Effect of Supplementary Pre-Harvest LED Lighting on the Antioxidant and Nutritional Properties of Green Vegetables Z. Bliznikas and A. Žukauskas Institute of Applied Research, Vilnius University Saulėtekio al. 9, building III LT–10222 Vilnius, Lithuania

G. Samuolienė, A. Viršilė A. Brazaitytė, J. Jankauskienė, P. Duchovskis and A. Novičkovas Institute of Horticulture, Lithuanian Research Centre for Agriculture and Forestry Kauno g. 30, Babtai LT–54333 Kaunas district, Lithuania

Keywords: vitamin C, free radicals, nitrates, phenols, solid-state illumination Abstract We report on the application of supplementary solid-state lighting within an industrial greenhouse for pre-harvest treatment of various green vegetables (spinach, parsley, dill, mustard, rocket, and onion leaves) grown under high-pressure sodium lamps and natural solar illumination. For 3 days before harvesting, supplementary lighting from red 638-nm light-emitting diodes (LEDs) was applied within a 19-h photoperiod in such a way that the net photosynthetically active flux density of at least ~300 µmol.m–2.s–1 was maintained. Such a pre-harvest treatment was found to remarkably enhance antioxidant and nutritional properties of green vegetables due to the increased activity of the metabolic system for the protection from a mild photooxidative stress. However, the effect of supplementary red light was found to be species dependent. The sensitivity of a species to the lighting conditions was determined by the natural level of phenolic compounds accumulated in the leaves. Supplemental lighting evokes a metabolic unbalance in green vegetables that accumulate low amounts of antioxidant compounds, therefore the flux of red light even diminish the nutritional value of spinach and rocket. Meanwhile, application of supplemental LED lighting to dill and parsley results in the accumulation of vitamin C and carbohydrates and in the enhancement of free radical binding activity and the activity of nitrate-reducing enzymes. INTRODUCTION Nutritional factors are widely considered to be critical for human health. Nutritional value of vegetables is defined by the presence of essential substances that are important to support life. For instance, all grain products are recommended for healthy diets as being recognized as sources of dietary fiber and antioxidant substances. A large array of compounds contained in human diets may function as antioxidants, such as vitamins C and E, carotenoids, flavonoids and other phenolic compounds, and may be helpful in the daily challenge against free radical and reactive oxygen species which are produced physiologically by our tissues (Ames et al., 1993). Products high in vitamin A, E and C, and β-carotene contents (as onion leaves, spinach or parsley) are believed to be the most

beneficial source of antioxidants. Besides, the total antioxidant power as an ‘integrated parameter of antioxidants present in a complex sample’ (Ghiselli et al., 2000) is often more meaningful to evaluate health beneficial effects because of the cooperative action of antioxidants. Individual antioxidant compounds act in combination with other antioxidants, as interactions among them can affect total antioxidant capacity, producing synergistic or antagonistic effects (Niki and Nogutchi, 2000). One of important nutritional quality factors of vegetables is low nitrate content. Gangolli with co-workers (1994), present contradictory data of nitrate for potential long-term health risk. Some national and international regulatory agencies are setting maximal allowed levels for nitrate in vegetables, which constitute the main dietary intake of nitrate (Santamaria, 2006). According to Hord, very high amounts (>250 mg/100 g fresh weight (FW)) of nitrate may be present in leaves and petioles of many leafy vegetables such as spinach (Spinacia oleracea L.), celery (Apium graveolens L.), rocket (Eruca sativa, Mill.), or green onion (Allium cepa L.); parsley (Petroselinum L.) and dill (Anethum graveolens L.) accumulate high (100 to 250 mg/100 g FW) and middle (50 to 100 mg/100 g FW) amounts of nitrate, respectively (Hord et al., 2009). Commonly, the nitrate content in vegetables increases with increasing the geographical latitude of an agricultural region as a result of substitution of electric light sources for deficient sunlight and use of increased concentration of fertilizers in soil. Low concentration of nitrate and the presence of vitamin C, which could reduce the negative health effect of nitrate, are important factors that determine the biochemical quality of green vegetables (Premuzic et al., 2001). In addition, vitamin C accumulated in metabolically active tissues, such as leaves, acts as a signaling molecule that coordinates a protective mechanism of the oxidative system (Pastori et al., 2003). In some growth environments, precise control of fertilization and nitrate supply in accordance with the lighting conditions can be performed without loss in productivity. Moreover, one of the approaches to stimulate the reduction of nitrates is the use of light. An attractive tool for the purposeful treatment of plants by light is solid-state lighting, which is based on narrow-bandwidth light-emitting diodes (LEDs) and offers possibilities in tailoring the illumination spectrum and controlling morphogenesis and productivity of plants (Folta and Childers, 2008; Massa et al., 2008; Morrow, 2008). Quite a number of studies have examined photomorphogenetic responses (Brown et al., 1995; Kevin 2000; Devlin et al., 2007; Folta and Childers, 2008; Massa et al., 2008) or phytochemical composition (Li and Kubota, 2009) of plants to different spectra of LED-based illumination. The main energy for photosynthesis and signaling for morphogenesis in natural illumination spectra, which plants are adapted for, is provided by red light. Pepper (Brown et al., 1995), lettuce (Hoenecke et al., 1992) has been successfully grown under red LEDs for limited time periods. However, a high photosynthetic photon flux density (PPFD) may result in a photooxidative stress. Besides, plant morphogenetic changes, functioning of the photosynthetic apparatus, and the trend of metabolic reactions can be determined by complex and multiple photoreception system (Chen et al., 2004; Devlin et al., 2007), which respond to light quantity and quality, duration and intensity of red/far red light, blue light, UV-A or UVB (Kevin, 2000). Moreover, lighting conditions might evoke the photooxidative changes in plants, which lead to the altered action of antioxidant defense system: increased contents and activity of antioxidative enzymes, carotenoid, tocopherol, flavonoid, ascorbate. However,

