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. 2019 May 14;25(4):905–919. doi: 10.1007/s12298-019-00672-6

24-epibrassinolide and spermidine alleviate Mn stress via the modulation of root morphology, stomatal behavior, photosynthetic attributes and antioxidant defense in Brassica juncea

Anjuman Hussain 1, Faroza Nazir 1, Qazi Fariduddin 1,✉
PMCID: PMC6656853  PMID: 31404216

Abstract

Brassinosteroids and polyamines are generally used to surpass different abiotic stresses like heavy metal toxicity in plants. The current study was conducted with an aim that 24-epibrassinolide (EBL) and/or spermidine (Spd) could modify root morphology, movement of stomata, cell viability, photosynthetic effectiveness, carbonic anhydrase and antioxidant enzyme activities in Brassica juncea under manganese (Mn) stress (30 or 150 mg kg−1 soil). EBL (10−8 M) and/or Spd, (1.0 mM) were applied to the foliage of B. juncea plants at 35 days after sowing (DAS), grown in the presence of Mn (30 or 150 mg kg−1 soil). High Mn concentration (150 mg kg−1 soil) altered root morphology, affected stomatal movement, reduced the viability of cells and photosynthetic effectiveness and increased the production of reactive oxygen species (O·−2 and H2O2) in the leaves and antioxidant defense system of B. juncea at 45 DAS. Furthermore, exogenous treatment of EBL and Spd under stress and stress- free conditions improved the aforesaid traits while decreased the O·−2 and H2O2 production. Therefore, EBL and Spd could be applied to the foliage of B. juncea plants for the better growth under metal stress.

Keywords: Epibrassinolide, Manganese, Photosynthesis, Root morphology, Spermidine, Stomata

Introduction

In agro-ecosystems, various micronutrients like iron (Fe), zinc (Zn), nickel (Ni), cadmium (Cd), copper (Cu), and manganese (Mn), are regarded as heavy metals. At adequate quantities, these trace elements are crucial for plants but higher concentrations of most of them are harmful (Srivastava and Dubey 2011). Mn is one of the essential trace metal needed by the plants for the normal metabolism and development (Marschner 2012). Mn is a main constituent of oxygen evolving complex of photosystem II and catalyze water splitting reaction in photosystem II, generating electrons that drive photosynthesis (Schmidt et al. 2016). It acts as a cofactor of several enzymes involved in a variety of processes including plant defense against ROS-induced oxidative stress (Goussias et al. 2002). However, when present in higher concentrations, it can become toxic to plants which results in disorganized protein structure, deactivated enzyme activity by attaching to thiols, declined rate of photosynthesis, inhibited uptake and transport of various mineral elements (Srivastava and Dubey 2011; Fernando and Lynch 2015).

Polyamines (PAs) are the aliphatic cations with low molecular weight and are found throughout the life kingdoms. PAs either in free state (mainly putrescine, spermidine and spermine) or in conjugation with hydroxycinnamic acids are essential for the development of plants and take part in the biotic and abiotic stress responses (Tiburcio et al. 2014) and also trigger a wide response in various physiological processes (Silveira et al. 2013). PAs also play a major role in balancing ROS levels of cells, thus protecting the cells from oxidative stress (Saha et al. 2015). PAs also regulate stress tolerance through their interaction with other protective molecules like abscisic acid (ABA), salicylic acid (SA), and brassinosteroids (BR) (Hatmi et al. 2015; Serna et al. 2015).

BRs, a class of polyhydroxy steroid phytohormones have tremendous growth promoting impact (Bajguz and Piotrowska-Niczyporuk 2014; Vardhini 2017). They are remarkably efficient in mitigating the damaging consequences of environmental factors and improving the metabolism of plants (Surgun et al. 2016). 24-epibrassinolide (EBL) and 28-homobrassinolide (HBL) are the active analogues of BRs that are extensively applied exogenously to the plants to improve growth and development (Khripach et al. 2000). Application of EBL has the potential to increase activity of antioxidant enzymes and accretion of osmoprotectives (Ozdemir et al. 2004), and enhance photosynthetic productivity and the transport of photosynthates from source to the sink (Shahbaz et al. 2008). Exogenously applied BRs confer tolerance to HM stress (Yusuf et al. 2012), waterlogging (Bajguz and Hayat 2009), and drought stress (Fariduddin et al. 2009).

