Mitigating effect of γ-aminobutyric acid and gibberellic acid on tomato plant cultivated in Pb-polluted soil

Abstract

Soil heavy metal pollution poses a significant environmental risk to human health and plant growth. Gibberellic acid (GA) and γ-aminobutyric acid (GABA) are effective methods for resolving this issue. GA regulates growth mechanisms such as seed germination, flowering, and stem elongation. Plants use GABA, a signaling molecule, to control physiological processes, growth, and responses to stress. This substance plays a crucial role in the interactions between hormones and plant defense, as evidenced by its effects on photosynthesis, food absorption, and stomatal behavior. The study aimed to determine how GABA and GA amendments affected tomato plants under no toxicity and Pb toxicity. The study included four treatments (0, GA, GABA, and GA + GABA) in four replications following a completely randomized design. Notably, the GA + GABA treatment led to considerable enhancements in fresh weight (88.98%), dry weight (68.28%), shoot length (39.98%), and root length (115.43%) compared to the control under Pb toxicity. Moreover, GA + GABA treatment significantly increased tomato chlorophyll a (161.72%), chlorophyll b (93.33%), and total chlorophyll content (112.45%) under Pb stress toxicity, confirming the effectiveness of GA + GABA treatment. In conclusion, GA + GABA is recommended as the best amendment to mitigate Pb stress in tomato plants. Our findings have broader implications for GA + GABA application, offering a potential technology to enhance sustainable crop production by improving plant growth and yield in Pb-contaminated soils. More investigations are suggested at field levels under different agroclimates on different crops for the declaration of GA + GABA as the best amendment for alleviating different heavy metal pollutions and sustainable agriculture productions.

Introduction

Lead (Pb) is a heavy metal that is becoming an acute soil pollutant. It is a non-essential element that causes plants’ stunted growth by disturbing their physiological and molecular processes^1,2. When plants uptake Pb beyond the threshold limit, it damages the photosynthetic machinery, decreasing their chlorophyll contents and photosynthetic efficiency. Inhibition of the electron transport chain decreased the ATP and NADPH production, providing essential energy for carbon assimilation and metabolism^3–6. Higher production of reactive oxygen species (ROS), i.e., superoxide anions, hydrogen peroxide, and hydroxyl radicals, induced oxidative stress in plants, another adverse effect of Pb toxicity^7,8. These ROS caused damage to DNA, lipid peroxidation, protein oxidation, and cellular dysfunction^9,10. To overcome these issues, time is needed to explore new strategies that can mitigate Pb-induced damage and are environment-friendly. In this context, γ-aminobutyric acid (GABA) and gibberellic acid (GA) can enhance stress tolerance by regulating antioxidant defenses and maintaining ion homeostasis against Pb stress.

GABA is a non-protein amino acid composed of four water-soluble carbons¹¹. It is a signaling molecule that aids in root growth, nutrient uptake, and overall plant growth¹². Its antioxidant properties effectively neutralize reactive oxygen species (ROS), thereby protecting cells from oxidative stress under stress conditions^13,14. Its signaling in plants is regulated by its interaction with aluminum-activated malate transporters (ALMTs), which modulate the anion flux and root growth under stress. Such regulation provides plants a chance to survive under stress conditions^15,16. In another study, improvement in plant vigor is also reported due to its positive influence on endogenous molecules when applied as exogenous foliar amendment^17,18.

As a growth hormone, GA role is vital in improving plant growth and development^19–22. Foliar application of GA can promote several physiological and developmental functions in plants, including root initiation, blooming, cell division, chlorophyll content, photosynthetic rates, and seed germination^5,23. It improves the antioxidants-based defense mechanism when plants are subjected to Pb toxicity^24,25. Furthermore, improved mitotic activity by GA counteracts the negative impacts of heavy metals in plants²⁶.

