Positive effects of nitric oxide on Solanum lycopersicum

Abstract

The present work examines benzoic acid (BA) induced stress in Solanum lycopersicum and explores the possible mechanism through which sodium nitroprusside (SNP) protects plants. Tomato seeds were soaked for 3 h in 250 µM SNP and 0.5, 1.0, and 1.5 mM BA with and without SNP. Seeds soaked in distilled water were treated as control. Seed germination (SG) and radicle length (RL) were recorded in Petri plate culture. SG and RL inhibition was concentration dependent in BA. SNP enhanced SG and RL. Twenty-one-days old seedlings were grown in hydroponic culture in Hoagland solution and BA at 0.5, 1.0, and 1.5 mM with and without SNP. The morphological and biochemical parameters of seedlings were assayed. Average growth rate of root and shoot, dry weight, sugar, pigment and protein content, and activity of nitrate reductase decreased in seedlings treated with BA while increased in SNP with and without BA. Lipid peroxidation increased in seedlings treated with BA but decreased in BA+SNP. SNP protected the seedlings against BA induced oxidative stress by scavenging reactive oxygen species. SNP demonstrated a positive role against BA toxicity which was evident from increased activities of antioxidant enzymes.

Keywords:

• allelopathy
• antioxidant enzymes
• benzoic acid
• lipid peroxidation
• nitric oxide
• sodium nitroprusside

Introduction

Nitric oxide (NO) is a signaling molecule (Duner et al. 1999), which participates in different physiological processes of plant (He et al. 2004; Delledonne 2005; De Pinto et al. 2006). Plants produce NO via the oxidation of arginine to citrulline (Neill et al. 2003a). NO formation from nitrite is also induced by phenolic acids in acidic condition (Bethke et al. 2004). It induces seed germination (SG) by breaking seed dormancy (Zhang et al. 2003; Bethke et al. 2004; Sarath et al. 2006). Under unfavorable conditions plants elevate the level of reactive oxygen species (ROS). NO is known to interact with ROS in various ways and alleviates the oxidative stress in plants (Beligni and Lamattina 1999; Neill et al. 2003b; Kopyra & Gwóźdź 2004; Crawford & Guo 2005). NO-treated wheat seedlings exhibited enhanced activities of antioxidant enzymes under osmotic stress such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and proline content (Zhang et al. 2003). NO induced root growth by inducing cell elongation just like auxin (Gouvêa 1997). SNP is a source of NO as it releases NO (Delledonne et al. 1998; Duner et al. 1999; Pedroso et al. 2000; De Pinto et al. 2002; Hu et al. 2007). In natural and agro-ecosystems, interaction among plants is frequent. Plants produce allelochemicals which affect growth and development of neighboring plants. Studies have shown that allelochemicals affect plant growth by altering physiological and biochemical parameters, viz. SG, pigment and protein contents, amount of sugar, photosynthesis, respiration, and water uptake (Inderjit and Duke 2003; Singh & Thapar 2005; Weir et al. 2003). Allelochemicals affect the activities of antioxidant enzymes that ameliorate oxidative stress. Impairment of metabolic activity ultimately affected crops yield. Benzoic acid (BA) and its derivatives are reported in plant and soil (Rice 1984; Vaughan & Ord 1991; Baziramakenga et al. 1995). BA in excess amount inhibits SG and seedling growth (Maffei et al. 1999), decreases respiration and photosynthesis by inhibiting electron transport (Zhou et al. 2006), affects chlorophyll biosynthesis and protein content (Baziramakenga et al. 1994), and interferes with sugar metabolism and nitrate reductase (NR) activity in several crop plants (Naguib 1965; Robert et al. 1982). It causes membrane damage by inducing electrolyte leakage and lipid peroxidation (LP) (Baziramakenga et al. 1995). Activity of several antioxidant enzymes increases under BA stress (Baziramakenga et al. 1995). BA has shown inhibitory effects on crop plants. NO stimulates growth and adaptation of plants under stress. Protective effects of NO against various stresses like metal stress (Kopyra et al. 2006; Hu et al. 2007; Zhang et al. 2008), osmotic stress (Lei et al. 2007), salt stress (Uchida et al. 2002), and chilling stress (Neill et al. 2003a) have been reported. NO supports defense system of plant, and thereby increases plant adaptation in unfavorable conditions. The survey of pertinent literature reveals that there are no studies on the combined effect of BA and SNP on plants. The present study was undertaken to investigate the effect of these two chemicals on growth and metabolism on tomato seedlings.