Recent advancements in interaction of plant hormones have depicted a viable role of plant growth regulators in the regulation of several abiotic stresses (Fariduddin et al. 2014). But the studies dissecting the roles of PAs and BRs in mitigating Mn stress is completely lacking. So the present study was conducted with the objective how exogenous application of EBL and/or Spd to stress free and stressed plants could improve root morphology, regulate stomatal behavior, increase cell viability and photosynthetic attributes, reduce the generation of superoxide radicals,  and enhance growth characteristics and antioxidant enzyme activities in B. juncea.

Materials and methods

Plant material

Mustard (B. juncea L. var. varuna) seeds 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 double distilled water at least thrice.

EBL and Spd preparation

The stock solutions of EBL or Spd have been made individually by dissolution of the desired amount of Spd and EBL in 5 mL of ethanol, in volumetric flasks (100 mL) and the final volume was kept up to the mark by double distilled water (DDW). The specified concentrations of EBL or Spd were formulated by diluting stock solution with DDW. Before its application, 5 mL of surface-active agent “Tween-20” was added to it.

Treatment pattern and experimental design

The disinfected seeds were sown in earthen pots of 20-cm diameter, loaded with re-established soil (sandy loam soil and farmyard manure; blended in the proportion of 6:1) and placed in the naturally lit net house of the Botany Department, Aligarh Muslim University, Aligarh (India). On the 7th day, subsequent to sowing (DAS), thinning was carried out and three plants for each pot were maintained. The experiment was accomplished in a totally randomized block layout. Forty five pots were arranged into 9 sets of 5 pots each (replicates) corresponding to one treatment. At 25-day phase of development, seedlings were subjected to Mn, given as MnSO4 (30 or 150 mg kg−1) through the soil and then permitted to grow. At 35-day period, plants were sprayed with DDW (control) or 1.0 mM Spd or 10−8 M EBL. Each plant was sprinkled thrice. The spout of the sprayer was balanced so that in one sprinkle it drew out 1 mL. The growth parameters as well as physiological and biochemical traits of plants have been accessed after harvesting at 45-day stage. Rest of the plants were allowed to grow up to maturity and were harvested at 120 DAS to study the yield attributes.

Growth traits

Plants with adhering soil were uprooted from the pots with utmost care and washed to remove the adhering soil. With the aid of the scale with metric units, centimeters (cm), length of root and shoot was measured. Fresh mass of root and shoot of each plant was calculated with the assistance of electronic weighing machine. The samples of root and shoot were put in an oven kept running at 80 °C for 72 h and their dry mass was noted. Leaf area was calculated with the assistance of leaf area meter (ADC Bioscientific, UK).

Determination of SPAD (soil and plant analysis development) value of chlorophyll

With the aid of SPAD chlorophyll meter (SPAD-502; Konica, Minolta sensing, Inc., Japan), SPAD value of chlorophyll in intact leaves of the plant was determined.

Analysis of leaf gas-exchange parameters

The net 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 μmol mol−1 and 800 μmol mol−2 s−1, respectively.

Estimation of leaf electrolyte leakage

Quantification of total ions spilled from the foliage was executed by the procedure depicted by Sullivan and Ross (1979). Leaf segments (25) were placed in a boiling test tube which contained 10 mL of deionized water and 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). Subsequently, the samples had been boiled at 100 °C for 10 min, and EC was again noted (ECc). The leaf electrolyte leakage was ascertained by the formula:

EL%=ECb-ECa/ECc×100.

Assay for carbonic anhydrase (CA) and nitrate reductase (NR) activity

CA activity was determined with the procedure 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. 0.05 N HCl was used as the titrating agent in the titration processes. The enzyme activity was formulated on per gram fresh mass basis.

NR activity 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-1-naphthylethylenediamine hydrochlorides were added. The intensity of the pink colour generated was recorded at 540 nm on a spectrophotometer.

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 in chilled mortar and pestle. The centrifugation of homogenates was done at 12,000 × g for 20 min at 4 °C and the supernatant generated was utilized for estimation of antioxidant enzyme (CAT, POX, and SOD) activities.