Tomatoes (Lycopersicon esculentum Mill.) are ranked third globally in globally^27,28. Tomatoes are grown on 4.81 million hectares globally, producing 163.02 million tonnes annually^29,30. Tomatoes can be grown in both open fields and greenhouses. This crop is rich in essential nutrients such as vitamins, minerals, and antioxidants, that are crucial for a balanced diet for humans³¹. However, lead toxicity has impaired tomato plant growth, root formation, and biomass. This Pb toxicity also results in structural abnormalities, oxidative stress, nutritional imbalances, and disruptions to photosynthesis that reduce fruit production and quality^32,33.

Therefore, the current study aimed to explore the potential of GA and GABA as combined amendments to mitigate the Pb stress on tomato plants. This study covers the knowledge gap regarding the limited availability of literature on the combined use of GABA and GA as an amendment against Pb toxicity. The novelty of the current study lies in introducing a new combination of GABA and GA as an amendment for the improvement of tomato plant growth under stress. It is hypothesized that the combined application of GABA and GA might potentially improve tomato plant growth under Pb stress.

Materials and methods

γ-Aminobutyric acid and gibberellic acid

GA and GABA solutions were prepared with various concentrations of analytical-grade salts. GABA is a distinct-looking white powder that Sigma Aldrich supplied under Product Number A2129 and Batch Number BCCJ0874. Foliar spraying was used to apply GABA treatments to plants at a measurement of 0.50 mM. Each pot got 25 mL of the separate arrangement, and this application was made multiple times at explicit spans: 15, 17, 19, 21, and 24 days after the transplantation of the plants. A standard handheld pressure sprayer equipped with a flat-fan nozzle (commonly used for uniform foliar application) was used to ensure even distribution of the solution. The sprayer was operated at a pressure of 0.2 MPa (2 bar), producing fine droplets for optimal leaf coverage and absorption. For our experimental purposes, we applied GA at 100 mg/L. The following information helped identify the product used in this experiment: Product Number G7645; Brand: SIGMA; Batch Number BCCJ9719; CAS Number 77-06-5.

Lead (Pb) toxicity

The toxicity of Pb (500 mg/kg soil)^34,35 was developed by spiking the soil with PbSO₄ (CAS Number: 7446-14-2, Batch Number: MKCR0778, Product Number: 254258, Brand: ALDRICH, Color: White; MDL Number: MFCD00011166, Form: Powder) for 21 days. For soil spiking, 1-inch layers of soil were used. The total number of layers was 3, and moisture was maintained at 65% FC. The soil was mixed manually after 7 and 14 days.

Fertilizer and irrigation

Tomatoes require varying amounts of nitrogen, phosphorous, and potassium at 32, 23, and 58 kg/acre for nutritional needs. The phosphorus supplementation was carried out using a single superphosphate, while the nitrogen source was urea, following the prescribed standards. Potassium (K) was not introduced to the soil due to its pre-existing enrichment. The experiment maintained a soil moisture content of 70% of the field capacity. A soil moisture meter was used for regular monitoring and adjustments to maintain a consistent soil moisture content of 70% of the field capacity (FC). The field capacity was first determined by saturating the soil and allowing excess water to drain for 24 h. The moisture level at this point was recorded as 100% FC.

A digital soil moisture meter probe was inserted at a uniform depth in each pot to measure real-time moisture content. The readings were taken daily at the same time to minimize variability. When the moisture level dropped below 70% FC, calculated volumes of water were added gradually using a graduated measuring cylinder to restore the target moisture level. A soil moisture meter was used for regular monitoring and adjustments to maintain a consistent soil moisture content of ~ 65–70% of the field capacity (FC). The field capacity was first determined by saturating the soil and allowing excess water to drain for 24 h. The moisture level at this point was recorded as 100% FC. A digital soil moisture meter (YIERYI 4 in 1; Shenzhen, Guangdong Province, China)³⁶ equipped with probes was inserted in each pot at a uniform depth (15 cm rhizosphere). The readings were taken daily at the same time to minimize variability. When the moisture level dropped below moderate (~ 65–70% FC) towards dry (< 60% FC), irrigation water was added gradually using a graduated measuring cylinder to restore the target moisture level.

