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Impact of nitrogen supply limitation on tomato fruit composition
Scientia Horticulturae 264 (2020) 109173
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Impact of nitrogen supply limitation on tomato fruit composition
T
Virginia Hernández, Pilar Hellín, José Fenoll, Pilar Flores* Fruit and Vegetable Sustainability and Quality Team, Murcia Institute of Agri-Food Research and Development (IMIDA), c/ Mayor s/n. La Alberca, 3150, Murcia, Spain
A R T I C LE I N FO
A B S T R A C T
Keywords: Bioactives Antioxidants Mineral nutrition Abiotic stress Nutritional quality Solanum lycopersicum
Controversial results exist about the effect of N availability on tomato yield and the subsequent increase or decrease in fruit quality. The aim of this work was to evaluate the impact of reducing the optimum N dose (14 mM) on tomato yield and composition, depending on when the reduction was imposed. Treatments consisted of three N doses (14, 7 and 3 mM) imposed from transplant or when the first trusses began to flower. The decrease in the N dose from transplant only affected the yield in the most drastic treatment (3 mM) but a decrease in sugar and carotenoid concentrations were observed from 7 mM N. By contrast, lowering the N dose from anthesis did not affect the total yield or the concentration of most of the studied carotenoids, except lycopene and phytoene. Finally, the concentration of vitamin C and the main phenolic groups increased as the dose of N decreased, regardless of the time of application of the treatment. The results show the feasibility of reducing the dose of N in tomato without compromising the yield, and the importance of choosing the most appropriate stage to impose this reduction without affecting, or even improving, the nutritional quality of the fruit.
1. Introduction
results in the literature. The interest that has been generated in the last 20 years by this type of compound lies in their role in human health, and the potential economic advantages of products with high levels of health-promoting compounds. The main bioactive compounds of tomatoes are vitamin C, phenolic compounds and carotenoids. The role of vitamin C in human health has been widely studied and includes the synthesis of collagen and muscle carnitine and the enhancement of iron bioavailability, among other many biological functions (Naidu, 2003). Its importance is also based on its protecting effect against chronic diseases that have their origins in oxidative stress (Davey et al., 2000). In the case of hydroxycinnamic acids, anticancer activities and diabetic nephropathy induced by inflammation have been recently described (Bao et al., 2018, Gouthamchandra et al., 2017). Naringenin, the predominant flavonone found in tomato in both free and glycoside forms, has gained increasing interest because of its positive health effects in cancer prevention and also in non-cancer diseases (Mir and Tiku, 2015). Moreover, flavonols have been widely studied for their multiple pharmacological activities such as anticancer effects, analgesic and antiarthritic activities and beneficial effects on endocrine, excretory, cardiovascular, gastrointestinal, respiratory and reproductive systems (Ganeshpurkar and Saluja, 2017). Finally, carotenoids constitute the major fraction of lipophilic antioxidant compounds in tomato. Lycopene and β-carotene, the main pigments responsible for the characteristic red color, are potent antioxidants that reduce the risk of cardiovascular diseases and some forms
The correct management of nitrogen nutrition is crucial for the proper development of crops and for the sustainability of the environment (Albornoz, 2016). While a deficiency of N has been correlated with significant losses in crop production (Silva et al., 2016), the application of high doses of this essential nutrient can lead to a decrease in the shelf life of fruits, producing physiological alterations and an increasing risk of tissue rupture and senescence (Hewett, 2006). In addition, over-applying N in intensive production systems increases nitrate leaching through the soil profile (Flores et al., 2005; Sylvestre et al., 2019), causing serious environmental problems, such as groundwater pollution and eutrophication of the environment (Cameron et al., 2013). In tomatoes, an increase in the dose of N supplied leads, in general, to an increase in fruit production up to a certain limit, above which vegetative growth is favored to the detriment of productive growth (Zotarelli et al., 2009; Elia and Conversa, 2012). This increase in yield implies, in some cases, a decrease in the organoleptic quality of the fruits, as a result of the decrease in their sugar and organic acid content (Parisi et al., 2006; Wang et al., 2007). However, in some cases, an increase in the dose of N has resulted in an increase in yield accompanied by an increase in the content of sugars and total acidity (Kuscu et al., 2014). The effect of N on the content of bioactive compounds is a less studied aspect and concerning which there are also contradictory
⁎
Corresponding author. E-mail address: mpilar.fl[email protected] (P. Flores).
https://doi.org/10.1016/j.scienta.2020.109173 Received 13 June 2019; Received in revised form 18 October 2019; Accepted 2 January 2020 0304-4238/ © 2020 Published by Elsevier B.V.