Materials and methods

Seeds and chemicals

Seeds of tomato (Solanum lycopersicum) variety Pusa ruby were procured from seed agency in Allahabad, India. Sodium nitroprusside (SNP) (Na₂ [Fe (CN)₅ NO].2H₂O) [Sodium pentacyanonitrosylferrate (II)] (molecular weight: 297.95 g/mol) was purchased from Merck, and BA (molecular weight: 122.12) from LOBA Chemie.

Petri plate culture

The seeds of tomato were surface sterilized with 0.01% (w/v) HgCl₂ solution for 3 min and washed extensively with distilled water. Doses of BA were decided before the experiment. One mM concentration caused 50% inhibition of SG. Graded concentrations, 0.5, 1.0, and 1.5 mM of BA, prepared in sodium phosphate buffer (pH 7.0), were used for the study. The most effective concentration, 250 µM of SNP, was used in the experiment as it caused maximum SG during preliminary experiment. The seeds were soaked for 3 h in 250 µM SNP and 0.5, 1.0 and 1.5 mM BA with and without SNP. The seeds soaked in distilled water were taken as control. Ten seeds of each treatment in replication of three were placed at equal distance in sterilized Petri plates (diameter: 9 cm and depth: 1.5 cm) lined with moistened Whatman No. 1 filter paper. The Petri plates were incubated at 28±2°C for germination in growth chamber. Germination was initiated next day after sowing (DAS). Germination was recorded till 5 DAS at the interval of 24 hours. Radicle length (RL) was recorded till 5 DAS.

Hydroponic culture

The seeds were sown in November, 2010, in a nursery bed (1 m×1 m) in the Department of Botany, University of Allahabad, Allahabad, India. The seed bed was irrigated as and when required. Twenty-one-day old seedlings of uniform height were transferred in 24 plastic pots (length: 23 cm, width: 17 cm, and height: 9 cm) filled with 2 L half strength Hoagland solution (Hoagland & Arnon 1950). In each pot, six seedlings were placed at equal distance. The pots were divided into eight sets each containing three pots. In one set, Hoagland solution was replaced by fresh Hoagland solution (2 L) and was taken as control. In the second set Hoagland solution was replaced by Hoagland solution (2 L) containing 250 µM SNP. In other three sets, Hoagland solution was replaced with Hoagland solution (2 L) containing graded concentrations of 0.5, 1.0, and 1.5 mM of BA. In remaining sets Hoagland solution was replaced with Hoagland solution (2 L) with the graded concentrations of BA with SNP (250 µM). Each pot was aerated for 12 h a day with the help of bubbler. The pots were covered with black sheet to avoid the growth of algae in the medium. On the 11th day the sampling was done. The first fully expanded leaves of the seedlings were sampled for biochemical analyses. Dry weight (DW) and average growth rate (AGR) of root and shoots were recorded.

Pigment and protein contents

The leaves (10 mg) were homogenized with 10 ml of 80% acetone. Chlorophylls and carotenoids were extracted and quantified following the method of Lichtenthaler (1987). Protein content was determined according to the method by Lowry et al. (1951). The amount of protein was calculated with reference to the standard curve obtained from bovine serum albumin.

Sugar content

The quantification of total soluble sugars (TSS) was done following the method by Hedge and Hofreiter (1962). About 0.1 g of fresh leaf tissue was homogenized in 5 ml of 95% (v/v) ethanol. After centrifugation, 1 ml of supernatant was mixed with 4 ml anthrone reagent and heated on boiling water bath for 10 min. Absorbance was recorded at 620 nm after cooling. The amount of sugar was determined by the standard curve prepared from glucose.

Nitrate reductase activity

Nitrate reductase (NR) activity was measured by following the procedure of Jaworski (1971). Fresh leaf tissue (0.25 g) was incubated in 4.5 ml medium which contained 100 mM phosphate buffer (pH 7.5), 3% (w/v) KNO₃ and 3 N HCl and 0.02% (w/v) N-(1-Naphthyl) ethylene diamine dihydrochloride. The absorbance was recorded at 540 nm. NR activity was measured with standard curve prepared from NaNO₂ and expressed as µmol NO₂ mg protein⁻¹ h⁻¹.

