Changes induced by Cu2+ and Cr6+ metal stress in polyamines, auxins, abscisic acid titers and antioxidative enzymes activities of radish seedlings

RESEARCH ARTICLE

Changes induced by Cu²⁺ and Cr⁶⁺ metal stress in polyamines, auxins, abscisic acid titers and antioxidative enzymes activities of radish seedlings

Sikander P. Choudhary^I; Renu Bhardwaj^I,*; Bishan D. Gupta^II; Prabhu Dutt^II; Rajinder K. Gupta^II; Mukesh Kanwar^I; Pitamber Dutt^I

^IDepartment of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar 143005, Punjab, India

^IINatural Product Chemistry Division, Indian Institute of Integrative Medicine, Jammu 180003 (J&K), India

ABSTRACT

The present study determined the effects of copper and chromium metals on the endogenous titers of polyamines, auxins, abscisic acid and antioxidative enzyme activities in Raphanus sativus L. cv. Pusa chetki seedlings. Among polyamines, putrescine and spermidine contents were enhanced by Cu²⁺ metal to 62.44 and 402.8 µg g^-1 f.w. respectively over control. Spermine which was not observed in control was recorded in highest concentration (1287.9 µg g^-1 f.w.) in Cu²⁺ metal stressed seedlings. On the other hand Cr⁶⁺ metal treated seedlings showed reduced contents of putrescine (1.43 µg g^-1 f.w.), cadaverine (0.09 µg g^-1 f.w.), spermidine (277.99 µg g^-1 f.w.) and spermine (2.29 µg g^-1 f.w.) when compared to control and Cu²⁺ metal treated seedlings. Significant decline in free and bound IAA concentration were found under Cu²⁺ and Cr⁶⁺ metal stress when compared to control. Naphthalene acetic acid not recorded in control seedlings was detected in seedlings treated with Cu²⁺ and Cr⁶⁺ metal. Activities of guaiacol peroxidase and catalase were reduced significantly under Cu²⁺ and Cr⁶⁺ metal stress in comparison to control. Superoxide dismutase activity enhanced significantly under Cr⁶⁺ rather than Cu²⁺ metal treatment. In addition the phytotoxicity of Cu²⁺ and Cr⁶⁺ metal on seedling growth was also determined. The data suggest that Cr⁶⁺ is more phytotoxic than exposure to Cu²⁺ metal.

Key words: catalase, chromium, copper, guaiacol peroxidase, polyamines (PAs), superoxide dismutase

INTRODUCTION

Heavy metals are major environmental contaminants occurring in soils and other habitats. Toxic levels of some of them (Cd, Cu, Pb, Hg and Cr) could appear in natural and agricultural areas as a result of human activities. Among these metals, copper is widely distributed in nature due to multifarious use for industrial and agronomic purposes. Soil contamination with Cu²⁺ has become an important environmental threat to human life through its penetrance to the food chain (Chary et al., 2008). It is required in small quantities for normal plant growth and development (Mengel and Kirkby, 1987), but leads to phytotoxicity at higher concentrations (Berenguer et al., 2008). Copper toxicity is mediated by the formation of free radicals (Luna et al., 1994) and by the catalysis of the Haber-Weiss reaction (Halliwel and Gutteridge, 1984). These radicals are highly toxic and can oxidize biological macromolecules such as nucleic acids, proteins and lipids, thereby disturbing the cell stability and membrane permeability (Schutzendubel and Polle, 2002). Similarly, Chatterjee et al. (2006) also documented oxidative damages and changes in radish physiology under copper stress. On the other hand, reactive oxygen species (ROS) producing capacity of Cr³⁺ or Cr⁶⁺ is mainly attributed to its redox character. Excess of Cr³⁺ or Cr⁶⁺ metal induces the production of ROS such as H₂O₂ to form OH^•, O₂^-directly by interacting with glutathione, NADPH and H₂O₂ (Shanker et al., 2005) and thus causes oxidative damage to biomolecules of immense importance like DNA, proteins and pigments. It also stimulates lipid peroxidation (Shanker et al., 2005).

Plants have strong defense system consisting of antioxidative enzymes, like catalase (Willekens et al., 1997), peroxidase (Csiszar et al., 2008), glutathione reductase (Romero-Puertas et al., 2006) and superoxide dismutase (Alscher et al., 2002), ascorbate peroxidase (Davletova et al., 2005) and antioxidants (vitamins, NAD, FAD, ascorbic acid). The phytohormones like auxins (Park et al., 2007), cytokinins (Zhang and Ervin, 2008), abscisic acid (Staneloni et al., 2008), ethylene (Tamaoki et al., 2008), salicylates (Flors et al., 2008), jasmonic acid (Maksymiec et al., 2005), brassinosteriods (Haubrick and Assmann, 2006; Clouse, 2008) and polyamines (Tassoni et al., 2008) are also known to confer resistance in plants against various abiotic and biotic stresses.

Auxins are growth regulators required for normal plant growth and development. Recent studies also indicate their stress protective property in plants. They are now widely used to confer resistance in plants against salinity (Iqbal and Ashraf, 2007), drought, chilling stress (Posymk et al., 2009), heat stress and heavy metals stress (Khan and Chaudhary, 2006; Dimpka et al., 2008).

