In-situ localization and biochemical analysis of bio-molecules reveals Pb-stress amelioration in Brassica juncea L. by co-application of 24-Epibrassinolide and Salicylic Acid

Lead (Pb) toxicity is a major environmental concern affirming the need of proper mitigation strategies. In the present work, potential of combined treatment of 24-Epibrassinolide (24-EBL) and Salicylic acid (SA) against Pb toxicity to Brassica juncea L. seedlings were evaluated. Seedlings pre-imbibed in EBL (0.1 mM) and SA (1 mM) individually and in combination, were sown in Pb supplemented petri-plates (0.25, 0.50 and 0.75 mM). Various microscopic observations and biochemical analysis were made on 10 days old seedlings of B. juncea. The toxic effects of Pb were evident with enhancement in in-situ accumulation of Pb, hydrogen peroxide (H2O2), malondialdehyde (MDA), nuclear damage, membrane damage, cell death and polyamine. Furthermore, free amino acid were lowered in response to Pb toxicity. The levels of osmoprotectants including total carbohydrate, reducing sugars, trehalose, proline and glycine betaine were elevated in response to Pb treatment. Soaking treatment with combination of 24-EBL and SA led to effective amelioration of toxic effects of Pb. Reduction in Pb accumulation, reactive oxygen content (ROS), cellular damage and GSH levels were noticed in response to treatment with 24-EBL and SA individual and combined levels. The contents of free amino acid, amino acid profiling as well as in-situ localization of polyamine (spermidine) was recorded to be enhanced by co-application of 24-EBLand SA. Binary treatment of 24-EBL and SA, further elevated the content of osmoprotectants. The study revealed that co-application of combined treatment of 24-EBL and SA led to dimination of toxic effects of Pb in B. juncea seedlings.

pb Localization. The roots of B. juncea L. seedlings were stained using Pb specific flouroscent probe Leadmium TM Green AM dye (Invitogen, ThermoFisher Scientific). The roots were placed in Na 2 -EDTA (disodium ethylene diamine tetra acetic acid) (20 mM) for 15 minutes and kept at room temperature. The sections were then washed with double distilled water three times for 10 min each. The stock solution of probe was prepared by adding 50 µL dimethyl sulfoxide directly to the vial of the dye, followed by diluting it 1:10 with 0.85% NaCl (sodium chloride) 52 . The roots were then immersed in this diluted stock for 2 hr at 37 °C. It was followed by washing three times with 0.85% NaCl. The roots were then visualized under confocal laser scanning microscope (Nikon AIR). The observations were made at the excitation wavelength of 488 nm and emission wavelength of 590 nm.
Oxidative Damage. The oxidative damage was evaluated by estimating contents of superoxide anion, H 2 O 2 and MDA spectrophotopmetrically and by in-situ localization of H 2 O 2 , nuclear damage, membrane damage, glutathione (GSH), MDA and cell viability employing confocal laser scanning microscope and visible compound microscope. Superoxide anion content. The level of superoxide anion was determined by following the method of Wu, et al. 53 . The seedling extract was prepared in 50 mM potassium phosphate buffer (PPB) with pH 7.8 and supernatant was obtained by centrifugation at 13, 000 × g for 15 minute at 4 °C. To 0.5 ml of the supernatant 0.5 ml PPB, 0.1 ml of 10 mM hydroxylamine hydrochloride was added. The reaction mixture was incubated for 30 minutes at 25 °C, followed by addition of equal volumes of 7 mM 1-napthylamine and 58 mM of 3-aminobenzenesulphonic acid and was incubated for another 20 minutes. The absorbance of the reaction mixture was read at 530 nm. A standard equation was derived to calculate the content of superoxide anion and standard used was sodium nirite (NaNO 2 ). H 2 O 2 estimation. H 2 O 2 content was estimated