Protoplasma (2012) 249:139–153 DOI 10.1007/s00709-011-0273-6
ORIGINAL ARTICLE
Cumulative effect of nitrogen and sulphur on Brassica juncea L. genotypes under NaCl stress Manzer H. Siddiqui & Firoz Mohammad & M. Masrooor A. Khan & Mohamed H. Al-Whaibi
Received: 13 December 2010 / Accepted: 22 March 2011 / Published online: 9 April 2011 # Springer-Verlag 2011
Abstract In the present study, N and S assimilation, antioxidant enzymes activity, and yield were studied in N and S-treated plants of Brassica juncea (L.) Czern. & Coss. (cvs. Chuutki and Radha) under salt stress. The treatments were given as follows: (1) NaCl90 mM+N0S0 mg kg-1 sand (control), (2) NaCl90 mM+N60S0 mg kg-1 sand, (3) NaCl90 mM+N60S20 mg kg-1 sand, (4) NaCl90 mM+N60S40 mg kg-1 sand, and (5) NaCl90 mM+N60S60 mg kg-1 sand. The combined application of N (60 mg kg−1 sand) and S (40 mg kg−1 sand) proved beneficial in alleviating the adverse effect of salt stress on growth attributes (shoot length plant−1, fresh weight plant−1, dry weight plant−1, and area leaf−1), physio-biochemical parameters (carbonic anhydrase activity, total chlorophyll, adenosine triphosphate-sulphurylase activity, leaf N, K and Na content, K/Na ratio, activity of nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase, catalase, superoxide dismutase, ascorbate peroxidase and glutathione reductase, and content of glutathione and ascorbate), and yield attributes (pods plant−1, seeds pod−1, and seed yield plant−1). Therefore, it is
Handling Editor: Friedrich W. Bentrup M. H. Siddiqui (*) : M. H. Al-Whaibi Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia e-mail:
[email protected] F. Mohammad : M. M. A. Khan Plant Physiology Section, Department of Botany, Aligarh Muslim University, Aligarh, UP 202002, India
concluded that combined application of N and S induced the physiological and biochemical mechanisms of Brassica. The stimulation of antioxidant enzymes activity and its synergy with N and S assimilation may be one of the important mechanisms that help the plants to tolerate the salinity stress and resulted in an improved yield. Keywords Antioxidants . Enzyme activities . Osmoprotectants . Salt stress . N assimilation
Introduction Salt stress is the major environmental limiting factor for plant growth and development. Salinity alters the plant metabolisms, like osmotic adjustment, ion uptake, protein and nucleic acid synthesis, photosynthesis, organic solutes accumulation, enzymes activity, hormonal balance, injury to tissue, alteration in respiration rates, interaction of salt with microbial activity, and reduced water availability to crop plants. High salt stress can develop nutrient imbalance in plants (Maathuis 2006). High salinity of sodium (Na+) causes osmotic and metabolic problems and alters the activities of many enzymes related to nitrogen (N) and sulphur (S) assimilation through the degradation of several N and S macromolecules and reduction of nutrients uptake (Khan and Srivastava 1998; Chakrabarti and Mukherji 2003; Nathawat et al. 2005; Siddiqui et al. 2008a, b). It is well established that N improves plant growth and yield. In many studies, researchers set out to test the hypothesis that N fertilizer additions alleviate, at least to some extent, the deleterious effect of salinity on plants (Lunin and Gallatin 1965; Soliman et al. 1994). Reduced N is incorporated into
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many essential compounds, such as amino acids, proteins, nucleic acids, plant growth regulators, vitamins, and chlorophylls (Beevers and Hageman 1969; Marschner 2002). N application gives a regulatory and stimulatory influence on protein, sucrose, glucose, proline (Pro), and glycinebetaine (GB) synthesis; hence, these solutes may play a role in osmotic adjustment. Plants demand for reduced S, which is essential for function of protein, oligopeptides, and many coenzymes, by reduction of inorganic sulphate (Kopriva et al. 2002). S-assimilation pathway, being a source of reduced S for various cellular processes and for the synthesis of cystein which is metabolized to methionine or directly incorporated into proteins or glutathione (GSH), is a major factor in plant stress defense (Kopriva et al. 2002; Kopriva and Rennenberg 2004; Noctor et al. 1998). Many reports have shown that S assimilation is well coordinated with the assimilation of N (Ahmad et al. 1999; Kopriva and Rennenberg 2004; Siddiqui et al. 2008b). It has long been known that, during nitrate deficiency, sulphate assimilation is reduced and that the capacity to reduce nitrate is diminished in plants starved