Magnesium deficiency induced oxidative stress and antioxidant responses in mulberry plants

Scientia Horticulturae 108 (2006) 7–14 www.elsevier.com/locate/scihorti Magnesium deficiency induced oxidative stress and antioxidant responses in mulberry plants Rajesh Kumar Tewari, Praveen Kumar, Parma Nand Sharma * Department of Botany, University of Lucknow, Lucknow 226007, Uttar Pradesh, India Received 17 December 2004; received in revised form 24 November 2005; accepted 9 December 2005 Abstract The aim of the study was to implicate induction of oxidative stress and antioxidative responses with the effects of Mg deficiency in mulberry plants. Mulberry (Morus alba L.) cv. Kanva-2 plants grown in hydroponics were subjected to deficiency of Mg. Mg-deficient plants developed visible symptoms—deep interveinal chlorotic mottling and necrosis in the older and middle leaves. The decreases in the dry matter yield of plants and concentrations of sugars and starch in the leaves of Mg-deficient plants are suggestive of decreased photosynthetic activity. Mg-deficiency decreased concentrations of photosynthetic pigments, and increased concentrations of H2O2 and ascorbate and activities of antioxidative enzymes—peroxidase (POD), ascorbate peroxidase (APX) and superoxide dismutase (SOD). The results suggest induction of oxidative stress by enhancing generation of ROS and inducing alterations in redox status, accompanied by activation of antioxidant machinery including induction of some new SOD isoforms in Mg-deficient mulberry plants. Despite significant increase in H2O2, lipid peroxidation was decreased in Mg-deficient plants. # 2005 Elsevier B.V. All rights reserved. Keywords: ROS; SOD; Morus alba; Mg-deficiency stress; Lipid peroxidation; Antioxidants 1. Introduction Apart from being a structural constituent of chlorophyll molecule, Mg also acts as activator or regulator of several kinases, ATPases, RUBP carboxylase/oxygenase and several other enzymes of carbohydrate metabolism (Marschner, 1995). Thus, deficiency of Mg is likely to decrease the efficiency of Calvin cycle, reduce the utilization of reductive power (NADPH) and cause over-saturation of photosynthetic electron transport system. Under such highly reduced condition, electrons could pass-on to O2 generating O2 and other reactive oxygen species (ROS). Plants are equipped with Abbreviations: APX, ascorbate peroxidase; AsA, ascorbic acid; ASC, total ascorbate; CAT, catalase; DHA, dehydroascorbate; DTT, dithiothreitol; EDTA, ethylenediamine tetraacetic acid; MDA, malondialdehyde; POD, peroxidase; ROS, reactive oxygen species; SOD, superoxide dismutase; TBA, thiobarbituric acid; TBARS thiobarbituric acid reactive substance; TCA, trichloroacetic acid * Corresponding author. Tel.: +91 522 2761506. E-mail addresses: rajesh_bot@rediffmail.com (R. Kumar Tewari), praveen_botany@yahoo.co.in (P. Kumar), sharmapn@sify.com (P. Nand Sharma). 0304-4238/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2005.12.006 effective antioxidant defence system comprising of antioxidant enzymes [superoxide dismutase (SOD), peroxidases (POD), ascorbate peroxidase (APX) and other enzymes of ascorbate– glutathione pathway], and molecular antioxidants [ascorbate, a-tocopherol, carotenoids and glutathione] (Noctor and Foyer, 1998; Foyer and Noctor, 2003; Apel and Hirt, 2004). However, severe or prolonged stress conditions can disturb intricate balance between ROS generation and its scavenging machinery, which initiates signaling responses, enzyme activation, gene expression, cellular damage and programmed cell death (PCD) (Neill et al., 2002; Mahalingam and Fedoroff, 2003). Mg-deficient leaves are reported to be highly photosensitive and an increase in light intensity causes severe chlorosis and photooxidation of thylakoid constituents of these leaves by generation of ROS (Cakmak and Marschner, 1992). Enhanced activities of antioxidative enzymes and concentrations of antioxidant molecules have been reported earlier in Mgdeficient bean (Cakmak and Marschner, 1992; Cakmak, 1994), Mentha (Candan and Tarhan, 2003) and maize (Tewari et al., 2004) plants. Moreover, Mg-deficient Mentha (Candan and Tarhan, 2003) and maize (Tewari et al., 2004) plants have also been shown to accumulate significantly higher amount of malondialdehyde (MDA, an index of lipid peroxidation). Polle 8 R. Kumar Tewari et al. / Scientia Horticulturae 108 (2006) 7–14 et al. (1994), on the other hand, did not find any effect of Mg deficiency on the concentrations of ascorbate or glutathione and activities of APX, monodehydroascorbate reductase, GR, POD or NADH oxidase in Norway Spruce, a gymnospermous tree species, grown with nitrate as the predominant source of nitrogen. These workers, however, observed increase in SOD activity in this Mg-deficient plants and suggested that loss of pigments was correlated with SOD activity in these plants. Although these studies suggest that Mg deficiency induces oxidative stress, information regarding responses of mulberry plants to Mg deficiency could not be found in literature. The study presented here is an attempt to relate the effects of Mg deficiency with induction of oxidative stress and antioxidative responses in mulberry plants under hydroponic culture conditions. 