Nutritional quality of greenhouse lettuce at harvest and after storage in relation to N application and cultivation season

Scientia Horticulturae 125 (2010) 93.e1–93.e5

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Nutritional quality of greenhouse lettuce at harvest and after storage in relation to N application and cultivation season Eleni Konstantopoulou a , Georgios Kapotis a , Georgios Salachas a , Spyridon A. Petropoulos b , Ioannis C. Karapanos b , Harold C. Passam b,∗ a Department of Greenhouse Cultures and Floriculture, Faculty of Agricultural Technology, Technological Educational Institute of Messolonghi, Nea Ktiria, 30200 Messolonghi, Greece b Laboratory of Vegetable Production, Agricultural University of Athens, 75, Iera Odos, 11855 Athens, Greece

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Article history: Received 7 January 2010 Received in revised form 2 March 2010 Accepted 18 March 2010 Keywords: Leafy vegetables Nitrogen Lactuca sativa L. Nitrates Chlorophyll Ascorbic acid

a b s t r a c t The effect of five levels of nitrogen fertilization on the growth and nutritional quality of Cos lettuce (Lactuca sativa L. cv. Parris Island) at harvest and after storage was studied during autumn and winter in South-West Greece. Plants were cultivated hydroponically in a greenhouse and the nitrate, chlorophyll and ascorbic acid (vitamin C) concentrations within the plant tissues were measured at harvest and following storage at 5 or 10 ◦ C for 10 days. Nitrate accumulated in the leaves with increasing amounts of N within the nutrient solution and was higher in the winter than in the autumn. At the lowest N level (20 mg L−1 ), the inner leaves accumulated more nitrate than the outer leaves, whereas at higher N levels (140, 200 or 260 mg L−1 ) nitrate accumulation was higher in the outer leaves. Overall, the highest nitrate concentrations were detected in the petiole and the proximal end of the leaf, but at the lowest N application rate (20 mg L−1 ) nitrate accumulated in the distal region of the leaf too. Although the nitrate concentrations within the leaves did not change significantly during 10 days storage at 5 or 10 ◦ C, the chlorophyll and vitamin C concentrations decreased. Chlorophyll loss was higher in lettuce that was grown under low N levels and was higher at 10 ◦ C than at 5 ◦ C, but was reduced by enclosure of the lettuce in polyethylene film. It is concluded that the optimum N application rate for Cos lettuce grown hydroponically under cover during autumn and winter in South-West Greece, and in other areas with a similar climate, is 200 mg N L−1 because at this N rate yield is satisfactory and leaf nitrate concentrations are below the maximum acceptable level for human consumption. Nutritional value (vitamin C concentration) and market quality (chlorophyll content) are highest at harvest and decrease during storage, but quality in terms of nitrate concentration does not change. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Nitrate uptake and accumulation in plants primarily relates to the amount (Chen et al., 2004; Mantovani et al., 2005) and form of N-fertilizer applied to the crop (Breimer, 1982; Lairon et al., 1984; Premuzic et al., 2002; Abu-Rayyan et al., 2004). Other factors influencing this process include the plant genotype, nitrate reductase activity, plant age and the cultivation season and environment (Drews, 1996; Drews et al., 1997; Boroujerdnia et al., 2007). Leafy vegetables such as lettuce and spinach, are especially prone to nitrate accumulation (Siomos et al., 2002a). Looseleaf, cos and iceberg varieties appear to accumulate higher amounts of nitrate than butterhead lettuce (Escobar-Gutierrez et al., 2002) especially, but not always, in the older, outer leaves (Cardenas-

∗ Corresponding author. Tel.: +30 210 5294535; fax: +30 210 5294504. E-mail address: [email protected] (H.C. Passam). 0304-4238/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2010.03.003