by following method of Velikova, et al. 54 . The seedling extract was prepared by homogenizing 100 mg fresh plant samples in 1.5 ml of tri-chloroacetic acid (0.1%). The supernatant was obtained by centrifugation of extract at 12,000 × g at 4 °C for 15 minutes. To 0.4 ml of supernatant, 400 µL of MDA content. The level of MDA was determined by the protocol of Heath and Packer 55 . Fresh seedlings were homogenized in tri-chloroacetic acid (0.1%) and were centrifuged for 20 minutes at 4 °C at 13, 000 × g. To the supernatant, 0.5% thiobarbituric acid and tri-chloroacetic acid (20%) were added. The reaction mixture was then kept in water bath at 95 °C for about 95 °C for 30 minutes, followed by cooling the mixture on an ice bath. The absorbance of the reaction mixture was read at 532 nm and 600 nm and content was calculated using extinction coefficient i.e. 155/ mM/ cm. H 2 O 2 localization. The method given by Ortega-Villasante, et al. 56 was followed for localization of H 2 O 2 which was done with 2 1 -7 1 -dichloroflourcien diacetate and was incubated for 30 minutes. The roots were then washed three times for 5 minutes with double distilled water and were mounted on a glass slide. The excitation wavelength was 488 nm and emission wavelength was 530 nm respectively.
Visualization of membrane damage. For visualization of membrane damage, propidium iodide (PI) a fluorescent adduct was used. A 50 µM solution of PI was prepared according to the method of Gutierrez-Alcala, et al. 57 . The root samples were dipped in PI fluorescent probe for 15 minutes, followed by washing with double distilled water and were then mounted on glas slide. The excitation wavelength was 535 nm and emission wavelength was 617 nm respectively.
Nuclear damage. The nuclear damage in the root cells was examined by employing a fluorescent dye i.e. 4,6-diamino-2-phenylindole (DAPI) and was prepared following the method of Callard, et al. 58 . The dye was prepared by dissolving 0.1 gm of DAPI in 100 ml of phosphate buffer saline (PBS). The roots were mounted on glass slide and visualized at excitation wavelength of 358 nm and emission wavelength of 461 nm respectively.
MDA localization. MDA was visualized by employing schiff 's reagent (a visible dye) following the protocol of Wang and Yang 59 . The roots of 10 days old seedlings were excised and dipped in schiff 's reagent for 15 minutes and was followed by washing with 0.5% potassium metabisulphite prepared in 0.05 M HCl. The washed roots were mounted on glass slide and visualized under visible compound microscope.
Cell Viability. The method of Romero-Puertas, et al. 60 was used for analyzing cell viability of root cells. The non-viable cells were localized by 0.25% Evan's blue dye. The roots were dipped in visible probe and were kept at room temperature for 10 minutes, followed by washing with double distilled water. The prepared slides were visualized under visible compound microscope. Quantitative and Qualitative estimation of Amino Acids. The quantitative estimation of total free amino acids was done by using spectrophotometer, while qualitative profiling of different amino acids was done by employing Amino acid analyzer (Shimadzu, Nexera X 2 ).
Total free amino acid content. Total free amino acid content was estimated by following the method of Lee and Takahashi 62 . 100 mg of dried plant sample was extracted by dipping the plant material in 5 ml of 80% ethanol (extractant). The samples were then centrifuged for 20 minutes at 4 °C at 2000 × g, followed by addition of 3.8 ml of ninhydrin reagent to 0.2 ml of supernatant. The reaction mixture was boiled for 12 minutes in a water bath. After boiling, the sample were cooled at room temperature followed by reading the absorbance at 570 nm. The betaine hydrochloride was used as standard for preparation of standard curve.
