Introduction

Green leafy vegetables are a rich source of various health-promoting compounds such as vitamins, minerals, antioxidants, and dietary fibers and are low in calories (Umar et al. 2013a). Moreover, they have low sodium content and no trans fatty acids and saturated fat (EFSA 2008). They also contain nitrate and nitrite, which are harmful to humans when taken in excess (Gupta et al. 1999a, b, 2008; Sharma and Sharma 2012). The most important nutrient, which limits the yield of plants, is nitrogen (Ekbic et al. 2010; Xia et al. 2011) and therefore N fertilization has a key role in plant growth and development (Liu et al. 2014). However, an improper and unbalanced fertilizer application generally leads to nitrate accumulation, often to a toxic level. High rates of N fertilization increase the carotene level and decrease the vitamin C level in plants (Rakocevic et al. 2012). Moreover, increased water content increases nitrate accumulation in plants (Qiu et al. 2014), whereas increased concentration of vacuolar carbohydrate decreases nitrate accumulation (Anjana et al. 2007a).

A high-nitrate diet may cause methemoglobinemia in infants and cancer in adults (L’hirondel and L’hirondel 2002). Although nitrate is non-toxic and is involved in many life processes (Mensinga et al. 2003), part of it is transformed to nitrite and N-nitroso compounds, which have harmful effects on health (Gupta et al. 2008). Nitrite may further react with amines and amides and form cancer-causing compounds such as nitrosamines and nitrosamides (Santamaria 2006). Interest has gathered around the dietary nitrate intake because of its detrimental impact on human health (Pegg and Shahidi 2000; Cavaiuolo and Ferrante 2014).

Most of the green leafy vegetables are considered nitrophilic, as they have a tendency to absorb high levels of NO3 −, this finally results in excessive nitrate accumulation which is more than required for growth and development (Grindlay 1997). At high soil nitrate, nitrate assimilation in most plants, including Arabidopsis, occurs in the shoot (Umar et al. 2013a; Krapp et al. 2014). Nitrate transport activity has been demonstrated for about 16 out of 53 NPF (nitrate transporter 1/peptide transporter family) protein in Arabidopsis (Hsu and Tsay 2013; Leran et al. 2014). The Arabidopsis gene Chl 1 is identified as the first gene to encode nitrate transporter from higher plants (Tischner 2005).

Vegetables provide almost 40–92 % of the average daily intake (ADI) of nitrate (Ximenes et al. 2000; Giola et al. 2013) and a daily vegetable diet supplies about 72–94 % of the total nitrate N to humans (Dich et al. 1996). Thus, the highest level of nitrate is supplied to humans by green leafy vegetables (Prasad and Chetty 2008); the other sources include air and drinking water (Umar et al. 2013b). According to the US Environmental Protection Agency, the limit for daily intake of nitrate by man is 7.0 mg kg−1 body weight. In 1995, the European Commission’s Scientific Committee for Food (SCF) proposed 3.65 mg kg−1 body weight as the nitrate intake limit per day (SCF 1997), whereas the Joint Expert Committee on Food and Agriculture in the WHO suggested an ADI range of 0–3.7 mg kg−1 body weight (Santamaria 2006).

Several factors, including the environmental, agricultural, and genetic factors such as temperature, humidity, light intensity, photoperiod, diurnal rhythm, forms and doses of N, genotypic variability, presence of other macro and micronutrients, and the use of insect and pest repellents modulate the ill effects of nitrate uptake and accumulation (Santamaria et al. 2001). Light intensity and nitrogen fertilization along with many nutrients are the major factors that influence N accumulation in plants (Cometti et al. 2011; Ozturk et al. 2013). Nitrate content is supposed to vary within plant species, cultivars, and genotypes with different ploidy levels (Blom-Zanstra 1989) and also in different plant parts (Anjana et al. 2007b). In a decreasing order of nitrate content, different vegetable parts may be arranged as: petiole > leaf > stem > root > inflorescence > tuber > bulb > fruit > seed (Santamaria et al. 1999).