for sulphate (Kopriva et al. 2002; Kopriva and Rennenberg 2004). Nitrate reductase (NR), a key enzyme of nitrate assimilation, is induced by sulphate and adenosine triphosphate-sulphurylase (ATP-S) activity, key enzyme of sulphate assimilation is regulated by nitrate (Smith 1980; Brunold and Suter 1984). The biosynthesis of GSH depends on the linking and the availability of its constituent amino acids cysteine, glutamic acid, and glycine. As we know, glutamic acid, the primary product of the glutamine synthetase (GS)-glutamate synthase (GOGAT) pathway of N assimilation, and cysteine are end products of S assimilation. GSH is central in plant defense against oxidative stress. It has been well documented that availability of cysteine has an influence on control of GSH levels (Kopriva and Rennenberg 2004; Mullineaux and Rausch 2005). Several studies have established regulatory interactions between sulphate and nitrate assimilation in plants (Brunold 1993; Takahashi and Saito 1996; Kim et al. 1999; Koprivova et al. 2000). The activities of ATP-S, adenosine 5′-phosphosulfate reductase, and O-acetyl-L-serine (thiol) lyase decreased under N-deficient condition in Lemma minor and cultured tobacco (Nicotiana tabacum) cells (Reuveny et al. 1980; Smith 1980; Brunold and Suter 1984). Addition of nitrate or ammonia to the N-deficient medium quickly restored the activity of these enzymes. To fight against salinity, plants have developed complex mechanism, such as antioxidant defense system that is usually accomplished by the activity of antioxidant enzymes that are able to metabolize reactive oxygen species (ROS) to less toxic compounds (Noctor et al. 2002). One aspect, still little studied, is the regulation of the activity of antioxidant enzymes, by the supply of N and S to the plants (Medici et al. 2004). The relationship between salinity and
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mineral nutrition of plants are extremely complex, and a full understanding of these interactions would be required for plants to survive under stressful environment. Today, a challenging task for alleviating the adverse effects of salt stress is extremely important to fulfill the demand of the increasing population across the globe. Like crops, Brassica genotypes are distinct in salt tolerance via different physiological and biochemical adjustment at different levels of organization (Ashraf and McNeilly 2004). Keeping in view the sulphur-rich nature of Brassica plant, the present experiment was planned to study the interactive effect of N and S on the activity of N and S-assimilation enzymes and oxidative stress enzymes and yield attributes under salt stress.
Materials and methods Plant materials and growth conditions Seeds of two genotypes of mustard [Brassica juncea (L.) Czern. & Coss.] [Brassica juncea (L.) Czern. & Coss.] Chuutki (salt sensitive) and Radha (salt tolerant) were selected from our earlier experiment (Siddiqui et al. 2009a). The earthen pots of 30 cm diameter, lined with polythene bags (to avoid contamination), were filled with sand, washed with acid, followed by tap and distilled water. The pots were arranged in a simple randomized design with a single factor and four replicates. Before sowing, seeds were surface sterilized with 1% sodium hypochlorite for 10 min, then vigorously rinsed with sterilized double distilled water (DDW) and sown in sand-filled pots supplied with Raukura's nutrient solution (Smith et al. 1983). Analytical grade chemicals were used to make up the nutrient solutions. After 2 weeks of sowing, thinning was done and three healthy plants of uniform size were maintained in each pot. When the plants were at the stage of two to three true leaves, NaCl solution was added to the pots with experimental Brassica plants to attain the final concentration. The half doses of N and S were applied basally at the time of sowing, and the remaining half doses were given as a spray treatment after 1 week of NaCl treatment. Nutrient solutions containing NaCl were supplied to plants at the rate of 50 mL three times a week per pot. The experimental pots were irrigated daily with DDW (100–200 mL) to keep the sand moist. The treatments were given as follows: (1) NaCl90 mM+N0S0 mg kg-1 sand (control), (2) NaCl90 mM+N60S0 mg kg-1 sand, (3) NaCl90 mM+N60S20 mg kg-1 sand, (4) NaCl90 mM+N60S40 mg kg-1 sand, and (5) NaCl90 mM+N60S60 mg kg-1 sand. The concentration of NaCl and N was selected on the basis of earlier findings (Siddiqui et al. 2009a, b). The sources of applied N and S were supplied as urea and single superphosphate, respectively.
