Spatiotemporal changes of nitrate and Vc contents in hydroponic lettuce treated with various nitrogen-free solutions

Abstract

Nowadays, off-season leafy vegetables are generally characterized by low vitamin C (Vc) content and high nitrate accumulation due to low light intensity inner protected systems and high-level nitrogen supply. A glasshouse experiment was conducted to investigate the effects of three kinds of nitrogen-free solution treatments before harvest on Vc and nitrate contents in expanded leaf blades, expanded leaf petioles and old leaves of hydroponic lettuce. The results showed that using nitrogen-free solutions could decrease nitrate contents of expanded leaf blades, expanded leaf petioles and old leaves, also nitrate contents in above parts reduced increasingly with treatment time extending. In addition, three nitrogen-free solution treatments increased the Vc content of expanded leaf blades instead of expanded leaf petioles and old leaves. There were instead of significant positive correlations between nitrate contents but Vc contents of expanded leaf blades, expanded leaf petioles and old leaves. To conclude, nitrogen disruption treatment before harvest could effectively reduce nitrate contents in edible parts of lettuce, and also improve Vc content in expanded leaf blade.

Keywords:

• Leaf blade
• nitrogen-free solution
• nutritional quality
• petiole
• vitamin C

Introduction

Vegetables are the indispensable staple produce providing humans with many beneficial substances such as vitamins, minerals, amino acids, carotenoids. Nowadays, vegetables are increasingly required to contain these beneficial substances and less contaminants (nitrate, nitrite, heavy metals and pesticide residues) to meet dietary requirements of human health (Liu et al. 2009). However, vegetables, particularly the off-season leafy vegetables grown in protected facilities, are readily contaminated by nitrate due to overuse of nitrogen fertilizer (Wang and Li 2003, Chen et al. 2004, Boroujerdnia 2007, Konstantopoulou 2010) and environmental weak light (Müller and David 1987, Riens and Heldt 1992, Ysart et al. 1999). In addition, vitamin C (Vc) content in leafy vegetables also can be mainly controlled by nitrogen application (Sørensen et al. 1994, Biesiada and Kolota 2010, Konstantopoulou et al. 2010) and light conditions (Lee and Kader 2000, Dumas et al. 2003). Generally, off-season leafy vegetables are characterized by high nitrate accumulation and low Vc content (Poulsen et al. 1995, Konstantopoulou et al. 2010).

The levels of Vc and nitrate in leafy vegetables are two key indices for evaluating their nutritional quality (Konstantopoulou et al. 2010). Human beings have to obtain Vc from foods since they cannot synthesize Vc. It was reported that more than 90% Vc of dietary ingestion was from vegetables and fruits (Lee and Kader 2000). On the other hand, more than 80% nitrate intake by people was from vegetables (Gangolli et al. 1994, Eichholzer and Gutzwiller, 1998). Excessive intake of nitrate poses a potential hazard to human health, especially for infants. The toxic effects of nitrate are due to its endogenous conversion to nitrite, which is implicated in the occurrence of methaemoglobinaemia, gastric cancer and many other diseases (Wolff and Wasserman 1972, Santamaria 2006). Many studies had shown that Vc could inhibit nitrosamine formation (Fan and Tannenbaum 1974, Gray and Dugan, Jr 1975, Sen et al. 1976, Tannenbaum et al. 1991). Therefore, Vc is used extensively in food industries not only for its nutritional value but for its many functional qualities as antioxidant and inhibitor of N-nitrosamine formation (Okafor and Nwogbo 2005). Based on this, low Vc and high nitrate accumulation of leafy vegetables may raise the risks of diseases to humans. Therefore, increasing the Vc content and lowering the nitrate content are effective ways to improve the quality of leafy vegetables.

