full play to the effect of the mixed preparation of serotonin and rhamnolipin.

[0017] (4) Existing technologies all focus on improving overall stress resistance during the seedling stage, full bloom stage, or the entire growth period. It is generally believed in the field that intervention should be carried out as early as possible to enhance the overall heat resistance of the plant, rather than precise intervention during the fruit development stage. This invention breaks with the conventional thinking of existing technologies that intervene during the seedling stage or vegetative growth stage, and chooses to spray after the first inflorescence sets fruit (early reproductive development). This period is the key quality formation period for fruit cell proliferation, cell enlargement, sugar accumulation, and large-scale synthesis of carotenoids, and the distribution of photosynthetic products to the fruit reaches its peak. During this window period, exogenous serotonin-rhamnolipin intervention can precisely concentrate the regulatory effect on the fruit itself, minimize the consumption of the agent by the plant's vegetative growth, and achieve targeted quality regulation. Therefore, this invention can specifically improve the internal nutritional quality of the fruit (soluble solids, soluble sugar, lycopene and β-carotene content) without significantly affecting the appearance traits such as single fruit weight and fruit size.

[0018] In summary, this invention forms a systematic protective network for tomato fruit quality under high-temperature stress through a triple synergistic mechanism of biochemical regulation of serotonin, physical delivery of rhamnolipin, and precise targeting during fruit development. This effectively solves the technical problems of poor fruit flavor quality regulation, inappropriate timing of regulation, and single mechanism of action in existing technologies.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] (1) This invention, through exogenous spraying of a serotonin-rhamnolipin mixture, can effectively alleviate the inhibitory effect of high temperature on fruit sugar accumulation and carotenoid biosynthesis. Experiments show that after spraying the 'Zheyingfen No. 1' cherry tomato with the preparation of this invention during the high-temperature season, the soluble solids content of mature fruit increased by 21.57%, the soluble sugar content increased by 35.53%, the lycopene content increased by 53.42%, and the β-carotene content increased by 42.7%, and the fruit flavor and quality were significantly improved.

[0021] (2) This invention selects to spray during the fruit development period after the first inflorescence sets fruit, precisely focusing the regulatory effect on the fruit development and ripening stages. Experimental results show that after spraying the formulation of this invention, there are no significant differences in appearance traits such as single fruit weight, transverse diameter, longitudinal diameter, and fruit shape index compared with the control, achieving a selective regulatory effect of "specifically improving internal nutritional quality without changing appearance and marketability". This selective regulatory effect of specifically improving internal quality without affecting appearance traits, as well as the synergistic effect of the combination of serotonin and rhamnolipin, both exceed the reasonable expectations of those skilled in the art.

[0022] (3) This invention utilizes rhamnolipin as a biosurfactant to significantly reduce the interfacial tension between the leaf and fruit surfaces of the spray solution, enhancing the penetration and transfer efficiency of serotonin into the internal tissues of the fruit, and enabling serotonin to reach an effective concentration in the target organs. In addition, rhamnolipin also improves the stability of serotonin by maintaining an acidic buffer environment. With the synergistic effect of the two, ideal quality control effects can be achieved with a lower spray concentration, reducing production costs and avoiding the adverse stress that high concentrations may cause to the fruit.

[0023] (4) This invention uses foliar and / or fruit spraying, requiring no complex equipment and is convenient for field operation. Serotonin has a metabolic degradation pathway in plants, leaving no harmful residues after spraying. The method of this invention is low-cost and environmentally friendly, making it suitable for large-scale application in greenhouse tomato production during the high-temperature summer season.

[0024] In summary, this invention effectively solves the technical problems of decreased sugar content, flavor deterioration, and reduced nutritional quality in tomato fruits under high-temperature stress through the triple synergistic effect of biochemical regulation of serotonin, physical delivery of rhamnolipin, and precise targeting during fruit development. It significantly improves the internal flavor quality while ensuring the marketability of the fruit appearance, providing a reliable technical solution for high-quality tomato production during the hot summer season.

