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Triadimefon induced salt stress tolerance in Withania somnifera and its relationship to antioxidant defense system
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South African Journal of Botany 74 (2008) 126 – 132 www.elsevier.com/locate/sajb
Triadimefon induced salt stress tolerance in Withania somnifera and its relationship to antioxidant defense system C. Abdul Jaleel ⁎, G.M.A. Lakshmanan, M. Gomathinayagam, R. Panneerselvam ⁎ Stress Physiology Lab, Department of Botany, Annamalai University, Annamalainagar 608 002, Tamilnadu, India Received 4 April 2007; received in revised form 25 September 2007; accepted 2 October 2007
Abstract The mitigative effects of triadimefon (5 mg/L) on the germination, early seedling growth, photosynthetic pigments, non-enzymatic antioxidant contents and activities of antioxidant enzymes were studied in salt stressed (40 mM NaCl) Withania somnifera Dunal plants. Salinity stress decreased the germination percentage, early seedling growth and chlorophyll contents. Similarly it affected severely the antioxidants like ascorbic acid (AA), reduced glutathione (GSH) and α-tocopherol (α-toc) in all plant parts (root, stem and leaf). Moreover, it caused changes in the activities of antioxidant enzymes like superoxide dismutase (SOD), peroxidase (POX), polyphenol oxidase (PPO) and catalase (CAT). Triadimefon partially mitigated the salinity stress by enhancing all studied parameters to a considerable extent. © 2007 SAAB. Published by Elsevier B.V. All rights reserved. Keywords: Antioxidant enzymes; Antioxidants; Salinity stress; Triadimefon; Withania somnifera
1. Introduction Soil salinity is one of the most hazardous environmental pollution, possibly which can induce an oxidative stress to the plants (Zhu, 2000). Salt stress alters various biochemical and physiological responses in plants, and thus affects almost all plant processes including photosynthesis, growth and development (Iqbal et al., 2006). The major effect of salinity is the inhibition of plant growth possibly by the reduced hormone delivery from roots to leaves (Azooz et al., 2004), as well as reduced enzymatic activities and biochemical constituents (Jaleel et al., 2007a). High salinity is known to cause hyperosmotic effects in plants, leading to membrane disorganization and metabolic toxicity (Hasegawa et al., 2000). In addition, an important consequence of salinity stress in plants is the excessive generation of reactive oxygen species (ROS) such as superoxide radicals (O2U˜), hydrogen peroxide (H2O2) and hydroxyl radical
⁎ Corresponding authors. Tel.: +91 41 44 238248x354; fax: +91 41 44 222265. E-mail addresses: [email protected] (C. Abdul Jaleel), [email protected] (R. Panneerselvam).
(OHU), particularly in chloroplast and mitochondria (Mittler, 2002). Active oxygen species (AOS) are highly reactive in the absence of any protective mechanism. They can seriously disrupt normal metabolism through oxidative damage to membrane lipids, proteins, pigments and nucleic acids (Misra and Gupta, 2006). The alleviation of oxidative damage and increased resistance to salinity and other environment stresses are often correlated with an efficient antioxidative system (Jaleel et al., 2007a,b; Manivannan et al., 2007). The major ROS scavenging activities include complex non-enzymatic antioxidant molecules viz., ascorbic acid (AA), reduced glutathione (GSH), α-tocopherol (α-toc), and antioxidant enzymes like catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR) and superoxide dismutase (SOD) (Jaleel et al., 2007b). Every year more and more land becomes non-productive owing to salt accumulation (Azooz et al., 2004). Application of PGR has been reported to mitigate the adverse effects of salinity on the growth and yield of some crops (Singh and Jain, 1982; Sankar et al., 2006a,b). Triazole compounds have both fungicidal and PGR properties, and thus can protect plants from several types of abiotic stresses (Fletcher and Hofstra, 1990; Kishorekumar et al., 2006). The PGR properties of triazoles are mediated by their interference with the isoprenoid pathway and
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subsequent shift in balance of important plant hormones, including gibberellins, abscisic acid (ABA) and cytokinins (Fletcher et al., 2000). Triadimefon (TDM) [1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazole-1-yl)-2-butanone] is a triazole derivative, which has PGR properties (Fletcher et al., 2000; Jaleel et al., 2007c,d). TDM has been shown to protect plants from water stress (Fletcher and Nath, 1984) and heat stress (Asare-Boamah and Fletcher, 1986). However, the influential mechanism of TDM on medicinal plants under salinity-induced stress is not studied much. Our previous works have shown the influences of triazoles on pigment composition antioxidant metabolism and alkaloid production in a medicinal plant, namely, Catharanthus roseus (Jaleel et al., 2006a,b). The effect of salinityinduced oxidative stress on agricultural crops is well studied (Sankar et al., 2006a,b; Kishorekumar et al., 2007), however, such type of information is lacking with respect to medicinal plants. Withania somnifera Dunal, known as ashwagandha, has been an important herb in the Ayurvedic and indigenous medical systems for centuries in India (Ganzera et al., 2003). In view of its varied therapeutic potential, it is the subject of considerable modern scientific attention. A perusal of the literature showed that there is a lack of information on the responses of this plant to salinity and PGRs. The present investigation was therefore undertaken to study the effect of TDM on the germination percentage, early seedling growth, chlorophyll content, non-enzymatic antioxidants viz., AA, GSH and α-toc contents and activities of antioxidant enzymes like SOD, peroxidase (POX), polyphenol oxidase (PPO) and CAT of W. somnifera plants under salinity stress. 