Active involvement of ABA in physiological processes such as stomatal closure, embryo morphogenesis, synthesis of storage proteins, lipids and seed germination is well understood. ABA plays an important role in stress protection by triggering plant responses to adverse environmental stimuli. Exogenous application of ABA has been reported to enhance chilling stress tolerance (Zhou et al., 2005). ABA induced adaptation to salt stress in suspension cultures of tobacco (Nicotiana tabacum L. Cv. Wisconsin) has been also reported by Etehadnia et al. (2008).

Another class of nitrogen-rich metabolites known as polyamines [such as putrescine (Put), cadaverine (Cad), spermidine (Spd) and spermine (Spm)] are low molecular weight organic cations, ubiquitously distributed in all living organisms (Groppa and Benavides, 2008). They are actively involved in the regulation of a large number of physiological processes like embryogenesis, cell division, morphogenesis and development (Minocha and Minocha, 2005; Groppa and Benavides, 2008; Kumar et al., 2008). They are now accepted as an important component of plant stress responses (Alcazar et al., 2006; Tassoni et al., 2008). Changes in the endogenous titers of polyamines have been associated with the retardation of senescence, and various environmental stresses like osmotic, salinity (Pirintsos et al., 2004; Tang et al., 2005; Shevyakova et al., 2006; Liu et al., 2007; Wen et al., 2008) and heavy metal stress (Shevyakova et al., 2006; Liu and Moriguchi, 2007; Zapata et al., 2008). They have the capacity for scavenging free radicals and reactive oxygen species (Ha et al., 1998), thus exerting a strong antioxidant function during various types of stress (Groppa and Benavides, 2008).

Raphanus sativus L. plants are facing variety of abiotic stresses. The prominent stress among these is heavy metal stress. Literature survey revealed scanty information on the comparative account of Cu²⁺ and Cr⁶⁺ metal stress on the endogenous levels of polyamines, auxins, and ABA and antioxidant system of Raphanus sativus. So keeping this in mind the present investigation was designed to find out the effects of Cu²⁺ and Cr⁶⁺ metal stress on the biosynthesis of polyamines, auxins and ABA in this economically and medicinally important root vegetable. In addition to this, antioxidant system (antioxidative enzyme activities, lipid peroxidation and proline content), and seedling growth were also assessed under metal stress.

MATERIAL AND METHODS

Plant material and treatments: Seeds of Raphanus sativus L. cv. ‘Pusa chetki' used in the present investigation were procured from Punjab Agriculture University, Ludhiana, India. Seeds were surface sterilized with 0.01% sodium hypochlorite and then rinsed with 3-4 times with distilled water. Seeds were grown in autoclaved Petriplates lined with Whatman No.1 filter paper at 25±2ºC with a photoperiod of 16 h under fluorescent white light (175 µmol/m²/s) in a controlled environmental growth chamber maintained at 70-80% relative humidity. Seeds were subjected to IC₅₀ concentrations of 0.2 mM CuSO₄•5H₂O (Cu²⁺ ions) and 1.2 mM K₂CrO₄ (Cr⁶⁺ ions) stress, whereas control seedlings were raised in distilled water alone. Seedlings were harvested on the 7^th day (3-3.5 cm hypocotyls length) for the analysis of PAs (putrescine, cadaverine, spermidine and spermine), auxins (Indole acetic acid, Naphthalene acetic acid and Indole butyric acid) ABA and antioxidative enzyme activities and seedling growth.

Isolation and characterization of polyamines: Polyamines were isolated and characterized by following the method proposed by Fontaniella et al. (2001). About 2g of seedlings were extracted with 5% perchloric acid (PCA) (v/v) in a pre-chilled pestle and mortar at 4º C. Homogenized extracts were centrifuged at 15,000 r.p.m. for 25 min at 4º C. Supernatant used as a source of PAs was dried in vacuum at 45ºC and residue was redissolved in 5% PCA. Impurties like polyphenolics were removed by stirring the residue with polyvinylpolypyrrolidone (PvPP) (50 mg/ml). Dansylation of the testing samples was carried out in derivatization vials. Samples after dansylation were cleaned according to Seiler and Knodgen (1979). They were finally dissolved in 1 ml of methanol (HPLC grade) for HPLC analysis.

HPLC analysis of polyamines: Analysis of PAs was carried out on Waters (515) Chromatograph HPLC equipped with Waters (717) Plus Autosampler and Photodiode Array Detector (2996).For the assessment of endogenous PAs, samples were injected in to 20 µl injector loop in RP-C18 Column (15 cm × 4 mm i.d.), 5 µm particle size, reversed phased column at 30ºC using Methanol: Water linear gradient from 50:50 (v/v) to 80:20 (v/v) for 30 min. The last proportion was maintained at 1 ml/min. The detection of PAs was carried out by measuring the fluorescence intensity at 254 nm of samples and making their comparisons with the peaks and retention times of standard PAs (Put, Cad, Spd and Spm). The standard PAs were prepared at a concentration of 1mM as described earlier.