[0025] The present invention will be explained in detail below with reference to the accompanying drawings and specific embodiments. Attached Figure Description

[0026] Figure 1 Figure 1 shows the results of the high-temperature stress treatment experiment in the artificial climate chamber of this invention. (A) Plant phenotypes with and without exogenous serotonin spraying under normal temperature (NT) or high temperature (HT) conditions, scale bar = 5 cm. (B) Chlorophyll fluorescence imaging (Fv / Fm) under heat stress. The pseudo-color bar scale (right) ranges from 0 (black) to 1 (purple) to represent the degree of leaf damage. Scale bar = 2 cm. (C, D) Hydrogen peroxide (H2O2) content in leaves under heat stress (C), histochemical staining of hydrogen peroxide (DAB), and superoxide anion (O2⁻, NBT) (D); scale bar = 2 cm. (E, F) Relative electrolyte leakage (REL) (E) and malondialdehyde (MDA) content in leaves under heat stress (F). (G) Activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) in leaves of plants with and without serotonin treatment under control and heat stress (HS) conditions. Data are presented as mean ± standard deviation (n=3). Different letters indicate significant differences (P<0.05, Tukey test).

[0027] Figure 2 This study describes the effects of exogenous spraying of a mixed formulation during the seedling stage on the quality of tomato fruits in the later stages of the invention. The study included the content of single-fruit weight (A), firmness (B), vitamin C (C), soluble solids (D), lycopene (E), and β-carotene (F) under high-temperature stress (HS). Data are presented as mean ± standard deviation (n=5). * indicates significant difference (P<0.05).

[0028] Figure 3This invention illustrates the effect of exogenous mixed formulations applied during the flowering and fruiting period on improving tomato fruit quality under heat stress. (A) Fruit phenotype under high-temperature stress (HS) (with and without serotonin application). Scale bar = 1 cm. (B, C) Fruit weight (B) and firmness (C) under HS; data are the mean ± standard deviation of three biological replicates from at least five independent fruits. (DG) Fruit quality parameters under high-temperature conditions: ascorbic acid (AsA) (D), soluble sugar (E), lycopene (F), and β-carotene (G) content. (H, I) Fruit phenotype (H) and damage index (I) after 7 days of high-temperature treatment. Data are presented as mean ± standard deviation (n=5 in Figures B and C, n=3 in Figures E, F, G, and I). Different letters indicate significant differences (P<0.05, Tukey test). Asterisks indicate significant differences (**, P<0.01; ***, P<0.001, t-test).

[0029] Figure 4 This figure illustrates the effect of spraying a mixed formulation during high-temperature seasons on the soluble solids and sugar content of tomato fruits. The data in the figure are the mean ± standard error of three replicate experiments. * and ** indicate significant (P<0.05) or highly significant (P<0.01) differences between the treatment and control.

[0030] Figure 5 This figure illustrates the effect of spraying a mixed formulation during high-temperature seasons on the lycopene and carotenoid content of tomato fruits. The data in the figure represent the mean ± standard error of three replicate experiments. ** indicates a highly significant difference between the treatment and the control (P < 0.01).

[0031] Figure 6 This figure illustrates the effect of spraying a mixed formulation during high-temperature seasons on the vitamin C content of tomato fruits. The data in the figure are the average of three replicate experiments ± standard error.

[0032] Figures 1-6 In this paper, CK represents the water control group, and Serotonin and SER both represent the serotonin-rhamnolipin mixture of the present invention. Detailed Implementation

[0033] To facilitate understanding of the present invention, a more comprehensive description of the present invention will be given below with reference to the accompanying drawings, which illustrate several embodiments of the present invention. However, the present invention can be implemented in different forms and is not limited to the embodiments described in the text. Rather, these embodiments are provided to make the disclosure of the present invention more thorough and comprehensive.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0035] 1. Plant materials

[0036] The tomato variety used in the artificial climate chamber experiment was "Micro-Tom". Tomato seedlings were cultivated on a substrate (peat moss:vermiculite:perlite in a volume ratio of 3:1:1) and then grown in a greenhouse. The photoperiod was set to 16h / 8h (light / dark), the air humidity was maintained between 70% and 85%, and the light intensity was 300 μmol·m⁻¹. -2 s -1 The normal growth temperature cycle for the plants is set at 25℃±1℃ / 20℃±1℃ (day / night). Water and fertilizer are applied as usual to grow the tomatoes to the budding stage under normal temperature.

