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Protection of Vigna unguiculata (L.) Walp. plants from salt stress by paclobutrazol
Available online at www.sciencedirect.com
Colloids and Surfaces B: Biointerfaces 61 (2008) 315–318
Short communication
Protection of Vigna unguiculata (L.) Walp. plants from salt stress by paclobutrazol P. Manivannan, C. Abdul Jaleel, A. Kishorekumar, B. Sankar, R. Somasundaram, R. Panneerselvam ∗ Division of Plant Physiology, Department of Botany, Annamalai University, Annamalainagar 608002, Tamil Nadu, India Received 18 July 2007; received in revised form 11 August 2007; accepted 4 September 2007 Available online 8 September 2007
Abstract A pot culture experiment was conducted to estimate the stress ameliorating ability of paclobutrazol, a triazole fungicide in Vigna unguiculata (L.) Walp. plants. Treatments were given as 80 mM NaCl, 80 mM NaCl + 15 mg l−1 paclobutrazol and 15 mg l−1 paclobutrazol alone. The samples were collected on 60 and 80 days after sowing (DAS). NaCl stress inhibited the root and stem length, total leaf area, fresh weight (FW), dry weight (DW) and activities of antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX). Plants treated with NaCl with paclobutrazol increased these parameters to a larger extent when compared to NaCl stressed plants. The results showed that the paclobutrazol significantly ameliorated the adverse effects of NaCl stress in V. unguiculata plants. © 2007 Elsevier B.V. All rights reserved. Keywords: Paclobutrazol; Vigna unguiculata; NaCl stress; Fungicide; Antioxidant enzymes
1. Introduction Salinity stress is a widespread environmental problem. Although considerable effort has been devoted to solve this problem, the application of plant growth regulator (PGR) attracted a little attention. Salt stress can affect plant survival, biomass, plant height and affect the capacity of plants to collect water and nutrients [1]. Soil salinity is known to reduce crop yield drastically and of serious concern to the agriculturists. Triazole compounds are used as fungicides, which have plant growth regulating as well as stress protecting properties. Protection of plants from apparently unrelated stress by triazoles is mediated by a reduction in free-radical damage and increase in antioxidant potential [2–4]. Triazoles affect the isoprenoid pathway and alter the levels of certain plant hormones by inhibiting gibberellin synthesis, reducing ethylene evolution and increasing cytokinin levels [5]. Triazole treated plants have a more efficient free-radical scavenging system that enables them to detox-
ify active oxygen [6]. Some of the previous works carried out in our lab revealed the morphological and physiological changes associated with triazole treatment in various plants, include the inhibition of plant growth, decreased internodal elongation, increased chlorophyll levels, enlarged chloroplasts, thicker leaf tissue, increased root to shoot ratio, increased antioxidant potentials and an enhancement in alkaloid production [7–11]. Paclobutrazol (PBZ) [(2RS; 3RS)-1-(4-chlorophenyl)-4,4dimethyl-2-(1H-12,4-triazol-1-yl)-pentan-3-ol], a triazole, fungicide, having plant growth regulator (PGR) properties, is reported to inhibit gibberellic acid (GA) biosynthesis and increase in abscisic acid (ABA) and cytokinin contents[12–14,7]. The present investigation deals with the salt stress ameliorating properties of paclobutrazol in Vigna unguiculata plants under pot culture. 2. Materials and methods 2.1. Plant materials and cultivation
∗
Corresponding author. Tel.: +91 4144 238248x354. E-mail addresses: [email protected] (P. Manivannan), [email protected] (C.A. Jaleel), [email protected] (R. Panneerselvam). 0927-7765/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2007.09.007
The seeds of V. unguiculata (L.) Walp. were obtained from Central Pulses Research Station, Tamil Nadu. Seeds were surface sterilized with 0.2 per cent HgCl2 solution for 5 min with
316
P. Manivannan et al. / Colloids and Surfaces B: Biointerfaces 61 (2008) 315–318
frequent shaking and thoroughly washed with deionised water to remove HgCl2 . Two seeds were sown in each pot of 30 cm diameter and 30 cm height containing 3 kg of soil mixture composed of red soil, sand and the farmyard manure (FYM) at 1:1:1 ratio. All the pots were watered to the field capacity with groundwater upto 14 days after sowing (DAS). Pots were irrigated with groundwater (control), 100 mM NaCl, 100 mM NaCl + 15 mg l−1 paclobutrazol and 15 mg l−1 paclobutrazol alone, respectively, on 55, 75 days after sowing (DAS). 2.2. Morphological parameters Root and stem length were recorded on 60 and 80 DAS. The root length and the stem length were expressed in cm plant−1 . The FW and DW were taken in electronic balance and expressed in g plant−1 . Total leaf area was measured with LICOR photoelectric leaf area meter (Model L1-3100, Lincoln, USA) and expressed in cm2 plant−1 .
