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Brassinosteroid enhanced the level of antioxidants under cadmium stress in Brassica juncea
Environmental and Experimental Botany 60 (2007) 33–41
Brassinosteroid enhanced the level of antioxidants under cadmium stress in Brassica juncea S. Hayat ∗ , B. Ali, S. Aiman Hasan, A. Ahmad Department of Botany, Aligarh Muslim University, Aligarh 202002, India Received 22 October 2005; received in revised form 26 January 2006; accepted 14 June 2006
Abstract The change in plant growth, photosynthesis, carbonic anhydrase, nitrate reductase and antioxidative enzymes resulting from the feeding of cadmium and/or 28-homobrassinolide (HBL) to Brassica juncea were studied in 60-day-old plants. One-week old seedlings were supplied with 50, 100 or 150 M of cadmium along with the nutrient solution. Subsequent seedlings, at day 30, were sprayed with 0.01 M of HBL to their foliage. The plants fed with cadmium alone exhibited a decline in growth, the levels of carbonic anhydrase (E.C. 4.2.1.1) and chlorophyll pigments and net photosynthetic rate. Moreover, nitrate content, the activity of nitrate reductase (E.C. 1.6.6.1) and the level of carbohydrate, both in the leaves and roots, decreased as the concentration of cadmium, in nutrient solution, increased. Compared with the leaves, roots possessed a larger quantity of nitrate. However, the trend was reversed in case of nitrate reductase and the level of carbohydrates in the two plant organs. The toxic effect, generated by cadmium was overcome if the stressed plants were sprayed with HBL. The activities of antioxidative enzymes [viz. catalase (E.C. 1.11.1.6), peroxidase (E.C. 1.11.1.7) and superoxide dismutase (E.C. 1.15.1.1)] and the contents of proline increased, over the control, irrespective of the treatment. Their level increased further, if the plants supplied with cadmium were also supplemented with HBL, both in the roots and the aerial parts. Nevertheless, the contents of proline in roots were higher than the leaves. © 2006 Elsevier B.V. All rights reserved. Keywords: Brassinosteroid; Carbonic anhydrase; Catalase; Peroxidase; Photosynthesis; Superoxide dismutase
1. Introduction The brassinosteroids occur in all plant parts, including roots (Bajguz and Tretyn, 2003). These chemicals elicit a wide range of physiological responses in plants, including stem elongation, pollen tube growth, leaf bending and epinasty, root growth inhibition, induced synthesis of ethylene, activation of proton pump, xylem differentiation, synthesis of nucleic acids and proteins, activation of enzymes and photosynthesis (Clouse and Sasse, 1998; Khripach et al., 2003; Hayat and Ahmad, 2003; Sasse, 2003; Yu et al., 2004). It is proposed that the changes induced by BRs are mediated through the repression and/or depression of specific genes (Felner, 2003). Besides this, BRs are also recognized to have an ameliorative role in plants, subjected to various biotic and abiotic stresses (Clouse and Sasse, 1998). The treatAbbreviations: BR, brassinosteroid; CA, carbonic anhydrase; HBL, 28homobrassinolide; NR, nitrate reductase; PN , net photosynthetic rate; RWC, relative water content; SOD, superoxide dismutase ∗ Corresponding author. Tel.: +91 9412328593. E-mail address: [email protected] (S. Hayat). 0098-8472/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2006.06.002
ment of the plants of rice and tomato (Kamuro and Takatsuto, 1991), maize (He et al., 1991), cucumber (Katsumi, 1991) and brome grass (Wilen et al., 1995), with BRs improved their capacity of resistance to low temperature. Similarly, BRs increased the degree of tolerance, to high temperature, in wheat (Kulaeva et al., 1991) and brome grass (Wilen et al., 1995). Brassinosteroids also countered the drought stress in sugarbeet (Schilling et al., 1991), moisture stress in wheat (Sairam, 1994) and favoured seed germination and seedling growth in Eucalyptus (Sasse et al., 1995) and rice (Anuradha and Rao, 2001), under saline conditions. Moreover, BRs activate antioxidative enzymatic defense system in rice seedlings, grown under salt stress (Nunez et al., 2003). Cadmium is extremely toxic to plants that retards biosynthesis of chlorophyll (Singh and Tewari, 2003), alters water balance (Barcelo and Poschenrieder, 1990), decreases activity of various enzymes (Siedlecka et al., 1997; Gouia et al., 2003), favours stomatal closure (Poschenrieder et al., 1989) and finally slows down the rate of photosynthesis (Sheoran et al., 1990; Chugh et al., 1992). Cadmium stress also reduces the uptake of essential mineral nutrients (Harnandez et al., 1996), decreases normal
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H+ /K+ exchange and the activity of ATPase, at the level of cellular plasma membrane (Obata et al., 1996). This experiment was planned with an aim to relate a change in the level of antioxidative enzymes, in cadmium fed plants of Brassica juncea, with the induced resistance and to neutralize the effects of stress by the application of 28-homobrassinolide. In this study, HBL was used because it is more active and stable in field trials than some other brassinosteroids (Khripach et al., 2000). We tested the hypothesis that the application of HBL will ameliorate the toxic effect of cadmium on the growth of B. juncea plants. 2. Materials and methods 2.1. Plant material and growth conditions Authentic, healthy seeds of B. juncea Czern & Coss cv. T-59, were surface sterilized with 0.5% (v/v) sodium hypochlorite and were sown in sand, moistened with deionized water in plastic pots of 6 in. diameter. The sand, before being used, was washed five times with deionized water. The pots were placed in net house, under natural conditions. The plants from day 5 were supplied with 100 ml of nutrient solution daily in the morning and an equal volume of deionized water, in the evening upto day 20. Thereafter the volume of the solution was increased to 500 ml. The composition of nutrient solution was 3 mmol KNO3 , 0.5 mmol Ca(NO3 )2 , 0.5 mmol MgSO4 , 2.5 mmol KH2 PO4 , 2 mmol NH4 Cl, 100 mol Fe–K–EDTA, 30 mol H3 BO3 , 5 mol MnSO4 , 1 mol CuSO4 , 1 mol ZnSO4 and 1 mol (NH4 )6 MO7 O24 per liter. The subsequent, 1week old, seedlings were supplied with 0, 50, 100 or 150 M of cadmium in the form of CdCl2 , with nutrient solution. The higher concentrations (200 and 250 M) proved lethal for growth of the plants. The plants were allowed to grow and at day 30, the foliage was applied with 0 or 0.01 M of HBL. The concentration of HBL was based on our earlier studies (Hayat et al., 2000, 2001; Fariduddin et al., 2004). Sixty-day-old plants were sampled with intact roots to assess the following parameters. The experiment was conducted according to simple randomized block design. The whole experiment was conducted twice. The data of both the years were pooled. The analysis of variance (ANOVA) was performed as described by Gomez and Gomez (1984) and the S.E. was calculated. 2.2. Fresh and dry mass per plant Plants were removed along with the sand and dipped in a bucket, filled with tap water, to remove the adhering sand particles, ensuring the safety of the roots. The plants were blotted and weighed to record their fresh mass and then placed in an oven, run at 80 ◦ C, for 24 h. The samples were weighed again after allowing them to cool to room temperature, in a desicator, to record their dry mass.