Lipid peroxidation

Lipid peroxidation was measured as the amount of malondialdehyde (MDA) determined by thiobarbituric acid reactive substance as described by Heath and Packer (1968). Fresh leaf (0.2 g) was ground in 0.1 w/v trichloroacetic acid (TCA) and centrifuged at 10,000g for 10 min. One milliliter supernatant was mixed with 4 ml of 0.5% thiobarbituric acid prepared in 20% (w/v) TCA. The mixture was then heated at 95°C for 30 min and again centrifuged after cooling. The absorbance of the supernatant was recorded at 532 nm and corrected by subtracting the non-specific absorbance at 600 nm. The MDA concentration was calculated using the extinction coefficient of 155 mM⁻¹ cm⁻¹ and expressed as nmol g⁻¹ FW.

Extraction and assay of antioxidant enzymes

For the extraction and assay of enzymes, fresh leaves (0.25 g) were homogenized with 0.1 M sodium phosphate buffer containing 1% (w/v) polyvinyl pyrrolidone (pH 7.0) in a pre-cooled mortar and pestle. The extract was centrifuged at 4°C at 14,000g for 30 min in cooling centrifuge (Remi instruments C 24). The supernatant was used for the assay of SOD, CAT, APX, and guaiacol peroxidase (POX).

Assay of SOD

The activity of SOD (EC 1.15.1.1) was estimated by the nitroblue tetrazolium (NBT) photochemical assay following Beyer and Fridovich (1987). The reaction mixture (4 ml) consisted of 20 mM methionine, 0.15 mM ethylene diamine-tetra acetic acid (EDTA), 0.12 mM NBT, and 0.5ml supernatant. The test tubes were exposed to fluorescent lamp for 30 min and identical unilluminated assay mixture served as blank. One unit of enzyme was measured as the amount of enzyme which caused 50% inhibition of NBT reduction.

Assay of CAT

CAT (EC1.11.1.6) activity was assayed following the method by Cakmak and Marschner (1992). Assay mixture (2 ml) contained 25 mM potassium phosphate buffer (pH 7.0), 10 mM H₂O₂, and 0.5 ml enzyme extract. The rate of H₂O₂ decomposition for 1 min was monitored at 240 nm and calculated using extinction coefficient of 39.4 mM⁻¹ cm⁻¹ and expressed as enzyme unit mg⁻¹ protein. One unit of CAT was determined as the amount of enzyme required to oxidize 1 µM H₂O₂ min⁻¹.

Assay of APX

APX (EC1.11.1.11) was assayed following the method by Nakano and Asada (1981). Assay mixture (2 ml) contained 25 mM potassium phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.25 mM ascorbate, 1.0 mM H₂O₂, and 0.2 ml enzyme extract. H₂O₂ was the last component to be added. The absorbance was recorded for 1 min at 290 nm (extinction coefficient of 2.8 mM⁻¹ cm⁻¹). Enzyme specific activity was measured as enzyme unit per one milligram protein as the amount of enzyme required to oxidize 1 µM H₂O₂ min⁻¹.

Assay of guaiacol POX

Guaiacol POX (EC 1.11.1.7) was assayed following the method of Hemeda and Klein (1990). The reaction mixture (2 ml) contained 25 mM phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.05% (v/v) guaiacol, 1.0 mM H₂O₂, and 0.2 ml of enzyme extract. The increase in absorbance due to oxidation of guaiacol was monitored at 470 nm. The enzyme activity was measured using extinction coefficient of 26.6 mM⁻¹ cm⁻¹ and expressed as enzyme unit per one milligram protein.

Statistical analysis

Treatments were arranged in a randomized block design with three replications. Data were statistically analyzed using analysis of variance (ANOVA) by using SPSS software (Ver. 10; SPSS Inc., Chicago, IL, USA). Appropriate standard error of means (±SEM) was calculated for presentation with tables and graphs. The treatment means were analyzed by Duncan's multiple range test (DMRT) at p<0.05.

