Polyamines (spermidine and putrescine) mitigate the adverse effects of manganese induced toxicity through improved antioxidant system and photosynthetic attributes in Brassica juncea

Chemosphere 236 (2019) 124830

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Polyamines (spermidine and putrescine) mitigate the adverse effects of manganese induced toxicity through improved antioxidant system and photosynthetic attributes in Brassica juncea Anjuman Hussain, Faroza Nazir, Qazi Fariduddin* Plant Physiology and Biochemistry Section, Department of Botany, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, 202 002, India

h i g h l i g h t s
Mitigative role of polyamines {spermidine (Spd) and putrescine (Put)} was established in manganese (Mn) stressed Brassica juncea plants. 
Mn (150 mg kg1) diminished photosynthetic attributes and growth, enhanced the production of ROS like H2O2 and O·2 content in plants.
Spd or Put improved stomatal behavior, photosynthetic attributes, growth and biochemical traits in Mn stressed plants.
Out of the two polyamines (Spd or Put), Spd proved more effective and decreased the accumulation of Mn in plants.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 May 2019 Received in revised form 5 September 2019 Accepted 9 September 2019 Available online 12 September 2019

Polyamines (PAs) are recognized as plant growth regulators that are involved in the stress management in various crops. In the current study, mitigative roles of spermidine (Spd) and putrescine (Put) were assessed in manganese (Mn) stressed Brassica juncea plants. Spd or Put (1.0 mM) were applied to the foliage of Brassica juncea at 35 days after sowing (DAS) grown in the presence of Mn (30 or 150 mg kg1 soil). The higher level of Mn (150 mg kg1) diminished photosynthetic attributes and growth, enhanced the production of reactive oxygen species (ROS) like hydrogen peroxide (H2O2) and superoxide anion  (O$2 ) content, affected stomatal movement and increased the Mn concentration in roots and shoots of the plant at 45 DAS, whereas it enhanced the activities of various antioxidant enzymes and proline content in the foliage of Brassica juncea plants. On the other hand, treatment of PAs (Spd or Put) to Mn stressed as well as non-stressed plants resulted in a remarkable improvement in the stomatal behaviour, photosynthetic attributes, growth and biochemical traits, decreased the production of ROS (H2O2 and  O$2 ) and concentration of Mn in different parts of plant. It is concluded that out of the two polyamines (Spd or Put), Spd proved more efficient and enhanced growth, photosynthesis, and metabolic state of the plants which bestowed tolerance and helped the plants to cope efficiently under Mn stress. © 2019 Published by Elsevier Ltd.

Handling Editor: T Cutright Keywords: Antioxidant enzymes Manganese toxicity Photosynthesis Polyamines Reactive oxygen species Stomata

1. Introduction Manganese (Mn) is considered as an essential micro nutrient which is necessary for plant growth regulation and is also responsible for activating various enzymes of several metabolic processes like protein and carbohydrate synthesis, respiration, photosynthesis, etc (Boojar and Goodarzi, 2008). Mn forms an important component of the water-splitting complex of photosystem II (PSII), which is bound to the D1 reaction center protein of

* Corresponding author. E-mail address: [email protected] (Q. Fariduddin). https://doi.org/10.1016/j.chemosphere.2019.124830 0045-6535/© 2019 Published by Elsevier Ltd.

PSII, providing necessary electrons that drive the electron transfer chain of photosynthesis (Goussias et al., 2002). Mn is also required for chlorophyll biosynthesis (by activating specific enzymes), biosynthesis of tyrosine (aromatic amino acid), diphenolic compounds (lignans and flavonoids), take part in the biosynthesis of isoprenoids, nitrate assimilation, and also detoxifies superoxide  (O$2 ) and hydrogen peroxide (H2O2) radicals (Ducic and Polle, 2005). Being a cofactor of antioxidant enzyme (superoxide dismutase; SOD), Mn is involved in conferring tolerance to the plants against oxidative burst, which is generated by an increased concentration of active oxygen and reactive oxygen species (ROS), that are deleterious to plants. However, when the concentration of Mn builds up in the soil, it could generate stress, like other heavy