50 mM potassium phosphate buffer (pH 7.0), 0.4 ml of 15 mM H2 O2 and 0.04 ml of enzyme extract. The decomposition of H2 O2 was followed by the decline in absorbance at 240 nm. The enzyme activity was expressed in U mg−1 protein (U = 1 mM of H2 O2 reduction min−1 mg−1 protein). 2.4. Statistical analysis The experiment was laid out in a completely randomized block design (CRBD) with six replicates for each treatment. Statistical analysis was performed using one way analysis of variance (ANOVA) followed by Duncan’s Multiple Range Test (DMRT). The values are mean ± S.D. for six samples in each group. P values ≤ 0.05 were considered as significant. 3. Results and discussion 3.1. Effect of salt, paclobutrazol and their combination on morphological parameters
2.3. Antioxidant enzyme assays 2.3.1. Superoxide dismutase (SOD, EC 1.15.1.1) Crude enzyme extract was prepared for assay of SOD by the method of Hwang et al. [15]. The enzyme protein was determined Bradford, [16] for all the three enzymes for expressing the specific activity of enzymes. SOD (EC 1.15.1.1) activity was assayed according to Beauchamp and Fridovich [17]. The reaction mixture contained 1.17 × 10−6 M riboflavin, 0.1 M methionine, 2 × 10−5 M potassium cyanide (KCN) and 5.6 × 10−5 M nitroblue tetrazolium salt (NBT) dissolved in 3 ml of 0.05 M sodium phosphate buffer (pH 7.8). Three milliliters of the reaction medium was added to 1 ml of enzyme extract. The mixtures were illuminated in glass test tubes by two sets of Philips 40 W fluorescent tubes in a single row. Illumination was started to initiate the reaction at 30 ◦ C for 1 h. Identical solutions that were kept under dark served as blanks. The absorbance was read at 560 nm in the spectrophotometer against the blank. SOD activity was expressed in units (U mg−1 protein). One unit is defined as the amount of change in the absorbance by 0.1 h−1 mg−1 protein. 2.3.2. Peroxidase (POX, EC 1.11.1.7) POX was assayed by the method of Kumar and Khan [18]. Assay mixture of POX contained 2 ml of 0.1 M phosphate buffer (pH 6.8), 1 ml of 0.01 M pyrogallol, 1 ml of 0.005 M H2 O2 and 0.5 ml of enzyme extract. The solution was incubated for 5 min at 25 ◦ C after which the reaction was terminated by adding 1 ml of 2.5 N H2 SO4 . The amount of purpurogallin formed was determined by measuring the absorbance at 420 nm against a blank prepared by adding the extract after the addition of 2.5 N H2 SO4 at zero time. The activity was expressed in U mg−1 protein. One unit is defined as the change in the absorbance by 0.1 min−1 mg−1 protein. 2.3.3. Catalase (CAT, EC 1.11.1.6) CAT was measured according to Chandlee and Scandalios [19] with modification. The assay mixture contained 2.6 ml of
The results of the present study showed that sodium chloride treatment decreased the root length to a larger extent when compared to control (Table 1). Salinity can inhibit root growth, external water potential, ion toxicity and ion imbalance [1]. NaCl with paclobutrazol treatment increased the root length when compared to NaCl treated plants. Similarly paclobutrazol treatment increased the root length at NaCl stressed Catharanthus plants [3]. The increase was associated with increased endogenous cytokinin levels under triazole application [12]. Stem length was inhibited by the NaCl treatment to a larger extent when compared to control (Table 1). Similar results were observed in Catharanthus roseus [20]. Abiotic stress like drought decreased the stem length in Abelmoschus esculentus [21]. Combination of NaCl with paclobutrazol increased the stem growth to a level higher than the NaCl treated plants. Similar observations NaCl stressed C. roseus [3] treated with paclobutrazol. The total leaf area, fresh and dry weight was very much inhibited by the NaCl stressed cowpea (Table 1). Similar results were observed in C. roseus under salt stress [20] and drought stressed Helianthus annuus [22]. Paclobutrazol treatment to the NaCl stressed plants showed increased total leaf area, FW and DW when compared to NaCl stressed plants. Similar results were observed in CaCl2 treated NaCl stressed C. roseus seedlings [23]. 