2. Materials and methods 2.1. Plant material and culture conditions Mulberry (Morus alba L. cv. Kanva-2) plants were grown in hydroponics culture under glasshouse conditions: maximum light ranged from 1175–1355 mmol m 2 s 1 at 12:00 noon, maximum and minimum temperature during this period ranged 34.3–40.5 8C and 24.4–30.6 8C, respectively. Relative humidity in the glasshouse in this period ranged from 70–80%. The plantlets initially were raised from cuttings obtained from a single plant in acid-washed sand-bed and were supplied glassdistilled water. After 30 days, when roots in the cuttings were sufficiently induced, plantlets were transplanted to 20 L plastic buckets containing aerated nutrient solution having composition (Hewitt, 1966): 2.0 mM KNO3, 2.0 mM Ca(NO3)2, 2.0 mM MgSO4, 0.67 mM NaH2PO4, 0.05 mM NaCl, 0.05 mM Fe–K2EDTA, 5.0 mM MnSO4, 0.5 mM CuSO4, 0.5 mM ZnSO4, 16.5 mM H3BO3, 0.1 mM Na2MoO4, 0.05 mM CoSO4 and 0.05 mM NiSO4. The pH of the nutrient solution was maintained at 6.7  0.2. Seven days after transplantation (DAT), pots were grouped into two lots having three pots each. Whereas plants in lot 1 continued to receive complete nutrient solution (control), those in lot 2 were supplied nutrient solution deficient in Mg. Deficiency of Mg was created by withholding the supply of MgSO4 and replacing it with equivalent moles of Na2SO4 in order to maintain the supply of sulphur and the osmoticum of the nutrient solution. The volume of nutrient solution was made-up daily by deionised water. The nutrient solution was refreshed every alternate day. After 27 days of deficient supply of Mg (34 DAT) when the deficiency symptoms become fairly perceptible, the fully expanded young (third leaf from apex) or old (seventh leaf from apex; wherever it applicable) leaves of plants were sampled for metabolic studies. The plants were harvested 50 DAT. At the harvest time, the plants were separated into young leaves, old leaves, stem and roots, washed in running glass distilled water, blotted, dried in a forced draught oven for 48 h and the material used for determinations of dry matter yield and concentrations of carbohydrates and mineral nutrients. The data given here are the mean of three experimental replicates. 2.2. Sugars and starch Young leaves harvested for sugar analysis were immediately dried in a microwave oven. Sugars were determined in an aliquot from the water-extract and starch in the water insoluble material of leaf tissue. The aliquot for sugar determination was treated with saturated lead acetate solution and centrifuged at 5000  g for 5 min to remove the pigments. The residual lead in the supernatant was removed by precipitating with the saturated solution of sodium phosphate and again centrifuged at 5000  g for 5 min. The supernatant was made to a volume and sugars were estimated after Nelson (1944). Total sugars were estimated as reducing sugars after hydrolysis of non-reducing sugars by invertase. Non-reducing sugars were found out by subtracting reducing sugar from total sugars. Starch was estimated in water insoluble fraction after maceration and hydrolysis with 50% (v/v) HClO4. The supernatant obtained after centrifugation, was made to volume with distilled water. Starch was estimated in an aliquot from the solution by the method of Montgomery (1957). 2.3. Mineral nutrients Tissue concentrations of nutrient elements were measured in the solution after wet digestion (HNO3:HClO4, 10:1, v/v, mixture) of the oven dried plant material. Magnesium was estimated after Wallace (1951) and iron, manganese, copper and zinc were estimated atomic absorption spectrophotometrically. 2.4. Chlorophylls and carotenoids Concentrations of chlorophylls and carotenoids were determined in 80% (v/v) acetone extract of the young fully expanded third leaf using the method of Lichtenthaler (1987). The absorbance of cleared extract was measured at 663.2, 646.8 and 470 nm for chlorophyll a, chlorophyll b and total carotenoids, respectively. 