Scientia Horticulturae 264 (2020) 109173
V. Hernández, et al.
first group of plants was cultivated with 14 mM N from transplant (seedlings with four true leaves, approximately 10−15 cm high) until the end of the experiment. The second group was initially cultivated with a 14 mM N dose and after the first three trusses had flowered, the different treatments were imposed until the end of the study. The treatments were distributed in the greenhouse according to a generalized randomized complete block design (Snedecor and Cochran, 1956) with two blocks and three repetitions per block (in total six repetition per treatment), each repetition formed by three plants (18 plants per treatment). To determine the total production, the number and mean weight of the fruits, each fruit was collected and weighed individually. For the analysis of quality parameters fruits were selected from trusses 2–4, when completely red and ripe, discarding those that did not have a homogeneous color or had a defect. The level of ripeness of the fruits was supervised daily to prevent them from over-ripening. For metabolite analysis, the fruits belonging to a same repetition were cut into small pieces and mixed, constituting a sample and frozen in liquid N2 and kept at −80 °C for further analysis. Soluble sugars and carotenoids were analyzed according to the method described by Flores et al. (2016). For sugars, an Agilent 1100 liquid chromatograph (HPLC) (Waldbronn, Germany) equipped with a refraction index detector and a 300 × 7.8 mm i.d., CARBOSep CHO682 LEAD column was used. Carotenoids were determined using a Hewlett-Packard mod. 1200 HPLC system (Santa Clara, CA, USA) with a photodiode array detector and a 250 mm x 4.6 mm i.d., 3 μm Prontosil C30 column (Bischoff, Leonberg, Germany). Vitamin C and phenolic compounds were analyzed following the methodology described by Fenoll et al. (2011) and Vallverdu-Queralt et al. (2010), respectively, using a HPLC-MS/MS (Agilent Series 1200, Agilent Technologies, Santa Clara, CA, USA) with an ESI interface operating in negative ion mode. The results were statistically analyzed by analysis of variance (ANOVA), using the IBM SPSS Statistic 25. Values were compared with Tukey’s range test.
of cancer (Gammone et al., 2015). In addition, β-carotene is one of the most widely studied carotenoids for its pro-vitamin A activity. Furthermore, tomato is a major source of lutein, which plays a fundamental role in the protection of vision (Sommerburg et al., 1998) and other less studied compounds, such as phytoene and phytofluene, which are attributed with an inhibitory role in the progression of atherosclerosis (Riso et al., 2006). Most authors agree that an increase in the availability of N causes a decrease in the content of vitamin C (Lee and Kader, 2000; Reboucas et al., 2015) and phenolic compounds (Li et al., 2008) in fruits. As regards the carotenoid content, in a review of the existing literature, Poiroux-Gonord et al. (2010) concluded that high doses of N favor the accumulation of carotenoids in the fruit. However, in a study conducted by Bènard et al. (2009), in which restrictions of N (6 and 4 mM) were applied in tomato plants from the flowering of the fourth truss until the end of the crop cycle, the results showed no significant differences in the lycopene and β-carotene content of the fruits compared with those cultivated with 12 mM. The differences found in the literature on the effect of the restriction of N on the concentration of metabolites of interest could be largely due to the doses of mineral N applied and the phenological time at which the reduction is applied. The proper management of nitrogen nutrition is decisive to avoid the overuse of nitrogen fertilizers that could endanger the sustainability of crops. In view of this necessity and the contradictory results found in the literature on the effect of decreased N doses on the production and composition of tomato, this work evaluates the effect on fruit quality of reducing the dose of N applied starting at from two different stages of growth: transplant and anthesis.
2. Materials and methods The experiment was carried out from winter to summer seasons in a polycarbonate greenhouse (even-spam type) from the Agrarian and Food Research & Development Institute of Murcia (IMIDA) located in La Alberca (Murcia), SE Spain. Tomato plants (Solanum lycopersicum L.) were cultivated in 20 L-pots filled with a mix of coconut fiber and perlite (85:15) and irrigated by drip irrigation at a rate based on FAO methodology (Doorenbos and Pruitt, 1997) partially modified by Keller and Bliesner (1990). The plants were watered with Hoagland nutrient solution which was modified in order to obtain three different N doses, by means of total or partial substitution of KNO3 and Ca(NO3)2, adding Cl2Ca and K2SO4 if necessary. The treatments consisted of the application of different doses of N (14, 7, 3 mM) from two stages of plant development (transplant and anthesis) to the end of experiment (Fig. 1). The concentration of 14 mM N was used as a control of the N dose while the other N treatments consisted of a 50 % and 78 % reduction in N, respectively. This control N dose was selected according to previous literature (Letard et al., 1995; Peet and Welles, 2005). A
3. Results and discussion 3.1. Fruit yield and sugar content The effect of N restriction on yield and sugar content depended to a large extent on the time from which restriction was imposed. Applied from transplant, a decrease in the dose of N only reduced fruit yield significantly compared with the control in the case of 3 mM N, as a result of a decrease in the number of fruits (Fig. 2). In the case of 7 mM N, yield was similar to that obtained in the 14 mM N treatment but there was a significant decrease in the concentration of sugars (glucose and fructose) in fruit (Table 1), compounds directly related to the organoleptic quality of tomatoes. On the other hand, a decrease of the N dose imposed from anthesis of the first trusses did not affect the total yield in either of the treatments (3 and 7 mM N). In the 3 mM N treatment, there was an increase in the number of fruits compared with the control (14 mM), accompanied by a decrease in their fruit mean weight, which was attributable to a lower source/sink ratio. Contrary to that observed in plants with N restrictions applied from transplant, in the treatments applied from anthesis a significant increase in the concentration of sugars in fruits cultivated with the lowest concentration of N was observed. Most of the previous studies describe an accumulation of sugars in tomato fruits as a result of a restricted N application (Wang et al., 2007). According to Bènard et al. (2009), this accumulation is due to the decrease of vegetative growth, which results in an increase in the irradiance of the fruit, thus increasing its photosynthetic activity and therefore their sugar content. In addition, the decrease in vegetative growth would also cause an increase in the temperature of the fruits and, consequently, the flow of C and the hexose content (Walker and Ho, 1977). Finally, Wingler et al. (2006) attributed the accumulation of sugars under N restrictions to a decrease
Fig. 1. Scheme of the application of the different N dose restrictions in the treatments. 2
