Chemosphere 85 (2011) 1574–1584

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Chemosphere

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28-Homobrassinolide mitigates boron induced toxicity through enhancedantioxidant system in Vigna radiata plants

Mohammad Yusuf, Qazi Fariduddin ⇑, Aqil AhmadPlant Physiology and Biochemistry Section, Department of Botany, Aligarh Muslim University, Aligarh 202 002, India

a r t i c l e i n f o

Article history:Received 7 April 2011Received in revised form 28 July 2011Accepted 3 August 2011Available online 6 September 2011

Keywords:Boron toxicityBrassinosteroidsPhotosynthesisLipid peroxidationAntioxidant enzymesOxidative stress

0045-6535/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.chemosphere.2011.08.004

⇑ Corresponding author. Tel.: +91 9412 172 134; faE-mail address: qazi_farid@yahoo.com (Q. Faridud

a b s t r a c t

The objective of this study was to establish relationship between boron induced oxidative stress and anti-oxidant system in Vigna radiata plants and also to investigate whether brassinosteroids will enhance thelevel of antioxidant system that could confer tolerance to the plants from the boron induced oxidativestress. The mung bean (V. radiata cv. T-44) plants were administered with 0.50, 1.0 and 2.0 mM boronat 6 d stage for 7 d along with nutrient solution. At 13 d stage, the seedlings were sprayed with deionizedwater (control) or 10�8 M of 28-homobrassinolide and plants were harvested at 21 d stage to assessgrowth, leaf gas-exchange traits and biochemical parameters. The boron treatments diminished growth,water relations and photosynthetic attributes along with nitrate reductase and carbonic anhydrase activ-ity in the concentration dependent manner whereas, it enhanced lipid peroxidation, electrolyte leakage,accumulation of H2O2 as well as proline, and various antioxidant enzymes in the leaves of mung beanwhich were more pronounced at higher concentrations of boron. However, the follow-up applicationof 28-homobrassinolide to the boron stressed plants improved growth, water relations and photosynthe-sis and further enhanced the various antioxidant enzymes viz. catalase, peroxidase and superoxide dis-mutase and content of proline. The elevated level of antioxidant enzymes as well as proline couldhave conferred tolerance to the B-stressed plants resulting in improved growth, water relations and pho-tosynthetic attributes.

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1. Introduction

Boron (B) is well documented as an essential micronutrient foroptimum growth of vascular plants. However, when B is presentabove the permissible limit in the soil or ground water, plantgrowth and reproduction can be affected, limiting crop productiv-ity throughout the world (Stangoulis and Reid, 2002; Reid et al.,2004). B toxicity is extensively located in the agricultural areas ofAustralia, North Africa, and West Asia characterized by alkalineand saline soils together with a low rainfall and very scare leach-ing. In addition to this, B-rich soils also occur as a consequenceof over fertigation and/or irrigation with water containing high lev-els of B (WHO, 1998; Parks and Edwards, 2005).

The exclusively established symptoms shown by plants exposedto excess B are reduced root cell division (Liu and Yang, 2000), de-creased shoot and root growth, lower stomatal conductance (Lov-att and Bates, 1984; Nable et al., 1990) and content ofchlorophyll, inhibition of photosynthesis, deposition of lignin andsuberin (Ghanati et al., 2002), reduced proton extrusion from roots(Roldan et al., 1992), increased membrane leakage, peroxidation of

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x: +91 571 270 2016.din).

lipids and altered activities of antioxidant pathways (Karabal et al.,2003; Keles et al., 2004).

It is now well acceptable fact that most of the ionic stressesincluding B trigger the formation of reactive oxygen species(ROS) which lead to the establishment of oxidative stress in plants.Moreover, during oxidative stress, excess production of ROS suchas superoxide radical (O��2 ) and hydroxyl radical (OH��) which arestrong oxidizers of lipids, proteins and nucleic acids causing mem-brane damage that eventually leads to cell death (Del Rio et al.,2003). Plants possess multifunctional enzymatic and non-enzy-matic antioxidant defense mechanisms to combat the oxidative ef-fects of ROS. Among these, superoxide dismutase (SOD) dismutatesO��2 to O2 and H2O2, which are further oxidized to molecular oxygenand H2O by peroxidases (POX), catalase (CAT) and ascorbate–gluta-thione pathway enzymes like ascorbate peroxidase (APOX) andglutathione reductase (GR) (Mathews et al., 1984). It was observedthat high supply of B invoked the formation of ROS which inducedoxidative damage by lipid peroxidation and accumulation ofhydrogen peroxide in leaves (Karabal et al., 2003; Molassiotiset al., 2006). It has been suggested that an efficient antioxidant sys-tem reduced the damage caused by excess B in grapevine (Guneset al., 2006) and tomato (Cervilla et al., 2007), indicating a crucialrole of antioxidant system in conferring tolerance to B stress inplants.