Isolation and characterization of auxins and ABA: Assessment of endogenous auxins and ABA content in the seedlings subjected to Cu²⁺ and Cr⁶⁺ stress was done by the method of Nagar and Sood (2003, 2006). 2 g of seedlings harvested on the 7^th day were homogenized with 80% methanol (20 ml/g) containing butylated hydroxytoulene (BHT) (100 mg/l). Homogenates were kept overnight at 4ºC in the dark, filtered, and re-extracted (4×) with 80% methanol. Resulting solution was frozen at -20ºC, thawed and centrifuged at 9000 r.p.m for 30 min at 4ºC to remove suspended impurities. Supernatants were dried in vacuo and taken up in 0.1 M potassium phosphate buffer (pH 8.1) and then applied to polyvinylpyrrolidone (PvP) column. Eluate obtained was evaporated to dryness, taken up in water and pH adjusted to 2.5 with 1 N HCl and then partitioned (4×) with diethyl ether. Combined ether phases were evaporated to dryness in vacuo and the residue was dissolved in methanol for the estimation of free auxins. The remaining aqueous phase was hydrolyzed at pH 11.0 for 1 h at 60ºC. The hydrolysate was cooled and acidified with 1 N HCl upto pH 3 and finally partitioned against diethyl ether. The combined ether phases were evaporated in vacuo, and taken up in methanol (HPLC grade) for the estimation of bound auxins.

HPLC analysis of auxins and ABA: For the determination of endogenous auxins and ABA concentrations, methanolic extracts ready for HPLC analysis were filtered through 0.45 µM Millipore filters. Elution was carried out with Methanol (40%) in 30 mM acetic acid (HPLC grade) at a flow rate of 1 ml/min. The solvents were filtered and degassed. Column eluants were passed through Uv detector (2996 PDA detector) at 280 nm and auxins (IAA, NAA and IBA) and ABA were characterized and quantified by making comparisons of retention times, fluorescence intensity and peak heights of extracted auxins with reference to their standards (IAA, NAA, IBA, and ABA, Sigma Aldrich Ltd., India), prepared at a concentration of 1 mM.

Determination of protein contents and antioxidative enzyme activities.

Preparation of plant extracts: For the estimation of protein content and activities of superoxide dismutase, catalase and guaiacol peroxidase, and glutathione reductase, 2 g of plant tissue was homogenized in pre-chilled pestle and mortar with 6ml of 100 mM potassium phosphate buffer (pH 7.0) under ice-cold conditions. The homogenate was centrifuged at 15000×g at 5ºC for 20 min and the supernatants were used for determining protein content and enzyme activities.

Protein estimation: The protein content was determined by following the method proposed by Lowry et al. (1951).

Guaiacol peroxidase (GPOX) (eC1.11.1.7) activity: The GPOx activity was determined by the method of Putter (1974). The reaction mixture consisted of 3 ml of phosphate buffer, 50 µl guaiacol solution, 100 µl of enzyme sample and 30 µl of H₂O₂ solution. The rate of formation of guaiacol dehydrogenation product (GDHP) was determined spectrophometrically at 436 nm.

Catalase (CAt) (eC 1.11.1.6) activity: The activity was determined as per the method of Aebi (1983). The reaction mixture consisted of 300 µl of enzyme extract taken in the test cuvette and to this 1.5 ml of phosphate buffer and 1.2 ml of H₂O₂ solution was added. This was followed by decrease in absorbance per min at 240 nm.

Superoxide dismutase (SOD) (eC 1.15.1.1) activity: The SOD activity was determined by the method of Kono (1976). In this method, reaction mixture containing 1.9 ml of sodium carbonate buffer, 750 µl NBT and 150 µl Triton x-100 were taken in a test cuvette and reaction was started by adding 150 µl hydroxylamine hydrochloride. After 2 min, 70 µl of enzyme extract was added. The percentage inhibition in the reduction rate of NBT was recorded with increase in absorbance at 540 nm.

Glutathione reductase (GR) (eC 1.6.4.2) activity: The GR activity was determined by following the Foyer and Halliwell method (1976) as described earlier.

Seedling growth: The effects of Cu²⁺ and Cr⁶⁺ metal stress on 7 days old Raphanus seedlings were determined. The growth was assessed in the terms of shoot length, root length and fresh biomass which were assessed for the metal treated and untreated control seedlings.

Statistical analysis: Three repetitions were designed for each experiment and data was expressed as mean values ± SE. One way analysis of variance (ANOvA) was carried and data was presented at significance of p<0.05.

RESULTS

Endogenous contents of Polyamines: Putrescine (Put) content in 7d old untreated control seedlings was 36.64 µg g^-1f.w., whereas under Cu²⁺ metal stress an increase in Put content was observed (62.44 µg g^-1 f.w.) in comparison to control. Cadaverine (Cad) synthesis showed varied pattern (Table 1) in its synthesis in comparison to other PAs. Significant decrease in Cad level was found in Cu²⁺ metal stressed seedlings (6.71 µg g^-1 f.w) (Table1) when compared to control ones (57.22 µg g^-1 f.w). Enhanced content of spermidine (Spd) level was observed in seedlings treated with Cu²⁺ metal (402.8 µg g^-1 f.w.) when compared to Spd content found in control seedlings (178.4 µg g^-1 f.w.). The synthesis of spermine (Spm) revealed drastic changes in its production under Cu²⁺ metal stress. A radical increase in Spm content (1287.9 µg g^-1 f.w.) was found in seedlings exposed to Cu²⁺ metal stress whereas no Spm was detected in control seedlings (Table1).

