Effects of the application of exogenous NO at different growth stage on the physiological characteristics of peanut grown in Cd-contaminated soil

Abstract

A pot experiment was conducted to study the effects of sodium nitroprusside (SNP, a nitric oxide [NO] donor) on cadmium (Cd) toxicity in peanut plants. SNP solution was poured into Cd-contaminated soil at sowing, seedling, flowering, and pod setting stages, respectively. The results showed that exogenous NO increased the biomass and yield of peanuts and improved chlorophyll content, photosynthesis (P_n), and transpiration (T_r). Cd-induced oxidative damages were eliminated by exogenous NO, reflected by decreased accumulation of superoxide anion (), hydrogen peroxide (H₂O₂), and malondialdehyde (MDA). Moreover, exogenous NO increased Cd-decreased activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and soluble protein and proline contents also reached a normal level. Furthermore, exogenous NO improved the uptake of nutrient elements and reduced Cd accumulation in the roots, shoots, and kernels under Cd stress. These data suggest that SNP application at sowing and seedling stages had better alleviation effects on Cd toxicity in peanut plants.

Keywords:

• alleviation effects
• cadmium (Cd)
• exogenous NO
• peanut (Arachis hypogaea L.)
• SNP

Abbreviations
CAT	=	catalase
Cd	=	cadmium
H2O2	=	hydrogen peroxide
MDA	=	malondialdehyde
NO	=	nitric oxide
	=	superoxide anion radical
POD	=	peroxidase
ROS	=	reactive oxygen species
SNP	=	sodium nitroprusside
SOD	=	superoxide dismutase

1. Introduction

Cadmium (Cd) is a heavy metal widely recognized as a very dangerous environmental pollutant due to its high toxicity to both plants and animals (Xiong et al. 2010; Magdalena et al. 2011). It can be readily absorbed by plant roots and concentrated by many cereals, potatoes, vegetables, and fruits (Wagner 1993). Daily consumption of crop products containing high Cd level results in increased toxic accumulation in organs, such as the kidneys and liver, and may impair their functions (Zhang et al. 2011). Numerous experimental studies have demonstrated the highly toxic effects of Cd on many plant metabolic processes such as photosynthesis (P_n), respiration, nitrogen metabolism, transpiration (T_r), and nutrient uptake (Sanita di Toppi & Gabbrielli 1999; Clemens 2006). At the cellular level, Cd has numerous negative effects, such as membrane distortion, production of reactive oxygen species (ROS), inhibition of enzyme activities, and ion leakage (Romero-Puertas et al. 2004; Hsu & Kao 2007). To survive against Cd stress, plants have evolved protective mechanisms of mitigating and repairing its damages, including increased antioxidant capacity, metal exclusion, active excretion, restricted distribution of toxic metal in sensitive tissue, metal binding to the cell wall, chelation by organic molecules, compartmentalization in vacuoles, and the regulation of root cell wall composition (Wang et al. 2008; Xiong et al. 2009).

Efforts have been made to develop practical techniques for alleviating the Cd stress on plants. One of the strategies is to develop or select the species or genotypes with high Cd tolerance. Moreover, the application of some plant growth regulators also plays important roles in activating the defense system of the plants against Cd stress, such as supply with salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA), gibberellins (GA), silicon (Si), and nitric oxide (NO). As a gaseous signaling molecule in plants, NO plays significant roles in regulating many crucial physiological processes (Besson-Bard et al. 2008). Sodium nitroprusside (SNP) was often used as a source of NO in many studies. The effects of exogenous NO alleviating Cd toxicity were reported in different plant species. For instance, Cd-induced oxidative stress on rice and sunflower leaves was alleviated by the addition of exogenous NO (Hsu & Kao 2004; Laspina et al. 2005). Similarly, Cd-induced oxidative stress on wheat roots was alleviated by supplementation with exogenous NO (Singh et al. 2008). Xiong et al. (2009) demonstrated that exogenous NO enhanced Cd tolerance of rice by increasing the pectin and hemicellulose content in the cell wall of roots. In addition, our previous study also indicated that NO supply can alleviate Cd toxicity by increasing antioxidant capacity, improving uptake of nutrition elements, and restricting Cd translocation from roots to shoots in perennial ryegrass (Wang et al. 2013).

In China, peanut is a kind of edible food, oilseed crop, and raw material used as food additives, with a high nutritional and economic value. In the shortage of protein and serious cases of oil supply security, peanut is expected to be of more use in the future (Wan 2003). It is known that peanut is one of four major oil crops with high capacity to tolerate Cd stress and can accumulate high Cd level in vegetative parts and seeds (McLaughlin et al. 2000; Shi & Cai 2009). Some phosphorus and trace element fertilizers may contain elevated amounts of Cd and repeated uses of the fertilizers at high rates over time may increase Cd uptake by plants (Huang et al. 2003). A wide use of phosphorous fertilizers could accumulate Cd in the peanut field. Therefore, it is necessary to find an approach to alleviate Cd toxicity in peanut plants and decrease Cd accumulation in peanut seeds. To our knowledge, most of the studies on exogenous NO alleviating Cd toxicity were carried out in a hydroponic experiment. However, little knowledge is reported regarding the effects of application of exogenous NO in Cd-contaminated soil on plant growth and development in the whole growth period, and further, or how the growing plants cope with Cd toxicity through physiological and biochemical adaptations.