The values were placed in the following formula, proposed by Arnon (1949), to compute chlorophyll content [(A645 × 28.2) + (A663 × 8.3)] × [(V/1000) × W]. The net photosynthetic rate (PN ) in intact leaves was measured by LI-6200 portable photosynthetic system (LI-COR Licoln, NE, USA) at light saturating intensity between 11:00 and 12:00 h. The atmospheric conditions during the experiments were: PAR, 1065 ± 30 mol m−2 s−1 ; Ci, 280 ± 12 mol mol−1 ; atmospheric CO2 , 355 ± 5 mol mol−1 ; relative humidity, 65 ± 5%; atmospheric temp., 22 ± 2 ◦ C. 2.4. Relative water content (RWC) The RWC was determined in fresh leaf discs of 2 cm diameter, excluding midrib. Discs were weighed quickly and immediately floated on double distilled water, in petridishes to saturate them with water for the next 24 h, in dark. The adhering water of the discs was blotted and turgor weight was taken. Dry mass of these discs was obtained, after dehydrating them at 70 ◦ C for 48 h. Relative water content was calculated by placing the observed values in the following formula (Jones and Turner, 1978): RWC =
fresh mass − dry mass × 100. turgor mass − dry mass
2.5. Estimation of carbonic anhydrase (CA) activity The activity of CA was determined following the procedure described by Dwivedi and Randhawa (1974). The leaf samples were cut into small pieces and suspended in cystein hydrochloride. These samples were incubated at 4 ◦ C for 20 min. The pieces were blotted and transferred to the test tube, containing phosphate buffer (pH 6.8) followed by the addition of alkaline bicarbonate solution and bromothymol blue, indicator. The test tube was incubated at 5 ◦ C for 20 min. The reaction mixture was titrated against 0.05N HCl, after the addition of few drops of methyl red, indicator. The results were expressed as mol (CO2 ) kg−1 (leaf fresh mass)s−1 . 2.6. Estimation of nitrate reductase (NR) activity The activity of NR was measured following the method adopted by Jaworski (1971). The fresh leaf samples were cut into small pieces and transferred to plastic vials, containing phosphate buffer (pH 7.5) followed by the addition of potassium nitrate and isopropanol solutions. The reaction mixture was incubated at 30 ◦ C, for 2 h followed with the addition of N-1naphthylethylenediamine dihydrochloride and sulphanilamide. The absorbance of the colour was read at 540 nm and was compared with that of the calibration curve. The activity of NR (nmol NO2 g−1 h−1 ) was computed on fresh mass basis. 2.7. Assay of antioxidative enzymes
2.3. Chlorophyll content and net photosynthetic rate Leaf chlorophyll was extracted in 80% acetone and the absorbance was read spectrophotometrically at 663 and 645 nm.
Leaf tissue (0.5 g) was homogenized in 5 ml of 50 mmol phosphate buffer (pH 7.0) containing 1% insoluble polyvenylpyrolidone. The homogenate was centrifuged at 15,000 rpm for
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10 min and the supernatant was used as the source of enzyme. This extraction was carried out at 4 ◦ C. Peroxidase and catalase were assayed following the procedure described by Chance and Maehly (1955). Catalase was estimated by tirating the reaction mixture, consisting of phosphate buffer (pH 6.8), 0.1 M H2 O2 , enzyme extract and 2% H2 SO4 , against 0.1N potassium permanganate. The reaction mixture for peroxidase consisted of pyrogallol phosphate buffer (pH 6.8), 1% H2 O2 and enzyme extract. Change in absorbance, due to catalytic conversion of pyrogallol to perpurogallin, was noted at an interval of 20 s for 2 min at 420 nm. A control set was prepared by using distilled water instead of enzyme extract. The activity of superoxide dismutase was assayed by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) using the method of Beauchamp and Fridovich (1971). The reaction mixture contained 50 mmol phosphate buffer (pH 7.8), 13 mmol methionine, 74 mol NBT, 2 mol riboflavin, 0.1 mmol EDTA ad 0–50 l enzyme extract, and was placed under 15 W fluorescent lamp. The reaction was started by switching on the light and was allowed to run for 10 min. The reaction was stopped by switching off the light. Fifty per cent inhibition by light was considered as one enzyme unit. 2.8. Estimation of nitrate and carbohydrate content The nitrate content in the leaves and roots was determined by the method described by Singh (1988). 100 mg of dried tissue powder was digested with 5 ml of 2% acetic acid for 20 min and was filter through Whatman filter paper. The extract was diluted to 10 ml, to which 500 mg of powder mixture, consisting of citric acid, manganese sulphate monohydrate, sulphanilamide, N-1naphthylethylenediamine dihydrochloride and powdered zinc, was added. The reaction mixture was centrifuged and the colour of the supernatant was read at 540 nm. Nitrate content was calculated by comparing the value of the sample with the calibration curve, drawn by using standard nitrate solution. Total carbohydrate was extracted following the method of Yih and Clark (1965) and estimated by adopting the procedure of Dubois et al. (1956). Dried (leaf or root) powder was transferred to glass centrifuge tube containing 1.5N H2 SO4 . The sample was centrifuged at 4000 rpm, for 10 min. One millilitre of the extract was taken in a test tube to which 1 ml of 5% distilled phenol was added. The absorbance was read at 490 nm, using spectrophotometer and compared with the calibration curve, using pure glucose. 2.9. Determination of proline content The proline content in fresh leaf and root samples was determined by adopting the method of Bates et al. (1973). Sample was extracted in sulphosalicylic acid. To the extract, an equal volume of glacial acetic acid and ninhydrine solutions were added. The sample was heated at 100 ◦ C, to which 5 ml of toluene was added. The absorbance of toluene layer was read at 528 nm, on a spectrophotometer.
