Growth and nutritional properties of lettuce affected by different alternating intervals of red and blue LED irradiation

Scientia Horticulturae 223 (2017) 44–52

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Growth and nutritional properties of lettuce affected by different alternating intervals of red and blue LED irradiation

MARK

⁎

Xiao-li Chena,b, Qi-chang Yangb, Wen-pin Songa, Li-chun Wanga, Wen-zhong Guoa, , ⁎ Xu-zhang Xuea, a b

Beijing Research Center for Information Technology in Agriculture, Beijing Academy of Agriculture and Forestry Sciences, Beijing, 100097, China Institute of Environment and Sustainable Development in Agriculture, Chinese Academy of Agricultural Sciences, Beijing, 100081, China

A R T I C L E I N F O

A B S T R A C T

Keywords: Red light Blue light LED Alternating irradiation Plant factory Photoreceptor

In this study, effects of alternating red light (R) and blue light (B) provided by LEDs on the growth and nutritional properties of ‘Green Oak leaf’ lettuce were examined. Four alternating light treatments (R/B) had the same 8.64 μmol m−2 daily light integral (DLI) and similar R:B ratio (2:1), but different R/B alternating intervals that were respectively 8 h, 4 h, 2 h, and 1 h during a 16 h photoperiod. Two simultaneous light treatments, one of which (RB) had the same DLI and energy consumption with the alternating light treatments, while the other (RB’) had the same photoperiod with the alternating light treatments were set up to compare the concurrently and alternately provided R and B. Results showed that different types of radiation led to obvious morphological changes, plants with simultaneous RB appeared the most sparse while those with RB’ looked the most compact. Plant height/width and leaf length/width were all the highest under R/B (8 h) followed by R/B (1 h). Lettuce biomass under RB’ was significantly higher than others, more than twice that under RB, R/B (4 h) and R/B (2 h), but less than twice that under R/B (8 h) and R/B (1 h). RB’ significantly decreased the soluble sugar content by 9%–32% while increased crude fiber content by 14%–39% compared with others. Significantly higher ascorbic acid content as well as lower nitrate content were detected in lettuce under R/B (4 h) and R/B (2 h), while significantly lower ascorbic acid content as well as higher nitrate content were detected in lettuce under R/B (8 h) and R/B (1 h). In all, based on the same energy consumption, R/B (8 h) and R/B (1 h) resulted in higher yield, while R/B (4 h) and R/B (2 h) brought about higher nutritive value compared with the cocurrent light RB. Therefore, we conclude that lettuce growth and qualities can be purposely adjusted by adopting different alternating intervals of red and blue light. Meanwhile, the alternating modes will provide methods for deeply studying the relationship of red and blue light when acting on plants.

1. Introduction Light is not only energy source but also important environmental signal for plant growth and development, morphological and physiological adaptations of plants can be mediated through morphogenetic responses and through light-dependent adjustments in photosynthesis (Abidi et al., 2013). Light quality shows much more complex effects on plant morphology and physiology compared with light intensity and photoperiod (Taiz and Zeiger, 1991). Light quality affects the formation and accumulation of leaf photosynthetic pigments which might either increase light harvest under low-light conditions, or act as screening pigments and free-radical scavengers under high-light conditions (Carvalho et al., 2011). In addition, light quality affects gene expression of plants through initiating the signaling cascade of photoreceptors like phytochromes, cryptochromes and phototropins (Lillo and Appenroth, ⁎

Corresponding authors. E-mail addresses: [email protected] (W.-z. Guo), [email protected] (X.-z. Xue).

http://dx.doi.org/10.1016/j.scienta.2017.04.037 Received 23 November 2016; Received in revised form 2 April 2017; Accepted 3 April 2017 0304-4238/ © 2017 Elsevier B.V. All rights reserved.

2001; Giliberto et al., 2005). In terms of light quality, the effects of red (R) and blue light (B) on plant growth and development attract most of the attentions since these wavelengths are predominantly absorbed by photosynthetic pigments and have the largest impact on plant architecture and development (Pfündel and Baake, 1990; Massa et al., 2008; Abidi et al., 2013). As a crucial part of the light spectrum for normal plant growth, R affects plant morphogenesis by inducing transformations in phytochrome, and is also crucial for developing the photosynthetic apparatus as well as regulating the synthesis of phytochemicals such as phenolics and oxalate (Saebo et al., 1995; Furuya, 1993; Qi et al., 2007; Choi et al., 2015). B is important for chloroplast development, stomatal opening and photomorphogenesis, as well as for regulating the biosynthesis of chlorophyll and anthocyanin. (Cosgrove, 1981; Senger, 1982; Giliberto et al., 2005; Li and Kubota, 2009).

