Impact of sun-simulated white light and varied blue:red spectrums on the growth, morphology, development, and phytochemical content of green- and red-leaf lettuce at different growth stages

Scientia Horticulturae 264 (2020) 109195

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Impact of sun-simulated white light and varied blue:red spectrums on the growth, morphology, development, and phytochemical content of greenand red-leaf lettuce at different growth stages

T

Hans Spalholza, Penelope Perkins-Veazieb, Ricardo Hernándeza,* a b

Department of Horticultural Sciences, NC State University, Raleigh NC, 27696, United States Plants for Human Health Institute, Kannapolis NC, 28081, United States

A R T I C LE I N FO

A B S T R A C T

Keywords: Light-emitting diode Plant factory Indoor farming Bolting Solar spectrum Transplant Baby-leaf lettuce Head lettuce

Light drives photosynthesis and regulates plant morphology, physiology, and phytochemical content. Using light emitting diodes (LEDs), customized spectra can be created, including spectrum that simulates solar light. The aim of this study was to assess the growth, development, and phytochemical content at three marketable stages of lettuce (transplant, baby-leaf, and head-lettuce) under a sun-simulated spectrum and common light spectra used in indoor growing systems. Oakleaf red (Salanova® ‘Red Oakleaf’) and green (Salanova® ‘Green Oakleaf’) lettuce were grown under seven spectra. A sun-simulated light treatment (SUN) was created with 5 % ultravioletA (UV-A), 20 % blue (B), 26 % green (G), 26 % red (R), and 23 % far-red (FR) light as percent photon flux density (PFD). In addition, five treatments of differing blue:red (B:R) ratios were evaluated: 0B:100R (100R), 20B:80R, 50B:50R, 80B:20R, and 100B:0R (100B) and fluorescent white light was used as a control (6500 K). Plants were provided with 200 ± 0.7 μmol·m−2·s−1 biologically active radiation (300–800 nm) for 18 h and grown at 20.0 ± 0.2 °C temperature. Fresh mass of lettuce in the SUN treatment was not significantly different when compared to B:R light treatments in all harvest dates despite the 36 % greater photosynthetic photon flux density (PPFD) in B:R treatments. Plant dry mass on day 17 of’ Green Oakleaf’ and ‘Red Oakleaf’ grown under 20B:80R was 15–39 % greater than those grown in 100B and SUN. When calculating total dry mass accumulation to cumulative yield photon flux density (YPFD), plants in SUN treatment accumulated the same dry mass per YPFD input (mg mol−1). Leaf area at day 42 of plants in 100B, SUN, and FL was 39–78 % greater than plants in B:R treatments. At final harvest (day 42), plant stem length in SUN was 2.1–4.4 times longer than in all other treatments, indicating bolting and flowering initiation. Both total phenolic and anthocyanin concentrations were greater in the B:R treatments than in SUN, 100R, and 100B treatments. This study presents baseline information for lettuce responses under LED-simulated SUN spectrum when compared to common B:R treatments and offers insights on lettuce growth and morphology under different spectra at multiple growth stages.

1. Introduction

μmol J−1) (Mitchell et al., 2015), have relative high quantum efficiency (RQE), and coincide with chlorophyll absorption peaks (McCree, 1972). Furthermore, as LED technology has become more versatile in terms of efficacy and diode availability, it is possible to formulate a wide range of spectral combinations including spectra that resembles solar radiation. White light used for plant growth is classified by the percent photon flux density (PPFD) of ultraviolet (UV), B, green (G), R, and far-red (Fr), to the total photon flux density (typically on a 100 nm increments). Also, white light can be classified by its correlated-color-temperature (CCT); the lower the color (warm-light) the more yellow (Y) and R light. In contrast, the higher the color temperature (cool-light) the more B

Light is a critical factor that drives photosynthesis and regulates plant morphology, physiology, and phytochemical content. Improvements in light emitting diode (LED) fixtures, has allowed both researchers and growers to study plant-light physiology and apply this knowledge to crop production systems. One such application is vertical farming (VF), a rapidly expanding agricultural sector that relies solely on electrical lighting as source for photosynthesis. Leaf lettuce and head lettuce are the main crops currently grown in VF. Most VF growers use fixtures that contain blue (B) and red (R) diodes since they are the most efficacious in terms of photon flux per watt of power (μmol s−1 W−1 or

⁎

Corresponding author. E-mail address: [email protected] (R. Hernández).

https://doi.org/10.1016/j.scienta.2020.109195 Received 23 May 2019; Received in revised form 6 November 2019; Accepted 9 January 2020 0304-4238/ © 2020 Elsevier B.V. All rights reserved.