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3. Results 3.1. Growth and relative water content The growth (as expressed by fresh and dry mass) of the shoot and the root and their relative water contents, decreased in the plants supplemented with cadmium and its rate of loss was proportional to the concentration of the metal (Fig. 1a and b). However, the treatment with HBL to the foliage favoured growth and also partially overcame the toxic effects generated by cadmium. The response of the plants to 50 mol of cadmium was completely overcome by the application of HBL, the values are, therefore, at par with the control. 3.2. Chlorophyll pigment and net photosynthetic rate It is evident from Fig. 1c and d that both chlorophyll contents and net photosynthetic rate decreased, if the plants were supplied with cadmium in the nutrient medium. Maximum damage was noticed in the plants receiving highest concentration (150 mol) of the metal. The effect was to some extent neutralized if the plants, at 30 days stage, were given a followup treatment with 0.01 M of HBL. This treatment generated values comparable with the control in those plants that were already exposed to cadmium (50 mol) stress. 3.3. CA activity The supply of cadmium to the seedlings significantly decreased the activity of the enzyme (Fig. 2a). The loss was more visible as the concentration of the metal was raised from 50 to 150 mol. However, the foliage of the plants sprayed with 0.01 M of HBL alone possessed significantly higher level of CA activity. Moreover, HBL nullified the effects of the metal, to a limited extent, in the plants fed with higher concentrations of cadmium but completely, if given 50 mol of the metal with a follow up treatment of the hormone. 3.4. Nitrate content and NR activity The leaves possessed lesser quantity of the nitrate and higher activity of NR than the roots. However, the pattern of response to the treatment by both these plant organ was very much similar (Fig. 2b and c). A sharp decrease was observed as the concentration of cadmium in the medium was enhanced from 50 to 150 mol. Application of HBL not only increased their values but also completely overcame the toxic effects of the metal (50 mol) and to a limited extent at its higher concentrations (100/150 mol). 3.5. Proline content Proline level was higher in the roots than the leaves (Fig. 2d). The concentration increased in both the major organs of the plants, if supplied with cadmium and/or HBL. The values
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Fig. 1. Effect of HBL on the changes in fresh and dry mass (a), relative water content (RWC) (b), total chlorophyll content (c) and net photosynthetic rate (PN ) (d) in 60-d-old plants of Brassica juncea induced by cadmium.
are much higher if the metal concentration was increased to 150 mol and that too supplied with HBL. 3.6. Activity of antioxidative enzymes The data depicted in Fig. 3a–c clearly revealed significant increase in the activity of antioxidative enzymes (viz. catalase, peroxidase and superoxide dismutase) in response to cadmium and/or HBL. Interestingly, the plants fed with highest concentration of the metal (150 mol) in association with its foliage supplied with HBL exhibited highest levels of all the enzymes whose values were 34%, 94% and 90% more than the control for catalase, peroxidase and superoxide dismutase, respectively. The control plants possessed lowest concentration of all the enzymes.
3.7. Carbohydrate content It was natural that the leaves had more carbohydrate than the roots (Fig. 3d) but decreased significantly under the impact of the cadmium. This loss was highly significant under the highest concentration of the metal (150 mol). The carbohydrate content in the plants, under stress was revived if supplemented with HBL, to their foliage. The best interaction of hormone was noticed with 50 mol of the metal where the values were comparable with those of the control. 4. Discussion The reversible interconversion of CO2 and HCO3 − is catalysed by the enzyme, carbonic anhydrase (CA), whose activity
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Fig. 2. Effect of HBL on the changes in carbonic anhydrase (CA) activity (a), nitrate content (b), nitrate reductase (NR) activity (c) and proline content (d) in 60-d-old plants of B. juncea induced by cadmium.
is largely determined by photon flux density, concentration of CO2 , the availability of Zn (Tiwari et al., 2005) and the genetic expression (Kim et al., 1994). The stress generated by cadmium decreases the partial pressure of CO2 in the stroma by inducing the stomatal closure (Barcelo and Poschenrieder, 1990) resulting in a loss in the activity of CA (Fig. 2a). Siedlecka et al. (1997) also reported low CA activity in Phaseolus vulgaris, exposed to Cd stress. However, the involvement of BRs in regulating transcription and/or translation (Kalinich et al., 1985) possibly favoured synthesis and/or activation of the enzyme protein (Fig. 2a) that has also been reported earlier in mustard (Hayat et al., 2001) and mung bean (Fariduddin et al., 2004). The plants exposed to cadmium stress exhibit an inhibition in the activity of protochlorophyllide reductase (Stabort et al., 1985) by possibly inactivating the sulphydryl site of the reductase protein (Ernst, 1980). This in addition to an increase in
chlorophyllase, the enzyme that activates the degradation of the chlorophyll (Reddy and Vora, 1986) could have contributed in lowering the total chlorophyll content (Fig. 1c). Similar observations have also been reported by Vassileve et al. (1998) in barley and Gadallah (1995) in sunflower grown under Cd stress. However, the application of HBL increased the chlorophyll content in the plants grown with/without the metal (Fig. 1c) by involving the expression of specific genes responsible for the synthesis of the enzymes determining chlorophyll generation. The observed decrease in the net photosynthetic rate under the impact of cadmium (Fig. 1d) may be a direct outcome of the response generated by metal through its ability to bind with specific proteins, to inactive them (Van Assche and Cligsters, 1990), in addition to the reasons (i.e. low CO2 and a decrease in photosynthetic pigments and the activity of carbonic anhydrase), mentioned earlier. Moreover, cadmium has an adverse
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Fig. 3. Effect of HBL on the changes in catalase (a), peroxidase (b), superoxide dismutase (c) and carbohydrate content (d) in 60-d-old plants of B. juncea induced by cadmium.
effect on the activity of Rubisco (Siedlecka et al., 1997) which will naturally have a negative impact on the CO2 reduction. In contrary to above, BR activate the rate of photosynthesis (Fig. 1d) by enhancing the level of the enzymes (Fariduddin et al., 2004; Yu et al., 2004) directly or indirectly involved in the process and thus also overcomes the ill effects generated by cadmium. The metal, in a natural course, exerts a damaging effect on the normal functioning of the plasma membrane by having an impact on the activity of inbuilt ATPase (Obata et al., 1996) and the fluidity of the membrane (Meharg, 1994). This could have checked the uptake and distribution of nitrate, leading to its decrease both in the roots and the shoot of the plants fed with cadmium (Harnandez et al., 1996; Fig. 2b). The availability of nitrate, in addition to the level and state of active sites of functional NR and overall metabolic conditions of the plants determines the status of the activity of nitrate reductase (Campbell, 1999).
