Phytohormones methyl jasmonate (MeJA) and gamma-aminobutyric acid (GABA) up-regulates growth and PS II photochemistry in brinjal and tomato seedlings exposed to cadmium toxicity

doi:10.21203/rs.3.rs-4340560/v1

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Phytohormones methyl jasmonate (MeJA) and gamma-aminobutyric acid (GABA) up-regulates growth and PS II photochemistry in brinjal and tomato seedlings exposed to cadmium toxicity

https://doi.org/10.21203/rs.3.rs-4340560/v1

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published 10 Aug, 2024

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Cadmium (cd) toxicity has become a major threat to the crop productivity and vegetables appeared to be on major risk. In present study, the potential of methyl jasmonate (MeJA, 0.015 µM) and gamma-aminobutyric acid (GABA 15 µM) was explored to alleviate the cd toxicity (12 µM) in tomato and brinjal seedlings. Cd declined fresh dry mass by 21% and 18% in tomato seedlings and 27% and 25% in brinjal seedlings. Cd significantly damage pigments contents (Chl a, Chl b and Car), PS II photochemistry (Chl a fluorescence kinetics) and photosynthetic gas exchange parameters in both seedlings. Furthermore, Cd exacerbated oxidative biomarkers and antioxidant enzymes CAT, SOD, POD and GST in both the seedlings. Phytohormones MeJA and GABA application to seedlings led to significant declined Cd uptake, oxidative biomarkers, antioxidative enzymes activity and up-regulation in leaves gas exchange parameters, photosynthetic performance and seedlings growth parameters. Additionally, biosynthetic inhibitors diethyldithiocarbamic acid (DIECA) of MeJA and 3-Mercaptopropionic acid (MPA) of GABA further raised Cd uptake, thereby excessive increase in oxidative biomarkers worsened Cd toxicity on photosynthesis, hence growth was greatly reduced. Thus, the study concludes that as compared to brinjal seedlings, tomato showed greater tolerance to Cd toxicity, and GABA plays a crucial role in mitigating the Cd toxicity, however, GABA and MeJA together more efficiently alleviated the toxicity.

Antioxidants

Oxidative biomarkers

Photosynthesis

Signalling molecules

Sustainable agriculture

Brinjal (Solanum melongena L.) and tomato (S. lycopersicum L.) are the important vegetables that are widely cultivated in Indian subcontinent and world as well. Vegetables are rich in vitamins, minerals, and antioxidants; hence they are important components of human food. The production and quality of vegetables are being compromised due to biotic and abiotic stresses. In recent years, heavy metal contamination is increased due to enhanced anthropogenic activities. Cadmium (Cd) is of particular concern as it is one of the most toxic metals, and due to mining, painting, industrial waste, excessive use of agricultural fertilizers and pesticides, irrigation practices with contaminated water, application in nickel-cadmium batteries as electrodes and in semiconducting solar panels (Wang et al. 2021; Genchi et al. 2020; Mir et al. 2022), its concentration rises above threshold limit. Cd pollution of soil and water is a major global threat to human health, agri-food systems and ecosystems. It is found in nature in the form of divalent metallic element and when present in excess amounts; it generates reactive oxygen species in excess which affect plant growth, metabolism, productivity, and vigour (Haider et al. 2021). Its concentrations in agricultural soil above 3 mg g^–1 is considered dangerous for crop cultivation (Lux et al. 2011). Although it is not essential for plant growth and development but plants absorb it by involving Ca²⁺ channels. Its excessive accumulation in plants may have major adverse effects on human health through the food chain (Haider et al. 2021). Cadmium toxicity caused serious threats to plants and disturbed normal physiological and biochemical activity and as a result shunted growth, decreased chlorophyll contents and efficiency of photosystem II in plants were observed (Piacentini et al. 2021). Furthermore, cadmium toxicity results into excessive production of reactive oxygen species (ROS) which harm plant membranes and destroys cell organelles and macromolecules (Unsal et al. 2020). Nowadays numerous studies suggest that various plant growth regulators (PGRs) and signalling molecules that are exogenously applied to plants significantly reduce the effects of abiotic and biotic stresses by modifying several biochemical pathways (Shah et al. 2021). Methyl jasmonate (MeJA) is one the key signalling molecules with a variety of roles in plants, including activating defence mechanisms, controlling growth and development, and interacting with other phytohormones (Mir et al. 2018). MeJA plays a critical role in the mitigation of environmental stresses under various conditions through various mechanisms like, improving the antioxidant mechanism and increasing the production of osmoregulators in plants (Raza et al. 2022; Mousavi et al. 2020). Previous studies showed that exogenous application of MeJA mitigates the chromium toxicity in calendula oficinalis (Barzin et al. 2022), enhanced cold tolerance in watermelon (Li et al. 2021), enhances the salinity tolerance in lemon seeds (Asadi and Jalilian 2021), drought tolerance in cowpea plant (Sadeghipour 2018), induced heat tolerance in the wheat plant (Fatima et al. 2021). However, there is currently limited information about the MeJA function in controlling Cd toxicity in vegetable crops.

Likewise, another plant growth regulator gamma-aminobutyric acid (GABA) is a four-carbon non-proteinogenic amino acid. Under various types of biotic and abiotic stresses, GABA quickly accumulates in plant tissues and works like an endogenous signaling molecule in plant growth and development (Li et al., 2021; Suhel et al., 2022). Exposure of a variety of stressful conditions: osmotic pressure, high temperatures, salinity stress, cold shock, and heavy metal toxicity have been linked to the accumulation of GABA (Seifikalhor et al. 2020; Sheteiwy et al. 2019). In recent years, it has been demonstrated that exogenously applied GABA enhanced the cold tolerance in tomato (Liu et al. 2020), improved heat tolerance in tea (Ren et al. 2021), enhanced salt stress tolerance in the mulberry (Zhang et al. 2022), drought stress reduction in Phaseolus vulgaris (Abd El-Gawad et al. 2021) and alleviated arsenic toxicity in tomato and brinjal seedlings (Suhel et al. 2022). The protective effects of GABA against stress-related oxidative damage in a variety of plant species have been exhibited (Choe et al. 2021).

Since GABA and MeJA alone has a distinct role in reducing abiotic stress in plants, however, the relationship between GABA and MeJA signalling under Cd stress has not yet been investigated in crops in general and vegetables in particular. Therefore, in present study an attempt has been made to investigate the crosstalk between GABA and MeJA in alleviating cadmium toxicity in two vegetable crops tomato and brinjal by examining growth, light harvesting pigments, PS II photochemistry, photosynthetic gas exchange, accumulation of intracellular Cd, oxidative biomarkers and antioxidant system.