Cr⁶⁺ metal treatment results in selective enhancement of the PAs levels, in comparison to control. Significant reduction in Put content (1.43 µg/g f.w.) was observed in Cr⁶⁺ metal stressed seedlings in comparison to control (36.64 µg g^-1f.w.) Cad concentration (0.090 µg/g f.w.) was observed to decrease in Cr⁶⁺ metal treated seedlings. Significantly higher Spd content (277.99 µg/g f.w.) was found in seedlings under Cr⁶⁺ metal stress. The Spm content which was not detected in over control seedlings was recorded in seedlings exposed to Cr⁶⁺ metal treatment (2.29 µg/g f.w.) (Table1).

Endogenous content of auxins: The exposure of seedlings to both Cu²⁺ and Cr⁶⁺ metal stress led to significant changes in the levels of auxins. Free IAA (2.56 µg/g f.w.) and bound IAA content (1.5 µg/g f.w.) observed in Cu²⁺ metal stressed seedlings was much lower than free IAA and bound IAA (7.29 µg/g f.w. and 2.07 µg/g f.w., respectively) found in control seedlings. No free and bound NAA was recorded in untreated control seedlings. However, higher contents of free NAA (35.01 µg/g f.w.) and bound NAA (22.37 µg/g f.w.) were detected in Cu²⁺ metal stressed seedlings. Free IBA (6.96 µg/g f.w.) and bound IBA (5.02 µg/g f.w.) content recorded in Cu²⁺ metal treated seedlings was higher than free IBA and bound IBA (1.01 µg/g f.w and 0.94 µg/g f.w., respectively) found in control seedlings.

Significant reduction in both free IAA (0.65 µg/g f.w.) and bound IAA (0.53 µg/g f.w.) was recorded in Cr⁶⁺ metal stressed seedlings in comparison to free IAA and bound IAA contents found in control seedlings. Cr⁶⁺ metal stress was observed to initiate synthesis of NAA, which was not detected in control seedlings. The concentration of both free NAA (7.20 µg/g f.w.) and bound NAA (5.77 µg/g f.w.) detected in Cr⁶⁺ metal stressed seedlings was higher than under Cu²⁺ stress. Similarly the concentration of free IBA (7.41 µg/g f.w.) and bound IBA (5.80 µg/g f.w.) observed in Cr⁶⁺ metal stressed seedlings was higher than IBA content found in control seedlings.

Endogenous contents of ABA: Significant increase in both free and bound forms of ABA were observed under Cu²⁺ and Cr⁶⁺ metal stress when compared to control. However, maximum rise in free ABA (13.51 µg/g f.w.) and bound (11.22 µg/g f.w.) was recorded under Cr⁶⁺ metal stress when compared to control and Cu²⁺ metal stressed seedlings.

Antioxidative enzyme activities: Copper stress has a drastic effect on radish physiology. The GPOx activity was reduced significantly from (0.270 U/mg protein f.w.) in control seedlings to (0.066 U/mg protein f.w.) in seedlings undergone Cu²⁺ metal treatment (Table 3). On the other hand CAT activity showed a decline from 0.171 U/mg protein f.w. (control) to 0.076 U/mg protein f.w. (Cu²⁺-metal treatment) (Table 3). GR activity was also severely affected by Cu²⁺metal stress, with decrease in activity (2.2 U/mg protein f.w.) in Cu²⁺ metal treatment, when compared to 10.5 U/mg protein f.w. in untreated control seedlings (Table 3). The SOD activity recorded enhancement from 3.56 U/mg protein f.w. to 8.65 U/mg protein f.w. in control and Cu²⁺-metal treated seedlings respectively (Table 3). A significant reduction in protein content from 20.78 mg/g f.w. (control) to 9.79 mg/f.w. (Cu²⁺ metal stress) has been also observed (Table 3).

Chromium (Cr⁶⁺) metal stress exhibits more phytotoxicity than Cu²⁺ metal in terms of antioxidative enzyme activity. A significant decrease in GPOx activity (0.0514 U/mg protein f.w.) in Cr⁶⁺ metal stressed seedlings when compared to GPOx activity (0.270 U/mg protein f.w.) found in control seedlings (Table 3). Similarly, a reduction in CAT activity under Cr⁶⁺ metal stress (0.053 U/mg protein f.w.) was observed in comparison to control. Drastic decrease in GR activity (1.21 U/mg protein f.w.) was found in Cr⁶⁺ metal stressed seedlings in compassion to 10.5 U/mg protein f.w. in control seedlings. SOD activity showed significant rise (12.4 U/mg protein f.w.) in comparison to control seedlings (Table 3). A significant reduction in protein content (7.62 mg/g f.w.) was observed in Cr⁶⁺ metal stressed seedlings in comparison to protein content (20.78 mg/g f.w.) recorded in control seedlings (Table 3).