2.5. Hydrogen peroxide and lipid peroxidation H2O2 concentration was determined as H2O2–titanium complex formed by reaction of tissue-H2O2 with titanium tetrachloride by the method of Brennan and Frenkel (1977). Lipid peroxidation was determined by method of Heath and Packer (1968) in terms of malondialdehyde (MDA) content by thiobarbituric acid (TBA) reaction. The amount of TBA reactive substance (TBARS) was calculated from the difference in absorbance at 532 and 600 nm using extinction coefficient of 155 mM 1 cm 1. 2.6. Ascorbate Fresh leaf tissue (250 mg) was homogenised in 2.0 mL of 10% (w/v) trichloroacetic acid (TCA) and centrifuged for 5 min at 10,000  g. Total ascorbate (ASC) [after reducing R. Kumar Tewari et al. / Scientia Horticulturae 108 (2006) 7–14 dehydroascorbate (DHA) to ascorbic acid (AsA) by dithiothreitol (DTT)] and AsA in the supernatant, were measured as Fe2+–bipyridyl complex (absorbance maximum at 525 nm) formed from the reduction of FeCl3 by AsA by the method of Law et al. (1983). DHA content was calculated from the difference between ASC and AsA. 2.7. Enzyme extraction and protein determination Fresh leaf tissue (2.5 g) was homogenized in 10.0 mL of chilled 50 mM potassium phosphate buffer (pH 7.0) containing 2.0% (w/v) insoluble polyvinylpolypyrrolidone 0.5% (v/v) triton X-100 and 1.0 mM phenylmethylsulfonylfluoride in chilled pestle and mortar kept in ice bath. The homogenate was filtered through two-fold muslin cloth and centrifuged at 20,000  g for 10 min at 2 8C. The supernatant was stored at 2 8C and used for enzyme assays within 4 h. For APX, 5.0 mM AsA and 1.0 mM DTT were also included in the extraction buffer. Protein concentration in the homogenate was determined in the TCA precipitate according to Lowry et al. (1951) using bovine serum albumin as standard. 2.8. Assays of enzymes The reaction mixtures for different enzymes were: CAT (EC 1.11.1.6), 500 mmoles H2O2 in 10 mL 100 mM phosphate buffer pH 7.0 and 0.1 mL tissue extract, H2O2 decomposed after 5 min reaction was assayed by titrating the reaction mixture with 0.5 M KMnO4 (Euler and Josephson, 1927, as modified by Bisht et al., 1989). CAT activity is expressed as mmol H2O2 decomposed mg 1 protein; POD (EC 1.11.1.7), 5 mL 100 mM phosphate buffer pH 6.5, 1 mL 0.5% p-phenylenediamine, 1 mL 0.01% H2O2 and 0.05 mL tissue extract, change in absorbance after 5 min was measured at 485 nm (Bisht et al., 1989, a modified method of Luck, 1963). The enzyme activity has been expressed as unit mg 1 protein. The enzyme unit is defined as DA485 of 0.01 between the blank and the sample min 1 of 9 reaction time; APX (EC 1.11.1.11), in 3 mL: 50 mM phosphate buffer pH 7.0, 0.5 mM AsA, 0.1 mM H2O2, 0.1 mM EDTA and 0.05 mL enzyme extract, both minus tissue-extract and minus H2O2 blanks were run and the changes in absorbance every 15 s were read at 290 nm (Nakano and Asada, 1981). The activity of APX was calculated in terms of mmol ASC oxidized min 1 mg 1 protein; SOD (EC 1.15.1.1), in 5 mL: 25 mM phosphate buffer pH 7.8, 65 mM p-nitroblue tetrazolium chloride (NBT), 2 mM riboflavin, 50 mL enzyme extract and 15 mL N,N,N0 ,N0 tetramethylethylenediamine (TEMED) and the reaction mixture was exposed to light of 350 mmol m 2 s 1 for 15 min. SOD activity was assayed by measuring its ability to inhibit the photochemical reduction of NBT. The change in absorbance was measured at 560 nm (modified from Beauchamp and Fridovich, 1971). The activity is expressed as unit min 1 mg 1 protein. Fresh matter equivalent of enzyme extract corresponding to 50% inhibition of reaction was considered as one enzyme unit. Native PAGE was carried out for separations of forms of SOD. Equal amount of protein in the extract was resolved on 10% native gel with a discontinuous buffer system and SOD was localized using photochemical method of Beauchamp and Fridovich (1971). The developed gels were scanned on a Bio-Rad Imaging Densitometer Model GS-690 and densitogram were recorded using Multi-Analyst software from Bio-Rad. The transmittance peaks through the achromatic zones of SOD activity in different lanes representing different treatments are provided along with an individual lane of the gel. In order to identify the different isoforms, either KCN (inhibitor of Cu/Zn-SOD) at 2 mM or H2O2 (inhibitor of Cu/Zn-SOD and Fe-SOD) at 5 mM concentration were used. 2.9. Statistical analysis The study was carried out thrice and the data were analysed by one-way analysis of variance (ANOVA) and tested for significance by Bonferroni t-test using Sigma-stat software. Fig. 1. Deficiency symptoms of Mg in mulberry (Morus alba L.) cv. Kanva-2 plants grown in solution culture; 45 DAT. (A) A twig showing interveinal chlorosis, (B) a leaf showing chlorosis and necrosis and (C) abaxial view leaf showing darkening near the veins. 