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spectrum at a specific wavelength, giving the possibility for real controls of light spectrum. Second in importance to the spectra is low heat output, because of which LEDs can be placed adjacent to or even within plant canopy, allowing more of the light radiation to be intercepted by the plant tissue and improving light use efficiency and energy (Okamoto et al., 1996; Ilieva et al., 2010). Although the development of LEDs have brought new opportunities for horticultural cultivation lighting, the cost of electricity for light irradiation is still a major factor which has limited the application and development of plant factories. It is necessary to explore new lighting strategies and irradiation patterns, which can well meet the growth of plants as well as reduce the electricity cost. As mentioned before, apart from the spectral compositions, radiation types such as continuous/intermittent and concurrent/alternate patterns may also affect the growth and physiological processes of plants. LED is a wavelength specific light source with a proper driver design, simultaneous or alternating light as well as continuous or pulse light, or both can be generated using an LED light source with specially designed driver (Jao and Fang, 2004). It is possible that different alternating modes of red and blue LED light which have the same DLI, spectral composition and power consumption can result in discrepancies in the growth or qualities of lettuce. Therefore, the focal point of the study was the comparison of red and blue light provided at the same time vs. provided separately with different alternating intervals based on the same daily light integral. The goal of the study was to determine the effects of different radiation modes of red and blue LED light on growth and quality of lettuce by investigating shoot growth, plant height, biomass, and accumulations of chlorophyll (Chl), carotenoids (Car), soluble sugars, ascorbic acid and nitrate. The results provide new ideas for the photo-physiology research as well as the possible lighting strategies in consideration of both plant and energy conservation during lettuce production.

The wavelengths of R and B always coexist in natural light environments, it was reported that monochromatic R or B could not meet the requirement for normal plant growth. For instance, plants under R alone displayed abnormal morphology and reduced net photosynthetic rate (Pn) compared with white light or R supplemented with B (Goins et al., 1998; Wang et al., 2015; Hogewoning et al., 2010). Also, B alone or a constant illumination with high amounts of B might have negative effects such as reduced Pn in many species due to chloroplast avoidance responses (Wada et al., 2003; Kim et al., 2004) and impaired mesophyll conductance (Loreto et al., 2009). It has been reported that mixed R and B resulted in increased Pn and shoot biomass compared to monochromatic R or B (Brown et al., 1995; Ohashi-Kaneko et al., 2006; Hogewoning et al., 2010; Nanya et al., 2012; Li et al., 2013). The optimal R:B ratio of mixed R and B differed with plant species, growth periods and the targeted qualities, for instance, the optimal R:B ratio for biomass accumulation in lettuce, strawberry, rapeseed and cucumber was respectively reported as 12 (Wang et al., 2016), 7/3(Nhut et al., 2003), 1/3(Li et al., 2013) and 9 (Hernández and Kubota, 2016). Moreover, some researchers suggested that blue light during plant growth was qualitatively required for normal photosynthesis and regulated plant responses quantitatively resembling those to irradiance intensity. Dougher and Bugbee (2001), who studied the growth and development of lettuce under high-pressure sodium (HPS) and metal halide (MH) lamps with yellow filters creating five fractions of blue light, reported that as little as 2% blue light added into red LED light increased lettuce biomass. Hogewoning et al. (2010) also reported that leaf photosynthetic machinery dysfunction was easily to happen for plants grown under R alone, and only 7% B was enough to prevent dysfunctional photosynthesis. That was, although lots of researches have been done on plant responses to R and B, the relationships between R and B when acting on plants still maintain unclear. All those studies mentioned above have not concerned the radiation type of alternative red and blue light. That was, either mixed R and B or monorchromic R and B was provided during the whole culture period. In fact, not only spectral compositions but also the types of radiation (continuous or intermittent, concurrent or alternate) affected the growth and physiological process of plants, and a few studies can be cited here. For example, Yamada et al. (2000) claimed that stepwise photosynthetic photon flux (PPF) control is a useful method for reducing the electricity consumption of lighting and increasing the electricity utilization efficiency. Sivakumar et al. (2006) who compared the effects of intermittent light with continuous light on sweet potato plantlets in vitro reported that dry weight and carbohydrate content were greater under intermittent blue or blue-plus-red light compared to corresponding continuous blue or blue-plus-red light. Hoffmann et al. (2016) reported that an intermittent illumination with high/low blue light enhanced the potential to accumulate epidermal flavonols and triggered the synthesis of anthocyanins and carotenoids, meanwhile, treatments with alternating high and low blue light intensities could be used to restrict negative effects of blue light on plant growth. Shimokawa et al. (2014) pointed that alternating R and B accelerated lettuce growth significantly based on the same total light intensity per day with the simultaneous irradiation of R and B. However, contrasting results with regard to potato plantlets were reported by Jao and Fang (2004) who found that plantlets illuminated with alternate R and B light had significantly less fresh/dry weight accumulation compared with concurrent R and B light treatment. Moreover, the alternating treatment of R and B may provide a possible way for deeply studying the relationship of red and blue light when acting on plants. As a new vegetable production system and platform for plant photophysiology research, plant factories with artificial light have been attracting more and more attentions. Conventional artificial lights such as metal halide, high-pressure sodium (HPS), incandescent and fluorescent lamps all have wide ranges of wavelengths that are impossible to be positioned for desired specific spectrums (Wheeler, 2008). In contrast, the most valuable characteristic of LEDs is the adjustable