[0037] The tomato varieties used by the Datian Experimental Institute were 'Zheyingfen No. 1' and 'Qingxia 66', which are representative varieties of pink and green cherry tomatoes commonly used in production. After substrate seedling cultivation under the above conditions, the seedlings were transplanted into plastic greenhouses at the end of April. Fertilization, plant adjustment, and pest and disease control were carried out according to conventional methods.

[0038] 2. Prepare serotonin stock solution and rhamnolipin solution separately:

[0039] First, prepare the serotonin stock solution. Taking 1L as an example, weigh 4.26 g of serotonin (5-hydroxytryptamine hydrochloride, Shanghai Yuanye Biotechnology Co., Ltd.), add ultrapure water and stir to dissolve. Add 17.6 mg of ascorbic acid, and then add ultrapure water to make up to 1L. Prepare a stock solution with a serotonin concentration of 20 mM. The final concentration of antioxidant in the serotonin stock solution is 0.1 mM. It is required to protect from light and keep at a low temperature during preparation.

[0040] Meanwhile, prepare a 20 g / L rhamnolipin stock solution. For example, weigh 20 g of rhamnolipin, add ultrapure water and stir to dissolve, and then bring the volume to 1 L.

[0041] Finally, the serotonin stock solution and rhamnolipin stock solution were mixed in an equal volume ratio of 1:1 to obtain the serotonin-rhamnolipin mixture.

[0042] A method for improving the nutritional quality of tomato fruit under high temperature includes the following steps:

[0043] Under high temperatures (referring to temperatures above 32℃ that affect normal coloring and sugar accumulation in tomatoes), after the tomatoes have developed their first inflorescence and fruiting, dilute the serotonin-rhamnolipin mixture 100 times with water to obtain a diluted serotonin-rhamnolipin solution (maximum serotonin concentration of 100 μM, final rhamnolipin concentration of 100 mg / L). Spray the diluted serotonin-rhamnolipin solution onto the leaves. The dosage per plant is 10 mL per spray, and the dosage per hectare is approximately 300 L. After the first spray, spray again every 7 days, for a total of 3 sprays during the high-temperature season.

[0044] Preliminary experiments revealed that when the final serotonin concentration was below 50 μM, the effect on increasing soluble sugars and lycopene in tomato fruits under high temperatures was not significant; when the concentration was above 100 μM, further increasing the concentration did not improve fruit quality and would increase application costs. Therefore, the preferred final serotonin concentration in this invention is 50–100 μM.

[0045] Similarly, the preferred final concentration of rhamnolipin in this invention is 50–100 mg / L; the application amount per spray for a single plant is 5–15 mL.

[0046] To verify the advantages of this invention, a comparative experiment is conducted below.

[0047] 1. Experiment on high temperature stress treatment in artificial climate chamber

[0048] (1) High temperature stress treatment and spraying treatment

[0049] A. High temperature stress treatment and serotonin spraying treatment during the seedling stage

[0050] When tomato plants cultivated at room temperature (25℃±1℃ / 20℃±1℃, day / night) reached five to six true leaves, the serotonin-rhamnolipin mixture prepared above was diluted 100 times, and a water control was sprayed onto the plant leaves at 10:00 AM for treatment. Three days after spraying, some of these plants were subjected to a four-day high-temperature treatment at 35℃ during the day and 30℃ at night. On the first and fourth days of heat stress, the serotonin-rhamnolipin mixture was sprayed again, with 5.0 mL of the mixture or water applied to each plant each time. Each treatment group contained 15 plants, with a total of 3 biological replicates. The remaining treated plants were kept at room temperature (25℃±1℃ / 20℃±1℃, day / night) as a temperature control.