The activity of the enzyme, therefore, decreased (Fig. 2c). However, BRs somehow activate the uptake of nitrate (Mai et al., 1989) and also favour transcription and/or translation (Kalinich et al., 1985; Bajguz, 2000). This could have been reason for the observed increase in the activity of NR (Fig. 2c) and also neutralized the impact of the metal stress because of the ameliorative characters of BRs (Hamada, 1986; Sasse, 2003). In a natural course, reactive oxygen species (ROS) includ• ing superoxide radical (O2 − ) hydrogen peroxide (H2 O2 ) and • − hydroxy radical (HO ) are produced in the plants under stress (Asada, 1999; Dat et al., 2000). These ROS, if generated in larger quantities, may oxidize proteins, lipids and nucleic acids leading to even mutation at the cellular level (Halliwell and Gutteridge, 1999). However, to neutralize the toxicity of ROS plants have endogenous system of enzymes (e.g. catalase, peroxidases, superoxide dismutase, glutathione reductase) and metabolites (e.g. ascorbate, glutathione, tocopherols and proline) to operate,
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if exposed to stress (Asada, 1999; Mittler, 2002) and also the fixation of the metals to phytochelatins (Benarides et al., 2005). Out of them a detailed account has been studied about the production of the stress proteins (proline) whose synthesis and degradation is a very guarded phenomenon, regulated by a set of enzymes. Sumithra and Reddy (2004) reported an elevation in the activity of two of the enzymes ( -pyroline-5-carboxylate synthetase and -pyroline-5-carboxylate reductase) responsible for proline synthesis and a decrease of that of proline dehydrogenase, the enzyme that degrades proline in cowpea, under stress. Moreover, the genes responsible for the transcription of these proteins were also over expressed in some transgenic plants, under stress (Kishore et al., 1995). The phytochelatins are small metal binding peptides (Grill et al., 1986a,b) that are synthesized in plants from tripeptide glutathione substrate, mediated by the enzyme phytochelatin synthase (E.C. 2.3.2.15), a constitutive enzyme requiring the presence of either of the heavy metals (Cobett, 2000). Biosynthesis of the phytochelatins continues by the time all the metal ions are chelated or by the addition of metal chelators such as EDTA (Loeffler et al., 1989). The physiological drought generated by cadmium, in the present study, resulted in the increase in the level of proline (Fig. 2d) and that of catalase, peroxidase and superoxide dismutase (Figs. 3a–c) to boost the resistance capacity of the plants to overcome the effects generated by the metal. The level of all these enzymes and that of proline is increased by the action of HBL (Figs. 2d and 3a–c). Nunez et al. (2003) also noted higher activity of antioxidative enzymes in rice, grown under salinity and supplemented with BRs. It gives an impression that BRs some how generate a system (transcription and/or translation) comparable with that of the stress that improves the degree of resistance in the plants to abnormal metabolism. Therefore, an amalgamated action of the stress and that of the HBL in inducing the activity of the enzymes and the synthesis of proline, is much higher than either of them applied alone. A loss in relative water content of the plants (Fig. 1b), supplemented with cadmium, is possibly an impact of the metal on the electrical potential of the plasma membrane that affected not only the absorption of ions (Harnandez et al., 1996) but also that of water, generating water stress (Barcelo and Poschenrieder, 1990). Therefore, physiological disorders are further expressed in the form of closure of stomata (Barcelo and Poschenrieder, 1990), slowing down of the photosynthesis (Prasad, 1995, Fig. 1d) associated with a shift in the proportion of various enzymes (Fig. 2a and c). This altered metabolism, under the impact of cadmium, resulted in the relocation of plant growth where not only the linear growth but also the fresh and dry mass of the plant was lost to a significant level (Fig. 1a). Deviating from the response generated by metal, HBL had a favourable impact on the RWC and fresh and dry mass of the plants, even if given as a followup treatment with cadmium (Fig. 1a and b). BRs generate such a response because of their involvement in the activation of ATPase pump (Khripach et al., 2003), synthesis of nucleic acids and proteins (Bajguz, 2000), repression/depression of genes (Felner, 2003), change in enzyme pattern (Figs. 2a and c and 3a–c) and speeding up of photosynthesis (Fig. 1d). These effects generated by HBL also neutralized
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the impact of cadmium to all of its concentrations but completely at 50 mol. 5. Conclusion The data generated in the present study gives a clear-cut impression that the presence of cadmium at a level that may cause stress in the plant adversely affects its metabolism and growth. Moreover, the metal also activates the system to improve the resistance capacity of the plants to the stress. HBL, on the other hand, has an added effect both on the general metabolism and the resistance capacity of the plants to overcome the toxic effect of the metal. Acknowledgements S. Hayat acknowledges Department of Science & Technology, Govt. of India, New Delhi for financial support (SR/FTP/LS-A-37/2002). We thank anonymous reviewers who made critical evaluation of this manuscript and suggested the incorporation of valuable points. We are also thankful to Dr. B.N. Vyas, General Manager, Godrej Agrovet Ltd., Mumbai, India for the generous gift of 28-homobrassinolide. References Anuradha, S., Rao, S.S.R., 2001. Effect of brassinosteroids on the salinity stress induced inhibition of germination and seedling growth of rice (Oryza sativa). Plant Growth Regul. 33, 151–153. Arnon, D.I., 1949. Copper enzyme in isolated chloroplast polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1–15. Asada, K., 1999. The water-water cycle in chloroplasts: scavenging of active oxygen and dissipation of excess photons. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 601–639. Bajguz, A., 2000. Effect of brassinosteroids on nucleic acid and protein content in cultured cells of Chlorella vulgaris. Plant Physiol. Biochem. 38, 209–215. Bajguz, A., Tretyn, A., 2003. The chemical structures and occurrence of brassinosteroids in plants. In: Hayat, S., Ahmad, A. (Eds.), Brassinosteroids: Bioactivity and Crop Productivity. Kluwer Academic Publishers, Dordrecht, pp. 1–44. Barcelo, J., Poschenrieder, C., 1990. Plant water relations as affected by heavy metal stress: a review. Plant Nutr. 13, 1–37. Bates, L.S., Waldeen, R.P., Teare, I.D., 1973. Rapid determination of free proline for water stress studies. Plant Soil 39, 205–207. Beauchamp, C.O., Fridovich, I., 1971. Superoxide dismutase: improved assays and assay applicable to acrylamide gels. Ann. Biochem. 44, 276–287. Benarides, M.P., Gallego, S.M., Tamaro, M.L., 2005. Cadmium toxicity in plants. Braz. J. Plant Physiol. 17, 21–34. Campbell, H.W., 1999. Nitrate reductase structure, function and regulation. Bridging the gap between biochemistry and physiology. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 277–303. Chance, B., Maehly, A.C., 1955. Assay of catalase and peroxidases. Meth. Enzymol. 2, 764–775. Chugh, L.K., Gupta, V.K., Sawhney, S.K., 1992. Effect of cadmium on enzymes of nitrogen metabolism in pea seedlings. Phytochemistry 31, 395–400. Clouse, S.D., Sasse, J.M., 1998. Brassinosteroids: Essential regulators of plant growth and development. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 427–451. Cobett, C.S., 2000. Phytochelatins and their roles in heavy metal detoxification. Plant Physiol. 123, 825–832. Dat, J.F., Van Breusegem, F., Vondenabeele, S., Vranova, E., Van Montagu, M., Inze, D., 2000. Dual action of active oxygen species during plant stress responses. Cell Mol. Life Sci. 57, 779–795.
