Jasmonic acid induced changes in physio-biochemical attributes and ascorbate-glutathione pathway in Lycopersicon esculentum under lead stress at different growth stages

Science of the Total Environment 645 (2018) 1344–1360

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Jasmonic acid induced changes in physio-biochemical attributes and ascorbate-glutathione pathway in Lycopersicon esculentum under lead stress at different growth stages Shagun Bali a, Parminder Kaur a, Sukhmeen Kaur Kohli a, Puja Ohri b, Ashwani Kumar Thukral a, Renu Bhardwaj a,⁎, Leonard Wijaya c, Mohammed Nasser Alyemeni c, Parvaiz Ahmad c,d,⁎⁎ a

Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar 143005, Punjab, India Department of Zoology, Guru Nanak Dev University, Amritsar 143005, Punjab, India c Department of Botany and Microbiology, Faculty of Science, King Saud University, Riyadh 11451, Saudi Arabia d Department of Botany, S.P. College, Srinagar 190001, Jammu and Kashmir, India b

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Exogenous application of JA reduced Pb uptake in tomato plants. • JA treatment improved photosynthetic efficiency in Pb-treated plants. • Application of JA modulated the ascorbate-glutathione cycle in tomato plants under Pb stress. • Treatment of JA enhanced the contents of osmolytes and metal chelating compounds in Pb treated tomato plants.

a r t i c l e

i n f o

Article history: Received 11 March 2018 Received in revised form 12 July 2018 Accepted 13 July 2018 Available online xxxx Keywords: Jasmonic acid Lead toxicity Photosynthetic parameters Ascorbate-glutathione cycle Osmolytes Metal chelating compounds Tomato

a b s t r a c t Lead (Pb) is one of most toxic heavy metals that adversely affect growth and developmental in plants. It becomes necessary to explore environment safe strategies to ameliorate its toxic effects. Phytohormones play an imperative role in regulating stress protection in plants. Jasmonic acid (JA) is recognized as a potential phytohormone which mediates immune and growth responses to enhance plant survival under stressful environment. The present study was undertaken to evaluate the effect of JA on the growth, metal uptake, gaseous exchange parameters, and on the contents of pigments, osmolytes, and metal chelating compounds in tomato plants under Pb stress during different stages of growth (in 30-, 45-, and 60-day-old plants). We observed a decrease in shoot and root lengths under Pb stress. Treatment of JA improved the shoot and root lengths in the Pb-treated plants. The Pb uptake was increased with the increasing concentrations of Pb, however, seeds pretreated with JA reduced the Pb uptake by the plants. The chlorophyll and carotenoid contents increased by JA treatment in plants under Pb stress. Pre-soaking of seeds in JA, improved gaseous exchange parameters, such as internal CO2 concentration, net photosynthetic rate, stomatal conductance, and transpiration rate under Pb stress. JA enhanced the

Abbreviations: JA, jasmonic acid. ⁎ Corresponding author. ⁎⁎ Correspondence to: P. Ahmad, Department of Botany and Microbiology, Faculty of Science, King Saud University, Riyadh 11451, Saudi Arabia. E-mail addresses: [email protected] (R. Bhardwaj), [email protected] (P. Ahmad).

https://doi.org/10.1016/j.scitotenv.2018.07.164 0048-9697/© 2018 Elsevier B.V. All rights reserved.

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enzyme activity of ascorbate-glutathione cycle and reduced H2O2 concentration in Pb-treated plants. The contents of osmolyte and metal chelating compounds (total thiols, and non-protein and protein-bound thiols) were increased with the increase in Pb stress. In seeds primed with JA, the contents of osmolytes and metal chelating compounds were further increased in the Pb-treated plants. Our results suggested that treatment of JA ameliorated the toxic effects of Pb stress by reducing the Pb uptake and improving the growth, photosynthetic attributes, activity of ascorbate-glutathione cycle and increasing the contents of osmolytes and metal chelating compounds in the tomato plants. © 2018 Elsevier B.V. All rights reserved.

1. Introduction Pb is an environmental pollutant present abundantly in the ecosystem. It is released through various anthropogenic activities like automobile exhaust, smelting of ores, discharge from batteries, paints, and burning of fossil fuel are among other sources that contribute Pb to the environment (Caboche et al., 2010; Chenery et al., 2012). It causes inhibition of seed germination, reduction in plant growth, root/shoot ratio, photosynthesis, respiration, and alterations in the organization of chloroplast lamellae and cell division, among other effects (Gupta et al., 2010; Maestri et al., 2010; Bharwana, 2013). It also inhibits the biosynthesis of plastoquinone, chlorophyll, and carotenoids, electron transport chain, the activities of enzymes involved in the Calvin cycle, and leads to insufficiency of CO2, eventually causing stomatal closure (Sharma and Dubey, 2005). Pb is known to trigger the formation of reactive oxygen species (ROS) which are counteracted by various antioxidative enzymes and metabolites (Singh et al., 2010). Plants have a complex antioxidant defense system consisting of enzymatic antioxidants (superoxide dismutase, catalase, guaiacol peroxidase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione reductase etc.) and non-enzymatic antioxidants (ascorbic acid, glutathione, polyphenols, tocopherols etc.) (Asadi karam et al., 2017). Ascorbate-glutathione cycle played a significant role in defense against abiotic stress (Paradiso et al., 2008; Shan et al., 2015). In ascorbate-glutathione cycle, ascorbate peroxidase (APOX) is the first enzyme that helps in the quenching of H2O2 by marinating ascorbate pool in reduced form (Foyer and Halliwell, 1976). Dehydroascorbate reductase (DHAR) is a crucial reducing enzyme, participated in ascorbate-glutathione pathway. DHAR converted dehydroascorbate into reduced ascorbate in the presence of glutathione which acts as reducing agent. Monodehydroascorbate reductase (MDHAR) is one of the important members of ascorbate-glutathione cycle that reduced monodehydroascorbate into reduced ascorbate. Glutathione reductase (GR) is a chief enzyme in this cycle which converts oxidized glutathione into reduced glutathione (Rennenberg, 1980). In this way, reduced ascorbate and glutathione are renewed in ascorbate-glutathione cycle and assist in the scavenging of H2O2. Plants have a complex network for identifying different stress signals that help them to survive under stress. Phytohormones play an important role in countering environmental stresses in addition to performing their functions in growth and development of plants. Jasmonates, such as JA and methyl jasmonate (MeJA), are cyclopentanone compounds that are biosynthesized from linolenic acid through the octadecanoic pathway. In plants, they assay essential roles under abiotic and biotic stresses (Fujita et al., 2006; Yan et al., 2015). JA is an important plant growth regulator, which is widely distributed in plant kingdom and plays important roles in various physiological processes in plants, such as seed germination, root growth, flowering, fruit ripening, tuber formation, senescence, and nutrient storage (Balbi and Devoto, 2007; Wasternack and Hause, 2013; Wasternack, 2014). It is one of the plant hormones that has the potential to protect plants from various stresses. In the recent past, JA has emerged as a signaling molecule under diverse environmental stresses, such as cold, light, desiccation, UV, and salt stress (Wasternack and Hause, 2013; Wasternack, 2014; Kamal and Komatsu, 2016).

JA modulated antioxidant capacity and decreasing the level of H2O2 and malondialdehyde and also improved the contents of photosynthetic pigments contents under Pb stress in Chlorella vulgaris (Piotrowska et al., 2009) and cadmium stress in Vicia faba (Ahmad et al., 2017). Tomato is one of the major vegetable crops, cultivated globally. It is an excellent source of vitamins, photochemicals, and folate and is used fresh or as processed fruit (Aldrich et al., 2010). Various reports showed that vegetable crops exposed to polluted water or soil with heavy metals because of rapid industrialization (Wu et al., 2016, 2017; Zeng et al., 2015). Reduction in plant growth and photosynthetic parameters was observed in tomato plants under cadmium stress (Jing et al., 2005). Metal uptake was increased in tomato seedlings with increasing concentration of cadmium and copper (Mediouni et al., 2006). Metal toxicity amelioration by plant growth regulators is an upcoming area of research which focuses on the potential of various plant growth regulators in abiotic stress tolerance. MeJA has positive effects on photosynthetic pigments and was reported to reduce cadmium toxicity by decreasing cadmium accumulation in Scenedesmus quadricauda (Kováčik et al., 2011). Keeping in mind the role of JA in providing metal stress protection to various plants, the present study was designed to evaluate the effects of JA in tomato plants exposed to different concentrations of Pb by measuring morphological and physiological parameters. 2. Materials and methods Seeds of tomato (Lycopersicon esculentum ‘Pusa ruby’) were surface sterilized by Tween 20. The soil was added to uniform-sized pots having mixture of clay and manure in the ratio of 3:1. The soil was amended with different concentrations of Pb solution (0, 0.25, 0.50, and 0.75 mM) in the form of Pb(NO3)2. Before sowing, the seeds were soaked for 4 h in solutions with different concentrations of JA (0, 0.01, 1, and 100 nM). The morphological and biochemical parameters were estimated after 30, 45, and 60 days of seed germination. 2.1. Estimation of growth and Pb uptake The shoot and root lengths were measured after harvesting the plants at different time intervals (30, 45, and 60 days). Plant materials (shoots and roots separately) were dried at 60 °C for 24 h. Dried plant material (200 mg) was digested using the method of Allen et al. (1976), with slight modifications. The digestion mixture comprised of H2SO4:HNO3:HClO4 in the ratio of 1:3:1. After digestion, the samples were filtered and diluted. The filtrate was analyzed for Pb content using atomic absorption spectrophotometer (Agilent Technologies, GTA 120, Shimadzu 6200). 2.2. Estimation of photosynthetic pigments The shoot and root lengths were measured after 30, 45, and 60 days of germination. The chlorophyll and carotenoid contents were determined using the procedures of Arnon (1949) and Maclachlan and Zalik (1963), respectively. The optical density for chlorophyll content was recorded at 645 and 653 nm and for carotenoid content, the absorbance was taken at 480 and 510 nm using a UV–visible