In this study, we poured SNP solution in Cd-contaminated soil to supply NO for peanut plants. SNP solution was supplied at sowing, seedling, flowering, and pod setting stages, respectively. This paper reported the toxic effects of Cd on the growth and development in peanut plants, the responses of plant to Cd stress, and the effects of application of exogenous NO at different growth stages on Cd toxicity in peanut plants. The aim of this study is to understand the mechanism of Cd detoxification in peanut plants, and explore the proper growth stages of supplying NO to promote plant growth in the fields with Cd contamination.

2. Materials and methods

2.1. Plant materials, growth, and treatment conditions

A pot experiment was conducted on brown clay loam soils (a soil group in Chinese genetic soil classification; named Hapli-Udic Argosols in Chinese soil taxonomy), at the College of Resources and Environment, Shandong Agricultural University, China. Soil in pots was taken from uncontaminated topsoil (0–20 cm) in Shandong Agricultural University's experimental field. The soil contains 14.1 g kg⁻¹ organic matter, 0.622 g kg⁻¹ total nitrogen, 46.5 mg kg⁻¹ available nitrogen, 22.5 mg kg⁻¹ available phosphorus, and 106.1 mg kg⁻¹ available potassium. Soil subsamples were mixed with appropriate amounts of analytical grade solid CdCl₂ in order to add 50 mg kg⁻¹ Cd of soil (oven-dry basis). Basal fertilizers applied were compound fertilizer, which were provided by the Shandong Kingenta Ecological Engineering Co., Ltd. The nutrient content of compound fertilizer was 15% N, 15% P₂O₅, and 15% K₂O. The mass of the fertilizer applied in each pot was 2.0 g, and the fertilizer was thoroughly mixed with the soil. Every pot (diameter: 30 cm and height: 40 cm) was full with 7.5 kg of soil.

Peanut seeds (Arachis hypogaea L.) were surface sterilized with 0.5% (v/v) sodium hypochlorite and washed with distilled water, then sowed on 28 April 2012. Seeds were sown in plastic pots (eight plants per pot). The pots were arranged in randomized block designs with three replicates. After the seedlings emerged, the pots were lessened to five seedlings per pot. During the growing season, plants were managed under commonly used agronomic and irrigation practices.

The experimental design is given as follows: CK, no Cd contaminated soil; Cd, 50 mg kg⁻¹ Cd-contaminated soil; S1, SNP solutions (200 µM, 50 mL) were poured into 50 mg kg⁻¹ Cd-contaminated soil at sowing stage; S2, SNP solutions (200 µM, 50 mL) were poured into 50 mg kg⁻¹ Cd-contaminated soil at seedling stage; S3, SNP solutions (200 µM, 50 mL) were poured into 50 mg kg⁻¹ Cd-contaminated soil at flowering stage; S4, SNP solutions (200 µM, 50 mL) were poured into 50 mg kg⁻¹ Cd-contaminated soil at pod setting stage. SNP was used as a donor of NO. At every growth stage, SNP was made into 200 µM of SNP solution in the lab. Before the SNP solution (200 µM, 50 mL) was poured into soil, it was stored in the dark, and the time interval of twice pour actions was five days.

2.2. Determination of chlorophyll content

The chlorophyll content was determined according to the method of Knudson et al. (1977). Young leaves were taken at the pod setting stage. Fresh leaves (0.5 g of fresh weight) were extracted in 2 mL 95% ethanol for 24 h in the dark, and the extracted solution was analyzed. The amounts of chlorophyll a, chlorophyll b, and carotenoids were determined spectrophotometrically (SHIMADZU UV-2450, Kyoto, Japan), by reading the absorbance at 665, 649, and 470 nm, respectively. The chlorophyll content results are expressed as milligram per gram fresh weight (mg g⁻¹ FW).

2.3. Determinations of P_n and T_r

Net P_n and T_r were monitored in vivo with a portable P_n system (LI-6400, LICOR, Lincoln, NB, USA). Measurements were made on three plants, in three replications per plant. The leaves were fully formed and located in the upper third part of the crown, oriented toward the south. Measurements were done in sunny and clear weather, in the period between 09:00 and 11:00 h. All the measurements were taken at a constant airflow rate of 500 µmol s⁻¹ and at saturation irradiance with incident photosynthetic photon flux density of 1200 µmol m⁻² s⁻¹. The ambient CO₂ concentration was approximately 350 cm³ m⁻³ and the temperature was approximately 25°C.

2.4. Determination of the generation rate

For the measurement of the generation rate, fresh leaf samples (0.3 g) were ground in liquid N₂ and extracted in 3 mL of ice-cold 50 mM sodium phosphate buffer (PBS) (pH 7.0). The generation rate was determined by monitoring the A₅₃₀ of the production of the hydroxylamine reaction following the modified method as described by He et al. (2005). One milliliter supernatant of fresh leaf extraction was added to 0.9 mL 65 mM phosphate buffer solution (pH 7.8) and 0.1 mL 10 mM hydroxyl ammonium chloride. The reaction was incubated at 25°C for 35 min. A 0.5 mL solution from the above reaction mixture was then added in 0.5 mL 17 mM sulfanic acid and 0.5 mL 7.8 mM α-naphthylamine solution. After 20 min of reaction, 2 mL of ether was added into the above solution, and then mixed well. The solution was centrifuged at 1500×g at 4°C for 5 min. The absorbance of the pink supernatant was measured at 530 nm with the spectrophotometer. Absorbance values were calibrated to a standard curve generated with known concentrations of HNO₂.