10 R. Kumar Tewari et al. / Scientia Horticulturae 108 (2006) 7–14 3. Results 3.2. Sugars and starch 3.1. Visible effects and dry matter yield While the concentration of non-reducing sugars increased by 32% in young leaves of Mg-deficient plants, the concentrations of reducing sugars, and starch decreased by 70 and 34%, respectively (Table 1). The visible effects of Mg-deficiency initiated after 25 days of the treatment. The effect of Mg-deficiency, first seen in the old leaves, initiated in the interveinal areas particularly along the margins, and progressed towards the midribs and from the old towards the young leaves. The symptoms started as mild interveinal chlorotic mottling that intensified with time. The more severely affected parts along the middle of the laminae developed minute pinhead sized brown necrotic spots (Fig. 1A). These spots later enlarged in size and coalesced, and resulted in irregularly shaped brown necrotic and scorched areas. The leaves eventually dried and withered (Fig. 1B). Deficiency of Mg also decreased dry matter yield of shoot (81.6%) and roots (86.6%) with reference to respective controls. The increased shoot/root ratio of Mg deficient plants signifies that roots were affected more severely than the shoots (Table 1). 3.3. Mineral nutrients The concentration of Mg was decreased by 83% in the young leaves of Mg-deficient plants in comparison to respective controls (Table 1). The decrease in Mg concentration was more marked—93%, in the older leaves of Mg-deficient plants (Table 1). While the concentrations of Fe, Mn and Zn increased, Cu was not affected by Mg-deficiency (Table 1). 3.4. Chloroplastic pigments The decreases in the concentrations of chlorophyll and carotenoids in young leaves of Mg-deficient plants were significant—46 and 24%, respectively (Fig. 2A). Mg-deficiency, though did not affect chlorophyll a/b ratio (Fig. 2A), increased carotenoids/chlorophyll ratio by 41% (Fig. 2B). 3.5. Lipid peroxidation, hydrogen peroxide and ascorbate Though, MDA equivalent of TBARS was decreased by 66% in the Mg-deficient plants, Mg deficiency caused significant (235%) increase in the concentration of H2O2 in the young leaves of mulberry plants (Fig. 3A). There was significant (63%) increase in the non-enzymatic antioxidants ASC. The increase in the ASC in the leaves of Mg-deficient plants was Table 1 Effect of deficient supply of magnesium on dry matter yield, shoot/root ratio, concentrations of carbohydrates (reducing, non-reducing sugars and starch); Mg and micronutrients (Fe, Mn, Cu, Zn) in the leaves of mulberry (Morus alba L.) var. Kanva-2 plants grown in solution culture; 50 DAT Control Dry matter yield (g plant 1) Shoot Root Shoot/Root 78.6  4.90a 8.2  0.81a 9.7  0.37a 14.5  0.27b 1.1  0.01b 13.7  0.17b Carbohydrates in young leaves (% of dry weight) Reducing sugar 1.10  0.03a Non-reducing sugar 0.25  0.03a Starch 25.76  0.35a 0.33  0.03b 0.33  0.00b 16.98  0.15b Magnesium (mg g Young leaves Old leaves Fig. 2. Effect of Mg-deficiency on the concentrations of photosynthetic pigments in the young leaves of mulberry (Morus alba L.) cv. Kanva-2 plants grown in solution culture; 34 DAT: (A) chlorophylls, open bars; chlorophyll a/b ratio, shaded bars and (B) carotenoids, open bars; carotenoids/chlorophyll ratio, shaded bars. The vertical bars represent mean  S.E. of three experimental replicates. Bars with different letters are significantly different (P  0.05) from respective controls by Bonferroni t-test. Mg 1 dry weight) 1.8  0.0a 2.9  0.0a Micronutrients in young leaves (mg g 1 dry weight) Fe 141.2  2.8a Mn 44.6  2.1a Cu 5.41  0.35a Zn 27.2  0.2a 0.3  0.9b 0.2  0.1b 189.9  10.3b 132.9  3.9b 5.41  0.35a 34.7  1.0b Data are mean  S.E. of three experimental replicates. The values of same rows having different letters (a and b) are significantly different (P  0.05) from control by Bonferroni t-test. R. Kumar Tewari et al. / Scientia Horticulturae 108 (2006) 7–14 11 Fig. 4. Effect of Mg-deficiency on the activity of SOD, open bars; ratio of Cu– Zn SOD/Non Cu–Zn SOD, shaded bars in the young leaves of mulberry (Morus alba L.) cv. Kanva-2 plants grown in solution culture; 34 DAT. The vertical bars represent mean  S.E. of three experimental replicates. Bars with different letters are significantly different (P  0.05) from respective controls by Bonferroni t-test. Fig. 3. Effect of Mg-deficiency on the concentrations of (A) H2O2, open bars; TBARS, shaded bars; (B) ascorbate, open bars; and the ratio of DHA/AsA, shaded bars in the young leaves of mulberry (Morus alba L.) cv. Kanva-2 plants grown in solution culture; 34 DAT. The vertical bars represent mean  S.E. of three experimental replicates. Bars with different letters are significantly different (P  0.05) from respective controls by Bonferroni t-test. mainly attributable to a significant increase in the reduced form, AsA that resulted in a marked (45%) decrease in DHA/AsA ratio (Fig. 3B). The DHA content was not affected significantly. 