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. 2014 Aug 13;20(4):449–460. doi: 10.1007/s12298-014-0259-x

Brassinosteroid-mediated evaluation of antioxidant system and nitrogen metabolism in two contrasting cultivars of Vigna radiata under different levels of nickel

Mohammad Yusuf 2, Qazi Fariduddin 2,✉, Iqbal Ahmad 1, Aqil Ahmad 2
PMCID: PMC4185052  PMID: 25320468

Abstract

The role of 28-homobrassinolide (HBL) in countering nickel-induced oxidative damage through overexpression of antioxidant enzymes and proline in Vigna radiata has been investigated. Two varieties of V. radiata, one sensitive to Ni (PDM-139) and the other tolerant to Ni (T-44), were sown in the soil fed with different levels (0, 50, 100 or 150 mg kg−1) of Ni, and at 29-day stage, foliage of plants was applied with deionized water (control), 10−8 or 10−6 M of HBL. The plants were sampled at 45-day stage of growth to assess various physiological as well as biochemical characteristics. The remaining plants were allowed to grow up to maturity to study the yield characteristics. The growth traits, leghemoglobin, nitrogen and carbohydrate content in the nodules, leaf chlorophyll content, photosynthesis efficiency, leaf water potential, activities of nitrate reductase, carbonic anhydrase and nitrogenase decreased proportionately with the increasing concentrations of nickel, whereas electrolyte leakage, various antioxidant enzymes viz. catalase, peroxidase and superoxide dismutase and accumulation of proline increased at 45-day stage. However, the exogenously applied HBL to the nickel-stressed or non-stressed plants improved growth, nodulation and photosynthesis and further enhanced the various antioxidant enzymes viz. catalase, peroxidase and superoxide dismutase and accumulation of proline. The deleterious impact of Ni on the plants was concentration dependent where HBL applied to the foliage induced overexpression of antioxidant enzyme and accumulation of proline (osmolyte) which could have conferred tolerance to Ni up to 100 mg kg−1, resulting in improved growth, nodulation, photosynthesis and yield attributes.

Keywords: Brassinosteroids, Nickel, Nitrogen metabolism, Oxidative stress, Proline

Introduction

Nutritional (mineral) stress has risen at an accelerating pace in recent decades due to various factors such as negligence of the farmer leading to nutrient deficiency or excess supply of nutrients. Supra-optimal concentrations of heavy metals such as Cu, Zn and Ni affect growth, development and yield of plants (Sresty and Rao 1999). However, Cu, Zn and Ni are essential micronutrients at low concentrations and it has been shown that plants cannot complete their life cycle in the absence of these elements (Barker 2006) but the level of these elements proved fatal above permissible limit and lead to the cellular injuries and in extreme cases death of plant which result into the loss of crop production worldwide.

Availability of Ni in the growing medium exerts adverse effect on plant growth and development (Chen et al. 2009; Yusuf et al. 2011). Moreover, visual symptoms of Ni toxicity are Fe-mediated deficiency such as chlorosis followed by yellowing and necrosis of leaves (Sinha and Pandey 2003). Low foliar Fe levels are closely associated with high concentrations of Ni in the growing medium. Plants grown in the environment containing Ni are iron deficient due to interaction of Ni-Fe, inhibiting Fe uptake and transportation in plants as well as preventing Fe to perform physiological functions (Rahman et al. 2005). Excessive amounts of Ni (10–1,000 mg/kg DW) alters many biochemical and physiological processes including photosynthesis and respiration (Llamas et al. 2008; Yusuf et al. 2011), mineral nutrition (Chen et al. 2009), membrane functions (Llamas et al. 2008) and water uptake. Jocsak et al. (2005) revealed that Ni toxicity mainly dealt with organic acids in plants in terms of transportation and detoxification, and similarly to the toxic response of other heavy metals, Ni toxicity also results in the induction of reactive oxygen species (ROS) such as ·OH and H2O2 (Gajewska and Sklodowska 2007) and this ROS has capability to oxidize lipids, proteins and nucleic acids causing membrane damage that eventually leads to programme cell death (Del Rio et al. 2003). In the recent years, it was well documented that proline accumulates in many plant species in response to environmental stress and plays pivotal role in maintaining cellular homeostasis, including redox balance and energy status. Proline can act as a signalling molecule to modulate mitochondrial functions, influence cell proliferation or cell death and trigger specific gene expression, which might play an essential role in recovery of plants from various abiotic stresses (Szabados and Savoure 2009).

A steroidal plant hormone known as brassinosteroids plays significant roles in various physiological processes of plant growth and development and also in various adaptive responses to environmental cues (Divi et al. 2010). Moreover, brassinosteroids (BRs) have been shown to increase photosynthesis and enhance tolerance against various stresses such as cold, salt, high temperature, oxidative and various heavy metal stresses—namely cadmium, aluminium and copper (Fariduddin et al. 2011). In addition to this, BRs have the ability to enhance biological yield and improve stress tolerance in plants so as to employ them in phytoremediation (Barbafieri and Tassi 2011). Several studies have also shown that BR-induced tolerance was associated with increased activity of antioxidants (Yusuf et al. 2012) and synthesis of heat shock proteins (HSPs) (Divi et al. 2010). However, the mechanisms by which exogenously applied BRs influence nitrogen metabolism, photosynthetic capacity and antioxidant system as well as proline accumulation under Ni stress in Vigna radiata are poorly understood.