2.5. Determination of H₂O₂ concentration

Fresh samples (1.0 g) were homogenized in 2 mL ice-cold acetone. Titanium reagent (2% TiCl₂ in conc. HCl) was added to a known volume of extract supernatant to give a Ti (IV) concentration of 2%. The Ti–H₂O₂ complex, together with unreacted Ti, was then precipitated by adding 0.2 mL 17 M ammonia solution for each 1 mL of extract. The precipitate was washed five times with ice acetone by resuspension, drained, and dissolved in 1 M H₂SO₄ (3 mL). The absorbance of the solution was measured at 410 nm against blanks, which had been prepared similarly but without plant tissue (Patterson et al. 1984).

2.6. Assay of antioxidant enzyme activities

For extraction of antioxidant enzymes, leaves and roots were homogenized with 50 mM Na₂HPO₄–NaH₂PO₄ buffer (pH 7.8) containing 0.2 mM EDTA and 2% insoluble polyvinylpyrrolidone (PVP) in a chilled pestle and mortar. The homogenate was centrifuged at 12,000×g for 20 min and the resulted supernatant was used for the determination of enzyme activities. The whole extraction procedure was carried out at 4°C. Protein content was estimated according to the method of Bradford (1976). Spectrophotometric analysis was conducted on a SHIMADZU UV-2450 spectrophotometer (Kyoto, Japan).

Superoxide dismutase (SOD) (EC 1.15.1.1) activity was measured in terms of its capacity to inhibit photochemical reduction of nitroblue tetrazolium (NBT), as described by Giannopolitis and Ries (1977). The reaction mixture (3 mL) contained 50 mM phosphate buffer (pH 7.8), 0.1 mM EDTA, 130 mM methionine, 0.75 mM NBT, 0.02 mM riboflavin, and 0.1 mL enzyme extract. Riboflavin was added as the last component and the reaction was initiated by placing the tubes under two 20 W fluorescent lamps. The reaction was terminated after 10 min by removing the reaction tubes from the light source. Nonilluminated and illuminated reactions without supernatant served as calibration standards. Absorbance of the reaction mixture was read at 560 nm. One unit of SOD activity (U) was defined as the amount of enzyme required to cause 50% inhibition of the NBT photoreduction rate.

Analysis of guaiacol peroxidase (POD, EC 1.11.1.7) capacity was based on the oxidation of guaiacol using hydrogen peroxide (Zhang & Kirham 1994). The enzyme extract (0.02 mL) was added to the reaction mixture containing 0.02 mL guaiacol solution and 0.01 mL hydrogen peroxide solution in 3 mL of phosphate buffer solution (pH 7.0). The addition of enzyme extract started the reaction and the increase in absorbance was recorded at 470 nm for 5 min. Enzyme activity was quantified by the amount of tetraguaiacol formed using its molar extinction coefficient (26.6 mM⁻¹ cm⁻¹).

Catalase (CAT, EC 1.11.1.6) activity was determined using the method of Cakmak and Marschner (1992), with minor modifications. The reaction solution (3 mL) consisted of 100 Mm phosphate buffer (pH 7.0), 0.1 µM EDTA, 0.1% H₂O₂, and 0.1 mL enzyme extract. The reaction was initiated by adding the enzyme extract. The decrease of H₂O₂ was monitored at 240 nm for at least 3 min and quantified by its molar extinction coefficient (36 M⁻¹ cm⁻¹).

2.7. Determination of lipid peroxidation

Lipid peroxidation was determined by measuring malondialdehyde (MDA), a major thiobarbituric acid reactive species (TBARS) and product of lipid peroxidation (Heath & Packer 1968). Samples (0.2 g) were ground in 3 ml of trichloroacetic acid (TCA) (0.1%, w/v). The homogenate was centrifuged at 10,000×g for 10 min and 1 mL of the supernatant fraction was mixed with 4 mL of 0.5% (w/v) thiobarbituric acid (TBA) in 20% (w/v) TCA. The mixture was heated at 95°C for 30 min, chilled on ice, and then centrifuged at 10,000×g for 5 min. The absorbance of the supernatant was measured at 532 nm. The value for nonspecific absorption at 600 nm was subtracted. The amount of MDA was calculated using the extinction coefficient of 155 mM⁻¹ cm⁻¹ and expressed as nmol g⁻¹ FW.

2.8. Soluble protein and proline assay

Proteins were estimated by Bradford's (1976) method. Fresh leaves (0.5 g) were homogenized in 1 mL phosphate buffer (pH 7.0). The crude homogenate was centrifuged at 5000×g for 10 min. Half milliliter of freshly prepared TCA was added and centrifuged at 8000×g for 15 min. The debris was dissolved in 1 mL of 0.1 N NaOH and 5 mL of Bradford's reagent was added. Absorbance was recorded photometrically at 595 nm (SHIMADZU UV-2450, Japan) using bovine serum albumin as a standard.

Proline concentration was determined using the method of Bates et al. (1973). Fresh leaves (0.3 g) were homogenized in 10 mL of 30% aqueous sulphosalicylic acid. The homogenate was centrifuged at 9000×g for 15 min. A 2 mL aliquot of the supernatant was mixed with an equal volume of acetic acid and acid ninhydrin (1.25 g ninhydrin in 30 mL acetic acid and 20 mL of 6N H₃PO₄) and incubated for 1 h at 100°C. The reaction was terminated in an ice bath and extracted with 4 mL of toluene. The extract was vortexed for 20 s and the chromatophore-containing toluene was aspirated from the aqueous phase and absorbance determined photometrically at 520 nm (SHIMADZU UV-2450, Japan) using toluene for a blank.