Seed collection, sterilization, and sowing

The certified seed dealer provided tomato seeds were used in this experiment. Seed quality was ensured by selecting strong, healthy seeds, while weakened or damaged seeds were excluded manually. The seeds were surface sterilized before sowing. For that 5% sodium hypochlorite solution and 95% ethanol were used. The seeds underwent three additional washes with sterilzed deionized water ³⁷.

Nursery sowing and transplantation

A total of 100 seeds were sown on 15th November 2020 for the establishment of nursery seedlings. Two healthy seedlings were then transplanted in each pots on 2 February 2021. The selection of seedlings was made on a visual basis. Only those plants that were healthy and almost equal in growth were selected based on the seedling’s height, number of leaves, and leaf area. Each pot (10-inch wide, 18 inches deep) containing 15 kg of soil was used to grow transplanted seedlings.

Harvesting and data collection

The data was collected 50 days after sowing and allowed to grow. After harvesting, the fresh weights of the roots and shoots were calculated using an analytical grade scale. Samples were dried in an oven at 65 °C for 72 h to determine shoot and root dry weights.

Chlorophyll contents, carotenoids, and anthocyanin

The study used Arnon’s method to measure chlorophyll content in freshly harvested tomato leaves³⁸. An 80% acetone solution was used in the extraction process. The absorbance of carotenoids was tested using varying wavelengths: 480 nm for carotenoids, 645 nm for chlorophyll b, and 663 nm for chlorophyll a. However, anthocyanin readings were taken at 530 nm wavelength on UV-1280 Spectrophotometer | SHIMADZU.

Antioxidants

The study determines the efficacy of superoxide dismutase (SOD) in inhibiting nitro blue tetrazolium (NBT) at a wavelength of 560 nm³⁹. The peroxidase (POD) activity was evaluated using a 420 nm approach⁴⁰. The catalase (CAT) activity was measured by analyzing the decrease in absorbance at 240 nm due to the breakdown of H₂O₂ ⁴¹. The activity of ascorbate peroxidase (APX) was assessed by observing a decrease in absorbance at a 290 nm wavelength following ascorbate oxidation in the presence of H₂O₂ ⁴². A colored complex was created from a sample extract and analyzed for MDA levels and NADPH oxidation rate, revealing a decrease in absorbance at 340 nm⁴³.

Electrolyte leakage

The leaves were thoroughly rinsed with deionized water to eliminate dust particles before being subjected to the analytical procedure. Uniform leaf segments weighing approximately one gram were obtained using a steel cylinder with a diameter of one centimeter. Each test tube containing leaf fragments contained 20 mL of deionized water. The test tubes were kept at 25 degrees Celsius for a day to facilitate the diffusion of electrolytes from leaf tissues into the water. After incubation, the water solution’s electrical conductivity (EC1) was measured using a calibrated EC meter. After heating the test tube in a 120 °C water bath for 20 min, the second electrical conductivity (EC2) was determined⁴⁴.

Lead (Pb) analysis

Dry leaf samples were digested using a di-acid nitric and perchloric acid solutions at 280 °C on a hot plate⁴⁵. The samples were diluted 1000-fold and then analyzed using a pre-calibrated atomic absorption spectrophotometer (Agilent 240 AA Atomic Absorption Spectrometer) to determine the lead (Pb) content⁴⁶.

Statistical analysis

Standard statistical analyses were performed on the collected data. The evaluation of treatment significance was conducted through a two-way ANOVA. The Tukey test was applied with paired comparisons to examine the treatment’s significance level at p ≤ 0.05. We utilized OriginPro software to create the convex hull, hierarchical cluster plots, and pearson correlation⁴⁷.

Results

Number of leaves, number of roots, leaf area

Under no toxicity, applying GA, GABA, and GA + GABA treatments resulted in a significant increase in the number of leaves (10.18%, 14.53%, and 29.84%), number of roots (16.19%, 25.37%, and 39.07%), and leaf area (14.68%, 25.88%, and 36.35%) over the control. Under Pb toxicity, the increase in the number of leaves (10.72%, 26.03%, and 41.14%), number of roots (55.80%, 90.80%, and 128.92%), and leaf area (27.78%, 43.85%, and 86.01%) were recorded over the control with GA, GABA, and GA + GABA treatments (Fig. 1A–C).