Scientia Horticulturae 264 (2020) 109173
V. Hernández, et al.
Fig. 2. Effect of decreasing N doses applied from transplant (A) and anthesis (B) on total fruit yield (kg plant-1), number of fruits and mean fruit weight (g). Values are mean ± SE (n = 6). Different letters in the bars indicate significant differences between means according to Duncan’s test at the 5 % level and without letters indicate non-significant differences at P = 5 %.
photoassimilates towards the fruits.
Table 1 Glucose, fructose and vitamin C concentrations (mg g−1) in tomato grown with different N doses supplied from transplant or anthesis. Stage
mM N
Glucose
Fructose
Vitamin C
Transplant
14 7 3
Anthesis
14 7 3
20.4b 17.7a 17.9a ** 20.4a 20.9ab 22.3b *
18.2b 16.0a 15.9a * 18.2a 18.7a 20.3b **
0.27a 0.31b 0.31b ** 0.25a 0.25a 0.29b *
3.2. Vitamin C A decrease in the dose of N increased the concentration of vitamin C, regardless of when the restriction was applied (Table 1). These results coincide with those found in a previous study on a commercial variety and the mutant dwarf Micro-Tom (Flores et al., 2016) and those described by Wang et al. (2008) for tomatoes and other vegetables. Some authors relate this increase, as in the case of sugars, with an increase in fruit irradiance (Dumas et al., 2003; Simmone et al., 2007). In plants, ascorbate is an antioxidant molecule and a key substrate for the detoxification of reactive oxygen species (ROS) that plays a key role modulating several fundamental plant functions both under stress and non-stress conditions (Akram et al., 2017). In particular, ascorbate is a secondary metabolite whose synthesis can increase under adverse environmental conditions, as a response of the plant to an abiotic stress (Gill and Tuteja, 2010; Gallie, 2013). Thus, the nutritional stress caused by the restriction of N and the increase of the temperature of the fruits as a result of lower vegetative growth, could lead to the accumulation of this metabolite in fruits.
*, ** Significant differences between means at 5 or 1 % level of probability, respectively; n.s., non-significant at P = 5 %. For each stage, different letters in the same column indicate significant differences between means according to Duncan’s test at the 5 % level.
in the demand for carbon skeletons for the synthesis of amino acids and proteins. These results coincide with those obtained in our experiment when the limitations of N were imposed from anthesis of the first trusses. However, in the N treatments applied from transplant the opposite effect was observed, probably because the longer restriction of N had a much more drastic effect, affecting photosynthesis during the first stages of development of the plant, thus interfering in the process of accumulation of reserves and therefore the subsequent distribution of
3.3. Phenolic compounds Hydroxycinnamic acid derivates were mainly represented in tomato 3
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carotenoids were not affected, except lutein, whose concentration was lower in the 3 mM N treatment compared to the rest of them (Table 3). The decrease in N availability causes, in the first place, a decrease in vegetative growth, which results in an increase in temperature and irradiation to which the fruit is exposed (Porto et al., 2016), which may have two adverse effects on the synthesis of carotenoids. On the one hand, the increase in irradiation would stimulate the synthesis of carotenoids while, on the other hand, the increase in temperature could cause its degradation (Dumas et al., 2003). According to Hernández et al. (2015), temperature affects the concentration of carotenoids in different ways, depending on the metabolic canalization between the different branches of the isoprenoid biosynthesis route. Thus, the results obtained for the effect of the limitation of N on the content of carotenoids in fruit can only be explained in the case of applying different treatments; from the moment of transplant, by a limitation in the flow and/or synthesis of photoassimilates towards the fruits, which translates into a lower content of sugars and carotenoids. Also, when N was limited at the time of anthesis, the differences found in the content of carotenoids would be due to the combination of the opposing effects that the increase in irradiation and temperature has on the synthesis and canalization of the different metabolites of the isoprenoids pathway.