2.9. Determination of Cd and mineral element concentrations

At harvest, the roots, shoots, and kernels were separated and oven-dried for 30 min at 105°C, then at 70°C till the materials reach their constant weights. The dried tissues were weighed and ground into powder for the determination of Cd and mineral element concentrations, which was measured by the flame atomic absorbance spectrometry (SHIMADZU AA-6300, Kyoto, Japan) after digestion with mixed acid [HNO₃+HClO₄ (3:1, v/v)] (Wang et al. 2013).

2.10. Statistical analysis

Statistical analyses were carried out by analysis of variance (ANOVA) using SAS software (SAS Institute, Cary, NC). Differences between treatments were separated by the least significant difference (LSD) test at a 0.05 probability level.

3. Results

3.1. Biomass and yield

Table 1 shows the biomass, yield, and water content of peanut at harvest time. Compared with CK, the reduction of root and shoot biomass grown in 50 mg kg⁻¹ Cd soil was 38.5% and 15.6%, respectively. Yield, kernel yield, and pods of Cd-treated plants were decreased by 29.6%, 31.7%, and 30.0%, respectively. In addition, Cd stress decreased the water content in roots and shoots significantly. Addition of exogenous NO at sowing, seedling, and flowering stages improved Cd-induced decrease of the biomass, yield, and water content, especially supplying NO at sowing and seedling stages. However, the application of NO at pod setting stage did not have an obvious effect on plant biomass, yield, and water content.

Table 1. Effects of the application of exogenous NO at different growth stages (S1, sowing stage; S2, seedling stage; S3, flowering stage; S4, pod setting stage) on biomass, yield, and water content of peanut plants grown in 50 mg kg⁻¹ Cd-contaminated soil at harvested time.

Treatments	Root biomass (g plant^−1 DW)	Shoot biomass (g plant^−1 DW)	Yield (g pot^−1 DW)	Kernel yield (g pot^−1 DW)	Pods per plant	Water content in roots (%)	Water content in shoots (%)
CK	0.39±0.01a	2.89±0.13a	62.37±4.23a	45.78±1.41a	13.33±0.58a	76.90±1.18a	75.33±2.01a
Cd	0.24±0.01d	2.44±0.17b	43.89±3.01c	31.27±2.14c	9.33±0.58c	70.53±1.63b	67.83±1.88b
S1	0.36±0.01b	2.89±0.11a	62.63±5.63a	44.77±3.19a	13.33±0.58a	76.10±1.56a	74.97±2.81a
S2	0.39±0.02a	3.12±0.12a	64.50±1.97a	45.53±1.37a	13.67±0.58a	76.80±1.54a	76.37±1.07a
S3	0.32±0.02c	2.72±0.15ab	52.53±2.51b	37.43±0.65b	10.67±0.58b	72.73±0.81b	69.97±1.37b
S4	0.25±0.01d	2.42±0.18b	46.37±2.90bc	33.73±1.32c	9.67±0.58bc	70.70±1.82b	67.87±1.99b

Notes: Values are the mean±SD of three replicates. The values followed by the different letter show statistically significant differences at p < 0.05.

3.2. Chlorophyll content

At pod setting stage, Cd-treated plants showed a significant decrease in contents of chlorophyll a, chlorophyll b, and carotenoids, while chlorophyll a/b was not affected significantly (Figure 1). SNP solutions added in soil at sowing, seedling, and flowering stages increased Cd-decrease in chlorophyll a, chlorophyll b, and carotenoids contents, especially the application of exogenous NO at seedling stage. NO supplementation at seedling stage increased chlorophyll a, chlorophyll b, and carotenoids by 50.1%, 68.3%, and 124.7%, respectively, compared with Cd treatment. However, SNP addition at pod setting stage had no effect on the chlorophyll contents of plants grown in Cd-contaminated soil. In addition, compared with CK, SNP supplementation at the four growth stages did not obviously change chlorophyll a/b.

Figure 1. Effects of the application of exogenous NO at different growth stages (S1, sowing stage; S2, seedling stage; S3, flowering stage; S4, pod setting stage) on chlorophyll a (A), chlorophyll b (B), chlorophyll a/b (C), and carotenoids (D) of peanut plant grown in 50 mg kg⁻¹ Cd-contaminated soil. The contents of chlorophyll in peanut leaves were investigated at pod setting stage. Values are the mean of three replicates. Bars with different letters are significantly different at p <0.05.

3.3. Photosynthesis and Transpiration

Nutrient uptake came to a climax at pod setting stage, when the pegs interposed into the soil to have pod, and the P_n was most vigorous (Zhang et al. 2012). Figure 2 shows P_n and T_r of peanut at pod setting stage. Cd stress induced an obvious decrease of P_n and T_r as relative to the normal plants. The decrease in P_n and T_r was alleviated by the addition of NO at sowing, seedling, and flowering stages significantly, especially NO was supplied at sowing and seedling stages. However, the addition of NO at pod setting stage had no obvious change on P_n and T_r compared with Cd treatment.