Most of the nitrate-accumulating leafy vegetables belong to families Brassicaceae, Amaranthaceae and Chenopodiaceae, and some to Asteraceae and Apiaceae (Jana and Moktan 2013). Brassica juncea (L.) Czern. of the Brassicaceae, generally known as Indian mustard, leaf mustard, mustard green, or brown mustard, is an economically important perennial herb. It has a good nutritional value and is cultivated as a vegetable and an oilseed crop. It follows a C3 mechanism, prefers bright sunlight, and flourishes well in irrigated or rainfed fertile soil. It grows best during the cool winter season (October–March) and responds effectively to the addition of nitrogen, with 30 % or more gain in yield (Squire 1990).

In view of the widespread use of leafy vegetables, the present study was planned to explore the nitrate accumulation pattern in different varieties of B. juncea used as leafy vegetable, and to find out the effect of various doses of nitrogen fertilizers on nitrate accumulation in different plant parts at varied ontogenetic age.

Materials and methods

Two field experiments were conducted in October 2011 and 2012 in the Herbal garden of Jamia Hamdard, New Delhi. The seeds were procured from Seed Division (NBPGR) and the Genetics and Oilseed division of IARI, New Delhi.

Genotype screening for nitrate content

Preliminary experiment was conducted in October 2011 with the aim to screen different genotypes for their nitrate-accumulating capacity. In the first experimental trial, 10 genotypes of B. juncea (listed in Table 1) were grown in triplicate. A field plot of 2 m × 2 m was prepared and seeds were sown in the first week of October. Prior to sowing, proper ploughing of plots was done and a uniform basal dose of NPK, Zn, and S was given to plots @ 120, 30, 80, 25, and 40 kg ha−1, using Urea, Single super phosphate, Murate of potash, and Zinc sulphate as sources. Soil analysis was done prior to sowing and after giving basal treatment to the soil. The soil texture was loamy sand (83.6 % sand, 6.8 % silt, and 9.6 % clay, pH 7.1), with available nitrogen (128 mg kg−1 of soil), phosphorous (4 mg kg−1), and potassium (158 mg kg−1 of soil). A population of 10–12 plants per plot was maintained by periodical thinning of the seedlings.

Table 1 List of Brassica genotypes used in the study
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Sampling

Sampling was done when plants had attained a commercially consumable size. These were then screened to identify the high-nitrate-accumulating (HNA) and low-nitrate-accumulating (LNA) genotypes. Samples were collected from Experiment 1 at the 3- and 6-week stages of seedlings in order to estimate nitrate content in the leaf. Diurnal rhythm of nitrate accumulation was studied at the 4-week stage, with samples collected thrice a day (at 7:00 am, 1:00 pm, and 7:00 pm).

The second experiment, conducted in 2012, was based on the results of the first experiment. Two genotypes, viz. RLM 619 and Pusa Bold, identified as HNA and LNA genotypes, respectively, were selected for further investigation. This experiment was focused on the effect of N fertilization on nitrate content in different plant parts of both the genotypes. Using the randomized complete block design, 24 plots of 4 m × 3 m were prepared for both genotypes (RLM-619 and Pusa Bold) designated as G5 and G8, respectively. Prior to sowing, a basal treatment of phosphorus, potassium, zinc, and sulphur was given to the sandy loam soil @ 30, 80, 40, and 25 kg per hectare, together with nitrogen (N) given @ 0, 40, 80, and 120 kg per hectare. These treatments were designated as T1, T2, T3, and T4, respectively, where T1 was the control. Urea, Single super phosphate, Murate of potash, Calcium sulphate, and Zinc sulphate were used as sources. Seeds of both the genotypes were sown 1–1.5 cm deep in the soil in triplicates on 5th October, 2012. Thinning and weeding of seedlings were done periodically. Crops were irrigated twice a week, based on the soil moisture content.