Legume crops are sensitive to excess heavy metal in the growing media (Ahmad et al. 2012) which are reflected as lower nitrogenase activity, reduced plant biomass, disrupted nodule ultrastructure, number of nodules and induced nodule senescence, reduced dry matter accumulation in roots, shoot and leaf and adversely affected metabolic activities like photosynthesis and respiration (Noriega et al. 2007). On the basis of the above reports, the objectives of this study were to examine whether 28-homobrassinolide (HBL) play a role in the mitigation of Ni-induced oxidative stress and how the equilibrium between antioxidant system and ROS will be affected by foliar HBL and also assess its potential to protect nitrogen metabolism, biomass production and various metabolic activities in Ni-tolerant and sensitive varieties of V. radiata.

Materials and methods

Experimental setup and treatment pattern

The surface-sterilized seeds of V. radiata cultivars T-44 (Ni-tolerant) and PDM-139 (Ni-sensitive) were inoculated with specific Rhizobium spp. and were sown in the earthen pots of 10 in. diameter filled with sandy loam soil and farmyard manure (mixed in the ratio 6:1) supplemented with different levels of Ni (0, 50, 100 or 150 mg Ni kg−1 of soil) in the form of NiCl2, at the rate of six seeds per pot. These two cultivars were selected on the basis of the study conducted by Yusuf (2011). On germination, three plants per pot were maintained after thinning and each treatment was replicated five times under completely randomized block design, in the net house, Department of Botany, Aligarh Muslim University, Aligarh, India, under natural environmental conditions. At 29-day stage of growth, foliage of plants were sprayed with 0 (double-distilled water; DDW), 10−8 or 10−6 M of HBL. These concentrations of HBL were selected on the basis of earlier studies conducted by Fariduddin et al. (2004). Tween-20 was added as surfactant prior to the foliar application. Each plant was sprinkled three times. The nozzle of the sprayer was adjusted in such a way that it pumped out 1 ml (approx.) in one sprinkle. Therefore, each plant received 3 ml of DDW or HBL solution. The plants were sampled at 45-day stage of growth to assess various growths and nodule-related traits as well as biochemical characteristics. The remaining plants were allowed to grow up to maturity to study the yield characteristics.

Determination of growth biomarkers and leaf water potential

The growth biomarkers (shoot and root length, shoot and root dry mass and leaf area) were determined by the method followed by Yusuf (2011).

The leaf water potential was monitored with the help of PSYPRO leaf water potential system (Wescor, Inc., USA) in the third fully expanded leaves of the plant as per manufacturer’s instruction.

Determination SPAD chlorophyll

The soil plant analysis development (SPAD) value of chlorophyll in the fresh leaf was measured by using the SPAD chlorophyll meter (SPAD-502; Konica, Minolta sensing, Inc., Japan).

Determination of photosynthetic parameters

Net photosynthetic rate (A) and its related attributes [stomatal conductance (gs), internal CO2 concentration (ci) and transpiration rate (E)] and chlorophyll fluorescence are determined by the method followed by Yusuf (2011).

Determination of nitrogen metabolism parameters

The nodule nitrogen content was estimated by employing the method of Lindner (1944). The plant material was digested with concentrated H2SO4, followed by its neutralization with NaOH and sodium silicate solutions. Nessler’s reagent was added to this solution and absorbance of samples were measured at 525 nm on a spectrophotometer (Spectronic 20D, Milton Roy, USA).

The carbohydrate was extracted from the sample, following the method of Yih and Clark (1965) and estimated by adopting the procedure of Dubois et al. (1956).

The leghemoglobin content, in fresh nodules, was estimated following the method described by Sadasivum and Manickam (1992).

Determination of biochemical parameters

The total inorganic ions leaked out of the leaves were measured by the method described by Sullivan and Ross (1979).

The activities of nitrate reductase (NR), carbonic anhydrase (CA), catalase (CAT), peroxidase (POX) and superoxide dismutase (SOD) were determined by the method followed by Jaworski (1971), Dwivedi and Randhawa (1974), Chance and Maehly (1956) and Beauchamp and Fridovich (1971), respectively.

The proline content in fresh leaf samples was determined by adopting the method of Bates et al. (1973).

Yield parameters

At harvest, 15 plants (three from each replicate) representing each treatment were randomly sampled and counted for the number of pods per plant. Twenty-five pods from each treatment were randomly selected and computed to get the number of seeds per pod. The pods from 15 plants representing each treatment were cleaned, crushed and computed to assess seed yield per plant. One hundred seeds were subsequently randomly picked and weighed to record 100-seed mass.

Statistical analysis

Data were statistically analysed using SPSS 17.0 for windows (IBM Corporation, 1 New Orchard Road, Armonk, New York 10504-1722, United States 914-499-1900). Standard error was calculated, and analysis of variance (ANOVA) was performed on the data to determine the least significance difference (LSD) between treatment means with the level of significance at P ≤ 0.05.