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Wilen, R.W., Sacco, M., Lawrene, V.G., Krishna, P., 1995. Effects of 24epibrassinolide on freezing and thermo tolerance of brome grass (Bromus inermis) cell cultures. Physiol. Plant 95, 195–202. Yih, R.Y., Clark, H.E., 1965. Carbohydrate and protein content of boron deficient tomato root tips in relation to anatomy and growth. Plant Physiol. 40, 312–315. Yu, J.Q., Huag, L.F., Hu, W.H., Zhou, Y.H., Mao, W.H., Ye, S.F., Nogues, S., 2004. A role of brassinosteroids in the regulation of photosynthesis in Cucumis sativus. J. Exp. Bot. 55, 1135–1143.
Brassinosteroid enhanced the level of antioxidants under cadmium stress in Brassica juncea S. Hayat ∗ , B. Ali, S. Aiman Hasan, A. Ahmad Department of Botany, Aligarh Muslim University, Aligarh 202002, India Received 22 October 2005; received in revised form 26 January 2006; accepted 14 June 2006
Abstract The change in plant growth, photosynthesis, carbonic anhydrase, nitrate reductase and antioxidative enzymes resulting from the feeding of cadmium and/or 28-homobrassinolide (HBL) to Brassica juncea were studied in 60-day-old plants. One-week old seedlings were supplied with 50, 100 or 150 M of cadmium along with the nutrient solution. Subsequent seedlings, at day 30, were sprayed with 0.01 M of HBL to their foliage. The plants fed with cadmium alone exhibited a decline in growth, the levels of carbonic anhydrase (E.C. 4.2.1.1) and chlorophyll pigments and net photosynthetic rate. Moreover, nitrate content, the activity of nitrate reductase (E.C. 1.6.6.1) and the level of carbohydrate, both in the leaves and roots, decreased as the concentration of cadmium, in nutrient solution, increased. Compared with the leaves, roots possessed a larger quantity of nitrate. However, the trend was reversed in case of nitrate reductase and the level of carbohydrates in the two plant organs. The toxic effect, generated by cadmium was overcome if the stressed plants were sprayed with HBL. The activities of antioxidative enzymes [viz. catalase (E.C. 1.11.1.6), peroxidase (E.C. 1.11.1.7) and superoxide dismutase (E.C. 1.15.1.1)] and the contents of proline increased, over the control, irrespective of the treatment. Their level increased further, if the plants supplied with cadmium were also supplemented with HBL, both in the roots and the aerial parts. Nevertheless, the contents of proline in roots were higher than the leaves. © 2006 Elsevier B.V. All rights reserved. Keywords: Brassinosteroid; Carbonic anhydrase; Catalase; Peroxidase; Photosynthesis; Superoxide dismutase
1. Introduction The brassinosteroids occur in all plant parts, including roots (Bajguz and Tretyn, 2003). These chemicals elicit a wide range of physiological responses in plants, including stem elongation, pollen tube growth, leaf bending and epinasty, root growth inhibition, induced synthesis of ethylene, activation of proton pump, xylem differentiation, synthesis of nucleic acids and proteins, activation of enzymes and photosynthesis (Clouse and Sasse, 1998; Khripach et al., 2003; Hayat and Ahmad, 2003; Sasse, 2003; Yu et al., 2004). It is proposed that the changes induced by BRs are mediated through the repression and/or depression of specific genes (Felner, 2003). Besides this, BRs are also recognized to have an ameliorative role in plants, subjected to various biotic and abiotic stresses (Clouse and Sasse, 1998). The treatAbbreviations: BR, brassinosteroid; CA, carbonic anhydrase; HBL, 28homobrassinolide; NR, nitrate reductase; PN , net photosynthetic rate; RWC, relative water content; SOD, superoxide dismutase ∗ Corresponding author. Tel.: +91 9412328593. E-mail address: [email protected] (S. Hayat). 0098-8472/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2006.06.002
ment of the plants of rice and tomato (Kamuro and Takatsuto, 1991), maize (He et al., 1991), cucumber (Katsumi, 1991) and brome grass (Wilen et al., 1995), with BRs improved their capacity of resistance to low temperature. Similarly, BRs increased the degree of tolerance, to high temperature, in wheat (Kulaeva et al., 1991) and brome grass (Wilen et al., 1995). Brassinosteroids also countered the drought stress in sugarbeet (Schilling et al., 1991), moisture stress in wheat (Sairam, 1994) and favoured seed germination and seedling growth in Eucalyptus (Sasse et al., 1995) and rice (Anuradha and Rao, 2001), under saline conditions. Moreover, BRs activate antioxidative enzymatic defense system in rice seedlings, grown under salt stress (Nunez et al., 2003). Cadmium is extremely toxic to plants that retards biosynthesis of chlorophyll (Singh and Tewari, 2003), alters water balance (Barcelo and Poschenrieder, 1990), decreases activity of various enzymes (Siedlecka et al., 1997; Gouia et al., 2003), favours stomatal closure (Poschenrieder et al., 1989) and finally slows down the rate of photosynthesis (Sheoran et al., 1990; Chugh et al., 1992). Cadmium stress also reduces the uptake of essential mineral nutrients (Harnandez et al., 1996), decreases normal
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H+ /K+ exchange and the activity of ATPase, at the level of cellular plasma membrane (Obata et al., 1996). This experiment was planned with an aim to relate a change in the level of antioxidative enzymes, in cadmium fed plants of Brassica juncea, with the induced resistance and to neutralize the effects of stress by the application of 28-homobrassinolide. In this study, HBL was used because it is more active and stable in field trials than some other brassinosteroids (Khripach et al., 2000). We tested the hypothesis that the application of HBL will ameliorate the toxic effect of cadmium on the growth of B. juncea plants. 2. Materials and methods 2.1. Plant material and growth conditions Authentic, healthy seeds of B. juncea Czern & Coss cv. T-59, were surface sterilized with 0.5% (v/v) sodium hypochlorite and were sown in sand, moistened with deionized water in plastic pots of 6 in. diameter. The sand, before being used, was washed five times with deionized water. The pots were placed in net house, under natural conditions. The plants from day 5 were supplied with 100 ml of nutrient solution daily in the morning and an equal volume of deionized water, in the evening upto day 20. Thereafter the volume of the solution was increased to 500 ml. The composition of nutrient solution was 3 mmol KNO3 , 0.5 mmol Ca(NO3 )2 , 0.5 mmol MgSO4 , 2.5 mmol KH2 PO4 , 2 mmol NH4 Cl, 100 mol Fe–K–EDTA, 30 mol H3 BO3 , 5 mol MnSO4 , 1 mol CuSO4 , 1 mol ZnSO4 and 1 mol (NH4 )6 MO7 O24 per liter. The subsequent, 1week old, seedlings were supplied with 0, 50, 100 or 150 M of cadmium in the form of CdCl2 , with nutrient solution. The higher concentrations (200 and 250 M) proved lethal for growth of the plants. The plants were allowed to grow and at day 30, the foliage was applied with 0 or 0.01 M of HBL. The concentration of HBL was based on our earlier studies (Hayat et al., 2000, 2001; Fariduddin et al., 2004). Sixty-day-old plants were sampled with intact roots to assess the following parameters. The experiment was conducted according to simple randomized block design. The whole experiment was conducted twice. The data of both the years were pooled. The analysis of variance (ANOVA) was performed as described by Gomez and Gomez (1984) and the S.E. was calculated. 2.2. Fresh and dry mass per plant Plants were removed along with the sand and dipped in a bucket, filled with tap water, to remove the adhering sand particles, ensuring the safety of the roots. The plants were blotted and weighed to record their fresh mass and then placed in an oven, run at 80 ◦ C, for 24 h. The samples were weighed again after allowing them to cool to room temperature, in a desicator, to record their dry mass.