Seedling growth: Cu²⁺ metal stress significantly reduced shoot length (1.5 cm) and root length (3.24 cm) of Raphanus seedlings in comparison to shoot (3.53 cm) and root length (6.50 cm) observed in control seedlings (Table 4). Decrease in fresh weight (0.150 g) was observed in Cu²⁺ metal stressed seedlings when compared to fresh weight (0.240 g) recorded in control seedlings. Similarly, Cr⁶⁺ metal stress reduced shoot length (2.23 cm) and root length (3.16 cm) in comparison to control seedlings. Moreover decrease in fresh weight (0.108 g) was more than Cu²⁺ metal induced decrease in fresh weight when compared to control seedlings.

DISCUSSION

Heavy metal stress leads to oxidative damage caused by the production of free radicals and ROS (Halliwel and Gutteridge, 1984). Both Cu²⁺ and Cr⁶⁺ metal induces oxidative stress in plant metabolism, ultimately resulting into the release of free radicals or oxidants (Luna et al., 1994). To counteract ill effects of ROS plants have evolved defense mechanisms consisting of antioxidants, antioxidative enzymes and phytohormones. The Cu²⁺ and Cr⁶⁺ metal phytotoxicity was observed in the present investigation in the form of reduced shoot and root growth. The phytotoxicity of Cu²⁺ metal in reducing shoot length was more than Cr⁶⁺ metal, which in turn showed its more toxicity on root length. Decrease in fresh weight of the seedlings was more pronounced under Cr⁶⁺ metal stress than Cu²⁺ metal stress. Decrease in shoot length and root length under metal stress may be due to improper chlorophyll synthesis and blockage of root hairs, which resulted in impaired uptake of nutrients and water (Barcelo et al., 1986; Shanker et al., 2005). Moreover impaired shoot and root system might be unable to undergo normal photosynthesis, cell division and cell elongation thus resulting in a visible reduction in fresh weight of the seedlings under Cu²⁺ and Cr⁶⁺ metal stress in comparison to untreated control seedlings (Table 4). The present investigation also revealed selective synthesis of high molecular weight PAs like Spm and Spd (Table 1) on account of more amine groups than low molecular weight Cad and Put (Table1) which has lesser number of amine groups clearly indicating faster scavenging of oxidants or the free radicals generated under Cu²⁺ and Cr⁶⁺ metal stress. Shevyakova et al. (2006) also recorded enhanced synthesis of Spd and Spm than Put and Cad in Mesembryanthemum crystalinum under salinity stress. The strong antioxidant character of high molecular weight PAs than low molecular weight PAs had also been recorded by Ha et al. (1998).

The reduction in Cad content has also been reported to induce oxidative burst which led to enhanced activity of superoxide dismutase (vladimir et al., 2009). Our findings are in agreement with these observations that reduction in Cad concentration acts as stress response for inducing the enhanced activity of SOD (vladimir et al., 2009). The enhanced synthesis of Put than Cad under Cu²⁺ metal stress indicated its effectiveness in oxidative stress management than Cad. However Cr⁶⁺ metal stress reduced Put content than control value, which in turn suggests selective induction of PAs biosynthesis under Cr⁶⁺ metal stress. These results clearly suggest that Cu²⁺ and Cr⁶⁺ metal stress has been very selective in the induction of polyamine synthesis. This has also signified the extraordinary ability of Spd and Spm in metal stress management than other PAs.

The active involvement of auxins in plant stress management is recently explored. Partial restoration of cambial activity in Luffa cylindrica L. under mercury stress on application of auxins was reported by Khan and Chaudhary (2006). Similarly, Dimpka et al. (2008) recorded the phytoremediation role of auxins and siderophores in Streptomyces spp. against heavy metals (Cu, Ni and Cd). Significant changes in auxins content under Cu²⁺ and Cr⁶⁺ metal stress revealed their significant involvement in metal stress management. Such that reduced synthesis of free and bound IAA under Cu²⁺ and Cr⁶⁺ metal stress could be correlated with reduced shoot and root growth and decreased fresh weight. On the other hand enhanced synthesis of free and bound NAA and IBA under Cu²⁺ and Cr⁶⁺ metal stress indicated there active involvement in metal stress management than IAA. Moreover Cr⁶⁺ metal induced synthesis of free and bound NAA and IBA more effectively than Cu²⁺ metal stress (Table 2). Similarly increased synthesis of free and bound ABA under Cu²⁺ and Cr⁶⁺ stress also showed their active involvement in metal stress tolerance (Table 2). Our findings are in agreement with Khan and Choudhary (2006) and Kurepin et al. (2008) who also showed increase in the contents of auxins and ABA in Luffa cylindrica and Brassica napus respectively.