Activity of 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 µL 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 µL 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.

The activity of SOD was measured by assessing its capability to lessen the photochemical reduction of nitro blue tetrazolium (NBT) following the procedure by Beauchamp and Fridovich (1971). The reaction mixture consisted of 50 mM phosphate buffer (pH 7.8), 9.9 mM L-methionine, 55 µM NBT, 2 mM EDTA, 0.02% Triton X-100 and 40 µL enzyme extract. Lastly, 1 mM riboflavin was added to reaction mixture. Control set was prepared with similar procedure. The activity was measured by reading the absorbance at 560 nm for 2 min at 25 °C. One unit of SOD activity was calculated as the measure of enzyme that brought about 50% reduction of NBT at 25 °C.

Leaf proline content

Proline level in fresh leaf samples was estimated followed the protocol of Bates et al. (1973). Sample extraction was done in sulfosalicylic acid, to which an equal volume of glacial acetic acid and ninhydrin solutions was 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.

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

Reactive oxygen species estimation

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) and 3, 3-diaminobenzidine (DAB) respectively, for staining leaf samples. The samples were immersed in 3,3′-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).

Confocal microscopic study for cell viability

Cleaned and washed roots of B. juncea were cut with the help of sharp knife and were dipped in propidium iodide dye (5 μM) for 30-35 min. After this, these stained roots were put on glass slide and were covered with cover glass and were studied under confocal microscope.

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 gold–palladium and the samples were studied using the JEOL JSM–JSM 6510 scanning electron microscope.

Statistical analysis

Data have been statistically interpreted via the usage of SPSS, 17.0 for windows (SPSS, Chicago, IL, USA). Analysis of variance (ANOVA) was computed on the data to calculate the least significant difference (LSD) between treatments implied with 5% level of probability.

Results

Growth characteristics

The growth characteristics increased slightly in the plants supplied with lower concentration of Mn, while, the higher dose of Mn (150 mg kg−1) caused a remarkable decrease of length of shoot and root by 29.81% and 35.30%, shoot and root fresh mass by 33.62% and 35.01%, shoot and root dry mass by 34.13% and 37.81%, and leaf area by 34.27% in comparison to their respective controls (Figs. 1, 2a–c). On the contrary, the foliar application of Spd prominently increased all the growth parameters, but this increase was lower than the effect of foliar applied EBL and their combination (EBL + Spd). Maximal increase in all the growth biomarkers was noted with the combined foliar application of EBL and Spd, over the stress free control plants. Furthermore, the additive effect of Spd and EBL substantially revived the damage caused by Mn toxicity.

Fig. 1.

Fig. 1

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Effect of Spd (1.0 mM) and EBL (10−8 M) on Mn-induced changes in a shoot length, b root length, c shoot fresh mass, and d root fresh mass of B. juncea plants at 45 DAS

Fig. 2.

Fig. 2

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Effect of Spd (1.0 mM) and EBL (10−8 M) on Mn-induced changes in a shoot dry mass, b root dry mass, c leaf area, and d chlorophyll content of B. juncea plants at 45 DAS

Physiological parameters

A remarkable increase in SPAD level (chlorophyll content) and Fv/Fm was generated by the foliar application of EBL and Spd (Figs. 2d, 4a) and the increase by EBL was 34.72% and 25.82% respectively and increase by Spd was 29.67% and 18.28%, respectively, as compared to respective controls. However, a decrease of 27.21% in chlorophyll content was observed in Mn (150 mg kg−1) fed plants against control plants. Additionally, the toxicity generated by Mn was totally restored by the combined dose of Spd and EBL. In the same way, PN and its related attributes showed a prominent decline in presence of high level of Mn (150 mg kg−1) and reduced the  PN by 33.21% from untreated control plants (Fig. 3). Application of EBL and Spd alone or in combination caused an appreciable increase in all the above traits compared with control and Mn treated plants. Furthermore, this combined dose effectively counteracted the Mn induced decline in PN and related traits.

Fig. 4.

Fig. 4

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Effect of Spd (1.0 mM) and EBL (10−8 M) on Mn-induced changes in a Maximum quantum yield of PSII, b electrolyte leakage, c carbonic anhydrase activity, and d nitrate reductase activity of B. juncea plants at 45 DAS

Fig. 3.