Table 2 Flavanones, flavonols and hydroxycinnamic acid concentration (μg g−1) in tomato grown with different N doses from transplant or anthesis. Stage
mM N
Flavanones
Flavonols
Hydroxycinnamic
Transplant
14 7 3
Anthesis
14 7 3
5.3a 7.1ab 9.0b ** 3.4a 4.1a 5.7b ***
7.5a 21.7b 35.1 c * 4.3a 5.2a 8.7b ***
19.1a 23.7b 27.6c *** 31.2a 34.6ab 37.2b *
*Significant differences between means at 5 % level of probability; n.s., nonsignificant at P = 5 %. For each stage, different letters in the same column indicate significant differences between means according to Duncan’s test at the 5 % level.
by coumaroylquinic acid (71 %) followed by p-coumaric acid (16 %), chlorogenic acid (7 %), cryptochlorogenic acid (1.4 %), caffeic acid-Ohexoside (2.2 %), ferulic acid-O-hexoside (1.5 %), dicaffeoylquinic (0.25 %), ferulic acid (0.05 %) and caffeic acid (0.17 %). The specific flavonols found in tomato were rutin (67 %), kaempferol-3-O-rutinoside (31 %), rutin-O-pentoside (1.1 %) and quercetin (0.28 %). Similar to the results observed for vitamin C, the concentrations of flavonones, hydroxycinnamic acids and flavonols found in tomato tended to increase as the N supply decreased, regardless of the time of application of the treatments (Table 2). For all the phenolic groups, at the lowest N concentration (3 mM), differences were significant when compared with the control (14 mM N). In addition, significant differences with regard the control were observed in flavononols and hydroxycinnamic derivates at 50 % N (7 mM) when that reduction was imposed from transplant. In addition to ascorbate, phenolic compounds are the main hydrophilic antioxidants that play a key role in the scavenging free radicals in plants by donating electron or hydrogen for the detoxification of H2O2 produced under stress conditions (Jaleel et al., 2009). The accumulation of these hydrophilic antioxidant compounds by a decreasing N supply correspond, as in the case of ascorbic acid, to a plant defense mechanism against the abiotic stress induced by N limitation (Ochoa-Velasco et al., 2016).
4. Conclusions In conclusion, the limitation of N favors the accumulation of hydrophilic antioxidants such as ascorbic and phenolic compounds, even under conditions of severe N limitation (78 % reduction starting at transplant). In addition, contrary to the effects observed in treatments in which the restriction of N was applied since transplant, a 50 % reduction in the dose of N since anthesis did not affect the production or concentration of the majority of carotenoids (lutein, violaxanthin, phytofluene, γ-carotene and β-carotene) and increased the content of compounds directly related to the organoleptic (sugars) and functional (phytoene and lycopene) quality of the fruit. It is concluded that it is feasible to reduce the dose of N in tomato without compromising the yield, and the importance of choosing the most appropriate stage to impose this reduction without affecting, while even improving, the nutritional quality of the fruit. At the same time, there are other possible advantages involved in reducing the N supplied in intensive growing conditions, including economic (saving of inputs) and environmental benefits.
3.4. Carotenoids The effect of N dose on the main carotenoids in tomato depended on each specific compound and on the time from which the restriction was imposed (Table 3). The severity of the limitation of N imposed from transplant, resulted in the concentration of all the analyzed carotenoids decreasing as the N dose decreased. However, the results obtained when the treatments were imposed from anthesis depended on the metabolite studied. While the concentration of lycopene and its precursor phytoene increased as a result of the reduction of N, most of the
Declaration of Competing Interest None. Acknowledges The authors are grateful to Inmaculada Garrido González, Juana
Table 3 Lutein, violaxanthin, phytoene, phytofluene, γ-carotene, β-carotene and lycopene, expressed as μg g−1, in tomato fruits grown wih different N doses from transplant or anthesis. mM N Transplant
14 7 3
Anthesis
14 7 3
Lutein b
14.1 9.0a 8.1a * 14.2b 14.4b 12.5a *
Violaxnthin b
2.5 1.4ab 1.0a * 2.5 2.9 2.4 n.s.
Phytoene b
Phytofluene b
24.9 20.8ab 16.5a * 24.9a 26.4b 26.9b *
6.0 5.5ab 3.6a * 6.0 7.1 6.9 n.s.
γ-caratone b
2.4 1.9ab 1.6a * 2.1 2.6 1.9 n.s.