Results

Seed germination of tomato under BA stress was adversely affected. Control group exhibited 68% germination and SNP slightly elevated the germination percentage. BA, an allelochemical, is produced as a byproduct of main metabolic pathway. Allelochemical decreased germination significantly with a maximum of 37.5% decrease in the highest dose (BA₃). SNP helped in detoxifying BA to such an extent that SG increased to maximum 47.05% in BA₃+SNP in comparison with other combinations. Increase in SG corresponded to the concentration of BA+SNP doses as the highest concentration of BA with SNP (BA₃+SNP) caused maximum elevation when compared to BA₃. RL was remarkably elevated in seeds treated with SNP. BA reduced RL in a dose-dependent manner. All treatments caused a decline in RL with a maximum of 44.16% reduction in BA₃ treatment as compared to SNP. However, in combined treatment, RL recorded a maximum of 1.32 fold elevation in BA₁+SNP as compared to BA₁ (Table 1).

Table 1. Effects of benzoic acid and SNP on seed germination, radicle length (Petri plate culture), average growth rate of root and shoot, dry weight, and sugar contents of Solanum lycopersicum.

Treatments	Seed germination (%)	Radicle length (cm)	AGR of root (cm/ day)	AGR of shoot (cm/day)	DW (g/ seedling)	Sugar (mg/g FW)
Control	67.99±2.602bc	2.26±0.233bc	0.35±0.01d	0.047±0.01b	0.197±0.001cde	141.99±1.78b
SNP	69.10±3.579bc	3.16±0.120a	0.95±0.01a	0.170±0.04a	0.299±0.001a	167.15±5.21a
BA1	69.16±3.367bc	2.16±0.120bc	0.29±0.02de	0.097±0.01b	0.198±0.001cd	106.39±1.31d
BA1+SNP	80.83±0.481a	2.86±0.437ab	0.47±0.03bc	0.102±0.01b	0.302±0.001a	161.70±2.33a
BA2	55.83±1.444e	1.86±0.066c	0.22±0.06ef	0.080±0.01b	0.186±0.001e	116.50±0.72c
BA2+SNP	72.50±0.481b	2.36±0.185bc	0.49±0.01b	0.100±0.02b	0.206±0.001bc	158.51±5.04a
BA3	42.50±1.443d	1.76±0.145c	0.17±0.04f	0.060±0.02b	0.188±0.001de	87.17±2.18f
BA3+SNP	62.50±2.405c	2.33±0.166bc	0.37±0.01cd	0.085±0.02b	0.214±0.009b	96.09±1.00e

Note: Mean±SE values followed by same letters within each column are not significantly different at 0.05 (ANOVA and Duncan's multiple range test), n=3. SNP=sodium nitroprusside (250 µM), BA₁=0.5, BA₂=1.0 and BA₃=1.5 mM of benzoic acid.

Average growth rate of root and shoot followed similar trends. The decrease in root and shoot growth corresponded to an increased concentration of BA. Root and shoot growth increased to maximum under the SNP treatment. In combined treatments (BA+SNP) both root and shoot growth was better in comparison with that of BA treatments. DW of seedling decreased significantly (p<0.05) in BA treatment in comparison with control with maximum decrease in BA₂. SNP enhanced DW of seedlings. DW decreased in all treatments except BA₁+SNP (Table 1).

TSS of seedlings was variously affected in all treatments. Significant decrease was observed in sugar content of tomato seedlings under BA treatments with a maximum of 38.6% in BA₃ as compared to control. TSS increased remarkably in SNP. SNP alleviated the affect of BA by increasing the sugar content of seedlings in combined treatment. In BA₂+SNP, TSS was higher than control. Sugar content in BA₃+SNP treatment recorded an increase over BA₃ but always lower than that of control (Table 1). BA affected the pigment content of tomato seedlings. A considerable loss of chlorophyll under the influence of BA with a maximum of 3.03 fold decrease was recorded in BA₃. The amount of chlorophyll increased 1.25 folds in SNP. Carotenoids exhibited greater loss due to BA with maximum 73.8% decrease in the highest concentration. Carotenoids followed the trend of chlorophyll (Table 2).

Table 2. Effects of benzoic acid and SNP on pigment contents, protein contents and nitrate reductase activity of Solanum lycopersicum.