3.2. Effect of salt, paclobutrazol and their combination on antioxidant enzyme activities The SOD activity has been lowered by the NaCl stress to a larger extent in cowpea plants Fig. 1, when compared to control. Similar observations were made in previous works [2–4]. Paclobutrazol treatment to the NaCl stressed and unstressed plants increased the SOD activity to a higher level than that of control. Triadimefon treatment [10] and treatment with gibberellic acid [24] increased the activity of SOD in C. roseus plants. Paclobutrazol treatment to the drought stressed Arachis
P. Manivannan et al. / Colloids and Surfaces B: Biointerfaces 61 (2008) 315–318
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Table 1 Effect of NaCl, paclobutrazol and their combination on growth and antioxidant enzyme activities in Vigna unguiculata (values are the mean ± S.D. of seven replicates) Parameters
DAS
Control
Root length (cm plant−1 )
70 80 70 80 70 80 70 80 70 80
187.39 202.51 23.11 27.1 95.61 137.41 6.683 12.62 1.51 2.99
Stem length (cm plant−1 ) Leaf area (cm2 plant−1 ) Fresh weight (g plant−1 ) Dry weight (g plant−1 )
100 mM NaCl ± ± ± ± ± ± ± ± ± ±
18.11 26.41 0.85 0.75 2.01 2.76 0.03 0.21 0.03 0.05
151.54 165.88 17.54 18.63 60.41 82.91 5.91 11.93 0.73 1.49
± ± ± ± ± ± ± ± ± ±
12.72 19.41 0.57 0.64 2.54 1.88 0.01 0.12 0.08 0.05
Fig. 1. Effect of NaCl, paclobutrazol and their combination on SOD activity in leaves of Vigna unguiculata. Values are given as mean ± S.D. of six samples in each group. Bar values are not sharing a common letters (a, b, and c) differ significantly at P ≤ 0.05 (DMRT).
hypogaea plants, retained more SOD activity than control plants [25]. The CAT activity has been lowered by the NaCl stress to a larger extent in cowpea plants (Fig. 2) when compared to control. Similar observations were made in previous works with abiotic stresses [26–29]. Paclobutrazol treatment to the NaCl stressed and unstressed plants increased the CAT activity to a higher level than that of control. These results are in accordance with previous reports on Arachis plants under combined treat-
100 Mm NaCl + 20 mg l−1 paclobutrazol 170.41 187.32 19.46 23.64 86.51 125.44 6.43 12.47 1.42 2.84
± ± ± ± ± ± ± ± ± ±
16.95 27.25 0.91 0.84 1.36 2.66 0.05 0.18 0.02 0.05
20 mg l−1 paclobutrazol 201.41 227.41 21.84 26.43 88.04 126.43 7.12 13.03 1.61 3.07
± ± ± ± ± ± ± ± ± ±
26.31 26.45 1.06 0.70 2.60 2.69 0.05 0.24 0.04 0.06
Fig. 3. Effect of NaCl, paclobutrazol and their combination on POX activity in leaves of Vigna unguiculata. Values are given as mean ± S.D. of six samples in each group. Bar values are not sharing a common letters (a, b, and c) differ significantly at P ≤ 0.05 (DMRT).
ments of drought and paclobutrazol [25]. Triadimefon treatment increased the activity of CAT in C. roseus plants [10]. POX activity has been reduced by the NaCl stress in cowpea when compared with control (Fig. 3). Salinity decreased the activity of POX in C. roseus [26]. Combination of paclobutrazol with NaCl stressed plants increased the POX activity when compared to control. The paclobutrazol treatment increased the POX activity significantly when compared to other treatment. Stress protection by triazole is mediated by a reduction in freeradical damage and increase in antioxidant potential [12]. From the results of this investigation, it can be concluded paclobutrazol moderately ameliorate the effect of NaCl stress in cowpea and increase the growth and antioxidant enzyme activities. References
Fig. 2. Effect of NaCl, paclobutrazol and their combination on CAT activity in leaves of Vigna unguiculata. Values are given as mean ± S.D. of six samples in each group. Bar values are not sharing a common letters (a, b, c, and d) differ significantly at P ≤ 0.05 (DMRT).
