Effects of temporally shifted irradiation of blue and red LED light on cos lettuce growth and morphology

Scientia Horticulturae 198 (2016) 227–232

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Effects of temporally shifted irradiation of blue and red LED light on cos lettuce growth and morphology Tomohiro Jishi ∗ , Keisuke Kimura, Ryo Matsuda, Kazuhiro Fujiwara Department of Biological and Environmental Engineering, Graduate School of Agricultural and Life Sciences, the University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan

a r t i c l e

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Article history: Received 5 August 2015 Received in revised form 5 November 2015 Accepted 2 December 2015 Keywords: Lactuca sativa Leaf area Photomorphogenesis Spindly growth

a b s t r a c t This study examined the growth and morphology of cos lettuce (Lactuca sativa L.) to ascertain the effects of irradiation patterns with combinations of blue and red LED light, with spectral photon flux density changing with a period of 24 h. In experiment 1, plants were grown under irradiation patterns in which plants were irradiated with blue and red LED lights at a PPFD of 90 ␮mol m−2 s−1 for 14 h per day respectively. The red LED light irradiation starting time was simultaneous to or delayed 1, 4, or 7 h from the blue LED light irradiation starting time. Results showed that the shoot fresh weight of plants grown under irradiation patterns with red LED light irradiation being delayed 4 or 7 h from blue LED light irradiation was significantly greater than that under irradiation patterns with blue and red LED light irradiation starting simultaneously. This result showed that the growth of plants can be promoted merely by temporally shifting the irradiation hours of blue and red LED lights. We then conducted experiments to assess the mechanism of the observed growth-promoting effect from viewpoints of the diurnal PPFD change and duration of blue and red light monochromatic irradiation. The results of these experiments revealed that the diurnal PPFD change contributed to the growth-promoting effect and that blue and/or red light monochromatic irradiation increased the total leaf area of plants. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Considerable research effort has been devoted to ascertaining the effects of spectral photon flux density distribution (SPD) of light on plant growth and morphology with a fixed SPD (e.g., Bula et al., 1991; Hogewoning et al., 2010; Stuefer and Huber, 1998 Vince-Prue, 1977). However, little research has been undertaken to evaluate the effects of irradiation patterns in which SPD changes over time, mainly because it is difficult to change SPD with time using traditional light sources such as fluorescent lamps and highpressure sodium lamps. Recently, it has become possible to change the SPD of light for plant cultivation with time using light emitting diodes (LEDs). Several reports of the literature have described growth promotion by changing SPD for half an hour or longer immediately before

Abbreviations: BL, blue LED light; PPFD, photosynthetic photon flux density; RSPD, relative spectral photon flux density distribution; RL, red LED light; SPD, spectral photon flux density distribution. ∗ Corresponding author. Fax: +81 3 5841 8172. E-mail addresses: [email protected] (T. Jishi), [email protected] (R. Matsuda), [email protected] (K. Fujiwara). http://dx.doi.org/10.1016/j.scienta.2015.12.005 0304-4238/© 2015 Elsevier B.V. All rights reserved.

or after the light period. Sung and Takano (1997) reported that the shoot fresh weight and total leaf area of cucumber seedlings grown under natural sunlight were significantly greater with irradiation of blue fluorescent lamp (FL) light immediately before dawn compared with irradiation of red FL light immediately before dawn or without irradiation. Hanyu and Shoji (2002) reported that the total dry weight of spinach grown under white FL light with irradiation of blue FL light immediately before the light period was greater than that with irradiation of red FL light immediately before the light period, or without irradiation. They also reported that the total dry weight of spinach with irradiation of red FL light immediately after the light period was greater than that with irradiation of blue FL light immediately after the light period or without irradiation. Based on these studies which described growth promotion, we investigated the effects on cos lettuce growth and morphology of irradiation patterns in which SPD changes with a combination of blue and red LED light on a 24-h cycle. For this study, based on experimentally obtained results of Sung and Takano (1997) and Hanyu and Shoji (2002), we considered that cos lettuce growth can be promoted by the irradiation patterns in which we start blue LED light (BL) irradiation first and start red LED light (RL) irradiation at some time after BL irradiation. Therefore we shifted the red light irradiation hours late from blue light irradiation hours, and

