Low level of selenium increases the efficacy of 24-epibrassinolide through altered physiological and biochemical traits of Brassica juncea plants

Food Chemistry 185 (2015) 441–448

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Low level of selenium increases the efficacy of 24-epibrassinolide through altered physiological and biochemical traits of Brassica juncea plants Fatima Salva Naz, Mohammad Yusuf ⇑, Tanveer A. Khan, Qazi Fariduddin, Aqil Ahmad Plant Physiology & Biochemistry Section, Department of Botany, Aligarh Muslim University, Aligarh 202002, India

a r t i c l e

i n f o

Article history: Received 22 December 2014 Received in revised form 14 March 2015 Accepted 7 April 2015 Available online 11 April 2015 Keywords: Antioxidant system Brassinosteroid Photosynthesis Selenium

a b s t r a c t This study was conducted to provide an insight into the effect of Se (through soil) induced changes in Brassica juncea plants in the presence and absence of 24-epibrassinolide (EBL; foliar). The Se treatments showed dual response, 10 lM of Se significantly increased growth, water relations, photosynthetic attributes along with carbonic anhydrase activity whereas its higher concentrations proved inhibitory in concentration dependent manner. The follow-up application of EBL to the Se stressed plants improved growth, water relations, photosynthesis and simultaneously enhanced the various antioxidant enzymes viz. catalase, peroxidase and superoxide dismutase with the excess accumulation of proline. In addition to this, 10 lM Se increases the efficacy of 108 M of EBL and both in combination showed maximum increase for the growth and photosynthetic traits of plants. On the other hand, the elevated level of antioxidant enzymes as well as proline could have conferred tolerance to the Se-stressed plants resulting in improved growth, water relations and photosynthesis. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Elements that stimulate growth and may be essential to particular species are defined as beneficial elements. The five most investigated beneficial elements are Al, Co, Na, Se and Si. It is well documented that all of these elements promote growth of various taxa, under certain environmental conditions. However, the function and concentration varies for each element with plant species. Out of these, selenium (Se) is not a very abundant element whose soil levels are typically below 1 ppm (mg/kg soil), but 4–100 ppm can be found in seleniferous soils. Selenium is chemically similar to sulfur (S) and its metabolism follows the same mechanisms and the main bioavailable form of Se in soils is selenate, which can be taken up by plants via sulfate transporters and assimilated into selenocysteine (SeCys) and selenomethionine (SeMet). While Se is generally metabolized by sulfur pathways, there is some evidence that plants have evolved Se-specific enzymes that facilitate Se accumulation, perhaps to serve an ecological or physiological function (Pilon-Smits, Quinn, Tapken, Malagoli, & Schiavon, 2009). Among higher plants, the largest beneficial effects of Se on growth (up to 2.8-fold higher biomass with Se) have been observed ⇑ Corresponding author. Tel.: +91 9415 840 350. E-mail addresses: [email protected] (M. Yusuf), [email protected] (Q. Fariduddin). http://dx.doi.org/10.1016/j.foodchem.2015.04.016 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

in the Se hyperaccumulator plants, and Se has been suggested to be essential for these species (Shrift, 1969). Trace amounts of Se also stimulated growth in a variety of non-hyperaccumulator species including ryegrass, lettuce, potato, and duckweed (Hartikainen, 2005). Recently, it has been shown that selenium can regulate the water status of plants under conditions of water deficiency and thereby performs a protective role (Kuznetsov, Kholodova, Kuznetsov, & Yagodin, 2003). The mechanism of this apparent positive effect of Se on antioxidant capacity may be direct, owing to antioxidant activity of seleno-compound, or indirect, via Se-induced up-regulation of general stress tolerance mechanisms. It was suggested that Se can alleviate oxidative stress in chloroplasts. The responses of potato to Se supplementation were investigated by monitoring chlorophyll fluorescence and the transcription of antioxidative enzymes (Seppanen, Turakainen, & Hartikainen, 2003). Brassinosteroids (BRs) is a group of naturally occurring plant steroidal compounds that are ubiquitously distributed in plant kingdom. BRs play prominent role in various physiological processes such as cell division, elongation and expansion, vascular differentiation, pollen tube growth, seed germination, proton pump activation, membrane polarization, source/sink relationships, reproductive development, ions uptake into the plant cell, regulation of gene expression, nucleic acid and protein synthesis, enzymes activation and photosynthesis (Sasse, 2003). Moreover,