Fig. 3

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Effect of Spd (1.0 mM) and EBL (10−8 M) on Mn-induced changes in a net photosynthetic rate, b stomatal conductance, c internal CO2 concentration, and d transpiration rate of B. juncea plants at 45 DAS

Electrolyte leakage

Plants subjected to Mn stress (150 mg kg−1) had more EL (24.05%) compared to the control (Fig. 4b). Application of EBL and Spd to non- stressed plants reduced electrolyte leakage, however, prominent decrease of 22.28% was observed in the plants supplied with EBL compared to control plants. Additionally, EBL and Spd application to the stressed plants (150 mg kg−1) also resulted in a significant decrease in the electrolyte leakage.

CA and NR activity

A subtle increase in the activities of CA and NR has been observed in plants exposed to lower level of Mn (30 mg kg−1) (Fig. 4c, d), but lower activities of these were noted in the plants that were fed with higher level of Mn (150 mg kg−1). Application of EBL alone or in combination with Spd increased the activity of both CA and NR over their respective controls. Treatment of EBL enhanced CA and NR activity by 38.17% and 42.31%, respectively, while Spd enhanced these activities by 26.38% and 33.12%, respectively, than untreated control plants. However, a maximal increase of 44.34% and 48.31% in CA and NR activity was observed in plants that received the combined dose of EBL and Spd along with 30 mg kg−1 Mn. Additionally, the combined treatment of EBL and Spd efficiently overcome the toxicity generated by Mn (150 mg kg−1), thus, enhancing the activities of NR and CA.

Antioxidant enzymes

A remarkable enhancement in the activity of antioxidant enzymes (CAT, POX, and SOD) was noticed by the treatment with EBL, Spd and Mn (Fig. 5a–c), over the control plants. A remarkable increase of 77.03% for CAT, 85.28% for POX and 87.56% for SOD was observed in the plants treated with Mn (150 mg kg−1) stress pursued by the combined treatment of Spd and EBL.

Fig. 5.

Fig. 5

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Effect of Spd (1.0 mM) and EBL (10−8 M) on Mn-induced changes in a Catalase activity, b peroxidase activity, c superoxide dismutase activity, and d proline content of B. juncea plants at 45 DAS

Proline content

Mn stress increased the levels of proline compared to control plants. Application of Spd and EBL to the foliage of stress free plants also exhibited an enhanced level of proline (Fig. 5d). However, the maximal increase in proline content (70.21%) was noticed in the leaves of plant that were subjected to 150 mg Mn kg−1 and subsequently treated with combined dose of Spd and EBL, compared to control plants.

Mn accumulation in root and shoot

The accumulation of Mn was higher in the root in comparison to the shoot (Fig. 6a, b). But foliar application with EBL and Spd reduced Mn accumulation in roots and shoots, and the greatest reduction was noted with the application of combined dose of Spd and EBL against control plants.

Fig. 6.

Fig. 6

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Effect of Spd (1.0 mM) and EBL (10−8 M) on Mn-induced changes in a root Mn content (μg g−1 DW), and b shoot Mn content of B. juncea plants at 45 DAS

Production of superoxide (O·−2) and hydrogen peroxide (H2O2)

O·−2 and H2O2 level in leaves was respectively depicted by blue and brown coloured spots (Fig. 7a, b). Leaf discs from Mn fed plants exhibited more pronounced spots as compared to control plants. Moreover, the leaves of plants supplied with EBL and Spd had less pronounced spots as compared to stressed plants.

Fig. 7.