Results

Growth biomarkers

Soil supplemented with different levels of Ni (50, 100 or 150 mg Ni kg−1 soil) generated a significant decrease in growth biomarkers (shoot and root length, dry mass and leaf area) in a concentration-dependent manner (Tables 1 and 2). Out of the two varieties, PDM-139 experienced more reduction than T-44 in the presence of 150 mg Ni kg−1 of soil. However, foliar application of HBL (10−8 M) significantly increased the values for shoot length (74.8 %), root length (64.9 %), shoot dry mass (71.5 %) and leaf area (35.6 %) over the non-treated control plants in T-44. The follow-up treatment with HBL (10−8 M) to the Ni-stressed plants completely neutralized the toxicity of 50 and 100 mg Ni kg−1 of soil and partially that of 150 mg Ni kg−1 soil in T-44 at 45-day stage of growth for growth biomarkers except leaf area. In leaf area, 50 mg Ni kg−1 soil was neutralized by the HBL (10−8 M) in T-44 at 45-day stage of growth. The degree of neutralization was more pronounced in the variety T-44 than PDM-139, at 45-day stage of growth.

Table 1.

Effect of foliage applied 28-homobrassinolide (HBL; 10−8, or 10−6 M; 29-day stage) and/or soil applied nickel (Ni; 50, 100 or 150 mg kg−1) on shoot and root length (cm) and shoot dry mass (g) in two varieties (T-44 and PDM-139) of Vigna radiata L. Wilczek at 45-day stage of growth

Shoot length Root length Shoot dry mass
T-44 PDM-139 Mean T-44 PDM-139 Mean T-44 PDM-139 Mean
Control 30.61 25.70 28.15 14.45 11.56 13.00 3.68 2.94 3.31
HBL10−8 M 53.56 41.89 47.72 23.84 17.10 20.47 6.31 4.70 5.50
HBL10−6 M 45.91 35.2 40.55 20.23 14.21 17.22 5.80 4.26 5.03
Ni 50 26.01 21.37 23.69 12.21 8.38 10.29 3.40 2.23 2.81
Ni 100 23.87 17.99 20.93 10.84 6.76 8.80 2.59 1.82 2.20
Ni 150 18.97 14.52 16.74 8.99 5.02 7.00 2.24 1.44 1.84
Ni 50 + HBL10−8 M 44.21 32.05 38.13 20.26 12.65 16.45 5.04 3.56 4.30
Ni 100 + HBL10−8 M 38.66 26.08 32.37 16.58 9.53 13.05 4.19 2.73 3.46
Ni 150 + HBL10−8 M 27.5 20.03 23.76 12.58 6.62 9.60 3.42 2.10 2.76
Ni 50 + HBL10−6 M 38.23 28.63 33.43 17.70 11.31 14.50 5.44 3.27 4.35
Ni 100 + HBL10−6 M 33.41 22.84 28.12 14.74 8.24 11.4 3.91 2.54 3.22
Ni 150 + HBL10−6 M 25.41 17.56 21.48 11.41 6.02 8.71 3.18 1.90 2.54
Mean 33.86 25.32 15.31 9.78 4.10 2.79
LSD at 5 % V = 0.60 (Sig) V = 0.59 (Sig) V = 0.31 (Sig)
T = 1.71 (Sig) T = 1.72 (Sig) T = 0.87 (Sig)
V × T = 2.42 (Sig) V × T = NS V × T = NS
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V varieties, T treatments, Sig significant, NS non-significant

Table 2.

Effect of foliage applied 28-homobrassinolide (HBL; 10−8, or 10−6 M; 29 days stage) and/or soil applied nickel (Ni; 50, 100, or 150 mg kg−1) on root dry mass (g), leaf area (cm2) and water potential (−Mpa) in two varieties (T-44 and PDM-139) of Vigna radiata L. Wilczek at 45 days stage of growth

Root dry mass Leaf area Leaf water potential
T-44 PDM-139 Mean T-44 PDM-139 Mean T-44 PDM-139 Mean
Control 2.81 2.30 2.55 22.71 18.50 20.60 0.63 0.74 0.68
HBL10−8 M 4.72 3.70 4.21 30.88 24.05 27.46 0.41 0.54 0.47
HBL10−6 M 4.83 3.54 4.18 28.16 21.83 24.99 0.51 0.63 0.57
Ni 50 2.27 1.58 1.92 20.66 14.80 17.73 0.73 0.92 0.82
Ni 100 1.88 1.28 1.58 18.62 13.50 16.06 0.79 0.97 0.88
Ni 150 1.65 1.08 1.36 15.66 11.19 13.42 0.83 1.03 0.93
Ni 50 + HBL10−8 M 3.63 2.29 2.96 27.68 19.09 23.38 0.48 0.65 0.56
Ni 100 + HBL10−8 M 2.74 1.74 2.24 23.27 16.20 19.73 0.57 0.75 0.66
Ni 150 + HBL10−8 M 2.27 1.40 1.83 18.63 12.64 15.63 0.63 0.86 0.74
Ni 50 + HBL10−6 M 3.35 2.02 2.68 24.58 16.28 20.43 0.57 0.76 0.66
Ni 100 + HBL10−6 M 2.51 1.54 2.02 21.22 14.58 17.90 0.63 0.84 0.73
Ni 150 + HBL10−6 M 2.11 1.26 1.68 17.22 11.86 14.54 0.73 0.94 0.83
Mean 2.89 1.97 22.44 16.21 0.62 0.80
LSD at 5 % V = 0.33 (Sig) V = 0.38 (Sig) V = 0.18 (Sig)
T = 0.95 (Sig) T = 1.09 (Sig) T = 0.45 (Sig)
V × T = NS V × T = NS V × T = NS
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V varieties, T treatments, Sig significant, NS non-significant