3.6. Antioxidative enzymes and soluble protein Mg-deficiency increased SOD activity from 8.46 to 10.05 units mg 1 protein (Fig. 4). Also, the leaves of Mg- deficient plants showed increases in the numbers and intensities of achromatic bands on native PAGE. Four new isoforms numbered as 3, 4, 5 and 8 were observed in Mg-deficient plants (Fig. 5A and B). Moreover, the ratio of Cu–Zn-SOD to non-Cu– Zn-SOD increased significantly (>25%) in Mg deficient plants (Fig. 4). While a marked decrease from 1.58 to 0.87 mmol H2O2 mg 1 protein was observed in the CAT activity, the activities of other H2O2-scavenging enzymes (POD, APX) were increased by >88 and >35%, respectively, in the leaves of Mg-deficient plants (Fig. 6). The soluble protein concentration, however, was not affected by Mg deficiency (Fig. 6). 4. Discussion Plants grown with nil supply of Mg showed significant decreases in the tissue concentration of Mg in the leaves and in growth and dry matter production of mulberry plants. A more marked (93%) decrease in the older leaves compared to a relatively smaller (83%) decrease in the concentration of Mg in the younger leaves corroborates the relative severity of Mgdeficiency effects in the older leaves. Accelerated senescence observed in the older parts of Mg-deficient plants may be Fig. 5. Effect of Mg-deficiency on isoforms of SOD in the young leaves of mulberry (Morus alba L.) cv. Kanva-2 plants grown in solution culture; 34 DAT. Densitogram of SOD along with respective lanes of (A) control and (B) Mg-deficient plants and (C) native gels showing identification of SOD isoforms by inhibition treatments—2 mM KCN for Cu–Zn-SOD and 5 mM H2O2 for Cu–Zn-SOD and Fe-SOD. 12 R. Kumar Tewari et al. / Scientia Horticulturae 108 (2006) 7–14 Fig. 6. Effect of Mg-deficiency on the activities of antioxidative enzymes. (A) CAT, (B) POD, (C) APX and (D) concentration of soluble protein in the young leaves of mulberry (Morus alba L.) cv. Kanva-2 plants grown in solution culture 34 DAT. The vertical bars represent mean  S.E. of three experimental replicates. Bars with different letters are significantly different (P  0.05) from respective controls by Bonferroni t-test. treated as a strategy for long-term sustenance under conditions of Mg stress as the nutrient elements released from the mature and gradually senescing organs have been shown to be utilized by young developing parts (Bleecker, 1998). Leaf senescence is the final stage of leaf development. Though, senescence is regulated by autonomous (internal) factors (age, reproductive development and phytohormone levels), the environmental signals—drought, ozone, nutrient deficiency, pathogen infection, wounding and shading also contribute to the development of senescence (Cao et al., 2003). The gradual loss of photosynthetic pigments (an inherent symptom of leaf senescence), and development of interveinal chlorotic mottling and necrosis in the old leaves of Mg-deficient plants may be the consequences of enhanced generation of ROS (Cakmak and Marschner, 1992; Cakmak, 1994). The contention is substantiated by intense (over 200%) increase in the H2O2 concentration in these leaves. The accumulation of H2O2 (an ROS) may not only inhibit Calvin cycle enzymes (Kaiser, 1976), but also induce stomatal closure (Desikan et al., 2004), which consequently may accentuate ROS generation by limiting CO2 uptake efficiency. Furthermore, retarded CO2 fixation in Mg-deficient plants due to decreased CO2 uptake and restricted activity of RUBP-carboxylase (Seftor et al., 1986) is likely to decrease NADPH/NADP ratio leading to enhanced generation of ROS (Cakmak and Marschner, 1992). Decreases in reducing sugars and starch and accumulation of nonreducing sugars in Mg-deficient plants imply disturbed carbohydrate metabolism and impaired phloem transport. Contrary to the present observations, Hermans et al. (2005) have reported accumulation of starch in youngest expanded leaves of Mg-deficient sugar beet