Figure 2. Effects of the application of exogenous NO at different growth stages (S1, sowing stage; S2, seedling stage; S3, flowering stage; S4, pod setting stage) on photosynthesis and transpiration of peanut plants grown in 50 mg kg⁻¹ Cd-contaminated soil. Photosynthesis and transpiration of peanut plants were investigated at pod setting stage. The contents were investigated at pod setting stages. Values are the mean of three replicates. Bars with different letters are significantly different at p <0.05.

3.4. production rate, H₂O₂accumulation, and MDA content

Figure 3 shows production rate, H₂O₂ accumulation, and MDA content of peanut leaves at pod setting and maturity stages. At pod setting stage, Cd stress enhanced the production rate compared with CK (Figure 3A). Addition of exogenous NO at sowing and seedling stages decreased Cd-induced production rate of significantly. However, NO supplied at pod setting stage did not reduce Cd-increased production rate. Similar trends of production rate were detected at maturity stage.

Figure 3. Effects of the application of exogenous NO at different growth stages (S1, sowing stage; S2, seedling stage; S3, flowering stage; S4, pod setting stage) on generation rate (A), H₂O₂ (B), and MDA content (C) in leaves of peanut grown in 50 mg kg⁻¹ Cd-contaminated soil. These indexes were investigated at seedling, flowering, and pod setting stages. Values are the mean of three replicates. Bars with different letters are significantly different at p <0.05.

Compared with the control, Cd stress caused a significant increase in H₂O₂ accumulation at pod setting and maturity stages (Figure 3B). Addition of exogenous NO at sowing and seedling stages decreased Cd-induced accumulation of H₂O₂ at pod setting and maturity stages. Compared with Cd treatment, NO supplementation at flowering stage reduced H₂O₂ accumulation at pod setting stage but had no change at maturity stage. In addition, NO supplied at pod setting stage did not inhibit Cd-caused increase in H₂O₂ accumulation at pod setting and maturity stages.

MDA, a product of lipid peroxidation, has been considered an indicator of oxidative damage. MDA content was investigated at pod setting and maturity stages (Figure 3C). Cd stress induced MDA accumulation at pod setting and maturity stages compared with CK. At pod setting stage, the application of exogenous NO at sowing, seedling, flowering, and pod setting stages did not decrease Cd-induced MDA accumulation. At maturity stage, compared with Cd-stressed plants, SNP supplementation at sowing and seedling stages inhibited MDA content. However, the addition of exogenous NO at flowering and pod setting stages had no effect on MDA accumulation.

3.5. Antioxidant enzymes

Activities of several representative antioxidant enzymes, including SOD, POD, and CAT in peanut seedlings were determined to assess the physiological mechanism of exogenous NO in regulation of these antioxidant enzymes upon Cd stress. As shown in Figure 4, compared with CK, Cd stress decreased SOD, POD, and CAT activities dramatically when investigating SOD activity at pod setting and maturity stages. However, the application of exogenous NO at sowing and seedling stages increased Cd-decreased SOD activity of peanut plants (Figure 4A). In addition, SNP solution supplied at flowering and pod setting stages had no obvious effect on increasing Cd-decreased activity of SOD at pod setting and maturity stages.

Figure 4. Effects of the application of exogenous NO at different growth stages (S1, sowing stage; S2, seedling stage; S3, flowering stage; S4, pod setting stage) on activities of superoxide dismutase (SOD) (A), peroxidase (POD) (B), and catalase (CAT) (C) in leaves of peanut grown in 50 mg kg⁻¹ Cd-contaminated soil. The contents of chlorophyll in peanut leaves were investigated at seedling, flowering, and pod setting stages. Values are the mean of three replicates. Bars with different letters are significantly different at p <0.05.

At pod setting stage, SNP supplementation at sowing and seedling stages enhanced Cd-decreased activity of POD activity, while the addition of exogenous NO at flowering and pod setting stages did not improve Cd-inhibited POD activity (Figure 4B). When investigating POD activity at maturity stage, the application of exogenous NO at sowing, seedling, flowering, and pod setting stages increased POD activity compared with Cd treatment, especially at seedling stage.

The application of exogenous NO at sowing, seedling, flowering, and pod setting stages increased CAT activity dramatically compared with Cd-treated plants, especially at seeding stage (Figure 4C).

3.6. Soluble protein and proline

The Cd treatment declined the soluble protein content of peanut plants to 54.0% and 56.5% at pod setting and maturity stages compare with CK, respectively (Figure 5A). SNP solutions added in soil at sowing, seedling, and flowering stages increased the content of soluble protein at pod setting stage significantly. However, the application of exogenous NO at pod setting stage did not increase Cd-decreased content of soluble protein. At maturity stage, NO application at sowing and seedling stages had obvious effects on increasing soluble protein content compared with Cd treatment. However, the addition of NO at flowering and pod setting stages did not improve the content of soluble protein.

Figure 5. Effects of the application of SNP at different growth stages (S1, sowing stage; S2, seedling stage; S3, flowering stage; S4, pod setting stage) on soluble protein (A), and proline content (B) in leaves of peanut grown in 50 mg kg⁻¹ Cd-contaminated soil. The contents of chlorophyll in peanut leaves were investigated at pod setting and maturity stages. Values are the mean of three replicates. Bars with different letters are significantly different at p <0.05.

As shown in Figure 5B, at pod setting and maturity stages, Cd stress decreased proline content significantly, compared with CK. The application of exogenous NO at sowing, seedling, and flowering stages increased Cd-decreased content of proline dramatically, especially at seedling stage. However, SNP supplementation at pod setting did not improve proline content compared with Cd treatment.