β-carotene b
19.3 17.1ab 13.1a * 34.9 35.4 36.2 n.s
Lycopene 113.4b 90.4ab 70.3a * 122.5a 150.1b 155.5b *
* Significant differences between means at 5 % level of probability; n.s., non-significant at P = 5 %. For each stage, different letters in the same column indicate significant differences between means according to Duncan’s test at the 5 % level. 4
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Impact of nitrogen supply limitation on tomato fruit composition
T
Virginia Hernández, Pilar Hellín, José Fenoll, Pilar Flores* Fruit and Vegetable Sustainability and Quality Team, Murcia Institute of Agri-Food Research and Development (IMIDA), c/ Mayor s/n. La Alberca, 3150, Murcia, Spain
A R T I C LE I N FO
A B S T R A C T
Keywords: Bioactives Antioxidants Mineral nutrition Abiotic stress Nutritional quality Solanum lycopersicum
Controversial results exist about the effect of N availability on tomato yield and the subsequent increase or decrease in fruit quality. The aim of this work was to evaluate the impact of reducing the optimum N dose (14 mM) on tomato yield and composition, depending on when the reduction was imposed. Treatments consisted of three N doses (14, 7 and 3 mM) imposed from transplant or when the first trusses began to flower. The decrease in the N dose from transplant only affected the yield in the most drastic treatment (3 mM) but a decrease in sugar and carotenoid concentrations were observed from 7 mM N. By contrast, lowering the N dose from anthesis did not affect the total yield or the concentration of most of the studied carotenoids, except lycopene and phytoene. Finally, the concentration of vitamin C and the main phenolic groups increased as the dose of N decreased, regardless of the time of application of the treatment. The results show the feasibility of reducing the dose of N in tomato without compromising the yield, and the importance of choosing the most appropriate stage to impose this reduction without affecting, or even improving, the nutritional quality of the fruit.
1. Introduction
results in the literature. The interest that has been generated in the last 20 years by this type of compound lies in their role in human health, and the potential economic advantages of products with high levels of health-promoting compounds. The main bioactive compounds of tomatoes are vitamin C, phenolic compounds and carotenoids. The role of vitamin C in human health has been widely studied and includes the synthesis of collagen and muscle carnitine and the enhancement of iron bioavailability, among other many biological functions (Naidu, 2003). Its importance is also based on its protecting effect against chronic diseases that have their origins in oxidative stress (Davey et al., 2000). In the case of hydroxycinnamic acids, anticancer activities and diabetic nephropathy induced by inflammation have been recently described (Bao et al., 2018, Gouthamchandra et al., 2017). Naringenin, the predominant flavonone found in tomato in both free and glycoside forms, has gained increasing interest because of its positive health effects in cancer prevention and also in non-cancer diseases (Mir and Tiku, 2015). Moreover, flavonols have been widely studied for their multiple pharmacological activities such as anticancer effects, analgesic and antiarthritic activities and beneficial effects on endocrine, excretory, cardiovascular, gastrointestinal, respiratory and reproductive systems (Ganeshpurkar and Saluja, 2017). Finally, carotenoids constitute the major fraction of lipophilic antioxidant compounds in tomato. Lycopene and β-carotene, the main pigments responsible for the characteristic red color, are potent antioxidants that reduce the risk of cardiovascular diseases and some forms
The correct management of nitrogen nutrition is crucial for the proper development of crops and for the sustainability of the environment (Albornoz, 2016). While a deficiency of N has been correlated with significant losses in crop production (Silva et al., 2016), the application of high doses of this essential nutrient can lead to a decrease in the shelf life of fruits, producing physiological alterations and an increasing risk of tissue rupture and senescence (Hewett, 2006). In addition, over-applying N in intensive production systems increases nitrate leaching through the soil profile (Flores et al., 2005; Sylvestre et al., 2019), causing serious environmental problems, such as groundwater pollution and eutrophication of the environment (Cameron et al., 2013). In tomatoes, an increase in the dose of N supplied leads, in general, to an increase in fruit production up to a certain limit, above which vegetative growth is favored to the detriment of productive growth (Zotarelli et al., 2009; Elia and Conversa, 2012). This increase in yield implies, in some cases, a decrease in the organoleptic quality of the fruits, as a result of the decrease in their sugar and organic acid content (Parisi et al., 2006; Wang et al., 2007). However, in some cases, an increase in the dose of N has resulted in an increase in yield accompanied by an increase in the content of sugars and total acidity (Kuscu et al., 2014). The effect of N on the content of bioactive compounds is a less studied aspect and concerning which there are also contradictory
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Corresponding author. E-mail address: mpilar.fl[email protected] (P. Flores).
https://doi.org/10.1016/j.scienta.2020.109173 Received 13 June 2019; Received in revised form 18 October 2019; Accepted 2 January 2020 0304-4238/ © 2020 Published by Elsevier B.V.