2.1 Experimental plants and growth condition

The healthy seeds of two vegetables, brinjal (Solanum melongena L. var. KSP-1307 ARUN) and tomato (Solanum lycopersicum L. var. BSS-791) were procured from the certified supplier of Prayagraj district, UP, India. Uniform-sized seeds of both vegetables were selected and surface sterilization was done with a 10% (sodium hypochlorite) solution for 20 min and rinsed with sterilized distilled water several times. Thereafter, seeds were wrapped with muslin cloth and kept in darkness at 25 ± 2°C for 24 hrs under sterilized conditions. Sprouted seeds were sown in a plastic tray containing sterilized sand and kept in the dark until seedlings emerged. The tray containing seedlings was transferred into the growth chamber (CDR model GRW-300Ge, Athens) under a light intensity of 240 ± 30 mol photons m^− 2 s^− 1, 16:8 hrs of day and night period and relative humidity maintained at 65–70% at 25 ± 2°C for 21 days. During the growth period, seedlings were irrigated with water and half-strength Hoagland's nutrition solution alternatively. On the emergence of the secondary leaf, the seedlings were uprooted carefully and washed under running water and thereafter they z were placed in half-strength Hoagland solution for 24 h for acclimatization. Finally, seedlings were grown hydroponically in growth medium containing different combinations.

2.2 Treatment of seedlings with GABA, MeJA, and Cd

The seedlings of both the experimental plants were treated with 12 µM Cd (CdCl₂), 15 µM GABA and 0.015 µM MeJA (Sigma-Aldrich) separately and in combination as required. The concentrations of Cd, GABA, and MeJA were selected after a series of screening experiments for a details study. Furthermore, to understand the role of the signalling molecules GABA and MeJA, 20µM MPA (3-mercaptopropionic acid; biosynthetic inhibitors of GABA) and 5 µM DIECA (diethyledithiocarbamic acid; biosynthetic inhibitors of MeJA) were applied. With different combinations, the treatments were given for five days under hydroponics conditions and the experimental setups consisted of: CK (control), Cd, Cd + GABA, Cd + MeJA, Cd + GABA + MeJA, Cd + GABA + DIECA, Cd + MeJA + MPA. After 5 days of treatments various growth, physiological and biochemical attributes in both seedlings were analysed.

2.3 Determination of growth attributes

Growth was measured as fresh and dry mass of both plant seedlings by digital electronic balance (Mettler Toledo, Switzerland, min. accuracy 0.1 mg). The seedlings of each setup were harvested and after determining fresh mass, seedlings were dried in a hot oven at 80°C for 48 h before the measurement of dry mass.

2.4 Estimation of Cd content

Samples from each set were digested in a tri acid mixture (5:1:1 ratio, v/v) (HNO₃, H₂SO_4, and HClO₄ in 5:1:1 ratio, v/v) at 80°C until a transparent solution was formed to determine the amount of Cd present in the roots and shoots (Allen et al. 1986). Following cooling, Whatman No. 42 was used to filter the digested material, and double distilled water was used to maintain the filtrate up to 15 milliliters. Using a suitable drift blank and an atomic absorption spectrometer (iCE 3000 Series, Thermo Scientific, UK) equipped with a specific metal lamp, the amount of Cd in the digested samples was determined.

2.5 Determination of photosynthetic pigments

The 50 mg leaf from each sample was crushed and the pigments were extracted in 80% acetone. The absorbance of transparent suspension was recorded at 663, 646, and 470 nm spectrophotometrically (Shimadzu double beam UV–Visible spectrophotometer-1700, Japan) and the amount of chlorophyll a (Chl a) and b (Chl b) and carotenoids (Car) was calculated by the equation given by Lichtenthaler (1987).

2.6 Determination of chlorophyll a fluorescence kinetics (JIP test)

Chlorophyll a fluorescence transient was assayed in intact leaves of treated and untreated seedlings with the help of a handheld leaf fluorometer (FluorPen FP 100, Photon System Instruments, Czech Republic). Prior to the measurement, the seedlings were preincubated for half an hour dark. The performance of photochemistry of photosystem II was determined according to the method given by Strasser et al. (2000) by analysing the status of quantum yield of primary photochemistry (F_V/Fm or ϕP₀), maximum quantum efficiency of PS II photochemistry (Ψ₀), yield of electron transport per trapped exciton (ϕE₀), performance index of PS II (PI_ABS) and also the energy fluxes per active reaction centre (ABS/RC, TR₀/RC, ET₀/RC and DI₀/RC).

2.7 Estimation of photosynthetic gas exchange parameters

The LCiSD portable photosynthesis system (L. MAN-LCI-SD, biosynthetic Ltd, EN11 0NT, UK) was applied to analyse the net photosynthetic rate (A), stomatal conductance (Gs), gas exchange indices including intercellular CO₂ concentration (Ci), and transpiration rate (E) of a fully exposed secondary leaves in seedlings. Throughout the measurements, the ambient CO₂ (380 ± 10 µmol mol^− 1) concentration, leaf temperature (25 ± 2°C), photosynthetic photon flux density (1200 ± 250 µmol m^− 2 s^− 1) and relative humidity (70%) were maintained.

2.8 Estimation of oxidative stress indices

2.8.1 Biochemical estimation of oxidative stress biomarkers

Superoxide radical (SOR; O₂•‾) and hydrogen peroxide (H₂O₂) contents in treated and untreated leaf samples were estimated by adopting the methods of Elstner and Heupel (1976) and Velikova et al. (2000), respectively. The assay of superoxide radical (SOR) was performed in in each sample based on the formation of NO₂⁻ from hydroxylamine in the presence of O₂^·−. The content of O₂^·− was quantified with the help of a standard curve prepared by the graded solution of NaNO₂. Likewise, for H₂O₂ content the absorbance in each reaction mixture was recorded at 390 nm and the amount was calculated by using a standard curve prepared with graded solution of H₂O₂. The lipid peroxidation as malondialdehyde (MDA) contents was estimated according to method of Heath and Packer (1968). The absorbance of the supernatant was read at 532 and 600 nm and the value for non-specific absorbance of each sample at 600 nm was subtracted from absorbance recorded at 532 nm, and malondialdehyde equivalents content was calculated by using an extinction coefficient of 155 mM^− 1cm^− 1.

2.8.2 Determination of membrane stability index

Membrane stability index (MSI) of leaf samples of both seedlings was measured according to the method of Gong et al. (1998). Fresh leaf (200 mg) was cut into equal size pieces and placed in a test containing 20 ml of deionized water and incubated at 30°C for 2h. The test tubes containing each sample were placed on a rotary shaker and at the end electric conductivity of the solution was recorded (C₁) using an electric conductivity meter. Likewise, an equal amount of leaf discs from untreated seedlings were placed in another test tube and placed in a boiling water bath at 100°C for 15 min. After cooling, the electric conductive of samples was determined (C₂) as earlier. The membrane stability index was calculated using the following equation.