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metals (Mukhopadhyay and Sharma, 1991). Mn toxicity is possibly the most important factor that limits plant productivity in acidic soils after aluminum (Al) (Foy, 1984). Toxicity of Mn is more evident in wet acidic soils (pH < 5.5) (Lynch and St. Clair, 2004) where it prevents the uptake and transport of various other essential plant nutrients like Mg, Fe, Ca, and P, because of their ionic radius resemblance and ligand binding ability (Marschner, 2012; Millaleo et al., 2013). Furthermore, Mn stress inhibits functioning of PSII and induces the aggregation of oxidized form of Mn and various phenolic compounds in the apoplast of leaf (Marschner, 2012). This leads to the development of traits of Mn toxicity which comprises the yellowing of leaves, and tissue necrosis, manifesting as brown spots on the plant foliage, eventually reducing plant biomass (Marschner, 2012). Polyamines (PAs) are aliphatic organic compounds which contain variable hydrocarbon chains with two or more amino groups and have an overwhelming biological activity (Takahashi and Kakehi, 2010; Vuosku et al., 2018). PAs find wide distribution in eukaryotic and prokaryotic cells (Liu et al., 2017). Initially, biological roles of PAs were assumed to be only structural as the polycationic nature of PAs allows the binding and stabilization of anionic macromolecules in the cell. However, later studies unveiled the involvement of PAs in several cellular activities like cell division, cell differentiation, transcriptional and translational regulation, gene expression, homeostasis and signal transduction (Anwar et al., 2015; Pegg, 2016). Genetic analyses further corroborated that the most plentiful PAs in plants such as putrescine (Put), spermidine (Spd), and spermine (Spm) are involved in the endurance of plants to different abiotic factors (Romero et al., 2018). In addition, these PAs also serve as an important sinks for assimilated nitrogen (Moschou, 2012). Exogenous treatment of PAs could confer protection against various biotic and abiotic stresses (Sheteiwy et al., 2017; Chen et al., 2018). PAs have been exploited in regulating the defense system in plants against various environmental stresses (Hasanuzzaman et al., 2019) including drought stress (Ebeed et al., 2017), heavy metal stress (Hussain et al., 2019), chilling stress (Sheteiwy et al., 2017) and salt stress (Nahar et al., 2016). Keeping in view the multifaceted roles of PAs, it was hypothesized that the exogenous application of Spd or Put could improve the growth of plants and productivity under Mn stress. The present study was conducted with the objectives that how Spd or Put could regulate stomatal behavior, photosynthetic attributes, growth traits and antioxidant enzyme activities in Brassica juncea under Mn stress. 2. Materials and methods 2.1. Plant material Seeds of mustard (Brassica juncea L. var. varuna) were bought from National Seed Corporation Ltd., Pusa, New Delhi, India and were surface sterilized with 1% solution of sodium hypochlorite, accompanied by rinsing with deionized water at least thrice. 2.2. Preparation of polyamines solution The stock solutions of Spd and Put have been prepared by dissolving the desired amount of Spd or Put in 5 mL of ethanol, in 100 mL volumetric flasks and the final volume was kept up to the mark by double distilled water (DDW). The specified concentrations of Spd or Put were formulated by diluting stock solution with DDW. 5 mL of surface-active agent “Tween-20” was added to it just before application. The desired concentrations of PAs were selected and based on the studies of Fariduddin et al. (2014) and Mir et al. (2015).

2.3. Experimental design and treatment pattern The surface sterilized seeds were sown in 20-cm diameter earthen pots loaded up with reestablished soil (sandy loam soil and farmyard manure; blended in the proportion of 6:1) and kept in the naturally lit up net house of the Botany Department, Aligarh Muslim University, Aligarh (India). Thinning was done on the 7th day subsequent to sowing (DAS) and three plants for each pot were maintained. The experiment was accomplished in a totally randomized block layout. Forty five pots were divided into 9 sets of 5 pots each (replicates) representing one treatment. At 25 d phase of development, seedlings were subjected to Mn, given as MnSO4 (30 or 150 mg kg1) through the soil and afterward permitted to grow. At 35 d period, plants were sprayed with DDW (control) or 1.0 mM Spd or Put for three days. Each plant was sprinkled thrice. The spout of the sprayer was balanced so that in one sprinkle it drew out 1 mL. The growth traits as well as physiological and biochemical characteristics of plants have been accessed after harvesting at 45 d stage. Selection of specific concentrations of Mn was based on the preliminary experiment for screening of different concentrations. 2.4. Growth traits Plants along with adhering soil were uprooted from the pots with utmost care and washed to remove the adhering soil. The plants had been then taken out tenderly, and with the aid of the scale with metric units, centimeters (cm), root and shoot length was measured. Fresh mass of root and shoot of each plant was calculated with the assistance of electronic balance. The root and shoot samples were put in an oven kept running at 80  C for 72 h and afterward their dry mass was noted. Leaf area was estimated with the assistance of leaf area meter (ADC Bioscientific, UK). 2.5. Determination of SPAD value of chlorophyll With the aid of SPAD chlorophyll meter (SPAD-502; Konica, Minolta sensing, Inc., Japan), SPAD value of chlorophyll in intact fully expanded leaves of the plant was determined. 2.6. Analysis of leaf gas-exchange parameters The net rate of photosynthesis (PN) and its associated variables, i.e., stomatal conductance (gs), transpiration rate (E), and intercellular CO2 concentration (Ci) were estimated between 11:00 and 13:00 h on the third completely expanded leaves with the assistance of an infrared gas analyzer (IRGA) portable photosynthetic system (Li-COR 6400, Li-COR, and Lincoln, NE, USA), by keeping up the air temperature, relative humidity, CO2 concentration and photosynthetic photon flux density (PPFD) at 25  C, 85%, 600 mmol mol1 and 800 mmol mol2 s1, respectively. 2.7. Compound microscopy and scanning electron microscopy For compound microscopic study, the epidermal peeling of the lower surface of leaf was removed, and was extended very carefully on a glass slide, and then the peeling of the leaf was seen under the compound microscope outfitted with NIKON digital camera. Leaf and root samples were first fixed in 0.1 M sodium cacodylate and 2.5% glutaraldehyde buffer (pH 7.3) for 2 h and were then post firmed with 1% osmium oxide. Finally, dehydration of the samples was done by the graded ethanol series (50%, 70%, 80%, 90%, and 100%). The dehydrated specimens were smeared with goldpalladium and the samples were studied using the JEOL JSM-JSM 6510 scanning electron microscope.