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first group of plants was cultivated with 14 mM N from transplant (seedlings with four true leaves, approximately 10−15 cm high) until the end of the experiment. The second group was initially cultivated with a 14 mM N dose and after the first three trusses had flowered, the different treatments were imposed until the end of the study. The treatments were distributed in the greenhouse according to a generalized randomized complete block design (Snedecor and Cochran, 1956) with two blocks and three repetitions per block (in total six repetition per treatment), each repetition formed by three plants (18 plants per treatment). To determine the total production, the number and mean weight of the fruits, each fruit was collected and weighed individually. For the analysis of quality parameters fruits were selected from trusses 2–4, when completely red and ripe, discarding those that did not have a homogeneous color or had a defect. The level of ripeness of the fruits was supervised daily to prevent them from over-ripening. For metabolite analysis, the fruits belonging to a same repetition were cut into small pieces and mixed, constituting a sample and frozen in liquid N2 and kept at −80 °C for further analysis. Soluble sugars and carotenoids were analyzed according to the method described by Flores et al. (2016). For sugars, an Agilent 1100 liquid chromatograph (HPLC) (Waldbronn, Germany) equipped with a refraction index detector and a 300 × 7.8 mm i.d., CARBOSep CHO682 LEAD column was used. Carotenoids were determined using a Hewlett-Packard mod. 1200 HPLC system (Santa Clara, CA, USA) with a photodiode array detector and a 250 mm x 4.6 mm i.d., 3 μm Prontosil C30 column (Bischoff, Leonberg, Germany). Vitamin C and phenolic compounds were analyzed following the methodology described by Fenoll et al. (2011) and Vallverdu-Queralt et al. (2010), respectively, using a HPLC-MS/MS (Agilent Series 1200, Agilent Technologies, Santa Clara, CA, USA) with an ESI interface operating in negative ion mode. The results were statistically analyzed by analysis of variance (ANOVA), using the IBM SPSS Statistic 25. Values were compared with Tukey’s range test.
of cancer (Gammone et al., 2015). In addition, β-carotene is one of the most widely studied carotenoids for its pro-vitamin A activity. Furthermore, tomato is a major source of lutein, which plays a fundamental role in the protection of vision (Sommerburg et al., 1998) and other less studied compounds, such as phytoene and phytofluene, which are attributed with an inhibitory role in the progression of atherosclerosis (Riso et al., 2006). Most authors agree that an increase in the availability of N causes a decrease in the content of vitamin C (Lee and Kader, 2000; Reboucas et al., 2015) and phenolic compounds (Li et al., 2008) in fruits. As regards the carotenoid content, in a review of the existing literature, Poiroux-Gonord et al. (2010) concluded that high doses of N favor the accumulation of carotenoids in the fruit. However, in a study conducted by Bènard et al. (2009), in which restrictions of N (6 and 4 mM) were applied in tomato plants from the flowering of the fourth truss until the end of the crop cycle, the results showed no significant differences in the lycopene and β-carotene content of the fruits compared with those cultivated with 12 mM. The differences found in the literature on the effect of the restriction of N on the concentration of metabolites of interest could be largely due to the doses of mineral N applied and the phenological time at which the reduction is applied. The proper management of nitrogen nutrition is decisive to avoid the overuse of nitrogen fertilizers that could endanger the sustainability of crops. In view of this necessity and the contradictory results found in the literature on the effect of decreased N doses on the production and composition of tomato, this work evaluates the effect on fruit quality of reducing the dose of N applied starting at from two different stages of growth: transplant and anthesis.
2. Materials and methods The experiment was carried out from winter to summer seasons in a polycarbonate greenhouse (even-spam type) from the Agrarian and Food Research & Development Institute of Murcia (IMIDA) located in La Alberca (Murcia), SE Spain. Tomato plants (Solanum lycopersicum L.) were cultivated in 20 L-pots filled with a mix of coconut fiber and perlite (85:15) and irrigated by drip irrigation at a rate based on FAO methodology (Doorenbos and Pruitt, 1997) partially modified by Keller and Bliesner (1990). The plants were watered with Hoagland nutrient solution which was modified in order to obtain three different N doses, by means of total or partial substitution of KNO3 and Ca(NO3)2, adding Cl2Ca and K2SO4 if necessary. The treatments consisted of the application of different doses of N (14, 7, 3 mM) from two stages of plant development (transplant and anthesis) to the end of experiment (Fig. 1). The concentration of 14 mM N was used as a control of the N dose while the other N treatments consisted of a 50 % and 78 % reduction in N, respectively. This control N dose was selected according to previous literature (Letard et al., 1995; Peet and Welles, 2005). A