[0051] B. High temperature stress treatment and serotonin spraying treatment during flowering and fruiting period

[0052] (a) During the budding stage of the first inflorescence of tomatoes, the serotonin-rhamnolipin mixture prepared above was diluted 100 times and the control solution was sprayed on the leaves and young fruits of different plants at 10:00 am. The amount sprayed on a single plant was 5.0 mL each time. Three days later, the plants were subjected to a 4-day high-temperature treatment with a daytime temperature of 35°C and a nighttime temperature of 30°C. Then, on the day of the high-temperature treatment and the day after the treatment, the mixed formulation or the control formulation was sprayed once each, with the formulation sprayed directly onto the flower buds, and 5.0 mL was sprayed on each single plant. Each treatment had 15 tomato plants, and a total of 3 biological replicates were performed.

[0053] (b) Normal temperature control conditions: Flower buds were treated using the method described in (a), except that the temperature was maintained at the normal growth temperature of tomato plants for a period of 25℃±1℃ / 20℃±1℃ (day / night), and all other conditions remained unchanged.

[0054] (2) Experimental measurement methods

[0055] A. Seedling heat resistance test

[0056] (a) Chlorophyll fluorescence (Fv / Fm)

[0057] The image-maxi system (Heinz Walz, Germany) was used. Specifically, the removed leaves were placed in a 96-well plate lined with filter paper moistened with distilled water and kept in the dark for 15 minutes. Minimum fluorescence (Fo) was measured using weak photochemical light, and maximum fluorescence (Fm) was measured using light at an intensity of 4000 μmol·m⁻¹. -2 s -1 The fluorescence was obtained by saturated pulsed light irradiation for 0.8 s. The maximum photochemical quantum yield of PSII is expressed as Fv / Fm, where the variable fluorescence (Fv) is calculated from the difference between Fm and Fo, i.e., Fv = Fm − Fo.

[0058] (b) ROS reaction

[0059] The accumulation of hydrogen peroxide (H2O2) and superoxide anion (O2⁻) was detected using diaminobenzidine (DAB) and nitrotetrazole blue (NBT) staining methods, respectively. The specific steps were as follows: Intact leaves were placed in a DAB (1 mg / mL) solution, completely immersed, and then vacuumed for 30 min. After incubation overnight at room temperature, the stain was discarded, and the solution was decolorized with 95% ethanol in an 80°C water bath until clear. The solution was then photographed and observed. Separately, leaves were placed in an NBT (0.5 mg / mL) solution, vacuumed for 30 min, and allowed to stand at room temperature for 60 min. The stain was then discarded, and the solution was decolorized with 95% ethanol in an 80°C water bath (the ethanol was changed every 10 min). The results were observed after the green color completely disappeared. Hydrogen peroxide content was determined using a Solarbio assay kit (BC3595), and the detailed operating procedures were performed strictly according to the kit instructions.

[0060] (c) Relative Electrolyte Leakage (REL) Measurement

[0061] The procedure was performed following the description by Lin et al. (2023) with slight modifications. 0.2 g of tomato leaves from the same functional leaf position after both the room temperature control and high-temperature stress treatments were weighed. After removing surface contaminants and the main vein, the leaves were cut into thin strips approximately 0.2 cm wide and placed in 50 mL centrifuge tubes containing 20 mL ddH₂O. The solution was shaken at 28℃ and 200 rpm for 1.5–2 h, and the conductivity was measured using a conductivity meter and recorded as EC1. The centrifuge tubes were then boiled in a 95℃ water bath for 10 min to completely kill the cells. After cooling to room temperature, the conductivity was measured again and recorded as EC2. The relative conductivity (ECL,%) was calculated using the following formula: ECL (%) = (EC1 / EC2) × 100.

[0062] (d) Peroxidase activity analysis

[0063] The activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) were measured using a Solarbio assay kit purchased from Solarbio, and the procedure was performed according to the kit instructions.