Cumulative effect of nitrogen and sulphur on Brassica juncea
Measurement of growth characteristics Sampling was done after 50 days of sowing. The combined effect of NaCl, N, and S on growth parameters was studied in terms of shoot length (SL) plant−1 fresh weight (FW) plant-1,dry weight (DW) plant-1 and area (A) leaf -1. The leaf area was obtained directly using a graph paper. The area of three leaves (upper, middle, and lower) of each plant of the sample (consisting of five plants) was determined with the help of graph paper.
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difference between the two figures (Lappartient and Touraine 1996). Determination of leaf nitrogen, potassium, and sodium concentration
Determination of physiological and biochemical parameters
Dried plant material was milled to pass through a 0.42-mm sieve, and 100 mg samples were analyzed by a Kjeldahl method, which included 0.5% selenium as a catalyst and salicylic acid to reduce nitrate (Eastin 1978). The total N concentration was determined according to the method of Lindner (1944). Concentration of leaf potassium (K) and Na was estimated using flame photometer.
Assay of carbonic anhydrase (EC 4.2.1.1) activity
Assay of glycinebetaine concentration
The activity of carbonic anhydrase (CA) was determined by the method of Dwivedi and Randhawa (1974). The leaf samples were cut into small pieces and suspended in cystein hydrochloride solution. The samples were incubated at 40°C for 20 min. After the addition of 0.2 mL of methyl red indicator, the reaction mixture was titrated against 0.05 N HCl. The results were expressed as [micromoles (CO2) per kilogram (f.m.) per second].
Glycinebetaine concentration was estimated by the method of Grieve and Grattan (1983). Leaves were weighed and oven-dried at 75°C, the dried leaves were finely ground with deionized water at 100°C for 60 min. GB concentration was determined spectrophotometrically at 365 nm, using aqueous extracts of dry-ground leaf material after reaction with KI-I2.
Assay of total chlorophyll The youngest fully expanded leaves were extracted with 80% acetone and the absorbance was read spectrophotometrically (Spekol-1500, Analytikjena, Germany) at 663 and 645 nm. The total chlorophyll (Chl) was determined by using the formula of Arnon (1949).
Assay of proline concentration Proline concentration was determined spectrophotometrically by adopting the ninhydrin method of Bates et al. (1973). We first homogenized 300 mg fresh leaf samples in sulphosalicylic acid. To the extract, 2 mL each of acid ninhydrin and glacial acetic acid were added. The samples were heated at 100°C. The mixture was extracted with toluene, and the free toluene was quantified spectrophotometrically at 528 nm using L-proline as a standard.