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spectrophotometer (Thermo Electron Corporation, Genesys 10 UV). The anthocyanin content was determined using the method of Macinelli (1984). The absorbance was measured at 530 and 657 nm using UV– visible spectrophotometer (Thermo Electron Corporation, Genesys 10 UV)·The flavonoid content was determined using the method of Zhishen et al. (1999). The absorbance was measured at 510 nm. Rutin (1 mg/mL) was used as a standard for the estimation of the flavonoid content. The content of xanthophylls was determined following the procedure of Lawrence (1990). The absorbance was taken at 474 nm using UV–visible spectrophotometer (Thermo Electron Corporation, Genesys 10 UV). 2.3. Determination of gaseous exchange parameters Gaseous exchange parameters, like photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 (Ci), and transpiration rate (Et) were determined using a portable photosynthesis system (LI-COR LI6400XT) at maximum light intensity on 30, 45, and 60 days of seed germination. 2.4. Estimation of H2O2 content H2O2 content was estimated by method described by Velikova et al. (2000). Absorbance was noted at 390 nm. 2.5. Determination of non-enzymatic (ascorbic acid and glutathione) and enzymatic antioxidants involved in ascorbate-glutathione cycle (APOX, MDHAR, DHAR and GR) The method for determination of ascorbic acid content was given by Roe and Kuether (1943). Absorbance was recorded at 540 nm. Glutathione content was determined by the method given by Sedlak and Lindsay (1968). Absorbance was taken at 412 nm.APOX activity was estimated by the method given by Nakano and Asada (1981). Change in absorbance was recorded at 290 nm (ε = 2.8 mM−1 cm−1). MDHAR activity was assayed according to the method of Hossain et al. (1984). Change in absorbance was taken at 340 nm (ε = 6.2 mM−1 cm−1). Activity of DHAR was determined by the method described by Dalton et al. (1986). Change in absorbance was noted at 265 nm (ε = 14 mM−1 cm−1). GR activity was estimated by the method of Carlberg and Mannervik (1975). Change in absorbance was taken at 340 nm (ε = 9.6 mM−1 cm−1). 2.6. Estimation of osmolytes The total carbohydrate content was measured following the method of Yemm and Willis (1954). The absorbance was taken at 630 nm. The total carbohydrate content was determined from the standard glucose curve. The proline content was determined using the procedure of Bates et al. (1973). The absorbance was recorded at 520 nm. The concentration of proline was calculated using a standard curve made with Lproline. The concentration of glycine betaine was determined using the method described by Grieve and Grattan (1983). The absorbance was taken at 365 nm. The concentration of glycine betaine was determined using a standard curve made with betaine hydrochloride.

solution was taken at 412 nm. The total thiol content was calculated using the molar extinction coefficient (ε = 13,100 M−1 cm−1). The procedure of Israr et al. (2006) was followed for estimation of non-protein thiols. The absorbance was recorded at 412 nm and the content was calculated using the molar extinction coefficient (ε = 13,100 M−1 cm−1). The content of protein-bound thiols was determined by subtracting the content of non-protein thiols from that of total thiols. 2.8. Statistical analysis Statistical analysis was done using two-way ANOVA test, Tukey's Honestly Significant Difference (HSD) and multiple linear regression (MLR). Using analysis by two-way ANOVA it was observed that in most cases the main effects as well as interactions between Pb and JA were significant. In order to quantify the relative effects of the main effects as well as interaction between Pb and JA, β-regression analysis was carried out. MLR was applied as mentioned in the equation: y ¼ a þ b1 x1 þ b2 x2 þ b3 x1 x2 where,x1 = independent variable (Pb); x2 = independent variable (JA), a = y-intercept, b1 = partial regression coefficient for x1 on y eliminating effect of x2, b2 = partial regression coefficient for x2 on y eliminating effect of x 1 , b3 = partial regression coefficient for interaction between Pb and JA. β-Regression coefficients are unitless standardized partial regression coefficients which give the relative effects of independent variables on the dependent variables. β-coefficients were calculated as follows: β ¼ b−Sx=Sy where Sx = Standard derivation of x, Sy = Standard derivation of y. 3. Results 3.1. JA improved the growth of tomato plants under Pb stress The shoot and root lengths were found to decrease with the increasing concentration of Pb in 30-, 45-, and 60-days-old tomato plants. The maximum decrease in shoot length was observed in plants treated with 0.75 mM Pb; the decrease was by 42.08%, 45.68%, and 47.73% after 30, 45, and 60 days, respectively, compared to that in the untreated plants (Fig. 1). The application of 100 nM JA was effective in enhancing the shoot and root lengths in the metal treated plants. Maximum improvement was found in the 30-days-old plants. The shoot length was significantly increased by 52.83%, 49.67%, and 29.89% compared to the length in the plants treated with 0.75 mM Pb alone at 30, 45, and 60 days (Fig.1). Root length was significantly decreased by 53.19% (30 days), 61.65% (45 days) and 64.41% (60 days) in 0.75 mM Pb treated plants as compared to untreated ones. 100 nM JA treatment enhanced root length by 77.27% (30 days), 66.67% (45 days) and 52.38% (60 days) in 0.75 mM Pb treated plants in comparison to 0.75 mM treatment alone. Negative β-regression coefficients for Pb showed reduction in shoot and root length due to Pb. Positive β-regression coefficients values for interaction Pb x JA depicted improvement in growth which was adversely affected by Pb treatment. 3.2. JA reduced the uptake of Pb by tomato plants

2.7. Determination of the contents of metal-chelating compounds (total thiols, non-protein thiols, and protein thiols) The content of total thiols was determined using the method described by Nagalakshmi and Prasad (2001). The absorbance of the

The Pb uptake was increased with the increasing concentration of Pb in the roots and shoots of 30-, 45-, and 60-days-old tomato plants. In plants exposed to 0.75 mM Pb, the application of 100 nM JA significantly reduced the Pb uptake in roots by 42.67%, 31.3% and 23.76% at 30, 45,

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Fig. 1. Effect of JA on growth parameters of 30, 45 and 60 days old tomato plants grown in Pb amended soil (mean ± SD, two-way ANOVA, Tukey's HSD and β-coefficients). r, correlation coefficient. *, ** and *** indicates significant at p ≤ 0.05, p ≤ 0.01 and p ≤ 0.001 respectively.

and 60 days, respectively (Fig. 2). JA treatments reduced the Pb uptake by tomato plants at the different growth intervals (30, 45, and 60 days). The soaking of seeds in 100 nM JA significantly decreased the Pb uptake in shoots by 44.71%, 33.84%, and 26.52% at the different time intervals in the plants treated with 0.75 mM Pb (Fig. 2). The maximum reduction in Pb uptake was observed in the roots and shoots of 30-days-old plants. Pb uptake was positively regressed with increasing concentrations of Pb in tomato plants during different growth phases (30 days, 45 days and 60 days). The interaction between Pb x JA was found to be negative which indicated that JA reduced Pb uptake in tomato plants (Fig. 2).

3.3. JA increased the pigment content in tomato plants under Pb stress Pb treatment significantly lowered the total chlorophyll, chlorophyll a, chlorophyll b, and carotenoid contents in the 30-, 45-, and 60-daysold tomato plants. Compared to the untreated plants, there was 58.59%, 64.15%, and 66.14% reduction in the total chlorophyll content in the 30-, 45-, and 60-days-old plants, respectively, treated with 0.75 mM Pb (Table 1). Pb treatment decreased the chlorophyll and carotenoid contents whereas the soaking of seeds in JA significantly increased the contents of these pigments. The application of JA increased the total chlorophyll, chlorophyll a, and chlorophyll b

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Fig. 2. Effect of JA on Pb uptake in roots (mg g−1 DW) and shoots (mg g−1) of 30, 45 and 60 days old tomato plants grown in Pb amended soil (mean ± SD, two-way ANOVA, Tukey's HSD and β-coefficients). r, correlation coefficient. *,** and *** indicates significant at p ≤ 0.05, p ≤ 0.01 and p ≤ 0.001 respectively.

contents in the Pb-treated plants. The contents of total chlorophyll, chlorophyll a, and chlorophyll b were significantly increased in the 30, 45-, and 60-days-old plants treated with 100 nM JA. However, the increase was maximum in the 30-days-old plants compared to that in the 45- and 60-days-old plants. The total chlorophyll content was increased by 65.92% (30 days), 57.94% (45 days), and 43.77% (60 days) in the 0.75 mM Pb-treated plants compared to that in the plants treated only with Pb (0.75 mM) (Table 1). In the 0.75 mM Pb treatment, the carotenoid content was reduced by 45.37%, 57.66%, and 64.03% in the 30-, 45-, and 60-days-old plants, respectively. The seeds primed with 100 nm JA showed significant increase in the carotenoid content (42.86%, 24.14%, and 23.85%) in the 0.75 mM Pb-treated plants on the

respective days as compared to the increase in the plants treated only with 0.75 mM Pb (Table 1). Negative β-regression coefficients revealed that Pb decreased the contents of chlorophyll and carotenoids. There was a positive interaction between Pb x JA, which showed that JA enhanced the contents of chlorophyll and carotenoids in tomato plants (Table 1). The contents of flavonoid, anthocyanin, and xanthophyll were increased with the increasing concentration of Pb. The flavonoid content was increased by 24.83%, 29.04% and 46.06% in the 30-, 45-, and 60days-old plants, respectively. The anthocyanin content was increased by 40%, 38.1%, and 50% during the different time intervals at the highest concentration of Pb in comparison to the content in the untreated plants