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they confer tolerance to plants against various abiotic and biotic stresses, including those caused by salt, chilling, heat, drought, and pathogens (Bajguz & Hayat, 2009). Exogenously applied 24epibrassinolide (EBL) has the ability to substantially enhance wheat yield and its stress tolerance by inducing cellular changes that are related to stress tolerance, like stimulate nucleic acid and protein synthesis (Dhaubhadel, Browning, Gallie, & Krishna, 2002), activate ATPase pump (Khripach, Zhabinskii, & Khripach, 2003), increase antioxidant enzyme activities and osmoprotectants accumulation (Ozdemir, Bor, Demiral, & Turkan, 2004), induce other hormone responses (Vert, Nemhauser, Geldner, Hong, & Chory, 2005), regulate stress-responsive genes expression (Kagale, Divi, Krochko, Keller, & Krishna, 2007) and induce photosynthetic efficiency and the translocation of photosynthates to the sink (Shahbaz, Ashraf, & Athar, 2008). Great efforts have been made to develop this phytohormone as a plant growth regulator for widespread utilization in agricultural production; however, the mechanisms by which BRs influence plant productivity and stress tolerance are still poorly understood (Bajguz & Hayat, 2009). In addition to this, role of BRs in the presence of beneficial elements need to be explored through various physiological and biochemical approaches. This study was conducted to investigate the response of 24epibrassinolide on the selenium induced changes in Brassica juncea in terms of physiological and biochemical characterization under different levels of selenium and also explore the possibility to establish the selenium as quasi-essential or essential elements for the growth and productivity of B. juncea. 2. Materials and methods 2.1. Plant materials The seeds of B. juncea cv. Krishna Kranti were procured from National Seed Corporation Ltd., New Delhi, India. The healthy looking and uniform size seeds were surface sterilized with 1% sodium hypochlorite solution for 10 min, followed by repeated washing with double distilled water (DDW). 2.2. Hormone preparation 24-Epibrassinolide (EBL) was obtained from Sigma–Aldrich Chemicals Pvt. Ltd. India. A stock solution of EBL (104 M) was prepared by dissolving required quantity of the EBL in 5 ml of ethanol in a 100 ml volumetric flask and final volume was made up to the mark by using double distilled water (DDW). The desired concentration of EBL i.e. 108 M was prepared by the dilution of stock solution and the concentration of EBL was based on the study of Hayat, Ahmad, Mobin, Hussain, and Fariduddin (2000). Tween-20 was added as surfactant prior to the foliar application.

sown in pots and allowed to germinate under natural environmental conditions in the net house of Department of Botany, Aligarh Muslim University, Aligarh, India. Fifty pots were divided into 10 sets of 5 pots each (replicates) representing one treatment. The treatments pattern are as follows:  Set I: served as control (-EBL and -Se) and foliage at 21 and 22 days stage of growth sprayed with deionized water.  Set II: foliage at 21 and 22 days stage of growth sprayed with 108 M of EBL.  Set III: at 10, 12 and 14 days stage, plants exposed to 10 lM of Se solution through soil.  Set IV: at 10, 12 and 14 days stage, plants exposed to 20 lM of Se solution through soil.  Set V: at 10, 12 and 14 days stage, plants exposed to 40 lM of Se solution through soil.  Set VI: at 10, 12 and 14 days stage, plants exposed to 80 lM of Se solution through soil.  Set VII: a combination of set II and set III (108 M EBL + 10 lM Se).  Set VIII: a combination of set II and set IV (108 M EBL + 20 lM Se).  Set IX: a combination of set II and set V (108 M EBL + 40 lM Se).  Set X: a combination of set II and set VI (108 M EBL + 80 lM Se). The foliage of each plant was sprinkled thrice. The nozzle of the sprayer was adjusted in such a way that it pumped out 1 ml (approx.) in one sprinkle. Therefore, each foliage of plants received 3 ml EBL solution. The plants in all the sets were harvested at 30 days stage of growth to assess various growth and leaf gas exchange traits as well as biochemical parameters. These assays were repeated 5 times with the utilization of 15 plants per treatment (03 plants per pot). 2.5. Morphological traits and leaf water potential The study of morphological traits i.e. root-shoot length, dry mass of plant, leaf area and leaf water potential were followed as described in our previous study Parashar, Yusuf, Fariduddin, and Ahmad (2014). 2.6. Chlorophyll content (SPAD level) The SPAD values of chlorophyll in the leaf was measured, under natural conditions by using the SPAD chlorophyll meter (SPAD-502; Konica, Minolta sensing, Inc., Japan). 2.7. Photosynthetic traits