plants. However, accumulation of sucrose (non-reducing sugars) in these leaves observed by these workers is in agreement with our observations. Hermans et al. (2005) attributed sucrose accumulation in Mgdeficient sugarbeet plants to impaired ploem loading. ROS generation due to accumulation of sugars (sucrose) that result from impaired phloem transport has been suggested earlier in Mg-deficient bean plants (Cakmak et al., 1994a,b; Cakmak, 1994). The greater decrease in the dry matter of roots as implied by over 40% increased shoot/root ratio in Mg-deficient plants and accumulation of non-reducing sugars (sucrose) corroborate impaired functioning of phloem in Mg-deficient plants (Cakmak et al., 1994a,b; Cakmak, 1994). A significant, over 40%, increase in carotenoids/chlorophylls ratio in Mg-deficient plants may be an adaptive response to the increased ROS generation, as carotenoids are known to detoxify ROS (PérezGálvez and Mı́nguez-Mosquera, 2002). Moreover, induction of oxidative stress and antioxidative responses due to greater accumulation of Fe and Zn in Mg deficient plants could not be discounted, as increased tissue concentrations of these elements have been shown to enhance ROS generation (Marschner, 1995). However, higher concentration of reduced form of ascorbate as also indicated by decreased DHA/AsA ratio in the Mg deficient plants might be a mechanism of keeping ROS and redox status of cells under control (Noctor and Foyer, 1998). Cakmak and Marschner (1992) have reported increased concentration of AsA in Mg-deficient bean plants. Increases in the activities of antioxidative enzymes—SOD, POD and APX also corroborates induction of oxidative stress in Mg-deficient plants. The observations are in agreement with those of Candan and Tarhan (2003), Cakmak and Marschner (1992) and Tewari et al. (2004) who have recorded increases in the activities of these antioxidative enzymes in herbaceous Mg-deficient plants—beans, mentha and maize, respectively. R. Kumar Tewari et al. / Scientia Horticulturae 108 (2006) 7–14 Polle et al. (1994) suggested induction of oxidative stress in Mg-deficient Norway spruce—a tree species, taking chlorosis in the needles as the index. Although, in this experiment, Polle et al. (1994) did not observed any significant effect of Mgdeficiency on the activities of POD, APX, monodehydroascorbate reductase, glutathione reductase and NADPH oxidase and concentrations of ascorbate and glutathione in Norway spruce plants grown with predominantly NO3 -nitrogen. A negative relationship between chloroplastic pigments and SOD activity observed in the present study is also at variance with the relationship between the two suggested by Polle et al. (1994) in Norway spruce. Despite of increased generation of ROS as evident from observed increases in concentration of H2O2 and activities of antioxidative enzymes (SOD, APX, POD), decreased TBARS level (lipid peroxidation) in young leaves of Mg-deficient mulberry is contrary to the observation of Candan and Tarhan (2003) in Mg-deficient Mentha pulegium. While increase in TBARS accumulation in Mg-deficient plants could be expected in older leaves that exhibited severe Mg-deficiency symptoms, the observed decrease in TBARS accumulation in younger leaves of Mg-deficient plants could possibly result from low functional Fe in these leaves. Low functional Fe, indicated by significantly decreased activity of CAT in Mg-deficient leaves, despite a mild increase in total Fe, could result from precipitation with phosphates (Becker et al., 1995), or sequestration in ferritin and/or nicotinamine, the synthesis of which is induced by Fe overload (Pich et al., 2001) and oxidative stress (Cairo et al., 1995). Phosphorus concentration in the young Mg-deficient leaves was found to be significantly higher than the controls (data not shown). Higher sensitivity of Mg-deficient leaves to photooxidative damages (Cakmak and Marschner, 1992), increased ASC pool, and induction of H2O2- and CN-sensitive isoforms of SOD indicate towards chloroplasts as the most likely sites of increased ROS generation. Generation of ROS in the cytoplasm of Mg-deficient leaves can not, however, be discounted as H2O2-scavenging enzymes of AsA-GSH cycle are also present in cytoplasm and the activity of non-specific POD [predominantly present in cytoplasm] showed significant, 88%, increase compared to a relatively smaller, 36% increase in the APX activity [ascorbate specific peroxidase predominantly located in the chloroplasts] (Cakmak and Marschner, 1992). Induction of additional isoforms of SOD in Mg-deficient plants having very high H2O2 