Navarro et al., 1999; Siomos et al., 2002a; Mun and Lee, 2002; Krohn et al., 2003). Nitrate accumulation tends to be higher in the petioles than in the laminae (Maynard and Barker, 1979) and is usually higher in the winter than in other seasons (Burns et al., 2004; Ysart et al., 1999) due to the reduction of nitrate reductase activity within the leaves under low light intensity and low temperatures (Riens and Heldt, 1992). Numerous studies have been directed towards the effects of nitrate intake on human health. Although earlier reports implicating nitrate in the occurrence of cancer are largely unsubstantiated, other nitrate-induced syndromes, such as methaemoglobinaemia in infants (blue baby syndrome) have been confirmed (Addiscott and Benjamin, 2004; Fewtrell, 2004). Moreover, the reduction of nitrates to nitrites and the subsequent formation of carcinogenic nitrosamines within the gastroenteric system has been reported and this observation together with those indicated above have contributed to the adoption of maximum acceptable nitrate levels in lettuce and spinach by the European Union (E.U.) (Siomos

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et al., 2002a). However, in contrast with the foregoing, there may also be a beneficial role for nitrates and nitrites in human health, where according to DASH (Dietary Approach to Stop Hypertension) diets rich in vegetables and even exceeding the recommended daily intake of nitrates cause vasodilation, decrease blood pressure and support cardiovascular function (Hord et al., 2009). The nutritional and market quality of lettuce relates not only to head size and appearance but also to the vitamin and mineral content and the maintenance of nitrate and nitrite concentrations within the foliage at levels below the E.U. maxima. Changes in nutritional value occur both during plant growth (Drews et al., 1997) as well as subsequent storage. For example, during lettuce storage at high temperatures nitrate may be reduced to nitrite, but at temperatures below 5 ◦ C, nitrate and nitrite concentrations are either not affected (Chung et al., 2004) or nitrate may even increase (Poulsen et al., 1995). Factors affecting the storage life and quality of lettuce include the type or cultivar (López-Gálvez et al., 1996), temperature (Bolin et al., 1977), ethylene (Ritenour and Saltveit, 2008) and the relative humidity (RH) and synthesis of the storage environment. Storage life and quality may be improved by enclosure of lettuce in polyethylene film (Martinez and Artés, 1999) or the implementation of modified atmospheres (Escalona et al., 2006; López-Gálvez et al., 1996). However, concentrations of CO2 within the storage environment higher than 2–3% may induce browning, depending on the temperature and variety (Brecht et al., 1973). Storage can also lead to oxidative loss of chlorophyll (Yamauchi and Watada, 1993) and vitamin C (Lee and Kader, 2000), processes that are exacerbated with increasing temperature (Lee and Kader, 2000). In the present paper we investigate the distribution of nitrate in the leaf tissues of greenhouse lettuce at harvest in relation to the N concentration in the nutrient medium and the cultivation season. In addition we examine the quality traits of the lettuce (chlorophyll, nitrate, vitamin C concentrations) during subsequent storage at 5 and 10 ◦ C in closed and open packages. 2. Materials and methods 2.1. Plant material and growth conditions Cos lettuce (Lactuca sativa L. cv. Parris Island) was cultivated in an automated glasshouse of the Technological Educational Institute of Messolonghi, Greece, during autumn (22 September–14 December) and winter (18 December–23 March). Mean minimum and maximum temperatures in the greenhouse were 15.7 ± 2.0 and 26.6 ± 4.3 ◦ C, respectively, for the autumn crop and 12.9 ± 1.9 and 23.9 ± 4.4 ◦ C for the winter crop. Solar radiation varied between 700–1400 ␮mol m−2 s−1 PAR (autumn) and 700–1350 ␮mol m−2 s−1 PAR (winter). Seeds were sown in a peat-based compost (peat: perlite 1:1, v/v) containing 2 g L−1 N, P, K (14–10–18) and trace elements and maintained in a growth cabinet at 21 ± 1 ◦ C, 90 ± 1% RH. On emergence, seedlings were transferred to the greenhouse (20 ± 1 ◦ C) and on full expansion of the cotyledons plants were transplanted to peat pots (2.5 cm × 2.5 cm) containing the same substrate. Two days after transplantation, the pots were placed in 4–5 cm deep openings in rockwool slabs (100 cm × 15 cm × 7.5 cm, Grodan supplied by Agrosystems A.E., Crete) in an open hydroponic system. The experimental layout consisted of 15 independent galvanized metal channels (4.7 m × 0.3 m × 0.15 m, inclined at 1%) lined with black and white polyethylene film. The rockwool slabs were enclosed within the polyethylene so as to exclude light from the rooting medium. Each channel contained 20 plants and the nutrient media were applied to the plants via individual drippers. Plants were treated with five different nitrogen application rates, namely 20, 80, 140, 200 and 260 mg N L−1 . The composi-