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Table 1 Effect of JA on the contents of chlorophyll and carotenoid of 30, 45 and 60 days old tomato plants grown in Pb amended soil (mean ± SD, two-way ANOVA, Tukey's HSD). Pb (mM) 30 days 0 0 0 0 0.25 0.25 0.25 0.25 0.50 0.50 0.50 0.50 0.75 0.75 0.75 0.75 F-ratio

β Coefficients

45 days 0 0 0 0 0.25 0.25 0.25 0.25 0.50 0.50 0.50 0.50 0.75 0.75 0.75 0.75 F-ratio

β Coefficients

60 days 0 0 0 0 0.25 0.25 0.25 0.25 0.50 0.50 0.50 0.50 0.75 0.75 0.75 0.75 F-ratio

JA (nM)

Chl-a (mg g−1FW)

Chl-b (mg g−1FW)

Total Chl (mg g−1FW)

Carotenoid (mg g−1FW)

0 0.01 1 100 0 0.01 1 100 0 0.01 1 100 0 0.01 1 100 FPb FJA FPb×JA HSD βPb βJA βPb×JA r

0.319 ± 0.007 0.279 ± 0.005 0.305 ± 0.008 0.326 ± 0.015 0.216 ± 0.004 0.263 ± 0.007 0.282 ± 0.004 0.303 ± 0.012 0.151 ± 0.006 0.2 ± 0.005 0.225 ± 0.006 0.236 ± 0.005 0.132 ± 0.006 0.166 ± 0.005 0.199 ± 0.006 0.221 ± 0.003 841.8⁎⁎ 213.5⁎⁎ 26.9⁎⁎

0.106 ± 0.0023 0.099 ± 0.0016 0.112 ± 0.003 0.108 ± 0.005 0.077 ± 0.0014 0.087 ± 0.0021 0.096 ± 0.0012 0.108 ± 0.0041 0.055 ± 0.002 0.073 ± 0.0018 0.074 ± 0.0019 0.078 ± 0.0016 0.044 ± 0.0018 0.056 ± 0.0017 0.071 ± 0.0019 0.074 ± 0.002 816.4⁎⁎ 188.3⁎⁎ 20.5⁎⁎

0.425 ± 0.0093 0.378 ± 0.0061 0.417 ± 0.0112 0.434 ± 0.0201 0.293 ± 0.0054 0.350 ± 0.0086 0.377 ± 0.0047 0.411 ± 0.0157 0.206 ± 0.0075 0.273 ± 0.0068 0.299 ± 0.0078 0.314 ± 0.0066 0.176 ± 0.0074 0.222 ± 0.0067 0.270 ± 0.0074 0.292 ± 0.0081 823.2⁎⁎ 198.6⁎⁎ 22.7⁎⁎

0.205 ± 0.005 0.170 ± 0.004 0.197 ± 0.003 0.213 ± 0.013 0.160 ± 0.008 0.192 ± 0.003 0.206 ± 0.003 0.225 ± 0.005 0.133 ± 0.008 0.183 ± 0.004 0.199 ± 0.003 0.206 ± 0.003 0.112 ± 0.009 0.135 ± 0.002 0.145 ± 0.003 0.160 ± 0.007 264.8⁎⁎ 154.4⁎⁎ 24.9⁎⁎

0.019 −0.8918 0.2276 0.1266 0.9122⁎⁎⁎

0.007 −0.9142 0.1692 0.1424 0.9139⁎⁎⁎

0.028 −0.8992 0.2163 0.1211 0.9153⁎⁎⁎

0.016 −0.6611 0.4114 0.0182 0.7811⁎⁎⁎

0 0.01 1 100 0 0.01 1 100 0 0.01 1 100 0 0.01 1 100 FPb FJA FPb×JA HSD βPb βJA βPb×JA r

0.448 ± 0.008 0.388 ± 0.025 0.397 ± 0.023 0.414 ± 0.017 0.302 ± 0.009 0.358 ± 0.009 0.377 ± 0.009 0.395 ± 0.01 0.213 ± 0.009 0.322 ± 0.006 0.344 ± 0.008 0.376 ± 0.012 0.161 ± 0.01 0.199 ± 0.01 0.224 ± 0.008 0.251 ± 0.011 573.5⁎⁎ 83.9⁎⁎ 28.3⁎⁎

0.149 ± 0.0027 0.138 ± 0.0087 0.147 ± 0.0083 0.138 ± 0.0056 0.108 ± 0.0032 0.119 ± 0.0029 0.129 ± 0.0031 0.140 ± 0.0035 0.078 ± 0.0031 0.119 ± 0.0022 0.114 ± 0.0025 0.125 ± 0.004 0.053 ± 0.0031 0.068 ± 0.0032 0.079 ± 0.0026 0.086 ± 0.0038 552.9⁎⁎ 74.03⁎⁎ 20.4⁎⁎

0.597 ± 0.0111 0.526 ± 0.0332 0.544 ± 0.0309 0.552 ± 0.0227 0.410 ± 0.0123 0.478 ± 0.0117 0.506 ± 0.0124 0.536 ± 0.0134 0.292 ± 0.0118 0.441 ± 0.0082 0.459 ± 0.0103 0.501 ± 0.0161 0.214 ± 0.0126 0.267 ± 0.0127 0.303 ± 0.0101 0.338 ± 0.0149 567.3⁎⁎ 81.03⁎⁎ 25.17⁎⁎

0.274 ± 0.003 0.228 ± 0.009 0.245 ± 0.009 0.267 ± 0.003 0.165 ± 0.002 0.190 ± 0.005 0.211 ± 0.004 0.225 ± 0.005 0.144 ± 0.003 0.169 ± 0.004 0.212 ± 0.007 0.221 ± 0.006 0.116 ± 0.003 0.128 ± 0.003 0.138 ± 0.005 0.144 ± 0.002 1234.7⁎⁎ 174.9⁎⁎ 48.0⁎⁎

0.03 −0.9255 0.0963 0.1977 0.9016⁎⁎⁎

0.009 −0.9531 0.0335 0.2281 0.9109⁎⁎⁎

0.04 −0.9353 0.0802 0.2079 0.9063⁎⁎⁎

0.013 −0.8715 0.2538 0.0114 0.9066⁎⁎⁎

0 0.01 1 100 0 0.01 1 100 0 0.01 1 100 0 0.01 1 100 FPb FJA FPb×JA HSD

0.622 ± 0.015 0.577 ± 0.01 0.601 ± 0.012 0.628 ± 0.01 0.350 ± 0.008 0.473 ± 0.011 0.5 ± 0.012 0.526 ± 0.013 0.253 ± 0.01 0.384 ± 0.009 0.423 ± 0.009 0.451 ± 0.011 0.211 ± 0.009 0.227 ± 0.009 0.257 ± 0.01 0.3 ± 0.013 2360.1⁎⁎ 260.9⁎⁎ 51.01⁎⁎

0.207 ± 0.005 0.206 ± 0.0034 0.222 ± 0.0042 0.209 ± 0.0033 0.124 ± 0.0028 0.157 ± 0.0035 0.172 ± 0.0041 0.187 ± 0.0047 0.093 ± 0.0037 0.142 ± 0.0032 0.141 ± 0.0029 0.150 ± 0.0037 0.070 ± 0.0028 0.078 ± 0.0031 0.091 ± 0.0034 0.103 ± 0.0043 2336.1⁎⁎ 245.7⁎⁎ 34.7⁎⁎

0.830 ± 0.0200 0.783 ± 0.0129 0.823 ± 0.0157 0.837 ± 0.0133 0.475 ± 0.0107 0.630 ± 0.0141 0.672 ± 0.0163 0.713 ± 0.0181 0.346 ± 0.0139 0.526 ± 0.0121 0.564 ± 0.0116 0.601 ± 0.0148 0.281 ± 0.0113 0.305 ± 0.0122 0.348 ± 0.0129 0.404 ± 0.0170 2376.9⁎⁎ 257.9⁎⁎ 44.4⁎⁎

0.303 ± 0.008 0.229 ± 0.013 0.253 ± 0.012 0.269 ± 0.01 0.205 ± 0.007 0.234 ± 0.015 0.268 ± 0.008 0.285 ± 0.016 0.159 ± 0.005 0.168 ± 0.004 0.225 ± 0.007 0.240 ± 0.008 0.199 ± 0.008 0.122 ± 0.003 0.125 ± 0.005 0.135 ± 0.003 573.3⁎⁎ 59.8⁎⁎ 27.5⁎⁎

0.030

0.019

0.042

0.027 (continued on next page)

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Table 1 (continued) Pb (mM)

JA (nM)

Chl-a (mg g−1FW)

Chl-b (mg g−1FW)

Total Chl (mg g−1FW)

Carotenoid (mg g−1FW)

β Coefficients

βPb βJA βPb×JA r

−0.9445 0.1535 0.0819 0.9445⁎⁎⁎

−0.9659 0.0933 0.1171 0.9472⁎⁎⁎

−0.9531 0.1370 0.0931 0.9468⁎⁎⁎

−0.8769 0.1929 0.0481 0.8915⁎⁎⁎

r, correlation coefficient. ⁎⁎ Significant at p ≤ 0.01. ⁎⁎⁎ Significant at p ≤ 0.001.