[1] C.A. Jaleel, R. Gopi, P. Manivannan, R. Panneerselvam, Turk. J. Bot. 31 (2007) 245–251. [2] P. Manivannan, C.A. Jaleel, A. Kishorekumar, B. Sankar, et al., Colloids Surf. B: Biointerf. 57 (2007) 69–74. [3] C.A. Jaleel, R. Gopi, P. Manivannan, R. Panneerselvam, Acta Physiol. Plant. 29 (2007) 205–209. [4] C.A. Jaleel, P. Manivannan, B. Sankar, A. Kishorekumar, et al., Colloids Surf. B: Biointerf. 60 (2007) 201–206. [5] A.P. Kamountsis, A.G. Chronopoulon-Sereli, Hortic. Sci. 34 (1999) 674–675. [6] M. Kopyra, E.A. Gwozdz, Biol. Lett. 40 (2003) 61–69.
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[7] A. Kishorekumar, C.A. Jaleel, P. Manivannan, B. Sankar, et al., Acta Biol. Szegediensis 50 (2006) 127–129. [8] A. Kishorekumar, C.A. Jaleel, P. Manivannan, B. Sankar, et al., Colloids Surf. B: Biointerf. 60 (2007) 207–212. [9] M. Gomathinayagam, C.A. Jaleel, G.M.A. Lakshmanan, R. Panneerselvam, C. R. Biol. 330 (2007) 644–655. [10] C.A. Jaleel, R. Gopi, G.M. Alagu Lakshmanan, R. Panneerselvam, Plant Sci. 171 (2006) 271–276. [11] C.A. Jaleel, R. Gopi, P. Manivannan, A. Kishorekumar, et al., Indian J. Appl. Pure Biol. 21 (2006) 369–372. [12] R.A. Fletcher, A. Gilley, N. sankhla, T.M. Davis, Hortic. Rev. 24 (2000) 56–138. [13] C.A. Jaleel, R. Gopi, P. Manivannan, A. Kishorekumar, et al., J. Zhejiang Univ. Sci. B 8 (2007) 283–288. [14] C.A. Jaleel, A. Kishorekumar, P. Manivannan, B. Sankar, et al., Plant Growth Regul. 53 (2007) 7–16. [15] S.Y. Hwang, H.W. Lin, R.H. Chern, H.F. Lo, et al., Plant Growth Regul. 27 (1999) 167–172. [16] M.M. Bradford, Ann. Biochem. 72 (1976) 248–253. [17] C. Beauchamp, I. Fridovich, Ann. Biochem. 44 (1971) 276–287. [18] K.B. Kumar, P.A. Khan, Ind. J. Exp. Bot. 20 (1982) 412–416.
[19] J.M. Chandlee, J.G. Scandalios, Theor. Appl. Genet. 69 (1984) 71–77. [20] C.A. Jaleel, R. Gopi, B. Sankar, P. Manivannan, et al., S. Afr. J. Bot. 73 (2007) 190–195. [21] B. Sankar, C.A. Jaleel, P. Manivannan, A. Kishorekumar, et al., Acta Bot. Croat. 66 (2007) 43–56. [22] P. Manivannan, C.A. Jaleel, B. Sankar, A. Kishorekumar, et al., Colloids Surf. B: Biointerf. 59 (2007) 141–149. [23] C.A. Jaleel, P. Manivannan, A. Kishorekumar, B. Sankar, et al., C. R. Biol. 330 (2007) 674–683. [24] B. Karthikeyan, C.A. Jaleel, R. Gopi, M. Deiveekasundaram, J. Zhejiang Univ. Sci. B 8 (2007) 453–457. [25] B. Sankar, C.A. Jaleel, P. Manivannan, A. Kishorekumar, et al., Colloids Surf. B: Biointerf. 60 (2007) 229–235. [26] C.A. Jaleel, P. Manivannan, A. Kishorekumar, B. Sankar, et al., Colloids Surf. B: Biointerf. 59 (2007) 150–157. [27] C.A. Jaleel, P. Manivannan, B. Sankar, A. Kishorekumar, et al., Colloids Surf. B: Biointerf. 60 (2007) 7–11. [28] C.A. Jaleel, R. Gopi, P. Manivannan, B. Sankar, et al., Colloids Surf. B: Biointerf. 60 (2007) 195–200. [29] C.A. Jaleel, P. Manivannan, B. Sankar, A. Kishorekumar, et al., Colloids Surf. B: Biointerf. 60 (2007) 110–116.