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2.8. Determination of leaf electrolyte leakage Quantification of total inorganic ions spilled from the leaves was executed by the procedure depicted by Sullivan and Ross (1979). Leaf segments (25) were placed in a boiling test tube containing 10 mL of deionized water and afterward electron conductivity (EC) was estimated (ECa). After that, the substances have been warmed at 45 and 55  C for 30 min each in a water bath, and EC was estimated (ECb). Afterward, the tubes had been boiled at a 100  C for 10 min, and EC was again noted (ECc). The leaf electrolyte leakage was ascertained with the aid of formula: EL (%) ¼ [(ECb  ECa)/ (ECc)]  100

2.9. Assay for nitrate reductase (NR) and carbonic anhydrase (CA) activity Activity of CA was determined with the aid of the method proposed by Dwivedi and Randhawa (1974). Small pieces of the leaf samples had been taken in test tubes containing solution of cystein hydrochloride. Each of these samples had been smudged and then relocated in the test tubes containing phosphate buffer (pH 6.8), 0.2 M NaHCO3, and bromothymol blue. The methyl red used as an indicator was added in the last. 0.05 N HCl was used as the titrating agent in the titration processes. The activity of enzyme was formulated on per gram fresh mass basis. Activity of NR quantification was followed by the method of Jaworski (1971). The fresh leaves were cut into small segments and then relocated to plastic vials, having phosphate buffer (pH 7.5), KNO3 and isopropanol and incubated at 30  C for 2 h. The tubes were then removed and solutions of sulfanilamide and N-1naphthylethylenediamine hydrochlorides were added. The intensity of the pink colour generated was recorded at 540 nm on a spectrophotometer. 2.10. Estimation and localization of reactive oxygen species 2.10.1. Estimation of hydrogen peroxide and superoxide anion content For the estimation of hydrogen peroxide content in the leaves, protocol of Patterson et al. (1984) was used. Fresh leaf sample (0.5 g) homogenized in acetone was centrifuged at 5000g for 15 min. Supernatant obtained was put in test tubes and in each tube 20% titanium chloride prepared in conc. HCl and 17 M ammonia solution was added. The precipitate obtained was washed with acetone and then dissolved in 2 N H2SO4. Absorbance was measured at 410 nm. H2O2 content was determined by using the standard curve of H2O2 and expressed as m mole g1 FM. Superoxide anion content was estimated by the method of Wu et al. (2010). 1 g of leaf was crushed in 65 mM phosphate buffer containing 1% PVP. The sample was centrifuged at 5000g for 15 min and to the supernatant, phosphate buffer (65 mM) and hydroxylamine hydrochloride (10 mM) were added and incubated at 25  C for half an hour. After incubation, metanilic acid (58 mM) and 1-napthylamine (7 mM) were added and the mixture was again incubated at 25  C for 20 min. The absorbance was measured at 530 nm. The superoxide content was determined from standard curve and the content was expressed as m mole g1 FM. 2.10.2. Localization of hydrogen peroxide and superoxide anions  The degree of production of O$2 and H2O2 was estimated by validating the histochemical staining protocol of Kaur et al. (2016) with small modification using nitro blue tetrazolium chloride (NBT)

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and 3, 3-diaminobenzidine (DAB) respectively, for staining leaf samples. The samples were immersed in 3, 30 - diaminobenzidine solution (pH 3.8) at room temperature under light for 8 h. Then, samples were immersed in absolute alcohol and subsequently boiled at 100  C until the removal of chlorophyll. The samples were cooled and then immersed in 20% glycerol. Pictures were taken with NIKON digital camera (COOLPIX110). 2.11. Activities of antioxidant enzymes For the estimation of activities of antioxidant defense enzymes, fresh leaf tissues (1 g) were homogenized with an extraction buffer consisting of 70 mM phosphate buffer (pH 7.0), 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethanesulfonylflouride (PMSF), 0.5% (v/v) Triton X-100 and 2% (w/v) polyvinyl pyrollidone (PVP) and ground with the aid of chilled mortar and pestle. The centrifugation of homogenates was done at 12,000g for 20 min at 4  C and the supernatant generated was utilized for estimation of an antioxidant enzyme (catalase, peroxidase, and superoxide dismutase). CAT enzyme was determined by evaluating the underlying rate of H2O2 vanishing following the procedure of Aebi (1984) with slight adjustment. 50 mM phosphate buffer (pH 7.0), 15 mM H2O2 and 100 mL enzyme extract was taken to set up the reaction mixture for the sample used for the assay of CAT activity. The reduction in H2O2 was pursued as decrease in optical density at 240 nm for 2 min with the interim of 30 s at 25  C. A test tube that served as control was prepared using the above solutions except enzyme extract. POX activity was estimated by using the protocol described by Sanchez et al. (1995) with slight changes. 50 mM phosphate buffer (pH 7.0), 20 mM guaiacol, 15 mM H2O2 and 100 mL enzyme extract were taken for the preparation of reaction mixture. Control set contained all the solutions except the enzyme extract. The activity was assessed by measuring the absorbance at 436 nm for 1 min at 25  C. SOD activity was measured by assessing its capability to lessen the photochemical reduction of nitrobluetetrazolium (NBT) following the procedure depicted by Beauchamp and Fridovich (1971). The reaction mixture consisted of 50 mM phosphate buffer (pH 7.8), 9.9 mM L-methionine, 55 mM NBT, 2 mM EDTA, 0.02% Triton X-100 and 40 mL enzyme extract. Lastly, 1 mM riboflavin was added to reaction mixture. Control set was set up with similar procedure. The activity was measured by measuring the absorbance at 560 nm for 2 min at 25  C. One unit of SOD activity was calculated as the measure of enzyme necessitated that brought about 50% reduction of NBT at 25  C. 2.12. Leaf proline content The proline content in fresh leaf samples was estimated followed the protocol of Bates et al. (1973). Sample extraction was executed in sulfosalicylic acid to which an equal volume of glacial acetic acid and ninhydrin solutions have been added. The sample was heated at 100  C for 20 min and then subsequently cooled. After that, 5 mL of toluene was added to each tube. The wavelength of the upper most layer was measured at 520 nm with the help of a spectrophotometer. 2.13. Mn accumulation in root and shoot Harvested samples of root and shoot were washed with deionized water and were put in an incubator, run at 80  C for 48 h. The dried tissue was ground to fine powder. Content of Mn in root and shoot was determined by digesting the samples with concentrated