2. Materials and methods 2.1. Plant material, growth, stress conditions The seeds of W. somnifera were obtained from Herbal Folklore Research Centre, Andhra Pradesh, India. The seeds were surface sterilized with 0.1% HgCl2 for 2 min, washed thoroughly with distilled water and soaked in 40 mM NaCl, 40 mM NaCl + 5 mg/L TDM solution or in distilled water (control) for 12 h. After soaking, the seeds were sown in acid-washed sand in 40× 30 × 7 cm (length, width and depth, respectively) plastic trays placed in a seed germinator maintained at 25 ± 1 °C, 80–85% relative humidity. An irradiance of 450 μE m− 2 s− 1 was given for 14 h/day till the end of the experiment. The trays were irrigated with 100 mL of 40 mM NaCl and 40 mM NaCl + 5 mg/L TDM
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solutions daily. Thirty days after sowing (DAS), the seedlings were uprooted randomly and washed with deionized water to remove adhering sand and separated into root, stem and leaf for analyses. 2.2. Estimation of growth and chlorophyll content The germination percentage was calculated up to 12 DAS. The root and shoot lengths were determined. The total leaf area was calculated with LICOR Photoelectric Area Meter (Model LI-3100, Lincoln, USA). Chlorophyll contents were estimated by the standard method of Arnon (1949) and expressed in mg/g fresh weight (FW). 2.3. Estimation of non-enzymatic antioxidants Ascorbic acid (AA) content was estimated as described by Omaye et al. (1979), and the results are expressed in mg/g FW. The reduced glutathione (GSH) content was estimated as described by Griffith and Meister (1979), and is expressed in μg/g FW. α-Tocopherol (α-toc) content was determined as described by Backer et al. (1980), and is expressed in mg/g FW. 2.4. Antioxidant enzyme assays Crude enzyme extract was prepared for assay of SOD and POX by the method of Joshi et al. (1984). The enzyme protein was determined by Bradford (1976) method for expressing the specific activity of enzymes. Superoxide dismutase (SOD; EC 1.15.1.1) activity was assayed as described by Beauchamp and Fridovich (1971) and expressed in unit (U)/mg protein. One U is defined as the amount of change in the absorbance by 0.1/h/mg protein. Peroxidase (POX; EC 1.11.1.7) and polyphenol oxidase (PPO; EC 1.10.3.1) were assayed according to the method of Kumar and Khan (1982) and expressed in U/mg protein. One U is defined as the change in the absorbance by 0.1/min/mg protein. Catalase (CAT; EC 1.11.1.6) was measured according to the method of Chandlee and Scandalios (1984) and expressed in U/mg protein (U = 1 mM of H2O2 reduction/min/mg protein). 2.5. Statistical analysis Statistical analysis was performed using one way analysis of variance (ANOVA) followed by Duncan's Multiple Range Test (DMRT). The values are mean ± SD for six samples in each group. P values ≤ 0.05 were considered as significant.
Table 1 Individual and combined effects of salinity and triadimefon (TDM) on germination, root length, shoot length, leaf area and chlorophyll (CHL) content in W. somnifera seedlings Treatments
Control 40 mM NaCl 40 mM NaCl + 5 mg L− 1 TDM
Germination (%)
Seedling growth Root length (cm plant− 1)
Shoot length (cm plant− 1)
Leaf area (cm2 plant− 1)
Chlorophyll content CHL ‘a’ (mg g− 1 FW)
CHL ‘b’ (mg g− 1 FW)
Total CHL (mg g− 1 FW)
70.00 ± 2.80a 51.36 ± 1.07b 68.61 ± 2.46a
4.50 ± 0.15a 2.12 ± 0.07b 5.34 ± 0.18c
5.20 ± 0.17a 3.41 ± 0.12b 4.44 ± 0.15c
4.21 ± 0.14a 1.24 ± 0.04b 2.37 ± 0.07c
0.018 ± 0.005a 0.006 ± 0.002b 0.019 ± 0.006a
0.021 ± 0.007a 0.017 ± 0.004b 0.034 ± 0.001c
0.039 ± 0.006a 0.023 ± 0.005b 0.053 ± 0.006c
Values are given as mean ± SD of six experiments in each group. Values not sharing a common superscript (a,b,c) differ significantly at P ≤ 0.05 (DMRT).
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3. Results 3.1. Effect of salinity, TDM and their combination on growth parameters Salinity inhibited and delayed the germination of W. somnifera to a large extent (Table 1). The germination percentage was slightly increased due to the TDM treatment. The root growth of W. somnifera was mainly affected due to salinity. Addition of TDM to the NaCl-stressed seedlings increased the root length to a greater extent even to a level higher than that of NaCl-stressed control. The plants showed a reduction in vegetative growth, with respect to plant height and leaf area when under salt stress (Table 1). TDM treatment increased these parameters, but only to a slight extent when compared with the salt stressed plants. 3.2. Effect of salinity, TDM and their combination on chlorophyll content Salinity caused a decline in chlorophyll content to a large extent (Table 1). Chlorophyll ‘a’ was most affected as compared with chlorophyll ‘b’. TDM increased the chlorophyll content both in stressed and non-stressed plants.