Colloids and Surfaces B: Biointerfaces 61 (2008) 315–318
Short communication
Protection of Vigna unguiculata (L.) Walp. plants from salt stress by paclobutrazol P. Manivannan, C. Abdul Jaleel, A. Kishorekumar, B. Sankar, R. Somasundaram, R. Panneerselvam ∗ Division of Plant Physiology, Department of Botany, Annamalai University, Annamalainagar 608002, Tamil Nadu, India Received 18 July 2007; received in revised form 11 August 2007; accepted 4 September 2007 Available online 8 September 2007
Abstract A pot culture experiment was conducted to estimate the stress ameliorating ability of paclobutrazol, a triazole fungicide in Vigna unguiculata (L.) Walp. plants. Treatments were given as 80 mM NaCl, 80 mM NaCl + 15 mg l−1 paclobutrazol and 15 mg l−1 paclobutrazol alone. The samples were collected on 60 and 80 days after sowing (DAS). NaCl stress inhibited the root and stem length, total leaf area, fresh weight (FW), dry weight (DW) and activities of antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX). Plants treated with NaCl with paclobutrazol increased these parameters to a larger extent when compared to NaCl stressed plants. The results showed that the paclobutrazol significantly ameliorated the adverse effects of NaCl stress in V. unguiculata plants. © 2007 Elsevier B.V. All rights reserved. Keywords: Paclobutrazol; Vigna unguiculata; NaCl stress; Fungicide; Antioxidant enzymes
1. Introduction Salinity stress is a widespread environmental problem. Although considerable effort has been devoted to solve this problem, the application of plant growth regulator (PGR) attracted a little attention. Salt stress can affect plant survival, biomass, plant height and affect the capacity of plants to collect water and nutrients [1]. Soil salinity is known to reduce crop yield drastically and of serious concern to the agriculturists. Triazole compounds are used as fungicides, which have plant growth regulating as well as stress protecting properties. Protection of plants from apparently unrelated stress by triazoles is mediated by a reduction in free-radical damage and increase in antioxidant potential [2–4]. Triazoles affect the isoprenoid pathway and alter the levels of certain plant hormones by inhibiting gibberellin synthesis, reducing ethylene evolution and increasing cytokinin levels [5]. Triazole treated plants have a more efficient free-radical scavenging system that enables them to detox-
ify active oxygen [6]. Some of the previous works carried out in our lab revealed the morphological and physiological changes associated with triazole treatment in various plants, include the inhibition of plant growth, decreased internodal elongation, increased chlorophyll levels, enlarged chloroplasts, thicker leaf tissue, increased root to shoot ratio, increased antioxidant potentials and an enhancement in alkaloid production [7–11]. Paclobutrazol (PBZ) [(2RS; 3RS)-1-(4-chlorophenyl)-4,4dimethyl-2-(1H-12,4-triazol-1-yl)-pentan-3-ol], a triazole, fungicide, having plant growth regulator (PGR) properties, is reported to inhibit gibberellic acid (GA) biosynthesis and increase in abscisic acid (ABA) and cytokinin contents[12–14,7]. The present investigation deals with the salt stress ameliorating properties of paclobutrazol in Vigna unguiculata plants under pot culture. 2. Materials and methods 2.1. Plant materials and cultivation
∗
Corresponding author. Tel.: +91 4144 238248x354. E-mail addresses: [email protected] (P. Manivannan), [email protected] (C.A. Jaleel), [email protected] (R. Panneerselvam). 0927-7765/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2007.09.007
The seeds of V. unguiculata (L.) Walp. were obtained from Central Pulses Research Station, Tamil Nadu. Seeds were surface sterilized with 0.2 per cent HgCl2 solution for 5 min with
316
P. Manivannan et al. / Colloids and Surfaces B: Biointerfaces 61 (2008) 315–318
frequent shaking and thoroughly washed with deionised water to remove HgCl2 . Two seeds were sown in each pot of 30 cm diameter and 30 cm height containing 3 kg of soil mixture composed of red soil, sand and the farmyard manure (FYM) at 1:1:1 ratio. All the pots were watered to the field capacity with groundwater upto 14 days after sowing (DAS). Pots were irrigated with groundwater (control), 100 mM NaCl, 100 mM NaCl + 15 mg l−1 paclobutrazol and 15 mg l−1 paclobutrazol alone, respectively, on 55, 75 days after sowing (DAS). 2.2. Morphological parameters Root and stem length were recorded on 60 and 80 DAS. The root length and the stem length were expressed in cm plant−1 . The FW and DW were taken in electronic balance and expressed in g plant−1 . Total leaf area was measured with LICOR photoelectric leaf area meter (Model L1-3100, Lincoln, USA) and expressed in cm2 plant−1 .