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HNO3/HClO4 (3:1, v:v) and was expressed in terms of mg g1 dry mass (DM) by the atomic absorption spectrophotometer (GBC, 932 plus; GBC Scientific Instruments, Braeside, Australia).

2.14. Confocal microscopic study for cell viability Cleaned and washed roots of Brassica juncea were cut with the help of sharp knife and were dipped in propidium iodide dye (5 mM) for 30e35 min. After this, these stained roots were put on glass slide and were covered with cover glass and were studied under confocal microscope.

2.15. Statistical analysis Statistical analysis of the data was done by SPSS ver. 17 for windows (IBM Corporation, 1 New 171 Orchard Road, Armonk, New York 10504-1722, United States 914- 499-1900). Standard error was computed and analysis of variance (ANOVA) was executed on the data having 5 replicates to evaluate the least significance difference (LSD) of the treatments at the 5% level of probability. Means were separated by using respective LSD values.

3. Results 3.1. Growth traits All Growth traits (i.e. length of root and shoot, fresh and dry weight of root and shoot and leaf area) were markedly declined in the presence of higher concentration (150 mg kg1 soil) of Mn against control plants (Figs. 1 and 2A) However, a modest increase in the above parameters was noted at the lower concentration of Mn (30 mg kg1 soil). Of the two Mn concentrations, the higher level (150 mg kg1 soil) of metal proved destructive that caused a reduction in the root and shoot length by 35.70% and 29.20%, root fresh and dry weight by 35.89% and 38.04%, shoot fresh and dry weight by 33.22% and 32.32%, and decreased leaf area by 32.11% in contrast to the respective control plants. Plants which received the foliage treatment of Spd (1.0 mM) or Put (1.0 mM) alone showed a prominent increase in all the aforesaid parameters than the control plants. However, the application of Spd excelled over Put in all the above parameters. Nevertheless, a maximum increase in the growth biomarkers was prominent in the plants which received the combined application of Spd (1.0 mM) and Mn (30 mg kg1 soil) in comparison to control plants. Moreover, toxicity caused by the higher concentration of Mn (150 mg kg1) was partially nullified by the application of Spd or Put to the stressed plants.

3.2. SPAD value of chlorophyll and maximum quantum yield of PS II (Fv/Fm) Mn (150 mg kg1) stress decreased both the SPAD value of Chl and Fv/Fm (Figs. 2B and 3A) by 24.75% and 27.13%, respectively, than the respective controls. Application of Spd or Put alone in stress free plants resulted in the enhanced Chl content and Fv/Fm compared to the respective controls. However, a prominent increase in both the parameters was generated by the treatment of Spd (1.0 mM). Moreover, the combined dose of Mn (30 mg kg1 soil) and Spd (1.0 mM) proved very effective and enhanced the chlorophyll content by 35.7% and Fv/Fm by 20.31% against the respective control plants. The follow-up application to the Mn stressed plants with Spd (1.0 mM) partially overcame the toxicity triggered by Mn (150 mg kg1 soil).