2. Materials and methods 2.1. Experimental set-up and growth conditions After incubation at 4 °C on moistened gauze for 5 d, germinated lettuce seeds (Lactuca sativa var. crispa ‘Green Oak Leaf’) were sown in sponge cubes (2.0 cm × 2.0 cm × 2.0 cm) and hydroponically grown in an environmentally controlled growth room. Environmental conditions in the experiment were maintained at 24/20 °C (day/night), 65% relative humidity (RH) and 400 μmol mol−1 CO2 level. 36 plants spaced 13 cm apart were planted in each hydroponic box (80 cm × 80 cm × 10 cm). The Hoagland’s solution (Hoagland and Arnon, 1950) was used and renewed per week, the electrical conductivity (EC) and pH were adjusted to 0.11–0.12 S m−1 and 5.8–6.0 respectively. The plants were irradiated with six light treatments with different radiation modes described below and harvested at 48 days after sowing (DAS). 2.2. Light quality treatments Illumination treatments were performed using two-color LED panels (80 cm × 80 cm) intentionally designed and assembled by NERCITA, Beijing, China. The two-color LED panels provides R with peak wavelength of 660 nm and B with peak wavelength of 450 nm (Fig. 1), which were determined by a spectrophotometer (Ocean Optics, model-SD 650, USA). Irradiation intensity of R and B could be individually controlled by regulating electric current of power DC supply for each treatment, and alternating intervals of R and B could be adjusted by the built-in timing switches. The distance between LED panels and plant canopy were 20 cm, red and blue light were respectively set at 200 ± 5 μmol m−2 s−1 and 100 ± 3 μmol m−2 s−1 in all the treatments as measured with a light quantum meter at plant canopy level (LI-250A, LI-COR, USA). As shown in Fig. 2, four alternating light patterns as well as two simultaneous light 45

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Fig. 1. Light spectral of red and blue LED used in the experiment. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2. The alternating modes of red (R) and blue (B) LED light during 24 h in different treatments of RB, RB’, R/B (8 h), R/B (4 h), R/B (2 h), R/B (1 h). The simultaneous irradiation provided by RB and RB’ respectively had a photoperiod of 8 h and 16 h. The alternating irradiation provided by R/B(8 h) was 8 h:8 h (i.e., 8 h of R and 8 h of B during a 16 h photoperiod), and by R/B (4 h), R/B (2 h), R/B (1 h) was respectively 4 h:4 h, 2 h:2 h and 1 h:1 h.

2.4. Determination of chlorophyll and carotenoid

patterns of R and B were examined. The alternating light treatments (R/B) had the same 8.64 μmol m−2 daily light integral (DLI) and similar R:B ratio (2:1), but different R/B alternating intervals that was respectively 8 h, 4 h, 2 h, and 1 h during a 16 h photoperiod. That was, the alternating irradiation provided by R/B(8 h) was 8 h:8 h (i.e., 8 h of R and 8 h of B for a 16 h photoperiod), and by R/B(4 h), R/B(2 h), R/B(1 h) was respectively 4 h:4 h, 2 h:2 h and 1 h:1 h. Two simultaneous light treatments, one of which (RB) had the same DLI and energy consumption with the alternating treatments, while the other (RB’) had the same photoperiod (16 h) with the alternating treatments were set up to compare the concurrently and alternately provided R and B. That was, the DLI and the energy consumption in RB’ was twice that of other treatments, while the photoperiod in RB was half of that in other treatments.

A total of 0.2 g fresh samples from the mature leaves of lettuce were ground in a mortar, and then the ground were washed using 80% acetone and subsequently filtered (repeated until the leaf turned white). The filtrates were diluted to a total volume of 100 mL with distilled water. The absorbance of the extraction at 470 nm, 645 nm, and 663 nm was respectively measured by a TU-1810s spectrophotometer (PERSEE, Beijing, China). Concentrations of the chlorophyll and carotenoid were determined using the following equations (Lichtenthaler and Wellburn, 1983): − 2.59 × OD645)V Chl a (mg/g) = (12.72 × OD663 1000 W − 4.67 × OD663)V Chl b (mg/g) = (22.88 × OD645 1000 W

2.3. Measurements of plant growth and morphology × Chl.a − 104 × Chl.b) / 229)V Car (mg/g) = ((1000 × OD470 − 3.271000 W

Eight plants randomly taken from each treatment were regarded as a repetition for biometric and nutritional measurements. Among which, plant height/width, leaf length/width were measured once every 6 days while other indexes were measured at harvest (48 DAS). The fresh weight (FW), as well as the contents of chlorophyll, carotenoid, nitrate and ascorbic acid were all determined using fresh lettuce samples. The dry weight (DW) as well as the contents of soluble sugar and crude fiber were determined using the oven-dried lettuce samples (60 °C for 40 h). The ratio of shoot and root (S/R) was determined from shoot/root DW. In addition, several representative plants were chosen from each treatment for plant morphology description, which was done mainly in terms of the shape and color of the harvested lettuce.