little published information on the variations in free-radical scavenging activity, total phenols concentration and interaction with other antioxidants, as well as on nitrate or carbohydrate accumulation in leafy vegetables under purely red light is available. The objective of this study was to evaluate the effect of supplemental narrow-band red light generated by LEDs on the antioxidant and nutritional properties of green vegetables during short-term pre-harvest illumination. MATERIALS AND METHODS White Mustard (Sinapis alba ‘Yellow mustard’), spinach (Spinacia oleracea ‘Geant d’hiver’), rocket (Eruca sativa ‘Rucola’), dill (Anethum graveolens ‘Mammouth’), parsley (Petroselinum ‘Plain Leaved’) and green onions (Allium cepa ‘White Lisbon’) were grown in peat (pH ≈ 6, accuracy ± 0.01 pH units) substrate within an industrial greenhouse (April, Lithuania, lat. 55° N). The amount of nutrients (mg/L) in substrate was as follows: N 70, P 30, K 160, Ca 200, Mg 50. Plants were fertilized with 0.2% ammonium nitrate solution once a week. The growth up to the harvesting time of the plants (about 30 days) was performed under natural daylight (averaging about 300 µmol m-2 s-1 PPFD within 14 h photoperiod and up to 1500 µmol.m-2.s-1 peak PPFD; daily integral of lighting of about 15 mol m-2). Daylight was supplemented by high-pressure sodium lamps (HPS) (Son-T Agro, Philips) at a PPFD of about 130 µmol.m-2.s-1 (12 h photoperiod; daily integral of lighting of about 8.5 mol m-2). The temperature conditions were maintained at 17-22º/14-17º C at day/night time. The relative air humidity was about 75-85 %. At the pre-harvest stage of 3 days, plants were supplementary illuminated by red (638 nm) LEDs (170 µmol m-2 s-1 peak PPFD) from 5 till 10 a.m. and from 5 till 12 p.m. The custom-made solid-state illuminator for greenhouse lighting (Bliznikas et al., 2009) contained high-power (3 W) red AlGaInP LEDs (LUXEON III Star, model LXHL-LD3C, Philips Lumileds Lighting Company, USA) with the peak wavelength of 638 nm. The LEDs were mounted on an oblong heat sink and protected against humid environment. The LEDs were driven by a custom-made power supply. The PPFD was measured using a portable photosynthesis system (CI-310, CID, Inc., Camas, USA). Antioxidant properties of the treated and reference plants were assessed after harvesting by measuring the 2,2–diphenyl–1–picrylhydrazyl (DPPH) free radical scavenging capacity and the concentrations of total phenols and vitamins C and E; nutritional quality was evaluated by measuring the concentrations of nitrate and carbohydrates. Vitamin C (L-ascorbic acid) content was evaluated using a spectrophotometric method (Janghel et al., 2007). The total content of phenolic compounds was determined in methanolic extracts of fresh leaves using a spectrophotometric Folin method (Ragaee et al., 2006). The antioxidant activity of methanolic extracts of the investigated green vegetables was evaluated spectrophotometricaly as the 2,2–diphenyl–1–picrylhydrazyl (DPPH) free radical scavenging capacity (Ragaee et al., 2006). A Genesys 6 spectrophotometer was used for the analysis (Thermospectronic, USA). Nitrate content was evaluated by a potentiometric method using ion-meter (Oakton, USA) with nitrate ion selective electrode (Cole-Parmer, USA) (Geniatakis et al., 2003). Vitamin E (alpha tocopherol, (α-T)) content was evaluated using high-performance liquid chromatographic (HPLC) method (Fernández-Orozco et al., 2003) with fluorescence detector (Shimadzu 10A, Japan) on Pinacle II silica column, 5 µm particle size, 150 x 4.6 mm (Restek, USA). The mobile phase was 0.5% isopropanol in