Leaf water potential

The foliar application of HBL (10−8 M) increased leaf water potential compared with that of the control (Table 2) at 45-day stage of growth and the value in T-44 by 34.9 % over the control plants. However, the highest level of Ni (150 mg kg−1) showed most inhibitory action and reduced the leaf water potential in T-44 by 31.7 % less than their control at 45-day stage of growth. In addition to this, the loss of water potential caused by 50 and 100 mg kg−1 of Ni was successfully restored by the follow-up application of HBL (10−8 M) in T-44 at 45-day stage of growth.

Leaf electrolyte leakage

The plants raised in the soil fed with Ni (50, 100 or 150 mg kg−1) showed marked increase in the leaf electrolyte leakage (Table 3). However, the maximum increase was reported in PDM-139 by 43.9 % at 45-day stage of growth, over the control. Moreover, the follow-up treatment with HBL (10−8 M) to the metal (Ni; 50 and 100 mg kg−1) stressed plants completely neutralized their ill effects at 45-day stage.

Table 3.

Effect of 28-homobrassinolide (HBL; 10−8, or 10−6 M) as foliar spray (29-day stage) and/or soil applied nickel (Ni; 50, 100 or 150 mg kg−1) on carbonic anhydrase (mol CO2 kg−1 Fm s−1), electrolyte leakage (%) and proline content (m mol g−1 Fm) in two varieties (T-44 and PDM-139) of Vigna radiata L. Wilczek at 45-day stage of growth

Carbonic anhydrase activity Electrolyte leakage Proline content
T-44 PDM-139 Mean T-44 PDM-139 Mean T-44 PDM-139 Mean
Control 1.85 1.51 1.68 6.40 7.64 7.02 12.86 9.64 11.25
HBL10−8 M 2.59 2.02 2.30 4.48 5.80 5.14 18 13.11 15.55
HBL10−6 M 2.38 1.88 2.13 5.12 6.87 5.99 15.94 11.18 13.56
Ni 50 1.7 1.22 1.46 6.94 8.97 7.95 17.48 11.66 14.57
Ni 100 1.48 1.08 1.28 7.68 9.70 8.69 19.03 12.24 15.63
Ni 150 1.29 0.96 1.12 8.70 11.00 9.85 20.19 13.3 16.74
Ni 50 + HBL10−8 M 2.31 1.58 1.94 4.23 6.45 5.34 24.29 15.39 19.84
Ni 100 + HBL10−8 M 1.92 1.35 1.63 5.14 7.37 6.25 26.64 16.76 21.70
Ni 150 + HBL10−8 M 1.63 1.61 1.62 7.26 9.58 8.42 28.87 18.62 23.74
Ni 50 + HBL10−6 M 2.14 1.47 1.80 4.99 7.35 6.17 22.02 14.22 18.12
Ni 100 + HBL10−6 M 1.79 1.25 1.52 6.14 8.14 7.14 24.54 15.30 19.92
Ni 150 + HBL10−6 M 1.53 1.07 1.30 6.87 9.68 8.27 26.65 17.15 21.90
Mean 1.88 1.41 6.16 8.21 21.37 14.04
LSD at 5 % V = 0.73 (Sig) V = 0.50 (Sig) V = 0.24 (Sig)
T = 0.20 (Sig) T = 0.84 (Sig) T = 0.66 (Sig)
V × T = 0.29 (Sig) V × T = NS V × T = 0.96 (Sig)
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V varieties, T treatments, Sig Significant, NS non-significant

SPAD chlorophyll content and chlorophyll fluorescence (Fv/Fm)

Foliar application of HBL (10−8 M) enhanced the values for SPAD chlorophyll in T-44 (34.9 %) and PDM-139 (27.9 %) significantly, over unstressed control plants, at 45-day stage of growth (Table 4). However, the plant grown in the soil supplemented with Ni (50, 100 or 150 mg kg−1) possessed less chlorophyll than the control plants. The variety PDM-139 experienced more damage than T-44 in terms of chlorophyll content under Ni stress. The loss caused by the Ni stress was effectively recovered by the application of HBL (10−8 M), more prominent in T-44 than PDM-139.

Table 4.