3.3. Effect of salinity, TDM and their combination on nonenzymatic antioxidants The AA (non-enzymatic antioxidant) increased in root, stem and leaf parts of W. somnifera treated with 40 mM NaCl when compared with control (Fig. 1a). Due to TDM treatment, there was again a slight increase in the content of this antioxidant. A sudden and significant decrease in GSH was found in salt stressed roots (Fig. 1b), whereas in stem and leaf there was a slight reduction in GSH content when compared with control. Only the roots had increased GSH content after TDM treatment in comparison to the control. Salinity altered α-tocopherol content significantly when compared with control plants. However, TDM treatment increased α-toc content in different plant tissues of both salt stressed and control plants (Fig. 1c). 3.4. Effect of salinity, TDM and their combination on antioxidant enzyme activities In different parts of the plants, the enzyme activities varied greatly. Salinity reduced SOD activity in roots, stems and leaves when compared with control. The TDM at 5 mg/L increased the
Fig. 1. Individual and combined effects of salinity and TDM on the contents of AA (a), GSH (b) and α-toc (c) in different parts (root, stem, leaf) of W. somnifera seedlings on 30 DAS. Values are given as mean ± SD of six experiments in each group. Bar values that do not share a common superscript (a,b,c) differ significantly at P ≤ 0.05 (DMRT).
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Fig. 2. Individual and combined effects of salinity and TDM on the activities of SOD (a), POX (b), PPO (c) and CAT (d) in different parts (root, stem, leaf) of W. somnifera seedlings on 30 DAS. Values are given as mean ± SD of six experiments in each group. Bar values that do not share a common superscript (a,b,c) differ significantly at P ≤ 0.05 (DMRT).
SOD activity in all parts of the plants. The increase was even greater than that of control (Fig. 2a). Salt stress reduced the activity of POX in all parts of W. somnifera plants when compared with control (Fig. 2b). However, the TDM application was very effective in increasing the POX activity even greater than that of control. The activity of PPO decreased in roots, stems and leaves in response to salinity stress in W. somnifera plants when compared with control (Fig. 2c). TDM treatment increased the PPO activity of salt stressed plants very close to that of control plants. Under saline conditions, CAT activity decreased in the roots, but increased in the stem and leaves when compared with control. Treatment with TDM increased the activity of CAT parallel with that of the roots of control plants. In stem and leaf, the CAT activity increased significantly due to TDM treatment (Fig. 2d). 4. Discussion Salinity reduced the germination percentage of W. somnifera plants, but the treatment with TDM nullified this reduction. Salinity has been shown to reduce the imbibition of water because of lowered osmotic potentials of the medium and cause changes in metabolic activities, thereby leading to the reduction in germination percentage (Meneguzzo et al., 1999; Jaleel et al.,
2007e). NaCl caused an overall reduction in growth of W. somnifera plants when compared with control. Decreased vegetative growth with respect to plant height and leaf area has also been shown in Zizyphus mauritiana (Hooda et al., 1990). TDM treatment partially ameliorated the hazardous effects of salinity by increasing the growth parameters. Saline stress amelioration through triazole application has earlier been reported in sunflower and mungbean seedlings, by pretreatment of seeds with LAB 150978 (a triazole compound) which counteracted the inhibitory effect of salinity on root growth, but it inhibited hypocotyls growth (Saha and Gupta, 1993). Our results showed a reduction in chlorophyll content under NaCl treatment in W. somnifera, which is in accordance with our previous findings (Jaleel et al., 2007a) in C. roseus under salinity stress. The reduction of leaf chlorophyll under salinity has been attributed to the destruction of chlorophyll pigments and the instability of the pigment protein complex (Levitt, 1980). The addition of TDM to the NaCl-stressed plants markedly increased the chlorophyll content. The increased chlorophyll content under TDM treatment might be due to the ability of TDM to increase the cytokinin production and thereby stimulate the chlorophyll biosynthesis (Fletcher et al., 2000). Plants under stress produce some defense mechanisms to protect themselves from the harmful effect of salinity-induced oxidative stress. ROS scavenging is among one of the common
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defense responses against abiotic stresses (Vranova et al., 2002). ROS scavenging depends on the detoxification mechanisms provided by an integrated system of non-enzymatic reduced molecules like AA and GSH as well as enzymatic antioxidants (Srivalli et al., 2003; Jaleel et al., 2007f). Although many changes have been found in the activities of antioxidant enzymes in plants under salinity, their mechanisms are not yet clear (Jaleel et al., 2007b). In W. somnifera plants, the salinity stress induced drastic changes in the antioxidant metabolism. The non-enzymatic antioxidants i.e. AA, GSH and α-toc varied in their contents in root, stem and leaf. The antioxidant enzymes activities were altered due to salinity stress when compared with control. However, TDM treatment enhanced the antioxidants metabolism, and thus partially ameliorated the negative effects of salinity mediated injury. A significant increase