3.7. P, K, Ca, and Fe absorption

At maturity stage, P, K, Ca, and Fe concentrations in the leaves and roots were measured (Table 2). Cd stress decreased P, K, Ca, and Fe concentrations in the leaves and roots significantly compared with CK. NO addition at sowing, seedling, and flowering stages had effects on improving P and K contents in leaves and roots under Cd stress. However, the application of NO at the pod setting stage did not obviously change absorption of P and K. The addition of NO at sowing, seedling, flowering, and pod setting stages had obvious positive effects on increasing Ca concentration in leaves and roots under Cd stress. Fe concentration in leaves and roots were significantly increased by the addition of NO at sowing and seedling stages. In addition, NO supply at flowering stage increased Fe content in leaves. However, the addition of NO at pod setting stage did not increase Fe absorption.

Table 2. Effects of the application of exogenous NO at different growth stages (S1, sowing stage; S2, seedling stage; S3, flowering stage; S4, pod setting stage) on the contents of mineral nutrients (P, K, Ca, and Fe) in roots and leaves of peanut grown in 50 mg kg⁻¹ Cd-contaminated soil. The contents were investigated at maturity stages.

		Treatments
Items		CK	Cd	S1	S2	S3	S4
P (mg g^−1 DW)	Roots	35.63±2.98a	22.86±1.44bc	35.67±3.28a	35.67±3.79a	26.88±0.56b	22.26±1.57c
	Leaves	20.42±1.65a	13.16±1.12d	19.65±1.15ab	18.23±0.71b	16.18±0.98c	13.81±0.74d
K (mg g^−1 DW)	Roots	57.20±2.80a	32.97±2.28c	52.37±2.45a	53.11±2.83a	46.78±3.06b	35.34±2.90c
	Leaves	11.12±0.80a	5.19±0.19d	10.71±0.50ab	10.19±0.36b	6.78±0.34c	5.72±0.41d
Ca (mg g^−1 DW)	Roots	4.12±0.30a	2.08±0.17d	3.99±0.31a	3.45±0.22b	3.10±0.11b	2.60±0.22c
	Leaves	15.66±0.83a	5.52±0.39d	13.04±0.74b	12.92±0.91b	8.12±0.25c	7.23±0.40c
Fe (mg g^−1 DW)	Roots	1.29±0.05a	0.84±0.04d	1.11±0.10b	1.02±0.06bc	0.92±0.05cd	0.84±0.05d
	Leaves	0.55±0.15a	0.32±0.01d	0.53±0.03a	0.46±0.01b	0.42±0.01c	0.34±0.02d

Notes: Values are the mean±SD of three replicates. The values followed by the different letter show statistically significant differences at p < 0.05.

3.8. Cd accumulation in the roots, shoots, and seeds

Cd accumulation in the roots, shoots, and seeds were measured at harvest time of plants (Table 3). Cd treatment dramatically increased Cd concentration in the roots, shoots, and seeds compared with CK. In root tissue, NO addition at sowing and seedling stages reduced Cd absorption by the roots. However, NO supplementation at flowering and pod setting stages had no effect on Cd content. In the shoots, the addition of NO at the four stages decreased Cd concentration effectively. In seeds, SNP supplementation at sowing, seedling, and flowering stages had an obvious effect on inhibiting Cd accumulation. However, the application of NO at pod setting stage did not diminish Cd accumulation in the seeds.

Table 3. Effects of the application of exogenous NO at different growth stages (S1, sowing stage; S2, seedling stage; S3, flowering stage; S4, pod setting stage) on Cd concentration in the roots, shoots and kernels of peanut grown in 50 mg kg⁻¹ Cd-contaminated soil. The contents were investigated at harvest time.

	Cd concentration (mg kg^−1)
Treatments	Roots	Shoots	kernels
CK	5.13±0.21d	1.27±0.06e	0.00±0.00d
Cd	738.33±32.81a	145.00±6.56a	30.62±7.71a
S1	612.33±39.11c	92.00±7.00d	14.99±0.79c
S2	642.67±39.55bc	100.67±2.52d	15.14±0.87c
S3	689.67±18.45ab	125.33±5.86c	22.90±1.53b
S4	722.00±23.52a	135.33±6.11b	28.11±1.48ab

Notes: Values are the mean±SD of three replicates. The values followed by the different letter show statistically significant differences at p < 0.05.

4. Discussion

Increasing evidence indicates that exogenous NO plays important roles in plant tolerance to Cd stress (Hsu & Kao 2004; Laspina et al. 2005; Xiong et al. 2009; Wang et al. 2013). In our lab, Zhang et al. (2012) supplied SNP (a donor of NO) for peanut plants growing on calcareous soil and their study found that the SNP added into the soil effectively increased peanut tolerance to Fe deficiency stress. In addition, Gao et al. (2011) demonstrated that SNP solutions poured into Cd-contaminated soil alleviated Cd toxicity in seedlings of Malus hupehensis Rehd. under continuous cropping. In this study, we also supplied NO for peanut grown in Cd-contaminated soil by adding SNP solution in soil, and exogenous NO was applied at different growth stages of peanut. By investigating the physiological characteristics of peanut, we detected that the effect of the application of NO at different growth stages varied. The addition of NO at sowing and seedlings stages had better alleviation effects.