Sodium selenate (Na2SeO4) was used as the source of Se. A stock solution of Se (1.0 mM) was prepared by dissolving the required quantity of Na2SeO4 in 10 ml of DDW in a 100 ml volumetric flask and final volume was made up to the mark by using deionized water. The required concentrations (10, 20, 40, 80 lM) of Se were prepared by the dilution of stock solution.

Photosynthetic traits were determined on the third fully expanded leaves between 11:00 and 12:00 h by using an infrared gas analyzer (IRGA) portable photosynthetic system (LI-COR 6400, LI-COR, and Lincoln, NE, USA). To measure net photosynthetic rate (PN) and its related attributes [stomatal conductance (gs), internal CO2 concentration (Ci), water use efficiency (WUE)], the air temperature, relative humidity, CO2 concentration and PPFD were maintained at 25 °C, 85%, 600 lmol mol1 and 800 lmol mol2 s1, respectively.

2.4. Treatment pattern and experimental design

2.8. Determination of carbonic anhydrase activity

The experiment was conducted under randomized block design with 50 earthen pots (10-in. in diameter), filled with sandy loam soil and farmyard manure (3:1). The surface sterilized seeds were

The activity of carbonic anhydrase (CA) in the leaves was measured following the method described by Dwivedi and Randhawa (1974). The leaf samples were cut into small pieces in cysteine

2.3. Source of selenium (Se)

F.S. Naz et al. / Food Chemistry 185 (2015) 441–448

hydrochloride solution. These leaf samples were blotted and transferred in a test tube, followed by the addition of phosphate buffer (pH 6.8), 0.2 M NaHCO3, bromothymol blue, and the methyl red indicator, at the last. This reaction was titrated against 0.5 N HCl. The activity of the enzyme was expressed on a fresh mass basis. 2.9. Biochemical analysis 2.9.1. Extraction for leaf protein content and activities of antioxidant enzymes 1 g of fresh leaves were weighed and homogenized in cold extraction buffer (70 mM phosphate buffer; pH 7.0, 1 mM EDTA, 1 mM PMSF, 0.5% Triton X-100 and 2% PVP) with the help of precooled mortar and pestle. The homogenate was centrifuged at 12,000g for 20 min at 4 °C and the supernatant was stored at 20 °C. This supernatant was utilized for analysis of protein content and activities of antioxidant enzymes (catalase, peroxidase, and superoxide dismutase).

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X-100 and 40 ll enzyme extract. 1 mM Riboflavin was added to the last reaction mixture. Control set was prepared in the same manner with phosphate buffer and NBT. The activity was determined by measuring the absorbance at 560 nm for 2 min at 25 °C. One unit of activity was determined as amount of enzyme required to inhibit the photoreduction of NBT to blue formazan by 50% and was expressed as SOD units lg protein1. 2.10. Proline accumulation The proline content in fresh leaf samples was determined by adopting the method of Bates, Aldren, and Teare (1973). Sample was extracted in sulphosalicylic acid. To the extract an equal volume of glacial acetic acid and ninhydrin solutions were added. The sample was heated at 100 °C to which 5 ml of toluene was added. The absorbance of toluene layer was read at 528 nm on a spectrophotometer. 2.11. Statistical analysis

2.9.2. Assay for leaf protein content Total protein content of leaves was determined by the method followed by Bradford (1976). 2 ml Bradford reagent was added to 100 ll of supernatant and mixed gently and thoroughly. Incubate the sample at 25 °C for 5–10 min and read the absorbance at 595 nm on spectrophotometer. A graph of absorbance versus different known concentrations for standard solutions of bovine serum albumin (BSA) was plotted and a standard linear equation was derived. The amount of protein in the samples was calculated from the standard linear equation. The amount of protein expressed as mg g1 fresh mass. 2.9.3. Assay for catalase (CAT, EC 1.11.1.6) activity Catalase activity was assayed by measuring the initial rate of H2O2 disappearance using the method of Aebi (1984). Reaction mixture for sample was prepared by 50 mM phosphate buffer (pH 7.0), 15 mM H2O2 and 100 ll enzyme extract. The decrease in hydrogen peroxide was followed as decline in optical density at 240 nm for 2 min with the interval of 30 s at 25 °C. Control set was prepared in the same manner excluding enzyme extract. The activity of catalase was expressed as:

Specific activity ðUA mg1 proteinÞ 1

¼ Unit Activity ðU min

g1 FMÞ=Protein content ðmg g1 FMÞ

Data were statistically analyzed using SPSS, 17.0 for windows (SPSS, Chicago, IL, USA). 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 6 0.05. 3. Results 3.1. Growth biomarkers Compared to the control, EBL (108 M) and Se (10 lM) significantly increased the growth biomarkers (root and shoot length, dry mass of plant and leaf area; Tables 1 and 2) individually. On the other hand, Se at higher concentrations (20, 40 or 80 lM) decreased the growth biomarkers in a concentration dependent manner. Moreover, 80 lM of Se decreased the root length (30%), shoot length (40%), dry mass (40%) and leaf area (28.6%) of plant in comparison to non-treated control plants. However, the Se stressed plants treated with EBL (108 M) as follow up treatment reduced the damaging effect of Se in a concentration dependent manner. Interestingly, combined treatment of EBL (108 M) and Se (10 lM) proved most significant in increasing the growth biomarkers over all the treatments. 3.2. Leaf water potential (LWP)

2.9.4. Assay for peroxidase (POX, EC 1.11.1.7) activity Peroxidase activity was assayed by the method followed by Sanchez, Revilla, and Zarra (1995) with some modifications. Reaction mixture for sample was prepared by 50 mM phosphate buffer (pH 7.0), 20 mM guaiacol, 15 mM H2O2 and 100 ll enzyme extract. The activity was determined by measuring the absorbance at 436 nm for 1 min at 25 °C. Control set was prepared in the same manner excluding enzyme extract. The activity of peroxidase was expressed as:

Specific activity ðUA mg1 proteinÞ 1

¼ Unit Activity ðU min

As depicted in Table 2, the leaf water potential of the plants increased with Se (10 lM) treatment by 12.5% over the control. The other three concentrations decreased leaf water potential along with their concentration gradient (20, 40 or 80 lM). EBL (108 M) treatment under stress-free conditions resulted in an increase in LWP by 27.5%. Moreover, a higher significant increase of 30% in LWP, compared to control, was observed with EBL along with Se (10 lM). Follow-up treatment of EBL with Se (20, 40 or 80 lM) increased LWP, over the control by 25%, 17.5% and 15.8% respectively.

g1 FMÞ=Protein content ðmg g1 FMÞ 3.3. Chlorophyll content (SPAD level)

2.9.5. Assay for superoxide dismutase (SOD, EC 1.15.1.1) activity Superoxide dismutase activity was assayed by measuring the ability of the enzyme extract to inhibit the photochemical reduction of nitrobluetetrazolium (NBT) (Kono, 1978). Reaction mixture for sample was prepared by 50 mM phosphate buffer (pH 7.0), 2 mM EDTA, 9.9 mM L-methionine, 55 lM NBT, 0.02% Triton

Foliar spray of EBL (108 M) generated a significant increase in chlorophyll content (SPAD values) which was 43.6% more than that of control (Fig. 1A). Out of various concentrations (10, 20, 40 or 80 lM) of Se treatments, 10 lM also increased chlorophyll content by 19.9% in comparison to the other three concentrations (20, 40 or 80 lM), that significantly reduced the values. However, the

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F.S. Naz et al. / Food Chemistry 185 (2015) 441–448 Table 1 Effect of 24-epibrassinolide (EBL; 108 M) and/or selenium (0, 10, 20, 40 or 80 lM) induced changes on root and shoot length, and dry mass plant1 of Brassica juncea at 30 days. Treatment

Root length (cm)

Shoot length (cm)

Dry mass plant1 (g)

Control EBL (108 M) Se (10 lM) Se (20 lM) Se (40 lM) Se (80 lM) Se (10 lM) + EBL Se (20 lM) + EBL Se (40 lM) + EBL Se (80 lM) + EBL LSD@5%