By now, some agronomic methods have been extensively developed to alter nitrate and Vc content in protected leafy vegetables, attempting to improve their nutritional quality. Particularly, previous studies found that preharvest treatments with nitrogen-free solutions could decrease nitrate contents of lettuce (Liu and Yang 2011, Liu et al. 2011), Pakchoi (Dong and Li 2003) and spinach (Mozafar 1996), and also alter their Vc content (Mozafar 1996, Dong and Li 2003). Apparently, preharvest nitrogen disruption treatment is a promising method to improve nutritional quality of leafy vegetables in hydroponics in terms of low costs and high efficiency. However, few investigations have been carried out to examine the response differences of nitrate and Vc contents in the various edible parts of leafy vegetables to preharvest nitrogen disruption treatment, especially the differences between expanded leaves and old leaves, expanded leaf blades and petioles. In this study, a glasshouse experiment was conducted to investigate the impacts of three kinds of nitrogen-free hydroponic solutions on Vc and nitrate contents in expanded leaves and old leaves during an eight-day treatment period.

Materials and methods

Experimental materials and experimental design

Lettuce (Lactuca sativa) seedlings, an Italian cultivar, were transplanted and cultivated in a cultivation bed (2 m × 0.5 m × 0.1 m) supplied with full nutrient solution after 22 days growth in a seedling tray (vermiculite used as substrate) on 15 November 2010. The cultivation bed was one metre high, and the seedlings were supplied nutrient solution every other 15 minutes with a pump. The nutrient solution composition was as follows: KNO₃, 5.94, MgSO₄, 1.42, NH₄H₂PO₄, 1.00; KH₂PO₄, 0.44; Ca(NO₃)₂ 2.12, EDTA-Fe, 4.29 × 10⁻², H₃BO₃, 4.839 × 10⁻², MnSO₄, 1.325 × 10⁻², ZnSO₄, 1.35 × 10⁻³, CuSO₄, 5 × 10⁻⁴, (NH₄)₆Mo₇O₂₄, 4 × 10⁻⁴ mmol·L⁻¹. On 4 January 2011, six uniform lettuce plants were selected and transplanted into each experimental pot. The length × width × height of the experimental pot was 32 cm × 24 cm × 11 cm. The experimental pots were aerated into nutrient solutions with an air-pump (Atman EP-9000) to ensure soluble oxygen concentration around roots. Four treatments were designed, including full nutrient solution, nitrogen-free nutrient solution, distilled water and 1mM KCI solution. Each treatment was replicated three times. The full nutrient solution is composed of 0.75 K₂SO₄, 0.5 KH₂PO₄, 0.65 MgSO₄, 0.1 KCl, 5.0 Ca (NO₃)₂, 1.0 × 10⁻³ H₃BO₃, 1.0 × 10⁻³ MnSO₄, 1.0 × 10⁻⁴ CuSO₄, 5.0 × 10⁻⁶ (NH₄)₆M_O7O₂₄, 1.0 × 10⁻³ ZnSO₄, 0.1 EDTA-Fe mmol·L⁻¹. All the hydroponic solutions were prepared with distilled water and analytical reagents. The experiment was conducted in the experimental glasshouse of the Laboratory of Vegetable Nutrition and Quality Physiology of Institute of Environment and Sustainable Development in Agriculture, Chinese Academy of Agricultural Sciences. The weather of all the eight days was sunny, and the temperature ranged from 15–30 °C in the glasshouse.

Harvest and determination

During the eight-day treatment, sampling was conducted four times at 2-day intervals. One lettuce plants at 2 days and 4 days, and two lettuce plants at 6 days and 8 days after treatment were randomly sampled from each pot at 8.30 in the morning. Sampling was completed within 30 minutes, and samples were bagged, then placed into refrigeration at 4 °C for ten minutes to cooling for determination. Before determination, the lettuce was separated into expanded leaves and old leaves, also the expanded leaves were divided into expanded leaf blades and petioles. Vc content in lettuce was determined by the 2, 6-dichlorophenolindophenol method, and nitrate content was examined with the concentrated sulphuric acid-salicylic acid method (Li 2000).

Statistical analyses

Variance analysis of data was conducted using SAS software 6.12. Significant differences between means were established by using the least significance difference (LSD) test.