(Fig. 3). The xanthophyll content was increased by 35.75%, 48.63%, and 49.3% in the 30-, 45-, and 60-days-old plants treated with 0.75 mM Pb. Application of JA further increased the contents of flavonoid, anthocyanin, and xanthophylls in the Pb-treated plants. The maximum increase was observed in the 30-days-old plants compared to the increase in the 45- and 60-days-old plants. The soaking of seeds in 100 nM JA increased the flavonoid content to 65.19%, 52.04%, and 40.6% in the 30-, 45-, and 60-days-old plants, respectively, treated with 0.75 mM Pb. The anthocyanin content was increased by 47.62%, 31.03%, and 30.56% and the xanthophyll content was increased by 45.15%, 37.42%, and 32.64%, respectively, in the 30-, 45-, and 60-days-old plants treated with 0.75 mM Pb (Fig. 3). Positive β- coefficients for interaction Pb x JA depicted that JA enhanced the contents of flavonoids, anthocyanins and xanthophylls in Pb treated plants (Fig. 3). 3.4. JA enhanced the gaseous exchange parameters in tomato plants under Pb stress The increasing concentration of Pb significantly lowered the gaseous exchange parameters (Pn, Ci, Gs and Et) in the 30-, 45-, and 60-days-old tomato plants (Table 2). Pb significantly reduced the gaseous exchange parameters. The maximum reduction in the Pn was observed in the 0.75 mM Pb treatment; it was about 55.24%, 60.85%, and 64.83% in the 30-, 45-, and 60-days-old plants, respectively (Table 2). Treatment with the highest concentration of Pb decreased the Gs by 47.63% (30 days), 59.55% (45 days), and 65.88% (60 days). Similarly, the Ci was found to reduce by 58.22%, 63.29%, and 70.43% in the 30-, 45-, and 60-days-old plants, respectively, in the plants treated with 0.75 mM Pb, compared to that in the untreated plants (Table 2). Et rate was lowered in the 0.75 mM Pb treated plants by 52.36%, 54.08%, and 57.05% in the 30-, 45-, and 60-days-old plants. JA treatment improved the gaseous exchange parameters during all the stages of growth in the Pb-treated plants; however, the maximum increase was observed in the 30-days-old plants. The maximum increase in Pn was observed upon treatment with 100 nM JA; the increase was by 75.94%, 66.85%, and 56.47% in the 0.75 mM Pb-treated plants at 30, 45, and 60 days. The application of 100 nM JA increased Ci (74.46%, 60.46%, and 56.38%) in the 0.75 mM Pb-treated plants on the respective days. Et was increased to 57.94% (30 days), 49.57% (45 days), and 43.39% (60 days) in the 0.75 mM Pb-treated plants. The JA (100 nM) treatment enhanced Gs in 30 (43.14%), 45 (37.91%), and 60 days (32%) in the 0.75 mM Pb-treated plants (Table 2). Negative β-coefficients for Pb caused reduction in gaseous exchange parameters in tomato plants. Interaction between Pb x JA was positively regressed for gaseous exchange parameters in 30 and 45 days of tomato plants (Table 2).

between Pb x JA was negatively regressed which showed that JA decreased the level of H2O2 in tomato plants under Pb toxicity (Fig. 4). 3.6. JA enhanced the activities of non-enzymatic and enzymatic antioxidants of ascorbate-glutathione cycle under Pb stress in tomato plants Ascorbic acid and glutathione contents were significantly enhanced under Pb treatment during different growth phases. JA supplementation further elevated the contents of ascorbic acid and glutathione in Pb treated plants (Fig. 4). 100 nM JA significantly elevated the ascorbic acid content by 58.29%, 45.31% and 29.72% in 30, 45 and 60 days old Pb (0.75 mM) treated plants in comparison to plants treated with Pb alone. Treatment of JA (100 nM) significantly enhanced glutathione content by 45.22% after30 days, 32.48% after 45 days and 26.56% after 60 days in 0.75 mM Pb treated plants as compared to 0.75 mM Pb treatment alone. The activity of antioxidant enzymes viz., APOX, MDHAR, DHAR and GR activities were increased under Pb treatment. APOX activity was significantly enhanced by 58.89%, 60.35% and 51.66% after 30, 45 and 60 days respectively in 0.75 mM Pb treated plants over control plants. JA (100 nM) treatment further significantly enhanced the activity of APOX by 42.17% (30 days), 37.49% (45 days) and 30.89% (60 days) in 0.75 mM Pb treated plants as compared to 0.75 mM Pb treatment alone (Table 3). The MDHAR activity was significantly enhanced by 52.26%, 57.95% and 52.94% after 30, 45 and 60 days respectively in 0.75 mM Pb treated plants over control plants. Treatment of 100 nM JA further significantly enhanced the MDHAR activity by 52.1% in 30 days, 41.94% in 45 days and 37.08% in 60 days in 0.75 mM Pb treated plants in comparison of Pb (0.75 mM) treated plants alone (Table 3). Pb (0.75 mM) treated plants after 30, 45 and 60 days showed significant increase in DHAR activity by 48.86%, 46.14% and 45.76% respectively over control. Pb treated plants supplied with JA further enhanced the DHAR activity by 37.83% (30 days), 24.43% (45 days) and 19.17% in 0.75 mM Pb treated plants in contrast to 0.75 mM Pb alone treated plants (Table 3). GR activity was found to increase by 63.29% (30 days), 68.24% (45 days) and 71.47% (60 days) relative to control plants. Application of 100 nM JA to 30, 45 and 60 days old Pb (0.75 mM) treated plants, significantly elevated the activity of GR by 58.68%, 48.41% and 26.78% respectively as compared to 0.75 mM Pb treatment alone. Positive βregression coefficients showed that Pb enhanced the activity of ascorbate-glutathione cycle in tomato plants. Interaction between Pb x JA was positively regressed for ascorbate-glutathione cycle which indicated that JA enhanced the activity of ascorbate-glutathione cycle under Pb stress (Table 3).

3.5. JA decreased H2O2 content in tomato plants under Pb stress The H2O2 content increased with increasing concentration of Pb at 30, 45 and 60 days. Treatment of JA significantly reduced the content of H2O2 in Pb treated plants. However, application of JA (100 nM) decreased the H2O2 content by 40.48% (30 days), 39.46% (45 days) and 33.49% (60 days) in 0.75 mM Pb treated plants in comparison to 0.75 mM Pb only treatment (Fig. 4). Positive β-coefficients for Pb indicated that H2O2 content was increased in Pb treated plants. Interaction

3.7. JA increased the contents of osmolytes (total carbohydrates, proline, and glycine betaine) under Pb stress in tomato plants The contents of osmolytes were significantly increased in the Pbtreated plants. The JA treatment further significantly increased the osmolyte contents at different growth phases (30, 45, and 60 days) whereas the maximum increase was found in the 30-days-old plants under Pb stress (Fig. 5). The total carbohydrate content was increased

S. Bali et al. / Science of the Total Environment 645 (2018) 1344–1360 Fig. 3. Effect of JA on the contents of flavonoid, anthocyanin and xanthophyll of 30, 45 and 60 days old tomato plants grown in Pb amended soil (mean ± SD, two-way ANOVA, Tukey's HSD and β-coefficients). r, correlation coefficient. *,** and *** indicates significant at p ≤ 0.05, p ≤ 0.01 and p ≤ 0.001 respectively. 1351

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Table 2 Effect of JA on gaseous exchange parameters of 30, 45 and 60 days old tomato plants grown in Pb amended soil (mean ± SD, two-way ANOVA, Tukey's HSD). Pb (mM) 30 days 0 0 0 0 0.25 0.25 0.25 0.25 0.50 0.50 0.50 0.50 0.75 0.75 0.75 0.75 F-ratio

β Coefficients

45 days 0 0 0 0 0.25 0.25 0.25 0.25 0.50 0.50 0.50 0.50 0.75 0.75 0.75 0.75 F-ratio

β Coefficients

60 days 0 0 0 0 0.25 0.25 0.25 0.25 0.50 0.50 0.50 0.50 0.75 0.75 0.75 0.75 F-ratio

JA (nM)

Net photosynthetic rate (Pn: μmole CO2 m−2S−1)

Stomatal conductance (Gs: mmole H2O m−2S−1)

Intercellular CO2 (Ci; μmole CO2 mole−1)

Transpiration rate (Et, mmole H2O m−2 S−1)

0 0.01 1 100 0 0.01 1 100 0 0.01 1 100 0 0.01 1 100 FPb FJA FPb×JA HSD βPb βJA βPb×JA r

16.71 ± 0.944 13.71 ± 0.735 15.69 ± 1.027 18.11 ± 0.715 10.09 ± 0.935 11.6 ± 0.377 12.62 ± 0.463 14.54 ± 0.681 8.54 ± 0.463 10.06 ± 0.586 11.41 ± 0.406 14.04 ± 0.638 7.48 ± 0.457 8.38 ± 0.561 8.83 ± 0.930 13.16 ± 0.525 203.1⁎⁎ 97.9⁎⁎ 7.40⁎⁎

0.779 ± 0.021 0.632 ± 0.02 0.716 ± 0.018 0.744v0.021 0.529 ± 0.03 0.608 ± 0.013 0.657 ± 0.033 0.715 ± 0.018 0.447 ± 0.032 0.480 ± 0.021 0.563 ± 0.037 0.663 ± 0.032 0.408 ± 0.016 0.514 ± 0.016 0.530 ± 0.016 0.584 ± 0.017 192.2⁎⁎ 81.58⁎⁎ 17.53⁎⁎

275.5 ± 6.09 248 ± 21.9 256.3 ± 7.55 264.3 ± 14.35 180.7 ± 8.95 212.8 ± 15.94 247.4 ± 10.13 266 ± 10.83 149.1 ± 6.08 184.8 ± 7.07 214.8 ± 13.9 255.4 ± 9.45 115.1 ± 11.04 146.9 ± 8.95 174.4 ± 7.63 200.8 ± 11.53 168.1⁎⁎ 77.18⁎⁎ 11.96⁎⁎