V is the total volume of acetone extract (mL) and W is the fresh weight (g) of the sample. 2.5. Determination of soluble sugar and crude fiber Soluble sugar was measured by the method of Fairbairn (1953). A mixture of 0.5 g (DW) lettuce shoot sample and 5 mL deionized water were heated in a 80 °C water bath for 30 min and subsequently cooled and filtered (repeated twice), a constant total volume of 10 mL was achieved using the filtrate and deionized water. The soluble sugar content was determined with the sulfuric acid anthrone method at a wavelength of 620 nm using TU-1810s spectrophotometer (PERSEE, Beijing, China). Crude fiber content was measured by digestion and gravimetric technique (Antial et al., 2006). Crude fiber was estimated 46

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than twice that of RB, R/B(4 h) and R/B(2 h), but was less than twice that of R/B(8 h) and R/B(1 h). The shoot/root (S/R) ratio of plants was significantly decreased under R/B(4 h) and R/B(2 h) compared with other treatments, no significant difference was observed under the other treatments.

from the loss in weight on ignition of dried residue remaining after digestion of fat-free samples with 1.25% sulphuric acid and 1.25% sodium hydroxide. Crude fiber content was determined using the following equation: of weight on ignition × 100 Fiber (%) = loss weight of sample used

3.2. Chlorophyll and carotenoid contents 2.6. Determination of ascorbic acid

Fig. 5 indicates the chlorophyll and carotenoid contents of lettuce plants cultured with different lighting modes. The content of Chla was approximately three times as much as that of Chlb irrespective of the various light treatment. Compared with RB, the content of Chla, Chlb, total Chl and Car under RB’ were significantly increased by 33.3%, 29.7%, 31.4% and 25% respectively. No significant difference was detected among R/B(2 h), R/B(1 h) and RB’ for the pigment contents mentioned above. For the five treatments which provided the same DLI, the content order of chlorophyll and carotenoid pigments was approximately RB < R/B(8 h) < R/B(4 h) < R/B(2 h) ≈ R/B(1 h), indicating that the synthesis of pigments were promoted to a certain extent with the decreasing alternating intervals of R and B.

Ascorbic acid content was determined according to a modified method reported by Gahler et al. (2003). A mixture of 0.2 g (FW) lettuce shoot samples and 15 mL of 4.5% aqueous phosphoric acid were shaken at 300 rpm for 30 min in the darkness and then centrifuged at 16,000g for 10 min. The contents of ascorbic acid was determined using the supernatants via the HPLC system (Agilent, model-1100, USA) equipped with C18 column (inner diameter 4.6 mm, length 250 mm, particle diameter 5 μm, Restek USA, Bellefonte, PA, USA) at 30 °C. Extract was eluted with 0.21% phosphoric acid at a flow rate of 0.8 mL/ min, and the concentrations were determined at 254 nm against ascorbic acid standards (Standard substance center, China).

3.3. Soluble sugar and crude fiber contents

2.7. Determination of nitrate

As shown in Fig. 6, the proportion of soluble sugar and crude fiber in the fresh weight of lettuce had been obviously affected by different radiation patterns. Plants grown under RB’ had the highest crude fiber content of 17.7 mg g−1 and the lowest soluble sugar content of 6.6 mg g−1, the differences reached significant level (p ≤ 0.05) compared with other treatments. Moreover, the minimum crude fiber content was detected under R/B(8 h) and R/B(1 h), while the highest soluble sugar content was observed under RB treatment. Compared with RB, soluble sugar content with R/B(4 h) was significantly decreased by 24.7%, while no significant difference was observed when compared with R/B(8 h), R/B(2 h) or R/B(1 h) treatment. Meanwhile, compared with RB, no significant difference for crude fiber content was brought about by R/B(8 h), R/B(4 h), R/B(2 h) nor R/B(1 h) treatment.

Nitrate content was measured using the modified method of Cataldo et al. (1975). A mixture of 0.5 g (FW) lettuce shoot samples and 6 mL deionized water were heated in a 80 °C water bath for 30 min and subsequently cooled and filtered (repeated twice), a constant total volume of 100 mL was achieved using the filtrate and deionized water. 0.1 mL of the solution was mixed with 0.4 mL of 5% (w/v) salicylic acid (in pure H2SO4) and 9.5 mL of 8% NaOH, finally the absorbance of the extract at a wavelength of 410 nm was measured with TU-1810s spectrophotometer (PERSEE, Beijing, China) and the nitrate content was calculated on a basis of the measured absorbance and the nitrate calibration curve. 2.8. Statistical analysis

3.4. Ascorbic acid and nitrate contents The experiment was repeated three times with means derived from 8 plants per repetition. A one-way analysis of variance (ANOVA, P ≤ 0.05) was performed using SPSS statistic software (PASW statistics version 11.0, SPSS Inc., Chicago, USA). The differences among the six treatments were determined by Tukey’s multiple range test at the 0.05 level.