50 mM potassium phosphate buffer (pH 7.0), 0.4 ml of 15 mM H2 O2 and 0.04 ml of enzyme extract. The decomposition of H2 O2 was followed by the decline in absorbance at 240 nm. The enzyme activity was expressed in U mg−1 protein (U = 1 mM of H2 O2 reduction min−1 mg−1 protein). 2.4. Statistical analysis The experiment was laid out in a completely randomized block design (CRBD) with six replicates for each treatment. Statistical analysis was performed using one way analysis of variance (ANOVA) followed by Duncan’s Multiple Range Test (DMRT). The values are mean ± S.D. for six samples in each group. P values ≤ 0.05 were considered as significant. 3. Results and discussion 3.1. Effect of salt, paclobutrazol and their combination on morphological parameters
2.3. Antioxidant enzyme assays 2.3.1. Superoxide dismutase (SOD, EC 1.15.1.1) Crude enzyme extract was prepared for assay of SOD by the method of Hwang et al. [15]. The enzyme protein was determined Bradford, [16] for all the three enzymes for expressing the specific activity of enzymes. SOD (EC 1.15.1.1) activity was assayed according to Beauchamp and Fridovich [17]. The reaction mixture contained 1.17 × 10−6 M riboflavin, 0.1 M methionine, 2 × 10−5 M potassium cyanide (KCN) and 5.6 × 10−5 M nitroblue tetrazolium salt (NBT) dissolved in 3 ml of 0.05 M sodium phosphate buffer (pH 7.8). Three milliliters of the reaction medium was added to 1 ml of enzyme extract. The mixtures were illuminated in glass test tubes by two sets of Philips 40 W fluorescent tubes in a single row. Illumination was started to initiate the reaction at 30 ◦ C for 1 h. Identical solutions that were kept under dark served as blanks. The absorbance was read at 560 nm in the spectrophotometer against the blank. SOD activity was expressed in units (U mg−1 protein). One unit is defined as the amount of change in the absorbance by 0.1 h−1 mg−1 protein. 2.3.2. Peroxidase (POX, EC 1.11.1.7) POX was assayed by the method of Kumar and Khan [18]. Assay mixture of POX contained 2 ml of 0.1 M phosphate buffer (pH 6.8), 1 ml of 0.01 M pyrogallol, 1 ml of 0.005 M H2 O2 and 0.5 ml of enzyme extract. The solution was incubated for 5 min at 25 ◦ C after which the reaction was terminated by adding 1 ml of 2.5 N H2 SO4 . The amount of purpurogallin formed was determined by measuring the absorbance at 420 nm against a blank prepared by adding the extract after the addition of 2.5 N H2 SO4 at zero time. The activity was expressed in U mg−1 protein. One unit is defined as the change in the absorbance by 0.1 min−1 mg−1 protein. 2.3.3. Catalase (CAT, EC 1.11.1.6) CAT was measured according to Chandlee and Scandalios [19] with modification. The assay mixture contained 2.6 ml of
The results of the present study showed that sodium chloride treatment decreased the root length to a larger extent when compared to control (Table 1). Salinity can inhibit root growth, external water potential, ion toxicity and ion imbalance [1]. NaCl with paclobutrazol treatment increased the root length when compared to NaCl treated plants. Similarly paclobutrazol treatment increased the root length at NaCl stressed Catharanthus plants [3]. The increase was associated with increased endogenous cytokinin levels under triazole application [12]. Stem length was inhibited by the NaCl treatment to a larger extent when compared to control (Table 1). Similar results were observed in Catharanthus roseus [20]. Abiotic stress like drought decreased the stem length in Abelmoschus esculentus [21]. Combination of NaCl with paclobutrazol increased the stem growth to a level higher than the NaCl treated plants. Similar observations NaCl stressed C. roseus [3] treated with paclobutrazol. The total leaf area, fresh and dry weight was very much inhibited by the NaCl stressed cowpea (Table 1). Similar results were observed in C. roseus under salt stress [20] and drought stressed Helianthus annuus [22]. Paclobutrazol treatment to the NaCl stressed plants showed increased total leaf area, FW and DW when compared to NaCl stressed plants. Similar results were observed in CaCl2 treated NaCl stressed C. roseus seedlings [23]. 