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In plants, coordination of growth and development is regulatedby both external and internal stimuli and related mechanisms. Inmost multicellular organisms, steroids act as internal modulatorfor regulating physiological and development processes. Brassinos-teroids (BRs) are a specific class of low-abundance plant steroidalhormone of ubiquitous occurrence in plants (Belkhadir and Chory,2006; Bajguz and Hayat, 2009). BRs induce a broad spectrum of re-sponses including stem elongation, pollen tube growth, leaf bend-ing and epinasty, xylem differentiation, and syntheses of nucleicacids and proteins (Clouse and Sasse, 1998) however; stimulationof growth via cell elongation and cell division is a major biologicaleffect of BRs (Zurek et al., 1994; Hu et al., 2000). Moreover, furthergenetic and biochemical approaches have contributed to animpressive progress in our understanding the precise role of BRsin the plant metabolism (Choe et al., 1999; Noguchi et al., 2000),and also in BR-induced signaling including, the identification ofBR receptors, key signaling elements, and BR-induced gene expres-sion (Choe et al., 2002; Geldner et al., 2007;). Numerous studieshave also reported that BRs are able to increase the plant’s abilityto cope with stress, such as water stress (Fariduddin et al., 2009a),pathogen attack (Krishna, 2003), chilling stress (Wilen et al., 1995;Fariduddin et al., 2011), salinity (Ali et al., 2007), and various heavymetal stresses-namely cadmium (Hayat et al., 2007), nickel (Yusufet al., 2011), aluminum (Ali et al., 2008), and copper (Fariduddinet al., 2009b). In addition to this, BRs have ability to enhance theyield and stress tolerance in plants and also the positive interac-tions of BRs with the plants under various abiotic stresses couldbe exploited for phytoremediation technologies (Barbafieri andTassi, 2011).

It is believed that B-deficiency could be resolved by applicationof fertilizers enriched in B, whereas, toxicity is more difficult prob-lem to address. So, it is the pressing need to explore easy, efficientand farmer’s-friendly tool to minimize the toxicity generated byexcess levels of B. With this aim, the present study was designedto explore the anti-stress response of brassinosteroids under differ-ent levels of B and also to investigate the antioxidant defensemechanisms for combating the oxidative stress. The hypothesistested is that brassinosteroids will elevate the level of antioxidantsystem that could protect the plants from the B-stress which fur-ther manifested in terms of improvement in growth andphotosynthesis.

2. Materials and methods

2.1. Plant material

The seeds of Vigna radiata L. Wilczek cv. T-44 were obtainedfrom National Seed Corporation Ltd., New Delhi, India. The healthylooking and uniform size seeds were surface sterilized with 1% so-dium hypochlorite solution for 10 min, followed by repeated wash-ing with double distilled water (DDW).

2.2. Hormone preparation

28-Homobrassinolide (HBL) was obtained from Godrej-AgrovetPvt. Ltd. Mumbai, India. A stock solution of HBL (10�4 M) was pre-pared by dissolving required quantity of the HBL in 5 mL of ethanolin a 100 mL volumetric flask and final volume was made up to themark by using double distilled water (DDW). The desired concen-tration of HBL i.e. 10�8 M was prepared by the dilution of stocksolution and the selection of concentration of HBL was based onthe earlier work carried by us (Fariduddin et al., 2004). Tween-20was added as surfactant prior to the foliar application.

2.3. Source of boron (B)

Boric acid (H3BO3) was used as the source of B. A stock solutionof B (1.0 M) was prepared by dissolving required quantity of the Bin 10 mL of DDW in a 100 mL volumetric flask and final volumewas made up to the mark by using deionized water. The requiredconcentrations (0.50, 1.0, and 2.0 mM) of B were prepared by thedilution of stock solution and the selection of concentration wasbased on the work carried out by Cervilla et al. (2007).

2.4. Treatment pattern and experimental design

The surface sterilized seeds were sown in acid washed sand,moistened with deionized water and were allowed to germinatein a plant growth chamber. On 6 d stage, the seedlings were sup-plemented with different concentrations (0.50, 1.0, and 2.0 mM)of B along with nutrient solution (Hewitt, 1966) for 7 d. At 13 dstage, the seedlings were sprayed with deionized water (control)or 10�8 M of HBL. Each seedling was sprinkled thrice. The nozzleof the sprayer was adjusted in such a way that it pumped out1 mL (approx.) in one sprinkle. Therefore, each seedling received3 mL of DDW or HBL solution. The plants were then allowed togrow under controlled environmental conditions; 25/20 �C (day/night); 70/80 % RH (day/night) and 14 h photoperiod and were irri-gated with deionized water and nutrient solution on alternatedays. Plants were harvested at 21 d stage to assess the variousgrowth and leaf gas exchange traits as well as biochemical param-eters. The experiment was conducted in five independent repli-cates, under completely randomized block design with 40 cups of350 mL size. Each treatment was replicated five times and eachreplicate (one cup) contained three plants.