9.0 ± 0.208 11.2 ± 0.252 10.0 ± 0.473 8.1 ± 0.404 7.2 ± 0.321 6.3 ± 0.306 12.0 ± 0.351 9.8 ± 0.361 10.6 ± 0.306 8.7 ± 0.265 0.50

21.0 ± 1.041 27.2 ± 0.473 24.0 ± 0.458 18.5 ± 0.361 15.6 ± 0.737 12.6 ± 0.436 28.9 ± 0.436 25.0 ± 0.361 23.0 ± 0.231 19.6 ± 0.321 1.15

0.85 ± 0.205 1.12 ± 0.095 1.02 ± 0.175 0.71 ± 0.137 0.64 ± 0.123 0.51 ± 0.089 1.14 ± 0.112 0.99 ± 0.131 0.96 ± 0.146 0.79 ± 0.099 0.34

(108 M) (108 M) (108 M) (108 M)

ANOVA table Root length Source of variation

DF

Replication Treatment Error Total

2 9 18 29

F-calculated 175.162

Source of variation

DF

Replication Treatment Error Total

2 9 18 29

Table 2 Effect of 24-epibrassinolide (EBL; 108 M) and/or selenium (0, 10, 20, 40 or 80 lM) induced changes on leaf area plant1 and leaf water potential of Brassica juncea at 30 days. Treatment

Leaf area plant1 (cm2)

Leaf water potential (MPa)

Control EBL (108 M) Se (10 lM) Se (20 lM) Se (40 lM) Se (80 lM) Se (10 lM) + EBL (108 M) Se (20 lM) + EBL (108 M) Se (40 lM) + EBL (108 M) Se (80 lM) + EBL (108 M) LSD@5%

58.6 ± 0.777 65.8 ± 0.351 58.6 ± 0.405 52.7 ± 0.351 48.0 ± 0.379 41.8 ± 0.436 67.3 ± 0.379 63.4 ± 0.306 61.2 ± 0.361 54.8 ± 0.404 0.54

1.20 ± 0.122 0.87 ± 0.122 1.05 ± 0.151 1.33 ± 0.080 1.44 ± 0.105 1.54 ± 0.125 0.84 ± 0.085 0.90 ± 0.171 0.99 ± 0.141 1.01 ± 0.095 0.24

ANOVA table Leaf area plant1 (cm2) Source of variation

DF

Replication Treatment Error Total

2 9 18 29

Dry mass plant1

Shoot length F-calculated 111.661

Source of variation

DF

Replication Treatment Error Total

2 9 18 29

F-calculated 3.389

neutralized the deleterious effect even that of 80 lM Se and completely neutralized the damaging effect of 20 and 40 lM Se. 3.5. Carbonic anhydrase activity As depicted in Fig. 1F, the carbonic anhydrase (CA) activity was significantly enhanced by the EBL (108 M) treatment and was higher by 40%, over the control. The treatment of the plants with Se (10 lM) also enhanced the activity of the enzyme (18.1% over the control) whereas; the remaining treatments (20, 40 or 80 lM) proved deleterious and decreased the activity in a concentration dependant manner. Moreover, EBL (108 M) spray on Se (10 lM) treated plants elevated the enzyme activity by 46.7%, compared to the untreated plants. Also, EBL (108 M) completely recovered the toxic effects generated by Se (20 and 40 lM) treatment and partially that of 80 lM Se.

Leaf water potential (MPa) F-calculated

1988.727

Source of variation

DF

Replication Treatment Error Total

2 9 18 29

F-calculated

8.974

follow-up treatment of EBL (108 M) completely neutralized the toxic effects generated by 20 and 40 lM Se and partially recovered that of 80 lM Se. EBL (108 M) along with Se (10 lM) increased the chlorophyll content (SPAD values) to a maximum by 47.92%.

3.6. Protein content Under stress free conditions, the treatment of the plants with EBL (108 M) increased the content of protein in the leaves by 24.9%, over the control (Fig. 2C). However, in plants which received Se (20, 40 and 80 lM), protein content dropped significantly with increasing concentrations of Se. The foliar spray of EBL (108 M) completely overcame the ill effect generated by 20 and 40 lM Se increasing the values by 16.8% and 5.1%, respectively. The toxic effect of 80 lM Se was partially neutralized by EBL spray. In addition to this, synergistically EBL (108 M) and Se (10 lM) proved best and had value for maximum protein content, over all the other treatments.