Results

Effects of three nitrogen-free solutions on Vc content in lettuce

Three nitrogen-free solution treatments improved the Vc content of the expanded leaf blades of lettuce compared with the full nutrient solution treatment, and their efficiencies varied with nitrogen-free solution species and sampling time (Table I). However, three nitrogen-free solutions didn't function significantly in improving Vc content of the old leaves of lettuce (Table I). Only nitrogen-free nutrient solution and distilled water treatments increased Vc content of the old leaves at the 4-day sampling.

Table I. Vc content in expanded leaf blades and old leaves of lettuce treated with three hydroponic nitrogen-free solutions (mg g⁻¹).

	Treatment time
	Two days		Four days		Six days		Eight days
Treatments	Leaf blades	Old leaves	Leaf blades	Old leaves	Leaf blades	Old leaves	Leaf blades	Old leaves
Full nutrient solution	0.290b	0.165a	0.150b	0.165b	0.331ab	0.155a	0.197b	0.167a
Nitrogen-free nutrient solution	0.365ab	0.178a	0.310ab	0.219ab	0.287b	0.141a	0.357a	0.199a
Distilled water	0.414a	0.204a	0.498a	0.316a	0.417a	0.143a	0.398a	0.199a
KCI solution	0.417a	0.189a	0.386a	0.203b	0.388ab	0.146a	0.386a	0.213a
Notes: Means with different letters within each column are significantly different at p < 0.05; Old leaf sample is made up of leaf blades and petioles.

Effects of three nitrogen-free solutions on nitrate content in lettuce

Nitrate contents of expanded leaf blades and petioles of lettuce treated with three nitrogen-free solutions were markedly reduced at three sampling times compared with the full nutrient solution treatment, and the decline extents were gradually elevated with the elongation of treatment time (Table II). Similarly, nitrate contents in old leaves treated with three nitrogen-free solutions were significantly reduced at three sampling times compared with the full nutrient solution treatment, and the decline extents were also gradually elevated with the elongation of treatment time (Table II).

Table II. Nitrate contents of expanded leaf blades and petioles, and old leaves of lettuce treated with three hydroponic nitrogen-free solutions at three sampling times (mg kg⁻¹).

	Two days			Six days			Eight days
Treatments	Leaf blade	Petiole	Old leaves	Leaf blade	Petiole	Old leaves	Leaf blade	Petiole	Old leaves
Full nutrient solution	3538.7a	3030.9a	6298.5a	1161.9a	2775.6a	2187.4a	1213.6a	2118.8a	1796.2a
Nitrogen-free nutrient solution	1843.2b	1935.3a	3491.4b	434.9b	2239.1ab	2066.5ab	377.7b	731.4b	806.8b
Distilled water	2674.6ab	3143.6a	4788.2ab	670.8b	1948.6b	2103.9a	346.7b	1000.2b	1179.9ab
KCI solution	3357.7a	3305.4a	3903.9b	539.0b	1571.6b	1452.8b	230.4b	633.7b	909.9b
Notes: Means with different letters within each column are significantly different at p < 0.05; Old leaf sample is made up of leaf blades and petioles.