Determination of S-assimilation enzyme activity Determination of malonaldehyde concentration Fresh tissue was rapidly ground at 4°C in a buffer consisting of 10 mM Na2EDTA, 20 mM Tris–HCl (pH 8.0), 2 mM DDT, and approximately 0.01 g/mL insoluble PVP, using 1:4 (w/v) tissue to buffer ratio. ATP-S activity was measured using molybdate-dependent formation of pyrophosphate. The reaction was started by adding 0.1 mL of crude extract to 0.5 mL of the reaction mixture, which contained 7 mM MgCl 2 , 5 mM Na 2 MoO 4 , 2 mM Na 2 ATP, and 0.032 units mL−1 of sulphate-free inorganic pyrophosphate in 80 mM Tris–HCl buffer (pH 8.0). Another aliquot from the same extract was added to the same reaction mixture except that Na2MoO4 was absent. Incubation was carried out side by side at 37°C for 15 min after which phosphate was determined spectrophotometrically. A unit of enzyme was defined as the amount that produces 1 μmol/L inorganic phosphate in 1 min at 37°C. The ATP-sulphurylasedependent formation of pyrophosphate was estimated from
Malonaldehyde (MDA) content was determined according to the method of Heath and Packer (1968). Leaves were weighed and the homogenates, containing 10% trichloroacetic acid, 0.65% 2-thiobarbituric acid were heated at 95°C for 60 min, then cooled to room temperature and centrifuged at 10,000×g for 10 min. The absorbance of the supernatant was read at 532 and 600 nm against a reagent blank. Determination of hydrogen peroxide concentration Content of hydrogen peroxide (H2O2) was determined based on the method of Okuda et al. (1991) by homogenizing 0.5 g leaf samples in ice-cold 200 mm perchloric acid. After centrifugation at 1,200×g for 10 min, perchloric acid of the supernatant was neutralized with 4 mM KOH.
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The insoluble potassium perchlorate was eliminated by centrifugation at 500×g for 3 min. The reaction was started by the addition of peroxidase and the increase in the absorbance was recorded at 590 nm for 3 min. Determination of N-assimilation enzymes Nitrate reductase (EC 1.6.6.1) activity was estimated by the intact tissue method of Jaworski (1971). Fresh leaf samples were placed in the incubation mixture containing 2.5 mL phosphate buffer (pH 7.5), 0.2 M potassium nitrate, and 5% isopropanol solutions. The colorimetric determination of the reaction was achieved by adding 1% sulphanilamide and 0.2% N-1-naphthylethylene-diamine dihydrochloride. The absorbance was read at 540 nm and was compared using a calibration curve. The activity of NR was expressed as nanomoles NO2 per gram fresh weight (FW) per hour. Nitrite reductase (NiR, EC 1.7.7.1) activity was assayed from the rate of disappearance of nitrite from the reaction mixture as described by Sawhney and Naik (1973). The assay mixture in a final volume of 2 mL contained the following: 100 μM phosphate buffer (pH 7.5), 0.4 μM methyl viologen, 1.0 μmol NaNO2, 0.4 μM H2O, and 0.4 mL of the enzyme extract. The reaction was started with 0.01 mL of sodium dithionite solution prepared in 0.29 M NaHCO3 solution. The assay mixture without dithionate served as the control. After incubation for 30 min at 30°C, the reaction was stopped by shaking the tubes vigorously to oxidize completely the methyl viologen as indicated by the disappearance of blue color. The amount of residual nitrite in a suitable aliquot (0.1 mL) of reaction mixture was then determined spectrophotometrically according to the procedure of Nicholas and Nanson (1957) as described above. The NiR activity was expressed as micromoles NO2 per gram FW per hour. Glutamine synthetase (GS, EC. 6.3.1.2) activity was determined by measuring the amount of γ-glutamyl hydroxymate formed as described by Washitani and Sato (1977). The standard assay mixture (1 mL) for transferase activities of GS consisted of 100 μM Tris–HCl buffer (pH 8.0), 10 μmol glutamine, 60 μM hydroxylamine hydrochloride (neutralized with NaOH, pH 7.0), 1 μM ADP, 20 μM sodium hydrogen arsenate, 1 μM MnCl2, and 0.3 mL enzyme extract. The reaction was started by adding glutamine. Glutamine was omitted in the control. After incubation for 30 min at 37°C, the γ-glutamyl hydroxamate formed was determined by adding 2 mL of the FeCl3 reagent (equal volumes of 0.37 M FeCl3, 0.67 M HCl, 0.2 M trichloroacetic acid) and measuring the absorbance at 540 nm. The activity of GS was expressed as micromoles γ-glutamyl hydroxymate released per gram FW per hour. Glutamate synthase (EC 2.6.1.53) activity was assayed spectrophometrically following the oxidation of NADH at