Amino acid profiling. The samples for amino acid profiling were prepared by method of Iriti, et al. 63 with minor modifications. 1 gm of fresh seedlings was crushed in 5 ml of 80% methanol. The samples were then centrifuged at 10,000 × g at 4 °C for 20 minutes. To 1 ml of supernatant, 1 ml of sulphosalicylic acid (6%) was added followed by centrifugation at 10,000 × g at 4 °C for 20 minutes. The prepared samples were filtered through 0.22 µm syringe filter. The 1 µL of this sample was injected in the sample vials of amino acid analyzer and were quantified. polyamine (spermidine) localization. Synthesis of Probe. The fluorescent probe employed for localization of spermidine was synthesized by following already reported procedure 64 . Reaction mixture comprising of formyl bonic acid, potassium carbonate, Pd (0) in 60 ml of dioxane-H 2 O and tetrabromo 18-crown-6/tetrabromobennzene were kept in N 2 atmosphere for 24 hrs and was continuously stirred at 80 °C. After 24 hrs, when the reaction was complete, the mixture was removed under pressure, to procure a residue to which water was added. Extraction of the aqueous layer was carried out by using 20 ml chloroform and the process was repeated thrice. A solid residue was obtained from the chloroform fraction by washing the fraction with water followed by drying by using sodium sulphate and finally was distilled under low pressure. Solid residue was then subjected to column chromatography to obtain pure compound by using ethyl acetate as an effluent. The probe was then www.nature.com/scientificreports www.nature.com/scientificreports/ crystallized by employing ethanol. The synthesized compound was characterized by 1 H NMR, 13 C NMR and Mass spectroscopic studies.
Confocal imaging. A 5 mM concentration of probe (molecular weight-815.2) was prepared in dimethyl sulfoxide (DMSO). Localization of spermidine was done by following the method proposed by Singh et al. 65 with slight modification. The roots of 10 days old seedlings of B. juncea were excised and incubated in the fluorescent probe for 30 minutes, followed by washing twice with phosphate buffer saline. The washed roots were then mounted on glass slides and visualized under confocal microscope.
Total carbohydrate content. Total carbohydrate content was assessed by method of Hodge and Hofreiter 66 . 100 mg of dried plant samples were dipped in 2.5 N HCl and boiled in a water bath for 3 hr. The samples were then cooled and neutralized by addition of sodium carbonates. The sample volume was made upto 100 ml, followed by centrifugation for 20 minutes at 4 °C at 13, 000 × g. To 1 ml of above extract 4 ml of Anthrone reagent and was heated for 8 minutes. The samples were then cooled and observations were made at 630 nm. Glucose was used as standard.
Reducing sugar estimation. The protocol proposed by Miller 67 was used for estimation of reducing sugar levels. 0.1 gm of dried plant material was extracted with 80% ethanol. 3 ml of 3,5-dinitrosalicylic acid (DNSA) was added to 3 ml of plant extract. DNSA reagent was prepared by dissolving 50 mg sodium sulphite, 1 gm DNSA and 200 mg of phenol crystals in 100 ml NaOH (1%) and the reaction mixture was stored at 4 °C. 1 ml of 40% potassium sodium tartarte was added to the reaction mixture. The absorbance of sample was read at 510 nm. Standard glucose concentrations were used for preparation of graph for estimation of reducing sugar levels.
Trehalose Content. The method proposed by Trevelyan and Harrison 68 were used for estimation of trehalose levels. 500 mg of dried plant material was crushed in 80% ethanol, followed by centrifugation at 5000 × g for 10 minutes at 4 °C. 2 ml of tri-chloroacetic acid (0.5 M) and 4 ml of Anthrone reagent was added to 0.1 ml of supernatant. The absorbance of yellow green color was read at 620 nm. Trehalose was used as standard for preparation of graph of absorbance vs. standard trehalose concentration.