T. Jishi et al. / Scientia Horticulturae 198 (2016) 227–232

investigated the effects of the shifted time duration on growth and morphology of cos lettuce plants (Experiment 1). We then assessed the mechanism of the effects of irradiation pattern on growth and morphology observed in experiment 1 from viewpoints of diurnal photosynthetic photon flux density (PPFD) change and the durations of blue and red monochromatic light irradiation. In experiment 2, to examine the effects of diurnal PPFD changes, we grew plants under irradiation patterns with different diurnal PPFD changes depending on the irradiation pattern, as in experiment 1. The relative SPD (RSPD) was maintained constant during the light period in all irradiation patterns. In experiment 3, to examine the effects of the duration of blue and red monochromatic light irradiation, we grew plants under irradiation patterns with different durations of blue and red monochromatic light irradiation depending on the irradiation pattern as in experiment 1. PPFD were maintained constant during the light period in all irradiation patterns. Based on the results of experiments 1–3, we respectively discuss the effects of diurnal PPFD change and duration of BL and RL monochromatic irradiation.

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Fig. 1. Irradiation patterns of experiment 1. Plants were irradiated with blue LED light (BL) and red LED light (RL), respectively, with a PPFD of 90 ␮mol m−2 s−1 for 14 h per day. R irradiation starting times were simultaneous to (C) or delayed 1 (1D), 4 (4D), and 7 (7D) h from B irradiation starting time.

2. Materials and methods 2.1. Irradiation patterns

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2.2. Plant materials Approximately 100 cos lettuce (Lactuca sativa L. cv. Cos Lettuce) seeds were sown on filter paper (JUS-P-3801; Toyo Roshi Kaisha Ltd., Tokyo, Japan) and were watered with 20-mL distilled water in a Petri dish. The Petri dish was placed in a temperature-controlled chamber at 25 ± 1 ◦ C under a 14-h day and 10-h night cycle. Light was provided with white LEDs as described below at a PPFD of approximately 120 ␮mol m−2 s−1 . Three days after sowing, each seedling was transplanted to a urethane-foam cube. The cubes were placed in 20-mm holes in 25-mm thick polystyrene foam plates. Thirty seedlings were transplanted to the plate. Then the plate was floated in a 12-L plastic container filled with continuously aerated nutrient solution (half-strength Otsuka-A nutrient solution; OAT Agrio Co. Ltd., Tokyo, Japan) with electrical conductivity of 1.5 ± 0.1 dS m− 1 . The PPFD at the top surface of each urethanefoam cube was approximately 150 ␮mol m−2 s−1 . Four days after transplanting, seedlings with an approximately 1-cm first true leaf were used for cultivation experiments.

PPFD [ µmol m–2 s–1 ]

180 PPFD values presented below are those set at 2 cm above the top surface of urethane-foam cubes. Integral PPFD values of BL and RL were, respectively, equal in all irradiation patterns of experiments 1–3. Experiment 1: Plants were irradiated with BL and RL continuously for 14 h per day at a PPFD of 90 ␮mol m−2 s−1 respectively in all irradiation patterns. The RL irradiation starting time was simultaneous to (C) or delayed 1 (1D), 4 (4D) or 7 (7D) h from the BL irradiation starting time (Fig. 1). Experiment 2: RSPD was maintained as constant by controlling PPFD for BL and RL at the same level of 45 or 90 ␮mol m−2 s−1 during the light period in all irradiation patterns. Diurnal PPFD change patterns were equal to irradiation patterns in experiment 1. The durations of the lower-PPFD light irradiation at a PPFD of 90 ␮mol m−2 s−1 in all were 0 (C), 2 (1L), 8 (4L) or 14 (7L) h as in experiment 1 (Fig. 2). Experiment 3: PPFD was maintained as constant during the light period in all irradiation patterns in which plants were irradiated for 14 h per day at PPFD of 180 ␮mol m−2 s−1 in total. The durations of BL and RL monochromatic irradiation were 0 (C), 1 (1M), 4 (4M) or 7 (7M) h as in experiment 1 (Fig. 3).