As shown in Fig. 7, no significant differences existed between RB and RB’, R/B (8 h) and R/B (1 h), R/B (4 h) and R/B (2 h) for both ascorbic acid and nitrate contents. Moreover, nearly contrary trends of ascorbic acid and nitrate accumulations were observed with the change in irradiation modes. The maximum ascorbic acid content was found with R/B (4 h) and R/B (2 h) treatments, which was increased by 19.7% and 25.5% respectively in contrast to RB. Meanwhile, the lowest nitrate content was also found with R/B (4 h) and R/B (2 h) treatments, which was respectively 11.5% and 14.7% lower than RB. Similarly, the lowest ascorbic acid content as well as the highest nitrate content were observed under R/B (8 h) and R/B (1 h), among which, the ascorbic acid content was decreased by approximately 27%, while the nitrate content was increased by approximately 16% compared with RB.

3. Results 3.1. Plant morphology and growth characteristics As shown in Fig. 3 and Fig. 4, the average growth rate of plant height, plant width, leaf length and leaf width during the whole culture period was respectively 4.17, 8.69, 6.42 and 2.89 mm d−1 with R/B (8 h) treatment, all of which were the highest among all the six treatments, and followed by R/B(1 h). No plants exhibited excessive elongation irrespective of different lighting modes, however there were obvious morphological differences under various treatments. Plants with R/B(8 h) and R/B(1 h) looked large and vigorous while those with R/B(4 h) and R/B(2 h) looked relatively small, lettuce under RB’ had the most compact morphology with dark green leaves while plants with RB were detected sparse and fragile. Table 1 displays the growth parameters of lettuce at harvest. Compared with RB, the fresh weight of lettuce shoot under R/B(8 h) and R/B(1 h) treatment was significantly increased by respectively 94.9% and 69.4%, while that under R/B(4 h) and R/B(2 h) treatment was decreased by respectively 33.7% and 44.9%. The highest shoot biomass (fresh or dry weight) was detected under RB’, which was more

4. Discussion Crops grown in natural light are exposed to coexisted R and B, while the controllable LED precise separate R and B (Ilieva et al., 2010). Studies about R:B ratio and alternating R/B proved that R and B might not be independent of each other when acting on plants, but how R and B interacts with each other remains not clear yet. However, some possible reasons could be assumed from the mechanism of physiological response and signal transduction pathways. Sivakumar et al. (2006) observed that dry weight and carbohydrate content of potato plantlets treated with intermittent light were greater than those treated with continuous light, and therefore they assumed that exposure to intermittent light might trigger some morphogenetic mechanism in more 47

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Fig. 3. Morphology of ‘Green Oak Leaf’ lettuce under different light treatments of RB, R/B (8 h), R/B (4 h), R/B (2 h), R/B (1 h) and RB’ (at harvest). The simultaneous irradiation provided by RB and RB’ respectively had a photoperiod of 8 h and 16 h. The alternating irradiation provided by R/B (8 h) was 8 h:8 h (i.e., 8 h of R and 8 h of B during a 16 h photoperiod), and by R/B (4 h), R/B (2 h), R/B (1 h) was respectively 4 h:4 h, 2 h:2 h and 1 h:1 h. The bars indicate 5 cm.

brief pulse of green light could negate the effect of ultraviolet light on stomatal opening; they suggested that the entity that absorbs and responds to blue and green light may toggle between active and inactive states similar to the phytochrome Pfr and Pr states (zeaxanthin had been put forth as one candidate for the entity). Therefore, not only phytochromes (red and far-red) but also phototropins (blue and dark) and zeaxanthin (blue and green) show a “light-quality-dependent” reversible response, and under alternating red/blue irradiation, these reversible pathways may lead to faster growth. In addition, it was reported that constant illumination with B alone might have negative effects such as reduced Pn in many species due to chloroplast avoidance responses and impaired mesophyll conductance (Wada et al., 2003; Kim et al., 2004; Loreto et al., 2009). Thus, it is possible that monochromatic R or B can fully function without negative effects as long as a different light follows with a proper interval (e.g., from red to blue), which may be another possible explanation for the benefit generated by alternating irradiation. As known, light spectrum especially red and blue light could shape plant morphology (Hoenecke et al., 1992; Johkan et al., 2010). In the study, despite the fact that the light quantity and its source in treatment R/B (8 h), R/B (4 h), R/B (2 h) and R/B (1 h) were the same, morphology appeared obviously different. The quotable results have been reported by Shimokawa et al. (2014), who claimed that growth characters differed with the alternating intervals of R/B even when the total light intensity per day was the same, for example the maximum petiole length and stem length were respectively detected under 6 h:6 h and 1 h:1 h. Regarding the biomass accumulation, fresh and dry weight of lettuce were significantly increased with R/B (8 h) and R/B (1 h) while decreased under R/B (4 h) and R/B (2 h) compared with the simultaneous mode RB, indicating that apart from light qualities, assimilate accumulation could also be accelerated or inhibited by the alternating irradiation. In the study by Shimokawa et al. (2014), the order of lettuce fresh weight with different R/B alternating intervals was 12 h/12 h > 3 h/3 h > 24 h/24 h > 6 h/6 h ≈ 1 h/ 1 h > 48 h/48 h. Thus, we may conclude that alternating R/B could affect the growth of lettuce, and results differed with the alternating intervals. However, just like that we can never say higher R:B ratio leads to better cultivation results, similarly, we can not expect the cultivation results to