A third experiment with respect to N fertilization under light and shade condition was conducted in order to understand the role of light intensity on nitrogen remobilization. To study the nitrate-distribution pattern, sampling was done in the morning at 8:00–9:30 am. To analyze the diurnal rhythm of N fertilization effect on nitrate content in HNA and LNA genotypes, samples were collected thrice a day. A separate sampling was done for the light–shade experiment.

Clean plastic bags containing fresh samples were labeled, sealed, and placed in an ice box to bring to the laboratory. The samples were washed with double-distilled water, dried with the help of tissue paper, and separated into leaf, stem, and petiole. For nitrate estimation, fresh plants were weighed, cut into small parts, and stored at 4 °C till their processing for analysis.

Methods used

Extraction and estimation of nitrate

Using the method of Grover et al. (1978), 0.1 g of fresh plant material, cut into small pieces, was added with 50 mg of activated charcoal and 50 ml distilled water, free from nitrate, and then boiled on a hot plate for 10 min to extract the nitrate. Filtration was done through Whatman No. 42 filter paper and the residue on filter paper was washed 3–4 times with 10 ml of hot water each time. The flask was allowed to cool down to room temperature and the volume was made to 100 ml. Nitrate in these extracts was estimated by the chemical reduction procedure, following the Downes’s improved reduction hydrazine method (Downes 1978). An aliquot of 0.6 ml of the sample was drawn, mixed with 0.1 ml each of catalyst (0.0354 g CuSO4·5H2O + 0.9 g ZnSO4·7H2O in one liter of water), sodium hydroxide (1 N), and hydrazine sulphate (1.71 g l−1) solution in a series. The reduction time was 7.5 min at 33 °C. The final reagent contents were: Cu2+—1.0 mg l−1, Zn+2—20.0 mg l−1, and hydrazine sulphate—190 mg l−1. Following the reduction of nitrate to nitrite, it was diazotized by adding 0.32 ml of sulphanilamide (10 g in 1 liter; 10 % hydrochloric acid) and 0.1 ml of naphthyl ethylene diamine dihydrochloride (1 g l−1) and the pink-colored solution was examined on a UV–Vis Spectrophotometer 119 (Systronic, India) at 540 nm.

Statistical analysis

The data obtained as mean ± SE in three independent experiments were worked out by One-way Anova, using Graphpad Instat and Tukey–Krammer Posttest at P < 0.05 to compare significant differences among treatment means.

Results

Comparative status of nitrate content in ten genotypes

Different genotypes of Brassica showed different patterns of nitrate accumulation, as is evident from Fig. 1. At 3-week stage, nitrate content was within safe limits in all genotypes except for G5, in which it exceeded ADI by about 2 % only. Among all the genotypes studied, the maximum (2266.32 ± 22.18 mg kg−1 fresh wt) and the minimum (1286.64 ± 14.62 mg kg−1 fresh wt) nitrate contents were found in genotypes G5 and G8, respectively. Nitrate estimation was repeated at 6-week stage in order to authenticate the results of the 3-week stage. At 6-week stage, the nitrate content exceeded the ADI manifolds in both G5 and G8, with a value of 4509.76 ± 32.38 mg kg−1 fresh wt of leaves in G5 and 3299.48 ± 25.02 mg kg−1 fresh wt of leaves in G8.

Fig. 1
figure 1

Genotypic variation in nitrate content in the leaves of 10 Brassica genotypes (designated as G1–G10) at 3- and 6-week stages. Bars with different letters are significantly (P < 0.05) different from each other in a single growth stage (Tukey–Krammer Posttest)

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Diurnal variation of nitrate content

The pattern of nitrate accumulation varied among different genotypes. A decrease in nitrate content from morning to noon was common in all the genotypes. However, the decrease was comparatively less in genotypes G3, G4, G5, G7, and G8 than in G1, G2, G6, G9, and G10. Similarly, an increase in nitrate content from noon to evening was common to all the genotypes, and the level of nitrate content was greater in the evening samples than in the morning samples in most of the genotypes. The highest nitrate accumulation was observed in G5 with a value of 2910.04 ± 27.09 mg kg−1 fresh wt of leaves. Different genotypes exhibited different nitrate accumulation pattern, as shown in Fig. 2.