Fig. 7

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Production of Superoxide ion by tetrazolium chloride (NBT) (a), hydrogen peroxide by 3, 3-diaminobenzidine (DAB) staining of leaves (b) and confocal micrographs showing propidium iodide staining of cell wall and nuclei of damaged root cells (c) of B. juncea at 45 DAS under (A) control, (B) Spd (1.0 mM), (C) EBL (10−8 M), (D) EBL + Spd and (E) Mn 150 mg kg−1

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 EBL and/or Spd increased cell viability of B. juncea roots where the combination of EBL + Spd induced maximum effect as compared to the cells of control plants sprayed with double distilled water. Moreover, higher level of Mn (150 mg kg−1) resulted in lesser number of living cells as more red fluorescent spots are visible in these cells (Fig. 7c)

Compound microscopy and SEM imaging

Stomatal behavior was significantly affected in the plants treated with Mn (30 or 150 mg kg−1). The leaves of Mn (150 mg kg−1) stressed plants had reduced stomatal aperture (Fig. 8a, b). Foliar application of EBL and Spd in the absence of Mn stress exhibited wide stomatal aperture compared to the control, however, EBL proved more effective for widening the stomatal width aperture. These observations were further corroborated by SEM observation (Fig. 8a).

Fig. 8.

Fig. 8

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Stomatal response of B. juncea at 45 DAS under (A) Control, (B) Spd (1.0 mM), (C) EBL (10−8 M), (D) EBL + Spd, and (E) Mn 150 mg kg−1 using scanning electron microscope  at 2000 ×) (a), compound microscope (40 ×) (b), and root response (c) of B. juncea at 45 DAS under (A) control, (B) Mn 30 mg kg−1 soil, and (C) Mn 150 mg kg−1 soil at 4000 × using scanning electron microscope

SEM images of root samples (Fig. 8c) revealed that the plants which received higher concentration of Mn (150 mg kg−1) had distorted root morphology, while intact root morphology was observed in the control plants.

Yield characteristics

Plants raised from Mn stress (150 mg kg−1) had significant decrease in all the yield parameters (number of pods per plant, number of seeds per pod, 100 seed mass or seed yield per plant). Stress free plants subjected to EBL and/or Spd or their combination (EBL + Spd) had remarkably higher number of pods and seed yield per plant compared to the control plants, at harvest (Fig. 9a, b). Moreover, the higher number of pods and the seed yield per plant were exhibited by the plants which received the combined dose of EBL + Spd under stress free conditions and the increase was 41% and 39.58%, respectively, over their controls. Additionally, EBL + Spd applied to foliage as a follow-up treatment to stressed plants completely neutralized the adverse effects of Mn (150 mg kg−1) stress in case of number of pods and seed yield per plant, at harvest.

Fig. 9.

Fig. 9

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Effect of Spd (1.0 mM) and EBL (10−8 M) on Mn-induced changes in a pods plant−1, b seed yield of B. juncea plants at 120 DAS

Discussion

Present study showed a remarkable decrease in all the growth characteristics of the plants grown in presence of Mn (150 mg kg−1). Diminished growth characteristics has been ascribed to Mn induced variation in various metabolic activities like photosynthesis (Wang et al. 2013) and to a gradual accumulation of Mn in shoot and root (Fig. 9a, b). A similar decrease in all growth characteristics due to Mn excess was described by Parashar et al. (2014) in B. juncea. But, supplication of EBL and/or Spd to the metal treated plants decreased deleterious effects of Mn and sustained the growth of plants (Figs. 1, 2a–c). Stimulation of growth by EBL in presence of metal stress was also observed in Vigna radiata by Yusuf et al. (2012). BR triggered such responses of plants could have been mediated through advancement and enhancement in cell division (Shahid et al. 2011) and also due to alteration in structure and permeability of plasma membrane under stressed conditions (Choudhary et al. 2012a, b). Further, growth enhancement by PAs might be due to their involvement in various plant processes like replication of DNA, gene transcription, and cell multiplication (Bais and Ravishankar 2002). Interestingly, the combined dose of EBL + Spd resulted in the prominent increase in all the growth traits under Mn stress which may be because of their additive effects.