content is in agreement with the observations of Kaminaka et al. (1999) who demonstrated that differential expression of SOD isoforms in rice was regulated by ROS in association with phytohormones. Moreover, induction of additional SOD isoforms by Mg-deficiency has also been recorded earlier in maize (Tewari et al., 2004). These observations indicate that Mg-deficient plants generate certain intrinsic cellular signals, probably ROS (O2 , H2O2), which consequently induce certain new SOD isoforms in plants. Recently, Shin and Schachtman (2004) showed accumulation of H2O2 in the roots and leaves of K-deficient Arabidopsis and maize and implicated ROS production in an early root response to K-deficiency that modulates gene expression and physio- 13 logical changes in the kinetics of K+ uptake. ROS have earlier been shown to serve as subcellular messengers in gene regulatory and signal transduction pathways (Allen and Tresini, 2000; Neill et al., 2002; Mori and Schroeder, 2004) and have been implicated in the modulation of gene expression (DineshKumar et al., 1995). Furthermore, recently it has been reported that nutrient deprivation triggers distinct redox changes and induce oxidative stress with a rather defined pattern in the context of nutrient-specific alterations in metabolism (Kandlbinder et al., 2004). At variance with the observations of Polle et al. (1994) that oxidative stress in Mg-deficient Norway spruce was attributable to low SOD rather than insufficient H2O2 detoxification, the present study suggests that in mulberry, like in most other plants, Mg deficiency induces oxidative stress by enhancing generation of ROS and triggering distinct redox changes in the cellular metabolism and is accompanied by activation of antioxidant machinery including induction of distinct SOD isoforms. Acknowledgements Authors are thankful to Council of Scientific and Industrial Research (CSIR), New Delhi, India for providing financial assistance. Authors are also grateful to Dr. Neetu and Dr. P.K. Singh for their sincere supports. References Allen, R.G., Tresini, M., 2000. Oxidative stress and gene regulation. Free Radic. Biol. Med. 28, 463–499. Apel, K., Hirt, H., 2004. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 55, 373–379. Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assays and assays applicable to acrylamide gels. Anal. Biochem. 44, 276–287. Becker, R., Fritz, E., Manteuffel, R., 1995. Subcellular localization and characterization of excessive iron in the nicotianamine-less tomato mutant chloronerva. Plant Physiol. 108, 269–275. Bisht, S.S., Sharma, A., Chaturvedi, K., 1989. Certain metabolic lesions of chromium toxicity in radish. Indian J. Agric. Biochem. 2, 109–115. Bleecker, A.B., 1998. The evolutionary basis of leaf senescence: method to the madness? Curr. Opin. Plant Biol. 1, 73–78. Brennan, T., Frenkel, C., 1977. Involvement of hydrogen peroxide in regulation of senescence in pear. Plant Physiol. 59, 411–416. Cairo, G., Tacchini, L., Pogliaghi, G., Anzon, E., Tomasi, A., Bernelli-Zazzera, A., 1995. Induction of ferritin synthesis by oxidative stress-transcriptional and post-transcriptional regulation by expansion of the ‘‘free’’ iron pool. J. Biol. Chem. 270, 700–703. Cakmak, I., 1994. Activity of ascorbate-dependent H2O2-scavenging enzymes and leaf chlorosis are enhanced magnesium and potassium-deficient leaves, but not in phosphorus-deficient leaves. J. Exp. Bot. 45, 1259–1266. Cakmak, I., Hengeler, C., Marschner, H., 1994a. Partitioning of shoot and root dry matter and carbohydrates in bean plants suffering from phosphorus, potassium and magnesium deficiency. J. Exp. Bot. 45, 1245–1250. Cakmak, I., Hengeler, C., Marschner, H., 1994b. Changes in phloem export of sucrose in leaves in response to phosphorus, potassium and magnesium deficiency in bean plants. J. Exp. Bot. 45, 1251–1257. Cakmak, I., Marschner, H., 1992. Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase and glutathione reductase in bean leaves. Plant Physiol. 98, 1222–1227. Candan, N., Tarhan, L., 2003. Relationship among chlorophyll-carotenoid content, antioxidant enzyme activities and lipid peroxidation levels by 14 R. Kumar Tewari et al. / Scientia Horticulturae 108 (2006) 7–14 Mg2+ deficiency in the Mentha pulegium leaves. Plant Physiol. Biochem. 