MSI = [1- (C₁/C₂)] x100

2.8.3 In-vivo analysis of oxidative stress biomarkers

In-vivo visualization of reactive oxygen species (ROS: O₂• ‾ and H₂O₂) was performed using nitroblue tetrazolium (NBT) and 3,3ʹ-Diaminobenzidine (DAB) as per the methods of Frahry and Schopfer (2001) and Thordal-Christensen et al. (1997), respectively. For visualizing O₂•‾and H₂O₂, leaves, and root tips were incubated in nitro blue tetrazolium (NBT, prepared in potassium phosphate buffer pH 6.4) containing 10 mM Na-azide (Sigma Aldrich), and 1% solution of 3, 3′-diaminobenzidine (DAB) (Sigma Aldrich) in the light and dark until blue and brown spots respectively appeared. After this, leaves and root tips were bleached by immersing in boiling ethanol to visualize blue and brown spots, respectively, and photographed. Lipid peroxidation and membrane damage were visualized using Schiff’s reagent and Evans's blue according to the methods of Pompella et al. (1987) and Yamamoto et al. (2001), respectively. To localize lipid peroxidation products and plasma membrane integrity, leaves and root tips were stained with Schiff’s reagent and Evans blue (0.025%, w/v prepared in 100 µM of CaCl₂, pH 5.6), respectively for 20 min. Thereafter, leaves and root tips were bleached by immersing in boiling ethanol to clearly visualize pink and blue spots, respectively, and photographed.

2.9 Estimation of enzymatic antioxidants activities

The activity of superoxide dismutase (SOD; EC 1.15.1.1) in leaf samples was measured using the method ascribed by Giannopolitis and Ries (1977), and one unit of SOD activity was defined as the quantity of enzyme required to prevent 50% of NBT reduction by O₂•‾. The activity of peroxidase (POD; EC 1.11.1.7) was determined using the method described by Zhang (1992) and 1 nmol guaiacol oxidized min^− 1 is considered as one unit of POD activity. Catalase (EC 1.11.3.6) activity was measured by monitoring the dissociation of H₂O₂ at 240 nm for 1 min with a UV-visible spectrophotometer (Shimadzu, Japan) and CAT activity was calculated by using an extinction coefficient of 39.4 mM^− 1 cm^− 1 (Aebi 1984). Further, 1 nmol H₂O₂ dissociated min^− 1 is defined as one unit (U) of enzyme activity. The activity of glutathione-S-transferase (GST, EC 2.5.1.18) was measured using the Habig et al. (1974) method. One unit (U) of enzyme activity denotes as one nmol of CDNB-conjugates produced min^− 1.

2.10 Statistical analysis

For the analysis of the data, a one-way analysis of variance (one-way ANOVA) was used. As a post hoc test to examine significant differences across several treatments at p˂0.05 significance level, Duncan's multiple range test (DMRT) was used. For each treatment, three separate biological duplicates for each treatment were performed.

3.1 GABA and MeJA alleviate Cd toxicity on growth

Results about growth attributes of fresh and dry mass of tomato and brinjal seedlings were displayed in Fig. (1,2). Cd treatment (12 µM) caused diminished growth as the reduction in fresh and dry mass was 21% and 18% in tomato and 27% and 25% in brinjal seedlings, respectively over the values of the control. Under similar stress, the exogenous supplementation of GABA (15 µM) induced significant alleviatory effects on growth attributes as the decrease in fresh and dry biomass remained to be 5% and 2% in tomato and 8% and 3% in brinjal seedlings, respectively (Fig. 1,2). Likewise, MeJA (0.015 µM) when applied alleviated the Cd toxicity as the suppression in fresh was only 8% and 10%) and in dry mass, it was only 4% and 6% in tomato and brinjal seedlings, respectively. Combined application of GABA and MeJA completely restored the damaging effect of Cd on tested growth attributes, besides there was a marginal increase in growth as compared to the values recorded for control plants.

To understand the ameliorative role of endogenous GABA and MeJA against Cd toxicity on growth attributes the biosynthetic inhibitors of MeJA (DIECA, 5 µM) and GABA (MPA, 20 µM) were applied in Cd-stressed seedlings (Fig. 1,2). The results revealed that with DIECA (Cd + GABA + DIECA) and MPA (Cd + MeJA + MPA) the alleviation of inhibitory effects due to Cd on fresh mass was reversed showing the reduction of 25% and 35% in tomato and 30% and 38% in brinjal seedlings, respectively. Similar trends were also observed in the reduction of dry mass in both the seedlings under similar treatments with inhibitors (DIECA and MPA) (Fig. 1, 2).

3.2 Gamma-aminobutyric acid and methyl jasmonate down regulate intracellular cadmium

Cd treatment resulted in a substantial accumulation of Cd in both the seedlings and the content was greater in brinjal (72%) than that of tomato (58%) compared to nontreated control. The seedlings exogenously treated with GABA exhibited a considerable decrease in 22% and 20% and MeJA declined 14% and 6%, in brinjal and tomato compared to Cd treated seedlings. Combined treatment (Cd + MeJA + GABA) caused further reduction in the accumulation of Cd and it was found to be 33% and 18% in brinjal and tomato compared to Cd treated seedlings. Contrary to this, when tested plants were exposed to Cd + MeJA + MPA then the Cd content enhanced 16% and 8% in brinjal and tomato compared to Cd treated seedlings. In Cd + GABA + DIECA treatments the intracellular accumulation of Cd was increased further 8% and 3% in brinjal and tomato compared to Cd treated seedlings (Fig. 4).

3.3 Gamma-aminobutyric acid and methyl jasmonate protect photosynthetic pigments against Cd stress

One of the most important factors for determining how plants respond to metal stress is the analysis of photosynthetic pigments which play an important role in the photosynthetic processes of the plants. Photosynthetic pigments (Chl a, Chl b, and Car) were reduced by 14%, 23% and 11% in tomato and 18%, 26%, and 14% in brinjal seedlings, respectively under Cd toxicity in comparison to the respective control values (Table 1). Unlike to this, on exogenous application of GABA the negative impact of Cd was alleviated, and the reduction in values of Chl a, Chl b, and Car were decreased by only 3%, 5%, and 2% in tomato and 5, 7, and 3% in brinjal seedlings (Table 1). Similar ameliorative results were also obtained with MeJA treatments on Chl a, Chl b, and Car under Cd stress in both seedlings, however, the recovery against Cd toxicity on pigment contents (Chl a, Chl b, and Car) was slightly lower than that observed with GABA (Table 1). The GABA and MeJA together alleviated more effectively as the negative effect of Cd was completely restored and the values were even greater than that of respective controls. Furthermore, to analyze the alleviatory effects caused by GABA and MeJA on Cd induced toxicity their specific biosynthetic inhibitors were used (Table 1). With the combinations of Cd + GABA + DIECA and Cd + MeJA + MPA the alleviation in the levels of pigment contents (Chl a, Chl b, and Car) was further reversed depicting the decline 16%, 26%, 14% and 20%, 34%, 19% in tomato and 21%, 30%, 17% and 24%, 36%, 23% in brinjal seedlings respectively over the values of control.