The values were placed in the following formula, proposed by Arnon (1949), to compute chlorophyll content [(A645 × 28.2) + (A663 × 8.3)] × [(V/1000) × W]. The net photosynthetic rate (PN ) in intact leaves was measured by LI-6200 portable photosynthetic system (LI-COR Licoln, NE, USA) at light saturating intensity between 11:00 and 12:00 h. The atmospheric conditions during the experiments were: PAR, 1065 ± 30 mol m−2 s−1 ; Ci, 280 ± 12 mol mol−1 ; atmospheric CO2 , 355 ± 5 mol mol−1 ; relative humidity, 65 ± 5%; atmospheric temp., 22 ± 2 ◦ C. 2.4. Relative water content (RWC) The RWC was determined in fresh leaf discs of 2 cm diameter, excluding midrib. Discs were weighed quickly and immediately floated on double distilled water, in petridishes to saturate them with water for the next 24 h, in dark. The adhering water of the discs was blotted and turgor weight was taken. Dry mass of these discs was obtained, after dehydrating them at 70 ◦ C for 48 h. Relative water content was calculated by placing the observed values in the following formula (Jones and Turner, 1978): RWC =
fresh mass − dry mass × 100. turgor mass − dry mass
2.5. Estimation of carbonic anhydrase (CA) activity The activity of CA was determined following the procedure described by Dwivedi and Randhawa (1974). The leaf samples were cut into small pieces and suspended in cystein hydrochloride. These samples were incubated at 4 ◦ C for 20 min. The pieces were blotted and transferred to the test tube, containing phosphate buffer (pH 6.8) followed by the addition of alkaline bicarbonate solution and bromothymol blue, indicator. The test tube was incubated at 5 ◦ C for 20 min. The reaction mixture was titrated against 0.05N HCl, after the addition of few drops of methyl red, indicator. The results were expressed as mol (CO2 ) kg−1 (leaf fresh mass)s−1 . 2.6. Estimation of nitrate reductase (NR) activity The activity of NR was measured following the method adopted by Jaworski (1971). The fresh leaf samples were cut into small pieces and transferred to plastic vials, containing phosphate buffer (pH 7.5) followed by the addition of potassium nitrate and isopropanol solutions. The reaction mixture was incubated at 30 ◦ C, for 2 h followed with the addition of N-1naphthylethylenediamine dihydrochloride and sulphanilamide. The absorbance of the colour was read at 540 nm and was compared with that of the calibration curve. The activity of NR (nmol NO2 g−1 h−1 ) was computed on fresh mass basis. 2.7. Assay of antioxidative enzymes
2.3. Chlorophyll content and net photosynthetic rate Leaf chlorophyll was extracted in 80% acetone and the absorbance was read spectrophotometrically at 663 and 645 nm.
Leaf tissue (0.5 g) was homogenized in 5 ml of 50 mmol phosphate buffer (pH 7.0) containing 1% insoluble polyvenylpyrolidone. The homogenate was centrifuged at 15,000 rpm for
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10 min and the supernatant was used as the source of enzyme. This extraction was carried out at 4 ◦ C. Peroxidase and catalase were assayed following the procedure described by Chance and Maehly (1955). Catalase was estimated by tirating the reaction mixture, consisting of phosphate buffer (pH 6.8), 0.1 M H2 O2 , enzyme extract and 2% H2 SO4 , against 0.1N potassium permanganate. The reaction mixture for peroxidase consisted of pyrogallol phosphate buffer (pH 6.8), 1% H2 O2 and enzyme extract. Change in absorbance, due to catalytic conversion of pyrogallol to perpurogallin, was noted at an interval of 20 s for 2 min at 420 nm. A control set was prepared by using distilled water instead of enzyme extract. The activity of superoxide dismutase was assayed by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) using the method of Beauchamp and Fridovich (1971). The reaction mixture contained 50 mmol phosphate buffer (pH 7.8), 13 mmol methionine, 74 mol NBT, 2 mol riboflavin, 0.1 mmol EDTA ad 0–50 l enzyme extract, and was placed under 15 W fluorescent lamp. The reaction was started by switching on the light and was allowed to run for 10 min. The reaction was stopped by switching off the light. Fifty per cent inhibition by light was considered as one enzyme unit. 2.8. Estimation of nitrate and carbohydrate content The nitrate content in the leaves and roots was determined by the method described by Singh (1988). 100 mg of dried tissue powder was digested with 5 ml of 2% acetic acid for 20 min and was filter through Whatman filter paper. The extract was diluted to 10 ml, to which 500 mg of powder mixture, consisting of citric acid, manganese sulphate monohydrate, sulphanilamide, N-1naphthylethylenediamine dihydrochloride and powdered zinc, was added. The reaction mixture was centrifuged and the colour of the supernatant was read at 540 nm. Nitrate content was calculated by comparing the value of the sample with the calibration curve, drawn by using standard nitrate solution. Total carbohydrate was extracted following the method of Yih and Clark (1965) and estimated by adopting the procedure of Dubois et al. (1956). Dried (leaf or root) powder was transferred to glass centrifuge tube containing 1.5N H2 SO4 . The sample was centrifuged at 4000 rpm, for 10 min. One millilitre of the extract was taken in a test tube to which 1 ml of 5% distilled phenol was added. The absorbance was read at 490 nm, using spectrophotometer and compared with the calibration curve, using pure glucose. 2.9. Determination of proline content The proline content in fresh leaf and root samples was determined by adopting the method of Bates et al. (1973). Sample was extracted in sulphosalicylic acid. To the extract, an equal volume of glacial acetic acid and ninhydrine solutions were added. The sample was heated at 100 ◦ C, to which 5 ml of toluene was added. The absorbance of toluene layer was read at 528 nm, on a spectrophotometer.