3.4. Leaf gas exchange parameters

3.7. Catalase (CAT) activity

The Fig. 1B–E showed that 10 lM Se and EBL (108 M) alone, increased the photosynthetic attributes [net photosynthetic rate (PN), stomatal conductance (gs), internal CO2 concentration (Ci), and water use efficiency (WUE)] whereas; other concentrations (20, 40 or 80 lM) of Se decreased the values of all the parameters in a concentration dependent manner. However, the follow-up treatment of Se-stressed plants with EBL (108 M) partially

The activity of the antioxidative enzyme, catalase (CAT) increased on exogenous application of EBL (108 M) by 29.6% compared to the control (Fig. 2B). However, Se (20, 40 or 80 lM) decreased the activity of this enzyme in a concentration dependant manner. The follow-up treatment of EBL (108 M), increased the CAT activity in comparison to the control as well as to EBL treatment alone. The maximum increase (59.9%) in the activity of CAT

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Fig. 1. Effect of 24-epibrassinolide and/or selenium induced changes in (A) SPAD chlorophyll content, (B) net photosynthetic rate, (C) stomatal conductance, (D) internal CO2 concentration, (E) water use efficiency, and (F) carbonic anhydrase activity of Brassica juncea at 30 days stage of growth. [T1 = control; T2 = EBL (108 M); T3 = Se (10 lM); T4 = Se (20 lM); T5 = Se (40 lM); T6 = Se (80 lM); T7 = Se (10 lM) + EBL (108 M); T8 = Se (20 lM) + EBL (108 M); T9 = Se (40 lM) + EBL (108 M); T10 = Se (80 lM) + EBL (108 M). All the data are the mean of five replicates (n = 5) and vertical bars shows standard errors (±SE)]. Asterisks indicate a significant difference between control and treatment.

was found in plants treated with Se (40 lM) along with foliar spray of EBL (108 M). 3.8. Peroxidase (POX) activity The treatment of plants with various concentration of Se (10, 20, 40 and 80 lM) increased the activity of POX by 29.9%, 30.2%, 38.3% and 26.9% over the control (Fig. 2A). Moreover, the exogenous application of EBL (108 M) increased the activity of the enzyme by 39.9%. The metal stressed plants sprayed with EBL (108 M), had higher POX activity which was much more than in those plants treated with either Se or EBL alone. Se (40 lM) along with EBL (108 M) exhibited maximum activity of POX (86.9% more than the control).

significantly increased SOD activity where the values were much higher than by EBL alone. The most significant increase of 74.2%, over that of the control, was found in plants treated with Se (40 lM) and EBL (108 M). 3.10. Proline content Proline content in the leaves increased in response to various concentrations of Se (Fig. 2C). The values of proline were raised by 19.9%, 25.4%, 27.9% and 32.3%, over the control, respectively in presence to 10, 20, 40 and 80 lM Se. The follow-up treatment of EBL (108 M) further increased the proline content. Maximum accumulation of proline (76.9%), compared to the control was found in the plants subjected to 80 lM Se and subsequently given EBL (108 M) treatment.

3.9. Superoxide dismutase (SOD) activity 4. Discussion Various concentrations of Se (10, 20, 40 or 80 lM) increased the activity of SOD (8.9%, 17.8%, 29.7% and 5.4% over that of control). Moreover, the treatment of EBL (108 M) also increased the enzyme activity by 39.1%, compared to the control (Fig. 2A). The follow up treatment by EBL (108 M), under different levels of Se,

In the present study, plants supplemented with lowest level (10 lM) of Se, exhibited an increase in growth biomarkers (root and shoot length, fresh and dry mass, and leaf area) and water relations (leaf water potential) but declined as the concentration was

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Fig. 2. Effect of 24-epibrassinolide and/or selenium induced changes in (A) activity of peroxidase and superoxide dismutase, (B) catalase activity, and (C) protein and proline content of Brassica juncea at 30 days stage of growth. [T1 = control; T2 = EBL (108 M); T3 = Se (10 lM); T4 = Se (20 lM); T5 = Se (40 lM); T6 = Se (80 lM); T7 = Se (10 lM) + EBL (108 M); T8 = Se (20 lM) + EBL (108 M); T9 = Se (40 lM) + EBL (108 M); T10 = Se (80 lM) + EBL (108 M). All the data are the mean of five replicates (n = 5) and vertical bars shows standard errors (±SE)].