Table 1

Regulation of photosynthetic pigments Chl a, Chl b, and Cars by GABA and MeJA in tomato and brinjal seedlings growing under Cd stress.
Treatment	Tomato			Brinjal
	Chl a (µg g^− 1 FM)	Chl b (µg g^− 1 FM)	Car (µg g^− 1 FM)	Chl a (µg g^− 1 FM)	Chl b (µg g^− 1 FM)	Car (µg g^− 1 FM)
Ck	1380 ± 22b	576 ± 9ab	480 ± 7ab	1272 ± 20b	535 ± 8ab	440 ± 7ab
Cd	1182 ± 19d	440 ± 7c	425 ± 6d	1040 ± 17d	396 ± 6 c	375 ± 6d
Cd + GABA	1338 ± 21bc	546 ± 8b	470 ± 7b	1250 ± 19bc	496 ± 8 b	425 ± 7b
Cd + MeJA	1324 ± 20c	535 ± 8bc	465 ± 7bc	1180 ± 19c	478 ± 7bc	417 ± 6bc
Cd + GABA + MeJA	1420 ± 22a	585 ± 9a	485 ± 8a	1298 ± 21a	541 ± 9a	448 ± 7a
Cd + GABA + DIECA	1155 ± 18de	423 ± 6cd	412 ± 6c	1005 ± 16de	380 ± 6cd	365 ± 6c
Cd + MeJA + MPA	1102 ± 17e	378 ± 5d	385 ± 6e	965 ± 15e	341 ± 5d	335 ± 5e
Data are means ± standard error of three independent biological replicates (n = 3). Values within the same column followed by different letters are statistically different at p˂0.05 significance level according to the Duncan’s multiple range test (DMRT)

3.4 Gamma-aminobutyric acid and methyl jasmonate alleviate the negative effect of Cd stress on PS II Photochemistry

To understand the impact of Cd and its regulation by GABA and MeJA on photosynthesis PS II photochemistry was analyzed by JIP test and results are depicted in Table 2. The results indicate that Cd toxicity exerted a negative impact on the values of chlorophyll a fluorescence kinetics parameters such as quantum yield of primary photochemistry (Phi_P₀), yield of electron transport per trapped exciton (Phi_E₀₎, the maximum quantum efficiency of PS П photochemistry (Psi_0), the performance index of PS II (PI_ABS) while the energy flux parameters [the energy for absorption of photon per active reaction center (ABS/RC), trapped energy per active reaction center (TR₀/RC), electron transport per active reaction center (ET₀/RC) and energy dissipation per active reaction center (DI₀/RC) ] were increased significantly under Cd toxicity. Exogenous usage of signalling molecules GABA and MeJA individually and both together alleviate the negative impact of Cd toxicity on these parameters (Table 2). Contrary to this, the MeJA inhibitor DIECA intensified the Cd toxicity on energy flux parameters (Table 2). Furthermore, the application of biosynthetic inhibitor (MPA) of GABA showed a greater increase in energy flux parameters even in the presence of MeJA.

Table 2

Regulation of chlorophyll a fluorescence characteristic by GABA and MeJA in tomato and brinjal seedlings exposed to Cd stress.
Treatments	Tomato
Treatments	Phi_P0	Phi_E0	Psi_0	PI_ABS	ABS/RC	ET0/RC	TR0/RC	DI0/RC
Ck	0.790 ± 0.013a	0.530 ± 0.011c	0.588 ± 0.008c	3.532 ± 0.050a	2.240 ± 0.042f	0.966 ± 0.018f	1.894 ± 0.029f	0.774 ± 0.011c
Cd	0.711 ± 0.011d	0.429 ± 0.013d	0.478 ± 0.006d	2.659 ± 0.031cd	2.783 ± 0.035c	1.212 ± 0.013c	2.436 ± 0.024c	0.885 ± 0.013bc
Cd + GABA	0.776 ± 0.013b	0.576 ± 0.009b	0.623 ± 0.008ab	3.101 ± 0.044b	2.538 ± 0.044d	1.056 ± 0.017d	2.198 ± 0.030e	0.731 ± 0.011d
Cd + MeJA	0.758 ± 0.013bc	0.588 ± 0.012ab	0.618 ± 0.009b	2.840 ± 0.040c	2.657 ± 0.045cd	1.140 ± 0.018cd	2.225 ± 0.028d	0.756 ± 0.011 d
Cd + GABA + MeJA	0.728 ± 0.011c	0.601 ± 0.008a	0.635 ± 0.012a	3.475 ± 0.050ab	2.496 ± 0.046e	0.994 ± 0.018 e	1.927 ± 0.031ef	0.720 ± 0.010e
Cd + GABA + DIECA	0.698 ± 0.013e	0.427 ± 0.014d	0.460 ± 0.013de	2.551 ± 0.036d	3.214 ± 0.031b	1.289 ± 0.013b	2.642 ± 0.020b	0.898 ± 0.009b
Cd + MeJA + MPA	0.647 ± 0.013f	0.412 ± 0.011e	0.431 ± 0.006e	2.296 ± 0.033e	3.782 ± 0.028a	1.332 ± 0.012a	2.886 ± 0.027a	0.935 ± 0.012a
Brinjal
Ck	0.775 ± 0.011a	0.527 ± 0.010c	0.626 ± 0.009c	2.431 ± 0.035a	2.198 ± 0.03f	0.845 ± 0.018f	1.745 ± 0.033f	0.671 ± 0.009c
Cd	0.671 ± 0.009d	0.435 ± 0.009d	0.425 ± 0.006d	1.661 ± 0.023cd	2.556 ± 0.049c	1.225 ± 0.010c	2.085 ± 0.028c	0.740 ± 0.010bc
Cd + GABA	0.731 ± 0.010b	0.496 ± 0.011b	0.675 ± 0.009ab	2.201 ± 0.031b	2.118 ± 0.049d	0.892 ± 0.019d	1.782 ± 0.037e	0.601 ± 0.008de
Cd + MeJA	0.742 ± 0.010bc	0.488 ± 0.009ab	0.655 ± 0.014b	2.284 ± 0.032c	2.322 ± 0.047cd	0.921 ± 0.018cd	1.811 ± 0.036d	0.635 ± 0.009d
Cd + GABA + MeJA	0.720 ± 0.010c	0.510 ± 0.014a	0.687 ± 0.115 a	2.675 ± 0.038ab	2.074 ± 0.048e	0.858 ± 0.021e	1.758 ± 0.038ef	0.598 ±. 0009e
Cd + GABA + DIECA	0.679 ± 0.009e	0.391 ± 0.009d	0.410 ± 0.009de	1.355 ± 0.019d	3.015 ± 0.041b	1.306 ± 0.008b	2.225 ± 0.026b	0.781 ± 0.011b
Cd + MeJA + MPA	0.626 ± 0.009f	0.422 ± 0.012e	0.391 ± 0.008e	1.296 ± 0.018e	3.315 ± 0.042a	1.328 ± 0.006a	2.554 ± 0.023a	0.795 ± 0.012a
Data are means ± standard error of three independent biological replicates (n = 3). The quantum yield of primary photochemistry (Phi_P0), yield of electron transport per trapped exciton (Psi_0), quantum yield of electron transport (Phi_E0), performance index of PS II (PI_ABS), the energy fluxes for absorption of photon per active reaction center (ABS/RC), trapped energy flux per active RC (TR0/RC), electron transport flux per active RC (ET0/RC) and energy dissipation flux per active RC (DI0/RC).