Glycine Betaine. Estimation of glycine betaine levels was done by following the protocol of Grieve and Grattan 69 . 0.5 gm of dried plant material was extracted with 5 ml of 0.05% of toluene and distilled water mixture. The reaction mixture was incubated in dark for 24 hr and was filtered. 2 N HCl and 0.1 ml of PI were added to 0.5 ml of extract. The samples were incubated for one and half hr in an ice bath. To the above mixture, 2 ml of ice cold water and 10 ml of 1,2-dichloroethane was added. Upper layer was discarded and absorbance of lower layer was read at 365 nm.
Proline content. Proline content was assessed by employing method of Bates, et al. 70 . 0.5 gm of fresh seedlings were crushed in 3% of sulphosalicylic acid, followed by centrifugation at 13, 000 × g 4 °C for 20 minutes. 2 ml of ninhydrin reagent (1.56 gm of ninhydrin was dissolved in glacial acetic acid and 6 M ortho-phosphoric acid and was warmed and stored at 4 °C), was added and warmed for 1 hr in water bath. To stop the reaction the test tubes were immediately shifted to ice-bath, followed by addition of 4 ml toluene and shook for 30-40 seconds vigorously. The absorbance of toluene layer was read at 520 nm. statistical Analysis. Data obtained was statistically analyzed by self-coded MS Excel software. The data is presented in the form of mean ± standard deviation (SD). The data was also subjected to two-way analysis of variance (ANOVA) and tukey's test (honestly significant difference, HSD). Multiple Linear Regression (MLR) was employed to analyze the response of independent variables i.e. Pb, 24-EBL and SA. β regression coefficient values implied relative effect of independent variables X 1 -Pb, X 2 -24-EBL and X 3 -SA. Following equation was used to evaluate the response: where,Y is parameter analyzed X 1 , X 2 , X 3 are independent variables i.e. Pb, 24-EBL and SA b 1 , b 2 , b 3 are partial regression coefficient due independent variables β 1 , β 2 , β 3 are regression coefficient values.

Results
pb localization. The localization of Pb metal ions by Leadmium TM Green AM dye showed green fluorescenece when visualized under confocal microscope. It was observed that the intensity of fluorescence was higher in Pb (0.75 mM) treated seedlings when compared to control. Metal treated seedlings pre-imbibed in 24-EBL and SA, alone and in combination lowered the intensity of green fluorescence. Combined treatment of 24-EBL and SA were found to be most effective (Fig. 1 (Fig. 1).
Membrane damage. PI fluorescent probe was used to observe membrane damage in B. juncea root cells. PI dye has red fluorescence and the intensity of red color was enhanced in Pb (0.75 mM) treated plants in contrast to control seedlings implying enhanced membrane damage. Pre-treatment of seedlings with 24-EBL, SA and their combination led to reduction in membrane damage as indicated by lowered intensity of red fluorescence (Fig. 1).
Nuclear Damage. Nuclear damage was assessed by employing DAPI and has blue fluorescence. The intensity of fluorescence was maximum in Pb (0.75 mM) treated seedlings. 24-EBL and SA alone and in combination showed relative lower intensity of blue color as compared to 0.75 mM Pb treated seedlings. Whereas, 24-EBL + SA combined treatment were the most effective (Fig. 2).
MDA visualization. The spectrophotometric results of MDA content were further confirmed by is visualization by schiff 's reagent using visible compound microscope. The Pb (0.75 mM) stressed roots showed higher intensity of pink color of schiff 's reagent in comparison to control seedlings. Priming of seedlings with 24-EBL and SA and their combination showed reduction in MDA levels as compared to metal (0.75 mM Pb) treated seedlings as suggested by lowered intensity of pink color (Fig. 2).
Cell Viability. The damaging effect of Pb (0.75 mM) was also assessed by using Evan's blue dye to visualize cell viability. Lowered viability of cells was observed in (0.75 mM) Pb treated seedlings when compared to control seedlings as implied by darkly stained cells. The viability of 24-EBL, SA and 24-EBL + SA treated seedlings was enhanced as indicated by lowered intensity of Evan's blue (Fig. 2). www.nature.com/scientificreports www.nature.com/scientificreports/ Glutathione tagging. The results of glutathione visualization revealed decline in accumulation of glutathione in 0.75 mM Pb stressed seedlings as evident by reduced fluorescence of MCB dye in comparison to control seedlings. Glutathione levels were enhanced in response to 24-EBL, SA and 24-EBL + SA pre-treatment as indicated by enhanced blue fluorescence (Fig. 3).