2.5. Analysis of growth biomarkers

The plants were removed from the pots along with the sand andwere dipped in a bucket filled with water. The plants were movedsmoothly to remove the adhering sand particles and the lengths ofroot and shoot were measured by using a meter scale. The plantswere blotted and weighed to record their root and shoot fresh massand then placed in an oven, run at 70 �C for 72 h. The samples wereweighed again after allowing them to cool at room temperature torecord their root and shoot dry mass. The leaf area was measuredby using a graph sheet, where the squares covered by the leaf werecounted to note the leaf area.

2.6. Determination of leaf relative water content (RWC) and leaf waterpotential (LWP)

The relative water content (RWC) was determined in fresh leafdiscs of 2 cm2 diameter, excluding midrib. Discs were weighedquickly and immediately floated on deionized water in petri dishesto saturate them with water for the next 24 h, in dark. The adher-ing water of the discs was blotted and turgor mass was noted. Drymass of the discs was recorded after dehydrating them at 70 �C for48 h. RWC was calculated by placing the values in the followingformula:

RWC = (fresh mass-dry mass)/(turgor mass-dry mass) � 100(Hayat et al., 2007).

The leaf water potential (LWP) was monitored with the help ofPsypro water potential system (Wescor, Inc. USA).

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2.7. Determination of chlorophyll (SPAD) value and fluorescence i.e.Maximum quantum yield of PS II (Fv/Fm)

The SPAD value of chlorophyll in the fresh leaf was measured byusing the SPAD chlorophyll meter (SPAD-502; Konica, Minoltasensing, Inc., Japan).

Chlorophyll fluorescence (Fv/Fm) was measured by using a leafchamber fluorometer (Li-COR 6400-40, Li-COR, and Lincoln, NE,USA). All the measurements were carried out at a photosyntheticphoton flux density (PPFD) of 1500 lmol m�2 s�1 with a constantairflow rate of 500 lmol s�1. The minimal fluorescence level (F0)was determined by modulated light, which was sufficiently low(<1 lmol m�2 s�1) not to induce any significant variable fluores-cence. The maximal fluorescence (Fm) was determined by a 0.8 ssaturation pulse at 4200 lmol m�2 s�1 on dark-adapted leaves(30 min). The sampled leaf was dark-adapted for 30 min prior tomeasurement of Fv/Fm.

2.8. Analysis of leaf gas exchange parameters

Gas-exchange parameters were determined on the third fullyexpanded leaves between 11:00 and 12:00 h by using an infraredgas analyzer (IRGA) portable photosynthetic system (Li-COR6400, Li-COR, and Lincoln, NE, USA). To measure net photosyn-thetic rate (A) and its related attributes [stomatal conductance(gs), internal CO2 concentration (ci), water use efficiency (WUE)],the air temperature, relative humidity, CO2 concentration andPPFD were maintained at 25 �C, 85%, 600 lmol mol�1 and800 lmol mol�2 s�1, respectively.

2.9. Determination of nitrate reductase (NR) and carbonic anhydrase(CA) activity

The activity of nitrate reductase (NR) was measured followingthe method laid down by Jaworski (1971). The fresh leaf sampleswere cut into small pieces and transferred to plastic vials, contain-ing phosphate buffer (pH 7.5), KNO3 and isopropanol wasincubated at 30 �C for 2 h. After incubation, sulfanilamide andN-1-naphthylethylenediamine hydrochlorides solutions wereadded. The absorbance was read at 540 nm on a spectrophotome-ter (Spectronic 20D; Milton Roy, USA).

The activity of carbonic anhydrase (CA) in the leaves was mea-sured following the method described by Dwivedi and Randhawa(1974). The leaf samples were cut into small pieces in cysteinehydrochloride solution. These leaf samples were blotted and trans-ferred in a test tube, followed by the addition of phosphate buffer(pH 6.8), 0.2 M NaHCO3, bromothymol blue, and the methyl redindicator, at the last. This reaction was titrated against 0.5 N HCl.The activity of the enzyme was expressed on a fresh mass basis.

2.10. Determination of lipid peroxidation

Lipid peroxidation rates were estimated by measuring the mal-ondialdehyde equivalents according to Hodges et al. (1999). 0.5 gof the leaf was homogenized in a mortar with 80% ethanol. Thehomogenate was centrifuged at 3000g for 10 min at 4 �C. The pelletwas extracted twice with the same solvent. The supernatants werepooled and 1 mL of this sample was added to a test tube with anequal volume of the solution comprised of 20% trichloroacetic acid,0.01% butylated hydroxy toluene and 0.65% thiobarbutyric acid.Samples were heated at 95 �C for 25 min and cooled to room tem-perature. Absorbance of the samples was recorded at 440, 532 and600 nm. Lipid peroxidation rates equivalent (n mol malondialde-hyde mL�1) were calculated by using the formula given by Hodgeset al. (1999).