increased (Tables 1 and 2). It is believed that soil enriched with Se can exert beneficial effects on plant growth characteristics but to a definite concentration (Hartikainen, 2005). On the other hand, the observed toxic effect of higher levels of Se on growth characteristics and water relations results from interferences of Se with sulfur metabolism and also from replacing sulfur-amino acids by corresponding Se-amino acids and their subsequent incorporation into proteins that alters metabolism of plant growth and development (Hajiboland & Amjad, 2007). Moreover, the dual response of Se on plant growth (beneficial or toxic) depends on its concentration was also observed in lettuce (Ramos et al., 2010), ryegrass (Hartikainen, Xue, & Piironen, 2000) and other plant species (Hajiboland & Amjad, 2007). Furthermore, EBL (108 M) in combination with Se (10 lM) significantly increased the growth characteristics and water relations over all the other treatments and the control plants (Tables 1 and 2). BRs are major growthpromoting hormones (Gudesblat & 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. Similar response of BRs is reported by various other workers, under different stress and stress free conditions (Fariduddin, Yusuf, Ahmad, & Ahmad, 2014). It is believed that improved growth and water relations are the result of efficient photosynthetic machinery and enhanced chlorophyll synthesis. The findings of present study revealed that net photosynthetic rate and its related attributes (Fig. 1B–E) along with chlorophyll content (SPAD value) increased significantly in the presence of EBL and/or Se (10 lM) whereas, increasing the concentration of Se deteriorated the photosynthetic machinery and also lowered the chlorophyll content (Fig. 1A). In addition to this,

follow up treatment of EBL countered the damage caused by the higher concentrations of Se. BRs had a positive effect on the activation of RuBisCO based on increased maximum RuBisCO carboxylation rates (Vc,max), total RuBisCO activity and, to a greater extent, initial RuBisCO activity induced by an enhanced expression of genes encoding other Calvin cycle genes (Xia et al., 2009). The BRs treatment might have also played a positive role in RuBP regeneration/(Jmax), thereby increasing maximum carboxylation rate of RuBisCO (Vc,max). Thus, BRs promote photosynthesis by positively regulating synthesis and activation of a variety of photosynthetic enzymes including RuBisCO (Xia et al., 2009). Furthermore, Yu et al. (2004) who reported that increase in net photosynthetic rate by BRs might be the result of the activation of ribulose 1,5-bisphosphatecarboxylase and/or enhanced activity of CA (Fig. 1F) and chlorophyll content (Fig. 1A). Our findings are in line with report of Fariduddin et al. (2014) that BRs improve the photosynthesis and related attributes, including quantum yield of PSII under various abiotic stresses. Earlier findings also corroborated that low doses of Se enhanced photosynthesis in rice seedlings (Wang, Wang, & Wong, 2012). However, Se toxicity induces the damage to photosynthetic apparatus, inhibits photosynthesis, and results in the overproduction of starch (Wang et al., 2012). Yu, Liu, Luo, and Peng (2003) showed that Se could increase the contents of Mg, Fe and Mn (involved in photosynthetic machinery) in soybean leaves when the application rate of Se was low and also revealed that soybean chloroplast membrane structure were in a good shape. Se at lower doses enhanced the activity of carbonic anhydrase (CA), whereas higher levels of selenium exhibited a decline in the enzyme activity (Fig. 1F). CA has been recognized as an important enzyme that is closely associated with photosynthesis. On the other hand, higher concentrations of Se replace sulfur in amino acids, with subsequent alteration of protein three-dimensional structure and impairment of enzymatic