Effect of foliage applied 28-homobrassinolide (HBL; 10−8 or 10−6 M; 29-day stage) and/or soil applied nickel (Ni; 50, 100 or 150 mg kg−1) on transpiration rate (mol H2O m−2 s−1), maximum quantum yield (Fv/Fm) and chlorophyll content (SPAD value) in two varieties (T-44 and PDM-139) of Vigna radiata L. Wilczek at 45 days stage of growth

Transpiration rate Maximum quantum yield (Fv/Fm) SPAD chlorophyll content
T-44 PDM-139 Mean T-44 PDM-139 Mean T-44 PDM-139 Mean
Control 2.96 2.51 2.73 0.912 0.729 0.820 45.37 30.85 38.11
HBL10−8 M 4.08 3.33 3.70 1.24 0.947 1.093 61.24 39.48 50.36
HBL10−6 M 3.78 3.01 3.39 1.14 0.874 1.007 52.17 34.51 43.34
Ni 50 2.72 2.18 2.45 0.738 0.539 0.638 38.56 22.49 30.52
Ni 100 2.6 2.03 2.31 0.684 0.488 0.586 36.29 20.66 28.47
Ni 150 2.42 1.88 2.15 0.583 0.393 0.488 29.49 16.96 23.22
Ni 50 + HBL10−8 M 3.86 2.87 3.36 1.107 0.765 0.936 56.22 34.07 45.14
Ni 100 + HBL10−8 M 3.43 2.61 3.02 0.978 0.683 0.830 50.11 30.03 40.07
Ni 150 + HBL10−8 M 3.09 2.29 2.69 0.752 0.506 0.629 44.24 27.11 35.67
Ni 50 + HBL10−6 M 3.69 2.68 3.18 1.033 0.711 0.872 45.01 27.11 36.06
Ni 100 + HBL10−6 M 3.35 2.47 2.91 0.923 0.605 0.764 40.24 24.29 32.26
Ni 150 + HBL10−6 M 2.95 2.21 2.58 0.705 0.463 0.584 36.60 20.11 28.35
Mean 3.24 2.50 0.899 0.641 44.62 27.30
LSD at 5 % V = 0.07 (Sig) V = 0.018 (Sig) V = 0.26 (Sig)
T = 0.20 (Sig) T = 0.052 (Sig) T = 0.76 (Sig)
V × T = 0.29 (Sig) V × T = 0.074 (Sig) V × T = 1.07 (Sig)
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V varieties, T treatments, Sig significant, NS non-significant

The follow-up treatment with 10−8 M of HBL to Ni-stressed plants significantly improved Fv/Fm values in both the varieties (T-44 and PDM-139) and also completely neutralized the damage caused by the lower concentration of metal (50 mg kg−1) in T-44 at 45-day stage.

Photosynthetic parameters

Exogenous application of HBL (10−8 M) significantly increased the net photosynthetic rate (37.9 %), stomatal conductance (73.0 %), internal CO2 concentration (21.8 %) and transpiration rate (37.8 %) in comparison to their control plants in T-44 at 45-day stage of growth (Table 5). On contrary, the highest concentration i.e. 150 mg Ni kg−1 of soil decreased the values of net photosynthetic rate (34.0 %), stomatal conductance (45.6 %), internal CO2 concentration (22.2 %) and transpiration rate (18.2 %) lower than the non-stressed control plants at 45-day stage of growth in T-44. Beside this, Ni (50 mg kg−1) stressed T-44 plants exposed to the follow-up treatment with 10−8 M of HBL completely neutralized the deleterious effect for net photosynthetic rate and its related attributes and partially for 100 and 150 mg kg−1 of Ni at 45-day stage of growth.

Table 5.

Effect of foliage applied 28-homobrassinolide (HBL; 10−8 or 10−6 M; 29-day stage) and/or soil applied nickel (Ni; 50, 100 or 150 mg kg−1) on net photosynthetic rate (μmol CO2 m−2 s−1), stomatal conductance (mol H2O m−2 s−1) and internal CO2 concentration (ppm) in two varieties (T-44 and PDM-139) of Vigna radiata L. Wilczek at 45-day stage of growth

Net photosynthetic rate Stomatal conductance Internal CO2 concentration
T-44 PDM-139 Mean T-44 PDM-139 Mean T-44 PDM-139 Mean
Control 15.92 11.39 13.65 0.046 0.039 0.042 338 248 293
HBL10−8 M 21.96 15.03 18.49 0.08 0.064 0.072 412 292 352
HBL10−6 M 20.37 13.89 17.13 0.075 0.06 0.067 398 282 340
Ni 50 13.05 8.54 10.79 0.034 0.025 0.029 308 220 264
Ni 100 12.09 7.06 9.57 0.031 0.023 0.027 270 193 231
Ni 150 10.50 5.92 8.21 0.025 0.018 0.021 263 186 224
Ni 50 + HBL10−8 M 20.09 12.08 16.08 0.055 0.036 0.045 391 268 329
Ni 100 + HBL10−8 M 16.80 9.31 13.05 0.046 0.031 0.038 326 229 277
Ni 150 + HBL10−8 M 13.86 7.57 10.71 0.035 0.023 0.029 312 212 262
Ni 50 + HBL10−6 M 18.27 10.76 14.51 0.04 0.034 0.037 369 261 315
Ni 100 + HBL10−6 M 15.83 8.82 12.32 0.039 0.028 0.033 313 221 267
Ni 150 + HBL10−6 M 13.02 6.74 9.88 0.029 0.019 0.024 297 206 251
Mean 15.98 9.75 0.044 0.033 333 234
LSD at 5 % V = 0.22 (Sig) V = 0.002 (Sig) V = 3.54 (Sig)
T = 0.62 (Sig) T = 0.006 (Sig) T = 10.02 (Sig)
V × T = 0.88 (Sig) V × T = NS V × T = NS
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V varieties, T treatments, Sig significant, NS non-significant