2.11. Determination of H2O2 content and electrolyte leakage (EL)

The hydrogen peroxide accumulation was determined by themethod proposed by Jana and Choudhari (1981). 500 mg plantsample was homogenized in 3.0 mL of phosphate buffer (50 mMand pH 6.8). The homogenate was centrifuged at 6000g for25 min. 3.0 mL of extract was mixed with 0.1% titanium chloridein 20% (v/v) sulphuric acid and the mixture was again centrifugedat 6000g for 15 min. The absorbance of the colour was read at410 nm, on a spectrophotometer and was compared with that ofthe calibration curve. The H2O2 content was computed on freshmass basis using a standard curve of known concentration of H2O2.

The total inorganic ions leaked out of the leaves were measuredby the method described by Sullivan and Ross (1979). Twenty leafdiscs were taken in a boiling test tube, containing 10 mL of deion-ized water and electron conductivity (EC) was measured (ECa). Thecontents were heated at 45 �C and 55 �C for 30 min each in a waterbath and EC was measured (ECb). Later the controls were boiled at100 �C for 10 min and EC was again recorded (ECc). The electrolyteleakage was calculated using the formula:

Electrolyte leakage ð%Þ ¼ ½ðECb� ECaÞ=ðECcÞ � 100�

2.12. Antioxidative enzymes assay

For the assay of antioxidant enzymes, the leaf tissue (0.5 g) washomogenized in 50 mM phosphate buffer (pH 7.0) containing 1%polyvinylpyrrolidone. The homogenate was centrifuged at27600g for 10 min at 4 �C and the supernatant was used as sourceof enzymes catalase, peroxidase and superoxide dismutase andglutathione reductase.

Peroxidase and catalase were assayed following the proceduredescribed by Chance and Maehly (1956). Catalase was estimatedby titrating the reaction mixture, consisting of phosphate buffer(pH 6.8), 0.1 M H2O2, enzyme extract and 2% H2SO4, against 0.1 Npotassium permagnate solution. The reaction mixture for peroxi-dase consisted of pyragallol, phosphate buffer (pH 6.8), 1% H2O2

and enzyme extract. Change in absorbance due to catalytic conver-sion of pyragallol to purpurogallin, was noted at an interval of 20 sfor 2 min, at 420 nm on a spectrophotometer. A control set wasprepared by using DDW instead of enzyme extract. The activityof superoxide dismutase was assayed by measuring its ability toinhibit the photochemical reduction of nitroblue tetrazolium fol-lowing the method of Beauchamp and Fridovich (1971). The reac-tion mixture contained 50 mM phosphate buffer (pH 7.8), 13 mMmethionine, 75 mM nitroblue tetrazolium, 2 mM riboflavin,0.1 mM EDTA and 0–50 mL enzyme extract and was placed under15 W fluorescent lamp. The reaction was started by switching onthe light and was allowed to run for 10 min. The reaction wasstopped by switching off the light. 50% inhibition by light was con-sidered as one enzyme unit.

Glutathione reductase was assayed as per the method of Smithet al. (1988). The reaction mixture contained, 66.67 mM potassiumphosphate buffer (pH 7.5), 0.33 mM EDTA, 0.5 mM 5,5-dithiobis-(2-nitrobenzoic acid) in 0.01 M potassium phosphate buffer (pH7.5), 66.67 mM NADPH, and 66.67 mM oxidized glutathione and0.1 mL enzyme extract. The reaction was started by adding oxi-dized glutathione and the increase in absorbance at 412 nm wasrecorded spectrophotometrically.

2.13. Determination of proline accumulation

The proline content in fresh leaf samples was determined byadopting the method of Bates et al. (1973). Sample was extractedin sulphosalicylic acid. To the extract an equal volume of glacialacetic acid and ninhydrin solutions were added. The sample was

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heated at 100 �C to which 5 mL of toluene was added. The absor-bance of toluene layer was read at 528 nm on a spectrophotometer.

2.14. Statistical analysis

Data were statistically analyzed using SPSS, 17.0 for windows(SPSS, Chicago, IL, USA). Standard error was calculated and analysisof variance (ANOVA) was performed on the data to determine theleast significance difference (LSD) between treatment means withthe level of significance at P 6 0.05.

