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Abstract The effect of paclobutrazol, a plant growth regulator, on antioxidant defense system was investi- gated in Catharanthus roseus (L.) G. Don. plants sub-.
Acta Physiol Plant (2007) 29:205–209 DOI 10.1007/s11738-007-0025-6

ORIGINAL PAPER

Responses of antioxidant defense system of Catharanthus roseus (L.) G. Don. to paclobutrazol treatment under salinity Cheruth Abdul Jaleel Æ Ragupathi Gopi Æ Paramasivam Manivannan Æ Rajaram Panneerselvam

Received: 9 May 2006 / Revised: 19 September 2006 / Accepted: 28 September 2006 / Published online: 25 January 2007  Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2007

Abstract The effect of paclobutrazol, a plant growth regulator, on antioxidant defense system was investigated in Catharanthus roseus (L.) G. Don. plants subjected to NaCl stress. The growth parameters were significantly reduced under 80 mM NaCl treatment; however, this growth inhibition was less in paclobutrazol-treated (15 mg l–1 plant–1) plants. The nonenzymatic antioxidants ascorbic acid and reduced glutathione were affected under NaCl stress and they increased significantly under paclobutrazol treatment when compared to NaCl treated as well as control plants (P £ 0.05). The activity of antioxidant enzyme ascorbate peroxidase showed a significant enhancement under salinity stress. The catalase activity decreased in roots of NaCl-treated plants, but recovered with paclobutrazol treatment. The results suggested that paclobutrazol have significant role in contributing salt stress tolerance of C. roseus by improving the components of antioxidant defense system. Keywords Ascorbate peroxidase  Ascorbic acid  Catalase  Catharanthus roseus  Glutathione  Paclobutrazol

Communicated by A. Tukiendorf. C. A. Jaleel  R. Gopi (&)  P. Manivannan  R. Panneerselvam Division of Plant Physiology, Department of Botany, Annamalai University, Annamalainagar 608 002, Tamil Nadu, India e-mail: [email protected]

Introduction Soil salinity continues to be one of the World’s most serious environmental problems in agriculture. Salt stress induces various biochemical and physiological responses in plants and affects almost all plant processes (Nemoto and Sasakuma 2002). Salinity can cause hyperionic and hyperosmotic effects on plants leading to membrane disorganization, increase in reactive oxygen species (ROS) levels and metabolic toxicity (Hasegawa et al. 2000). In order to survive under stress conditions, plants are equipped with oxygen radical detoxifying enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and antioxidant molecules like ascorbic acid (AA), a-tocopherol and reduced glutathione (GSH) (Prochazkova et al. 2001). Mechanisms of salt tolerance, not yet clear, can be to some extent explained by stress adaptation effectors that mediate ion homeostasis, osmolytic biosynthesis, toxic radical scavenging, water transport and long distance response coordination (Hasegawa et al. 2000). Chemical treatment and agronomical crop management practices have been tried to alleviate the salinity effects without much success and a wide spread practice to reduce salt content in soils is leaching (Sohan et al. 1999). With rising cost of water, this method may not continue to be a feasible method for the future. Application of plant growth regulators (PGR) has been reported to mitigate the adverse effects of salinity and results in a significant increase in the growth and yield of many crops under stress condition (Singh and Jain 1982). Triazole compounds are widely used as fungicides and they also possess varying degrees of PGR

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properties. Triazoles have been called plant multiprotectants because of their ability to induce tolerance in plants to environmental and chemical stresses (Fletcher et al. 2000). The ability of triazole compounds such as triadimefon, in the enhancement of antioxidant enzyme activities in Catharanthus roseus (L.) G. Don. is previously reported (Jaleel et al. 2006). Paclobutrazol (PBZ) [(2 RS, 3 RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1 H-1,2,4-trizol-1-yl)-pentan-3-ol] is a triazolic group of fungicide which has PGR properties. However, details of interaction between PBZ, ROS and antioxidant defense system are not clear yet. Being a triazole compound, the stress protection ability of PBZ is studied in the present work in a medicinal plant C. roseus belonging to the family Apocyanaceae. C. roseus contain many medicinally important alkaloids which have anticancer activities (Filippini et al. 2003). The aims of this study were to investigate the alleviation of salinity stress by PBZ treatment and to determine the antioxidant defense capacities of C. roseus under NaCl stress conditions.