3.5 Gamma-aminobutyric acid and methyl jasmonate ameliorate Cd toxicity on photosynthetic gas exchange parameters

Photosynthetic gas exchange parameters were also adversely affected by Cd toxicity in tomato and brinjal seedlings. (Table 3,4). Upon Cd exposure, leaves gas exchange parameters A, Ci, gs, E, were inhibited by 23%, 18%, 28% and 20% in tomato and by 25%, 21%, 31% and 22% in brinjal, compared to control. However, the application of exogenous GABA significantly alleviated the toxicity of Cd on A, Ci, gs, and E values as it was decreased by only 8%, 2%, 7%, and 3% in tomato and 11%, 4%, 9% and 5% in brinjal, respectively over the values of respective controls. Under similar stress conditions when the seedlings were treated with MeJA significant amelioration (11%, 5%, 10%, and 8% in tomato and 13%, 7%, 12%, and 9% in brinjal seedlings) was noticed in A, Ci, gs, and E respectively. Furthermore, when seedlings were subjected to GABA and MeJA treatments damaging effects of Cd on these parameters were overcome in both the seedlings completely (Table 3,4).

Table 3

Regulation of photosynthetic characteristics: A, Ci, gs, and E in tomato seedlings by GABA and MeJA growing under Cd toxicity.
Treatments	Tomato
Treatments	A (µmol CO₂ m^− 2 s ^− 1)	Ci (µmol CO₂ mol^− 1)	Gs (mol H₂O m^− 2 s ^− 1)	E (m mol H₂O m^− 2 s ^− 1)
Ck	10.49 ± 0.16b	280.66 ± 4.05b	0.14 ± 0.002a	3.11 ± 0.050ab
Cd	8.04 ± 0.12 e	232.67 ± 3.36e	0.10 ± 0.001d	2.49 ± 0.040d
Cd + GABA	9.60 ± 0.15c	274.37 ± 4.59c	0.13 ± 0.002b	3.01 ± 0.048b
Cd + MeJA	9.35 ± 0.15d	266.00 ± 4.47d	0.12 ± 0.002c	2.85 ± 0.046c
Cd + GABA + MeJA	10.91 ± 0.17a	292.67 ± 4.74a	0.14 ± 0.002a	3.15 ± 0.050a
Cd + GABA + DIECA	7.75 ± 0.12f	218.33 ± 3.15f	0.09 ± 0.001e	2.38 ± 0.038e
Cd + MeJA + MPA	7.12 ± 0.11g	191.33 ± 2.76g	0.75 ± 0.001f	2.25 ± 0.037f
Brinjal
Ck	9.54 ± 0.15ab	237.00 ± 3.83b	0.13 ± 0.002b	3.11 ± 0.063b
Cd	7.15 ± 0.11d	185.33 ± 2.99d	0.08 ± 0.001e	2.57 ± 0.049e
Cd + GABA	8.40 ± 0.13b	228.00 ± 3.68c	0.12 ± 0.001c	3.07 ± 0.060c
Cd + MeJA	8.25 ± 0.13c	220.33 ± 3.55cd	0.11 ± 0.001d	2.98 ± 0.057d
Cd + GABA + MeJA	9.79 ± 0.15a	252.67 ± 4.07a	0.14 ± 0.002a	3.32 ± 0.065a
Cd + GABA + DIECA	6.88 ± 0.11e	168.33 ± 2.72e	0.07 ± 0.001f	1.90 ± 0.047f
Cd + MeJA + MPA	6.18 ± 0.10f	139.67 ± 2.41f	0.06 ± 0.001g	1.35 ± 0.044g
Data are means ± standard error of six replicates (n = 6). Bars followed by different letters are significantly different at p < 0.05 according to the Duncan’s multiple range test. Data are means ± standard error of three independent biological replicates (n = 3). Values within the same column followed by letters are statistically different at p˂0.05 significance level according to the Duncan’s multiple range test.

To explore the relationship between GABA and MeJA regarding the alleviatory role, the biosynthetic inhibitors of GABA (MPA) and MeJA (DIECA) [in combinations (Cd + GABA + DIECA; Cd + MeJA + MPA)] were applied and the inhibitory effect of Cd became more prominent, however, this effect appeared to be more prominent with the inhibitor (MPA) of GABA.

3.6 Gamma-aminobutyric acid and methyl jasmonate down-regulate oxidative biomarkers and protect against damage under Cd stress

To pinpoint the damaging effect of Cd on growth and related parameters such as photosynthetic pigments, PS II photochemistry, and gas exchange parameters, and the role of GABA and MeJA on the oxidative stress biomarkers were analysed (Fig. 3, 4). The oxidative biomarkers SOR, H₂O_2, and MDA equivalents were found to increase by 20%, 33%, and 38% in tomato, respectively, while the corresponding rise in the values was 24%, 39%, and 46% in brinjal seedlings under Cd stress. A considerable decrease in SOR, H₂O₂, MDA was noticed in Cd-stressed tomato seedlings in the presence of GABA as 3%, 11% and 13% and MeJA i.e. 6%, 19% and 16%, respectively. Further, as on the application of both the signalling molecules, the negative effects of Cd were completely overcome in both the seedlings, thereby the levels of oxidative biomarkers were decreased to the extent even less than that of control (untreated samples) (Fig. 5). Together with the biosynthetic inhibitor (MPA) of GABA the levels of oxidative biomarkers were excessively risen, while with inhibitor (DIECA) of MeJA the rise in these markers were also increased but the levels were found to be comparatively low than those observed with MPA, an inhibitor of GABA (Fig. 5).

Further, the formation/accumulation of oxidative biomarkers in leaves under Cd stress and with different combinations were visualized by histochemical staining by blue formazan due to a reaction between NBT and SOR, by brown spots as s results of the reaction of hydrogen peroxide and DAB and by pink colour developed between the reaction of lipid peroxidation product and Shiff reagent (Fig. 7). The intensity of the blue colour was noticeable during Cd toxicity. With the biosynthetic inhibitors of GABA and MeJA the blue colour became more intense. The exogenously applied GABA and MeJA exhibited decreased colour intensity, hence showing the appreciable decrease in ROS content. Under Cd stress the biosynthesis inhibitors of MeJA and GABA exhibited more intense brown colour, and the increasing order is as Cd˂ MeJA˂ GABA as seen in Fig. 7. Under GABA and MeJA treatment, the formation of brown colour in cells was quite less. The results revealed that histochemical analysis supports the biochemical data regarding H₂O₂ generation under Cd toxicity and the subsequent effects of GABA and MeJA with and without their biosynthetic inhibitors. The lipid peroxidation as studied by histochemical staining with the Schiff reagent exhibited with the greater intensity of pink colour was noticed under Cd toxicity alone and also with inhibitors of GABA and MeJA. The exogenously used GABA / MeJA appreciably lowered the colour intensity, and this result indicates the ameliorative effect of GABA and MeJA under Cd toxicity.