11.23 ± 0.882 9.635 ± 0.154 10.36 ± 0.297 11.44 ± 0.455 8.439 ± 1.354 12.13 ± 0.294 12.87 ± 0.751 14.51 ± 0.502 7.687 ± 0.453 7.684 ± 0.605 8.263 ± 0.138 12.3 ± 0.559 5.359 ± 0.372 6.027 ± 0.156 6.911 ± 0.447 8.450 ± 0.584 184.8⁎⁎ 80.58⁎⁎ 14.35⁎⁎

2.072 −0.8318 0.3641 0.2097 0.9322⁎⁎⁎

0.067 −0.8166 0.2776 0.1983 0.8716⁎⁎⁎

34.71 −0.8711 0.1878 0.3088 0.8941⁎⁎⁎

1.76 −0.7081 0.3461 0.1689 0.8119⁎⁎⁎

0 0.01 1 100 0 0.01 1 100 0 0.01 1 100 0 0.01 1 100 FPb FJA FPb×JA HSD βPb βJA βPb×JA r

37.91 ± 2.683 31.47 ± 1.221 32 ± 2.381 38.91 ± 3.621 28.56 ± 1.289 30.17 ± 1.299 32.93 ± 1.863 34.93 ± 1.831 16.77 ± 0.984 24.49 ± 1.953 31 ± 1.845 34.19 ± 1.675 14.84 ± 1.064 16.76 ± 1.278 21.76 ± 1.093 24.76 ± 0.950 163.6⁎⁎ 55.1⁎⁎ 12.4⁎⁎

1.063 ± 0.043 0.892 ± 0.034 0.933 ± 0.031 1.07 ± 0.071 0.714 ± 0.037 0.754 ± 0.03 0.857 ± 0.035 0.933 ± 0.031 0.545 ± 0.049 0.658 ± 0.024 0.748 ± 0.04 0.856 ± 0.022 0.430 ± 0.033 0.478 ± 0.023 0.560 ± 0.024 0.593 ± 0.018 362.9⁎⁎ 61.3⁎⁎ 10.9⁎⁎

317.6 ± 7.74 276.9 ± 5.33 289 ± 7.09 325.9 ± 9.37 227.9 ± 6.029 254 ± 7.917 289.2 ± 5.363 327 ± 12.49 186.5 ± 4.57 214.4 ± 12.78 250.8 ± 7.76 282.6 ± 6.51 116.6 ± 9.15 145.3 ± 7.71 168.1 ± 6.31 187.1 ± 7.87 64.9⁎⁎ 172.5⁎⁎ 20.4⁎⁎

14.04 ± 0.498 11.3 ± 0.392 12.4 ± 0.548 13.89 ± 0.291 11.19 ± 0.311 12.95 ± 0.279 14.32 ± 0.398 15.76 ± 0.272 9.077 ± 0.135 11.29 ± 0.392 12.33 ± 0.413 13.46 ± 0.501 6.447 ± 0.432 7.403 ± 0.521 8.621 ± 0.596 9.643 ± 0.419 418.1⁎⁎ 124.5⁎⁎ 22.6⁎⁎

5.53 −0.8342 0.2924 0.1359 0.8861⁎⁎⁎

0.109 −0.8934 0.3094 0.0181 0.9445⁎⁎⁎

24.45 −0.8654 0.3248 0.0453 0.9235⁎⁎⁎

1.26 −0.7404 0.3305 0.0676 0.8141⁎⁎⁎

0 0.01 1 100 0 0.01 1 100 0 0.01 1 100 0 0.01 1 100 FPb FJA FPb×JA HSD

46.37 ± 1.78 37.53 ± 1.97 42.62 ± 0.71 49.4 ± 4.39 36.41 ± 1.15 39.74 ± 1.83 43.38 ± 1.31 46.64 ± 1.89 18.62 ± 1.15 21.86 ± 1.03 25.83 ± 1.11 29.15 ± 1.48 16.31 ± 0.81 21.7 ± 1.17 23.77 ± 1.12 25.52 ± 0.521 554.3⁎⁎ 59.4⁎⁎ 8.7⁎⁎

2.849 ± 0.157 2.832 ± 0.140 3.163 ± 0.167 3.289 ± 0.126 1.639 ± 0.231 1.972 ± 0.046 2.074 ± 0.197 2.097 ± 0.097 1.081 ± 0.063 1.24 ± 0.145 1.3 ± 0.183 1.554 ± 0.077 0.972 ± 0.023 1.118 ± 0.071 1.156 ± 0.059 1.283 ± 0.043 536.2⁎⁎ 23.3⁎⁎ 1.331 0.383

341.9 ± 7.22 312.8 ± 6.81 334.4 ± 12.57 365.9 ± 8.74 205.5 ± 5.62 223.7 ± 5.45 240.5 ± 4.53 255.3 ± 5.56 137.1 ± 7.62 156.1 ± 8.31 172.6 ± 4.87 186.7 ± 5.48 101.1 ± 10.82 119.5 ± 7.01 145.8 ± 4.82 158.1 ± 6.45 1887.5⁎⁎ 93.6⁎⁎ 7.14⁎⁎

35.41 ± 0.598 30.48 ± 1.94 31.82 ± 2.38 34.59 ± 1.93 22.7 ± 2.04 25.07 ± 1.25 26.94 ± 0.894 34.95 ± 2.04 18.46 ± 0.891 20.62 ± 0.627 22.62 ± 0.999 26.7 ± 0.327 15.21 ± 0.506 13.89 ± 0.511 16.24 ± 1.46 21.81 ± 2.95 247.6⁎⁎ 52.3⁎⁎ 7.03⁎⁎

22.18

4.66

5.15

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Table 2 (continued) Pb (mM)

JA (nM)

Net photosynthetic rate (Pn: μmole CO2 m−2S−1)

Stomatal conductance (Gs: mmole H2O m−2S−1)

Intercellular CO2 (Ci; μmole CO2 mole−1)

Transpiration rate (Et, mmole H2O m−2 S−1)

β Coefficients

βPb βJA βPb×JA r

−0.8563 0.3021 −0.0521 0.9127⁎⁎⁎

−0.9404 0.1791 −0.0315 0.9404⁎⁎⁎

−0.9428 0.1808 −0.0005 0.9601⁎⁎⁎

−0.9200 0.2956 0.1268 0.9641⁎⁎⁎

⁎⁎ Significant at p ≤ 0.01. ⁎⁎⁎ Significant at p ≤ 0.001.

with the increasing concentration of Pb. The maximum increase observed in the 0.75 mM Pb-treated plants was by 61.17% (30 days), 59.66% (45 days), and 56.34% (60 days) compared to that in the untreated plants (Fig. 5). The application of 100 nM JA increased the total carbohydrate content by 47.47%, 36.79%, and 31.83% in the 30-, 45-, and 60-days-old Pb-treated plants (Fig. 5). Pb treatment significantly increased the proline content in the 30-, 45-, and 60-days-old tomato plants. The increase was by 41.83%, 49.6%, and 57.83% in the 0.75 mM Pb-treated plants compared to that in the untreated plants at 30, 45, and 60 days of growth. The application of JA further increased the proline content. The JA (100 nM) increased the proline content by 60.97%, 52.81%, and 33.89% in the 0.75 mM Pb-treated plants at 30, 45, and 60 days, respectively. The increasing concentration of Pb significantly increased the content of glycine betaine. At 0.75 mM Pb concentration, there was an increase of 51.85%, 54.32%, and 58.99% in the glycine betaine content in comparison the content in the control at 30, 45, and 60 days, respectively (Fig. 5). The application of JA (100 nM) increased the glycine betaine content by 61.12% (30 days), 49.27% (45 days), and 38.71% (60 days) in the 0.75 mM Pb-treated plants (Fig. 5). Positive β-coefficients showed that Pb enhanced the contents of osmolytes in tomato plants. Significant positive interaction Pb x JA was observed in glycine betaine content in 30, 45 and 60 days old tomato plants (Fig. 5).

3.8. JA enhanced the contents of metal chelating compounds (total thiols, non-protein thiols, and protein thiols) in tomato plants under Pb stress The contents of total thiols, non-protein thiols, and protein thiols were significantly increased under Pb stress; total thiols content was increased by 19.77%, 15.3%, and 16.43% in the 0.75 mM Pb-treated plants, compared to that in the untreated plants, at 30, 45, and 60 days of growth (Table 4). The content of non-protein thiols was increased by 47.92%, 26.79%, and 18.07% in the 0.75 mM Pb-treated plants, compared to that in the untreated plants, at 30, 45, and 60 days of growth. The content of protein thiols as increased by 17.19% (30 days), 14.16% (45 days), and 16.12% (60 days) in the 0.75 mM Pb-treated plants, compared to that in the untreated plants (Table 4). JA treatment further increased the contents of total thiols, nonprotein thiols, and protein thiols. At 100 nM, JA increased the content of total thiols by 34.29%, 26.4%, and 24.29% in the 30-, 45-, and 60days-old plants, compared to the respective contents in the plants treated only with 0.75 mM Pb. The soaking of seeds in 100 nM JA increased the content of non-protein thiols by 30.99%, 26.76%, and 13.27% at 30, 45, and 60 days in 0.75 mM Pb-treated plants, compared to the respective contents in the plants treated only with 0.75 mM Pb (Table 4). The JA (100 nM) treatment enhanced the content of protein thiols by 34.7%, 26.2%, and 25.64% in the 0.75 mM Pb-treated plants at 30, 45, and 60 days of growth, compared to the respective contents in the plants treated only with 0.75 mM Pb (Table 4). Positive βcoefficients for Pb showed that metal chelating compounds were enhanced under Pb toxicity. Interaction between Pb x JA for metal chelating compounds was positively regressed in 30 and 45 days old tomato plants.