Carbonic anhydrase and nitrate reductase activity

The plants raised from the Ni-supplemented soil possessed significantly lower CA and NR activity in both the varieties than their respective control plants (Table 3 and Fig. 1a), and the loss was more pronounced with the highest level of Ni (150 mg kg−1). However, 10−8 M of HBL significantly increased the activity of NR in both the varieties i.e. T-44 and PDM-139 by 46.9 and 39.8 %, respectively, over their control plants at 45-day stage. In addition to this, foliar application of HBL (10−8 M) to the foliage of Ni-stressed plant completely nullified the effect of Ni (50 and 100 mg kg−1) and also partially neutralized the ill effect of higher level of Ni (150 mg kg−1) in T-44 at 45-day stage of growth for both the activity.

Fig. 1.

Fig. 1

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Effect of foliage applied 28-homobrassinolide (HBL; 10−8 or 10−6 M; 29-day stage) and/or soil applied nickel (Ni; 50, 100 or 150 mg kg−1) on a nitrate reductase activity, b nodule nitrogen content, c nodule carbohydrate content, d leghemoglobin content, e nitrogenease activity, and f seed yield per plant in two varieties (T-44 and PDM-139) of Vigna radiata L. Wilczek at 45-day stage of growth. *P < 0.05, significant difference between control and treatment

Nodule nitrogen, carbohydrate and leghemoglobin content

Of the various concentrations of HBL (10−8 or 10−6 M), 10−8 M significantly increased the nodule nitrogen, carbohydrate and leghemoglobin content in T-44 by 45.9, 27.9 and 33.9 %, respectively, at 45-day stage of growth, over their control plants. Moreover, the follow-up treatment with HBL (10−8 M) in T-44 at 45-day stage of growth partially neutralized the toxic effect generated by Ni (150 mg kg−1) and also completely recovered the loss generated by 50 and 100 mg kg−1 of Ni in terms of nodule nitrogen and carbohydrate content.

Nitrogenase activity

The foliar application of 10−8 M significantly increased the activity of nitrogenase in T-44 by 40.9 % at 45-day stage of growth, over the control plants (Fig. 1e). However, the varied levels (50, 100 or 150 mg kg−1) of Ni supplied in soil generated loss in nitrogenase activity in the concentration-dependent manner and the maximum loss was noted by the 150 mg Ni kg−1 of soil. The loss induced by the highest concentration of metal (150 mg kg−1) was partially neutralized by the follow-up treatment with HBL (10−8 M) more effectively at 45-day stage of growth.

Activity of antioxidative enzymes and proline accumulation

The activity of antioxidative enzymes (CAT, POX and SOD) significantly increased in response to the metal and/or HBL treatment in both the varieties (T-44 and PDM-139). Control plants possessed minimum activity of antioxidative enzymes (CAT, POX and SOD). Maximum increase in CAT, POX and SOD was recorded in the plants administered with metal (150 mg Ni kg−1 of soil) followed by foliar application with 10−8 M of HBL by 67.2, 113.5 and 165.2 %, in T-44 over the respective control, at 45-day stage of growth (Fig. 2).

Fig. 2.

Fig. 2

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Effect of foliage applied 28-homobrassinolide (HBL; 10−8 or 10−6 M; 29-day stage) and/or soil applied nickel (Ni; 50, 100 or 150 mg kg−1) on activities of a catalase, b peroxidase, c superoxide dismutase in two varieties (T-44 and PDM-139) of Vigna radiata L. Wilczek at 45-day stage of growth. *P < 0.05, significant difference between control and treatment

The proline accumulation in the leaves of both the varieties (T-44 and PDM-139) increased with Ni (50, 100, or 150 mg kg−1) and/or different concentrations (10−8 or 10−6 M) of HBL. Of the two varieties, T-44 possessed high proline content than PDM-139. Moreover, the maximum value for proline content was recorded in plants exposed to Ni (150 mg kg−1) and HBL (10−8 M) as a follow-up treatment at 45-day stage of 124.4 and 93.1 % for the varieties T-44 and PDM-139, respectively, over the control plants at 45-day stage.

Yield characteristics

Yield characteristic (number of pods and seed yield per plant) at harvest was significantly increased by exogenous application of HBL (10−8 M; Table 6 and Fig. 1f). However, the plants raised in the soil supplemented with highest level of Ni (150 mg kg−1) showed significant reduction in number of pods per plant (28.5 %), number of seeds per pod (30.1 %), seed yield per plant (39.1 %) and 100-seed mass (34.5 %), less than the respective control plants. However, the follow-up treatment with HBL (10−8 M) to the Ni-stressed (50 mg kg−1) plants neutralized the damaging effect of the metal and restored the values for number of pods per plant and seed yield.

Table 6.