3.2. Effect of salt, paclobutrazol and their combination on antioxidant enzyme activities The SOD activity has been lowered by the NaCl stress to a larger extent in cowpea plants Fig. 1, when compared to control. Similar observations were made in previous works [2–4]. Paclobutrazol treatment to the NaCl stressed and unstressed plants increased the SOD activity to a higher level than that of control. Triadimefon treatment [10] and treatment with gibberellic acid [24] increased the activity of SOD in C. roseus plants. Paclobutrazol treatment to the drought stressed Arachis
P. Manivannan et al. / Colloids and Surfaces B: Biointerfaces 61 (2008) 315–318
317
Table 1 Effect of NaCl, paclobutrazol and their combination on growth and antioxidant enzyme activities in Vigna unguiculata (values are the mean ± S.D. of seven replicates) Parameters
DAS
Control
Root length (cm plant−1 )
70 80 70 80 70 80 70 80 70 80
187.39 202.51 23.11 27.1 95.61 137.41 6.683 12.62 1.51 2.99
Stem length (cm plant−1 ) Leaf area (cm2 plant−1 ) Fresh weight (g plant−1 ) Dry weight (g plant−1 )
100 mM NaCl ± ± ± ± ± ± ± ± ± ±
18.11 26.41 0.85 0.75 2.01 2.76 0.03 0.21 0.03 0.05
151.54 165.88 17.54 18.63 60.41 82.91 5.91 11.93 0.73 1.49
± ± ± ± ± ± ± ± ± ±
12.72 19.41 0.57 0.64 2.54 1.88 0.01 0.12 0.08 0.05
Fig. 1. Effect of NaCl, paclobutrazol and their combination on SOD activity in leaves of Vigna unguiculata. Values are given as mean ± S.D. of six samples in each group. Bar values are not sharing a common letters (a, b, and c) differ significantly at P ≤ 0.05 (DMRT).
hypogaea plants, retained more SOD activity than control plants [25]. The CAT activity has been lowered by the NaCl stress to a larger extent in cowpea plants (Fig. 2) when compared to control. Similar observations were made in previous works with abiotic stresses [26–29]. Paclobutrazol treatment to the NaCl stressed and unstressed plants increased the CAT activity to a higher level than that of control. These results are in accordance with previous reports on Arachis plants under combined treat-
100 Mm NaCl + 20 mg l−1 paclobutrazol 170.41 187.32 19.46 23.64 86.51 125.44 6.43 12.47 1.42 2.84
± ± ± ± ± ± ± ± ± ±
16.95 27.25 0.91 0.84 1.36 2.66 0.05 0.18 0.02 0.05
20 mg l−1 paclobutrazol 201.41 227.41 21.84 26.43 88.04 126.43 7.12 13.03 1.61 3.07
± ± ± ± ± ± ± ± ± ±
26.31 26.45 1.06 0.70 2.60 2.69 0.05 0.24 0.04 0.06
Fig. 3. Effect of NaCl, paclobutrazol and their combination on POX activity in leaves of Vigna unguiculata. Values are given as mean ± S.D. of six samples in each group. Bar values are not sharing a common letters (a, b, and c) differ significantly at P ≤ 0.05 (DMRT).
ments of drought and paclobutrazol [25]. Triadimefon treatment increased the activity of CAT in C. roseus plants [10]. POX activity has been reduced by the NaCl stress in cowpea when compared with control (Fig. 3). Salinity decreased the activity of POX in C. roseus [26]. Combination of paclobutrazol with NaCl stressed plants increased the POX activity when compared to control. The paclobutrazol treatment increased the POX activity significantly when compared to other treatment. Stress protection by triazole is mediated by a reduction in freeradical damage and increase in antioxidant potential [12]. From the results of this investigation, it can be concluded paclobutrazol moderately ameliorate the effect of NaCl stress in cowpea and increase the growth and antioxidant enzyme activities. References
Fig. 2. Effect of NaCl, paclobutrazol and their combination on CAT activity in leaves of Vigna unguiculata. Values are given as mean ± S.D. of six samples in each group. Bar values are not sharing a common letters (a, b, c, and d) differ significantly at P ≤ 0.05 (DMRT).