3.3. Photosynthesis traits Photosynthetic traits (i.e., PN, gs, Ci and E) were considerably lowered by the treatment of Mn (150 mg kg1) and decreased these parameters by 32.32%, 32.18%, 34.27% and 31.44%, respectively, than the control plants (Fig. 2CeF). The plants treated with Spd (1.0 mM) or Put (1.0 mM) alone to stress-free plants had improved values of all these parameters. However, the application of Spd (1.0 mM) was most effectual in increasing the PN by 35.57%, gs by 33.33%, Ci by 28.97% and E by 28.04% against the respective control plants. The plants which were supplied with the combined dose of Spd (1.0 mM) and Mn (30 mg kg1soil) generated maximum increase in PN by 37.61%, gs by 36.28%, Ci by 31.15% and E by 30.58% than the control plants. However, Mn-stressed plants treated with Spd (1.0 mM) or Put (1.0 mM) moderately counteracted the toxicity produced by higher concentration of Mn (150 mg kg1 soil). 3.4. Compound microscopy and SEM imaging Stomatal behavior was significantly affected in the plants treated with Mn (150 mg kg1). Higher dose of Mn (150 mg kg1) resulted in the closure of stomata, whereas, exogenous application of Spd or Put (1.0 mM) in the absence of Mn increased stomatal width aperture compared to the control. Moreover, 1.0 mM Spd was more effective in widening the stomatal width aperture in comparison to the Put (1.0 mM) treated plants (Fig. 4.1). These observations were further corroborated by SEM observation (Fig. 4.2). Scanning electron micrographs of the roots of plants raised from Mn (30 or 150 mg kg1) had a deformed morphology. Higher level of Mn (150 mg kg1) proved more effective in damaging root morphology of plants (Fig. 4.3). Root morphology of the control, Spd or Put treated plants were not affected. 3.5. Electrolyte leakage Plants grown in Mn stress (150 mg kg1) had more EL (24.05%) than the control plants (Fig. 3B). The exogenous application of Spd (1.0 mM) or Put (1.0 mM) to stress-free plants decreased EL by 14.40% and 10.19%, respectively, than control. Application of Spd or Put to the stressed plants (150 mg kg1) followed a considerable decrease in the electrolyte leakage. 3.6. Carbonic anhydrase (CA) and nitrate reductase (NR) activity Plants grown in Mn stress (150 mg kg1) exhibited lower CA and NR activities compared to the respective control plants (Fig. 3CeD). Treatment of the plants with Spd or Put elicited enhanced activity of these enzymes compared to their respective controls. However, the treatment of Spd (1.0 mM) excelled over Put (1.0 mM) and enhanced the CA and NR activity by 29.2% and 35.15% respectively over the control plants. Moreover, the highest activity of these enzymes were observed in the plants which received the combined dose of Spd (1.0 mM) and Mn (30 mg kg1) and increased the CA and NR activities by 32.2% and 38.21%, against the control plants. Moreover, follow up treatment of Spd or Put partially triumphed over the damage caused by Mn stress (150 mg kg1). 3.7. Reactive oxygen species 3.7.1. Superoxide anion content and H2O2 content Higher level of Mn (150 mg kg1) caused a significant increase in  O$2 anion content and H2O2 accumulation by 33.14% (Fig. 5E), and 51.24% (Fig. 6E), respectively, in comparison to control. Application  of PAs, reduced the content of O$2 anion and H2O2 and Spd proved more effective than Put in this regard.

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Fig. 1. Effect of polyamines (Spd and Put, 1.0 mM) on Mn induced changes in (A) Shoot length, (B) Root length, (C) Shoot fresh mass, (D) Root fresh mass, (E) Shoot dry mass, (F) Root dry mass of Brassica juncea plants at 45-day stage of growth. All the data are the means of five replicates (n ¼ 5); vertical bars show standard errors (±SE). Means with different letters above the bars are significantly different between the treatments.



3.7.2. Superoxide anion (O$2 ) and hydrogen peroxide (H2O2) localization  O$2 and H2O2 level in leaves was respectively depicted by blue (Fig. 5) and brown coloured spots (Fig. 6). Leaf discs from Mn fed plants exhibited more pronounced spots as compared to control plants. Moreover, the leaves of Spd and Put treated plants had less pronounced spots as compared to stressed plants.

the presence of Mn (150 mg kg1) (Fig. 7AeC). The plants exposed separately to Spd or Put also induced the activities of these enzymes. Spd stimulated the activities by 33.49%, 37.67% and 35.07%, respectively, whereas Put by 23.02%, 30.45% and 25.37% respectively, over the respective controls. Furthermore, the maximum activities of CAT, POX, and SOD was exhibited by plants subjected to Mn stress (150 mg kg1) and subsequently treated with Spd (1.0 mM).

3.8. Antioxidant enzymes 3.9. Proline content A remarkable increase in the activities of antioxidant enzymes was observed. The activities of CAT, POX, and SOD were increased by 43.02%, 56.12%, and 54.47%, respectively, in the plants raised in

A higher content of proline was observed in the plants supplied with Mn (150 mg kg1) than control plants (Fig. 7D). In Mn stressed

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Fig. 2. Effect of polyamines (Spd and Put, 1.0 mM) on Mn induced changes in (A) Leaf area, (B) Chlorophyll content, (C)Net photosynthetic rate, (D) Stomatal conductance, (E) Internal CO2 concentration, (F) Transpiration rate of Brassica juncea plants at 45-day stage of growth.All the data are the means of five replicates (n ¼ 5); vertical bars show standard errors (±SE). Means with different letters above the bars are significantly different between thetreatments.

plants, proline content was increased by 41.67%, over control. Treatment with Spd or Put under stress free conditions also increased the proline content, with Spd (1.0 mM) triggering more promising response and increased the proline content by 32.76% as compared to control. The plants exposed to Mn stress (150 mg kg1) and subsequently treated with Spd (1.0 mM) were found to have more content of proline compared to control. 3.10. Mn accumulation in root and shoot The accumulation of Mn in the plants increased with plant age. In general, it was observed that root aggregated more Mn than the shoot. However, foliar spray of polyamines (Spd or Put; 1.0 mM) reduced the accumulation of Mn, with the greatest reduction generated by the treatment of Spd (1.0 mM) compared to the control plants (Fig. 7EeF) Moreover, a subtle reduction of Mn content in the root and the shoot was caused by the combined

treatment of Spd (1.0 mM) and Mn (150 mg kg1) in comparison to Mn (150 mg kg1) stressed plants. 3.11. Confocal studies Propidium iodide penetrate the damaged cell membrane and stains nucleic acids which is visible inside the dead cells as red fluorescent spots. In our study, foliar spray of Spd and/or Put increased cell viability of Brassica juncea roots as compared to the cells of control plants sprayed with double distilled water. Moreover, higher level of Mn (150 mg kg1) resulted in lesser number of living cells as more red fluorescent spots were visible in these cells (Fig. 8). 4. Discussion Our results in the current work revealed that subjection of