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Fig. 2. Irradiation patterns of experiment 2. Plants were irradiated with blue LED light (BL) and red LED light (RL) at a constant relative spectral photon flux density in which PPFD of B and R were equal. The low light irradiation durations with a PPFD of 90 ␮mol m−2 s−1 were 0 (C), 2 (1 L), 8 (4 L), and 14 (7 L) h.

2.3. Cultivation conditions Uniformly sized 16 seedlings were transplanted individually along with the urethane foam cubes to 20-mm holes in the white acrylic boards. Four seedlings were transplanted per board. Each of four boards was put on a 5-L plastic container filled with continuously aerated nutrient solution as described above. Internal spaces of temperature-controlled chambers were separated into upper and lower compartments with cardboards and black papers. Seedlings were cultivated in four compartments under different irradiation patterns. The four compartments in the two temperature-controlled chambers were maintained at 25 ± 1 ◦ C throughout the day. External air was introduced into the chambers with air pumps to maintain the inside CO2 concentration at approximately atmospheric CO2 concentration. Moreover, air of the four compartments was circulated with air pumps to equalize the CO2 concentrations of the four compartments, which were under different irradiation patterns. Four plants were cultivated under each irradiation pattern simultaneously. The cultivation experiments were repeated twice in the respective cultivation experiments (experiments 1–3).

T. Jishi et al. / Scientia Horticulturae 198 (2016) 227–232

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respectively, 656 nm and 20 nm. Eighty-four blue LEDs and 98 red LEDs were arrayed on 12 cm × 17 cm printed circuit boards (AQCB98FLS; Sozo Kagaku Y. K., Shizuoka, Japan) in a checkerboard design. Two LED arrays were connected side by side and were used for each group of four seedlings on one acrylic board. RSPDs of the white LED light, the BL and the RL are presented in Fig. 4. The LED arrays were connected to DC power supplies (PMC35-1A, PMC70-1A; Kikusui Electronics Corp., Yokohama, Japan). The voltages applied to the LED arrays were temporally changed to desired values by controlling the applied voltage to analog-remote-control terminals of DC power supplies with an external voltage control device. The external voltage control device consisted of an Arduino microcomputer, a digital–analog converter, and an operational amplifier.

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Fig. 3. Irradiation patterns of experiment 3. Plants were irradiated with blue LED light (BL) and red LED light (RL) at a total PPFD of 180 ␮mol m−2 s−1 s−1 for 14 h per day. The duration of blue and red monochromatic light irradiation was 0 (C), 1 (1 M), 4 (4 M), or 7 (7 M) h.

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2.5. Measurements Fourteen days after the start of the cultivation, plants were measured to record the shoot fresh weight, shoot dry weight, and total leaf area. The shoot fresh and dry weights were measured using an electronic scale. The shoot dry weight was measured after one-hour drying at 100 ◦ C and subsequent three-day drying at 80 ◦ C (partially modified using the method described by Raguse and Smith (1965)). Leaf area of leaves longer than 3 cm was measured using an automatic area meter (AAM-9; Hayashi Denko Co. Ltd., Tokyo, Japan).

0.6 3. Results

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Fig. 4. Relative spectral photon flux density (RSP) distribution of white LEDs used for rearing cos lettuce seedlings (A) and blue and red LEDs used for cultivating cos lettuce plants (B).