Treatments	Chlorophyll a (mg/g FW)	Chlorophyll b (mg/g FW)	Total chlorophyll (mg/g FW)	Carotenoid (mg/g FW)	Protein (mg/g FW)	NR (µmol NO2 mg protein^−1 h^−1)
Control	0.587±0.002b	0.160±0.001bcd	0.747±0.003b	0.356±0.001b	97.53±1.80b	12.29±0.27d
SNP	0.821±0.002a	0.119±0.001bcd	0.940±0.003a	0.457±0.010a	122.27±1.76a	13.01±0.22d
BA1	0.490±0.005de	0.137±0.002bcd	0.627±0.007cd	0.302±0.004c	74.27±1.85e	8.87±0.37bc
BA1+SNP	0.540±0.014c	0.182±0.077bc	0.722±0.091bc	0.388±0.011b	81.80±2.65c	8.08±0.29cd
BA2	0.244±0.006f	0.368±0.009a	0.612±0.003d	0.163±0.027d	66.80±2.30f	6.87±0.27d
BA2+SNP	0.506±0.001d	0.210±0.001b	0.716±0.003bc	0.382±0.001b	81.00±2.19cd	9.98±0.02c
BA3	0.177±0.010g	0.070±0.006d	0.246±0.004e	0.093±0.025e	61.60±1.38f	7.08±1.34d
BA3+SNP	0.470±0.003e	0.096±0.004cd	0.566±0.007d	0.264±0.007c	74.87±2.16de	12.60±0.28a

Note: Mean±SE values followed by same letters within each column are not significantly different at 0.05 (ANOVA and Duncan's multiple range test), n=3. SNP=sodium nitroprusside (250 µM), BA₁=0.5, BA₂=1.0, and BA₃=1.5 mM of benzoic acid.

BA caused significant decrease in protein content. The decline in protein was dose dependent. Higher amount of protein in SNP treatment was recorded as compared with control. Protein content increased in BA+SNP in comparison with that of BA. Maximum NR activity was recorded in SNP-treated plants followed by control. A graded loss in activity was seen in seedlings treated with BA with no significant difference in NR activity between BA₂ and BA₃. SNP in combined treatments (BA+SNP) increased the NR activity at the level lower than that of control and SNP (Table 2).

The extent of LP varied in response to BA. LP was measured in terms of MDA content. Lowest amount of MDA was recorded in SNP followed by control. A gradual increase in MDA content was recorded in BA treatments with maximum in BA₃. MDA content decreased relatively in combined (BA+SNP) treatments, but was still higher than that of control and SNP (Figure 1). The antioxidant enzymes were activated in response to BA stress. SOD activity increased in BA treatments. Lowest SOD activity was observed in control. SNP recorded higher SOD activity in comparison with control. SOD activity was highly elevated under combined treatment with maximum 513.19% increase in BA₃+SNP. A significant (p <0.05) increase in CAT activity was observed in BA₂ and BA₃ as compared to control and SNP. The difference of CAT activity between control and SNP was not significant. BA+SNP treatments increased CAT activity in comparison with control and SNP. The combined treatments elevated the activity of CAT in comparison with BA treatments except BA₂+SNP. Activity of APX was significant in BA₂ and BA₃ treatments as compared with control. Maximum APX activity was recorded in BA₃+SNP followed by BA₃ and BA₂. Activity of POX was not significant in BA and SNP treatments but combined treatments drastically increased POX activity over control and SNP (Figure 2).

Figure 1. Effect of benzoic acid and SNP on lipid peroxidation of Solanum lycopersicum. Mean±SE values followed by same letters within each column are not significantly different at 0.05 (ANOVA and Duncan's multiple range test), n=3. C=control, S=sodiumnitroprusside (250 µM), B₁=0.5, B₂=1.0, and B₃=1.5 mM of benzoic acid.

Figure 2. Effect of benzoic acid and SNP on superoxide dismutase, catalase, ascorbate peroxidase and guaiacol peroxidase of Solanum lycopersicum. Mean±SE values followed by same letters within each column are not significantly different at 0.05 (ANOVA and Duncan's multiple range test), n=3. C=control, S=sodiumnitroprusside (250 µM), B₁=0.5, B₂=1.0, and B₃=1.5 mM of benzoic acid.