3. Results

3.1. Growth biomarkers

All the growth biomarkers (root and shoot length; root andshoot fresh and dry mass and leaf area) affected by the exogenousapplication of HBL and presence of B in the soil in comparison tothe non-treated (Control) plant (Figs. 1A–F and 2A). Out of variousconcentrations (0.50, 1.0, or 2.0 mM) of B, 2.0 mM generated max-imum reduction in the root and shoot length by 43.4% and 42.1%;root fresh and dry mass by 30.3% and 59.1%; shoot fresh and drymass by 42.4% and 54.4% and leaf area by 43.2% compared withthe respective control (Figs. 1A–F and 2A). However, the foliageof plants received HBL (10�8 M) alone had more values than con-trol plants for the growth biomarkers and also the follow-up treat-ment with HBL partially neutralized the deleterious effect of 2 mMof B whereas, toxicity generated by 0.50 and 1 mM of B was com-pletely nullified and values were more than control in all the abovementioned parameters.

3.2. Leaf relative water content (RWC) and leaf water potential (LWP)

The plants raised in the presence of different concentrations(0.50, 1.0, or 2.0 mM) of B had lower RWC and LWP comparedwith unstressed control in concentration dependent manner andmaximum reduction was noted in 2.0 mM of B (Fig. 2C and D).Contrary to this, exogenous application of HBL (10�8 M) signifi-cantly increased the RWC as well as LWP over the control plants.In addition to this, follow-up application with HBL (10�8 M) to theB-stressed plants, partially recovered the loss of RWC and LWPgenerated by 2.0 mM of B and completely that of 0.50 and1.0 mM of B.

3.3. Chlorophyll content (SPAD level) and chlorophyll fluorescence i.e.Maximum quantum yield of PSII (Fv/Fm)

Out of three levels (0.50, 1.0, or 2.0 mM) of B, 0.50 mM did notgenerate any significant decline whereas, 2.0 mM of B generatedmaximum decrease in both chlorophyll content (SPAD level;Fig. 2B) and Fv/Fm (Fig. 3F) by 38.3% and 30.2% respectively, thanthe control plants. The foliar application of HBL alone increasedboth chlorophyll content (SPAD level) as well as Fv/Fm over thenon-treated plants and also the follow-up treatment of HBL tothe stressed plants minimized the ill effect generated by differentlevels of B.

3.4. Leaf gas exchange parameters

It is evident from Fig. 3B–D that the presence of different levels(0.50, 1.0, or 2.0 mM) of B decreased the net photosynthetic rate(Fig. 3B) and related attributes i.e. stomatal conductance (gs;Fig. 3C), internal CO2 concentration (Ci; Fig. 3D) and water use effi-ciency (WUE; Fig. 3E) where, 2.0 mM proved most deleterious. Onthe other hand, the application of HBL (10�8 M) significantly in-

creased the gas exchange parameters and the values for net photo-synthetic rate (32.1%) and stomatal conductance (30.8%) more thanthe control plants. Moreover, the follow-up treatment with HBL(10�8 M) neutralized the deleterious effect of B in concentrationdependent manner.

3.5. Carbonic anhydrase (CA) and nitrate reductase (NR) activity

It is depicted from Figs. 2F and 3A that there is significantdecline in the activity of both CA and NR in the plants grown inthe sand supplemented with 2.0 mM of B followed by 1.0 and0.50 mM. The foliar application of HBL (10�8 M) alone signifi-cantly increased the activity of both the enzymes by 26.4% and24.0%, respectively, over the control plants whereas, the follow-up treatment with HBL to the B-stressed plants completely over-came the toxicity generated by 0.50 and 1.0 and partially that of2.0 mM.

3.6. Lipid peroxidation (LPO), H2O2 content and electrolyte leakage(EL)

The Figs. 4A, B and 2E shows that both the LPO and H2O2 accu-mulation in leaves increased with the increasing concentration ofB. At 2.0 mM of B treatment, LPO and H2O2 accumulated by74.5% and 40.6% higher, respectively over the control plants. How-ever, the accumulation of H2O2 was relatively less than LPO at2.0 mM of B treatment. HBL alone decreased both the LPO andH2O2 accumulation (Fig. 4A and B) and in addition to this, it alsolowered down the values of LPO and H2O2 accumulation in B-stressed plants when applied as follow-up treatment.

EL also showed the pattern similar to that of LPO and H2O2

accumulation and the maximum leakage of ions was reported inthe plants subjected to 2.0 mM of B by 35.96% over the non-stressed control plants. However, the exogenous application ofHBL (10�8 M) alone lowered down the leakage of ions in compari-son to the control.

3.7. Antioxidative enzymes

The antioxidative enzymes, catalase, peroxidase, superoxidedismutase and glutathione reductase exhibited an increasing trendin response to both HBL and B treatment except for 0.50 mM of Bover the control plants (Fig. 4C–F). The HBL alone caused a signif-icant increase in the activities of all the enzymes. However, theassociation of B and HBL treatment in plants further improvedthe activities of above mentioned antioxidative enzymes. More-over, the maximum activities of antioxidative enzymes [CAT(62.4%), POX (64.8%), SOD (98.9%) and GR (51.9%)] were recordedin the plants exposed to 2.0 mM of B stress and subsequently re-ceived HBL.