3. Results and discussion 3.1. Fruit yield and sugar content The effect of N restriction on yield and sugar content depended to a large extent on the time from which restriction was imposed. Applied from transplant, a decrease in the dose of N only reduced fruit yield significantly compared with the control in the case of 3 mM N, as a result of a decrease in the number of fruits (Fig. 2). In the case of 7 mM N, yield was similar to that obtained in the 14 mM N treatment but there was a significant decrease in the concentration of sugars (glucose and fructose) in fruit (Table 1), compounds directly related to the organoleptic quality of tomatoes. On the other hand, a decrease of the N dose imposed from anthesis of the first trusses did not affect the total yield in either of the treatments (3 and 7 mM N). In the 3 mM N treatment, there was an increase in the number of fruits compared with the control (14 mM), accompanied by a decrease in their fruit mean weight, which was attributable to a lower source/sink ratio. Contrary to that observed in plants with N restrictions applied from transplant, in the treatments applied from anthesis a significant increase in the concentration of sugars in fruits cultivated with the lowest concentration of N was observed. Most of the previous studies describe an accumulation of sugars in tomato fruits as a result of a restricted N application (Wang et al., 2007). According to Bènard et al. (2009), this accumulation is due to the decrease of vegetative growth, which results in an increase in the irradiance of the fruit, thus increasing its photosynthetic activity and therefore their sugar content. In addition, the decrease in vegetative growth would also cause an increase in the temperature of the fruits and, consequently, the flow of C and the hexose content (Walker and Ho, 1977). Finally, Wingler et al. (2006) attributed the accumulation of sugars under N restrictions to a decrease
Fig. 1. Scheme of the application of the different N dose restrictions in the treatments. 2
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Fig. 2. Effect of decreasing N doses applied from transplant (A) and anthesis (B) on total fruit yield (kg plant-1), number of fruits and mean fruit weight (g). Values are mean ± SE (n = 6). Different letters in the bars indicate significant differences between means according to Duncan’s test at the 5 % level and without letters indicate non-significant differences at P = 5 %.
photoassimilates towards the fruits.
Table 1 Glucose, fructose and vitamin C concentrations (mg g−1) in tomato grown with different N doses supplied from transplant or anthesis. Stage
mM N
Glucose
Fructose
Vitamin C
Transplant
14 7 3
Anthesis
14 7 3
20.4b 17.7a 17.9a ** 20.4a 20.9ab 22.3b *
18.2b 16.0a 15.9a * 18.2a 18.7a 20.3b **
0.27a 0.31b 0.31b ** 0.25a 0.25a 0.29b *
3.2. Vitamin C A decrease in the dose of N increased the concentration of vitamin C, regardless of when the restriction was applied (Table 1). These results coincide with those found in a previous study on a commercial variety and the mutant dwarf Micro-Tom (Flores et al., 2016) and those described by Wang et al. (2008) for tomatoes and other vegetables. Some authors relate this increase, as in the case of sugars, with an increase in fruit irradiance (Dumas et al., 2003; Simmone et al., 2007). In plants, ascorbate is an antioxidant molecule and a key substrate for the detoxification of reactive oxygen species (ROS) that plays a key role modulating several fundamental plant functions both under stress and non-stress conditions (Akram et al., 2017). In particular, ascorbate is a secondary metabolite whose synthesis can increase under adverse environmental conditions, as a response of the plant to an abiotic stress (Gill and Tuteja, 2010; Gallie, 2013). Thus, the nutritional stress caused by the restriction of N and the increase of the temperature of the fruits as a result of lower vegetative growth, could lead to the accumulation of this metabolite in fruits.
*, ** Significant differences between means at 5 or 1 % level of probability, respectively; n.s., non-significant at P = 5 %. For each stage, different letters in the same column indicate significant differences between means according to Duncan’s test at the 5 % level.
in the demand for carbon skeletons for the synthesis of amino acids and proteins. These results coincide with those obtained in our experiment when the limitations of N were imposed from anthesis of the first trusses. However, in the N treatments applied from transplant the opposite effect was observed, probably because the longer restriction of N had a much more drastic effect, affecting photosynthesis during the first stages of development of the plant, thus interfering in the process of accumulation of reserves and therefore the subsequent distribution of
3.3. Phenolic compounds Hydroxycinnamic acid derivates were mainly represented in tomato 3
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carotenoids were not affected, except lutein, whose concentration was lower in the 3 mM N treatment compared to the rest of them (Table 3). The decrease in N availability causes, in the first place, a decrease in vegetative growth, which results in an increase in temperature and irradiation to which the fruit is exposed (Porto et al., 2016), which may have two adverse effects on the synthesis of carotenoids. On the one hand, the increase in irradiation would stimulate the synthesis of carotenoids while, on the other hand, the increase in temperature could cause its degradation (Dumas et al., 2003). According to Hernández et al. (2015), temperature affects the concentration of carotenoids in different ways, depending on the metabolic canalization between the different branches of the isoprenoid biosynthesis route. Thus, the results obtained for the effect of the limitation of N on the content of carotenoids in fruit can only be explained in the case of applying different treatments; from the moment of transplant, by a limitation in the flow and/or synthesis of photoassimilates towards the fruits, which translates into a lower content of sugars and carotenoids. Also, when N was limited at the time of anthesis, the differences found in the content of carotenoids would be due to the combination of the opposing effects that the increase in irradiation and temperature has on the synthesis and canalization of the different metabolites of the isoprenoids pathway.