Plant metabolism and development are primarily associated with the photosynthetic efficiency and increased formation of chlorophyll in plants. In the present study, excess Mn decreased chlorophyll content and various photosynthetic parameters (PN, gs, Ci, E, and Fv/Fm). The depletion in the level of chlorophyll might be due to Mn induced deficiency of Fe, Mg, and Ca which are essential for chlorophyll biosynthesis (Shi et al. 2005) and probable reason for decreased photosynthetic parameters could be the increased concentration of soluble hexose sugar in the leaves due to Mn excess which causes suppression of photosynthetic genes, thus diminishing the rate of CO2 assimilation and concentration of Rubisco (Sheen 1994). Similar decrease in the photosynthetic parameters was also reported in perennial ryegrass (Ribera et al. 2013). However, the SPAD value of chlorophyll and photosynthetic parameters significantly increased due to application of EBL and Spd individually and in combination under stress or non-stress condition. Increased photosynthetic efficiency by EBL has also been suggested by Jan et al. (2018) from Pisum sativum under Cd toxicity and Fariduddin et al. (2015) in B. juncea under Mn stress. This positive regulation of photosynthesis by EBL might be due to EBL induced stimulation of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and their protective role to over excitation of PSII, that might have resulted in the impairment from the loss of constancy in thylakoid membrane (Fariduddin et al. 2015).

Various abiotic stresses like HM stress have been reported to be responsible for ROS mediated oxidative stress (Adrees et al. 2015b). As revealed in the present study, Mn treatment increased the levels of ROS in the form of O·−2 and H2O2 as seen in the leaves, which could have led to photoinhibition of PSII and PSI directly affecting the plant productivity (Takagi et al. 2016). However, the exogenous treatment of EBL and Spd inhihibited this increase and decreased O·−2 and H2O2. A similar loss of O·−2 and H2O2 by the combined treatment of BRs and PAs has been reported by Choudhary et al. (2012a, b) in Raphanus sativus. To cope with ROS generated oxidative stress, plants also utilize an efficient enzyme operated defense mechanism like SOD, CAT, and POX as well as non-enzymatic defense system like proline (Gao et al. 2008). The present work unveiled an increase in the antioxidant system along with the levels of proline accumulation in presence of Mn and this increase was further enhanced by the application of EBL and/or Spd. Similar increase in activity of antioxidant enzyme system under Mn stress has been observed in Polish wheat (Sheng et al. 2016) and in Vigna radiata under boron toxicity (Yusuf et al. 2011a). Increased activities of antioxidant enzymes by EBL were also observed by Dalyan et al. (2018) in B. juncea under Pb stress. The most probable reasons for EBL induced protection against oxidative stress might be due to the expression of regulatory genes involved in defense, like RBOH (respiratory burst oxidase homologue), MAPK1 (mitogen-activated protein kinase 1), and MAPK3 (Mitogen-activated protein kinase 3) (Xia et al. 2009). Similarly, the PAs, especially Spd, provide tolerance against Mn toxicity by preventing the formation of ROS by inducing the activities of antioxidants enzymes, which might participate in instigating the antioxidant defense and by avoiding lipid peroxidation reactions (Mostofa et al. 2014). The increased levels of proline by the application of EBL may be because of the EBL induced expression of the genes involved in proline biosynthetic pathway (Ozdemir et al. 2004). In our present work, the treatment of EBL and/or Spd neutralized ROS by activating the antioxidant system and elevated level of proline resulted in enhanced capability of tolerance to Mn stress, well reflected by improved growth and photosynthesis.

In the present work, reduced CA activity was observed in B. juncea under Mn stress. The loss of CA activity due to Mn toxicity might be explained as a result of decreased internal CO2 concentration (Fig. 3c). However, foliar treatment with EBL and Spd individually or as a follow-up treatment to Mn stressed plants increased the CA activity. BR mediated enhanced activity of CA has also been reported in the cultivars of Triticum aestivum and Vigna radiata under Ni stress (Yusuf et al. 2011b, 2012). Increased activity of CA by BRs and Spd could be due to their role at transcriptional and/or translational levels of biosynthetic genes (Bajguz 2000; Perez-Leal et al. 2012), thus generating a prominent effect on the activity of CA (Fig. 4c). The similar results were obtained by Mir et al. (2015) and Rosales et al. (2012).

The present work demonstrated that the activity of NR was enhanced by lower level of Mn (30 mg kg−1). However, the higher level (150 mg kg−1) caused a decline in its activity which might be because of the Mn induced deficiencies of other elements like N, Ca, Mg, K, Fe, and Si (Abou et al. 2002), thereby limiting the nitrate uptake (Hernández et al. 1996) which acts as a substrate of NR. However, application of EBL and/or Spd to stressed and stress free plants increased the activity of NR. This increased activity of NR is because of the membrane stabilization by BRs and Spd which might promote the uptake of substrate for NR (NO3−), thus enhancing its activity (Kubis 2006). Same results have also been shown in Vigna radiata (Mir et al. 2015), Raphanus sativus (Ramakrishna and Rao 2015) when treated with BRs.