Fig. 1.

The study examines the effects of treatments on tomato plants grown in non-toxic and Pb-toxic environments in terms of the number of leaves (A), number of roots (B), and leaf area (C). Different letters on bars that reflect the average of four replicates indicate significant changes in the Tukey test at p < 0.05.

Chlorophyll A, chlorophyll B, total chlorophyll

A significant increase in chlorophyll a (34.19%, 47.91%, and 68.37%), chlorophyll b (18.95%, 44.87%, and 66.58%), and total chlorophyll (24.45%, 45.97%, and 67.23%) was recorded by adding GA, GABA, and GA + GABA treatments compared to the control under no toxicity. Applying GA, GABA, and GA + GABA treatments under Pb toxicity showed a rise in chlorophyll a (50.78%, 94.53%, and 161.72%), chlorophyll b (61.82%, 77.58%, and 93.33%), and total chlorophyll (58.73%, 82.31%, and 112.45%) over the control (Fig. 2A–C).

Fig. 2.

The study examines the effects of treatment on tomato grown under Pb toxicity and no toxicity conditions in terms of chlorophyll a (A), chlorophyll b (B), and total chlorophyll (C). Different letters on bars that reflect the average of four replicates indicate significant changes in the Tukey test at p < 0.05.

Carotenoid, anthocyanin, and lycopene

Under no toxicity, the increase in carotenoids (17.17%, 43.43%, and 59.60%), anthocyanin (19.94%, 29.60%, and 41.01%), and decrease in lycopene (9.06%, 18.89%, and 41.49%) were measured over the control with GA, GABA, and GA + GABA treatments. Under Pb toxicity, adding GA, GABA, and GA + GABA treatments exhibit an increase in carotenoids (78.57%, 139.29%, and 192.86%), anthocyanin (11.76%, 56.95%, and 98.90%), and caused decrease in lycopene (11.64%, 19.66%, and 36.76%) than the control (Fig. 3A–C).

Fig. 3.

Effect of treatments on carotenoids (A), anthocyanin (B), and lycopene (C), of tomato cultivated under no toxicity and Pb toxicity. Different letters on bars that reflect the average of four replicates indicate significant changes in the Tukey test at p < 0.05.

Total protein, total soluble sugar, total amino acids, and flavonoids

Under no toxicity applying GA, GABA, and GA + GABA treatments resulted in a significant increase in the total protein (9.12%, 18.37%, and 24.19%), total soluble sugar (3.95%, 6.67%, and 13.38%), total amino acids (6.45%, 13.47%, and 24.33%), and caused a decrease in flavonoids (8.82%, 17.93%, and 48.06%) over the control. Under Pb toxicity, the increase in the total protein (7.88%, 16.44%, and 27.63%), total soluble sugar (8.99%, 18.03%, and 21.57%), total amino acids (4.67%, 19.57%, and 34.62%), and decrease in flavonoids (7.36%, 17.49%, and 32.10%) were recorded over the control with GA, GABA, and GA + GABA treatments (Fig. 4A–C).

Fig. 4.

The study examines the effects of treatments on tomato plants grown under no toxicity and Pb toxicity, focusing on total protein (A), total soluble sugar (B), total amino acids (C), and flavonoids (D). Different letters on bars that reflect the average of four replicates indicate significant changes in the Tukey test at p < 0.05.

MDA, H₂O₂, and apx activity

Under no toxicity applying GA, GABA, and GA + GABA treatments resulted in a significant decrease in the MDA (10.63%, 26.04%, and 37.43%), APX (8.64%, 14.48%, and 26.59%), and H₂O₂ (12.68%, 21.48%, and 38.22%) over the control. Under Pb toxicity, the decrease in the MDA (4.02%, 13.61%, and 25.30%), APX (8.58%, 22.15%, and 36.09%), and H₂O₂ (15.19%, 24.18%, and 50.53%) were recorded compared the control with GA, GABA, and GA + GABA treatments (Fig. 5A–C).

Fig. 5.