Effect of foliage applied 28-homobrassinolide (HBL; 10−8 or 10−6 M; 29-day stage) and/or soil applied nickel (Ni; 50, 100 or 150 mg kg−1) on number of pods per plant, number of seeds per pod and 100-seed mass (g) in two varieties (T-44 and PDM-139) of Vigna radiata L. Wilczek at harvest

No of pods per plant No of seeds per pod 100-seed mass
T-44 PDM-139 Mean T-44 PDM-139 Mean T-44 PDM-139 Mean
Control 17.81 15.13 16.47 8.20 6.72 7.46 2.52 2.08 2.30
HBL10−8 M 24.22 19.97 22.09 8.51 6.83 7.67 2.57 2.10 2.33
HBL10−6 M 19.91 16.42 18.16 8.29 6.78 7.53 2.50 2.07 2.28
Ni 50 16.91 13.91 15.41 7.33 5.77 6.55 2.26 1.70 1.98
Ni 100 14.99 12.40 13.69 6.29 4.80 5.54 2.05 1.49 1.77
Ni 150 12.73 10.13 11.43 5.73 4.35 5.04 1.65 1.24 1.44
Ni 50 + HBL10−8 M 21.85 17.49 19.67 7.50 5.75 6.62 2.30 1.72 2.01
Ni 100 + HBL10−8 M 19.05 15.15 17.10 6.35 4.83 5.59 2.11 1.49 1.80
Ni 150 + HBL10−8 M 17.80 13.30 15.55 5.80 4.33 5.06 1.66 1.22 1.44
Ni 50 + HBL10−6 M 19.65 14.89 17.27 7.30 5.77 6.53 2.24 1.68 1.96
Ni 100 + HBL10−6 M 15.75 13.11 14.43 6.20 4.78 5.49 2.05 1.45 1.75
Ni 150 + HBL10−6 M 14.04 11.11 12.57 5.55 4.30 4.92 1.60 1.20 1.40
Mean 17.89 14.41 6.92 5.41 2.12 1.62
LSD at 5 % V = 0.58 (Sig) V = 0.45 (Sig) V = 0.15 (Sig)
T = 1.65 (Sig) T = 1.29 (Sig) T = 0.41 (Sig)
V × T = NS V × T = NS V × T = NS
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V varieties, T treatments, Sig significant, NS non-significant

Discussion

Foliar application of HBL (10−8 M) at 29-day stage of growth to the plants grown in the soil supplemented with different concentrations (50, 100 or 150 mg kg-1) of Ni decreased the deleterious effects and increased morphological traits (Tables 1 and 2) in comparison to the Ni-stressed plants. Excess Ni in soil is believed to disrupt various physiological functions (Marschner 2005) through inhibition of cell division (Chen et al. 2009) and cell elongation (Gajewska et al. 2006). Moreover, BRs are major growth-promoting hormones (Gudesblat and Russinova 2011) and genome-wide transcriptional profiling and ChIP-chip analyses highlight the importance of two master BR regulators, the transcription factors BZR1 and BES1/BZR2, in mediating multiple effects of BRs, including the coordination of growth and development and the interaction with other hormones and environment. On the other hand, excess Ni in soil also led to the loss of chlorophyll content (Table 4), maximum quantum yield of PS II (Fv/Fm; Table 4) and net photosynthetic rate (A; Table 5) along with related attributes, i.e. stomatal conductance and internal carbon dioxide concentration (Tables 5 and 4). Excess Ni is reported to damage the structure and/or integrity of chlorophyll molecules (Kupper et al. 1998) by displacing central Mg2+ ion (Chen et al. 2009) which hampers the functioning of photosynthetic electron transport chain (Singh and Pandey 2011), inhibition of enzymatic activity like malate dehydrogenase, ATP-ase and δ-aminolevulinic acid dehydrogenase (Carpentier 2001). The cumulative effect of all these altered processes under excess Ni lead to reduced activity of CA (Table 3) that could have finally reduced the net photosynthetic rate (Table 5). Moreover, the application of HBL (10−8 M) protected the photosynthetic machinery and increased the activities of carbonic anhydrase (key enzymes in photosynthesis) under Ni stress. BRs promote photosynthesis by positively regulating synthesis and activation of a variety of photosynthetic enzymes including Rubisco (Xia et al. 2009), and high CA activity increases the capacity of CO2 assimilation in the Calvin cycle which is mainly attributed to efficient functioning of Rubisco (Bajguz and Asami 2005) thereby improving the net photosynthetic rate (Table 5). Beside this, HBL also improved water relations, i.e. leaf water potential (Table 2), membrane structure and its stability (Nassar 2004) as evident from decreased electrolyte leakage (Table 3).

It may be believed that alteration in microbial activity of soil microorganism under excess metal (Simon 1999; Friedlova 2010) led to the decrease in nodule nitrogen and carbohydrate content (Fig. 1b, c), activity of nitrate reductase, nitrogenase and leghemoglobin content in nodules as the concentration of metal (Ni) increased from 50 to 150 mg kg−1 (Fig. 1a, d). Moreover, legume plants are capable of developing an association with nitrogen-fixing bacteria generally known as rhizobia to form nodules. This symbiotic process is activated by the presence of phytohormones (Hopkins 1995; Suzuki et al. 2004). The increase in the nodulation is reported in Cajanus cajan and Vigna sinensis (Bajguz 2000). Furthermore, in the present study, HBL (10−8 M) improved nitrogen metabolism, i.e. activity of nitrate reductase, nitrogenase (Fig. 1a, e) and nodule nitrogen, carbohydrate and leghemoglobin content (Fig. 1b–d) under Ni stress and stress-free conditions. HBL appeared to act synergistically with auxin and triggered a response similar to that of auxin bioassay, based on root formation in mung bean (Ashraf et al. 2010). Moreover, HBL manage to overcome the Ni-induced damage in terms of nodule nitrogen, carbohydrate and leghemoglobin content and activity of nitrate reductase and nitrogenase (Fig. 1a-e) that may be because of BR and ABA act antagonistically and co-regulate various developmental process related to nitrogen metabolism (Finkelstein et al. 2008; Chan and Gresshoff 2009) and the expression of hundreds of genes (Nemhauser et al. 2006).