A significant increment in electrolyte leakage was noticed in our present work in B. juncea under Mn stress (150 mg kg−1). The enhanced value of electrolyte leakage might be the consequence of metal binding to sulfhydryl group resulting in the formation of disulfide links, thereby destabilizing the organization and function of membrane ion channels (Aravind and Prasad 2005). However, application of EBL and/or Spd to the stressed plants restored the loss of EL across membrane. EBL mediated decrease in ionic leakage was also reported in Phaseolus vulgaris under salinity and Cd stress (Rady 2011). BRs and PAs maintain membrane integrity and stability by increasing the level of the antioxidant system that provides protection to plants against the oxidative stress (Groppa et al. 2007).The prospective function of EBL and Spd in regulation of stomatal responses in plants can be noteworthy. In our study, Mn fed plants exhibited closed stomata than control plants. However, normal open stomata in the Mn stressed plants were reinstated by the application of EBL and Spd. Stomatal responses due to PAs and BRs might be due to the induction of variations in H2O2 and redox state of guard cells as reported in tomato (Xia et al. 2014).

Plants exposed to Mn (150 mg kg−1) had higher content of Mn in roots than shoots. The possible reason for this may be the slow translocation of metals from roots to aerial parts thereby helping the plants to counter the damage caused by the metal (Adrees et al. 2015a). However, the foliar application of EBL and/or Spd individually or their combined application led to a decrease in accumulation of Mn in roots and shoots under stressed conditions, probably by forming various phytochelatins (Choudhary et al. 2012a, b).

Plants raised from soil supplied with higher concentration of Mn showed decline in yield parameters (number of pods per plant, number of seeds per pod, 100 seed mass and seed yield per plant). Similar reduction in yield was reported by Soltangheisi et al. (2014) in sweet corn under Mn stress. Decreased yield in the stressed plants could be related to the slower rate of photosynthesis and subsequent restriction on growth and leaf area in presence of metal stress. However, EBL or Spd application to the leaves of stressed and non-stressed plants enhanced the yield characteristics which could be because of the improved growth and photosynthesis.

Conclusion

The present study revealed that the high concentration (150 mg kg−1) of Mn supplied to B. juncea through soil reduced the growth, distorted root morphology and altered the stomatal behavior and further, decreased the photosynthetic and biochemical attributes. Application of EBL and Spd through foliage proved beneficial in terms of reduced ROS production, improved photosynthesis and also restored stomatal openings in mustard plants. Therefore, EBL and Spd can be applied to the foliage of B. juncea plants for the better growth under metal stress as they enhance the parameters studied which finds support by the data of microscopic studies.

Acknowledgements

Anjuman Hussain is thankful to UGC (New Delhi) India for providing the research fellowship. We are also grateful to University Sophisticated Instrumentation Facility (USIF) A.M.U., Aligarh for SEM analysis.

Abbrevations

ABA

Abscisic acid

ANOVA

Analysis of variance

BR

Brassinosteroid

CA

Carbonic anhydrase

CAT

Catalase

Cd

Cadmium

Ci

Intercellular CO2 concentration

Cu

Copper

DAS

Days after sowing

DDW

Double distilled water

E

Transpiration rate

EBL

24-epibrassinolide

EC

Electron conductivity

EL

Electrolyte leakage

Fe

Iron

gs

Stomatal conductance

HBL

28-homobrassinolide

HM

Heavy metals

LSD

Least significant difference

Mn

Manganese

NR

Nitrate reductase

Ni

Nickel

PA

Polyamines

POX

Peroxidase

ROS

Reactive oxygen species

SA

Salicylic acid

SEM

Scanning electron microscopy

SOD

Superoxide dismutase

Spd

Spermidine

SPAD

Soil and plant analysis development

Zn

Zinc

Funding

Funding was provided by UGC New Delhi, India, in the form of Non-NET fellowship (Grant No. gj1136) from Dec. 07, 2015.

Footnotes

Publisher's Note

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