tion of the nutrient solution, except for N, was as follows: 1 mM P, 3.07 mM Ca, 9.75 mM K, 1.26 mM Mg, 35 ␮M Fe, 5 ␮M Mn, 3 ␮M Zn, 0.5 ␮M Cu, 0.5 ␮M Mo, and 20 ␮M B. The appropriate N level was achieved by applying 1.43, 5.71, 10.00, 14.28 and 18.57 mM N in the form of Ca(NO3 )2 within the nutrient solution, with a proportional change in S concentration from 6.76 to 5.50, 3.36, 1.22 and 0.72 mM, respectively. The electrical conductivity ranged between 1.9 and 2.2 dS/m. Plants were harvested at the stage of market acceptance (14 December and 23 March for the autumn and winter crops, respectively) and immediately assayed for nitrate, chlorophyll and vitamin C content (day 0) or after storage in plastic boxes (23 cm × 12 cm × 7 cm) for 10 days at 5 or 10 ◦ C. Each box contained five leaves and were stored open or wrapped in plastic film (flexible vinyl film, General Plastic Extrusions, Prescott, USA), with an O2 permeability of 19000 cm3 m−2 24 h−1 and water vapour permeability of 190 g m−2 24 h−1 . 2.2. Nitrate content determination The nitrate concentration in the leaves was determined colorimetrically by the reduction of nitrate to nitrite through a cadmium column as described in the AOAC Official Methods of Analysis (AOAC, 1995), using a Shimadzu Spectrophotometer Model UV1601 VIS (Shimadzu, Kyoto, Japan). Ten plants from each replicate were harvested and separated into inner and outer leaves. Each group of leaves was further divided into three subgroups. For nitrate evaluation in the different parts of leaves, the same procedure was followed and the leaves of each subgroup were separated into petioles, distal and proximal ends of the laminae by cutting with a sharp knife. 2.3. Chlorophyll content determination The total chlorophyll content of the leaves was determined by the method of Arnon (1949) based on acetone extraction of the chlorophyll and colorimetric assay with the aid of a Shimadzu Spectrophotometer Model UV-1601 VIS. Five plants from each replicate were harvested and measurements were made on the outer, fully mature leaves. 2.4. Vitamin C determination Vitamin C content in leaves was determined by the method of Bajaj and Kaur (1981) using a Shimadzu Spectrophotometer Model UV-1601 VIS. Extracts were prepared from the outer, fully mature leaves of five plants per treatment replicate. 2.5. Statistical analysis The experiment was laid out in a completely randomised design with each channel comprising one replicate. Statistical analysis was performed with the aid of the Statgraphics 5.1.Plus statistical package (Statistical Graphics Corporation) and Microsoft Office Excel 2003. Data were evaluated by analysis of variance for the main effects and the means of values were compared by Duncan’s multiple range test (DMRT), or by the least significant difference (LSD) test in the case of pairs of means (p = 0.05). 3. Results and discussion 3.1. Effect of N application rate on leaf nitrate content at harvest The mean head weight of lettuce at harvest increased with N application rate in both seasons. Head weight was significantly