Table 2 Flavanones, flavonols and hydroxycinnamic acid concentration (μg g−1) in tomato grown with different N doses from transplant or anthesis. Stage
mM N
Flavanones
Flavonols
Hydroxycinnamic
Transplant
14 7 3
Anthesis
14 7 3
5.3a 7.1ab 9.0b ** 3.4a 4.1a 5.7b ***
7.5a 21.7b 35.1 c * 4.3a 5.2a 8.7b ***
19.1a 23.7b 27.6c *** 31.2a 34.6ab 37.2b *
*Significant differences between means at 5 % level of probability; n.s., nonsignificant at P = 5 %. For each stage, different letters in the same column indicate significant differences between means according to Duncan’s test at the 5 % level.
by coumaroylquinic acid (71 %) followed by p-coumaric acid (16 %), chlorogenic acid (7 %), cryptochlorogenic acid (1.4 %), caffeic acid-Ohexoside (2.2 %), ferulic acid-O-hexoside (1.5 %), dicaffeoylquinic (0.25 %), ferulic acid (0.05 %) and caffeic acid (0.17 %). The specific flavonols found in tomato were rutin (67 %), kaempferol-3-O-rutinoside (31 %), rutin-O-pentoside (1.1 %) and quercetin (0.28 %). Similar to the results observed for vitamin C, the concentrations of flavonones, hydroxycinnamic acids and flavonols found in tomato tended to increase as the N supply decreased, regardless of the time of application of the treatments (Table 2). For all the phenolic groups, at the lowest N concentration (3 mM), differences were significant when compared with the control (14 mM N). In addition, significant differences with regard the control were observed in flavononols and hydroxycinnamic derivates at 50 % N (7 mM) when that reduction was imposed from transplant. In addition to ascorbate, phenolic compounds are the main hydrophilic antioxidants that play a key role in the scavenging free radicals in plants by donating electron or hydrogen for the detoxification of H2O2 produced under stress conditions (Jaleel et al., 2009). The accumulation of these hydrophilic antioxidant compounds by a decreasing N supply correspond, as in the case of ascorbic acid, to a plant defense mechanism against the abiotic stress induced by N limitation (Ochoa-Velasco et al., 2016).
4. Conclusions In conclusion, the limitation of N favors the accumulation of hydrophilic antioxidants such as ascorbic and phenolic compounds, even under conditions of severe N limitation (78 % reduction starting at transplant). In addition, contrary to the effects observed in treatments in which the restriction of N was applied since transplant, a 50 % reduction in the dose of N since anthesis did not affect the production or concentration of the majority of carotenoids (lutein, violaxanthin, phytofluene, γ-carotene and β-carotene) and increased the content of compounds directly related to the organoleptic (sugars) and functional (phytoene and lycopene) quality of the fruit. It is concluded that it is feasible to reduce the dose of N in tomato without compromising the yield, and the importance of choosing the most appropriate stage to impose this reduction without affecting, while even improving, the nutritional quality of the fruit. At the same time, there are other possible advantages involved in reducing the N supplied in intensive growing conditions, including economic (saving of inputs) and environmental benefits.
3.4. Carotenoids The effect of N dose on the main carotenoids in tomato depended on each specific compound and on the time from which the restriction was imposed (Table 3). The severity of the limitation of N imposed from transplant, resulted in the concentration of all the analyzed carotenoids decreasing as the N dose decreased. However, the results obtained when the treatments were imposed from anthesis depended on the metabolite studied. While the concentration of lycopene and its precursor phytoene increased as a result of the reduction of N, most of the
Declaration of Competing Interest None. Acknowledges The authors are grateful to Inmaculada Garrido González, Juana
Table 3 Lutein, violaxanthin, phytoene, phytofluene, γ-carotene, β-carotene and lycopene, expressed as μg g−1, in tomato fruits grown wih different N doses from transplant or anthesis. mM N Transplant
14 7 3
Anthesis
14 7 3
Lutein b
14.1 9.0a 8.1a * 14.2b 14.4b 12.5a *
Violaxnthin b
2.5 1.4ab 1.0a * 2.5 2.9 2.4 n.s.
Phytoene b
Phytofluene b
24.9 20.8ab 16.5a * 24.9a 26.4b 26.9b *
6.0 5.5ab 3.6a * 6.0 7.1 6.9 n.s.
γ-caratone b
2.4 1.9ab 1.6a * 2.1 2.6 1.9 n.s.
β-carotene b
19.3 17.1ab 13.1a * 34.9 35.4 36.2 n.s
Lycopene 113.4b 90.4ab 70.3a * 122.5a 150.1b 155.5b *
* Significant differences between means at 5 % level of probability; n.s., non-significant at P = 5 %. For each stage, different letters in the same column indicate significant differences between means according to Duncan’s test at the 5 % level. 4
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