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340 nm (Boland et al. 1978). The reaction mixture (2.5 mL) contained 160 μM Tris–HCl buffer (pH 7.5), 10 μM αketoglutarate (pH 6.8–7.0, neutralized with Na2CO3), 0.4 μM NADH, 10 μM glutamine, and 0.15 μM enzyme extract. GOGAT activity was expressed as micromolar NADH oxidized per gram FW of leaf per hour. Determination of antioxidant enzymes activity Preparation of enzyme extracts A crude enzyme extract was prepared by homogenizing 500 mg of leaf tissue in extraction buffer containing 0.5% Triton X-100 and 1% polyvinylpyrrolidone in 100 mM potassium phosphate buffer (pH 7.0) using a chilled mortar and pestle. The homogenate was centrifuged at 15,000×g for 20 min at 4°C. The supernatant was used for the enzymatic assays. For ascorbate peroxidase (APX), extraction buffer was supplemented with 2 mM ascorbate. Catalase (CAT) activity (EC 1.16.1.6) was measured by the disappearance of H2O2 (Aebi 1984). The reaction mixture contained 50 mM phosphate buffer (pH 7.0), 10 mM H2O2, and 100 μL plant extract. The activity was estimated by monitoring the decrease in absorbance due to H2O2 reduction at 240 nm. The activity of superoxide dismutase (SOD, EC 1.15.1.1) was determined by measuring its ability to inhibit the photoreduction of nitro blue tetrazolium (NBT) according to the methods of Giannopolitis and Ries (1977), using the plant extract. One unit of SOD activity was defined as the amount of enzyme that inhibited 50% of NBT photoreduction. Ascorbate peroxidase (EC 1.11.1.11) activity was measured following the method of Nakano and Asada (1981), estimating the rate of ascorbate oxidation at 290 nm (absorbance coefficient at 2.8 mM−1 cm−1). Glutathione reductase (GR, EC 1.6.4.2) activity was also assayed as described by Foyer and Halliwell (1976), with minor modifications. The assay mixture consisted of 50 μL of the enzyme extract, 100 mM phosphate buffer (pH 7.8), 0.1 μM EDTA, 0.05 mM NADPH, and 3.0 mM oxidized glutathione in a total volume of 1.0 mL. NADPH oxidation rate was monitored by reading the absorbance at 340 nm at the moment of H2O2 addition and 1 min later. The difference in absorbance (A340) was divided by the NADPH molar extinction coefficient (6.22 mM cm−1). Determination of antioxidants Total reduced GSH was determined in homogenates spectrophotometrically at 340 nm using glutathione reductase, 5, 5′-dithio-bis-nitrobenzoic acid and NADPH. Oxidized glutathione (glutathione disulfide, GSSG) was
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assayed by the same method in the presence by 2-vinyl pyridine and GSH content was calculated as a difference between total glutathione (GSG + GSSG) and GSSG (Anderson 1985). Ascorbate (ASC) concentration was determined according to the method of Foyer et al. (1983) with some modifications. In liquid nitrogen, 0.1 g of leaves was ground, and 1 mL of 2.5 M perchloric acid was added. The crude extract was centrifuged at 2°C for 10 min at 10,000×g, and the supernatant was neutralized with saturated Na2CO3 using methyl orange as the indicator. The reduced ascorbate was assayed spectrophotometrically at 265 nm in 1 M NaH2PO4 buffer, pH 5.6, with 1 unit ascorbate oxidase. The total ascorbate was assayed after incubation in the presence of 30 mM dithiothreitol. Statistical analysis Each pot was treated as one replicate and all the treatments were repeated four times. The data were analyzed statistically with SPSS-11 statistical software (SPSS Inc., Chicago, IL, USA). Means were statistically compared by Duncan's multiple range test at p