Discussion

Plants compete for water, light, space, and nutrient. Besides, allelochemicals released into the surrounding by exudation, evaporation, leaching, and residue decomposition affect the neighboring plants. BA is a common allelochemical produced by plants which accumulates in the soil (Vaughan & Ord 1991) and alter the growth and metabolism of neighboring plants of same or different species and plants of next generation. Allelochemicals caused allelopathic stress. Stress is the result of environmental change which adversely affect growth and metabolism of plants. Allelochemicals are known to alter the plant metabolic processes (Cruz-Ortega et al. 2002) and cause oxidative damage which induce antioxidant enzymes (Weir et al. 2004). In the present study, SNP is used as NO donor that participates in physiological processes of plant. Hu et al. (2007) also used SNP as NO donor and in addition, they have chosen cPTIO (2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) as an NO scavenger and NaNO₃, NaNO₂, and K₄Fe(CN)₆ as additional controls for SNP decomposition. They concluded that NO, but not other compound derived from SNP was responsible for the promising promoting roles of SNP. NO is a lipophilic molecule that permeates the membrane and is an important signal molecule in plants growing under biotic and abiotic stresses (Tan et al. 2008). NO is reported to induce SG (Beligni and Lamattina 2000), regulate plant metabolism (Leshem et al. 1998) and photosynthesis (Takahashi & Yamasaki 2002), and alleviate the effect of oxidative stress (Beligni and Lamattina 1999; Crawford & Guo 2005).

Seed germination was greatly affected by BA with a maximum decrease in BA₃. SNP singly and in combination with BA increased SG. The decrease in germination may be due to a hindrance in activity of glycolytic enzymes (Muscolo et al., 2001) which was accelerated in response to increased respiration (Podesta and Plaxton 1994). Allelochemicals have been found to inhibit SG (Weir et al. 2003). However, NO is reported to induce SG of lettuce (Beligni and Lamattina 2000). Delayed germination in BA treatments may be the result of regulated elongation and expansion of cells of radicle and disrupted metabolic activities. RL greatly increased in seeds treated with SNP and combinations (BA+SNP) but decreased in BA. Allelochemicals inhibit elongation and division of cells which arrest the growth of radicle (Avers & Goodwin 1956).

The decrease in the AGR of root of tomato seedlings was dependent on the concentration of BA. The highest concentration of BA caused maximum inhibition of root growth. NO promoted root growth singly and in combination. BA inhibits root elongation and formation of secondary roots (Baziramakenga et al. 1994). BA might have interrupted the meristematic activity of root causing impaired cell division (Vaughan & Ord 1991) or hormonal imbalance (IAA) which affected growth (Rice 1984). NO induced root growth, which may be due to cell elongation (Gouvêa 1997) in root tips. This function of NO is somewhat similar to that of auxin causing cell elongation and induction of adventitious roots (Pagnussat et al. 2002). Alteration in root growth affected the nutrient availability to the seedlings hence affecting the growth (Baziramakenga et al. 1997). In SNP treatments the elevated seedling biomass under SNP supports the finding. Inhibited organ growth (roots and shoots) due to reduced production of NO in the Arabidopsis (Atnos I) could be reversed by treatment with SNP (Guo et al. 2003) The pigment content decreased significantly due to allelopathic stress caused by BA. NO alleviated the effect of the allelochemical. The plant dry matter is closely related to chlorophyll content (Buttery and Buzzell 1977). The lower chlorophyll content caused limited net photosynthesis and thus reduced plant growth. The allelochemicals caused degradation of chlorophyll or inhibition of chlorophyll biosynthesis (Kanchan & Jayachandra 1980). NO plays a protective role in case of chlorophyll. It may have some attribution in the availability of iron to plants for retention of chlorophyll (Graziano et al. 2002). Increase in chlorophyll content in pea by NO is reported by Leshem et al. (1997).

Sugar and protein content decreased in response to BA. The impairment of metabolic processes due to BA resulted in reduced seedlings growth. Decreased photosynthesis due to low pigment contents regulated the availability of photosynthates. In order to overcome the shortage of nutrients the plants resort to their food reserve. NO elevated pigments' content to restore normal protein and sugar content (Zhang et al. 2008).