E. Konstantopoulou et al. / Scientia Horticulturae 125 (2010) 93.e1–93.e5 Table 1 Mean head weight (g) of lettuce at harvest in relation to the cultivation season (autumn and winter) and nitrogen application rate. Level of N (mg N L−1 )

Autumn

Winter

20 80 140 200 260

17.6 e 153.7 d 263.3 c 301.8 b 356.0 e*

24.0 e 185.9 d 294.0 c 344.7 b 398.9 a*

Means that differ significantly within columns are followed by different letters; means that differ significantly within rows are indicated by an asterisk (*).

Table 2 The mean nitrate concentration (mg kg−1 fresh weight) of lettuce leaves in relation to the cultivation season (autumn and winter), leaf position (inner and outer leaves) and nitrogen application rate. Level of N (mg N L−1 )

Autumn

Winter

Outer leaves 20 80 140 200 260

(b)

107 e 842 d(a) 2535 c(a) 3019 b(a) 4873 a(a)

Inner leaves (a)

465 d 931 c(a) 2021 b(b) 2128 b(b) 2371 a(b)

Outer leaves (b)

111 e 728 d(a) 2675 c(a) 3942 b(a) 5771 a(a)

Inner leaves 291 e(a) 1039 d(a) 2098 c(a) 2718 b(b) 3230 a(b)

Means that differ significantly within columns are followed by different letters without parenthesis; means that differ significantly within rows are indicated by a different letter within parenthesis.

higher at 260 mg N L−1 in the winter than in the autumn, but did not differ with season at the lower N rates (Table 1). The increase in nitrogen application rates from 20 to 260 mg N L−1 , resulted in a significant increase in the nitrate concentration of lettuce (Table 2), similar to that reported previously in other studies (Mun and Lee, 2002; Chen et al., 2004; Mantovani et al., 2005; Boroujerdnia et al., 2007). At application rates of 140–260 mg N L−1 nitrate accumulation was higher in the outer leaves. However, at the lowest application rate (20 mg N L−1 ), nitrate concentrations were higher in the inner, younger leaves, while at 80 mg N L−1 , the nitrate concentration of the outer and inner leaves was not significantly different (Table 2). These differences in distribution of nitrate between the leaves were observed in both seasons of cultivation. In previous studies it has generally been asserted that nitrates accumulate more in the older, outer leaves of lettuce than in the inner leaves (Marsic and Osvald, 2002; Abu-Rayyan et al., 2004; Boroujerdnia et al., 2007), although Krohn et al. (2003) reported that in closed heads of a crisphead lettuce variety, nitrates may

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accumulate more in the inner leaves. In Cos varieties, the lettuce heads are less compact than those of crisphead varieties and at low N application rates (20 mg N L−1 in the present experiments) the leaves may even fail to form a head. In consequence, both the young and old leaves of this lettuce type are equally exposed to sunlight, whereas in the heads formed at higher N rates (>140 mg N L−1 ) the outer leaves are more exposed than the inner leaves. This means that in the small, open lettuce produced under low N rates, the inner and outer leaves are likely to photosynthesise at a similar rate, whereas in the heads produced at higher N rates the outer leaves are more photosynthetically active than the inner leaves. Nitrate accumulation under increasing N rates therefore appears to occur more in the leaves that are photosynthetically active. The distribution of nitrate within the tissues of the leaves varied with the N application rate. At the lowest N rate (20 mg N L−1 ), nitrate concentrations were highest in the distal region of the lamina of the outer leaves of the autumn crop, but in the outer leaves of the winter crop, nitrate concentrations at this N rate were highest in the petiole (Table 3). In the inner leaves, nitrate concentrations were higher in the petiole and the distal tissues of the lamina than in the proximal region of the lamina, irrespective of season. At higher N rates (80–260 mg N L−1 ), nitrate concentrations were highest in the petiole and lowest in the distal region of the lamina of both the inner and outer leaves, irrespective of season (Table 3). In all the leaf tissues nitrate concentration increased with increasing rates of N application. Although differences between different regions of the lamina of lettuce have not been previously reported, Breimer (1982) noted higher concentrations of nitrate in the petioles of spinach than in the laminae. With respect to human health considerations, the nitrate concentrations within whole lettuce or isolated leaf tissues at N rates of up to 200 mg N L−1 were lower than the maximum acceptable levels for human consumption (Siomos et al., 2002a). However, bearing in mind the controversial significance of nitrates with respect to human health (Fewtrell, 2004; Hord et al., 2009), there are clearly differences in nutritional value (expressed in terms of tissue nitrate content) between leaves within the lettuce head and between tissues within individual leaves.