In experiment 1, leaves were elongated in 4D and 7D (Fig. 5). Some plants in 7D toppled by 14 days after the start of the cultivation. The shoot fresh weights of plants in 4D and 7D were, respectively, 58% and 72% greater than that in C. Shoot fresh weights of plants in 4D and 7D were significantly greater than that in C and 1D (Fig. 6A). The shoot dry weight of plants in 7D was 46% greater than that in C. The shoot dry weight of plants in 7D was significantly greater than that in C (Fig. 6B). The total leaf areas of plants in 4D and 7D were, respectively, 54% and 67% greater than that in C. The total leaf areas of plants in 4D and 7D were significantly greater than in C or 1D (Fig. 6C). One plant inexplicably died in 7D. Therefore, statistical analyses used n = 7 only in 7D. This one plant death was considered not to affect other plants because plants did not shade each other in 7D. In experiment 2, no noticeable difference was found in the appearances of plants among all the irradiation patterns (Fig. 7). Shoot fresh weight, shoot dry weight, and total leaf area of plants tended to increase concomitantly with increasing duration of lower-PPFD light irradiation at 90 ␮mol m−2 s−1 (Fig. 8). In experiment 3, leaves were elongated in 4 M. Leaves in 7 M were more elongated than that in 4 M (Fig. 9). The shoot fresh weight and shoot dry weight were not significantly different among all irradiation patterns (Fig. 10A, B). Total leaf areas of plants in 7 M were significantly greater than in the other irradiation patterns (Fig. 10C).

2.4. Light sources and irradiation system

4. Discussion

White LED arrays (AL1411A-13S28P; Solidlite Corp., Taiwan) were used for irradiation of seedlings before cultivation experiments. The peak wavelengths of the white LED light were 443 (sharp peak by the LED-chip emission), 533 and 622 (broad peaks by wavelength-conversion phosphors) nm. Mold-type blue LEDs (HBL3-3S55-LE; Toricon, Shimane, Japan) and mold-type red LEDs (SRK1-3A80-LE; Toricon, Shimane, Japan) were used for the cultivation experiments. The peak wavelengths and the FWHM of the blue LED light were 463 nm and 22 nm. Those of the red LED light were,

Based on the results showing that shoot fresh weights of plants in 4D and 7D were significantly greater than in C (Fig. 6A), delaying the start of RL irradiation more than a certain duration from the start of BL irradiation is regarded as promoting the growth of cos lettuce plants. Hanyu and Shoji (2002) considered reasons why the 30-min BL irradiation before the light period promoted the growth of spinach as the promotion of photosynthesis resulting from an increased stomatal aperture. Although this growth-promoting effect resulting from enhancement of the stomatal aperture was

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Fig. 5. Cos lettuce plants grown under irradiation patterns portrayed in Fig. 1 (experiment 1).

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Fig. 6. Shoot fresh weight (A), shoot dry weight (B), and total leaf area (C) of cos lettuce plants under irradiation patterns portrayed in Fig. 1 (experiment 1). Bars represent standard errors of the means (n = 7–8). Means labeled with different small letters in each panel differ significantly from each other at the 5% level, as determined using Tukey–Kramer HSD test.

Fig. 7. Cos lettuce plants grown under irradiation patterns presented in Fig. 2 (experiment 2).

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T. Jishi et al. / Scientia Horticulturae 198 (2016) 227–232

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Fig. 8. Shoot fresh weight (A), shoot dry weight (B), and total leaf area (C) of cos lettuce plants under irradiation patterns presented in Fig. 2 (experiment 2). Bars represent standard errors of the means (n = 8). Means labeled with different small letters in each panel differ significantly from each other at the 5% level, as determined using the Tukey–Kramer HSD test.

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Fig. 9. Cos lettuce plants grown under irradiation patterns presented in Fig. 3 (experiment 3).

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Fig. 10. Shoot fresh weight (A), shoot dry weight (B), and total leaf area (C) of cos lettuce plants under irradiation patterns presented in Fig. 3 (experiment 3). Bars represent standard errors of the means (n = 8). Means labeled with different small letters in each panel differ significantly from each other at the 5% level, as determined by using Tukey–Kramer HSD test.