hexane. The peak was detected using an excitation wavelength of 295 nm and emission wavelength of 330 nm. Contents of carbohydrates (fructose, glucose and sucrose) were evaluated using the HPLC method with a refractive index detector (Shimadzu 10A, Japan) on the Shodex Sugar SC1011 column (5 µm particle size, 300×8.0 mm; Waters, Germany). The mobile phase was bi-distilled water, the oven temperature was maintained at 85º C. Data were processed using MS Excel (version 7.0). Conjugated biological sample of the green matter of 5 plants randomly selected were used for each analysis. Three analytical replications were done for each treatment. Standard errors of means are indicated in tables (p=0.05). RESULTS AND DISCUSSION The response of various plant species to treatment by solid-state lighting was different. Both positive and negative effects on the antioxidant properties and nutritional quality of green vegetables were observed (see Tables 1 and 2). The sensitivity of antioxidant system to light spectra of green vegetables depended on the naturally accumulated amount of protective phenolic compounds. Dill and parsley accumulated more phenolic compounds in comparison with other treated vegetables (see Table 1) and the supplemental red LED light resulted in an altered antioxidant activity, expressed as DPPH free radical scavenging capacity (see Table 1). Besides, a decrease in nitrate accumulation (see Table 2) under red LED treatment was observed. Wu and co-authors also noticed that antioxidant capacity of pea seedlings after 96 h radiation by various LED lights was significantly enhanced by red light radiation (Wu et al., 2007). Li and Kubota (2009) also stated that supplemental red light increased phenolics concentration in baby leaf lettuce. However supplemental red light had no influence on free radical scavenging capacity, total phenols (see Table 1) and nitrate (see Table 2) accumulation in spinach. Red LED lighting resulted in an increase of vitamin C content in all treated vegetables, except of parsley. However, it had no significant effect on vitamin E (α-T) accumulation but correlated with changes in vitamin C contents (see Table 1). Thus in agreement with Liu et al. (2008), free-radical scavenging capacity and total phenols act synergistically with vitamin C and vitamin E. Moreover, the total antioxidant power was substantially superior to the sum of the properties of individual antioxidants and was enhanced by the selecting of a proper lighting spectrum. Thus the interactions among them can affect total antioxidant capacity, producing synergistic or antagonistic effects (Niki and Nouguchi, 2000). The observed alternations in vitamin C concentration (see Table 1) indicated on the absence of a direct correlation of the vitamin metabolism with the reduction of nitrate (see Table 2). The same trend of red LED effect was observed in our previous investigations with lettuce, marjoram and green onions (Samuolienė et al., 2009). However, the tendency of dropping vitamin C content in some LED-treated species deserves discussion, since vitamin C is not only an important nutrient but also plays a major role in the protection of plants against photo-oxidative stress, as well as in photoprotection and action of phytohormones (Pastori et al., 2003). Ono et al. (2001) supposes that red light may induce the senescence but this is not related to photo-oxidative stress. Our data shows that no senescence was observed. The protective reaction of the metabolic processes was the response of the antioxidant