3.7 Gamma-aminobutyric acid and methyl jasmonate ameliorate Cd toxicity on membrane stability index

The data related to oxidative damage indices such as membrane stability index (MSI) are depicted in Fig. 3. Cd declined MSI by 15 and 20% in tomato and brinjal, respectively in comparison to their control values. After the supplementation of GABA under Cd toxicity, MSI is declined by 2% and 5% in tomato and brinjal, respectively to their control values, similarly on the addition of MeJA the decrement of MSI is 4% and 8% in tomato and brinjal seedlings, respectively. When we applied both together GABA + MeJA under Cd stress, the MSI is enhanced by 2% and 3% in tomato and brinjal seedlings, respectively to their respective control values. The addition of MPA and DIECA caused further decline in MSI in both vegetables. On the application of MPA the MSI was greatly decreased by 31% and 34% in tomato and brinjal seedlings, respectively with their respective control values (Fig. 3).

3.7 Gamma-aminobutyric acid and methyl jasmonate up-regulate enzymatic antioxidant system under stress

To keep the oxidants under control in the biological system, the plant possesses an array of antioxidant systems which include enzymatic and non-enzymatic antioxidants. The status of enzymatic antioxidants in test plants is displayed in Fig. 6. Cadmium treatment resulted in enhanced activity of SOD, POD, CAT, and GST by 15, 30, 16, and 11% in tomato and 19, 36, 25, 14% in brinjal seedlings, respectively with their respective control values. The increment of SOD, POD, CAT, and GST was further enhanced in the presence of exogenous GABA (activity raised by 5, 11, 3, and 2% in tomato and 7, 13, 5, and 5% in brinjal seedlings, respectively), and the corresponding increase in the enzyme activity with exogenous MeJA (activity enhanced by 8, 14, 6, and 5% in tomato and 11, 16, 7, and 7% in brinjal seedlings, respectively) was noticed (Fig. 6). In the combined treatments Cd + GABA + MeJA the activity of SOD, POD, CAT, and GST showed further decreasing trend in both the seedlings, however, it was still greater than that of respective controls. To understand the interaction between GABA and MeJA the specific inhibitors of GABA (Cd + MeJA + MPA) and MeJA (Cd + GABA + DIECA) were applied, the results revealed that the activity of SOD, POD, CAT, and GST was increased by 31, 43, 25, 22% in tomato and 34, 46, 35, 25% in brinjal seedlings exposed with Cd + MeJA + MPA. Under similar treatments the corresponding increase with Cd + GABA + DIECA combination was 20, 35, 19, 16% in tomato and 24, 40, 29, 20% in brinjal seedlings, respectively (Fig. 6).

The two vegetables tomato and brinjal are important crops, widely grown in Indian continent, facing serious threats due to rise in heavy metal contamination. In current study, the seedlings of tomato and brinjal exposed to Cd showed a significant reduction in growth (Fig. 1, 2) and such an adverse effect was more prominent in brinjal seedlings. Similar observations were also recorded in Trigonella (Bashri et al. 2021), rice (Huang et al. 2021) and wheat seedlings (Zeshan et al. 2022) under Cd stress. In both the seedlings Cd diminished the growth which could occur due to (i) damage caused to the light harvesting pigments Chl a, b and Cars (Bashri et sl. 2021) (ii) negative impact on PS II photochemistry (Liu et al. 2020), and (iii) excess accumulation of SOR and hydrogen peroxide that led to oxidation of nucleic acids (Sanghai et al. 2021) and proteins (Alamri et al. 2021) and peroxidation of lipids (Fig. 5). The exogenously applied signalling molecules GABA or MeJA significantly alleviated the Cd toxicity in both the seedlings as reduction in growth attributes was recovered to the greater extent (Fig. 1, 2). Many studies were earlier reported similar results (Zaid and Mohammad, 2018; Kaya et al. 2021; Wei et al. 2021). Combined application of GABA and MeJA was found to be more effective in recovering the loss of growth of tested vegetables due to Cd stress. Our results are in congruence with earlier findings where GABA has been shown to efficiently ameliorated the negative effect of arsenic on growth in tomato and brinjal seedlings (Suhel et al. 2024) and Cd on apples (Li et al. 2022). Likewise, exogenously applied MeJA mitigated the damaging effect of Cd stress on growth of Brassica oleracea L (Srihindi et al. 2020).

Tomato and brinjal seedlings (Suhel et al. 2022), Cd toxicity in maize plant (Seifikalhor et al. 2020) and tobacco (He et al. 2021) and phenanthrene phytotoxicity in cucumber (Guo et al. 2021). To study the impact of MeJA, its inhibitor DIECA in the presence of GABA exhibited significant decrease in the fresh/ dry mass of tomato and brinjal seedlings over the values to Cd stress. While with the inhibitor (MPA) of GABA a greater decrease was noticed in fresh mass/dry mass. These results reveal that GABA appears to be the main signalling molecule for alleviation of Cd toxicity, while MeJA regulates the GABA mediated toxicity amelioration in both the tested seedlings. In this study, it was noticed that accumulation of Cd in seedlings of tomato and brinjal (Fig. 3) exhibited severe oxidative burst as evidenced by enhanced levels of SOR (O₂ ^•−), H₂O₂ and MDA. Moreover, Cd can block ETC (electron transport chain) or displace the iron (Fe) molecule with other protein molecules that cause the abundant production of ROS inside the cell (Genchi et al. 2020). The growth of seedlings is mainly regulated by photosynthesis and status of light harvesting pigments. A significant decrease in photosynthetic pigment contents under Cd stress was observed and these results are in consistent with the earlier findings where Cd induces senescence in the leaf of pea plants and inhibited the biosynthesis of chlorophyll (Bashri et al. 2021; Hayat et al. 2021). Conversely, the exogenous application of GABA and MeJA alone or together significantly up-regulated Chl and Car contents under Cd stress. The considerable rise in Chl and Car contents by the application of GABA and MeJA alone and together might have direct correlation with an alleviating effect on the growth performance of seedlings under Cd toxicity. In consonance with our results, Suhel et al. (2022) have shown the involvement of GABA in protecting Chl and Car in plants grown under As stress. Furthermore, MeJA exposure increased 5-aminolevulinic acid production, thereby up-regulation of gene expression which is linked with chlorophyll biosynthesis (Ueda and Saniewski 2006). Therefore, MJA positively modulated the efficiency of the photosynthetic apparatus; hence induces the level of tolerance against Cd toxicity tolerance in tested plants. Besides this, Salavati et al. (2021) has suggested that down-regulation in the degrading process of Chl and Car by exogenous MeJA treatment significantly contributed to the improvement of pigment contents in Oryza sativa seedlings under Pb stress.