produced only in 30-min BL irradiation in their report, the growth of plants in 1D in the present experiment was not promoted. In addition, the growth of plants in 7D tended to be promoted more than in 4D. Therefore, the growth promotion mechanisms in 4D and 7D were regarded as different from those of growth promotion explained by Hanyu and Shoji (2002). We tried to clarify this mechanism from viewpoints of diurnal PPFD change and the duration of BL and RL monochromatic irradiation. In experiment 2, which examined the effect of diurnal PPFD change, no noticeable differences were found in the appearance

of plants among irradiation patterns (Fig. 7). Therefore, the effect of diurnal PPFD change on the morphology of cos lettuce plant is regarded as negligibly small. Shoot fresh and dry weights tended to be greater with increasing duration of lower PPFD light irradiation as in experiment 1. This result indicates that the growth promoting effect in 4D and 7D observed in experiment 1 can be attributed partly to the effects of the diurnal PPFD change. The diurnal PPFD change affects the growth of cos lettuce because of the light use efficiency of photosynthesis (gross photosynthetic rate per PPFD). The light use efficiency generally decreases concomitantly with

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increasing PPFD. Therefore, daily integral of net photosynthesis in irradiation patterns with longer duration of lower-PPFD light irradiation is regarded as greater than that in irradiation patterns with longer higher-PPFD light irradiation at the same daily light integral. Perhaps for this reason, the shoot fresh weight of plants was greater in irradiation patterns with longer duration of lower-PPFD light irradiation at a PPFD of 90 ␮mol m−2 s−1 in experiment 2. In view of light use efficiency, the daily integral of net photosynthesis is maximized when plants are irradiated at a constant PPFD during light periods under conditions in which the daily light integral is the same. The diurnal PPFD change is accompanied by the day length change. The difference in growth observed in experiment 2 might be affected not only by the daily integral of net photosynthesis but by the day length. The present experiments cannot distinguish the effect of the day length from that of the daily integral of net photosynthesis. In experiment 3, which examined the effects of the duration of BL and RL monochromatic irradiation, leaves were elongated in 4 M. Leaves in 7 M were more elongated (Fig. 9). Cos lettuce leaves are regarded as narrower and longer in irradiation patterns with longer duration of BL and RL monochromatic irradiation. In addition, durations of BL and RL monochromatic irradiation are considered not to have a strong effect on the shoot fresh weight of cos lettuce plants because no significant difference was found in the shoot fresh weight among irradiation patterns in experiment 3. However, the difference in shoot fresh weight among irradiation patterns in experiment 1 was greater than that in experiment 2, indicating that the growth-promoting effect in experiment 1 cannot be explained solely by diurnal PPFD changes. Leaves became long and large under irradiation patterns with longer duration of BL and RL monochromatic light irradiation, as observed in experiment 3, which might contribute to enhancement of the amount of light received. This enhancement of the amount of light received might contribute synergistically to the growth promoting effects in 4D and 7D observed in experiment 1. Plant morphology is known to be affected by the end-of-day red or far red light irradiation for a short time period of a few minutes (Blom et al., 1995; Chia and Kubota, 2010). This end-of-day effect is not expected to contribute substantially to growth promotion in experiment 1 because RL irradiation at the end of light periods continued for 1(1D), 4(4D), or 7(7D) h, during which phytochrome photostationary state was regarded as reaching the equilibrium irrespective of the duration of RL irradiation at the end of light periods (estimated using an equation by Sager et al., 1988). The specific leaf area (SLA) and the shoot/root ratio tended to increase concomitantly with increasing period of delay of RL irradiation in experiment 1, although those values were almost identical among the irradiation patterns in experiments 2 and 3 (data not shown). Although shoot fresh weight of plants in 4D and 7D were significantly greater than that in C, leaves were elongated in 4D and 7D. Some plants in 7D toppled (Fig. 5). Cos lettuce plants in 7D are predicted to grow abnormally when they are cultivated for longer than 14 days. Plant growth might be promoted in irradiation patterns with a long duration of BL and RL monochromatic irradiation, but overly long duration of BL and RL monochromatic irradiation is regarded as encouraging spindly growth. Low-PPFD light conditions generally bring spindly growth of plants (Johkan et al., 2012; Kozuka et al., 2005). Cos lettuce plants are possibly abnormally elongated when they are grown in a lower-PPFD condition although without BL and RL monochromatic irraditaion. In contrast, plants