photoreceptor cells than exposure to continuous light. Similar assumption had been made by Shimokawa et al. (2014), who said that the activation approaches for receptor pathway of R and B were possibly different between the alternating patterns and the simultaneous irradiation. It has been known that red and blue light affect the plant photoresponses via the photoreceptors. Five phytochromes (phyA through phyE) as red light receptors, three cryptochromes (cry1, cry2, cry3) and two phototropins (phot1, phot2) as blue-ultraviolet light receptors have been identified (Sánchez-Lamas et al., 2016; Gärtner, 2017; Liscum, 2016). In nature, sunlight simultaneously activates more than one photoreceptor, which both interact and depend on each other, and the relationship among photoreceptors may be related to the light environment. For instance, it was reported by Casal (2000) that under short photoperiods of simultaneous red and blue light, phyB and cry1 are synergistic, but under continuous exposure to the same light the actions of phyB and cry1 become independent and additive. Moreover, the relationship among photoreceptors may also be related to specific plant physiological activities. For example, cry2 and phyB are antagonistic in the induction of flowering, while cry1, cry2, phyA and phyB positively regulate phototropic bending of the shoot toward unilateral blue light, which is known to be mediated by phototropin. In addition, the blue-light receptor cry1 also shows functional dependence on phyA or phyB in Arabidopsis thaliana (Ahmad and Cashmore, 1997; Poppe et al., 1998). That is, the signal transduction pathways of R and B are independent in some cases but are interactive in other cases, an indication for the cross talk of the photoreceptor signaling pathways. Thus, if there is any conflict in photoresponses between R and B, alternating irradiation may resolve the conflict, thereby leading to more efficient growth. The following reports are examples of such similar conflicts, although not entirely about red and blue light. One was reported by Kozuka et al. (2013), who showed an antagonistic regulation of leaf flattering by phytochrome B and phototropin. Another was by Frechilla et al. (2000) who demonstrated that a green light pulse could preclude blue-lightmediated stomatal opening in Vicia faba epidermal peels. Similar results were observed by Eisinger et al. (2003), who showed that a 48

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Fig. 4. Plant height (a), plant width (b), leaf length(c) and leaf width (d) growths of lettuce cultivated under different light treatments of RB, R/B (8 h), R/B (4 h), R/B (2 h), R/B (1 h) and RB’ (n = 3). The simultaneous irradiation provided by RB and RB’ respectively had a photoperiod of 8 h and 16 h. The alternating irradiation provided by R/B (8 h) was 8 h:8 h (i.e., 8 h of R and 8 h of B during a 16 h photoperiod), and by R/B (4 h), R/B (2 h), R/B (1 h) was respectively 4 h:4 h, 2 h:2 h and 1 h:1 h.

difference for crude fiber content was caused by alternating R/B treatments compared with the simultaneous mode RB (Fig. 6). Generally, high soluble sugar and low crude fiber resulted in more enjoyable taste for lettuce (Lin et al., 2013). However, results mentioned above showed that alternating lighting in the study did not make additional positive effects as regards to soluble sugar and crude fiber, and responses in soluble sugar and crude fiber were not sensitive enough to the alternating irradiation patterns. Vitamin C is a potent water-soluble antioxidant in humans that protects the body from free

vary in an unidirectional trend with the increasing R/B alternating intervals. Regarding the other indexes involved in the study, it can be inferred from the pigment content order RB < R/B(8 h) < R/B(4 h) < R/B (2 h) ≈ R/B(1 h) (Fig. 5) that alternating modes promoted pigment accumulation, however the utilization efficiency of pigments that was related to biomass accumulation differed with alternating intervals. For soluble sugar content (Fig. 6), no significant difference was brought about by alternating R/B treatments except R/B (4 h) which resulted in an obvious decline compared with RB. Meanwhile, no significant