3.2. Effect of N application rate on leaf nitrate content during storage The nitrate concentration of the inner and outer leaves did not change significantly during storage for 10 days at 5 or 10 ◦ C, with the exception of the inner leaves of lettuce grown at 20 mg N L−1 in which the nitrate concentration decreased at both temperatures

Table 3 The mean nitrate concentration (mg kg−1 fresh weight) within the petiole and the proximal and distal regions of the lamina of inner and outer leaves of lettuce in relation to the cultivation season and rate of nitrogen application. Nitrogen rate (mg N L−1 )

Outer leaves

Inner leaves

Distal region of lamina

Proximal region of lamina

Petiole

Distal region of lamina

Proximal region of lamina

Petiole

Autumn crop 20 80 140 200 260

61 e(a) 300 d(b) 1203 c(c) 1629 b(c) 3021 a(c)

11 e(b) 466 d(b) 2056 c(b) 2480 b(b) 4013 a(b)

24 e(b) 1043 d(a) 3570 c(a) 4104 b(a) 6103 a(a)

50 e(a) 738 d(b) 1361 c(b) 1411 b(b) 1757 a(c)

19 d(b) 839 c(b) 1568 b(ab) 1957 a(a) 2018 a(b)

64 e(a) 1172 d(a) 1675 c(a) 2162 b(a) 2338 a(a)

Winter crop 20 80 140 200 260

23* e(b) 289 d(b) 804 c(c) 1280 b(c) 1702 a(c)

24 e(b) 329 d(b) 1312 c(b) 2041 b(b) 4325 a(b)

59 e(a) 582 d(a) 3247 c(a) 4155 b(a) 6438 a(a)

78 e(a) 472 d(b) 848 c(b) 1277 b(b) 1674 a(b)

36 e(b) 537 d(b) 1018 c(b) 1445 b(ab) 1784 a(b)

75 d(a) 933 c(a) 1498 b(a) 1732 ab(a) 2016 a(a)

Means that differ significantly within columns are followed by different letters without parenthesis; means that differ significantly within rows are indicated by a different letter within parenthesis.

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Table 4 The effect of nitrogen application rate on the mean nitrate concentration (mg kg−1 fresh weight) of inner and outer leaves of lettuce after storage at 5 or 10 ◦ C for 10 days and in relation to cultivation season. Leaf position

Nitrogen application rate (mg N L−1 ) 20

80

140

200

260

Autumn crop Outer leaves At harvest 10 days at 5 ◦ C 10 days at 10 ◦ C

107 a 98 a 101 a

842 a 953 a 804 a

2535 a 2487 a 2361 a

3019 a 2945 a 2922 a

4873 a 5033 a 5194 a

Inner leaves At harvest 10 days at 5 ◦ C 10 days at 10 ◦ C

465 a 301 b 342 b

931 a 895 a 823 a

2021 a 1935 a 1814 a

2128 a 2094 a 2091 a

2371 a 2267 a 2215 a

Winter crop Outer leaves At harvest 10 days at 5 ◦ C 10 days at 10 ◦ C

111 a 96 a 187 a

728 a 574 ab 501 b

2675 a 2730 a 2581 a

3942 a 3860 a 3932 a

5771 a 5737 a 5525 a

Inner leaves At harvest 10 days at 5 ◦ C 10 days at 10 ◦ C

291 a 117 b 120 b

1030 a 933 a 865 a

2098 a 2053 a 2171 a

2718 a 2631 a 2713 a

3230 a 3317 a 3436 a

Means that differ significantly within columns are followed by different letters.