Cd stress induced detrimental effects on plant growth and development, just as reported by Wang et al. (2008) on Bechmeria nivea (L.) Guad, Qiu et al. (2013) about wheat seedlings, and Wang et al. (2013) on perennial ryegrass. However, this work presented strong evidence supporting a role for exogenous NO in increasing Cd tolerance of peanut plants. Addition of exogenous NO to 50 mg kg⁻¹ Cd-contaminated soil at sowing and seedling stages effectively alleviated Cd-induced growth inhibition in peanut plants, described as its ability to prevent inhibition of biomass and yield, increase chlorophyll content, and improve P_n, achieving a similar growth condition as control (Table 1, Figures 1 and 2). Many studies suggested that decreasing leaf chlorophyll content was one of the most general toxicity effects of Cd on plants (Chen et al. 2008, 2010), which was confirmed in this study. However, more and more researches reported that exogenous NO could alleviate Cd-induced chlorophyll synthesis inhibition (Chen et al. 2010; Wang et al. 2013). In this study, Cd-decreased chlorophyll content was recovered to the level of normal plants when peanut plants were treated with exogenous NO at sowing and seedling stages (Figure 1), accompanied by the recovery of P_n and T_r, which was severely affected by 50 mg kg⁻¹ Cd (Figure 2). This indicated that NO-mediated improvement in P_n and T_r, partly due to increasing chlorophyll synthesis, is important for increasing Cd tolerance. Carotenoids are kinds of photosynthetic pigments, which are necessary for P_n. They can transfer the absorbed light energy to the chlorophyll a and capture light energy for the chlorophyll pigment effectively. The application of exogenous NO increased Cd-decreased content of carotenoids (Figure 1D), which was responsible for the improvement of P_n. Furthermore, carotenoids are regarded as molecules providing nonenzymatic protection against the oxidative damage (Tiryakioglu et al. 2006). The NO-mediated increase in carotenoids might play an important role in alleviating ROS damage, which might account for the enhancement of Cd tolerance in peanut plants.

Numerous data from different plant species have suggested that Cd is a potent pro-oxidant, evoking oxidized stress by promoting the generation of ROS (Laspina et al. 2005; Hsu & Kao 2007; Singh et al. 2008). MDA, a product of lipid peroxidation, has been considered an indicator of oxidative damage (Wang et al. 2008). In this experiment, we investigated the three molecules to evaluate Cd-induced oxidative damage. , H₂O₂, and MDA are generated excessively in peanut plants grown in Cd-polluted soil (Figure 3). The excess formation of ROS can oxidize various cellular components (for example, proteins, lipids, and nucleic acids) and result in lipid peroxidation, membrane leakage, and enzyme inactivation, which can finally lead to oxidative damages and alteration in cell structure (Romero-Puertas et al. 2007). The growth inhibition might be partly due to enhanced production and accumulation of ROS. However, NO-treated plants counteracted oxidative damages and had protective effects against stressful condition, which was concomitant with the decreased production rate of , and the concentrations of H₂O₂ and MDA (Figure 3). In particular, the application of exogenous NO at sowing and seedling stages eliminated the ROS damages effectively. This suggested that NO functions as a signaling molecule involving in a signal transduction pathway that could activate protective effects on lipid peroxidative to Cd stress. Many studies also demonstrated that the addition of exogenous NO alleviated Cd-caused oxidative damages (Hsu & Kao 2004; Xu et al. 2010; Wang et al. 2013). By investigating activities of antioxidant enzymes, NO-mediated improvement of SOD, POD, and CAT activities played an important role in ameliorating the ROS damages, which was consistent with the report by Wang et al. (2013).

For surviving against Cd stress, plants have evolved protective mechanisms of mitigating and repairing the ROS damages caused by environmental stresses. In many studies, it was found that the function of NO alleviation in oxidative stress was attributed to induction of various ROS-scavenging enzyme activities (Laspina et al. 2005; Hsu & Kao 2007; Singh et al. 2008; Wang et al. 2013). The addition of NO to plant tissues was demonstrated to act as a signaling molecule in the induction of gene expression of the enzymes, such as CAT, APX, POD, and GR (Chen et al. 2010; Xiong et al. 2010). Our present results clearly indicated that the application of exogenous NO raised SOD, POD, and CAT activities in peanut plants under Cd stress, especially the application of NO at sowing and seedling stages. These results are in accordance with our previous study in which we demonstrated that NO counteracted Cd-induced decrease in the activities of antioxidant enzymes. Qiu et al. (2013) have also reported that NO upregulated the components of the antioxidant defense machinery activities of SOD, POD, and CAT. Improved activities of SOD, POD, and CAT in Cd-stressed peanut plants prevented lipid peroxidation and MDA production, and thus the plant cells were protected from Cd stress damage. However, NO-mediated improvement in antioxidant enzymes needed proper NO concentrations. Many studies reported that higher concentrations of exogenous NO inhibited the activities of antioxidant enzymes in Dendrobium huoshanense (Fan et al. 2012) and perennial ryegrass (Wang et al. 2013). Therefore, different NO concentrations, crop types, disposal methods, and levels of stress can bring about different changes of antioxidant enzymes.