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Fig. 3. Effect of polyamines (Spd and Put, 1.0 mM) on Mn induced changes in (A) Maximum quantum yield of PSII, (B) Electrolyte leakage, (C) carbonic anhydrase activity, (D) nitrate reductase activity, of Brassica juncea plants at 45-day stage of growth. All the data are the means of five replicates (n ¼ 5); vertical bars show standard errors (±SE). Means with different letters above the bars are significantly different between thetreatments.

Brassica juncea plants to Mn stress (150 mg kg1) caused a pronounced decrease in all the growth traits than their control plants (Figs. 1 and 2A). Growth reduction by Mn stress could be due to disruption of photosynthesis and balance of nutrient elements (Sheng et al., 2015). Our findings are in conformity with that of Fariduddin et al. (2015) and Hussain et al. (2019), who also noticed a remarkable decrease in growth parameters of Brassica juncea plants in presence of Mn stress. The application of Spd or Put proved favourable to sustain growth and reduced the toxic effects of the higher dose of Mn (150 mg kg1) (Fig. 1AeF). A similar increase in the growth by the exogenous application of PAs was reported by Rady and Hemida (2015) in Triticum aestivum under Cd toxicity and Rady et al. (2016) in wheat under Pb-stress. Involvement of PAs in modulating physiology and biochemistry of plants in presence of HM toxicity has been documented by other workers (Fariduddin et al., 2014; Hasanuzzaman et al., 2019). PAs could have modulated numerous plant processes through alterations in cell division and root development, DNA replication, transcription of genes and signal transduction (Anwar et al., 2015) thereby accountable for overall growth enhancement. In the present study, excess Mn (150 mg kg1) supplied to plants significantly diminished the SPAD value of chlorophyll, however, a subtle increase in SPAD value was observed in the plants which received the lower dose of Mn (30 mg kg1). The decrease in chlorophyll content could be due to a gradual acquisition of a small amount of Mn in the thylakoids conjugated to the outer layer (Lidon et al., 2004), or due to the interference of Mn with the thylakoid stacking and accumulation of pigments (Lidon and Teixeira, 2000a, 2000b). These observations are further corroborated by the

findings of Mukhopadhyay and Sharma (1991) who reported the damage to photosynthetic apparatus in the presence of excess Mn. It is also believed that the toxicity of Mn in plants reduces the levels of Fe, Mg and Ca which induce the suppression of chlorophyll biosynthesis (Shi et al., 2005). Further, as Fe is necessary for chlorophyll synthesis, its deficiency could likely reduce chlorophyll concentrations in plants (Hauck et al., 2003). However, the foliar application of PAs (Spd or Put) increased the SPAD chlorophyll value (Fig. 2B). In the present study, Spd treatment improved Chl content and a similar increase was also described in Vigna radiata grown in presence of salt and zinc stress (Fariduddin et al., 2015). It could be due to the increase in Mg2þconcentration, which is required for Chl synthesis (Lakra et al., 2006). Moreover, Put treatment also improved the SPAD value in Brassica juncea raised from Cd or Pb and salinity stress (Lakra et al., 2016). The detrimental result of HM stress on the growth of plants has been ascribed due to their adverse effects on photosynthetic processes (Nazir et al., 2019a, 2019b). In our study, excess Mn significantly inhibited PN and other traits (gs, Ci and E). However, a slight increment in the aforesaid parameters was noticed in the plants which were supplied with 30 mg kg1 soil of Mn (Fig. 2CeF). A decline in gs and E was noted in soybean with an increasing Mn content and it was inferred because of the interference of Mn with stomatal regulation (Suresh et al., 1987). Excess Mn leads to impairment of the transfer of electrons in the PSII from its donor side up to the reduction of electron acceptors of PSI, restricting the generation of reducing equivalents and thus CO2 assimilation rate (Li et al., 2010). Therefore, in the present study, a decrease in photosynthesis by higher concentrations of Mn could be closely related to the significant damage to the

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4.1

4.2

4.3 Fig. 4. Stomatal response of Brassica juncea at 45 DAS under (A) control, (B) 1.0 mM Spd, (C) 1.0 mM Put, (D)Mn 150 mg kg1 (E) Mn 150 þ Spd, and (F) Mn 150 þ Put using (4.1) compound microscope (40X) (4.2) Scanning electron microscope (3000X).(4.3)root response of Brassica juncea at 45 DAS under (A) control, (B) 1.0 mM Spd, (C) 1.0 mM Put, (D) Mn 150 mg kg1 soil (E) Mn 150 þ Spd, and (F) Mn 150 þ Put at 1500 X using scanning electron microscope.