Table 1 Influences of alternating modes of red and blue LED lights on plant height, plant width, fresh weight (FW), dry weight (DW), leaf length, leaf width and shoot: root ratio (S/R) of lettuce at harvest (48 DAS). Light treatment

RB R/B(8 h) R/B(4 h) R/B(2 h) R/B(1 h) RB'

Plant height (cm)

9.93b 20.03a 10.83b 8.07b 15.13ab 9.10b

Plant width (cm)

32.67b 41.73a 24.57c 29.13bc 36.27b 31.10b

FW (g)

DW (g)

Shoot

Root

Shoot

Root

32.67c 63.67b 21.67cd 18.00d 55.33b 77.33a

7.00b 5.20c 4.58c 3.80d 7.94b 9.78a

1.63c 2.53b 1.15cd 0.82d 2.40b 3.40a

0.28d 0.5b 0.5b 0.38cd 0.54b 0.72a

Leaf length (cm)

Leaf width (cm)

S/R

19.83b 30.83a 15.93c 18.23b 25.40ab 19.77b

10.07b 13.87a 8.40c 8.47c 11.93b 11.27b

5.82a 5.06a 2.31c 2.15c 4.44ab 4.73ab

The simultaneous irradiation provided by RB and RB’ respectively had a photoperiod of 8 h and 16 h. The alternating irradiation provided by R/B (8 h) was 8 h:8 h (i.e., 8 h of R and 8 h of B during a 16 h photoperiod), and by R/B (4 h), R/B (2 h), R/B (1 h) was respectively 4 h:4 h, 2 h:2 h and 1 h:1 h. Values for the same parameter with different letters significantly differ at the 5% level (by Tukey’s test, n = 3).

49

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Fig. 5. Chlorophyll and carotenoid contents of plants grown under different light treatments of RB, R/B (8 h), R/B (4 h), R/B (2 h), R/B (1 h) and RB’ (at harvest). The simultaneous irradiation provided by RB and RB’ respectively had a photoperiod of 8 h and 16 h. The alternating irradiation provided by R/B (8 h) was 8 h:8 h (i.e., 8 h of R and 8 h of B during a 16 h photoperiod), and by R/B (4 h), R/B (2 h), R/B (1 h) was respectively 4 h:4 h, 2 h:2 h and 1 h:1 h. Different letters for the same parameter indicate significant differences at the 5% level, according to the Tukey’s test (n = 3). The bars represent the standard errors.

radicals, while nitrate is regarded as harmful substance for human health (Padayatty et al., 2003; Chen et al., 2014). In the study, R/B (4 h) and R/B (2 h) significantly stimulated the accumulation of ascorbic acid while inhibited the activities of nitrate uptake. On contrary, R/B (8 h) and R/B (1 h) decreased ascorbic acid content and showed no inhibition on nitrate accumulation. It indicated that R/B (4 h) and R/B (2 h) were more efficient than RB in terms of ascorbic acid and nitrate indexes. In addition, almost opposite trends of ascorbic acid and nitrate accumulation were detected with the change in irradiation modes, that was, an irradiation mode enhanced the accumulation of ascorbic acid and simultaneously degraded the nitrate content. It may be due to the role of ascorbic acid in modulating the effects of various nitrosating agents in plants, ascorbic acid can effectively remove nitrite via biochemical reaction thus acting on the nitrate-nitrite balance (Izumi et al., 1989; Tannenbaum et al., 1991). Moreover, it was necessary to give attention to the treatment RB’, which provided simultaneous R and B with twice DLI and energy

consumption than that of the treatment RB, R/B (8 h), R/B (4 h), R/B (2 h) and R/B (1 h). As shown in the results, the shoot biomass (fresh or dry weight) under RB’ was more than twice that of RB, R/B (4 h) and R/ B (2 h), but less than twice that of R/B (8 h) and R/B (1 h), particularly, the shoot FW was merely enhanced by 21.5% compared with R/B (8 h). Meanwhile, lettuce treated with RB’ contained too much crude fiber and little soluble sugar which probably led to bad taste. Compared with RB, R/B (8 h) and R/B (1 h) significantly increased lettuce shoot biomass by switching R and B at a certain frequency, while RB’ significantly increased shoot biomass by doubling the photoperiod. The pivotal difference between the two methods to increase biomass was that R/B (8 h) and R/B (1 h) did not add the energy consumption, but RB’ did. It indicated that alternating R/B modes with appropriate alternating intervals could enhance the energy efficiency. As an important system for precise agriculture, plant factories with artificial lighting have been applied in many fields (Shimizu et al., 2011; Christou, 2013; Pamungkas et al., 2014). It has been reported that artificial lighting made up 45% of the total electricity consumption in plant factories, therefore, LEDs which have the characteristic of

Fig. 6. Soluble sugar, crude fiber contents and shoot FW of plants grown under different light treatments of RB, R/B(8 h), R/B (4 h), R/B (2 h), R/B (1 h) and RB’ (at harvest). The simultaneous irradiation provided by RB and RB’ respectively had a photoperiod of 8 h and 16 h. The alternating irradiation provided by R/B (8 h) was 8 h:8 h (i.e., 8 h of R and 8 h of B during a 16 h photoperiod), and by R/B (4 h), R/B (2 h), R/B (1 h) was respectively 4 h:4 h, 2 h:2 h and 1 h:1 h. Different letters for the same parameter indicate significant differences at the 5% level, according to the Tukey’s test (n = 3). The bars represent the standard errors.