The study examines the effects of treatments on tomato grown under no toxicity and Pb toxicity in terms of MDA (Malondialdehyde) (A), APX (Ascorbate peroxidase) (B), and H₂O₂ (Hydrogen peroxide) (C). Different letters on bars that reflect the average of four replicates indicate significant changes in the Tukey test at p < 0.05.

POD, SOD, CAT

Under no toxicity, the decrease in POD (17.83%, 55.86%, and 79.20%), SOD (9.78%, 23.02%, and 45.64%), and CAT (9.40%, 29.59%, and 60.28%) were measured over the control with GA, GABA, and GA + GABA treatments. Under Pb toxicity, adding GA, GABA, and GA + GABA treatments showed a decrease in POD (12.76%, 24.93%, and 47.38%), SOD (10.62%, 17.48%, and 46.54%), and CAT (17.60%, 24.11%, and 34.09%) over the control (Fig. 6A–C).

Fig. 6.

The study examines the effects of POD (Peroxidase) (A), SOD (Superoxide dismutase) (B), and CAT (Catalase) (C) treatments on tomatoes grown under Pb toxicity and without toxicity conditions. Different letters on bars that reflect the average of four replicates indicate significant changes in the Tukey test at p < 0.05.

Plant fresh and dry weight, shoot and root length

Adding GA, GABA, and GA + GABA treatments under no toxicity caused an increase in plant fresh weight (10.50%, 17.01%, and 35.30%), plant dry weight (18.52%, 31.64%, and 45.25%), shoot length (9.91%, 21.69%, and 38.86%), and root length (17.05%, 35.73%, and 52.31%) over the control. Treatment GA, GABA, and GA + GABA also showed an increase in plant fresh weight (20.94%, 47.24%, and 88.98%), plant dry weight (20.69%, 44.48%, and 68.28%), shoot length (16.32%, 24.78%, and 39.98%), and root length (40.37%, 70.45%, and 115.43%) over the control under Pb toxicity (Table 1).

Table 1.

Effect of treatment on plant fresh and dry weight, root and shoot length, and uptake of Pb concentration in shoot and root of tomato.

Stress	Treatment	Plant fresh weight (g)	Plant dry weight (g)	Shoot length (cm)	Root length (cm)	Shoot Pb (µg/g)	Root Pb (µg/g)
No toxicity	Control	3.42a	1.52a	9.50a	17.49a	3.16a	3.78a
No toxicity	GA	3.78b	1.80b	10.45b	20.47b	2.61b	2.83b
No toxicity	GABA	4.13c	2.00c	11.57c	23.74c	2.12c	2.64b
No toxicity	GA + GABA	4.63d	2.21d	13.20d	26.64d	1.82d	2.03c
Pb toxicity	Control	1.58a	0.72a	5.85a	7.097a	5.26a	6.49a
Pb toxicity	GA	1.92b	0.87b	6.80b	9.962b	4.74b	5.57b
Pb toxicity	GABA	2.33c	1.04c	7.30b	12.09c	4.04c	4.83c
Pb toxicity	GA + GABA	3.00d	1.22d	8.19c	15.29d	3.58d	4.45d

Four replicates are averaged to get the values. Significant changes were observed at p < 0.05 for different letters in the Tukey Test. No Toxicity; = Pb Toxicity.

Pb uptake in shoot and root

Under no toxicity, the application of GA, GABA, and GA + GABA showed a decrease in Pb uptake in the shoot (21.31%, 48.78%, and 73.69%) and root (33.50%, 43.10%, and 86.07%) over the control. Under Pb toxicity, GA, GABA, and GA + GABA caused a decrease in Pb uptake in the shoot (11.11%, 30.43%, and 46.93%) and root (16.52%, 34.45%, and 45.64%) compared to the control (Table 1).