In the present study, exogenously applied HBL increased the activities of CAT, POX, SOD and proline content, i.e. osmolyte (Table 3). In addition to this, when plants were grown in the soil supplemented with different levels of nickel and consequently, follow-up treatment of HBL (10−8 M) led to the further increase of the activities of antioxidant enzymes and proline accumulation (Table 3). Ashraf et al. (2010) revealed that under higher accumulation of free radicals, BRs have an ability to regulate the activity of antioxidant system to scavenge the excess ROS. Recently, BR treatment conferred tolerance under abiotic stresses mediated through the induced expression of both regulatory genes, such as RBOH (respiratory burst oxidase homologue), MAPK1 (mitogen-activated protein kinase 1) and MAPK3 (mitogen-activated protein kinase 3) and genes involved in defence, antioxidant responses and also those that elevate H2O2 level resulting from enhanced NADPH oxidase activity involved in BR-induced stress tolerance (Xia et al. 2009). In addition to this, BRs induce stress tolerance by triggering the apoplast H2O2 accumulation, which subsequently upregulates the antioxidant system (Xia et al. 2011). Beside this, Gill and Tuteja 2010 believed that proline serves as persuasive inhibitor of PCD and also acts as non-enzymatic antioxidants that is known for stabilizing subcellular structures such as proteins and cell membranes, scavenging free radicals and buffering redox potential under stress conditions, and also have ability of molecular chaperones that protects the integrity of protein and enhances the activity of different enzymes (Szabados and Savoure 2009), such as protection of nitrate reductase during heavy metal stress (Sharma and Dubey 2005). These reports authenticate our observations where HBL treatment under Ni stress or stress-free conditions enhanced the accumulation of proline (Table 3). It has been reported earlier that BRs induced the expression of biosynthetic genes of proline (Ozdemir et al. 2004). Moreover, BRs also increased proline content as well as the activity of antioxidant enzymes in sorghum plants, exposed to osmotic, Cd, Al, Ni and Cu stresses (Bajguz and Hayat 2009).

Ni treatment significantly increased the yield gap of mung bean plants in the concentration-dependent manner which are reflected in decreased values for yield characteristics, including number of seeds/pod, 100-seed weight and seed yield per plant (Table 6 and Fig. 1f; Tripathy et al. 1981). The variety T-44 showed lower yield gap under stress conditions compared with PDM-139. This loss could be attributed to poor plant growth (Tables 1 and 2) resulting largely from the lower pace of photosynthesis (Table 5 and Chen et al. 2009) and the availability of nitrate (Chen et al. 2009). However, the application of BRs to the stressed and non-stressed plants favoured the rate of CO2 assimilation (Xia et al. 2009) and the net photosynthetic rate (Table 5 and Gomes et al. 2003). Higher BR induced photosynthetic CO2 assimilation rates, i.e. A (Table 5) could have resulted due to availability of more carbohydrates for metabolism and export to the sink (Bajguz and Asami 2005). Moreover, the key enzyme invertase involved in the source-sink regulation is also regulated by BRs (Bajguz and Asami 2005). Therefore, directed transportation of photosynthates to the sink in association with induced expression of genes encoding enzymes of carbohydrate metabolism (Roitsch 1999) and lowering the process of senescence before and/or after pollination (Iwahori et al. 1990) could have naturally helped the plant in extending the duration of photosynthetically active sites and also to prevent the premature loss of flowers and fruits leading to improved seed yield (Fig. 1f). The observation get additional support from Gomes et al. (2006) who noted higher yield in passion fruit plants correlating it with higher photosynthetic carbon assimilation under BRs (Gomes et al. 2003).

Conclusions

In conclusion, Ni-mediated damage to the mung bean plants was in the concentration-dependent manner with retarded growth, altered nitrogen metabolism, lowered photosynthetic rate and increased yield gap. However, the antioxidant systems and proline accumulation speed up to cope with damages triggered by Ni stress which was further accelerated by the follow-up treatment with HBL (10−8 M) and played a pivotal role in maintaining the equilibrium between excess free radicals and antioxidants system. Therefore, exogenous application of HBL (10−8 M) could be exploited as potent inhibitor of oxidative stress which made the mung bean plants tolerant to nickel up to 100 mg kg−1 of soil by protecting nitrogen metabolism, photosynthetic efficiency, overexpression of antioxidant enzymes and proline accumulation, and also increased the biomass production.

Acknowledgments

M. Yusuf gratefully acknowledges the financial assistance rendered by the Science and Engineering Research Board (SERB), Department of Science and Technology (DST), New Delhi, India, in a form of Young Scientists (SB/FT/LS-210-2012).

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