are expected to grow into normal morphology with duration of BL and RL monochromatic irradiation of 7 h or longer when those are grown at higher PPFDs. Additional work is in progress to reveal growth-promoting irradiation methods for leaf vegetables with consideration of daily light integral and the cultivation duration. 5. Conclusions The shoot fresh weights of cos lettuce plants grown under irradiation patterns with red LED light irradiation starting time delayed 4 and 7 h from the blue LED light irradiation starting time were significantly greater than those grown with blue LED light irradiation and red LED light irradiation starting simultaneously. This result showed that cos lettuce plant growth can be promoted merely by temporally shifting the irradiation times of blue and red LED lights. This growth promoting effect is regarded as caused primarily by the long duration of lower-PPFD irradiation. Moreover, the long of spindly and large leaves caused by longer duration of blue and red light monochromatic irradiation possibly contributed to growth promotion effects. References Blom, T.J., Tsujita, M.J., Roberts, G.L., 1995. Far-red at end of day and reduced irradiance affect plant height of Easter and Asiatic hybrid lilies. HortScience 30 (5), 1009–1012. Bula, R.J., Morrow, R.C., Tibbitts, T.W., Barta, D.J., Ignatius, R.W., Martin, T.S., 1991. Light-emitting diodes as a radiation source for plants. HortScience 26 (2), 203–205. Chia, P.L., Kubota, C., 2010. End-of-day far-red light quality and dose requirements for Tomato rootstock hypocotyl elongation. HortScience 45 (10), 1501–1506. Hanyu, H., Shoji, K., 2002. Acceleration of growth in spinach by short-term exposure to red and blue light at the beginning and at the end of the daily dark period. Acta Hortic. 580, 145–150. Hogewoning, S.W., Trouwborst, G., Maljaars, H., Poorter, H., van Ieperen, W., Harbinson, J., 2010. Blue light dose-responses of leaf photosynthesis, morphology, and chemical composition of Cucumis sativus grown under different combinations of red and blue light. J. Exp. Bot. 61 (11), 3107–3117, http://dx.doi.org/10.1093/jxb/erq132. Johkan, M., Shoji, K., Goto, F., Hahida, S., Yoshihara, T., 2012. Effect of green light wavelength and intensity on photomorphogenesis and photosynthesis in Lactuca sativa. Environ. Exp. Bot. 75, 128–133, http://dx.doi.org/10.1016/j. envexpbot.2011.08.010. Kozuka, T., Horiguchi, G., Kim, G.T., Ohgishi, M., Sakai, T., Tsukaya, H., 2005. The different growth responses of the Arabidopsis thaliana leaf blade and the petiole during shade avoidance are regulated by photoreceptors and sugar. Plant Cell Physiol. 46 (1), 213–223, http://dx.doi.org/10.1093/pcp/pci016. Raguse, C.A., Smith, D., 1965. Carbohydrate content in alfalfa herbage as influenced by methods of drying. J. Agric. Food Chem. 306, 306–309, http://dx.doi.org/10. 1021/jf60140a005. Stuefer, J.F., Huber, H., 1998. Differential effects of light quantity and spectral light quality on growth, morphology and development of two stoloniferous Potentilla species. Oecologia 117 (1–2), 1–8, http://dx.doi.org/10.1007/ s004420050624. Sager, J.C., Smith, W.O., Edwards, J.L., Cyr, K.L., 1988. Photosynthetic efficiency and phytochrome photoequilibria determination using spectral data. Trans. ASAE 31, 1882–1889. Sung, K.I., Takano, T., 1997. Effects of supplemental blue- and red-lights in the morning twilight on ghe growth and physiological responses of cucumber seedlings. Environ. Control Biol. 35 (4), 261–265, http://dx.doi.org/10.2525/ ecb1963.35.261. Vince-Prue, D., 1977. Photocontrol of stem elongation in light-grown plants of Fuchsia hybrida. Planta 133 (2), 149–156, http://dx.doi.org/10.1007/ bf00391913.