4. Discussion Pb enters in plants through root system. After sequestration in root cells little amount of Pb is transported from the roots to the shoots. Probst et al., 2009 observed that high concentration of Pb in the roots as compared to leaves and shoots of Vicia faba. Plant cell wall contains pectins and Pb may form complexes with carboxyl group of pectins and this phenomenon is considered as corner stone of plant cells which provide resistance to Pb toxicity. It may act as natural barrier and inhibit the movement of metal ions by plasma membrane (Eun et al., 2000). As Pb uptake increases, it may enter into symplastic pathway through water transport system and cause adverse effects on plants (Pourrut et al., 2011). The toxic effects induced serious morphological and physiological alterations such as decrease in seed germination, suppression of root growth, blackening of root tips and chlorosis. It also altered photosynthesis, nutrient uptake, water balance and activity of essential enzymes (Dogan et al., 2018). The inhibition of photosynthesis might be due to the interaction of Pb with the\\SH groups of enzymes involved in the biosynthesis of chlorophyll, hampered uptake of essential elements, like Mn and Fe, or replacement of divalent cations with Pb (Chatterjee et al., 2004; Cenkci et al., 2010). Being sessile organisms, plants impose to synthesize an enormous armory of chemical compounds to acclimatize and counter the environmental challenges. Plant hormones play a significant role in various physiological processes and complex signaling pathways in plants under diverse abiotic and biotic stresses. JA is recognized as a potential phytohormone which regulates immune and growth responses to enhance survival of plants under stressful environment. It plays an essential role in plants against various abiotic stresses like drought, salt, heavy metals, osmotic, heat and cold (Wasternack, 2014; Dar et al., 2015; Santino et al., 2013; Sharma and Laxmi, 2016). Interplay between jasmonoyl-L-isoleucine and CORONATINE INSENSITIVE receptor leads to proteolysis of JASMONATE ZIM-DOMAIN [JAZ proteins (transcriptional repressor)] and degrades JAZs proteins that eventually activate MYC2 transcription factors that further mediate JA responses by monitoring the expression of JA responsive genes which play crucial role in regulating physiological and defense responses in plants (Chini et al., 2007; Thines et al., 2007; Yan et al., 2009). JAZ-MYC (transcriptional modules) attributed to synthesis of various secondary metabolites, terpenoids, alkaloids and phenylpropanoids in plants (Goossens et al., 2017). These metabolites may provide protection to photosynthetic pigments under stress conditions. In the present study, the shoot and root lengths were decreased under Pb stress. The pre-treatment with JA improved the shoot and root lengths of stressed plants. Maximum improvement in growth was observed during the vegetative phase of 30 days old plants. Our results are in agreement with those of Aftab et al. (2010) who reported that shoot length was increased with the supplementation of 300 μM MeJA in boron treated Artemisia annua L. plants. Farooq et al. (2018) reported that treatment of MeJA improved root growth in arsenic stressed Brassica napus plants. JA may improve the uptake of essential minerals and photosynthetic efficiency of plants under Pb stress. It has been reported that Pb caused mitochondrial swelling, vacuolization of dictyosomes and endoplasmic reticulum, loss of cristae and distorted plasma membrane in Allium sativum roots (Jiang and Liu, 2010). JA may improve the functioning of these organelles under stress conditions.

1354 S. Bali et al. / Science of the Total Environment 645 (2018) 1344–1360 Fig. 4. Effect of JA on H2O2, ascorbic acid and glutathione contents of 30, 45 and 60 days old tomato plants grown in Pb amended soil (mean ± SD, two-way ANOVA, Tukey's HSD and β-coefficients). r, correlation coefficient. *, ** and *** indicates significant at p ≤ 0.05, p ≤ 0.01 and p ≤ 0.001 respectively.

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Table 3 Effect of JA on the activities of APOX, MDHAR, DHAR and GR of 30, 45 and 60 days old tomato plants grown in Pb amended soil (mean ± SD, two-way ANOVA, Tukey's HSD and βcoefficients). Pb (mM) 30 days 0 0 0 0 0.25 0.25 0.25 0.25 0.50 0.50 0.50 0.50 0.75 0.75 0.75 0.75 F-ratio

β Coefficients

45 days 0 0 0 0 0.25 0.25 0.25 0.25 0.50 0.50 0.50 0.50 0.75 0.75 0.75 0.75 F-ratio

β Coefficients

60 days 0 0 0 0 0.25 0.25 0.25 0.25 0.50 0.50 0.50 0.50 0.75 0.75 0.75 0.75 F-ratio

JA (nM)

APOX activity (UA min−1 mg−1 protein)

MDHAR activity (UA min−1 mg−1 protein)

DHAR activity (UA min−1 mg−1 protein)

GR activity (UA min−1 mg−1 protein)

0 0.01 1 100 0 0.01 1 100 0 0.01 1 100 0 0.01 1 100 FPb FJA FPb×JA HSD βPb βJA βPb×JA r

18.12 ± 1.157 14.68 ± 0.341 18.51 ± 1.559 20.06 ± 1.285 22.04 ± 3.242 25.44 ± 0.601 27.97 ± 1.264 31.2 ± 1.961 25.41 ± 2.196 27.94 ± 2.945 30.32 ± 2.952 38.14 ± 0.934 28.79 ± 1.314 32.99 ± 0.465 38.04 ± 3.791 40.93 ± 2.406 154.4⁎⁎ 45.43⁎⁎ 3.81⁎⁎

21.87 ± 1.3 16.22 ± 3.1 19.63 ± 2.494 21.97 ± 2.361 24.13 ± 3.337 28.25 ± 0.815 30.37 ± 1.219 34.52 ± 1.026 28.97 ± 2.613 31.99 ± 3.14 38.12 ± 2.064 41.4 ± 3.595 33.3 ± 2.161 38.42 ± 2.895 47.43 ± 1.709 50.65 ± 1.76 192.4⁎⁎ 45.81⁎⁎ 6.34⁎⁎

28.61 ± 1.181 23.05 ± 2.716 27.4 ± 0.975 28.73 ± 1.875 36.52 ± 2.06 41.74 ± 4.001 48.64 ± 3.277 49.05 ± 0.895 39.11 ± 1.446 44.91 ± 1.995 49.51 ± 2.155 52.09 ± 2.068 42.59 ± 2.204 51.64 ± 1.519 58.09 ± 1.016 58.7 ± 2.066 321.8⁎⁎ 63.07⁎⁎ 8.062⁎⁎

11.96 ± 0.529 9.437 ± 1.159 11.38 ± 1.577 12.74 ± 1.173 12.6 ± 1.185 15.3 ± 1.864 18.74 ± 1.452 19.13 ± 1.005 15.76 ± 1.381 17.48 ± 1.598 21.7 ± 2.058 25.1 ± 1.714 19.53 ± 2.086 21.61 ± 2.183 25.52 ± 1.38 30.99 ± 2.529 137.5⁎⁎ 46.78⁎⁎ 4.96⁎⁎

6.201 0.7688 0.2329 0.2126 0.9345⁎⁎⁎

7.201 0.7826 0.1518 0.2375 0.9323⁎⁎⁎

6.448 0.7827 0.1435 0.1412 0.8700⁎⁎⁎

4.939 0.7101 0.1238 0.3607 0.9415⁎⁎⁎

0 0.01 1 100 0 0.01 1 100 0 0.01 1 100 0 0.01 1 100 FPb FJA FPb×JA HSD βPb βJA βPb×JA r

21.79 ± 2.031 15.31 ± 2.376 18.88 ± 2.026 22.03 ± 1.983 25.45 ± 1.812 28.56 ± 2.283 28.48 ± 2.743 35.59 ± 1.03 29.81 ± 2.601 31.94 ± 1.49 38.73 ± 2.487 39.57 ± 3.744 34.94 ± 2.066 39.81 ± 3.421 46.04 ± 2.469 48.04 ± 3.346 166.8⁎⁎ 26.97⁎⁎ 4.33⁎⁎

26.42 ± 1.245 22.81 ± 1.285 25.37 ± 1.532 26.89 ± 3.706 32.79 ± 1.015 37.42 ± 1.733 38.59 ± 1.234 44.54 ± 1.436 38.28 ± 2.135 47.29 ± 1.038 49.35 ± 1.206 51.88 ± 2.121 41.73 ± 3.138 43.62 ± 2.902 52.99 ± 1.32 59.23 ± 2.095 355.9⁎⁎ 67.26⁎⁎ 10.3⁎⁎

33.14 ± 2.258 28.03 ± 0.562 28.52 ± 0.198 33.58 ± 0.902 35.73 ± 1.123 39.73 ± 1.898 43.6 ± 1.543 47.38 ± 1.61 39.33 ± 2.234 45.74 ± 1.841 50.49 ± 1.51 53 ± 0.887 48.43 ± 1.229 50.03 ± 1.365 55.25 ± 2.377 60.26 ± 3.291 374.2⁎⁎ 70.13⁎⁎ 9.9⁎⁎

13.79 ± 0.982 11.6 ± 1.46 16.37 ± 0.691 16.22 ± 1.084 16.3 ± 1.187 18.29 ± 1.076 19 ± 0.899 22.44 ± 1.178 21.74 ± 2.764 20.4 ± 1.616 25.5 ± 1.625 31.74 ± 1.53 23.2 ± 2.223 24.51 ± 2.189 28.95 ± 1.866 34.43 ± 1.687 165.9⁎⁎ 60.24⁎⁎ 4.6⁎⁎

7.776 0.8564 0.2180 0.1067 0.9424⁎⁎⁎

6.006 0.7646 0.1146 0.2614 0.9185⁎⁎⁎

5.215 0.8278 0.2141 0.1499 0.9403⁎⁎⁎

4.838 0.7132 0.1824 0.3384 0.9536⁎⁎⁎

0 0.01 1 100 0 0.01 1 100 0 0.01 1 100 0 0.01 1 100 FPb FJA FPb×JA HSD

27.43 ± 2.604 23.88 ± 3.731 24.17 ± 3.798 28.72 ± 3.308 31.91 ± 2.2 36.7 ± 2.499 40.18 ± 3.905 42.96 ± 1.783 36.31 ± 1.152 39.37 ± 4.79 43.32 ± 2.036 48.04 ± 1.681 41.6 ± 2.482 42.31 ± 1.056 49.42 ± 0.974 54.45 ± 2.271 126.1⁎⁎ 27.55⁎⁎