irrespective of season (Table 4). Therefore, from a food quality perspective, it is apparent that the nitrate content of Cos lettuce leaves at harvest (which is dependent on the rate of N application) is retained during storage without significant reduction to nitrite (Poulsen et al., 1995). Similarly, Siomos et al. (2002b) reported that the nitrate concentration of romaine and loose-leaf lettuce types did not change during storage at 1 ◦ C for 3–15 days, while in spinach the leaf nitrate content did not change during storage at 5 ◦ C for 7 days, but rapidly decreased at higher temperatures (Chung et al., 2004). 3.3. Chlorophyll and vitamin C concentrations in relation to N application rate and storage conditions It is known that the chlorophyll content of leafy plants such as parsley (Yamauchi and Watada, 1993), rocket, chicory, Swiss chard (Ferrante et al., 2004) and Valeriana lettuce (Ferrante and Maggiore, 2007) decreases during storage. In the present experiments, a loss in chlorophyll content was also observed in Cos lettuce irrespective of the cultivation season (autumn or winter). Since green colour is a major quality trait in leafy vegetables, this decrease in chlorophyll concentration represents a loss of market quality. As shown for winter-grown lettuce in Fig. 1, the percent decrease in chlorophyll concentration was higher at 10 ◦ C than at 5 ◦ C (Fig. 1), indicating a temperature related rate of chlorophyll degradation as noted by Ferrante and Maggiore (2007). A similar decrease was observed in the autumn crop (data not presented). The loss of chlorophyll at both storage temperatures was not affected by N application rates between 140 and 260 mg L−1 , but the percent decrease in chlorophyll concentration was highest at the lowest rate of N application (20 mg N L−1 ). The enclosure of lettuce in polyethylene film reduced the degradation of chlorophyll during storage (Fig. 1), thereby improving the retention of visual quality, as also indicated for broccoli inflorescences (Dan et al., 2005). Leafy vegetables, especially when consumed fresh, are a valuable source of vitamin C for the human diet (Drews, 1996; Drews et al., 1997). This is particularly true for lettuce, which is a major constituent of fresh salads. The vitamin C concentration of both crops was highest at harvest and decreased with increasing N application rates. Additionally, the vitamin C concentration declined

Fig. 1. The percent decrease in chlorophyll concentration of lettuce stored at 5 ◦ C (a) or 10 ◦ C (b) for 10 days in relation to packaging (open or enclosed in polyethylene) and the rate of N application during cultivation of the winter crop. Significant differences between the means for open and closed packing at each N application rate are indicated by different letters (p = 0.05).

during storage at both 5 and 10 ◦ C, but was less when enclosed in polyethylene film. Similar data were obtained for both the winter crop (Fig. 2) and the autumn crop (not presented). Poulsen et al. (1995) reported a decrease in the vitamin C content of crisphead

Fig. 2. The concentration of vitamin C in lettuce at harvest and after storage at 5 or 10 ◦ C for 10 days in relation to packaging (open or enclosed in polyethylene) and the rate of N application during cultivation (winter crop). Significant differences between the means at each N application rate are indicated by different letters (p = 0.05).