Fig. 7. Ascorbic acid and nitrate contents of plants grown under different light treatments of RB, R/B (8 h), R/B (4 h), R/B (2 h), R/B (1 h) and RB’ (at harvest). The simultaneous irradiation provided by RB and RB’ respectively had a photoperiod of 8 h and 16 h. The alternating irradiation provided by R/B(8 h) was 8 h:8 h (i.e., 8 h of R and 8 h of B during a 16 h photoperiod), and by R/B (4 h), R/B (2 h), R/B (1 h) was respectively 4 h:4 h, 2 h:2 h and 1 h:1 h. Different letters for the same parameter indicate significant differences at the 5% level, according to the Tukey’s test (n = 3). The bars represent the standard errors.

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relatively lower electricity consumption compared with other light sources have been regarded as ideal light sources for plant factories (Ikeda et al., 1992). However, for large-scale applications of plant factories, more measures still need to be explored to lower the electricity consumption of LED lamps. Li et al. (2016) reported a method to improve light distribution of LED by zoom lens, which saved over 50% of the light source electricity. Li et al. (2014) also showed a vertical and horizontal movable LED system which decreased the lighting consumption by 18.4%. Both measures above focused on improving the LED equipment. In fact, results in our study showed that optimized indexes of lettuce such as higher yield or higher nutritional value could be generated by alternating R/B irradiation patterns without additional electric energy consumption. As known, optimized indexes of lettuce mentioned above could also be realized by adjusting R:B ratio, however, due to different luminescence principle and processing technology, the red and blue LEDs have different luminous efficiency, that is, to emit the same intensity of illumination (PPFD), the energy consumption of red and blue LEDs differed a lot. Thus, different R:B ratio results in different plant responses as well as different energy consumption, but different R/B alternating frequency results in different plant responses with the same energy consumption, thus people can choose ideal R/B alternating intervals without adding energy consumption. Therefore, appropriate irradiation strategies could be another possible way to enhance the light use efficiency in plant factories. The study of alternating R/B irradiation not only provide a new idea for plant light formula research, but also exhibited large potential for the future applications in plant factories due to the fully consideration of energy consumption problem. 5. Conclusion Alternating irradiation of R and B influenced the growth and quality of lettuce, and plant responses differed with the alternating intervals. The alternating interval of 8 h and 1 h during a photoperiod accelerated lettuce growth speed in terms of plant height/width and leaf length/ width, also 8 h:8 h and 1 h:1 h significantly promoted the shoot biomass of lettuce compared with the concurrent irradiation RB. Meanwhile 4 h:4 h and 2 h:2 h treatments significantly raised ascorbic acid content and simultaneously decreased nitrate content, leading to higher nutritive value. Based on the above results, we conclude that lettuce growth and quality can be purposely adjusted by adopting different alternating intervals of red and blue light without adding new energy consumption. However, more experiments need to be conducted to determine the optimal R/B alternating intervals for the highest biomass or the best nutritional qualities of lettuce. Acknowledgements This work was supported by Beijing Natural Science Foundation (6174041), the National High Technology Research and Development Program of China (863 program) (2013AA103005) and the Youth Foundation of Beijing Academy of Agriculture and Forestry Sciences (QNJJ201421). References Abidi, F., Girault, T., Douillet, O., Guillemain, G., Sintes, G., Laffaire, M., Leduc, N., 2013. Blue light effects on rose photosynthesis and photomorphogenesis. Plant Biol. 15 (1), 67–74. Ahmad, M., Cashmore, A.R., 1997. The blue—light receptor cryptochrome 1 shows functional dependence on phytochrome A or phytochrome B in Arabidopsis thaliana. Plant J. 11 (3), 421–427. Antial, B.S., Akpanz, E.J., Okonl, P.A., Umorenl, I.U., 2006. Nutritive and anti-Nutritive evaluation of sweet potatoes. Pak. J. Nutr. 5 (2), 166–168. Brown, C.S., Schuerger, A.C., Sager, J.C., 1995. Growth and photomorphogenesis of pepper plants under red light-emitting diodes with supplemental blue or far-red lighting. J. Am. Soc. Hortic. Sci. 120 (5), 808–813. Carvalho, R.F., Takaki, M., Azevedo, R.A., 2011. Plant pigments: the many faces of light

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