Convex hull cluster plot

For the no toxicity condition, the scores primarily span positive values of PC 1, ranging from 0.24918 to 7.86004, with PC 2 scores between − 0.54237 and 3.09612. These scores reflect a consistent pattern in the absence of toxicity, clustering tightly in a specific region of the principal component space. In contrast, the Pb toxicity displays scores predominantly on the negative side of PC 1, ranging from − 8.46917 to -0.249. The PC 2 scores for this group are more narrowly clustered between − 0.19492 and 0.32762. This clustering indicates a distinct separation from the no toxicity condition, with scores for Pb toxicity being lower and more tightly grouped (Fig. 7A).

Fig. 7.

Cluster plot convex hull for lead (Pb) toxicity levels (A), treatments (B), and hierarchical cluster plot (C) for studied attributes.

For the Control treatment, the scores range from − 8.46917 to 1.83417 along PC1 and from − 0.16226 to 0.32762 along PC2. The distribution of the control samples is quite spread out, indicating variability within the treatment group. The GA treatment scores range from − 5.98881 to 4.10039 along PC1 and from − 0.2651 to 0.16654 along PC2. The GA treatment samples show a more concentrated score compared to the control group, indicating a more tightly clustered distribution. The GABA treatment scores are spread from − 3.94413 to 5.81589 along PC1 and from − 0.48436 to 0.08308 along PC2. This treatment shows a moderate spread, indicating some variability within the treatment group. The combined GA + GABA treatment shows scores ranging from − 1.73498 to 7.86004 along PC1 and from − 0.19492 to 3.09612 along PC2. The presence of high positive values in PC2 for GA + GABA treatment indicates a distinct separation from the other treatments, suggesting that the combination of GA and GABA has a unique effect compared to the individual treatments (Fig. 7B).

The root length and total protein have a high degree of similarity, as indicated by a similarity score of 0.24371. POD and shoot Pb also form a close cluster with a similarity value of 0.27364. Plant fresh weight and leaf area cluster together with a similarity of 0.2892. The correlation between total chlorophyll and chlorophyll b is strong, with a similarity value of 0.29235. The relationship between root length and total protein and plant dry weight is demonstrated by a similarity value of 0.32385. MDA forms a cluster with POD and shoot Pb with a similarity value of 0.33015. Chlorophyll an also clusters with this group at a similarity value of 0.37313. The relationship between H₂O₂ and APX is strong, as indicated by a similarity score of 0.45175. The study found that anthocyanins had a similarity value of 0.53132 when grouped with POD and shoot Pb. The similarity score between SOD and lycopene is 0.55213, indicating their proximity. The number of roots and roots Pb are strongly associated, as indicated by the similarity value of 0.63186. Flavonoids cluster with POD and shoot Pb at a similarity value of 0.6679. The shoot length, with a similarity score of 0.68161, is classified alongside total chlorophyll and chlorophyll b. The leaf clusters have a similarity value of 0.71294 with H₂O₂ and APX. Carotenoids, with a similarity value of 0.76442, form a distinct cluster with POD and shoot Pb. CAT shows a high similarity value of 1.46323, clustering closely with POD and shoot Pb. Total soluble sugar is grouped with POD and shoot Pb, showing a high similarity value of 2.04034. The total amino acid has the highest similarity value of 9.31948, indicating its distinct nature compared to the other measured variables (Fig. 7C).

Pearson correlation analysis

The analysis reveals the strength and direction of the linear relationships between pairs of variables. The correlation coefficients range from − 1 to 1, with positive values indicating positive correlations, negative values indicating negative correlations, and values closer to 0 indicating weaker correlations. The analysis indicates strong positive correlations between plant fresh weight and plant dry weight (r = 0.98624), shoot length (r = 0.98288), root length (r = 0.99261), number of leaves (r = 0.9903), number of roots (r = 0.99037), leaf area (r = 0.99433), chlorophyll a (r = 0.98616), chlorophyll b (r = 0.95987), total chlorophyll (r = 0.97874), carotenoids (r = 0.98431), and total protein (r = 0.98825). However, there are also negative correlations found, such as between lycopene and various variables like flavonoids (r = −0.99439), MDA (r = − 0.99344), H₂O₂ (r = − 0.98338), POD (r = − 0.9903), SOD (r = − 0.99128), CAT (r = −0.97485), and APx (r = − 0.98156) (Fig. 8).