BA decreased the NR activity while SNP elevated the activity. The elevation of the NR activity by SNP treatment is due to the alleviation of BA toxicity in combined treatments. The NR activity is known to be regulated by starvation (Kaiser and Huber 2001) or low availability of nitrate (Lin et al. 1994). BA decreased NR activity. The root damage by allelochemical decreased nitrate absorption (Abd-El Baki et al. 2000). The inhibition of root growth by BA supports our finding. NO is known to play an important role in plants' resistance to abiotic and biotic stresses by regulating organelle function (Guo & Crawford 2005) and tissue development (Leshem et al. 1998). Du et al. (2008) reported enhanced NR activity in Chinese pakchoi cabbage (Brassica chinensis L.) upon SNP treatment. They suggested that the increase in NR activity was due to NO which stimulated in electron transfer from haem to nitrate through activating the haem and molybdenum centers in NR.

Allelochemicals also exert a kind of abiotic stress on plants. In the abiotic stress, cellular homeostasis is disturbed causing oxidative stress and accumulation of ROS (Asada 2006). Allelochemicals are known to depolarize membrane and increase its permeability by inducing LP (Devi & Prasad 1996; Lin et al. 2000). During oxidative stress, a large amount of superoxide radicals are produced that increases MDA content, the index of LP. BA increased the production of MDA in a dose-dependent manner. In our results increased MDA content in response to BA treatment is in agreement with several studies (Smirnoff 1993; Baziramakenga et al. 1995). SNP maximally mitigates LP at BA₃ treatment by reducing MDA content. The decreased SOD activity evinced the fact that plants were not able to tolerate the oxidative stress. The allelochemical inhibited SOD activity (Zeng et al. 2001). SOD converts O₂⁻ into H₂O₂ which is changed into water by activity of CAT and peroxidase. CAT activity decreased in BA treatments. Thus accumulation of harmful H₂O₂ might have occurred. Lin et al. (2000) have also reported that SOD and CAT activity decreased in barnyardgrass in response to rice allelochemicals. It appears that APX and POX enzymes worked synergistically and detoxify H₂O₂ to some extent. APX activity increased in BA₃+SNP treatment and POX activity increased in combined (BA+SNP) treatments. NO alleviates the effect of oxidative stress. In our results antioxidant enzymes increased in combined treatments. NO is known to play an important role in maintaining cellular redox homeostasis by inducing O₂⁻ conversion to H₂O₂ and O₂ and also enhancing/promoting the H₂O₂ scavenging activities of enzymes (Lamattina et al. 2003). NO exhibits antioxidant properties (Karplus et al. 1991). There are reports that the toxicity of paraquat (PQ), a herbicide was diminished by NO in rice leaves (Hung et al. 2002). NO prevented PQ-induced reduction in protein content. It also prevented increase in level of MDA and decline in activity of antioxidant enzymes such as SOD, APX, and POX in PQ-treated rice and thus decrease the breakdown of protein by ROS. Cell death, ion leakage, and DNA fragmentation which are the ROS-mediated damages resulting from Phytophthora infestans were inhibited by NO donor (Beligni and Lamattina 1999). There are several reports that NO has positive effects on plants (Beligni and Lamattina 1999; Hung et al. 2002; Guo et al. 2003). SNP used as an NO donor in the experiments; if it degrades to cyanide group it will inactivate the terminal oxidase/cytochrome c oxidase of the respiratory chain (Wang et al. 2002). It will regulate transfer of electron to oxygen by alternative oxidase and will cause the loss of energy in the form of heat. The cyanide produced thus inhibits the respiratory process. But in the experiment most of the parameters have been enhanced which prove that cyanide is not produced, instead NO is produced which supports the plant metabolic processes. Further studies are required to investigate the conditions in which SNP produces NO and cyanide group and the mechanism behind it.

Conclusions

The tomato seedlings can well be adapted to the stress caused by BA when supplied with SNP. Solanum lycopersicum recorded a better growth and development in combined treatments, i.e. BA+SNP. SNP minimized the damage caused by BA and protect cells from oxidative stress by inducing antioxidant enzymes. SNP ameliorates the toxic effect of BA and buttresses the defense system of the plant for better survival.

Acknowledgments

The authors are thankful to the University Grant Commission, New Delhi, and University of Allahabad for providing financial assistance to Kavita Yadav.