3.8. Proline content

The level of proline exhibited an increase in response to B-stress, both in roots and leaves compared to the control (Fig. 5Aand B). The accumulation of proline was higher in roots than theleaves. The application of HBL on the unstressed plants broughtabout a change in the level of proline (Fig. 3C). However, in associ-ation with B, HBL further increased the quantity of proline, both inleaves and roots. The maximum accumulation of proline (119.7%)was found in the roots which were subjected to 2.0 mM B stressand subsequently received HBL, over the non-treated controlpants.

Fig. 1. Effect of 28-homobrassinolide (HBL; 10�8 M) on the boron (0.50, 1.0, and 2.0 mM) induced changes in (A) root length, (B) shoot length, (C) shoot fresh mass, (D) rootfresh mass (E) Shoot dry mass and (F) root dry mass in Vigna radiata plants at 21 d stage of growth. All the data are the mean of five replicates (n = 5) and vertical bars showsstandard errors (±SE). Asterisks indicate a significant difference between control and treatment (P < 0.05).

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4. Discussion

The foliage of the plants, developed in the sand supplementedwith different levels of B, exhibited a sharp decline in the activityof CA and NR, in a concentration dependent manner (Figs. 2F and

3A). The possible reason behind this may be that B has ability ofmetabolic disruption by binding to the ribose moieties of mole-cules such as ATP, NADH or NADPH (Reid et al., 2004) and alsoan inhibition and/or metabolic dysfunction of the enzyme protein(Hopkins, 1995). In addition to this, ionic metal also has a role in

Fig. 2. Effect of 28-homobrassinolide (HBL; 10�8 M) on the boron (0.50, 1.0, and 2.0 mM) induced changes in (A) leaf area, (B) chlorophyll content (SPAD level), (C) leafrelative water content, (D) leaf water potential, (E) electrolyte leakage and (F) carbonic anhydrase (CA) activity in Vigna radiata plants at 21 d stage of growth. All the data arethe mean of five replicates (n = 5) and vertical bars shows standard errors (±SE). Asterisks indicate a significant difference between control and treatment (P < 0.05).

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controlling the activity of plasmamembrane bound proton pump(Obata et al., 1996) and the fluidity of the membrane (Meharg,1993), restricting the uptake of nitrate (Harnandez et al., 1996),the inducer and the substrate of NR (Campbell, 1999). Conversely,the application of HBL in stress-free or as a follow up treatment in

B-stressed plants elevated the activity of both CA and NR that couldbe an expression of impact of BRs on translation and/or transcrip-tion (Khripach et al., 2003). The other possible reason may be theinvolvement of HBL in elevating the level of NO3 by acting at themembrane in case of NR (Mai et al., 1989) and speeding up

Fig. 3. Effect of 28-homobrassinolide (HBL; 10�8 M) on the boron (0.50, 1.0, and 2.0 mM) induced changes in (A) nitrate reductase (NR) activity, (B) net photosynthetic rate,(C) stomatal conductance, (D) internal CO2 concentrations, (E) water use efficiency and (F) maximum quantum yield of PS II (Fv/Fm) in Vigna radiata plants at 21 d stage ofgrowth. 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 andtreatment (P < 0.05).

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assimilation of CO2 (Yu et al., 2004) which is also supported by in-creased level of Ci and gs (Fig. 3C and D) in case of CA. An increasein NR and CA is in conformity with the finding of other workers un-der different ionic stress (Fariduddin et al., 2009a,b; Yusuf et al.,2011).

In the present study, chlorophyll value (SPAD level) was lowerin leaves of B-treated plants, but more pronounced at 2.0 mM ofB. This decrease of chlorophyll value might be due to the B inducereactive oxygen species (Reid et al., 2004; Camacho-Cristobal et al.,2008) to cause photo-oxidative damages in organic molecules

Fig. 4. Effect of 28-homobrassinolide (HBL; 10�8 M) on the boron (0.50, 1.0, and 2.0 mM) induced changes in (A) H2O2 content, (B) lipid peroxidation, (C) catalase activity, (D)peroxidase activity, (E) superoxide dismutase activity and (F) glutathione reductase activity in Vigna radiata plants at 21 d stage of growth. All the data are the mean of fivereplicates (n = 5) and vertical bars shows standard errors (±SE). Asterisks indicate a significant difference between control and treatment (P < 0.05).