in AA content in all the parts of W. somnifera plants was observed in response to NaCl stress. The increased AA content was correlated with the stress protecting mechanism of the plant under salinity conditions (Shalata et al., 2001). AA is an important antioxidant, which reacts not only with H2O2, but also with O2U˜, OHU and lipid hydroperoxidases (Reddy et al., 2004). TDM treatment increased the AA content in all parts of W. somnifera plants. Similar results were found in triazole-treated tomato seedlings (Senaratna et al., 1988). AA can function as the ‘terminal antioxidant’ because the redox potential of AA/monodehydro ascorbate (MDA) pair (+ 280 nm) is lower than that of most of the bio-radicals (Scandalios et al., 1997). However, very little is known about the regulation of AA biosynthesis in higher plants. It is conceivable that adaptations of intracellular AA to oxidative stress might clearly depend on the balance between the rates and capacity of AA biosynthesis and turnover related to antioxidant demand (Chaves et al., 2002). A high level of endogenous ascorbate is essential to effectively maintain the antioxidant system that protects plants from oxidative damage due to the biotic and abiotic stresses (Shigeoka et al., 2002). We noticed a great reduction in GSH content in different parts of W. somnifera plants under saline conditions. The main functions of GSH in the protection against oxidative stress are its involvement in the ascorbate–glutathione cycle and in the regulation of protein thiol–disulphide redox status (Alscher et al., 1997). GSH also plays a protective role in salinity tolerance by the maintenance of the redox status (Shalata et al., 2001). The decrease in GSH found in this study might be due to the predominant oxidation under salinity conditions. TDM treatment increased the GSH content to a great extent in accordance with the findings of Pinhero et al. (1997) in triazole-treated maize. In our study, we noticed a reduction in SOD activity under NaCl stress. The activity varied in different parts of the plant. U˜ SOD catalyses the dismutation of O2 with great efficiency, resulting in the production of H2O2 and O2 (Lin and Kao, 2000). The decrease in the SOD activity could activate the accumulation of ROS, which in turn causes oxidative damage in the salt stressed plants. The decrease in SOD activity could impair the U˜ U˜ O2 scavenging system of cells and favour accumulation of O2 and H2O2 which could lower the SOD activity. The TDM treatment resulted in an increase in the SOD activity. Increased
SOD activity has been reported in triazole-treated drought stressed Vigna unguiculata (Manivannan et al., 2007), and in TDM-treated Catharanthus plants (Jaleel et al., 2006a, 2007a). The activity of POX reduced due to the NaCl stress in all parts of W. somnifera plants when compared with control. Environmental stresses appear to cause changes in the content of cellular antioxidants. In some cases, the decrease in protection against oxidative stress attack was directly linked with lipid peroxidation and free radical formation (Bor et al., 2003; Jaleel et al., 2007g,h). Free oxygen radicals retard cell division, and thus cessation of cell elongation in plant tissues (Shalata et al., 2001). The inhibition of POX activity by salinity may interfere with the regulation of auxin levels and also with cell wall biosynthesis (Bor et al., 2003). Our results also showed that NaCl stress decreased the POX activity and growth of plants. Triazole application increased the POX activity in W. somnifera plants. Protection of stressed plants by triazole is mediated by a reduction in free radical damage and increase in antioxidant potential (Fletcher and Hofstra, 1990). TDM treatment protected cucumber from chilling stress (Feng et al., 2003). The enhancement in POX activities and AA and α-toc contents has been shown the basis of triazole mediated stress protection in plants (Pinhero et al., 1997). NaCl stress inhibited the activity of PPO in all parts of W. somnifera plants. A decrease in PPO could induce accumulation of total phenols and in turn indole acetic acid (IAA) catabolism by increasing the activity of IAA oxidase that in turn could retard the growth during salt stress (Smirnoff, 1993). TDM treatment to the NaCl-stressed and non-stressed plants increased the PPO activity to a large extent when compared with NaClstressed and control plants. This increased PPO activity may decrease the phenol content thereby protecting the level of IAA (Fletcher et al., 2000), and this can increase cell and cell wall growth, thereby inducing growth in the NaCl-stressed plants. The increased PPO activity was well correlated with the increase in the growth of TDM-treated plants. There was a significant decrease in CAT activity in roots and increase in the leaves under salinity stress when compared with control plants, which suggested the existence of an effective scavenging mechanism to remove ROS because roots are the first organs which come in contact with salt and are thus thought to play a critical role in plant salt tolerance. In contrary to our results, Rios-Gonzalez et al. (2002) reported increased antioxidant activity in roots of maize and sunflower under salinity and different nitrogen sources. The changes in CAT may vary according to the intensity of stress, time of assay after stress and induction of new isozyme(s) (Shim et al., 2003). The level of antioxidative response depends on the species, the development and metabolic state of the plant, as well as the duration and intensity of the stress (Reddy et al., 2004). An increase in CAT activity under triazole treatment was reported in banana (Biyan et al., 1995). We have reported increased CAT activity in response to TDM treatment in one of our previous findings in C. roseus (Jaleel et al., 2006a). From the results of this investigation, it is clear that TDM treatment ameliorated the injurious effects of NaCl stress by increasing the growth, photosynthetic pigments, non-enzymatic