Quantitative and Qualitative amino acids levels. Total free amino acid level. The endogenous levels of free amino acids were lowered by 60.49% in 0.75 mM Pb treated seedlings in contrast to un-treated seedlings. Priming of seeds with 24-EBL, SA and their combination led to elevation in free amino acid content by 90.26%, 16.24% and 124.10% respectively, in contrast to 0.75 mM Pb treated seedlings (Fig. 4).
Amino acid profiling. A total of 21 amino acids were detected as mentioned in Table 2. Pb metal treatment resulted in decline in levels of specific amino acid including glutamine, GABA, methionine and leucine in comparison to control seedlings. Elevations in content of amino acids were observed when pre-treated with 24-EBL and SA individually as well as in combination. Levels of serine, glutamine, histidine, β-Alanine, GABA, methionine, isoleucine and leucine were further enhanced by co-application of 24-EBL and SA. polyamine (spermidine) imaging. In-situ localization of spermidine in roots of B. juncea was done using chemical probe, which showed blue fluorescence. It was observed that, 0.75 mM Pb treated plants had higher levels of polyamine when compared to control seedlings as indicated by darkly stained cells. Individual as well as combined application of 24-EBL and SA led to further elevation in accumulation of polyamines as evidenced by darkly stained cells (Fig. 3).
osmoprotectants. Total Carbohydrates Content. Pb (0.75 mM) treatment led to significant enhancement (195.60%) in total carbohydrate content in comparison to control seedlings. Seedlings treated with 24-EBL and SA individually and in combination led to further enhancement in levels of total carbohydrates by 179.9%, 17.48% and 207.80% respectively. Combined treatment of 24-EBL and SA was most effective in enhancing total carbohydrate levels (Fig. 4).  Table 1. Effect of pre-treatment with combination of 24-EBL and SA on superoxide anion (µg g −1 FW), H 2 O 2 (µM g −1 FW) and MDA (mM g −1 FW) content in 10 days old B. juncea seedlings exposed to Pb. * Data is presented as mean ± SD. Two-way ANOVA, Tukey's test and MLR analysis was performed * and ** designated significant at P ≤ 0.05 and P ≤ 0.01 respectively. Statistical letters are mentioned HSD.
www.nature.com/scientificreports www.nature.com/scientificreports/ Reducing sugar content. The seedlings raised under 0.75 mM Pb treatment had elevated levels of reducing sugars by 51.25% in comparison to control. Seedlings primed with in 24-EBL and SA individual treatment led to increase in reducing sugar levels by 222.40% and 146.20% respectively in contrast to 0.75 mM Pb treatment. Co-application of 24-EBL and SA maximally enhanced reducing sugar levels by 246.25% in comparison to metal (0.75 mM) Pb treated seedlings (Fig. 4).
Trehalose content. Trehalose levels were significantly elevated in response to Pb treatment by 219.42% in contrast to control seedlings. Priming with 24-EBL, SA and 24-EBL + SA resulted in enhancement in trehalose levels by 28.72%, 8.06% and 140.80% respectively in comparison to 0.75 mM Pb treated seedlings (Fig. 4).  www.nature.com/scientificreports www.nature.com/scientificreports/ Glycine betaine content. Content of glycine betaine was enhanced by 64.15% in Pb (0.75 mM) treated seedlings in contrast to control seedlings. Priming of seeds with 24-EBL and SA individual treatment further enhanced the levels of glycine betaine by 21.07% and 10.92% respectively in comparison to 0.75 mM Pb treated seedlings. Pb (0.75 mM) treated seedlings pre-imbibed in combined treatment of 24-EBL and SA led to most significant elevation i.e. by 40.61% respectively in glycine betaine content (Fig. 4).