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3. Results 3.1. Growth and relative water content The growth (as expressed by fresh and dry mass) of the shoot and the root and their relative water contents, decreased in the plants supplemented with cadmium and its rate of loss was proportional to the concentration of the metal (Fig. 1a and b). However, the treatment with HBL to the foliage favoured growth and also partially overcame the toxic effects generated by cadmium. The response of the plants to 50 mol of cadmium was completely overcome by the application of HBL, the values are, therefore, at par with the control. 3.2. Chlorophyll pigment and net photosynthetic rate It is evident from Fig. 1c and d that both chlorophyll contents and net photosynthetic rate decreased, if the plants were supplied with cadmium in the nutrient medium. Maximum damage was noticed in the plants receiving highest concentration (150 mol) of the metal. The effect was to some extent neutralized if the plants, at 30 days stage, were given a followup treatment with 0.01 M of HBL. This treatment generated values comparable with the control in those plants that were already exposed to cadmium (50 mol) stress. 3.3. CA activity The supply of cadmium to the seedlings significantly decreased the activity of the enzyme (Fig. 2a). The loss was more visible as the concentration of the metal was raised from 50 to 150 mol. However, the foliage of the plants sprayed with 0.01 M of HBL alone possessed significantly higher level of CA activity. Moreover, HBL nullified the effects of the metal, to a limited extent, in the plants fed with higher concentrations of cadmium but completely, if given 50 mol of the metal with a follow up treatment of the hormone. 3.4. Nitrate content and NR activity The leaves possessed lesser quantity of the nitrate and higher activity of NR than the roots. However, the pattern of response to the treatment by both these plant organ was very much similar (Fig. 2b and c). A sharp decrease was observed as the concentration of cadmium in the medium was enhanced from 50 to 150 mol. Application of HBL not only increased their values but also completely overcame the toxic effects of the metal (50 mol) and to a limited extent at its higher concentrations (100/150 mol). 3.5. Proline content Proline level was higher in the roots than the leaves (Fig. 2d). The concentration increased in both the major organs of the plants, if supplied with cadmium and/or HBL. The values
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Fig. 1. Effect of HBL on the changes in fresh and dry mass (a), relative water content (RWC) (b), total chlorophyll content (c) and net photosynthetic rate (PN ) (d) in 60-d-old plants of Brassica juncea induced by cadmium.
are much higher if the metal concentration was increased to 150 mol and that too supplied with HBL. 3.6. Activity of antioxidative enzymes The data depicted in Fig. 3a–c clearly revealed significant increase in the activity of antioxidative enzymes (viz. catalase, peroxidase and superoxide dismutase) in response to cadmium and/or HBL. Interestingly, the plants fed with highest concentration of the metal (150 mol) in association with its foliage supplied with HBL exhibited highest levels of all the enzymes whose values were 34%, 94% and 90% more than the control for catalase, peroxidase and superoxide dismutase, respectively. The control plants possessed lowest concentration of all the enzymes.
3.7. Carbohydrate content It was natural that the leaves had more carbohydrate than the roots (Fig. 3d) but decreased significantly under the impact of the cadmium. This loss was highly significant under the highest concentration of the metal (150 mol). The carbohydrate content in the plants, under stress was revived if supplemented with HBL, to their foliage. The best interaction of hormone was noticed with 50 mol of the metal where the values were comparable with those of the control. 4. Discussion The reversible interconversion of CO2 and HCO3 − is catalysed by the enzyme, carbonic anhydrase (CA), whose activity
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Fig. 2. Effect of HBL on the changes in carbonic anhydrase (CA) activity (a), nitrate content (b), nitrate reductase (NR) activity (c) and proline content (d) in 60-d-old plants of B. juncea induced by cadmium.
is largely determined by photon flux density, concentration of CO2 , the availability of Zn (Tiwari et al., 2005) and the genetic expression (Kim et al., 1994). The stress generated by cadmium decreases the partial pressure of CO2 in the stroma by inducing the stomatal closure (Barcelo and Poschenrieder, 1990) resulting in a loss in the activity of CA (Fig. 2a). Siedlecka et al. (1997) also reported low CA activity in Phaseolus vulgaris, exposed to Cd stress. However, the involvement of BRs in regulating transcription and/or translation (Kalinich et al., 1985) possibly favoured synthesis and/or activation of the enzyme protein (Fig. 2a) that has also been reported earlier in mustard (Hayat et al., 2001) and mung bean (Fariduddin et al., 2004). The plants exposed to cadmium stress exhibit an inhibition in the activity of protochlorophyllide reductase (Stabort et al., 1985) by possibly inactivating the sulphydryl site of the reductase protein (Ernst, 1980). This in addition to an increase in
chlorophyllase, the enzyme that activates the degradation of the chlorophyll (Reddy and Vora, 1986) could have contributed in lowering the total chlorophyll content (Fig. 1c). Similar observations have also been reported by Vassileve et al. (1998) in barley and Gadallah (1995) in sunflower grown under Cd stress. However, the application of HBL increased the chlorophyll content in the plants grown with/without the metal (Fig. 1c) by involving the expression of specific genes responsible for the synthesis of the enzymes determining chlorophyll generation. The observed decrease in the net photosynthetic rate under the impact of cadmium (Fig. 1d) may be a direct outcome of the response generated by metal through its ability to bind with specific proteins, to inactive them (Van Assche and Cligsters, 1990), in addition to the reasons (i.e. low CO2 and a decrease in photosynthetic pigments and the activity of carbonic anhydrase), mentioned earlier. Moreover, cadmium has an adverse
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Fig. 3. Effect of HBL on the changes in catalase (a), peroxidase (b), superoxide dismutase (c) and carbohydrate content (d) in 60-d-old plants of B. juncea induced by cadmium.
effect on the activity of Rubisco (Siedlecka et al., 1997) which will naturally have a negative impact on the CO2 reduction. In contrary to above, BR activate the rate of photosynthesis (Fig. 1d) by enhancing the level of the enzymes (Fariduddin et al., 2004; Yu et al., 2004) directly or indirectly involved in the process and thus also overcomes the ill effects generated by cadmium. The metal, in a natural course, exerts a damaging effect on the normal functioning of the plasma membrane by having an impact on the activity of inbuilt ATPase (Obata et al., 1996) and the fluidity of the membrane (Meharg, 1994). This could have checked the uptake and distribution of nitrate, leading to its decrease both in the roots and the shoot of the plants fed with cadmium (Harnandez et al., 1996; Fig. 2b). The availability of nitrate, in addition to the level and state of active sites of functional NR and overall metabolic conditions of the plants determines the status of the activity of nitrate reductase (Campbell, 1999).