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South African Journal of Botany 74 (2008) 126 – 132 www.elsevier.com/locate/sajb
Triadimefon induced salt stress tolerance in Withania somnifera and its relationship to antioxidant defense system C. Abdul Jaleel ⁎, G.M.A. Lakshmanan, M. Gomathinayagam, R. Panneerselvam ⁎ Stress Physiology Lab, Department of Botany, Annamalai University, Annamalainagar 608 002, Tamilnadu, India Received 4 April 2007; received in revised form 25 September 2007; accepted 2 October 2007
Abstract The mitigative effects of triadimefon (5 mg/L) on the germination, early seedling growth, photosynthetic pigments, non-enzymatic antioxidant contents and activities of antioxidant enzymes were studied in salt stressed (40 mM NaCl) Withania somnifera Dunal plants. Salinity stress decreased the germination percentage, early seedling growth and chlorophyll contents. Similarly it affected severely the antioxidants like ascorbic acid (AA), reduced glutathione (GSH) and α-tocopherol (α-toc) in all plant parts (root, stem and leaf). Moreover, it caused changes in the activities of antioxidant enzymes like superoxide dismutase (SOD), peroxidase (POX), polyphenol oxidase (PPO) and catalase (CAT). Triadimefon partially mitigated the salinity stress by enhancing all studied parameters to a considerable extent. © 2007 SAAB. Published by Elsevier B.V. All rights reserved. Keywords: Antioxidant enzymes; Antioxidants; Salinity stress; Triadimefon; Withania somnifera
1. Introduction Soil salinity is one of the most hazardous environmental pollution, possibly which can induce an oxidative stress to the plants (Zhu, 2000). Salt stress alters various biochemical and physiological responses in plants, and thus affects almost all plant processes including photosynthesis, growth and development (Iqbal et al., 2006). The major effect of salinity is the inhibition of plant growth possibly by the reduced hormone delivery from roots to leaves (Azooz et al., 2004), as well as reduced enzymatic activities and biochemical constituents (Jaleel et al., 2007a). High salinity is known to cause hyperosmotic effects in plants, leading to membrane disorganization and metabolic toxicity (Hasegawa et al., 2000). In addition, an important consequence of salinity stress in plants is the excessive generation of reactive oxygen species (ROS) such as superoxide radicals (O2U˜), hydrogen peroxide (H2O2) and hydroxyl radical
⁎ Corresponding authors. Tel.: +91 41 44 238248x354; fax: +91 41 44 222265. E-mail addresses: [email protected] (C. Abdul Jaleel), [email protected] (R. Panneerselvam).
(OHU), particularly in chloroplast and mitochondria (Mittler, 2002). Active oxygen species (AOS) are highly reactive in the absence of any protective mechanism. They can seriously disrupt normal metabolism through oxidative damage to membrane lipids, proteins, pigments and nucleic acids (Misra and Gupta, 2006). The alleviation of oxidative damage and increased resistance to salinity and other environment stresses are often correlated with an efficient antioxidative system (Jaleel et al., 2007a,b; Manivannan et al., 2007). The major ROS scavenging activities include complex non-enzymatic antioxidant molecules viz., ascorbic acid (AA), reduced glutathione (GSH), α-tocopherol (α-toc), and antioxidant enzymes like catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR) and superoxide dismutase (SOD) (Jaleel et al., 2007b). Every year more and more land becomes non-productive owing to salt accumulation (Azooz et al., 2004). Application of PGR has been reported to mitigate the adverse effects of salinity on the growth and yield of some crops (Singh and Jain, 1982; Sankar et al., 2006a,b). Triazole compounds have both fungicidal and PGR properties, and thus can protect plants from several types of abiotic stresses (Fletcher and Hofstra, 1990; Kishorekumar et al., 2006). The PGR properties of triazoles are mediated by their interference with the isoprenoid pathway and
0254-6299/$ - see front matter © 2007 SAAB. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.sajb.2007.10.003
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subsequent shift in balance of important plant hormones, including gibberellins, abscisic acid (ABA) and cytokinins (Fletcher et al., 2000). Triadimefon (TDM) [1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazole-1-yl)-2-butanone] is a triazole derivative, which has PGR properties (Fletcher et al., 2000; Jaleel et al., 2007c,d). TDM has been shown to protect plants from water stress (Fletcher and Nath, 1984) and heat stress (Asare-Boamah and Fletcher, 1986). However, the influential mechanism of TDM on medicinal plants under salinity-induced stress is not studied much. Our previous works have shown the influences of triazoles on pigment composition antioxidant metabolism and alkaloid production in a medicinal plant, namely, Catharanthus roseus (Jaleel et al., 2006a,b). The effect of salinityinduced oxidative stress on agricultural crops is well studied (Sankar et al., 2006a,b; Kishorekumar et al., 2007), however, such type of information is lacking with respect to medicinal plants. Withania somnifera Dunal, known as ashwagandha, has been an important herb in the Ayurvedic and indigenous medical systems for centuries in India (Ganzera et al., 2003). In view of its varied therapeutic potential, it is the subject of considerable modern scientific attention. A perusal of the literature showed that there is a lack of information on the responses of this plant to salinity and PGRs. The present investigation was therefore undertaken to study the effect of TDM on the germination percentage, early seedling growth, chlorophyll content, non-enzymatic antioxidants viz., AA, GSH and α-toc contents and activities of antioxidant enzymes like SOD, peroxidase (POX), polyphenol oxidase (PPO) and CAT of W. somnifera plants under salinity stress. 