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(Papadakis et al., 2004). Beside this, Fig. 3B–E also revealed that Binduced decrease in net photosynthetic rate (A) and related attri-butes (gs, Ci and WUE) along with chlorophyll fluorescence i.e.maximum quantum yield of PSII (Fv/Fm) (Fig. 3F) in a concentrationdependent manner. One of the probable reason for the reduction ofA, gs, Ci, WUE and E is the structural damage of thylakoids, whichaffects the photosynthetic transport of electrons, as indicated bythe reduction of the ratio between variable fluorescence and initialfluorescence (Fv/F0) (Pereira et al., 2000). Further, the decrease inchlorophyll content (Fig. 2B), leading to reduction in net photosyn-thesis, could be attributed to oxidation of chlorophyll and chloro-plastic membranes, which might be exacerbated by the excess of

B (Lee, 2006; Ardic et al., 2009). The decrease in maximum quan-tum yield i.e. chlorophyll fluorescence (Fv/Fm) (Fig. 3F) can beattributed to oxidation of chlorophyll and chloroplastic mem-branes, which might be exacerbated by excess B, as reported inhot pepper (Lee, 2006) and apple rootstocks (Sotiropoulos et al.,2006). According to Sotiropoulos et al. (2002), B toxicity in kiwifruit induced a significant decrease of the photosynthetic rateand a significant increase of the intercellular CO2 concentration.Lovatt and Bates (1984) have further reported that net photosyn-thesis and stomatal conductance of summer squash leaves weresignificantly decreased, due to B toxicity. The above mentioneddetrimental effect of B was counteracted in the plants exposed to

Fig. 5. Effect of 28-homobrassinolide (HBL; 10�8 M) on the boron (0.50, 1.0, and 2.0 mM) induced changes in (A) leaf proline content, and (B) root proline content in Vignaradiata plants at 21 d stage of growth. All the data are the mean of five replicates (n = 5) and vertical bars shows standard errors (±SE). Asterisks indicate a significantdifference between control and treatment (P < 0.05).

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a follow up treatment with HBL and also completely neutralizedthe effect of the lower concentration of B (0.50 and 1.0 mM) andpartially that of higher concentration (2.0 mM). In addition to this,exogenous application of HBL to the non-stressed plant, signifi-cantly enhanced SPAD level, net photosynthetic rate and their re-lated attributes along with Fv/Fm (Fig. 3F) that is in support ofothers (Fariduddin et al., 2004; Hasan et al., 2008; Fariduddinet al., 2009a,b; Hayat et al., 2010; Yusuf et al., 2011). The reasonlooking most appropriate in defending the said observation is pos-sibly that the BR-induced impact on transcription and/or transla-tion (Bajguz, 2000) which is further supported by Yu et al. (2004)who reported that increase in net photosynthetic rate by HBLmight be the result of the activation of ribulose 1,5-bisphosphate-carboxylase (Rubisco) and/or enhanced activity of CA and chloro-phyll content (Fig. 2B). Moreover, BRs had a positive effect on theactivation of Rubisco based on increased maximum Rubisco car-boxylation rates (Vc,max), total Rubisco activity and, to a greater ex-tent, initial Rubisco activity induced by an enhanced expression ofgenes encoding other Calvin cycle genes after BRs treatment mightalso play a positive role in RuBP regeneration/(Jmax), therebyincreasing maximum carboxylation rate of Rubisco (Vc,max). Thus,BRs promote photosynthesis by positively regulating synthesisand activation of a variety of photosynthetic enzymes includingRubisco (Xia et al., 2009). Earlier reports also showed that exoge-nous application of BRs improved photosynthesis and related attri-butes, including quantum yield of PSII under various abioticstresses particularly mineral nutrition stress (Ali et al., 2008; Fari-duddin et al., 2009b; Yusuf et al., 2011).

It is a challenging task to determine which process is mostresponsible for the decline in overall growth of plant underB-stress conditions. In the present study, there is decrease in thegrowth traits i.e. root and shoot length, fresh and dry mass of rootand shoot, and leaf area in the plants supplemented with variedlevels of B and 2.0 mM of B proved to be highly toxic (Figs. 1A–Fand 2A). Under B toxic concentration, high internal concentrations

of B could conceivably inhibit normal biosynthetic activities or en-ergy transduction, inhibition of protein synthesis, accumulatedretardation of many cellular processes, enhancing in light byphoto-oxidative stress (Reid et al., 2004). Our findings are inaccordance with the reports on tomato (Alpaslan and Gunes,2001); Cervilla et al. (2007), cucumber (Alpaslan and Gunes,2001), and barley Karabal et al. (2003) in terms of retarded growthunder B toxicity. In addition to this, we found that exogenousapplication of HBL to the B-stressed and stressed-free plants re-sulted in enhanced growth traits (Fig. 1A–F). This is because sev-eral studies have shown that application of BRs led to theoverexpression of genes involved in the BR biosynthetic pathwayresults in enhanced vegetative growth in plant (Choe et al.,2000). Nakaya et al. (2002) discovered that exogenous applicationof BRs unregulated a particular cyclin gene, CycD3, which was in-volved in the cell cycle of Arabidopsis, leading to enhanced seedgermination by stimulating cell expansion and cell proliferation.These findings are further corroborated by other reports whereBRs substantially improved plant growth and development undervarious stress and stress-free conditions (Clouse and Sasse, 1998;Yu et al., 2004; Houimli et al., 2008; Fariduddin et al., 2009b; Hayatet al., 2010; Yusuf et al., 2011).