[1] C.A. Jaleel, R. Gopi, P. Manivannan, R. Panneerselvam, Turk. J. Bot. 31 (2007) 245–251. [2] P. Manivannan, C.A. Jaleel, A. Kishorekumar, B. Sankar, et al., Colloids Surf. B: Biointerf. 57 (2007) 69–74. [3] C.A. Jaleel, R. Gopi, P. Manivannan, R. Panneerselvam, Acta Physiol. Plant. 29 (2007) 205–209. [4] C.A. Jaleel, P. Manivannan, B. Sankar, A. Kishorekumar, et al., Colloids Surf. B: Biointerf. 60 (2007) 201–206. [5] A.P. Kamountsis, A.G. Chronopoulon-Sereli, Hortic. Sci. 34 (1999) 674–675. [6] M. Kopyra, E.A. Gwozdz, Biol. Lett. 40 (2003) 61–69.
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P. Manivannan et al. / Colloids and Surfaces B: Biointerfaces 61 (2008) 315–318
[7] A. Kishorekumar, C.A. Jaleel, P. Manivannan, B. Sankar, et al., Acta Biol. Szegediensis 50 (2006) 127–129. [8] A. Kishorekumar, C.A. Jaleel, P. Manivannan, B. Sankar, et al., Colloids Surf. B: Biointerf. 60 (2007) 207–212. [9] M. Gomathinayagam, C.A. Jaleel, G.M.A. Lakshmanan, R. Panneerselvam, C. R. Biol. 330 (2007) 644–655. [10] C.A. Jaleel, R. Gopi, G.M. Alagu Lakshmanan, R. Panneerselvam, Plant Sci. 171 (2006) 271–276. [11] C.A. Jaleel, R. Gopi, P. Manivannan, A. Kishorekumar, et al., Indian J. Appl. Pure Biol. 21 (2006) 369–372. [12] R.A. Fletcher, A. Gilley, N. sankhla, T.M. Davis, Hortic. Rev. 24 (2000) 56–138. [13] C.A. Jaleel, R. Gopi, P. Manivannan, A. Kishorekumar, et al., J. Zhejiang Univ. Sci. B 8 (2007) 283–288. [14] C.A. Jaleel, A. Kishorekumar, P. Manivannan, B. Sankar, et al., Plant Growth Regul. 53 (2007) 7–16. [15] S.Y. Hwang, H.W. Lin, R.H. Chern, H.F. Lo, et al., Plant Growth Regul. 27 (1999) 167–172. [16] M.M. Bradford, Ann. Biochem. 72 (1976) 248–253. [17] C. Beauchamp, I. Fridovich, Ann. Biochem. 44 (1971) 276–287. [18] K.B. Kumar, P.A. Khan, Ind. J. Exp. Bot. 20 (1982) 412–416.
[19] J.M. Chandlee, J.G. Scandalios, Theor. Appl. Genet. 69 (1984) 71–77. [20] C.A. Jaleel, R. Gopi, B. Sankar, P. Manivannan, et al., S. Afr. J. Bot. 73 (2007) 190–195. [21] B. Sankar, C.A. Jaleel, P. Manivannan, A. Kishorekumar, et al., Acta Bot. Croat. 66 (2007) 43–56. [22] P. Manivannan, C.A. Jaleel, B. Sankar, A. Kishorekumar, et al., Colloids Surf. B: Biointerf. 59 (2007) 141–149. [23] C.A. Jaleel, P. Manivannan, A. Kishorekumar, B. Sankar, et al., C. R. Biol. 330 (2007) 674–683. [24] B. Karthikeyan, C.A. Jaleel, R. Gopi, M. Deiveekasundaram, J. Zhejiang Univ. Sci. B 8 (2007) 453–457. [25] B. Sankar, C.A. Jaleel, P. Manivannan, A. Kishorekumar, et al., Colloids Surf. B: Biointerf. 60 (2007) 229–235. [26] C.A. Jaleel, P. Manivannan, A. Kishorekumar, B. Sankar, et al., Colloids Surf. B: Biointerf. 59 (2007) 150–157. [27] C.A. Jaleel, P. Manivannan, B. Sankar, A. Kishorekumar, et al., Colloids Surf. B: Biointerf. 60 (2007) 7–11. [28] C.A. Jaleel, R. Gopi, P. Manivannan, B. Sankar, et al., Colloids Surf. B: Biointerf. 60 (2007) 195–200. [29] C.A. Jaleel, P. Manivannan, B. Sankar, A. Kishorekumar, et al., Colloids Surf. B: Biointerf. 60 (2007) 110–116.