Materials and methods Plant materials and growth C. roseus seeds were obtained from the Department of Horticulture, Annamalai University, Tamil Nadu, India, and surface sterilized with 0.2% HgCl2 solution for 5 min with frequent shaking and then a thorough wash with deionized water. The seeds were pre-soaked in 500 ml of deionized water (control), 80 mM NaCl, 80 mM NaCl + 15 mg l–1 PBZ and 15 mg l–1 PBZ solutions for 12 h. Seeds were sown in plastic pots (300 mm diameter) filled with 3 kg of soil mixture containing red soil, sand and farmyard manure (FYM) at 1:1:1 ratio. Before sowing the seeds, the pots were irrigated with the respective treatment solutions and the electrical conductivity (EC) of the soil mixture was measured. Four seeds were sown per pot and the pots were watered to the field capacity with deionized water up to 90 days after sowing (DAS) and every care was taken to avoid leaching. The initial EC level of the soil was maintained by flushing each pot with required volume of corresponding treatment solution on 45, 60 and 75 DAS. The position of each pot was randomized at 4-day intervals to minimize spatial effects in the greenhouse, where the temperature was 28C during the day and 22C at night and the relative humidity (RH) varied between 60 and 70%. The seedlings were thinned to

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one per pot on 10 DAS. Plants were uprooted randomly on 90 DAS and analysed for estimating the growth and antioxidants status. In the preliminary experiments, 40, 60, 80, 100 and 120 mM NaCl solutions were used for treatments to estimate the concentration which reduces the dry weight (DW) significantly. Among these treatments, 80 mM NaCl produced a significant reduction in DW and this concentration was selected to produce NaCl stress to Catharanthus plants. In the PBZ treatment, 5, 10, 15, 20 and 25 mg l–1 concentrations were tried in combination with 80 mM NaCl, of which 15 mg l–1 was found to increase the DW more or less the level of control and the higher concentration slightly decreased the DW. Hence, 15 mg l–1 of PBZ concentration was used to ameliorate the NaCl stress. The same concentration was used to treat the unstressed plants to determine the effect of PBZ on the unstressed C. roseus plants. Growth parameters Morphological parameters such as root length, plant height, fresh weight (FW) and DW were measured in the samples. The total leaf area was calculated with LICOR Photoelectric Area Meter (Model L1-3100, Lincoln, USA). Non-enzymatic antioxidants Ascorbic acid (AA) content was assayed as described by Omaye et al. (1979). The extract was prepared by grinding 1 g of fresh material with 5 ml of 10% trichloro acetic acid (TCA), centrifuged at 3,500 rpm for 20 min, re-extracted twice and supernatant made up to 10 ml and used for assay. To 0.5 ml of extract, 1 ml of 6 mM 2,4-dinitrophenylhydrazine-thiourea-CuSO4 (DTC) reagent was added, incubated at 37C for 3 h and 0.75 ml of ice-cold 65% H2 SO4 was added, allowed to stand at 30C for 30 min and resulting colour was read at 520 nm in spectrophotometer (U-2001Hitachi). The AA content was determined using a standard curve prepared with AA. The reduced glutathione (GSH) content was assayed as described by Griffith and Meister (1979). Two hundred milligrams of fresh material was ground with 2 ml of 2% metaphosphoric acid and centrifuged at 17,000 rpm for 10 min. The supernatant was neutralized by adding 0.6 ml 10% sodium citrate. One millilitre of assay mixture was prepared by adding 100 ll extract, 100 ll distilled water, 100 ll of 6 mM 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) and 700 ll of