Fig. 2
figure 2

Diurnal variation in nitrate content in the leaves of 10 Brassica (G1–G10) genotypes at 4-week stage. Bars with different letters are significantly (P < 0.05) different from each other during a single sampling time (Tukey–Krammer Posttest)

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In general, nitrate content of leaves was higher in the evening than in the morning or noon samples. Observation on diurnal variation of nitrate content at a 4-week stage revealed that among the ten genotypes nitrate accumulation was minimum during the noon time, showing the importance of light intensity in the regulation of nitrate content in leaves. Light intensity showed a negative correlation with nitrate accumulation, as nitrate content was lowest at the noon. Among the genotypes, nitrate accumulation was highest in G5 and lowest in G8 at different timings of the day (Fig. 2). The mean nitrate content was 2448.48 ± 20.55 and 1395.52 ± 12.93 in the morning, 2209.92 ± 13.00 and 1083.21 ± 24.14 at noon, and 2910.04 ± 27.09 and 1317.06 ± 25.62 mg kg−1 fresh wt of leaves in the evening in G5 and G8 genotypes, respectively.

Effect of N fertilization on HNA and LNA genotypes

On the basis of experiment 1, genotypes G5 and G8 were identified as having the highest and the lowest nitrate contents, respectively. The nitrate contents of these genotypes decreased with increase in N doses up to treatment T3, beyond which (at T4) it showed a non-significant increase, as depicted in Fig. 3. Nitrate accumulation was the maximum in petioles, followed by leaves and then the stem in both the genotypes, with G5 exceeding the permissible ADI.

Fig. 3
figure 3

Nitrate accumulation pattern in different plant parts of screened genotypes at the 3-week stage. Bars with different letters are significantly (P < 0.05) different from each other in a single plant part of a single variety (Tukey–Krammer Posttest)

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In the petiole of G5 plants, the highest value of nitrate content (2785.86 ± 18.35 mg kg−1fresh wt) was seen in the control (T1), and the lowest (2469.40 ± 24.95) in treatment T3. Similarly, in the petiole of G8 plants, the lowest amount of nitrate (1988.51 ± 26.23 mg kg−1fresh wt of petiole) was recorded at T3, as compared to the control (2248.543 ± 20.34). In the G5 leaf, N content in control plants was 2421.15 ± 23.99 mg kg−1fresh wt of leaf, which successively decreased with increasing N dose up to treatment T3 (2010.93 ± 18.95), and then increased non-significantly in treatment T4. In the G8 leaf, the lowest nitrate content (1130.276 ± 27.48 mg kg−1fresh wt) was again at T3, compared with the control (1361.314 ± 20.02 mg kg−1fresh wt). A similar trend was observed for the stem. In G5 stem, it was 1586.61 ± 13.83 mg kg−1fresh wt at the control, the lowest (1435.23 ± 14.81) being at T3. In the case of G8, nitrate content in the stem was 1189.693 ± 17.86 mg kg−1 fresh wt at T1, and 852.09 ± 15.67 mg kg−1 fresh wt at treatment T3. Thus, the stem accumulated a relatively lesser nitrate than the leaf and the petiole, and the optimum dose of N fertilization (N @ 80 kg ha−1) was seen in treatment T3.

Thus, both G5 and G8 showed a similar trend of nitrate accumulation, but the extent of accumulation was less in G8 than in G5. Saturation in nitrate reduction was observed at T3 (N@ 80 kg ha−1), which is supposed to be the optimum dose of N fertilization for nitrate-accumulating leafy vegetables, because further increase in N doses (N @ 120 kg ha−1) increased the nitrate content in both genotypes.