Decrease in the activities of GPOx, CAT and GR under Cu²⁺ and Cr⁶⁺ metal stress were observed (Table 3). However significant enhancement in the activity of SOD recorded under Cu²⁺ and Cr⁶⁺ metal stress was observed to be accompanied by increase in the endogenous titers high molecular weight PAs (Put, Spd and Spm). Whereas reduction in Cad content observed under Cu²⁺ and Cr⁶⁺ metal stress might be able to enhance synthesis and activity of SOD. More recently, vladimir et al. (2009) observed that burst of Cad pool might act as a signal for initiating the enhanced activity of SOD, which may be able to protect the plants against oxidative stress induced by Cu²⁺ and Cr⁶⁺ metal stress along with PAs and auxins in a better way.

CONCLUSION

The present investigation revealed pronounced phytotoxicity of Cr⁶⁺ over Cu²⁺ stress in terms of reduced shoot and root growth, and fresh weight. The synthesis of PAs was more influenced by Cu²⁺ metal than Cr⁶⁺ metal. Moreover, Cu²⁺ was able to induce the synthesis of high molecular weight PAs (Spd and Spm) more effectively than Cr⁶⁺ metal. The synthesis of free and bound forms of IAA, NAA and IBA were observed to be significantly altered under Cr⁶⁺ metal stress than Cu²⁺ metal stress. Therefore the results obtained suggest that Cr⁶⁺ metal stress mainly targeted seedling growth and synthesis of auxins while Cu²⁺ metal showed its phytotoxicity in terms of enhanced synthesis of PAs.

Acknowledgements: The present study was supported by Council of Scientific and Industrial Research (CSIR), New Delhi, India. Authors are thankful to Dr. G.N. Qazi (Former Director) Indian Institute of Integrative Medicine (IIIM), Jammu, India for extending instrumentation facilities for HPLC analysis.