[0064] B. Analysis of Fruit Heat Resistance and Fruit Quality

[0065] (a) Analysis of fruit marketability and heat resistance

[0066] The fruits were weighed individually using an analytical balance, and the average weight for each treatment or genotype was calculated.

[0067] Fruit firmness was determined using a digital fruit firmness meter (TP-GY-4). For each genotype or treatment, eight fruits were collected from at least eight independent plants.

[0068] Record the symptoms of surface damage on the fruit (such as sunken lesions, browning, and surface pitting) 7 days after heat stress treatment.

[0069] The degree of fruit surface damage is graded on a 0-4 scale: 0: no visible damage; 1: damaged area <10%; 2: damaged area 11-25%; 3: damaged area 26-40%; 4: damaged area >40%.

[0070] The damage index is calculated using the following formula:

[0071] Damage Index = [Σ(Score × Number of Fruits Corresponding to That Score)] / (4 × Total Number of Fruits) × 100

[0072] (b) Analysis of fruit nutritional quality

[0073] Ascorbic acid (AsA) was quantitatively analyzed using a commercial kit (Jiancheng Biotechnology).

[0074] Carotenoid extraction was performed using a modified chloroform-methanol method. The specific procedure was as follows: A suitable amount of tomato fruit sample was thoroughly ground into powder in liquid nitrogen, and 100 mg was accurately weighed and transferred to a 2 mL centrifuge tube. All subsequent steps were performed on ice under light-protected conditions. First, 350 μL of methanol was added and vortexed for at least 30 seconds; then 700 μL of chloroform was added and vortexed for at least 30 seconds; subsequently, 350 μL of double-distilled water was added and vortexed for approximately 20 seconds. The mixture was centrifuged at 10,000 rpm for 10 min at 4 °C, and the lower chloroform phase (approximately 600 μL) was collected. 700 μL of chloroform was added to the remaining precipitate, vortexed, and centrifuged again to collect the chloroform phase. This step was repeated 2–4 times until the chloroform phase was colorless. All chloroform phases were combined and dried using a nitrogen evaporator. Add 350 μL of 6% KOH methanol solution (6 g KOH solid dissolved in 100 mL chromatographic grade methanol) to the dried sample, vortex for at least 30 s, and react in a 60°C metal bath in the dark for 30 min. After the reaction, add 350 μL of double-distilled water and 700 μL of chloroform, vortex for 15 s, and centrifuge at 4°C and 10,000 rpm for 10 min to collect the chloroform phase. Repeat the extraction with 700 μL of chloroform 2–4 times until the chloroform phase is colorless, and combine the chloroform phases. Then add 700 μL of double-distilled water to the combined chloroform phase, vortex for 30 s, and centrifuge at 4°C and 10,000 rpm for 5–10 min. Discard the upper aqueous phase, and repeat this washing step until the aqueous phase is colorless. Finally, collect the chloroform phase (approximately 700 μL), dry it with nitrogen, and store it at -80°C. Before the determination, add 200 μL LDMSO:isopropanol (1:1, v / v) mixed solvent to reconstitute, vortex for more than 30 seconds, centrifuge at the highest speed for 10 min, take 50 μL of supernatant and pack it into tubes to prepare two parallel samples for HPLC determination of lycopene and β-carotene, respectively.

[0075] Endogenous serotonin analysis: 0.2 g of ground tissue sample was mixed with 80% methanol and incubated overnight at 4°C with shaking. After centrifugation (13000 rpm, 15 min), the supernatant was filtered through a 0.22 µm filter and analyzed using an Agilent 1100 high-performance liquid chromatography (HPLC) system equipped with an Inertsil ODS-4 column. The mobile phase was a solution containing 0.1% trifluoroacetic acid, the flow rate was 0.8 mL / min, and the detection wavelength was 280 nm.

[0076] (3) Experimental results

[0077] A. Effects of serotonin-rhamnolipin mixture spraying during the vegetative growth stage on heat resistance of tomatoes

[0078] (a) Effects on heat tolerance during the seedling stage.