At 6-week stage, both G5 and G8 crossed the permissible ADI in all consumable plant parts. G5 showed a higher nitrate accumulation than G8, with the lowest occurring in treatment T3. The pattern of accumulation was the same as observed at 3-week stage, the highest accumulation occurring in the petiole, followed by the leaf and then the stem. Petioles of G5 accumulated 6891.92 ± 37.74 mg nitrate kg−1 fresh wt in the control, which was reduced to 5293.86 ± 36.77 mg nitrate kg−1 fresh wt after treatment T3, but this too was beyond the acceptable ADI limit. In G8, the nitrate content gradually decreased from T1 to T3, attaining a value of 4630.74 ± 18.11 mg kg−1fresh wt of petiole, compared to 5665.97 ± 28.92 mg kg−1 in the control (T1).

Nitrate content of the leaf was intermediate, the highest being in the petiole and the lowest in the stem. The maximum nitrate reduction was attained at treatment T3. Nitrate content also increased with the age of the plant (Figs. 3, 4), because at the 6-week stage it was high even in N-treated plants.

Fig. 4
figure 4

Nitrate accumulation pattern in different plant parts of screened genotypes at 6-week stage. Bars with different letters are significantly (P < 0.05) different from each other in a single plant part of a single variety (Tukey–Krammer Posttest)

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Effect of light intensity on nitrate content

A separate experiment, conducted to study the pattern of nitrate accumulation in plants grown under light and shade conditions, revealed that plants in full sunlight accumulated lesser nitrate, compared to those kept in shade (Fig. 5). Genotype G5 accumulated more nitrate than G8 under both light and shade conditions. The nitrate content decreased with increasing nitrogen dose till the treatment T3 in both genotypes. It was thus minimum in G5T3, being less (2053.43 ± 23.64) under light condition than under shade (3561.94 ± 24.70 mg kg−1fresh wt of leaf). With the optimum dose of N fertilization (T3), nitrate content was reduced significantly, but it was 42 % higher in plants grown under shade than in those exposed to sunlight. In G8, the nitrate content was 1854.99 ± 13.60 in the control plants and merely 1301.59 ± 11.51 mg kg−1 fresh wt at treatment T3 under light. Even under shade, the lowest nitrate level appeared with T3 (2189.56 ± 26.63), as compared to the control (2608.12 ± 22.16 mg kg−1 fresh wt). It was 40 % higher than in plants exposed to sunlight. At T4 treatment, the nitrate content increased non-significantly in both genotypes under light as well as shade conditions.

Fig. 5
figure 5

Nitrate accumulation pattern in the light and shade experiment at 4-week stage. The Bars with different letters are significantly (P < 0.05) different from each other in individual varieties and irradiation levels (Tukey–Krammer Posttest)

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Discussion

Our study has revealed a genotypic variability of nitrate accumulation in B. juncea, which may be linked to various genetic (Giola et al. 2013) and environmental factors. Among the genotypes examined, G5 came out to be a HNA at 3- and 6-week stages, whereas G8 was a LNA at these stages; the nitrate content increased with increasing age of the plant. As is evident from Fig. 1, G5 exceeded the ADI by about 2 % at early stage of plant growth, whereas all other genotypes had the nitrate range within the limits as set by the European Commission’s Scientific Committee on Food (SCF 1997) and hence are safe for consumption as leafy green vegetables. However, in a later stage of plant growth (4 week and later on) all genotypes became unsafe for consumption as food, because the nitrate content exceeded the ADI. This could be due to the difference in their N-metabolic pathways.

Different genotypes responded differently to the same light intensity at three different timings of the day, with a gap of 6 h. The percent decrease in nitrate level from morning to noon differed in different genotypes. Similarly, percent increase from morning to evening or from noon to evening also varied with genotype (Fig. 2). G5 accumulated the maximum and G8 the minimum nitrate in each sampling during the day. In all the genotypes nitrate content was highest at 7:00 pm in the evening and the lowest at 1:00 pm in the noon. This study substantiates some earlier findings on spinach leaves (Anjana et al. 2007b). The nitrate content seems to be correlated to light intensity rather than time duration, because it was at its minimum in all the genotypes when light intensity was the maximum. Thus, light intensity is likely to play a significant role in the regulation of nitrate content in vegetables (Cometti et al. 2011; Ozturk et al. 2013). Leafy vegetables are nitrate accumulators, but the nitrate intake by humans through these leafy greens can be minimized by harvesting them at appropriate time and plant age (Anjana et al. 2007b). Genotype G8 is safe for consumption at early age and can be harvested at any time during the day, because variation in nitrate accumulation is quite diminutive and negligible.