Proline level. Significant elevation in proline content was recorded in 0.75 mM Pb treated seedlings (237.91%) in comparison to control seedlings. Further increase in proline levels were recorded in 24-EBL, SA and 24-EBL + SA primed seedlings by 50.60%, 31.51% and 76.31% respectively when compared to 0.75 mM Pb treated seedlings (Fig. 4).

Discussion
Pb is a non-essential element that prominently perturbs plants physiology. It is a less available and low solubility metal which occurs in phosphates, nitrates and sulphate forms 71 . It is a extremely toxic metal and exerts certain critical effects on the physiological and biochemical attributes in plants 72 . In-situ localization of Pb ions in roots revealed enhancement in Pb content in response to increment in metal concentration. Similar elevation in Pb www.nature.com/scientificreports www.nature.com/scientificreports/ content was reported in Oryza sativa 73 , Nicotiana tabaccum 74 , Medicago sativa 75 and Triticum aestivum 76 . The bulk of Pb in soil is absorbed by roots of plants and bind to the carboxyl group of mucilage uronic acids 77 . A very small fraction of Pb is available for plants to accumulate due to the fact that it forms strong complexes with colloidal particles or organic matter 78 . The endodermis of the root cells act as a barrier for movement of Pb from roots to shoots. This may be a possible reason for higher accumulation of Pb in roots when compared to shoots 79 . Plant growth regulators (PGRs) are well studied for their anti-metal stress activities 80 . In present study pre-treatment of seedling with EBL, SA and their combination showed reduced Pb metal localization in root cells. This is attributed to BR stimulated activation of antioxidative defense system, lowered ROS content and improvement in growth of plants 21,27 . Several reports suggest BR-induced reduction in metal upake such as Al in Phaseolus aureus 81 , Ni in Brassica juncea 31 , Cr in Raphanus sativus 82 and Cu in Brassica juncea 83 . Similarly, SA application to B. juncea seedlings resulted in lowering of Pb ion deposition in root cells. Transportation and accumulation of several metals have been reported to be affected by exogenous application of SA. Decline in metal content i.e. Cd in Zea mays 84 , Pb in B. juncea 85 , As in Raphanus sativus 86 and Ni in Nicotiana tabaccum 22 was reported in SA pre-treated plants. Reduced uptake of Pb in Chlorella vulgaris cells has been reported under 24-EBL treatment by Bajguz 87 . He suggested that Pb metal in combination with EBL results in stimulation of phytochelatin de-novo synthesis. Similarly, Kaur et al. 88 suggested that SA treatment leads to reduced metal uptake, possibly due to its ability to reduce oxidative stress and increased membrane stability 88 .
Exposure of B. juncea plants to Pb in the present study resulted in enhanced synthesis of ROS which further caused cellular damage in plants. Pb treatment resulted in elevation in levels of superoxide anion, H 2 O 2 and MDA levels. ROS production in response to Pb stress is well documented 89,90 . H 2 O 2 is easily converted to hydroxyl ion (free radical) by fenton reaction and is membrane permeable and diffusible 91 . The enhanced production of ROS might also enhance the tolerance mechanism in plants 92 . Asada-Halliwell pathway, with the co-ordination of enzymatic and non-enzymatic antioxidants, also plays an instrumental role in the removal of H 2 O 2 . Histological studies carried out also supported the biochemical responses and affirmed enhancement in total ROS, H 2 O 2 and MDA levels. Furthermore, cellular damage and loss of viability of cells caused by oxidative burst was also observed. The results of present study corroborated with the previous studies conducted on Ocimum tenuiflorum 93 , Vigna radiata 94 and Zea mays 95 . BRs application resulted in reduction in ROS levels and hence aided in lowering the toxic effects of Pb. Similar findings of lowered ROS production were reported by Sharma and Bhardwaj 83 in Zea mays plants by Choudhary,et al. 96 in Raphanus sativus, by Yadav, et al. 27 and by Kohli,et al. 49 in B. juncea. Reduction in Pb induced MDA, H 2 O 2 and superoxide anion content by BRs pre-treatment is attributed to their ability to enhance activity of antioxidative defense system and hence scavenging of free radicals 97 . Interplay between SA signaling networks and other signaling cascade has been studied to regulate ROS stimulated

Amino acid content (µg −1 g of FW)
Aspartic  www.nature.com/scientificreports www.nature.com/scientificreports/ cell death 98 . The observation was also affirmed with histological studies which showed reduction in ROS content, nuclear and membrane damage and altered cell viability.