system. Interestingly, a noticeable increase in vitamin C content (see Table 1) was observed in green vegetables where red-LED treatment invoked an increase in the concentration of saccharides. The supplemental red light remarkably affected the accumulation of fructose and glucose but showed a diverse effect on the accumulation of sucrose (see Table 2). A significant increase in saccharides (especially of fructose and glucose) and decrease in nitrate content was observed in dill and parsley under supplemental red-LED treatment. In vegetables, where the increase of carbohydrates was not so remarkable, the accumulation of nitrate was increased (in mustard, rocket or onion leaves) or remained at the same level (in spinach) (see Table 2). Price et al. (2004) showed that glucose has a stronger effect on the regulation of genes associated with nitrogen metabolism than nitrogen supply. Besides, such an increase in carbohydrate concentration is favourable in terms of self-supporting nitrate reductase activity through the stimulation of gene expression (Lillo and Appenroth, 2001). Moreover, carbohydrates are able to compete light in stimulating expression of genes involved in nitrogen assimilation (Thum et al., 2003). However, the supplemental lighting with red LEDs results in an increased carbohydrate content, especially of sucrose in dill and parsley (see Table 2). Besides, sucrose metabolism is a physiological signal affecting further metabolic processes (Koch, 2004) as well as nutrient and gustative properties. Different lighting conditions affected various plant species and particular metabolic pathways in them in different way. In some cases such short-term lighting by supplemental narrow-band red light was not optimal for the improvement of antioxidant and nutritional properties of green vegetables. It caused an unbalance of the metabolic system in inherently less antioxidant compounds accumulating green vegetables and resulted in the decrease of nutritional quality (see Tables 1 and 2). This is in agreement with other authors who demonstrated that supplemental light of selected wavelengths (UV-A, blue, red, far red or their appropriate ratio) could be strategically used to improve phytochemical content and growth of vegetables (Li and Kubota, 2009). CONCLUSIONS Short-term pre-harvest treatment was found to remarkably enhance antioxidant and nutritional properties of green vegetables due to the increased activity of the metabolic system for the protection from a mild photooxidative stress. However, the effect of supplementary red light was found to be species dependent. The sensitivity of a species to the lighting conditions was determined by the natural level of phenolic compounds accumulated in the leaves. Supplemental lighting evokes a metabolic unbalance in green vegetables that accumulate low amounts of antioxidant compounds, therefore the flux of red light even diminish the nutritional value of spinach and rocket. Meanwhile, application of supplemental LED lighting to dill and parsley results in the accumulation of vitamin C and carbohydrates and in the enhancement of free radical binding activity and in the decrease in nitrate accumulation. ACKNOWLEDGEMENTS This work was partially supported by the Lithuanian State Science and Studies Foundation under PHYTOLED project.

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Tables Table 1 Antioxidant properties of green vegetables after 3 days of red-LED treatment before harvesting DPPH, Total phenols, Vitamin C, α-T, µg/g, FM µmol/g, FM mg/g, FM mg/g, FM HPS

HPS+LED

HPS

HPS+LED

HPS

Mustard

2.60 2.98 0.76 0.86 0.46 ±0.481 ±0.736 ±0.017 ±0.030 ±0.011 Spinach 1.16 1.01 0.37 0.35 0.34 ±0.672 ±0.261 ±0.030 ±0.010 ±0.017 Rocket 10.37 5.45 0.72 0.62 0.35 ±0.179 ±0.093 ±0.035 ±0.022 ±0.032 Dill 6.60 6.33 1.43 1.32 0.52 ±0.369 ±0.076 ±0.019 ±0.023 ±0.008 Parsley 7.94 8.06 1.07 1.00 0.53 ±0.138 ±0.102 ±0.023 ±0.034 ±0.021 Onion 5.41 6.40 0.56 0.42 0.33 leaves ±0.319 ±0.098 ±0.033 ±0.047 ±0.012 ± standard errors of means (p=0.05); FM – fresh mass

HPS+LED

HPS

HPS+LED

0.58 ±0.057 0.41 ±0.044 0.39 ±0.007 0.87 ±0.016 0.43 ±0.015 0.35 ±0.017

3.52 ±0.387 3.06 ±0.224 3.41 ±0.341 2.18 ±0.107 1.90 ±0.177 1.39 ±0.060

3.32 ±0.224 3.63 ±0.589 2.96 ±0.139 1.81 ±0.083 1.77 ±0.107 1.68 ±0.132

Table 2 Nutritional properties of green vegetables after 3 days of red-LED treatment before harvesting Carbohydrates Nitrate Fructose Glucose Sucrose mg/kg, FM mg/g, FM mg/g, FM mg/g, FM HPS

Mustard

HPS+LED

HPS

HPS+LED

HPS

3011 5361 0.33 ±160.6 ±363.6 ±0.027 Spinach 2989 2783 0.92 ±127.3 ±102.0 ±0.047 Rocket 4120 4491 0.26 1.61 0.61 ±188.8 ±80.4 ±0.034 ±0.316 ±0.016 Dill 4879 4313 4.11 10.37 2.48 ±80.9 ±154.5 ±0.360 ±0.141 ±0.610 Parsley 4486 2675 3.25 3.32 3.34 ±408.5 ±35.3 ±0.103 ±0.150 ±0.375 Onion 2142 2650 1.65 3.06 1.59 leaves ±26.1 ±33.2 ±0.262 ±0.608 ±0.019 ± standard errors of means (p=0.05); FM – fresh mass

HPS+LED

HPS

HPS+LED

0.22 ±0.011 0.98 ±0.003 3.76 ±0.084 13.82 ±2.177 7.30 ±0.266 2.86 ±0.101

0.03 ±0.07 0.06 ±0.53 -

0.58 ±0.068 -

4.04 ±0.113 2.51 ±0.033

0.95 ±0.002 9.16 ±0.092 16.12 ±0.050 2.36 ±0.236