Moreover, different plant parts have uneven nitrate accumulation and distribution, which may be linked to the genotypic variability of nitrate assimilation, based on different rate of NR activity (Black et al. 2002), and hence to the plant metabolic status (Stitt et al. 2002; Carelli and Fahl 2006). It is also evident that different plant parts had a different capacity to reduce nitrate and hence different nitrate contents. Fertilizer supply and N availability are the key factors that influence nitrate content in leafy vegetables (Guadagnin et al. 2005). At the 3-week stage, on increasing the N fertilization, nitrate content decreased in both HNA and LNA genotypes. However, petiole of G5 exceeded the ADI limit (at all treatments) for a 60-kg person (if consumed @ 100 g/day), whereas G8 genotype had the nitrate content within the permissible limits in all consumable plant parts and with all N treatments. In general, petioles that accumulate the maximum amount of nitrate should be removed while preparing these leafy vegetables for human consumption. According to Muramoto (1999), NO3–N was reduced by 31 % when midribs were detached from the ‘Iceberg’ and ‘Romaine’ lettuces.

Figure 3 indicates that N@ 80 kg ha−1 is the optimum dose for Brassica leafy vegetables at vegetative stage, because nitrate content increased at higher N dose in both HNA and LNA genotypes. This may be related to nitrate saturation and its lesser utilization in plants due to excess N application, which may also inhibit plant growth and the nitrate uptake from the soil.

However, six-week-old plants of both the genotypes were unfit for consumption as food, because nitrate content was far beyond the ADI and could cause toxicity. Harvesting age also affects nitrate content considerably, as the control plants of G5 at 3-week (20–25 days) stage, and both the genotypes harvested at 6-week (40–45 days) stage, possessed higher nitrate levels than the ADI limits (Figs. 3, 4). The effect of nitrogen fertilization and mineralization on nitrate reduction in plants was significant. This experiment indicates that nitrate content of vegetables can be maintained to consumable limits by harvesting the nitrate-accumulating vegetables at an early stage of 7–8 days. This goes in line with the findings of Ren et al. (1997).

Nitrate accumulation and high irradiance seem to be inversely related to each other (Fig. 5), possibly because high light intensity promotes nitrate absorption, its assimilation and translocation, and carbohydrate production, thus leaving little nitrate to accumulate. The nitrate content in vegetables is significantly influenced by nitrogen fertilization and light intensity. In the LNA genotype, it was low at all N treatments in plants grown under light, and safe enough for consumption as food, but exceeded the ADI in the shade-grown plants with all treatments except T3. Nitrogen @80 kg ha−1 (treatment T3) was the saturating dose for both the genotypes. In the light-grown HNA genotype, T3 and T4 plants had the nitrate content within ADI limit, whereas the control and T2 exceeded it. The nitrate content was beyond the ADI limits in all the control and treated plants, grown under shade, despite a gradual decrease detected till T3.

This study suggests that (a) only those leafy vegetables may be used for human consumption, which receive proper light intensity and are low-nitrate accumulators, and (b) an optimum dose of fertilizer should be provided to plants, depending on the crop type. Moreover, petioles of leafy vegetables accumulate several-fold higher nitrate than the leaf and the stem, and must therefore be removed while processing Brassica leaves as vegetable. Light intensity was a limiting factor in regulation of nitrate accumulation, which declined linearly with increase in light intensity during day time, and attained the maximum during night hours. Further, the nitrate accumulated to unsafe limits at high N fertilization and with increasing plant age. The fertilizers supplied and the N availability are the key factors in ensuring low-nitrate content and a high nutritive value of the plant.