Sulfur is one of the most imperative elements that is incorporated in several biomolecules including amino acids and antioxidants 99 . GSH is one of the sulfur containing antioxidant. Present work further revealed reduction in levels of glutathione in response to Pb toxicity. Our findings corroborated the studies of Okamoto, et al. 100 who reported lowered GSH in Gongauloux polyedea exposed to Pb stress. The decline in GSH levels is due to lowered de-novo production of GSH as well as over-utilization of GSH in the synthesis of phytochelatins, pre-requisite for chelation of toxic metal ions 101 . This antioxidant fortifies protein oxidation and acts as a substrate for production of glutathione peroxidase and glutathione-S-transferase 102 . In addition, heavy metals can alter the active sites of the enzymes, thus rendering them nonfunctional 103 . Exogenous application of combination of 24-EBL and SA resulted in enhancement in levels of GSH in the present study. Exogenous application of BRs to stressed plants elevated the contents of glutathione in Lycopersicon esculentum exposed to Cd and Pb 30 , Cicer arietinum exposed to Cd 104 and Vigna radiata exposed to Al 105 . The possible reasons for BR induced increase in the activities of antioxidative enzymes might be due to their ability to regulate the transcription and/or translation of genes that further mediate the activation, or de novo synthesis of antioxidative enzymes 87 . Similarly, application of SA was also reported to enhance endogenous levels of GSH in Medicago sativa plants under Mg stress. SA primed plants had enhanced tolerance to Cd in Triticum aestivum plants which was largely co-related to enhanced GSH synthesis. Furthermore, several studies suggested regulation of various enzymes of AsA-GSH (ascorbate-glutathione) cycle by SA application 106,107 . Another possible reason for enhanced activity of antioxidative defense system might be due to BR Signaling Kinase 1 (BSK 1) which is a positive regulator of SA accumulation in stressed plants and eventually results in amelioration of oxidative damage 108 .
Amino acids specifically S containing amino acids have anti-stress and antioxidant properties. These organic amino acids include glutathione and methionine 109 . In the present study, the levels of free amino acids and other amino acids was lowered in response to Pb stress. Amino acids have metal chelating, antioxidant and signaling ability under metal stress 110 . The probable reason for lowered amino acid levels might be due to enhanced chelation of bivalent metal ions with free amino acid which consequently lowers available amino acids 111 . Co-application of BRs and SA resulted in elevation in free amino acid levels. Our findings corroborated with the study carried out by Li, et al. 112 who reported enhancement in amino acids content in response to 24-EBL application. Similarly, application of SA resulted in elevation in levels of amino acids specifically proline, glycine betaine and glutathione in the present study. Few specific amino acids such as proline and glycine betatine also act as osmoprotectants 113 . Combined application of 24-EBL and SA led to enhancement in osmolyte levels in the present study. Similar elevation in glycine betaine levels were observed in response to CO, Fe and Zn stress 114 . Glycine betaine levels are known to alter the membrane permeability of stressed plant cells 115 . Enhanced levels of proline and glycine betaine resulted in further elevation in antioxidative capacities of berries 116 . Proline content significantly elevated by BRs application is well documented 29,82,96,117 . SA also resulted in further enhancement in proline and glycine betaine levels and they aid in detoxification of elevated ROS content, maintenance of membrane stability and enzyme activities 118 . It was observed by Parmar et al. 119 , that the main reason for increase in levels of proline content is associated with increase in synthesis of new amino acids under heavy metal stress. Elevation in concentration of osmolytes is considered as an important marker indicating metal stress and have important role in stress mitigation.