The gas exchange parameters viz. A, Ci, Gs, and E were used to determine the photosynthetic activities, and in tomato and brinjal seedlings these parameters were negatively affected under Cd toxicity (Table 3,4). Similar trends were also reported by Huarancca et al. (2018) in Chenopodium quinoa under UV stress. According to Mosadegh et al. (2019), the reduction in photosynthetic rate might be caused by the degradation of pigments. In current study Cd could restrain the stomatal apertures, hence negatively affected the A, Ci, Gs, and E parameters because of the photosynthetic interruption as noticed in other study (Zhao et al. 2021). Stomatal and non-stomatal factors are the two categories of factors that influence the rate of photosynthetic activity in plants. Papadakis et al. (2023) suggested that variations in stomatal conductance and intercellular carbon dioxide concentration should be examined concurrently in order to assess the factors that influence the rate of photosynthetic activity of plants.

It is a well-known fact that Cd decreases Gs and causes a real decrease in Ci, which in turn makes the plant more susceptible to photoinhibition (Zhao et al. 2021). In fact, under restricting states of CO₂ fixation, the rate of reducing power production could decrease the rate of its utilization in the photosynthetic electron transport chain, accordingly harming the photosynthetic mechanical apparatus. The significant decrease in net photosynthesis rate suggests that Cd toxicity can influence photosynthesis by influencing primary photochemistry, electron transport, enzyme activity, biochemical reactions of the Calvin cycle, and changes in chloroplast structure, as previously discovered in a few studies (Nwugo and Huerta 2008).

Cao et al. (2020) reported that E is important for the transport of inorganic nutrients over long distances via xylem tissues. In present study, under Cd toxicity the transpiration rate was decreased (Table 2), however, after applying GABA and MeJA the photosynthesis was improved because of an increment in CO₂ fixation by increasing the value of Ci, Gs, E and A. In another study, GABA enhanced these activities under heat and drought stress in the Helianthus annuus plant (Abdel Razik et al. 2021). Similarly, MeJA also enhanced the values of these attributes of photosynthesis which are reported in rice plant which was exposed to heat stress (Tang et al. 2022). When Gs values were found to increase, the mesophyll cell's capacity to take up CO₂ may increase and this would enhance the Calvin cycle’s capacity to take up CO₂ (Messinger et al. 2006).

A quick and non-destructive method to evaluate the photosynthetic efficiency of stressed plants is chlorophyll a fluorescence. Previous researches have demonstrated that Cd impacted photosynthetic pigments and net photosynthesis in tomato seedlings (Song et al. 2024; Yadav et al. 2022). Cd is known to inhibit PS II activity by interfering with electron flow at the oxidation side and harming the PS II reaction centre (Bashri and Prasad 2015), hence in the present study, a marked decrease in oxygen yield might have occurred due to a direct effect of Cd on light reactions including oxygen evolving complex. In current study, different fluorescence parameters were detected to observe the change in the photochemistry of photosystem PS II (Table 2). The values of Phi_E₀, Phi_0, Phi_P₀ and PI_ABS were decreased significantly, and the values of energy flux parameters ET₀/RC, TR₀/RC and DI₀/RC were increased under Cd toxicity (Table 2). Khan et al. (2019) also reported that decreased values of Phi_E₀, Phi_0, and Phi_P₀ under Cd stress suggested disturbed electron flow between the photosystems which ultimately reduced the performance index (PI_ABS) of tested tomato seedlings. An increase in the values of specific energy flux parameters such as ABS/RC, ET₀/RC, TR₀/RC, DI₀/RC suggested the dissipation of excess light energy under Cd stress. The application of exogenous GABA and MeJA alone and together exhibited alleviating effects on Phi_E₀, Phi_0, Phi_P₀, PI_ABS in both tested seedlings. These improved values may be correlated with the result of decreased Cd uptake. Specific energy flux parameters were reduced in response to the exogenous GABA and MeJA while inhibitors MPA and DIECA with Cd worsened the damage to the PS II.

ROS generation in living cells under aerobic condition is the results of metabolic reactions, and under stress ROS are produced at several sites of respiratory electron transport, photosynthetic electron transport, peroxisome, several sites of cytosol l and the apoplast in huge amount. Further, imbalance between ROS production and safe detoxification generates oxidative stress and its accumulation in high amount in cells becomes harmful to vital part of cells (Zulfiqar and Ashraf 2021). In current study, under Cd accelerated production/accumulation of ROS disturbed the metabolism of test plants thereby damaging impact on photosynthetic apparatus and growth of test seedlings was noticed (Fig. 5). Exogenous application of GABA and MeJA alone/together in both Cd stressed plants caused significant lowering in the level of ROS in tissues, and this effect was more pronounced GABA treated seedlings. As a result, significant alleviation in oxidative damage (lipid peroxidation and membrane damage) was observed. Contrary to this, when seedlings were treated with the inhibitors (MPA and DIECA) of GABA and MeJA the excess accumulation of ROS was seen, hence aggravated the damaging effects in both the seedlings (Fig. 5). Earlier findings have also demonstrated that GABA down regulates oxidative biomarkers hence significant improvement in the functioning of photosynthetic apparatus in tomato and brinjal seedlings under arsenic stress (Suhel et al. 2022) and in tobacco (He et al. 2021) under Cd stress by substantial reduction in oxidative stress. Likewise, MeJA also plays an important role in Cd toxicity alleviation on structure and function chloroplast in Pisum sativus L. (Manzoor et al. 2022) and Mentha arvensis (Zaid et al. 2018) exposed to Cd stress by decreasing oxidative damage. The biosynthetic inhibitor of GABA (MPA) and MeJA (DIECA) also induced O₂^•− and H₂O₂ production and together with Cd it further enhanced the production of ROS in the tomato and brinjal seedlings in comparison to Cd stress (Fig. 5). Histochemical analysis also revealed that excess accumulation of H₂O and O₂•‾ was also visualized in leaves of both the seedlings exposed Cd stress (Fig. 4), and the intensity of colours developed due to SOR and H₂O₂ was considerably decreased by the application of GABA and MeJA. Further, the intensity of colours became more intense when inhibitors (MPA and DIECA) of GABA and MeJA were applied in Cd stressed both the seedlings (Fig. 7). Furthermore, excessive rise in oxidative stress radicals due to Cd exposure to test plants enhanced the lipid peroxidation and membrane damage as shown by in vivo analysis where intensity of colour developed between the reaction of MDA equivalents and Schiff ‘reagent and also due to the staining with Evans’ blue reagent became more intense. However, GABA and MeJA application exhibited less intense colour in both the seedlings grown under Cd stress and again colour became more intense when treated with biosynthetic inhibitors and this effect was more prominent with the inhibitor of GABA. Enhanced level of H₂O₂ may be assigned due to the altered photosynthetic process that resulted into leakage of electrons to oxygen. As a consequence of this increased lipid and protein degradation and damage to the plasma membranes and its stability are observed. Decrease in membrane stability, in turn might be associated with an increased accumulation of Cd in tissues (Emamverdian et al. 2023). However, exogenous application of GABA and MeJA significantly lowered Cd-mediated rise in oxidative stress (Fig. 4) and thus membrane stability must have been improved.