2. Materials and methods 2.1. Plant material, growth, stress conditions The seeds of W. somnifera were obtained from Herbal Folklore Research Centre, Andhra Pradesh, India. The seeds were surface sterilized with 0.1% HgCl2 for 2 min, washed thoroughly with distilled water and soaked in 40 mM NaCl, 40 mM NaCl + 5 mg/L TDM solution or in distilled water (control) for 12 h. After soaking, the seeds were sown in acid-washed sand in 40× 30 × 7 cm (length, width and depth, respectively) plastic trays placed in a seed germinator maintained at 25 ± 1 °C, 80–85% relative humidity. An irradiance of 450 μE m− 2 s− 1 was given for 14 h/day till the end of the experiment. The trays were irrigated with 100 mL of 40 mM NaCl and 40 mM NaCl + 5 mg/L TDM
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solutions daily. Thirty days after sowing (DAS), the seedlings were uprooted randomly and washed with deionized water to remove adhering sand and separated into root, stem and leaf for analyses. 2.2. Estimation of growth and chlorophyll content The germination percentage was calculated up to 12 DAS. The root and shoot lengths were determined. The total leaf area was calculated with LICOR Photoelectric Area Meter (Model LI-3100, Lincoln, USA). Chlorophyll contents were estimated by the standard method of Arnon (1949) and expressed in mg/g fresh weight (FW). 2.3. Estimation of non-enzymatic antioxidants Ascorbic acid (AA) content was estimated as described by Omaye et al. (1979), and the results are expressed in mg/g FW. The reduced glutathione (GSH) content was estimated as described by Griffith and Meister (1979), and is expressed in μg/g FW. α-Tocopherol (α-toc) content was determined as described by Backer et al. (1980), and is expressed in mg/g FW. 2.4. Antioxidant enzyme assays Crude enzyme extract was prepared for assay of SOD and POX by the method of Joshi et al. (1984). The enzyme protein was determined by Bradford (1976) method for expressing the specific activity of enzymes. Superoxide dismutase (SOD; EC 1.15.1.1) activity was assayed as described by Beauchamp and Fridovich (1971) and expressed in unit (U)/mg protein. One U is defined as the amount of change in the absorbance by 0.1/h/mg protein. Peroxidase (POX; EC 1.11.1.7) and polyphenol oxidase (PPO; EC 1.10.3.1) were assayed according to the method of Kumar and Khan (1982) and expressed in U/mg protein. One U is defined as the change in the absorbance by 0.1/min/mg protein. Catalase (CAT; EC 1.11.1.6) was measured according to the method of Chandlee and Scandalios (1984) and expressed in U/mg protein (U = 1 mM of H2O2 reduction/min/mg protein). 2.5. Statistical analysis Statistical analysis was performed using one way analysis of variance (ANOVA) followed by Duncan's Multiple Range Test (DMRT). The values are mean ± SD for six samples in each group. P values ≤ 0.05 were considered as significant.
Table 1 Individual and combined effects of salinity and triadimefon (TDM) on germination, root length, shoot length, leaf area and chlorophyll (CHL) content in W. somnifera seedlings Treatments
Control 40 mM NaCl 40 mM NaCl + 5 mg L− 1 TDM
Germination (%)
Seedling growth Root length (cm plant− 1)
Shoot length (cm plant− 1)
Leaf area (cm2 plant− 1)
Chlorophyll content CHL ‘a’ (mg g− 1 FW)
CHL ‘b’ (mg g− 1 FW)
Total CHL (mg g− 1 FW)
70.00 ± 2.80a 51.36 ± 1.07b 68.61 ± 2.46a
4.50 ± 0.15a 2.12 ± 0.07b 5.34 ± 0.18c
5.20 ± 0.17a 3.41 ± 0.12b 4.44 ± 0.15c
4.21 ± 0.14a 1.24 ± 0.04b 2.37 ± 0.07c
0.018 ± 0.005a 0.006 ± 0.002b 0.019 ± 0.006a
0.021 ± 0.007a 0.017 ± 0.004b 0.034 ± 0.001c
0.039 ± 0.006a 0.023 ± 0.005b 0.053 ± 0.006c
Values are given as mean ± SD of six experiments in each group. Values not sharing a common superscript (a,b,c) differ significantly at P ≤ 0.05 (DMRT).
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3. Results 3.1. Effect of salinity, TDM and their combination on growth parameters Salinity inhibited and delayed the germination of W. somnifera to a large extent (Table 1). The germination percentage was slightly increased due to the TDM treatment. The root growth of W. somnifera was mainly affected due to salinity. Addition of TDM to the NaCl-stressed seedlings increased the root length to a greater extent even to a level higher than that of NaCl-stressed control. The plants showed a reduction in vegetative growth, with respect to plant height and leaf area when under salt stress (Table 1). TDM treatment increased these parameters, but only to a slight extent when compared with the salt stressed plants. 3.2. Effect of salinity, TDM and their combination on chlorophyll content Salinity caused a decline in chlorophyll content to a large extent (Table 1). Chlorophyll ‘a’ was most affected as compared with chlorophyll ‘b’. TDM increased the chlorophyll content both in stressed and non-stressed plants.