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Table 1 Effects of salinity (80 mM NaCl), PBZ (15 mg l–1 plant–1) and their combination on the growth of Catharanthus roseus on 90 DAS Root length (cm) Control NaCl NaCl + PBZ PBZ

26 18 21 27

± ± ± ±

0.88a 0.46b 0.53c 0.83d

Plant height (cm) 61 52 57 54

± ± ± ±

2.20a 1.80b 2.07c 2.18d

Leaf area (cm2) 162 107 134 154

± ± ± ±

6.11a 3.25b 4.40c 5.10d

Fresh weight (g) 28.48 26.12 28.02 29.86

± ± ± ±

0.94a 0.96b 0.90a 1.10c

Dry weight (g) 3.64 2.99 3.51 3.81

± ± ± ±

0.12a 0.10b 0.13a 0.13c

Values are given as mean ± SD of seven experiments in each group. Values are not sharing a common superscript (a, b, c, d) differ significantly at P £ 0.05 (DMRT)

0.3 mM NADPH. The mixture was stabilized at 25C for 3–4 min. Then 10 ll of glutathione reductase was added and read the absorbance at 412 nm in spectrophotometer.

multiple range test (DMRT). The values are mean ± SD for seven samples in each group. P values £0.05 were considered as significant.

Enzyme assays

Results and discussion

Ascorbate peroxidase (APX) (EC 1.11.1.1) activity was determined by the method of Asada and Takahashi (1987). A total of 0.5 g of plant tissue was ground in a pestle and mortar under liquid nitrogen, with 10 ml of 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA, 1% polyvinyl pyrollidone (PVP) and 1 mM ascorbic acid. The homogenate was filtered through a double-layered cheese cloth and centrifuged at 15,000 rpm for 20 min at 4C. The supernatant was used as enzyme source. The reaction mixture (1 ml) contained 50 mM potassium phosphate buffer (pH 7.0), 0.5 mM ascorbic acid, 0.1 mM H2 O2 and 200 ll of enzyme extract. The decrease in absorbance at 290 nm due to ascorbate oxidation was measured against the blank. Correction was done for the low, non-enzymatic oxidation of ascorbic acid by H2O2 (extinction coefficient 2.9 mM–1 cm–1). Catalase (CAT) (EC 1.11.1.6) activity was measured according the method of Chandlee and Scandalios (1984) with small modification. Frozen plant material weighing 0.5 g, was homogenized in a prechilled pestle and mortar with 5 ml of ice cold 50 mM sodium phosphate buffer (pH 7.5) containing 1 mM phenylmethylsulfonyl fluoride (PMSF). The extract was centrifuged at 4C for 20 min at 12,500 rpm. The supernatant was used for enzyme assay. The assay mixture contained 2.6 ml of 50 mM potassium phosphate buffer (pH 7.0), 400 ll of 15 mM H2 O2 and 40 ll of enzyme extract. The decomposition of H2O2 was followed by the decline in absorbance at 240 nm.