Abiotic stress may inhibit a synthesis of some proteins and promote others (Ericson & Alfinito 1984) with a general trend of decline in the overall content. Our studies coincide with John et al. (2008) who also reported a decrease in the soluble protein content under Cd stress in Lemna polyrrhiza L. It is likely that heavy metals have induced lipid peroxidation and fragmentation of proteins due to toxic effects of ROS, which led to the reduction of protein content (Davies et al. 1987). However, NO supplementation improved Cd-decreased soluble protein content. The probable reason was that exogenous NO enhanced the antioxidant ability to eliminate Cd toxicity on peanut growth, accompanied by the improvement of protein content. In addition, the NO-induced increase in soluble protein of peanut plants grown in Cd-contaminated soil was more obvious by the addition of NO at sowing and seedling stages. Proline, an amino acid, is regarded as a kind of antioxidant. Cd-caused decrease of proline was reversed by the addition of NO. This suggested that NO mediated the enhancement of antioxidant ability in peanut plants. In addition, proline also acts as a major reservoir of energy and nitrogen, which can be used in resuming the growth (Chandrashekhar & Sandhyarani 1996) after the stress removal, resulting in that plant growth under Cd stress was recovered.

Cd toxicity has been related to the interactions between uptake and translocation of mineral nutrients in plants (Jiang et al. 2004). It has been reported that Cd toxicity in plants is affected by other ions in the nutrient solution (Sanita di Toppi & Gabbrielli 1999; Xu et al. 2010; Wang et al. 2013). Cd toxicity inhibited the uptake of P, K, Ca, and Fe by plants, disturbing intracellular ion homeostasis and exerting a toxic effect on plants (Table 2). P is an essential element in the plasma membrane, and the uptake of mineral elements by plant root is subjected to selective properties of the plasma membrane (Gussarsson 1994). Heavy metals including Cd may interfere with nutrient uptake by altering the plasma membrane permeability, and by affecting element transport processes across the membrane (Gussarsson 1994), as a result that P, K, Ca, and Fe concentrations were reduced in Cd-treated plants. Our study indicated that NO supplementation increased the absorption of P element and reduced the ROS damages, indicating that the plasma membrane permeability was enhanced, which might improve the absorption of K, Ca, and Fe. K is an essential element that plays vital roles in various aspects of plant cell growth and metabolism and is needed in large quantities (Zhu et al. 1998). Appropriate concentration of K is advantageous for the accumulation of divalent cations (Kahn & Hanson 1957). NO-increased uptake of K was important for improving the amount of divalent cations and ameliorating the effects of Cd toxicity on intracellular ion equilibrium. NO stimulates Ca release from intracellular stores (Sokolovski & Blatt 2007) but can also act as a strong stimulator of Ca influx across the plasma membrane (Lamotte et al. 2006), as a result that Ca concentrations were increased by the application of exogenous NO. The stimulative effect of NO on Fe uptake might be the consequence of a close relation between NO and Fe metabolism. Furthermore, NO has high affinity toward the Fe-containing active sites of many proteins and thus participates in regulation of Fe transport in plants (Ramirez et al. 2010). In addition, it is well known that H⁺-ATPase in the plasma membrane plays an important role in the transport of multiple ions (Palmgren & Harper 1999), and there is the investigation indicating that NO could induce H⁺-ATPase activity (Cui et al. 2010; Wang et al. 2013), which might be responsible for NO increasing absorption of P, K, Ca, and Fe in peanut plants under Cd stress. These results indicated that NO could ameliorate ion equilibrium in peanut cells under Cd stress.

Plant root is the main part that is in direct contact with Cd in the soil, and the cell walls in roots plays a significant role in heavy metal tolerance in plants (Xiong et al. 2009). Our study demonstrated that Cd concentration in the roots was more than shoots, and Cd accumulation in the kernels was the least (Table 3). Cataldo et al. (1981) had also reported that most Cd was accumulated in the roots of soybean plants. Interestingly, addition of NO at sowing and seedling stages decreased Cd concentration in the roots, shoots, and kernels of peanut grown in Cd-contaminated soil. Rodriguez-Serrano et al. (2009) found that the addition of Ca inhibited Cd accumulation in peanut plant, suggesting that the competition between Ca and Cd was for the same transporters. In this study, the application of NO increased the uptake of Ca, which might be responsible for the decreased accumulation of Cd in peanut plants. The result indicated that NO-induced increase in Ca level was important for improving Cd tolerance in peanut plants. Like essential micronutrients, Cd with similar physicochemical properties, competes with and enters the plant via transport systems operating for micronutrient acquisition (Papoyan et al. 2007). NO-mediated increase in P, K, Ca, and Fe might also account for the decrease of Cd content in the roots, shoots, and kernels of peanut plants.

5. Conclusion

In this study, SNP solution was poured into Cd-contaminated soil at different growth stages, aimed at supplying NO for peanut. The results provided evidence for NO-mediated alleviated effects on Cd toxicity in peanut plants. Application of exogenous NO applied in soil at sowing and seedling stages had obvious effects on alleviating Cd-induced inhibition on peanut growth and development. The alleviation mechanism contains: (1) increased chlorophyll content and P_n; (2) improved antioxidant ability and reduced ROS damages; (3) increased uptake of P, K, Ca, and Fe and decreased Cd accumulation. This finding may provide a new approach to protecting crops against heavy metal toxicity with supplying NO for plant at the proper time.

Acknowledgments

Great thanks were given to Pingping Yang, College of Animal Science Technology, Shandong Agricultural University, China, for her supplying instruments and patient guidance. Special acknowledgments are given to the editors and reviewers.