3.3. Effect of salinity, TDM and their combination on nonenzymatic antioxidants The AA (non-enzymatic antioxidant) increased in root, stem and leaf parts of W. somnifera treated with 40 mM NaCl when compared with control (Fig. 1a). Due to TDM treatment, there was again a slight increase in the content of this antioxidant. A sudden and significant decrease in GSH was found in salt stressed roots (Fig. 1b), whereas in stem and leaf there was a slight reduction in GSH content when compared with control. Only the roots had increased GSH content after TDM treatment in comparison to the control. Salinity altered α-tocopherol content significantly when compared with control plants. However, TDM treatment increased α-toc content in different plant tissues of both salt stressed and control plants (Fig. 1c). 3.4. Effect of salinity, TDM and their combination on antioxidant enzyme activities In different parts of the plants, the enzyme activities varied greatly. Salinity reduced SOD activity in roots, stems and leaves when compared with control. The TDM at 5 mg/L increased the
Fig. 1. Individual and combined effects of salinity and TDM on the contents of AA (a), GSH (b) and α-toc (c) in different parts (root, stem, leaf) of W. somnifera seedlings on 30 DAS. Values are given as mean ± SD of six experiments in each group. Bar values that do not share a common superscript (a,b,c) differ significantly at P ≤ 0.05 (DMRT).
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Fig. 2. Individual and combined effects of salinity and TDM on the activities of SOD (a), POX (b), PPO (c) and CAT (d) in different parts (root, stem, leaf) of W. somnifera seedlings on 30 DAS. Values are given as mean ± SD of six experiments in each group. Bar values that do not share a common superscript (a,b,c) differ significantly at P ≤ 0.05 (DMRT).
SOD activity in all parts of the plants. The increase was even greater than that of control (Fig. 2a). Salt stress reduced the activity of POX in all parts of W. somnifera plants when compared with control (Fig. 2b). However, the TDM application was very effective in increasing the POX activity even greater than that of control. The activity of PPO decreased in roots, stems and leaves in response to salinity stress in W. somnifera plants when compared with control (Fig. 2c). TDM treatment increased the PPO activity of salt stressed plants very close to that of control plants. Under saline conditions, CAT activity decreased in the roots, but increased in the stem and leaves when compared with control. Treatment with TDM increased the activity of CAT parallel with that of the roots of control plants. In stem and leaf, the CAT activity increased significantly due to TDM treatment (Fig. 2d). 4. Discussion Salinity reduced the germination percentage of W. somnifera plants, but the treatment with TDM nullified this reduction. Salinity has been shown to reduce the imbibition of water because of lowered osmotic potentials of the medium and cause changes in metabolic activities, thereby leading to the reduction in germination percentage (Meneguzzo et al., 1999; Jaleel et al.,
2007e). NaCl caused an overall reduction in growth of W. somnifera plants when compared with control. Decreased vegetative growth with respect to plant height and leaf area has also been shown in Zizyphus mauritiana (Hooda et al., 1990). TDM treatment partially ameliorated the hazardous effects of salinity by increasing the growth parameters. Saline stress amelioration through triazole application has earlier been reported in sunflower and mungbean seedlings, by pretreatment of seeds with LAB 150978 (a triazole compound) which counteracted the inhibitory effect of salinity on root growth, but it inhibited hypocotyls growth (Saha and Gupta, 1993). Our results showed a reduction in chlorophyll content under NaCl treatment in W. somnifera, which is in accordance with our previous findings (Jaleel et al., 2007a) in C. roseus under salinity stress. The reduction of leaf chlorophyll under salinity has been attributed to the destruction of chlorophyll pigments and the instability of the pigment protein complex (Levitt, 1980). The addition of TDM to the NaCl-stressed plants markedly increased the chlorophyll content. The increased chlorophyll content under TDM treatment might be due to the ability of TDM to increase the cytokinin production and thereby stimulate the chlorophyll biosynthesis (Fletcher et al., 2000). Plants under stress produce some defense mechanisms to protect themselves from the harmful effect of salinity-induced oxidative stress. ROS scavenging is among one of the common
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defense responses against abiotic stresses (Vranova et al., 2002). ROS scavenging depends on the detoxification mechanisms provided by an integrated system of non-enzymatic reduced molecules like AA and GSH as well as enzymatic antioxidants (Srivalli et al., 2003; Jaleel et al., 2007f). Although many changes have been found in the activities of antioxidant enzymes in plants under salinity, their mechanisms are not yet clear (Jaleel et al., 2007b). In W. somnifera plants, the salinity stress induced drastic changes in the antioxidant metabolism. The non-enzymatic antioxidants i.e. AA, GSH and α-toc varied in their contents in root, stem and leaf. The antioxidant enzymes activities were altered due to salinity stress when compared with control. However, TDM treatment enhanced the antioxidants metabolism, and thus partially ameliorated the negative effects of salinity mediated injury. A significant increase in AA content in all the parts of W. somnifera plants was observed in response to NaCl stress. The increased AA content was correlated with the stress protecting mechanism of the plant under salinity conditions (Shalata et al., 2001). AA is an