photosynthetic pigments. Moreover, impairment of PSII reaction centers by the metal further aggravated the functioning of photosynthetic apparatus and led to the reduction in photosynthesis (Fig. 2CeF; Ashraf and Harris, 2013). The foliar application of Spd or Put improved the quantum yield of PSII (Fv/Fm) in Mn treated plants suggesting that Spd or Put helped in protecting PS II against over-excitation due to toxicity that could have provoked loss of unity of thylakoid membrane, the same could also be true in salinity and HM stress (Haldimann and Feller, 2005). Co-application of Spd and epibrassinolide improved the yield of PSII in Brassica juncea under Mn Stress (Hussain et al., 2019). Increase in the overall PN by Spd or Put could be due to the improved contents of chlorophyll and efficiency of PS II. It has been revealed that PAs could modulate the organization and functions of the photosynthetic apparatus by interacting with the thylakoid membrane (Yiu et al., 2009). Moreover, PAs reduce damage to large subunits of Rubisco and Chl from the leaf tissues and also reduces damage to D1, D2 and cyt f (Besford et al., 1993), this could have led to overall improvement in photosynthetic traits (Fig. 2CeF). The potential of Spd or Put in regulating stomatal responses of

plants could also be very significant. In the present study, stomata were found closed in Mn (150 mg kg1) treated plants, whereas the application of Spd or Put to the stressed plants reduced the impact of Mn towards closure of stomata. Put also triggered a slight opening of stomata in wheat (Szalai et al., 2017). The results were further validated by the study conducted through the scanning electron microscopy. The current study, therefore, revealed that PAs had been implicated in the regulation of stomata under Mn stress. The present study showed that the CA and NR activities were ameliorated by lower level of Mn (30 mg kg1). However, the higher level of Mn (150 mg kg1) caused a decline in their activities. The possible reason could be the lack of various nutrient elements like K, Mg, Ca, Fe, and Si triggered by excess Mn which becomes more prominent in the lower quantities of these nutrients (Abou et al., 2002). As a consequence of which these mineral elements check the proper functioning of the plant metabolism. Reduced activity of NR may also be due to limitation in reducing power and/ or low NO 3 availability to plants which occur because of the decreased carbon fixation, decreased uptake of NO 3 by roots and xylem translocation of NO (Kleinhofs and Warner, 1990). 3

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F

Fig. 5. Superoxide localization in the leaves of Brassica juncea plants (45DAS) by nitro blue tetrazolium (NBT) staining under (A) control, (B) Spd 1.0 mM,(C) 1.0 mM Put, and(D), Mn 150 mg kg1. (E) Represents the effect of PAs (Spd and Put, 1.0 mM) on the Mn induced changes in the superoxide content in the leaves of Brassica juncea plants at 45 DAS. All the data are the means of five replicates (n ¼ 5); vertical bars show standard errors (±SE). Means with different letters above the bars are significantly different between the treatments.

Fig. 6. Hydrogen peroxide localization in the leaves of Brassica juncea plants (45DAS) by 3,3- diaminobenzidine solution under (A) control, (B) Spd 1.0 mM,(C) 1.0 mM Put, and(D), Mn 150 mg kg1. (E) Represents the effect of PAs (Spd and Put, 1.0 mM) on the Mn induced changes in the hydrogen peroxide content in the leaves of Brassica juncea plants at 45 DAS. All the data are the means of five replicates (n ¼ 5); vertical bars show standard errors (±SE). Means with different letters above the bars are significantly different between the treatments.

Gajewska and Sklodowska (2009) have reported that decreased NR activity corresponds to the limited accessibility of NO 2 ions, which likely originated from the NO 3 reduction catalyzed by NR. Decreased CA activity could be due to decrease in the partial pressure of CO2 in the stroma, as a result of decreased stomatal conductance (Fig. 2D) and induced stomatal closure. Reduced activity of these enzymes could also be due to the reduction in energy supply because of suppression of photosynthetic electron transport, inhibition of CO2 incorporation, and an indirect inhibition of uptake of substrate (NO 3 ) of enzyme (Panda and Choudhury, 2005). However, foliar treatment with PAs alone or as a follow-up treatment to the stressed plants increased NR and CA activity (Fig. 3CeD). Increased activity of CA by PAs could be due to the impact of PAs on transcription and/or translation (Perez-Leal et al., 2012) and improved CO2 assimilation (Farooq et al., 2009). PAs also triggered the activity of Rubisco, a key enzyme in photosynthetic carbon fixation, in the leaves of cowpea under cinnamic acid stress (Huang and Bie, 2010). Moreover, Spd or Put, increased gs and Ci

levels, in Mn-stressed as well as stress-free plants (Fig. 2DeE) which improve the activity of CA by promoting CO2 assimilation. Our results are further confirmed by Mir et al. (2015) and Rosales et al. (2012). The enhanced activity of NR due to Spd or Put is ascribed to the feature that these stabilizes the plasma membrane integrity which enhanced nutrient uptake thereby, increasing NR activity (Kucera, 2003). In our study, it was revealed that higher concentration of Mn (150 mg kg1 soil) escalated electrolyte leakage. Increase in the EL could be due to the outcome of binding of metal to sulfhydryl group and thus destabilizing the membrane by forming disulfide bonds causing an alteration in organization and functioning of ion channels of membrane (Aravind and Prasad, 2005). However, treatment of PAs to stressed and non-stressed plants restored the loss in EL across the membrane. PAs are involved to stabilize the plant cell membrane by direct binding membrane phospholipids under stress conditions and thereby, provides protection against various stresses (Todorova et al., 2015; Igarashi and Kashiwagi, 2015), this