30.98 ± 2.276 23.69 ± 0.651 24.84 ± 2.931 26.93 ± 4.57 31.81 ± 1.452 33.29 ± 1.763 38.12 ± 1.723 41.65 ± 1.192 44.83 ± 2.685 52.48 ± 1.295 56.65 ± 1.126 61.5 ± 1.647 47.38 ± 3.498 55.73 ± 3.465 64.66 ± 2.574 64.95 ± 3.453 421.5⁎⁎ 39.18⁎⁎ 10.9⁎⁎

36.69 ± 2.348 29.39 ± 2.051 34.86 ± 2.444 35.33 ± 2.558 40.77 ± 1.079 41.54 ± 1.764 43.14 ± 1.103 45.23 ± 1.106 44.49 ± 3.301 48.22 ± 2.736 52.64 ± 1.2 53.78 ± 1.898 53.48 ± 3.042 55.33 ± 1.028 60.34 ± 2.199 63.73 ± 3.235 260.1⁎⁎ 20.94⁎⁎ 4.15⁎⁎

19.49 ± 1.176 17.28 ± 0.926 16.17 ± 1.36 19.2 ± 2.206 23.1 ± 2.129 25.79 ± 1.66 28.39 ± 1.295 31.87 ± 2.235 30.92 ± 2.064 34.02 ± 1.492 35.73 ± 1.286 41.14 ± 2.431 33.42 ± 2.214 31.27 ± 1.311 35.32 ± 2.559 42.37 ± 2.814 229.5⁎⁎ 33.46⁎⁎ 4.8⁎⁎

7.587

6.689

5.771

3.022 8.305

(continued on next page)

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Table 3 (continued) Pb (mM)

JA (nM)

APOX activity (UA min−1 mg−1 protein)

MDHAR activity (UA min−1 mg−1 protein)

DHAR activity (UA min−1 mg−1 protein)

GR activity (UA min−1 mg−1 protein)

β Coefficients

βPb βJA βPb×JA r

0.7817 0.1986 0.2086 0.9298⁎⁎⁎

0.8436 0.0745 0.1799 0.9328⁎⁎⁎

0.8836 0.0741 0.1729 0.9677⁎⁎⁎

0.7716 0.1354 0.2601 0.9312⁎⁎⁎

r, correlation coefficient. ⁎⁎ Significant at p ≤ 0.01. ⁎⁎⁎ Significant at p ≤ 0.001.

The Pb uptake was enhanced with its increasing concentrations and maximum accumulation was observed in the roots of tomato plants during the different stages of growth (30, 45, and 60 days). JA treatment lowered the Pb uptake in the 30-, 45-, and 60-days-old tomato plants. Maximum reduction in Pb uptake was observed in the 30-days-old plants. Ahmad et al. (2017) reported that JA reduced the Cd uptake in shoots by 60.58% and in roots by 57.46% as compared to the uptake in Vicia faba L. plants treated only with Cd. The JA (0.1 μM) treatment restricted the Pb uptake and improved the growth in Wolffia arrhiza L. under Pb stress (Piotrowska et al., 2009). JA may act as signaling molecule to upregulate the activity of phytochelatin biosynthetic pathway or may reduce the expression of heavy metal transporter proteins which may reduce the uptake of metal in plants. Maksymiec et al. (2007) reported that MeJA induced the accumulation of phytochelatins in Cu and Cd treated A. thaliana plants. In the present, the chlorophyll and carotenoid contents were lowered under Pb stress. Treatment of seeds with JA led to improvement in the contents of chlorophyll and carotenoid in the tomato plants under Pb stress. The 30 days-old-plants showed a higher increase than the 45- and 60-days-old tomato plants. Rezai et al. (2013) showed that application of MeJA significantly enhanced the chlorophyll content in pepper plants under salt stress. A study conducted by Piotrowska et al. (2009) showed that JA (0.1 μM) treatment restored the levels of carotenoids in W. arrhiza under Pb stress. MeJA may stimulate the transcript level of some crucial enzymes associated in chlorophyll synthesis by the generation of 5-aminolevulinic acid (Ueda and Saniewski, 2006). The xanthophyll, flavonoid, and anthocyanin contents were observed to increase under Pb stress in the present study. The soaking of seeds in JA further increased the contents of xanthophyll, flavonoid, and anthocyanin in Pb-stressed plants at the different growth phases. However, the vegetative phase of tomato plants showed maximum increase in the contents of these pigments. Xanthophylls provide protection against oxidative stress produced not only by high light intensity but also by other factors, such as ROS generation through various abiotic stresses (drought, heat, chilling, salinity, etc.). Anthocyanin accumulation provides tolerance in plants exposed to various abiotic stresses. They act as scavengers, stress signals, and photoprotectants. Flavonoids are secondary metabolites and are widely present in the plant kingdom. They decrease the generation of reactive oxygen species by interfering with the regulation of enzymes that are involved in the generation of ROS, repression of singlet oxygen, and reduction in free radical reaction during lipid peroxidation and help in the re-cycling of various antioxidants (Mierziak et al., 2014). The results obtained by Belhadj et al. (2008) showed that the application of MeJA/sucrose induced the regulation of phenylalanine ammonia lyase and chalcone synthase, which consequently lead to the accumulation of anthocyanins. It was reported by Smith and Banks (1986) that phenylalanine ammonia lyase and chalcone synthase are the two main enzymes of the phenylpropanoid biosynthesis pathway, which lead to the production of flavonoids, anthocyanins, and various secondary metabolites. In the present study, the gaseous exchange parameters (Pn, Gs, Ci, and Et) were decreased under Pb stress in tomato plants. The presowing treatment of seeds with JA enhanced the Pn, Gs, Ci and Et. Thirty-days-old tomato plants showed maximum improvement in the gaseous exchange parameters. Earlier studies on the application of

300 μM MeJA significantly improved the photosynthetic attributes in Artemisia annua L. under boron toxicity (Aftab et al., 2010). JA application improved the gaseous exchange parameters (Pn, Gs, Ci, and Et) in Cd exposed Brassica napus L. plants (Ali et al., 2018). JA may improve the contents of photosynthetic pigments by mediating the expression of key enzymes involved in the biosynthesis of these pigments which may consequently enhance the efficiency of gaseous exchange parameters. H2O2 content was increased and the activities of non-enzymatic and enzymatic antioxidants of ascorbate-glutathione cycle were enhanced under Pb stress during different growth stages of tomato plant (30, 45 and 60 days). JA treatment reduced the content of H2O2 and further enhanced the activity of ascorbate-glutathione cycle in Pb-stressed plants at 30, 45 and 60 days. Maximum improvement was observed in 30 days old plants. Treatment of MeJA reduced H2O2 content in rice seedlings under Cd stress (Singh and Shah, 2014). JA may activate the antioxidative defense system to quench free radicals that harm the membranes of cell organelles including chloroplast. MeJA increased the glutathione level in Cd-stressed rice seedlings as reported by Singh and Shah (2014). Enhancement in ascorbic acid content may be due to induction of MeJA responsive genes that are involved in ascorbic acid biosynthesis as studied in tobacco and Arabidopsis plants (Wolucka et al., 2005). A study conducted by Dar et al. (2015) reported that MeJA might be associated with signal transduction mechanisms which control the concentration of glutathione at various levels and increased its synthesis under heavy metal stress. In Agropyron cristatum, JA regulated the enzymes involved in ascorbate-glutathione cycle under abiotic stress (Shan and Liang, 2010). Application of JA enhanced the activities of APOX, MDHAR, DHAR and GR in wheat seedlings under drought stress (Shan et al., 2015). MeJA enhanced the transcript levels of APOX, DHAR and MDHAR in Brassica napus under arsenic stress (Farooq et al., 2016). It has been suggested by Santino et al. (2013) that enhancement in the activities these antioxidative enzymes might be attributed to de novo synthesis or modulation in the transcriptional or translational levels of defense related genes in response to MeJA treatment. In the present study, the contents of osmolytes (glycine betaine, proline, and total carbohydrate) were observed to increase under Pb stress and in the plants obtained from seeds that were pre-treated with JA, a further increase in the contents of osmolytes was observed during the different growth phases. The increase in the contents of the osmolytes under Pb stress was maximum during the vegetative phase in comparison to the other two stages of development. It has been reported that application of MeJA (0.01 μM) significantly enhanced the proline content in Cd-stressed Solanum nigrum plants (Yan et al., 2015). The exogenous application of JA significantly increased the glycine betaine content in pear leaves under drought stress by increasing the level of betaine aldehyde dehydrogenase (Gao et al., 2004). Jasmonic acid might regulate the expression of genes that are involved in the synthesis of sugars under stress conditions. The contents of metal chelating-compounds (total thiols, nonprotein, and protein bound thiols) in the present work were increased under Pb stress in the 30-, 45-, and 60-days-old plants. The JA treatment further increased the contents of metal-chelating compounds during the different growth phases under Pb stress. Enhancement in the total non-protein and protein-bound thiols might be due to the increased

S. Bali et al. / Science of the Total Environment 645 (2018) 1344–1360 1357

Fig. 5. Effect of JA on osmolytes (total carbohydrate, glycine betaine and proline) of 30, 45 and 60 days old tomato plants grown in Pb amended soil (mean ± SD, two-way ANOVA, Tukey's HSD and β-coefficients). r, correlation coefficient. *,** and *** indicates significant at p ≤ 0.05, p ≤ 0.01 and p ≤ 0.001 respectively.