Received: 10 November 2009

Accepted: 27 October 2010

• Aebi HE (1983) Catalase. In: Methods of Enzymatic Analysis, [H.U. Bergmeyer, ed.]. verlag Chemie Press, Weinham, vol.3, pp 273-282.
• Alcazar R, Francisco M, Jaun CC, Marcena P, Alejandro F, Pedro C, Antonio FT, Teresa A (2006) Involvement of polyamines in plant response to abiotic stress. Biotech. Lett. 28: 1867-1876.
• Alscher RG, Erturk N, Heath LS (2002) Role of Superoxide dismutases (SODs) in controlling oxidative stress in plants. J. Exp. Bot.. 53(372): 1331-1341.
• Baek S-H, Park M, Suh J-H, Choi H-S (2008) Protective Effects of an Extract of young Radish (Raphanus sativus L) cultivated with Sulfur (Sulfur-Radish Extract) and of Sulforaphane on Carbon Tetrachloride-Induced Hepatotoxicity. Biosci. Biotech. Biochem. 72 (5): 1176-1182.
• Barcelo J, Poschenrieder C, Gunse B (1986) Water relations of chromium vI treated bush bean plants (Phaseolus vulgaris L. cv. Contender) under both normal and water stress conditions. J. Exp. Bot.. 37: 178-87.
• Barillari J, Cervellati R, Costa S, Guerra MC, Speroni E, Utan A, Iori R (2006) Antioxidant and Choleretic Properties of Raphanus sativus L. Sprout (Kaiware Daikon) Extract. J. Agr. Food Chem. 54: 9773-9778.
• Barillari J, Iori R, Papi R, Orlandi M, Bartolini G, Gabbanini S, Pedulli GF, valgimigli L (2008) Kaiware Daikon (Raphanus sativus L.) Extract: A Naturally Multipotent Chemopreventive Agent. J. Agric. Food Chem. 56 (17): 78237830
• Berenguer P, Cela S, Santivera F, Boixadera J, Lloveras J (2008) Copper and Zinc soil accumulation and Plant Concentration in irrigated Maize fertilized with Liquid Swine Manure. Agron. J. 100: 1056-1061.
• Carlberg I, Mannervik B (1985) Glutathione reductase assay. Methods in Enzymology. Academic Press, Orlando FL, 113: 484495.
• Chary NS, Kamala CT, Raj DS (2008) Assessing risk of heavy metals from consuming food grown on sewage irrigated soils and food chain transfer. Ecotoxic. Env. Safety. 69(3): 513-524.
• Chatterjee C, Sinha P, Dube BK, Gopal R (2006) Excess Copper-induced Oxidative Damages and Changes in Radish Physiology. Comm. Soil Anal. Plant Sci. 37: 2069-2076.
• Clouse SD (2008) The molecular intersection of brassinosteriods-regulated growth and flowering in Arabidopsis. Proc. Natl. Acad. Sci. U. S.A. 105 (21): 7345-7346.
• Csiszar J, Pinter B, Kolbert Z, Erdel L, Tari I (2008) Peroxidase activities in root segments of wheat genotypes under osmotic stress. Acta Biol. Szegediensis 52(1): 155-156.
• Davletova S, Rizhsky L, Liang H, Shengqiang Z, Oliver DJ, Coutu J, Shulaev v, Schlauch K, Mittler R (2005) Cytosolic Ascorbate Peroxidase 1 is a central component of the Reactive oxygen gene Network of Arabidopsis. Plant Cell 17(1): 268-281.
• Dimpka CO, Svatos A, Dabrowska P, Schmidt A, Boland W, Kothe E (2008) Involvement of Siderophores in the reduction of metal-induced inhibition of auxin synthesis in Streptomyces spp. Chemosphere 74(1): 19-25.
• Etehadnia M, Waterer DR, Tannino KK (2008) The Method of ABA Application Affects Salt Stress Responses in Resistant and Sensitive Potato Lines. J. Plant. Growth Regul. 27(4): 333-341.
• Flors v, Ton Jurrinam, Doorn Rv, Jakab G, Augustin-Garcia P, Mauch-Mani B (2008) Interplay between JA, SA, and ABA signaling during basal and induced resistance against Pseudomonas syringae and Alternaria brassicola Plant J. 54: 81-92.
• Fontaniella B, Mateos JL, vicente C, Legaz M-E (2001) Improvement of the analysis of dansylated derivatives of polyamines and their conjugates by high-performance liquid chromatography. J. Chromatogr. A. 919: 283-288.
• Foyer CH, Halliwell B (1976) The presence of glutathione and glutathione reductase in chloroplasts: A proposed role in ascorbic acid metabolism. Planta 133(1): 21-25.
• Gao J, Igarashi K, Nukina M (1999) Three new phenylethanoid glycosides from Caryopteris incana and their antioxidative activity. Chem. Pharm. Bull. 48: 1075-1078.
• Groppa MD, Benavides MP (2008) Polyamines and abiotic stress: recent advances. Amino Acids 34: 35-45.
• Ha HC, Sirisoma NS, Kuppusamy P, Zweier JL, Woster PM, Casero RA (1998) The natural polyamine spermine functions directly as a free radical scavenger. Proc. Natl. Acad. Sci. USA 95: 11140-11145.
• Halliwel B, Gutteridge JMC (1984) Oxygen radicals, transition metals and diseases. Biochem. J. 219: 1-14.
• Haubrick LL, Assmann SM (2006) Brassinosteriods and plant function: some clues, more puzzles. Plant Cell Environ. 29: 446-457.
• Iqbal M, Ashraf M (2007) Seed Treatment with Auxins Modulates Growth and Ion Partitioning in Salt-stressed Wheat Plants. J. Integr. Plant Biol. 49(7): 1003-1015.
• Khan AS, Chaudhry Ny (2006) Auxins partially restore the cambial activity in Luffa cylindrica L. (Cucurbitaceace) under mercury stress. Int. J. Food Agr. Env. 4(1): 276-281.
• Kono y (1978) Generation of superoxide radical during autoxidation of hydroxylamine and an assay for superoxide dismutase. Arch. Biochem. Biophys.186 (1): 189-195.
• Kumar v, Giridhar P, Chandrashekar A, Ravishankar GA (2008) Polyamines influence morphogenesis and caffeine biosynthesis in in vitro cultures of Coffea canephora P. ex Fr. Acta Physiol. Plant. 30: 217-223.
• Kurepin Lv, Qaderi MM, Back TG, Reid DM, Pharis RP (2008) A rapid effect of applied brassinolide on abscisic acid concentrations in Brassica napus leaf tissue subjected to short-term heat stress. Plant Growth Regul. 55: 165-167.
• Kuznetsov vv, Stetsenko LA, Shevyakova NI (2009) Exogenous cadaverine induces oxidative burst and reduces cadaverine conjugate content in the common ice plant. J. Plant Physiol. 166: 40-51.