[0079] like Figure 1 As shown in Figure A, the growth status of the treated plants under normal conditions was not significantly different from that of the control, indicating that the exogenous serotonin-rhamnolipin mixture did not significantly affect normal growth. However, as... Figure 1 As shown in Figure B, after high temperature (HS) treatment, the control plants rapidly developed typical heat damage symptoms such as leaf curling, wilting, and chlorosis, while the plants treated with the exogenous serotonin-rhamnolipin mixture maintained relatively normal leaf morphology and high growth activity. This phenotypic improvement was closely related to enhanced photosynthetic efficiency, with the serotonin-rhamnolipin mixture treatment showing a higher Fv / Fm ratio.

[0080] like Figure 1 C Figure 1 As shown in Figure D, exogenous application of a serotonin-rhamnolipin mixture successfully reduced oxidative stress levels in cells under high-temperature conditions. This was evident from the decrease in histochemical staining intensity and the reduction in hydrogen peroxide (H2O2) and superoxide anion (O2) concentrations. 2- This was confirmed by the decrease in the accumulation of [ ]. Furthermore, as [ ]... Figure 1 E, Figure 1 As shown in F, in plants treated with the serotonin-rhamnolipin mixture, cell membrane integrity was better protected, as evidenced by significantly reduced REL and MDA levels. Similarly, as... Figure 1 As shown in G, regarding antioxidants, plants treated with the serotonin-rhamnolipin mixture exhibited increased activities of key antioxidant enzymes CAT, POD, and SOD under high-temperature conditions compared to the control plants, further demonstrating the reduction of oxidative damage. Figure 1(G). The results of this experiment further demonstrate that the serotonin-rhamnolipin mixture has a positive regulatory effect on the high-temperature adaptation of tomatoes. These results indicate that treatment with the exogenous serotonin-rhamnolipin mixture can significantly reduce the level of ROS accumulation and alleviate cell membrane damage under high temperature, further demonstrating its protective effect on the high-temperature adaptation of tomatoes.

[0081] (b) Impact on fruit quality in the later stages

[0082] like Figure 2 As shown in AF, although spraying a serotonin-rhamnolipin mixture during the seedling stage can significantly improve the heat resistance of tomato plant leaves, its effects on fruit development and quality formation, apart from significantly increasing the weight of individual fruits, have no significant impact on fruit firmness, soluble solids, vitamin C, lycopene, or carotenoid content. Therefore, seedling spraying may affect tomato plant yield, but it does not have a significant impact on the nutritional quality of tomato fruits under high temperatures.

[0083] B. Effects of spraying a serotonin-rhamnolipin mixture during the flowering and fruiting period on the heat resistance of tomatoes.

[0084] like Figure 3 As shown in Figure A, under normal temperature conditions, the overall phenotypic differences among the treated fruits were small. However, after high-temperature treatment, the control fruits showed obvious heat damage symptoms, including fruit surface dehydration and wrinkling, local tissue softening, and expansion of the damaged area. In contrast, the fruits treated with the serotonin-rhamnolipin mixture maintained a better overall appearance, and the heat damage symptoms were significantly reduced, indicating that the exogenous serotonin-rhamnolipin mixture can effectively improve the fruit's adaptability to high-temperature stress.

[0085] like Figure 3 B- Figure 3 As shown in G, further measurements of fruit quality-related indicators revealed that high-temperature treatment significantly inhibited normal fruit development, leading to a marked decrease in both fruit weight and firmness. However, the application of a serotonin-rhamnolipin mixture significantly alleviated this downward trend, allowing the treated fruit to maintain higher levels of weight and firmness. This indicates that the serotonin-rhamnolipin mixture helps mitigate the negative impact of high temperatures on fruit growth and tissue structure stability. Figure 3 B Figure 3 C). In addition to changes in fruit appearance and texture, high temperature also significantly affects the formation of fruit nutritional quality. Results showed that the contents of ascorbic acid (AsA), soluble sugars, lycopene, and β-carotene in the fruit were significantly reduced after high-temperature treatment, indicating that high temperature not only affects fruit development but also inhibits the accumulation of important nutrients and quality metabolites. Figure 3 D- Figure 3G). Notably, treatment with the exogenous serotonin-rhamnolipin mixture significantly mitigated the decline in these quality indicators. Compared to the high-temperature control, the serotonin-rhamnolipin mixture treatment maintained higher levels of AsA, soluble sugars, lycopene, and β-carotene in the fruit. Figure 3 D- Figure 3 (G) indicates that the serotonin-rhamnolipin mixture helps maintain the nutritional quality and metabolic homeostasis of fruit under high temperature conditions.