Every cell is empowered with antioxidant system; enzymatic and non-enzymatic. The antioxidants efficiently neutralize the oxidants, hence proved to be beneficial components of defense system as they limit the ROS under control. In current study under Cd stress considerable increase the activity of SOD, POD and CAT could not keep the ROS levels under control as a result of this increased damage to lipids and membrane caused substantial damage to photosynthesis (Tables 3 and 4), thus Cd toxicity significantly affected the growth of both the seedlings. When Cd stressed seedlings of both vegetables were treated with GABA and MeJA either alone or in combinations exhibited significant lowering in SOD, CAT and POD activity, however the enzymatic antioxidants kept the ROS levels under control and this could also be supported by improved activity of photosynthesis (Fig. 6, Table 3, 4). Notwithstanding to this, the treatment with Cd + GABA + DIECA and Cd + MeJA + MPA raised the activity of SOD, POD and CAT, however ROS contents were enhanced significantly high (Fig. 6) thereby inhibiting the photosynthetic activity of both the seedlings. Under similar experimental conditions GST activity exhibited similar trend as noticed with other studied enzymatic antioxidants this could support the plants by sequestering the xenobiotic and finally channelized these radicals into vacuoles. Various studies have also demonstrated that under metal toxicity, GST enzyme plays a major role in removing the reactive lipid derivatives and harmful carbonyl groups (Sharapov et al. 2021; Singh et al. 2013). Exogenously application of GABA and MeJA minimizes the build-up of ROS and reduces the negative effect on lipids and membranes. Several studies have found that SOD, POD, CAT, and GST activity increase under metal stress. (Suhel et al. 2022, Singh et al. 2020; Singh and Prasad 2019; Ahmad et al. 2018). In this study, the greater rise of SOD, POD, CAT, and GST activities greatly increased under Cd toxicity which reflected in the form of decreased growth characteristics. Exogenous application of GABA and MeJA lowered SOD, POD, CAT, and GST activities (Fig. 6). The improved growth of both the seedlings after exogenous application of GABA and MeJA suggested that the activity of SOD and POD was balanced and it sufficient to control the level of O₂^•− and H₂O₂ (Fig. 6). The activation of GST is required GABA and MeJA-mediated mitigation of Cd toxicity in both the seedlings.

The results conclude that the growth of tomato and brinjal seedlings were diminished by Cd, which could occur due to inhibitory effect on PSII photochemistry, light harvesting pigments and gas exchange parameters. Under Cd stress, despite of accelerated activity of SOD, CAT, POD and GST the oxidative biomarkers (O₂•‾, H₂O₂, and MDA) were greatly higher. Exogenous application of MeJA and GABA individually or in combinations alleviated Cd toxicity by significantly lowering the oxidative biomarkers, hence improved the photosynthetic efficiency and growth. The biosynthetic inhibitors of GABA and MeJA application suggested that GABA and MeJA alleviated the toxicity of Cd, however GABA appears to play greater role in minimizing Cd toxicity in both the seedlings. The study further demonstrated that tomato seedlings were found to show more tolerance to Cd as compare to brinjal which could be due to strong antioxidant system and GABA/MeJA alleviation that negatively affects plant growth. According to these findings, brinjal is more vulnerable to Cd toxicity than tomato. This study can be helpful for minimizing the effect of Cd toxicity in the food chain using the justified amount of MeJA and GABA.

Chl a Chlorophyll a

Chl b Chlorophyll b

Car Carotenoids

A Respiration rates

Ci Intracellular CO₂concentration

Gs Stomatal conductance

E Transpiration rate

Phi_P₀The quantum yield of primary photochemistry

Psi_0 Yield of electron transport per trapped exciton

Phi_E₀ Quantum yield of electron transport

PI_ABS Performance index of PS II

ABS/RC The energy fluxes for absorption of photon per active reaction center

TR₀/RC Trapped energy flux per active RC

ET₀/RC Electron transport flux per active RC

DI₀/RC Energy dissipation flux per active RC

SOR; O₂•‾ Superoxide radical

H₂O₂ Hydrogen peroxide

MDA Malondialdehyde equivalent content

SOD Superoxide dismutase

POD Peroxidase

CAT Catalase

GST Glutathione-S-transferase

MeJA Methyl jasmonate

GABA Gamma-aminobutyric acid

MPA 3-mercaptopropionic acid

DIECA Diethyledithiocarbamic acid

Acknowledgment

The authors are very thankful to Head, Department of Botany, University of Allahabad, for providing laboratory facilities. University Grant Commission, New Delhi, India is acknowledged for providing financial support to Varunendra Kumar Singh as UGC-AU research scholarship to carry out the present work.

Author Contributions SMP designed experiments. VKS performed experiments. SMP and VKS analysed data. VKS and SRV wrote the manuscript. SMP, SRV and VKS corrected the manuscript.

Competing intrests: Authors have no competing intrests.

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Phytohormones methyl jasmonate (MeJA) and gamma-aminobutyric acid (GABA) up-regulates growth and PS II photochemistry in brinjal and tomato seedlings exposed to cadmium toxicity

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Experimental plants and growth condition

2.2 Treatment of seedlings with GABA, MeJA, and Cd

2.3 Determination of growth attributes

2.4 Estimation of Cd content

2.5 Determination of photosynthetic pigments

2.6 Determination of chlorophyll a fluorescence kinetics (JIP test)

2.7 Estimation of photosynthetic gas exchange parameters

2.8 Estimation of oxidative stress indices

2.8.1 Biochemical estimation of oxidative stress biomarkers

2.8.2 Determination of membrane stability index

2.8.3 In-vivo analysis of oxidative stress biomarkers

2.9 Estimation of enzymatic antioxidants activities

2.10 Statistical analysis

3. Results

3.1 GABA and MeJA alleviate Cd toxicity on growth

3.2 Gamma-aminobutyric acid and methyl jasmonate down regulate intracellular cadmium

3.3 Gamma-aminobutyric acid and methyl jasmonate protect photosynthetic pigments against Cd stress

3.5 Gamma-aminobutyric acid and methyl jasmonate ameliorate Cd toxicity on photosynthetic gas exchange parameters

3.6 Gamma-aminobutyric acid and methyl jasmonate down-regulate oxidative biomarkers and protect against damage under Cd stress

3.7 Gamma-aminobutyric acid and methyl jasmonate ameliorate Cd toxicity on membrane stability index

3.7 Gamma-aminobutyric acid and methyl jasmonate up-regulate enzymatic antioxidant system under stress

4. Discussion

5. Conclusion

Abbreviations

Declarations

References

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