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lettuce leaves stored at 1 ◦ C for 1–2 weeks, regardless of N application (50–200 kg ha−1 ). Moreover, Nam and Kwon (1999) observed a 39% loss of vitamin C in leafy lettuce after 4 days storage at 20 ◦ C and 50% RH, compared to 59% during storage for 10 days at 4 ◦ C and 95% RH. In our experiments, the loss of vitamin C content of Cos lettuce stored in open packages for 10 days was clearly less than that recorded by Nam and Kwon (1999) at 4 ◦ C, indicating that the nutritional quality of Cos lettuce in terms of vitamin C content is preserved better during storage than in leafy lettuce. In conclusion, although the yield of Cos lettuce grown in a hydroponic system during autumn or winter increases with increasing N concentrations up to 260 mg N L−1 in the nutrient solution, for acceptable nitrate levels within the leaves an application rate of 200 mg N L−1 may be recommended. Nitrate distribution varies with leaf age and between leaf tissues, both with respect to season and N level. Nutritional value (vitamin C content) and market quality (chlorophyll content) are highest at harvest and decrease during storage at 5 or 10 ◦ C, but the nitrate content does not change. References Abu-Rayyan, A., Kharawish, B.H., Al-Ismail, K., 2004. Nitrate content in lettuce (Lactuca sativa L.) heads in relation to plant spacing, nitrogen form and irrigation level. J. Sci. Food Agric. 84 (9), 931–936. Addiscott, T.M., Benjamin, N., 2004. Nitrate and human health. Soil Use Manage. 20 (2), 98–104. AOAC, 1995. Nitrogen (nitrate and nitrite) in animal feed. Official Methods Anal. 4, 14–15. Arnon, D., 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24 (1), 3–4. Bajaj, L.K., Kaur, G., 1981. Spectrophotometric determination of l-ascorbic acid in vegetables and fruits. Analyst 106, 117–120. Bolin, H.R., Stafford, A.E., King Jr., A.D., Huxsoll, C.C., 1977. Factors affecting the storage stability of shredded lettuce. J. Food Sci. 42 (5), 1319–1321. Boroujerdnia, M., Ansari, N.A., Dehcordie, F.S., 2007. Effect of cultivars, harvesting time and level of nitrogen fertilizer on nitrate and nitrite content, yield in romaine lettuce. Asian J. Plant Sci. 6 (3), 550–553. Brecht, P.E., Kader, A.A., Morris, L.L., 1973. The effect of composition of the atmosphere and duration of exposure on brown stain of lettuce. J. Am. Soc. Hortic. Sci. 98 (6), 536–538. Breimer, T., 1982. Environmental factors and cultural measures affecting the nitrate content in spinach. Nutr. Cycl. Agroecosyt. 3 (3), 191–292. Burns, I.G., Lee, A., Escobar-Gutierrez, A.J., 2004. Nitrate accumulation in protected lettuce. Acta Hortic. 633, 271–278. Cardenas-Navarro, R., Adamowicz, S., Robin, P., 1999. Nitrate accumulation in plants: a role for water. J. Exp. Bot. 50 (334), 613–624. Chen, B.M., Wang, Z.W., Li, S.X., Wang, G.X., Song, H.X., Wang, X.N., 2004. Effect of nitrate supply on plant growth, nitrate accumulation, metabolic nitrate concentration and nitrate reductase activity in three leafy vegetables. Plant Sci. 167 (3), 635–643. Chung, J., Chou, S., Hwang, D., 2004. Changes in nitrate and nitrite content of four vegetables during storage at refrigerated and ambient temperatures. Food Addit. Contam. 21 (4), 317–322. Dan, K., Yamato, Y., Nagata, M., Yamashita, I., 2005. Production of volatile sulfur compounds in polyethylene film packaged broccoli held at different temperatures. Jpn. Agric. Res. Quart. 39 (4), 293–297. Drews, M., 1996. Nitrate, vitamin C, and sugar content of Lactuca sativa depending on cultivar and head development. Gartenbauwissenschaft 61 (3), 122–129. Drews, M., Schonhof, I., Krumbein, A., 1997. Content of minerals, vitamins, and sugars in iceberg lettuce (Lactuca sativa var. capitata L.) grown in the greenhouse

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