41, 35–40. Cao, J., Jiang, F., Sodmergen, Cui, K., 2003. Time-course of programmed cell death during leaf senescence in Eucommia ulmoides. J. Plant Res. 116, 7–12. Desikan, R., Cheung, M., Bright, J., Henson, D., Hancock, J.T., Neill, S.J., 2004. ABA, hydrogen peroxide and nitric oxide signalling in stomatal guard cells. J. Exp. Bot. 55, 205–212. Dinesh-Kumar, S.P., Witham, S., Choi, D., Hehel, R., Corr, C., Baker, B., 1995. Transposon tagging of tobacco mosaic virus resistance gene N: its possible role in the TMV-N-mediated signal transduction pathway. Proc. Natl. Acad. Sci. U.S.A. 192, 4175–4180. Euler, H.V., Josephson, K., 1927. Über Katalase I. Leibigs Ann. 452, 158–187. Foyer, C.H., Noctor, G., 2003. Redox sensing and signalling associated with reactive oxygenin chloroplasts, peroxisomes and mitochondria. Physiol. Plant. 119, 355–364. Heath, R.L., Packer, L., 1968. Photoperoxidation in isolated chloroplast. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys. 125, 180–198. Hermans, C., Bourgis, F., Faucher, M., Strasser, R.J., Delrot, S., Verbruggen, N., 2005. Magnesium deficiency in sugar beets alters sugar partitioning and phloem loading in young mature leaves. Planta 220, 541–549. Hewitt, E.J., 1966. Sand and Water Culture Methods Used in the Study of Plant Nutrition. Commonwealth Agricultural Bureaux, Farnham Royl. Bucks, England. Kaiser, M.W., 1976. The effect of hydrogen peroxide on CO2 fixation of isolated chloroplast. Biochim. Biophys. Acta 440, 476–482. Kaminaka, H., Morita, S., Tokumoto, M., Masumura, T., Tanaka, K., 1999. Differential gene expressions of rice superoxide dismutase isoforms to oxidative and environmental stresses. Free Radic. Res. 31S, 219–225. Kandlbinder, A., Finkemeier, I., Wormuth, D., Hanitzsch, M., Dietz, K.-J., 2004. The antioxidant status of photosynthesizing leaves under nutrient deficiency: redox regulation, gene expression and antioxidant activity in Arabidopsis thaliana. Physiol. Plant. 120, 63–73. Law, M.Y., Charles, S.A., Halliwell, B., 1983. Glutathione and ascorbic acid in spinach (Spinacia oleracea) chloroplasts. The effect of hydrogen peroxide and of paraquat. Biochem. J. 210, 899–903. Lichtenthaler, H.K., 1987. Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol. 148, 350–382. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with Folin–phenol reagent. J Biol. Chem. 193, 265–275. Luck, H., 1963. Peroxidase. In: Bergmeyer, H.U. (Ed.), Methods of Enzymic Analysis. Academic Press Inc., New York, pp. 895–897. Mahalingam, R., Fedoroff, N., 2003. Stress response, cell death and signalling: the many faces of reactive oxygen species. Physiol. Plant. 119, 56–68. Marschner, H., 1995. Mineral Nutrition of Higher Plants, second ed. Academic Press Inc., London. Montgomery, R., 1957. Determination of glycogen. Arch. Biochem. Biophys. 67, 378–386. Mori, I.C., Schroeder, J.I., 2004. Reactive oxygen species activation of plant Ca2+ channels, a signaling mechanism in polar growth, hormone transduction, stress signaling, and hypothetically mechanotransduction. Plant Physiol. 135, 702–708. Nakano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbate specific peroxidase in spinach chloroplast. Plant Cell Physiol. 22, 867–880. Neill, S., Desikan, R., Hancock, J., 2002. Hydrogen peroxide signaling. Curr. Opin. Plant Biol. 5, 388–395. Nelson, N., 1944. A photometric adaptation of Somogyi method for determination of glucose. J. Biol. Chem. 153, 375–380. Noctor, G., Foyer, C.H., 1998. Ascorbate and glutathione: keeping active oxygen under control. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 249–279. Pérez-Gálvez, A., Mı́nguez-Mosquera, M.I., 2002. Degradation of non-esterified and esterified xanthophylls by free radicals. Biochim. Biophys. Acta 1569, 31–34. Pich, A., Manteuffel, R., Hillmer, S., Scholz, G., Schmidt, W., 2001. Fe homeostasis in plant cells: does nicotinamine play roles in the regulation of cytoplasmic Fe concentration? Planta 213, 967–976. Polle, A., Otter, T., Mehne-Jacob, B., 1994. Effect of Mg-deficiency on antioxidative system of Norway spruce [Picea abies (L) Karst.] grown with different ratio of nitrate and ammonium as nitrogen sources. New Phytol. 128, 621–628. Seftor, R.E.B., Bahr, J.T., Jensen, R.G., 1986. Measurement of the enzyme– CO2–Mg2+ form of spinach ribulose 1,5-bisphosphate carboxylase/oxygenase. Plant Physiol. 80, 599–600. Shin, R., Schachtman, D.P., 2004. Hydrogen peroxide mediates plant root cell response to nutrient deprivation. Proc. Natl. Acad. Sci. U.S.A. 101, 8827– 8832. Tewari, R.K., Kumar, P., Tewari, N., Srivastava, S., Sharma, P.N., 2004. Macronutrient deficiencies and differential antioxidant responses-influence on the activity and expression of superoxide dismutase in maize. Plant Sci. 166, 687–694. Wallace, T., 1951. The Diagnosis of Mineral Deficiencies in Plants by Visual Symptoms: A Colour Atlas and Guide. Her Majesty’s Stationary Office, London.