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function (Amweg, Stuart, & Weston, 2003). This supports the results obtained in this study that higher concentrations of Se had lowered protein content whereas, EBL treatment improved the protein content (Fig. 2C) over the non-treated control plants and overcome the Se mediated loss. BR-mediated increase in stress tolerance is integrated with other hormone pathways and several hormone responsive genes are also BR-responsive. Generally, two types of antioxidants are triggered in plants to balance the excess reactive oxygen species (ROS). One type is the low molecular weight substances, such as glutathione (GSH), ascorbate (AsA) and tocopherol, and the other type is enzymes, such as, superoxide dismutase (SOD), peroxidase (POX), catalase (CAT), ascorbate peroxidase (APX), glutathione peroxidase (GSHPx), guaiacol peroxidase (GPOX) and glutathione reductase (GR) (Asada, 2006). These antioxidants can react with ROS directly or indirectly via enzyme catalysis to counteract the production of ROS, under stress conditions as Mittler (2002) believed that ROS, under control conditions act as signals for the activation of the stress response and defense pathways. In the present investigation, under excess Se, enzymatic (superoxide dismutase, catalase and peroxidase) antioxidant systems increased (Fig. 2A and B) to scavenge the Se induced excess ROS. Reports have shown that excess Se gives rise to the robust accumulation of ROS in plants, although the actual role of Se in plants has not yet been resolved (MroczekZdyrska & Wójcik, 2011). Feng, Chaoyang, and Tu (2013) proposed that the increased production of ROS at high Se levels may be partially related to an imbalance in the levels of GSH, thiols (SH), ferredoxins (Fdred) and/or NADPH, which can play vital roles in the assimilation of Se. If these substances are not sufficient to simultaneously meet the needs of Se-assimilation and ROS quenching, the addition of Se may lead to a ROS burst and the inhibition of plant growth (Tables 1 and 2). However, one innovative thing that emerged in the present study is that the treatment of plants with EBL both in the absence or presence of excess Se enhanced the activities of antioxidant enzymes (CAT, POX and SOD). Therefore, maximum values were recorded in the plants subjected to Se (40 lM), followed by application of EBL (108 M). The elevation in antioxidant enzymes by BRs was the consequence of enhanced expression of det2 gene, which enhanced the tolerance to oxidative stress in Arabidopsis (Cao et al., 2005) through antioxidant system. Our findings are in accordance with the reports which showed that the application of BRs modified activities of antioxidant enzymes, under various abiotic stresses (Fariduddin et al., 2014). Proline serves as persuasive inhibitor of PCD (Gill & Tuteja, 2010) and also acts as non-enzymatic antioxidant that is known to stabilize the sub cellular structures such as that of proteins and cell membranes, scavenging free radicals and buffering redox potential under stress conditions and also have the ability of molecular chaperones that protect the integrity of protein and enhances the activity of different enzymes, such as protection of nitrate reductase during heavy metal stress (Szabados & Savoure, 2009). In addition to this, among various compatible solutes, proline is the only molecule that has been shown to protect plants against singlet oxygen and free radical induced damages resulting from stress (Alia, Pardha, & Mohanty, 1997). These reports authenticate the present observations where application of EBL treatment under Se enhanced the accumulation of proline (Fig. 2C). 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 under various abiotic stresses (Fariduddin et al., 2014). Low Se (10 lM) concentration also resulted in greater plant growth (Tables 1 and 2) and photosynthetic pigments accumulation (Fig. 2A). Thus, it can be suggested that elevated proline level induced by Se supplementation may play some role in the growth

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and development. Increase of proline content in Se-treated soybean plants has also been reported by Djanaguiraman, Devi, Shanker, Sheeba, and Bangarusamy (2005). However, the mechanism and the reasons for proline accumulation in Se-supplied plants have not been fully investigated and still needs debate.

5. Conclusions In conclusion, selenium mediated response is concentration dependent and 10 lM of Se acts as quasi-essential micronutrient through altered physiological and biochemical traits which were reflected as improved growth and photosynthesis whereas, higher concentrations (20, 40, or 80 lM) of Se induced deleterious effect in B. juncea plants. On the other hand, Se (10 lM) synchronizes with EBL (108 M) and acts as an effective combination to enhance the growth and photosynthesis. Moreover, the antioxidant systems and proline accumulation speed up to cope with damages triggered by Se stress which was further accelerated by the follow up treatment with EBL (108 M). Therefore, it is believed that exogenous application of EBL (108 M) in combination with lower levels (10 lM) of Se could be exploited to improve the productivity and as potent inhibitor of oxidative stress in Indian mustard plants to resist the higher concentrations of Se.

Acknowledgments Fatima S. Naz gratefully acknowledges the Chairman, Department of Botany for providing all necessary facilities in carrying out this work and M. Yusuf also gratefully acknowledges the financial assistance rendered by the SERB (SB/FT/LS-210-2012), DST, New Delhi, India.

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