Salt stress caused significant decrease in plant height, root length, leaf area, FW and DW of C. roseus plants and the PBZ treatments to these plants increased these parameters (P £ 0.05) (Table 1). PBZ treatment to the unstressed plants caused a significant increase in root length, FW and DW (P £ 0.05). The plant height and leaf area showed a significant reduction under PBZ treatment (P £ 0.05) (Table 1). The reduction in growth may be an adaptive response to stress (Zhu 2001). A decrease in growth under unfavourable conditions allows the conservation of energy, thereby launching the appropriate defence response and also reducing the risk of heritable damage as reported in Calendula officinalis plants under salinity by Chaparzadeh et al. (2004). This decrease may be due to the ability of salinity to affect external water potential, ion toxicity or imbalance (Hasegawa et al. 2000). PBZ application improved the growth potential of C. roseus plants under salinity injury. Stimulation of root growth under salt stress by triadimefon, a triazole compound was reported in one of the previous studies conducted in our lab by Panneerselvam et al. (1997) in radish. Increasing of root growth by triazoles is also associated with increased level of endogenous cytokinin (Fletcher and Arnold 1986). Under salt stress, the non-enzymatic antioxidants like AA and GSH were affected significantly (P £ 0.05). The AA content (Fig. 1a) increased in both root and leaves, but the GSH content (Fig. 1b) decreased under salinity when compared with controls. PBZ caused a significant rise of these antioxidants both in NaCl-stressed and unstressed plants (P £ 0.05). Oxidative stress is the result of ROS, such as superoxide, H2O2 and hydroxyl molecules and cause

Statistics Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Duncan’s

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Fig. 1 Effects of salinity (80 mM NaCl), PBZ (15 mg l–1 plant–1) and their combination on the a ascorbic acid (AA) and b reduced glutathione (GSH) contents of Catharanthus roseus on 90 DAS. Values are given as mean ± SD of seven experiments in each group. Bar values are not sharing a common superscript (a, b, c, d) differ significantly at P £ 0.05 (DMRT)

Fig. 2 Effects of salinity (80 mM NaCl), PBZ (15 mg l–1 plant–1) and their combination on the a ascorbate peroxidase (APX) and b catalase (CAT) activities of Catharanthus roseus on 90 DAS. Values are given as mean ± SD of seven experiments in each group. Bar values are not sharing a common superscript (a, b, c, d) differ significantly at P £ 0.05 (DMRT)

rapid cell damage by triggering off a chain reaction (Imlay 2003). ROS scavenging is one among the common defense responses against abiotic stresses (Vranova et al. 2002). Changes of antioxidants and protective molecules reflect the impact of environmental stresses on plant metabolism (Herbinger et al. 2002). Increased AA and GSH contents were reported due to PBZ application in lemon (Jain et al. 2002). An increase in AA and GSH content due to PBZ treatment is the basis of the stress protection ability of PBZ (Fletcher et al. 2000). PBZ and NaCl treatments led to changes in the activities of APX (Fig. 2a) and CAT (Fig. 2b) in roots and leaves of C. roseus. The extractable specific activity of APX increased in salt-stressed root and leaves of C. roseus plants when compared to control. PBZ treatments to the unstressed plants caused an increase in APX activity in both roots and leaves. The activity of CAT was decreased under NaCl stress in roots. But in leaves, there was no significant change in the activity

when compared to control plants. PBZ treatments to the salt-stressed and unstressed plants caused an increase in CAT activity to a significant level when compared to NaCl stressed and control plants (P £ 0.05) (Fig. 2b). Increased APX activity was reported in wheat under long-term salinity stress (Sairam and Srivastava 2002). Increased activities of antioxidant enzymes with triadimefon treatment were reported in radish under salinity (Muthukumarasamy et al. 2000). In one of our previous works, we reported an enhancement in the activities of antioxidant enzymes and alkaloid content by triadimefon treatment in C. roseus plants (Jaleel et al. 2006). Triazole compounds like PBZ can inhibit GA biosynthesis and in turn make substrate available for the synthesis of other isoprenoid pathway compounds like ABA and/or cytokinin (Fletcher and Arnold 1986, Kabi and Pujari 1980). Cytokinin and ABA have been used to reverse the salinity induced deleterious effects (Katz et al. 1978, Singh et al. 1987). Protection of plants from apparently unrelated stress

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by triazoles is mediated by a reduction in free radical damage and increase in antioxidant potentials and enzyme activities (Fletcher and Hofstra 1990). From these results, it can be concluded that, the PBZ at 15 mg l–1 plant–1 application could well be used for partial amelioration of salinity stress in this medicinal plant.

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