BRs have also been known to improve water relations such asincrease in relative water content and water uptake (Ali et al.,2005), leaf water potential, water use efficiency, stomatal conduc-tance and thus the transpiration rate both in stressed as well asunstressed plants. BRs have also role in turgor driven cell expan-sion by enhancing the activity of aquaporins (Morillon et al.,2001), proton pumps thereby modulating the stress tolerance(Sakurai et al., 1999). Similar role of BRs, have also been reportedearlier in different abiotic stresses Hayat et al., 2007; Ali et al.,2008; Fariduddin et al., 2009b; Yusuf et al., 2011).

A different pattern of response was observed for the marker ofoxidative stress i.e. lipid peroxidation and H2O2 content (Mittler,2002) and electrolyte leakage. At 2.0 mM of B, there is increase

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of leakage of ions (Fig. 2E), peroxidation of lipids and accumulationof H2O2 (Fig. 2F). The reason behind this may be that membranedamage might be caused by high accumulation of H2O2, whichcould accelerate the Haber–Weiss reaction, resulting in the gener-ation of hydroxyl radical (OH��) and thus lipid peroxidation (Mit-tler, 2002). Others have also found that excess B increased MDAconcentrations in apple rootstock (Molassiotis et al., 2006), grape(Gunes et al., 2006) and tomato (Cervilla et al., 2007). This suggeststhat toxicity generated by excess B may be due to the B inducedoxidative stress in plants. However, the level of reactive oxygenspecies (ROS) in plant tissues is controlled by an antioxidant sys-tem that consists of antioxidant enzymes (superoxide dismutase,catalase, peroxidase and glutathione reductase) and non-ezymaticlow molecular weight antioxidants (glutathione, proline, carote-noids, tocopherols, etc.) (Schutzendubel and Polle, 2002). Althoughboth BRs and ROS act as vital secondary messengers for the induc-tion and regulation of antioxidant systems in plants under stress(Mazorra et al., 2002). The present study revealed that the treat-ment of plants with HBL both in absence and presence of stress en-hanced the activities of antioxidant enzymes (Figs. 4C–F and 5A–B)as well as the level of proline and GR (Fig. 3A–C). Therefore, max-imum values were recorded in the plants subjected to 2.0 mM of B-stress, followed by foliar spray of HBL. The elevation in the activi-ties of antioxidant enzymes by BRs is a gene regulated phenome-non. Cao et al. (2005) demonstrated on the basis of molecular,physiological and genetic approaches the elevation in antioxidantenzymes was the consequence of enhanced expression of DET2gene, which enhanced the resistance to oxidative stress in Arabid-opsis. The involvement of BRs in the regulation of ROS metabolismis evident as they can induce and regulate the expression of certainantioxidant genes and increase the activities of key antioxidant en-zymes, including superoxide dismutase (SOD), peroxidase (POD)and catalase (CAT) (Mazorra et al., 2002; Nunez et al., 2003; Caoet al., 2005; Ogweno et al., 2008). The enzyme superoxide dismu-tase is the first line of defense to counter superoxide (O��2 ) radical.It catalyzes the conversion of O��2 to H2O2 that is subsequently con-verted to H2O by enzyme peroxidase (Alscher et al., 2002). Catalasescavenges H2O2 by converting it to H2O and finally O2, and perox-idase reduce H2O2 using several reductants, such as ascorbate,guaiacol and phenolic compounds (Apel and Hirt, 2004). Glutathi-one reductase maintains the pool of glutathione in the reducedstate, which in turn reduces dehydroascorbate to ascorbate. In-creased expression of glutathione reductase enhances toleranceto oxidative stress (Noctor and Foyer, 1998). Proline, under stressconditions acts as osmoprotectant (Hartzendorf and Rolletschek,2001), membrane stabilizer (Bandurska, 2001), protection of ni-trate reductase reductase during heavy metal stress (Sharma andDubey, 2005) and ROS scavenger (Matysik et al., 2002). The accel-eration of the activities of antioxidant enzymes and increasedaccumulation of proline resulted in an increase in the capacity oftolerance to B-stress in the present study. The increased toleranceto the stress was manifested in terms of improved growth, waterrelations and photosynthesis (Fig. 2C).

5. Conclusions

The present piece of work revealed that B induced oxidativestress in a concentration dependent manner has been manifestedin restriction of plant growth and photosynthetic efficiency and alsomodified antioxidant system. HBL could be used as potent B-stressalleviator through an enhanced antioxidant system and osmolyte(proline), the ROS scavengers and finally increased the growth, pho-tosynthetic efficiency and water relations under B-stress condi-tions. This work therefore provides an efficient eco-friendly routefor the farmers to minimize the B-toxicity worldwide.

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