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Table 4 Effect of JA on metal chelating compounds (Total thiols, non-proteins bound thiol and protein bound thiols) of 30, 45 and 60 days old tomato plants grown in Pb amended soil (mean ± SD, two-way ANOVA, Tukey's HSD and β-coefficients). Pb (mM) 30 days 0 0 0 0 0.25 0.25 0.25 0.25 0.50 0.50 0.50 0.50 0.75 0.75 0.75 0.75 F-ratio

β Coefficients

45 days 0 0 0 0 0.25 0.25 0.25 0.25 0.50 0.50 0.50 0.50 0.75 0.75 0.75 0.75 F-ratio

β Coefficients

60 days 0 0 0 0 0.25 0.25 0.25 0.25 0.50 0.50 0.50 0.50 0.75 0.75 0.75 0.75 F-ratio

JA (nM)

Total thiol content (mmol g−1 FW)

Non-protein bound thiol content (mmol g−1 FW)

Protein-bound thiol content (mmol g−1 FW)

0 0.01 1 100 0 0.01 1 100 0 0.01 1 100 0 0.01 1 100 FPb FJA FPb×JA HSD βPb βJA βPb×JA r

0.526 ± 0.127 0.545 ± 0.0151 0.551 ± 0.0026 0.570 ± 0.0051 0.587 ± 0.0061 0.610 ± 0.0115 0.659 ± 0.0161 0.698 ± 0.0126 0.607 ± 0.0076 0.641 ± 0.0103 0.657 ± 0.0149 0.687 ± 0.0087 0.630 ± 0.0052 0.730 ± 0.0069 0.772 ± 0.0128 0.846 ± 0.0104 673.7⁎⁎ 234.7⁎⁎ 26.47⁎⁎

0.048 ± 0.0031 0.049 ± 0.0013 0.054 ± 0.0013 0.058 ± 0.0016 0.056 ± 0.0016 0.063 ± 0.0013 0.069 ± 0.0022 0.076 ± 0.0021 0.065 ± 0.0022 0.075 ± 0.0016 0.081 ± 0.0013 0.088 ± 0.0016 0.071 ± 0.0016 0.078 ± 0.0018 0.085 ± 0.0023 0.093 ± 0.0022 623.004⁎⁎ 236.6⁎⁎ 6.432⁎⁎

0.477 ± 0.0135 0.495 ± 0.0159 0.497 ± 0.0034 0.511 ± 0.0047 0.531 ± 0.0046 0.546 ± 0.0129 0.591 ± 0.0138 0.621 ± 0.0146 0.541 ± 0.0054 0.564 ± 0.0087 0.575 ± 0.0162 0.599 ± 0.0074 0.559 ± 0.0057 0.651 ± 0.0080 0.686 ± 0.0105 0.753 ± 0.0127 473.9⁎⁎ 157.2⁎⁎ 23.47⁎⁎

0.0317 0.6965 0.1571 0.2979 0.8981⁎⁎⁎

0.0055 0.7773 0.2927 0.1444 0.9214⁎⁎⁎

0.0329 0.6672 0.1231 0.3277 0.8793⁎⁎⁎

0 0.01 1 100 0 0.01 1 100 0 0.01 1 100 0 0.01 1 100 FPb FJA FPb×JA HSD βPb βJA βPb×JA r

0.621 ± 0.008 0.594 ± 0.006 0.615 ± 0.01 0.635 ± 0.005 0.616 ± 0.015 0.656 ± 0.016 0.689 ± 0.009 0.773 ± 0.005 0.652 ± 0.013 0.715 ± 0.03 0.732 ± 0.027 0.802 ± 0.008 0.716 ± 0.004 0.813 ± 0.005 0.848 ± 0.008 0.905 ± 0.007 503.1⁎⁎ 195.1⁎⁎ 19.80⁎⁎

0.056 ± 0.002 0.053 ± 0.003 0.056 ± 0.002 0.061 ± 0.002 0.059 ± 0.001 0.065 ± 0.003 0.072 ± 0.0009 0.077 ± 0.002 0.067 ± 0.002 0.069 ± 0.001 0.076 ± 0.002 0.08 ± 0.001 0.071 ± 0.001 0.084 ± 0.002 0.084 ± 0.001 0.09 ± 0.001 503.6⁎⁎ 183.6⁎⁎ 10.15⁎⁎

0.565 ± 0.006 0.541 ± 0.0067 0.558 ± 0.0108 0.573 ± 0.0027 0.556 ± 0.0139 0.590 ± 0.0131 0.618 ± 0.0084 0.695 ± 0.0025 0.585 ± 0.0151 0.646 ± 0.0308 0.656 ± 0.025 0.721 ± 0.0089 0.645 ± 0.005 0.734 ± 0.0041 0.764 ± 0.0071 0.814 ± 0.0059 411.34⁎⁎ 159.86⁎⁎ 17.98⁎⁎

0.039 0.7277 0.2527 0.2341 0.9221⁎⁎⁎

0.004 0.7861 0.3049 0.1288 0.9241⁎⁎⁎

0.0383 0.7198 0.2425 0.2443 0.9179⁎⁎⁎

0 0.01 1 100 0 0.01 1 100 0 0.01 1 100 0 0.01 1 100 FPb FJA FPb×JA HSD

0.852 ± 0.0071 0.792 ± 0.0134 0.835 ± 0.0128 0.873 ± 0.0034 0.907 ± 0.0029 0.933 ± 0.0221 0.952 ± 0.0099 1.393 ± 0.0181 0.927 ± 0.0135 0.956 ± 0.0153 1.009 ± 0.0116 1.045 ± 0.0111 0.992 ± 0.0146 1.091 ± 0.0112 1.159 ± 0.0146 1.233 ± 0.0280 810.57⁎⁎ 536.91⁎⁎ 145.08⁎⁎

0.083 ± 0.0023 0.063 ± 0.0018 0.069 ± 0.0009 0.074 ± 0.0021 0.089 ± 0.0013 0.095 ± 0.0011 0.098 ± 0.0012 0.101 ± 0.0016 0.094 ± 0.0009 0.099 ± 0.0013 0.102 ± 0.0009 0.105 ± 0.0014 0.098 ± 0.0013 0.103 ± 0.0012 0.104 ± 0.0013 0.111 ± 0.0013 898.47⁎⁎ 49.12⁎⁎ 35.15⁎⁎

0.769 ± 0.0085 0.728 ± 0.0115 0.766 ± 0.0135 0.798 ± 0.0015 0.818 ± 0.0038 0.838 ± 0.0214 0.854 ± 0.0111 1.292 ± 0.0194 0.832 ± 0.0129 0.857 ± 0.0167 0.907 ± 0.0107 0.939 ± 0.0125 0.893 ± 0.0132 0.988 ± 0.0103 1.054 ± 0.0138 1.122 ± 0.0266 649.86⁎⁎ 506.41⁎⁎ 141.90⁎⁎

0.0439

0.0048

0.0436

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Table 4 (continued) Pb (mM)

JA (nM)

Total thiol content (mmol g−1 FW)

Non-protein bound thiol content (mmol g−1 FW)

Protein-bound thiol content (mmol g−1 FW)

β Coefficients

βPb βJA βPb×JA r

0.5807 0.5519 −0.0342 0.7753⁎⁎⁎

0.7862 0.0971 0.1296 0.8548⁎⁎⁎

0.5450 0.5769 −0.0471 0.7576⁎⁎

r, correlation coefficient. ⁎⁎ Significant at p ≤ 0.01. ⁎⁎⁎ Significant at p ≤ 0.001.

chelation of metal ions with sulfhydryl group-containing compounds (Kandziora-Ciupa et al., 2016). The increase in the levels of nonprotein thiols might be because of the stimulation of glutathione synthesis and increase in the contents of metal-binding peptides (phytochelatins), which have significant function in the homeostasis and sequestration of various metals (Maserti et al., 2005). The antioxidants such as proline, cysteine, ascorbic acid and non-protein thiols play an essential role in metal detoxification (Singh and Sinha, 2005). JA may act as signaling molecule for the production of metal chelating compounds under heavy metal stress. 5. Conclusions The JA treatment of seeds ameliorated the Pb toxicity by reducing the accumulation of Pb in the roots and shoots and also had positive effects on the growth, pigment content, gaseous exchange parameters, ascorbate-glutathione cycle, contents of osmolytes and metal chelating compounds in tomato plants during different phases of growth. The study further revealed that improvement in all the assessed parameters was significant at all the stages of growth but was maximum in the 30days-old plants in the vegetative phase, compared to that in the other stages, which prepared the plants to enter into the reproductive and post-reproductive development phase. Conflict of interest No conflicts of interest exist. Author contributions RB, PO and PA designed the experimental work. SB, PK and SKK performed the experimental work. LW and MNA carried out the statistical analysis. SB, PK, PO wrote the manuscript. RB and PA revised the manuscript and helped in discussion part. All the authors have read the manuscript. Acknowledgements The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding (Research group No. RG-1438-039). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2018.07.164. References Aftab, T., Khan, M.M.A., Idrees, M., Naeem, M., Moinuddin, Hashmi, N., 2010. Methyl jasmonate counteracts boron toxicity by preventing oxidative stress and regulating antioxidant enzyme activities and artemisinin biosynthesis in Artemisia annua L. Protoplasma 248, 601–612. Ahmad, P., Alyemeni, M.N., Wijaya, L., Alam, P., Ahanger, M.A., Alamri, S.A., 2017. Jasmonic acid alleviates negative impacts of cadmium stress by modifying osmolytes and antioxidants in faba bean (Vicia faba L.). Arch. Agron. Soil Sci. 63, 1889–1899.

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