important antioxidant, which reacts not only with H2O2, but also with O2U˜, OHU and lipid hydroperoxidases (Reddy et al., 2004). TDM treatment increased the AA content in all parts of W. somnifera plants. Similar results were found in triazole-treated tomato seedlings (Senaratna et al., 1988). AA can function as the ‘terminal antioxidant’ because the redox potential of AA/monodehydro ascorbate (MDA) pair (+ 280 nm) is lower than that of most of the bio-radicals (Scandalios et al., 1997). However, very little is known about the regulation of AA biosynthesis in higher plants. It is conceivable that adaptations of intracellular AA to oxidative stress might clearly depend on the balance between the rates and capacity of AA biosynthesis and turnover related to antioxidant demand (Chaves et al., 2002). A high level of endogenous ascorbate is essential to effectively maintain the antioxidant system that protects plants from oxidative damage due to the biotic and abiotic stresses (Shigeoka et al., 2002). We noticed a great reduction in GSH content in different parts of W. somnifera plants under saline conditions. The main functions of GSH in the protection against oxidative stress are its involvement in the ascorbate–glutathione cycle and in the regulation of protein thiol–disulphide redox status (Alscher et al., 1997). GSH also plays a protective role in salinity tolerance by the maintenance of the redox status (Shalata et al., 2001). The decrease in GSH found in this study might be due to the predominant oxidation under salinity conditions. TDM treatment increased the GSH content to a great extent in accordance with the findings of Pinhero et al. (1997) in triazole-treated maize. In our study, we noticed a reduction in SOD activity under NaCl stress. The activity varied in different parts of the plant. U˜ SOD catalyses the dismutation of O2 with great efficiency, resulting in the production of H2O2 and O2 (Lin and Kao, 2000). The decrease in the SOD activity could activate the accumulation of ROS, which in turn causes oxidative damage in the salt stressed plants. The decrease in SOD activity could impair the U˜ U˜ O2 scavenging system of cells and favour accumulation of O2 and H2O2 which could lower the SOD activity. The TDM treatment resulted in an increase in the SOD activity. Increased
SOD activity has been reported in triazole-treated drought stressed Vigna unguiculata (Manivannan et al., 2007), and in TDM-treated Catharanthus plants (Jaleel et al., 2006a, 2007a). The activity of POX reduced due to the NaCl stress in all parts of W. somnifera plants when compared with control. Environmental stresses appear to cause changes in the content of cellular antioxidants. In some cases, the decrease in protection against oxidative stress attack was directly linked with lipid peroxidation and free radical formation (Bor et al., 2003; Jaleel et al., 2007g,h). Free oxygen radicals retard cell division, and thus cessation of cell elongation in plant tissues (Shalata et al., 2001). The inhibition of POX activity by salinity may interfere with the regulation of auxin levels and also with cell wall biosynthesis (Bor et al., 2003). Our results also showed that NaCl stress decreased the POX activity and growth of plants. Triazole application increased the POX activity in W. somnifera plants. Protection of stressed plants by triazole is mediated by a reduction in free radical damage and increase in antioxidant potential (Fletcher and Hofstra, 1990). TDM treatment protected cucumber from chilling stress (Feng et al., 2003). The enhancement in POX activities and AA and α-toc contents has been shown the basis of triazole mediated stress protection in plants (Pinhero et al., 1997). NaCl stress inhibited the activity of PPO in all parts of W. somnifera plants. A decrease in PPO could induce accumulation of total phenols and in turn indole acetic acid (IAA) catabolism by increasing the activity of IAA oxidase that in turn could retard the growth during salt stress (Smirnoff, 1993). TDM treatment to the NaCl-stressed and non-stressed plants increased the PPO activity to a large extent when compared with NaClstressed and control plants. This increased PPO activity may decrease the phenol content thereby protecting the level of IAA (Fletcher et al., 2000), and this can increase cell and cell wall growth, thereby inducing growth in the NaCl-stressed plants. The increased PPO activity was well correlated with the increase in the growth of TDM-treated plants. There was a significant decrease in CAT activity in roots and increase in the leaves under salinity stress when compared with control plants, which suggested the existence of an effective scavenging mechanism to remove ROS because roots are the first organs which come in contact with salt and are thus thought to play a critical role in plant salt tolerance. In contrary to our results, Rios-Gonzalez et al. (2002) reported increased antioxidant activity in roots of maize and sunflower under salinity and different nitrogen sources. The changes in CAT may vary according to the intensity of stress, time of assay after stress and induction of new isozyme(s) (Shim et al., 2003). The level of antioxidative response depends on the species, the development and metabolic state of the plant, as well as the duration and intensity of the stress (Reddy et al., 2004). An increase in CAT activity under triazole treatment was reported in banana (Biyan et al., 1995). We have reported increased CAT activity in response to TDM treatment in one of our previous findings in C. roseus (Jaleel et al., 2006a). From the results of this investigation, it is clear that TDM treatment ameliorated the injurious effects of NaCl stress by increasing the growth, photosynthetic pigments, non-enzymatic
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