In-situ localization of polyamines (spermidine) revealed elevation in polyamines accumulation in Pb stressed seedlings. Polyamines specifically spermine and spermidine have antioxidant properties and protect DNA against oxidative damage 120 . Similar to our findings, Groppa, et al. 121 reported elevation in polyamines content in response to Cu and Cd toxicity. Another report suggests similar decline in polyamine accumulation in Lacuta sativa plants under Zn stress 122 . Co-application of 24-EBL and SA significantly elevated in-situ deposition of polyamines in root cells of B. juncea in the present study. In our earlier study, Choudhary, et al. 123 reported positive crosstalk between BRs and polyamines in regulating Cu stress in Raphanus sativus plants. It was further suggested that synergistic interplay between BRs and polyamines (spermidine) led to enhancement in levels of other polyamines such as putrecine and spermine. Similarly, exogenous application of SA resulted in higher level of polyamines in Zea mays 124 and Lycopersicon esculentum 125 .
Sugars and sugar polyols accumulate in response to stress and they act as an imperative biomarker and osmoregulator 126 . Levels of total carbohydrate, reducing sugars and trehalose were significantly increased in response to Pb treatment. Sugars play a vital role in modulating plants osmotic balance and membrane stability in stressed plants 127 . It has been hypothesized that heavy metal toxicity might hinder the metabolic pathway of carbohydrates or it might play a role in the accumulation of photoassimilates because of reduced loading of veins 128 . Plethora of reports suggest enhancement in sugar levels due to Pb toxicity in B. juncea 48 and Raphanus sativus 129 . It was further suggested by Bhushan and Gupta 130 that enhanced levels of total carbohydrates and reducing sugar could be due to meddling of Pb ions with transportation through endodermis into plant cells causing severe toxicity. Pre-treatment with 24-EBL and SA led to further increase in content of sugars. Similar to amino acids, sugars also have stress mitigating properties via enhanced sequestration of ROS molecules as well as activation of antioxidant defense system 131 . It has been reported that sugars acts as osmolytes and provide protection to cells from metal toxicity 132,133 . These sugars not only help as osmolytes, participate in stabilization of cellular membranes and maintenance of turgor but also act as signaling molecules 134 .

Conclusion
The present study indicates that co-application of 24-EBL and SA to B. juncea L. plants may enhance their potential to overcome Pb stress. The positive interpay between 24-EBL and SA led to elevation in total free amino acids, content of various amino acids (glutathione, proline, methionine etc.), which act as antioxidants and metal chelating compounds. These metabolites scavenge ROS and also result in reduced lipid peroxidation, nuclear and www.nature.com/scientificreports www.nature.com/scientificreports/ membrane damage. From the results, it is further concluded that 24-EBL and SA combined treatment has a significant role in regulating the balance of total carbohydrates, reducing sugars and osmoprotectants like trehalose, glycine betaine and proline which are involved in osmoregulation of plant cells. The co-application of 24-EBL and SA reduced Pb accumulation in plants that consequently results in lowering oxidative stress. Therefore, combined treatment of 24-EBL and SA can counter adverse effects of Pb toxicity through regulating various physiological processes. Further insight into mechanisms of 24-EBL and SA crosstalk pertaining to metal stress amelioration might provide better understanding of stress tolerance strategies in plants.