Discussion

The data showed that nitrate contents in both expanded leaf blades, expanded leaf petioles and old leaves treated by nitrogen-free solutions were largely decreased compared with full nutrient treatment after 8-day cultivation. The results suggested that nitrogen disruption treatment is an effective method to significantly lower nitrate content in soilless cultivation lettuce in several days. However, there were apparent differences in nitrate reduction rate between three parts of lettuce to nitrogen disruption treatment, roughly following the sequence of expanded leaf blades > expanded leaf petioles > old leaves. This may be related to the organic reduction capacity of nitrate accumulated in various parts of lettuce. Moreover, the response time and reduction rate may also depend on the rates of nitrate remobilization and reutilization processes occurring in different parts of lettuce. These processes are driven by photosynthetic assimilation of nitrate in leaf blades. Expanded leaf blades are fundamental sites for nitrate reduction. Thus, both nitrate in expanded leaf petioles and in old leaf petioles should be transported into leaf blades for reduction. Our results also showed that there were significant positive correlations between nitrate contents of expanded leaf blades, petioles and old leaves (n=36) during nitrogen disruption treatment period, which reflected the linkage relationship in nitrate reduction. Many studies found that lettuce usually accumulated more nitrate in the older, outer leaves (Cardenas-Navarro et al. 1999, Escobar-Gutierrez et al. 2002, Mun and Lee 2002, Siomos et al. 2002, Krohn et al. 2003), and nitrate accumulation tends to be higher in the petioles than in the laminae (Maynard and Barker 1972). Mozafar (1996) and Dong and Li (2003) previously reported that nitrogen-free nutrient solution treatment could decrease nitrate contents in whole leaf of Pakchoi and spinach. Our results showed that nitrogen disruption treatment could jointly decrease nitrate content in all edible parts of lettuce.

More importantly, besides reducing nitrate content, nitrogen-free solution treatments are helpful in improving Vc content in expanded leaf blades of lettuce. The results showed that nitrogen-free solution treatments did only improve the Vc content in expanded leaf blades of lettuce instead of old leaves. Furthermore, Vc content in petioles is low and not affected by nitrogen-free solutions (unpublished data). Young leaves contained 3.3 times higher levels of total Vc than old leaves. Therefore, unfolded young leaves contained more Vc with high quality. Nitrate and total Vc contents increased as the nitrogen level increased (Mun and Lee 2002). Therefore, Vc content enhancement in leafy vegetables by a nitrogen-free solution treatment method is more efficient for those with low proportion petioles in biomass. Our results found that Vc contents in old leaves of lettuce treated with three nitrogen-free solutions, and Vc contents in expanded leaf blades of lettuce treated with full nutrient solution fluctuated up and down with the elongation of treatment time. The change in rhythm may relate to the metabolic processes and key enzymatic activities, needing further investigation. The results showed that there were no significant correlation was found between Vc contents of them, as well as between Vc contents and nitrate contents either of expanded leaf blade or old leaf.

The result showed that Vc content of expanded leaf blades of lettuce treated by nitrogen-free solutions did not change significantly among four sampling times. However, the Vc content in old leaves treated by distilled water and KCI solution changed significantly between four sampling times. Nitrate contents in expanded leaf blades, expanded leaf petioles and old leaves reduced increasingly with treatment time extending. The results showed that there were significant correlations between nitrate contents of expanded leaf blades and old leaves (r=0.90, n=36), expanded leaf blades and petioles (r=0.80, n=36), and expanded leaf petioles and old leaves (r=0.79, n=36), but no significant correlation was found between Vc content of expanded leaf blades and old leaves as well as between Vc and nitrate content either of expanded leaf blades or old leaves.

Leafy vegetables, especially when consumed fresh, are a valuable source of Vc in the human diet. This is particularly true for lettuce, which is a major constituent of fresh salads. Nowadays there is an increasing interest in hydroponics systems due to the advantage of cultivation of leafy vegetables for salads with very short cycles. Our study suggested that the practical significances of tap water (distilled water in experiment) and KCI solution were higher in term of their efficiencies in improving lettuce quality and the low cost. Nitrogen disruption treatments could effectively reduce nitrate contents in edible parts of lettuce, and improve Vc content in expanded leaf blades. The results suggested that nitrogen-free solution treatment is a feasible way to improve the quality of soilless-cultivation leafy vegetables, particularly those with high proportion of leaf blades, with low costs. Our study suggested that the practical significances of tap water (distilled water in experiment) and KCI solution were higher in terms of their efficiencies in improving lettuce quality and the low cost.

Acknowledgements

The authors want to thank the financial support of the Basic Scientific Research Fund of National Nonprofit Institutes (BSRF2009, BSRF201004), Institute of Environment and Sustainable Development in Agriculture, CAAS.