[0086] like Figure 3 H- Figure 3 As shown in Figure I, to further assess the cumulative damage to fruit caused by sustained high temperatures, we conducted phenotypic observations and statistical analysis of damage indices on fruit treated for 7 days. The results showed that with prolonged high-temperature treatment, the damage to control fruit worsened, with large areas of lesions and tissue necrosis appearing on the fruit surface, and the damage index significantly increasing. In contrast, fruit treated with the serotonin-rhamnolipin mixture exhibited stronger tolerance, with significantly reduced surface damage symptoms, a significantly smaller lesion area, and a significantly lower damage index than the high-temperature control group. These results indicate that the exogenous serotonin-rhamnolipin mixture can not only alleviate the adverse effects of short-term high temperatures on fruit quality formation but also effectively reduce tissue damage and quality deterioration caused by long-term high temperatures.

[0087] In conclusion, treatment with exogenous serotonin-rhamnolipin mixture can significantly enhance the tolerance of tomato fruits to high-temperature stress. While maintaining fruit weight, firmness, and nutritional quality, it can effectively reduce the degree and damage index induced by high temperature, thereby improving the quality stability and stress resistance of fruits under high-temperature conditions.

[0088] 2. Field spraying effect experiment

[0089] (1) Field Experiment Design. For two high-quality cherry tomato varieties, 'Zheyingfen No. 1' and 'Qingxia 66', two treatments were set up for each variety: serotonin-rhamnolipin mixed preparation spray treatment (SER) and water control (CK). Each treatment was replicated in 3 times, in a randomized block design, with a plot area of ​​no less than 30m². 2 Each community should plant at least 20 plants.

[0090] (2) High-temperature season spraying treatment: It can be used when the night temperature in the facility rises above 30℃. Generally, the first spraying is carried out after the first inflorescence sets fruit. The serotonin-rhamnolipin mixture is sprayed on the functional leaves. It can also be sprayed directly on the young fruit. Spray once every 7 days, for a total of 3 sprays. The amount used per single plant for each spray is about 1.0 mL. After spraying, samples are taken after the fruit matures. Eight plants are selected from each plot as test plants to analyze important traits such as fruit marketability and nutritional quality.

[0091] (3) Experimental results

[0092] The results of the comparative experiment on the appearance characteristics of tomato fruits treated with the method of this invention and the water control are shown in Table 1. The effects of spraying a serotonin mixture on the soluble solids and sugar content of cherry tomato fruits during high-temperature seasons are also shown in Table 1. Figure 4 As shown, the effects of spraying a serotonin mixture on the lycopene and carotenoid content of cherry tomato fruits during the high-temperature season are as follows: Figure 5 As shown, the effect of spraying a serotonin mixture on the vitamin C content of cherry tomato fruits during the high-temperature season is as follows: Figure 6 As shown.

[0093] Table 1. Comparison of appearance parameters of tomato fruits treated with the method of the present invention and the water control.

[0094]

[0095] As shown in Table 1, under high temperature conditions, the application of serotonin-rhamnolipin mixture had no significant effect on the single fruit weight, transverse diameter, longitudinal diameter, and fruit shape index of the two varieties 'Zheyingfen No. 1' and 'Qingxia 66'.