• Lin CC, Kao CH (1999) Excess copper induces an accumulation of putrescine in rice leaves. Bot. Bull. Acad. Sinica. 40: 213-218.
• Liu JH, Moriguchi T (2007) Changes in free polyamine titers and expression of polyamine biosynthetic genes during growth of peach in vitro callus. Cell Bio. Morphogen. 26: 125-131.
• Liu J-H, Kitashiba H, Wang J, Ban y, Moriguchi T (2007) Polyamines and their ability to provide environmental stress tolerance to plants. Plant Biotech. 24: 117-126.
• Lowry OH, Rosebrough MJ, Farr AL, Randall RJ (1951) Protein measurements with the Folin phenol reagent. J. Bio. Chem. 193: 265-275.
• Luna CM, Gonzalez CA, Trippi vS (1994) Oxidative damage caused by an excess of Copper in oat leaves. Plant Cell Physiol. 35(1): 11-15.
• Maksymiec W, Wianowska D, Dawidowicz AL, Radkiewicz S, Mardarowicz M, Krupa Z (2005) The level of jasmonic acid in Arabidopsis thaliana and Phaseolus coccineus under heavy metal stress. J. Plant Physiol. 16(12): 1338-1346.
• Mengel K, Kirkby EA (1987) Principles of plant nutrition 4^thed. Int. Potash Inst., Basel, Switzerland.
• Nagar PK, Sood S (2003) Changes in abscisic acid and phenols during flower development in two diverse species of rose. Acta Physiol. Plant. 25(4): 411-416.
• Nagar PK, Sood S (2006) Changes in endogenous auxins during winter dormancy in tea (Camellia sinensis L.) O. Kuntze. Acta Physiol. Plant. 28(2): 165-169.
• Park J-E, Park J-y, Kin y-S, Staswick P-E, Jeon J, yun J, Kim S-y, Kim J, Lee H-y, Park C-M (2007) GH3-medaited auxin homeostasis links growth regulation with stress adaptation response in Arabidopsis. J. Bio. Chem. 282 (3): 10036-10046.
• Pirintsos SA, Kotzabasis K, Loppi S (2004) Polyamine production in Lichens under Metal Pollution Stress. J. Atmosph. Chem. 49: 303-315.
• Posymk MM, Balabusta M, Wieczorek M, Sliwinska E, Janas KM (2009) Melatonin applied to cucumber (Cucumis sativus L.) seeds improves germination during chilling stress. J. Pineal Res. 46(2): 214-223.
• Putter J (1974) Peroxidase In: Methods of Enzymatic Analysis (Ed. Bergmeyer, H.U.) verlag Chemie, Weinhan, pp. 685-690.
• Romero-Puertas MC, Corpas FJ, Sandalio LM, Leterrier M, Rodríguez-Serrano MR, Luis A, Palma JM (2006) Glutathione reductase from pea leaves: response to abiotic stress and characterization of the peroxisomal isozyme. New Phytol 170 (1): 43-52(10)
• Schutzendubel A, Polle A (2002) Plant responses to abiotic stresses: heavy metal-induced oxidative stress and protection by mycorrhization. J. Exp. Bot.. 53(372): 1351-1365.
• Seiler N, Knodgen B (1979) Determination of the naturally occurring monoacetyl derivatives of di and polyamines. J. Chromatograp.164: 155-168.
• Sgherri C, Cosi E, Navari-izzo F (2002) Phenols and antioxidative status of Raphanus sativus in copper excess. Physiol. Plant. 118 (1): 21-28.
• Shanker AK, Cervantes TC, Loza-Taverac H, Avudainayagam S (2005) Chromium toxicity in plants. Env. Int. 31: 739-753.
• Shevyakova NI, Shorina Mv, Rakitin vy, Kuznetsov vv (2006) Stress- Dependent Accumulation of Spermidine and Spermine in the Halophyte Mesembryanthemum crystalinum under salinity conditions. Russ. J. Plant Physiol. 53(6): 739-745.
• Staneloni JR, Batiller-Rodriguez MJ, Casal JJ (2008) Abscisic acid, high-light, and oxidative stress down-regulate a photosynthetic gene via a promoter motif not involved in phytochrome-mediated transcriptional regulation. Mol. Plant 1 (1): 75-83.
• Tamaoki M, Freeman JL, Pilon-Smits EAH (2008) Cooperative ethylene and jasmonic acid signaling regulates selenite resistance in Arabidopsis Plant Physiol. 146: 1219-1230.
• Tang W, Newton RJ (2005) Polyamines reduce salt-induced oxidative damage by increasing the activities of antioxidant enzymes and decreasing lipid peroxidation in virginia pine. Plant Growth Regul. 46: 31-43.
• Taniguchi H, Muroi R, Kobayashi-Hattori K, Uda y, Oishi y, Takita T (2007) iffering Effects of Water-Soluble and Fat-Soluble Extracts from Japanese Radish (Raphanus sativus) Sprouts on carbohydrate and lipid metabolism in normal and Streptozotocin-Induced Diabetic Rats. J. Nutr. Sci. vitaminol. 53: 261-266.
• Tassoni A, Franceschetti M, Bagni N (2008) Polyamines and salt stress response and tolerance in Arabidopsis thaliana flowers. Plant Physiol. Biochem. 46(5-6): 607-613.
• villaluenga CM, Frias J, Gulewicz P, Gulewicz K, vidan-valverde C (2008) Food safety evaluation of broccoli and radish sprouts. Food Chem. Toxicol. 46 (5): 1635-1644.
• Wen x-P, Pang x-M, Matsuda N, Kita M, Inoue H, Hao y-J, Honda C, Moriguchi T (2008) Over-expression of the apple spermidine synthase gene in pear confers multiple abiotic stress tolerance by altering polyamine titers. Trans. Res. 17: 251- 263.
• Willekens H, Chamnogpol S, Davey M, Schraudner M, Langebartels C, Montagu Mv, Inze D, Camp Wv (1997) Catalase is a sink for H2O2 and is indispensable for stress defence in C3 plants. Eur. Mol. Biol. Org. J. 16(16): 4806-4816.
• Zapata PJ, Serrano M, Pretel MT, Botella MA (2008) Changes in free polyamine concentrations induced by stress in seedlings of different species. Plant Growth Regul. 56 (2): 167-177.
• Zhang x, Ervin EH (2008) Impact of seaweed extract-based cytokinins and zeatin riboside on creeping bentgrass heat tolerance. Crop Sci. 48: 364-370.
• Zhou B, Guo Z, Liu Z (2005) Effects of abscisic acid on antioxidant systems of Stylosanthes guainensis (Aublet) Sw. under chilling stress. Crop Sci. 45:599-605.

Publication Dates

• Publication in this collection
  06 May 2011
• Date of issue
  2010

History

• Accepted
  27 Oct 2010
• Received
  10 Nov 2009