The activity of the enzyme, therefore, decreased (Fig. 2c). However, BRs somehow activate the uptake of nitrate (Mai et al., 1989) and also favour transcription and/or translation (Kalinich et al., 1985; Bajguz, 2000). This could have been reason for the observed increase in the activity of NR (Fig. 2c) and also neutralized the impact of the metal stress because of the ameliorative characters of BRs (Hamada, 1986; Sasse, 2003). In a natural course, reactive oxygen species (ROS) includ• ing superoxide radical (O2 − ) hydrogen peroxide (H2 O2 ) and • − hydroxy radical (HO ) are produced in the plants under stress (Asada, 1999; Dat et al., 2000). These ROS, if generated in larger quantities, may oxidize proteins, lipids and nucleic acids leading to even mutation at the cellular level (Halliwell and Gutteridge, 1999). However, to neutralize the toxicity of ROS plants have endogenous system of enzymes (e.g. catalase, peroxidases, superoxide dismutase, glutathione reductase) and metabolites (e.g. ascorbate, glutathione, tocopherols and proline) to operate,
S. Hayat et al. / Environmental and Experimental Botany 60 (2007) 33–41
if exposed to stress (Asada, 1999; Mittler, 2002) and also the fixation of the metals to phytochelatins (Benarides et al., 2005). Out of them a detailed account has been studied about the production of the stress proteins (proline) whose synthesis and degradation is a very guarded phenomenon, regulated by a set of enzymes. Sumithra and Reddy (2004) reported an elevation in the activity of two of the enzymes ( -pyroline-5-carboxylate synthetase and -pyroline-5-carboxylate reductase) responsible for proline synthesis and a decrease of that of proline dehydrogenase, the enzyme that degrades proline in cowpea, under stress. Moreover, the genes responsible for the transcription of these proteins were also over expressed in some transgenic plants, under stress (Kishore et al., 1995). The phytochelatins are small metal binding peptides (Grill et al., 1986a,b) that are synthesized in plants from tripeptide glutathione substrate, mediated by the enzyme phytochelatin synthase (E.C. 2.3.2.15), a constitutive enzyme requiring the presence of either of the heavy metals (Cobett, 2000). Biosynthesis of the phytochelatins continues by the time all the metal ions are chelated or by the addition of metal chelators such as EDTA (Loeffler et al., 1989). The physiological drought generated by cadmium, in the present study, resulted in the increase in the level of proline (Fig. 2d) and that of catalase, peroxidase and superoxide dismutase (Figs. 3a–c) to boost the resistance capacity of the plants to overcome the effects generated by the metal. The level of all these enzymes and that of proline is increased by the action of HBL (Figs. 2d and 3a–c). Nunez et al. (2003) also noted higher activity of antioxidative enzymes in rice, grown under salinity and supplemented with BRs. It gives an impression that BRs some how generate a system (transcription and/or translation) comparable with that of the stress that improves the degree of resistance in the plants to abnormal metabolism. Therefore, an amalgamated action of the stress and that of the HBL in inducing the activity of the enzymes and the synthesis of proline, is much higher than either of them applied alone. A loss in relative water content of the plants (Fig. 1b), supplemented with cadmium, is possibly an impact of the metal on the electrical potential of the plasma membrane that affected not only the absorption of ions (Harnandez et al., 1996) but also that of water, generating water stress (Barcelo and Poschenrieder, 1990). Therefore, physiological disorders are further expressed in the form of closure of stomata (Barcelo and Poschenrieder, 1990), slowing down of the photosynthesis (Prasad, 1995, Fig. 1d) associated with a shift in the proportion of various enzymes (Fig. 2a and c). This altered metabolism, under the impact of cadmium, resulted in the relocation of plant growth where not only the linear growth but also the fresh and dry mass of the plant was lost to a significant level (Fig. 1a). Deviating from the response generated by metal, HBL had a favourable impact on the RWC and fresh and dry mass of the plants, even if given as a followup treatment with cadmium (Fig. 1a and b). BRs generate such a response because of their involvement in the activation of ATPase pump (Khripach et al., 2003), synthesis of nucleic acids and proteins (Bajguz, 2000), repression/depression of genes (Felner, 2003), change in enzyme pattern (Figs. 2a and c and 3a–c) and speeding up of photosynthesis (Fig. 1d). These effects generated by HBL also neutralized
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the impact of cadmium to all of its concentrations but completely at 50 mol. 5. Conclusion The data generated in the present study gives a clear-cut impression that the presence of cadmium at a level that may cause stress in the plant adversely affects its metabolism and growth. Moreover, the metal also activates the system to improve the resistance capacity of the plants to the stress. HBL, on the other hand, has an added effect both on the general metabolism and the resistance capacity of the plants to overcome the toxic effect of the metal. Acknowledgements S. Hayat acknowledges Department of Science & Technology, Govt. of India, New Delhi for financial support (SR/FTP/LS-A-37/2002). We thank anonymous reviewers who made critical evaluation of this manuscript and suggested the incorporation of valuable points. We are also thankful to Dr. B.N. Vyas, General Manager, Godrej Agrovet Ltd., Mumbai, India for the generous gift of 28-homobrassinolide. References Anuradha, S., Rao, S.S.R., 2001. Effect of brassinosteroids on the salinity stress induced inhibition of germination and seedling growth of rice (Oryza sativa). Plant Growth Regul. 33, 151–153. Arnon, D.I., 1949. Copper enzyme in isolated chloroplast polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1–15. Asada, K., 1999. The water-water cycle in chloroplasts: scavenging of active oxygen and dissipation of excess photons. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 601–639. Bajguz, A., 2000. Effect of brassinosteroids on nucleic acid and protein content in cultured cells of Chlorella vulgaris. Plant Physiol. Biochem. 38, 209–215. Bajguz, A., Tretyn, A., 2003. The chemical structures and occurrence of brassinosteroids in plants. In: Hayat, S., Ahmad, A. (Eds.), Brassinosteroids: Bioactivity and Crop Productivity. Kluwer Academic Publishers, Dordrecht, pp. 1–44. Barcelo, J., Poschenrieder, C., 1990. Plant water relations as affected by heavy metal stress: a review. Plant Nutr. 13, 1–37. Bates, L.S., Waldeen, R.P., Teare, I.D., 1973. Rapid determination of free proline for water stress studies. Plant Soil 39, 205–207. Beauchamp, C.O., Fridovich, I., 1971. Superoxide dismutase: improved assays and assay applicable to acrylamide gels. Ann. Biochem. 44, 276–287. Benarides, M.P., Gallego, S.M., Tamaro, M.L., 2005. Cadmium toxicity in plants. Braz. J. Plant Physiol. 17, 21–34. Campbell, H.W., 1999. Nitrate reductase structure, function and regulation. Bridging the gap between biochemistry and physiology. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 277–303. Chance, B., Maehly, A.C., 1955. Assay of catalase and peroxidases. Meth. Enzymol. 2, 764–775. Chugh, L.K., Gupta, V.K., Sawhney, S.K., 1992. Effect of cadmium on enzymes of nitrogen metabolism in pea seedlings. Phytochemistry 31, 395–400. Clouse, S.D., Sasse, J.M., 1998. Brassinosteroids: Essential regulators of plant growth and development. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 427–451. Cobett, C.S., 2000. Phytochelatins and their roles in heavy metal detoxification. Plant Physiol. 123, 825–832. Dat, J.F., Van Breusegem, F., Vondenabeele, S., Vranova, E., Van Montagu, M., Inze, D., 2000. Dual action of active oxygen species during plant stress responses. Cell Mol. Life Sci. 57, 779–795.
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