[0096] Depend on Figure 4 It can be seen that under high temperature, the soluble solids content of mature fruits of the two varieties 'Zheyingfen No. 1' and 'Qingxia 66' significantly increased after spraying with a serotonin-rhamnolipin mixture, increasing by 21.57% and 14.75% respectively compared with the control. Figure 4 a). Consistent with this, the content of soluble sugars in the fruit increased by 35.53% and 31.57% respectively compared with the control group sprayed with water. Figure 4 b).

[0097] Depend on Figure 5 It can be seen that the changes in carotenoid content differed between the two varieties. In the 'Zheyingfen No. 1' tomato variety, the lycopene content increased by 53.42% and the carotenoid content increased by 42.7% after spraying with serotonin, both showing highly significant improvements. However, in the green variety 'Qingxia 66', the fruit itself contained very low levels of lycopene. Although the lycopene content increased somewhat after high-temperature treatment, it did not reach a significant level, and the change in carotenoid content was also not obvious.

[0098] Depend on Figure 6 It can be seen that the changes in vitamin C content in the fruits of the two varieties, 'Zheyingfen No. 1' and 'Qingxia 66', after being sprayed with serotonin were not significant, indicating that the effect on vitamin C was minimal.

[0099] In summary, the results of this invention demonstrate that exogenous spraying of a serotonin mixture can significantly improve the high-temperature resistance of tomatoes and reduce high-temperature damage to the fruit. While spraying the serotonin mixture at high temperatures has no significant effect on the appearance of tomato fruits, it significantly increases the soluble solids content and soluble sugar content. Simultaneously, it also has a protective effect on the synthesis of nutrients such as lycopene. Therefore, the method of this invention can significantly improve the quality of tomato fruits under high temperatures and enhance the marketability of greenhouse tomatoes during the high-temperature season.

[0100] The present invention has been described by way of example in conjunction with the accompanying drawings. Obviously, the specific implementation of the present invention is not limited to the above-described manner. Any non-substantial improvement made by adopting the inventive concept and technical solution of the present invention, or the direct application of the inventive concept and technical solution of the present invention to other occasions without modification, shall be within the protection scope of the present invention.

Claims

1. A method for improving the nutritional quality of tomato fruit under high temperature, characterized in that: Under high temperature, after the tomatoes have grown to the first inflorescence and bear fruit, the serotonin-rhamnolipin mixture is diluted with water and sprayed on the leaves and / or fruits; in the diluted solution of the serotonin-rhamnolipin mixture, the concentration of serotonin is 50-100 μM and the concentration of rhamnolipin solution is 50-100 mg / L.

2. The method for improving the nutritional quality of tomato fruit under high temperature according to claim 1, characterized in that: The serotonin-rhamnolipin mixture was diluted with water by 100-200 times.

3. The method for improving the nutritional quality of tomato fruit under high temperature according to claim 1, characterized in that: The dosage for each spraying of a single plant is 5-15 mL.

4. The method for improving the nutritional quality of tomato fruit under high temperature according to claim 1, characterized in that: After the first application, apply again after 7 days.

5. The method for improving the nutritional quality of tomato fruit under high temperature according to claim 1, characterized in that: The preparation method of the serotonin-rhamnolipin mixture is as follows: first, prepare serotonin stock solution and rhamnolipin stock solution separately, and then mix them evenly in equal volumes to obtain the serotonin-rhamnolipin mixture.

6. The method for improving the nutritional quality of tomato fruit under high temperature according to claim 5, characterized in that: When preparing serotonin stock solution, first dissolve serotonin in ultrapure water by stirring, add an antioxidant, and then add ultrapure water to make up the volume to prepare a serotonin stock solution with a concentration of 20 mM. The preparation should be done in the dark and at a low temperature.

7. The method for improving the nutritional quality of tomato fruit under high temperature according to claim 6, characterized in that: The final concentration of antioxidant in the serotonin stock solution is 0.1 mM.

8. The method for improving the nutritional quality of tomato fruit under high temperature according to claim 5, characterized in that: When preparing rhamnolipin stock solution, add rhamnolipin to ultrapure water, stir to dissolve, and make up to a final volume to prepare a rhamnolipin stock solution with a final concentration of 20 g / L.

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