Fig. 8.

Pearson correlation for studied attributes.

Discussion

The current study’s hierarchical cluster plot clearly shows that improvement in shoot length due to GA + GABA was the most representative attribute that positively changes the tomato plants under Pb toxicity. Application of GA enhances cell expansion by modifying cell wall properties, i.e., wall loosening and activating the genes for cell wall modifications⁴⁸. It also signals the root for improvement in cell production rate and meristem size⁴⁹. Exogenous application of GA enhances the chlorophyll contents by inhibiting chlorophyllase activity⁵⁰. Furthermore, it also activates antioxidants SOD, CAT, and POD that neutralize reactive oxygen species (ROS), preventing oxidative damage to chloroplasts and preserving chlorophyll⁵¹. Plants produce organic acids, i.e., citric acid, malic acid, and oxalic acid, in response to stress conditions. When GABA is applied as a foliar amendment, it stimulates the production of these organic acids, which can immobilize or chelate Pb²⁺ ions in the rhizosphere or root cells^52–54. High synthesis of non-enzymatic antioxidants like glutathione and ascorbic acid via its application also reduces ROS-based oxidative stress. It also increased osmolyte accumulation, enhancing membrane stability and regulating polyamine metabolism under stress conditions^55,56. GABA also supports the biosynthesis of chlorophyll. It improves the metabolic pathways linked to chlorophyll precursors, i.e., δ-aminolevulinic acid (ALA). This ALA enhanced the intermediates, i.e., protoporphyrin IX, upregulating the key genes HEMA1 and CHLH⁵⁷. GA₃ and GABA also improve plant growth by enhancing cell division, photosynthetic efficiency, and nutrient uptake. This increased growth demand may result in a shift in carotenoid metabolism towards xanthophylls and β-carotene (which contribute to chlorophyll stability) rather than lycopene accumulation^58,59. In addition to the above, GABA improves ion homeostasis by increasing K⁺ retention and Na⁺ exclusion⁶⁰. Stimulation of polysaccharide, lignin, and pectin in the cell wall by application of GA and GABA bind the Pb ions. Both amendments can potentially immobilize Pb ions in the apoplast of roots and minimize their translocation in vascular tissues⁶¹. Similar results were also noted in the current study, where the application of GA + GABA caused significant improvement in chlorophyll content under Pb stress. When applied in combination, both amendments positively interact and improve the antioxidants, facilitating the plants’ survival under Pb toxicity. These were the main mechanisms by which our tomato plants decreased the oxidative stress and improved the growth attributes, i.e., shoot and root length.

Conclusion

In conclusion, GA + GABA can potentially improve shoot length, the most representative attribute for enhancing tomato growth under Pb stress. The GA + GABA treatments may regulate Pb antioxidant levels, potentially reducing the harmful effects of Pb stress on tomatoes. Further field research is recommended to justify the effectiveness of GA + GABA as a practical solution for reducing Pb stress in tomato plants. The GA + GABA applications into precision agriculture practices could further optimize their benefits, allowing tailored treatments based on contamination levels, crop type, and growth stages.

Author contributions

S.S.; U.Y.; contributed to the conceptualization and design of the study, as well as data collection, analysis, and interpretation. M.J.A.; A.A.A.; contributed to the statistical analysis during revision; S.A.A.; R.K.I.; H.A.; M.A.R.;interpretation of the data during revision. S.S.; U.Y.; contributed to the writing, statistical analysis, M.J.A.; A.A.A.; S.A.A.; R.K.I.; H.A.; M.A.R.; editing of the manuscript. All authors have reviewed and approved the final version of the manuscript.

Funding

The authors extend their appreciation to the Researchers Supporting Project number (RSP2025R98), King Saud University, Riyadh, Saudi Arabia, for financial support.

Data availability

All data generated or analyzed during this study are included in this published article.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

We all declare that manuscript reporting studies do not involve any human participants, human data, or human tissue. So, it is not applicable.

Study protocol must comply with relevant institutional, National, and international guidelines and legislation

Our experiment follows the with relevant institutional, national, and international guidelines and legislation.