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Fig. 7. Effect of polyamines (Spd and Put, 1.0 mM) on Mn induced changes in (A)Catalase activity, (B) Peroxidase activity, (C)Superoxide dismutase activity, (D) Proline content (E) Root Mn content (mg g1 DW), and (E) Shoot Mn content of Brassica juncea plants at 45 DAS. All the data are the means of five replicates (n ¼ 5); vertical bars show standard errors (±SE). Means with different letters above the bars are significantly different between the treatments.

seems to be reason for the decrease in EL in the present study. It is well known that HM trigger overproduction of ROS (Georgiadou et al., 2018), whose accumulation causes indirect or direct oxidative damage to the plants exposed to a number of stresses thus, causing significant damages to cellular constituents, especially membrane lipids. As shown in the present study, the  treatment of Mn increased the levels of ROS (O$2 and H2O2) in the leaves, which might have caused the photoinhibition of PSII and PSI which affected plant productivity directly (Takagi et al., 2016). However, the exogenous treatment of PAs (Spd and/or Put)  inhibited this increase and decreased O$2 and H2O2. Polyamines have been reported to elevate the destruction of ROS by scavenging free radicals (Gupta et al., 2013). Plants possess antioxidant enzyme system (e.g. CAT, POX, and SOD) and osmoprotectant, i.e., proline, that control ROS production and maintain cellular homeostasis in plant tissues if exposed to stress (Pandey et al., 2017). During stress

conditions, proline functions as a scavenger of ROS, osmoprotectant and acts as a membrane stabilizer (Kaur and Asthir, 2015). In our present study, it has been shown that higher concentration of Mn enhanced enzymes of antioxidant systems (Fig. 3EeF and 4A). A similar increase in antioxidant enzyme activity (CAT, POX and SOD) under Mn stress has been described in Polish wheat and Brassica juncea (Sheng et al., 2016; Hussain et al., 2019). This increase was further enhanced by treatment of Spd or Put to the Mn stressed plants. Exogenous PAs under HM stress have been shown to regulate antioxidant pathways which serve as an adaptive mechanism of plants for scavenging excessive ROS (Paul et al., 2018). The antioxidant effect of PAs is reported to be because of their anion and cation binding properties which bestow scavenging of free radicals (Groppa and Benavides, 2008). PA binding to anions (phospholipid membranes, nucleic acids) leads to a high local concentration at particular sites prone to oxidations, while cations binding ability

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Put to the stressed and stress-free plants modified the stomatal movement, improved photosynthetic and growth traits and also enhanced antioxidant system, whereas the accumulation of Mn and  production of superoxide radicle (O$2 ) and H2O2 were decreased. Out of the two PAs (Spd or Put), Spd was more prominent in restoring the damage caused by Mn (150 mg kg1) stress. Conflicts of interest The authors declare that No conflict of interest exists. Acknowledgements We acknowledge support by UGC New Delhi India as Non Net fellowship (No. 15PHDBT010) and Aligarh Muslim University, Aligarh, India. References

Fig. 8. Confocal micrographs showing propidium iodide staining of cell wall and nuclei of damaged root cells of Brassica juncea at 45 DAS (A) control, (B) Spd (1.0 mM), (C) Put (1.0 mM), (D)Mn 150 mg kg1, (E) Mn 150 þ Spd, and (F) Mn 150 þ Put.

effectively prevents generation of ‘‘active oxygen’’ (hydroxyl radicals and singlet oxygen). It is also well known that various abiotic stresses enhance the levels of PAs, such as Put and Spd in a plant tissue (Liu et al., 2015). Additionally, the exogenous application of PAs also increase the endogenous PAs because of the interconver
l et al., 2015). Oxidative deamination of PAs can sion of PAs (Pa generate ROS like H2O2 and may result in the cellular damage under stress conditions (Stewart et al., 2018). However, H2O2 is also considered a signaling molecule and can enter the signal transduction pathway causing an activation of antioxidant defense response (Nazir et al., 2019b). Therefore, PAs as regulators of redox homeostasis can play dual role in oxidative stress of plants (Saha et al., 2015). It is also obvious from our present study that the proline content enhanced in the plants treated with Mn and/or PAs (Spd or Put). Such an increase in proline was also observed under the effects of other metals such as Ni and Cu (Nazir et al., 2019a, 2019b). Proline has been described to detoxify free radicals and function as plasma membrane stabilizer and various other micromolecules and also act as a C and N source for immediate recuperation from the stress (Jain et al., 2001; Dar et al., 2016), thereby, providing protection to the plants under severe stress conditions. Plants grown with Mn had more content of Mn (Fig. 7EeF). Moreover, the accumulation of Mn in roots was prominently higher as compared to shoots because of slow translocation of metals from roots to aerial parts, thereby helping the plants to counter the damage caused by the metal (Adrees et al., 2015). Our study is further supported by findings of Singh and Sinha (2005). Application of PAs (Spd or Put) decreased the accumulation of Mn in root and shoots of mustard plants under both stress and stress free condition (Fig. 7EeF).

5. Conclusion The present study revealed that Mn stress (150 mg kg1) reduced the stomatal movement, photosynthetic traits, and biochemical parameters in Brassica juncea. Application of Spd or

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