Scientia Horticulturae 264 (2020) 109195

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vertical farm conditions and earlier studies have shown the influence of B:R treatments on plant growth and morphology. For example, Son and Oh (2013) evaluated two lettuce cultivars (‘Sunmang’, ‘Grand Rapids TBR’) under various B:R treatments for 4-weeks and found that plant fresh mass decreased as the ratio of B increased. Son and Oh (2015) grew lettuce (‘Sunmang’ and ‘Grand Rapid TBR’) under 10B:90R, 20B:80R and 30B:70R light treatments and found that plants grown under 20B:80R and 30B:70R had lower fresh mass, dry mass, and leaf area than in 10B:90R treatment. Wang et al. (2016) grew lettuce (cultivar name not provided) under different B:R treatments including 0B:100R, 11B:89R, 8B:92R, 50B:50R and 100B:0R and found no correlation between photosynthetic rate and shoot dry mass. Wang et al. (2016) showed increased leaf photosynthetic capacity, photosynthetic rate, and stomata density with the increase of B PFD (except 100B:0R); however, shoot dry mass decreased with the increase B PFD. Wang et al. (2016) attributed the increase in dry mass to greater leaf area and number of leaves (increase light capture) with the decrease of B PFD. Spectrum with different B:R light can also have a large role in the phytochemical content of lettuce. For example, Amoozgar et al. (2017) found that lettuce (‘Grizzly’) grown under 100B had higher levels of vitamin C than in 100R, 30B:70R, or white LED light, while 30B:70R enhanced chlorophyll and carotenoid content. Lettuce (‘Grand Rapid TBR’ and ‘Sunmang’) grown under monochromatic B, G, or R light only, had the highest total phenolic concentration when grown in B (100B) treatment; for example, lettuce in 100B light had 2–5 times higher phenolic concentration than in 100R (Son et al., 2012). When comparing different B:R treatments, Son and Oh (2015) found that total phenolic content in lettuce (‘Sunmang’) under 34B:66R was 30–57 % greater than in 13B:87R and 24B:76R. Plant growth, including lettuce, follows a sigmoidal curve trajectory for biomass accumulation (Liu et al., 2018; Seginer et al., 1991; van Henten, 1994). Choice of plant growth stage for light spectrum experiments can greatly affect response, and conversely light spectrum can be used to create desired morphological chacteristics. Lettuce seedlings should be compact well-rooted plant with high leaf mass area; this morphology can be elucidated by a higher percent B PFD (Spalholz and Hernández, 2018). After seedling establishment, rapid leaf expansion is desirable to increase light capture; green light (Johkan et al., 2012), Fr (Li and Kubota, 2009), or monochromatic B (Hernández and Kubota, 2016) can be used to increase leaf expansion. After canopy closure, canopy establishment can be achieved by increasing the number of leaves and leaf mass area (LMA: leaf thickness). Finally, increased pigmentation and phytochemical concentration can be achieved by high B:R treatments (Owen and Lopez, 2015). As light spectrum can affect plant physiological responses, understanding the effect of several “fixed” spectrums during the different growth stages will establish the basis for future “dynamic spectral manipulation” (changing light recipes throughout the growth cycle). The present study provides novel results on green and red lettuce responses to SUN simulated spectrum in indoor conditions (VF) and establishes baseline data for growth stage specific plant responses to different spectra.

and green (G) light (Han et al., 2017). Different color white light can have different effects on plant growth and morphology. For example, Red-leaf lettuce (‘Banchu Red Fire’) transplants (17 days) exposed to white fluorescent lights (4200 K) and three G lights (peaks: 510 nm, 520 nm, 530 nm) at 200 or 300 μmol m−2·s−1 for seven days responded differently with light and intensity (Johkan et al., 2012). After light treatment at 200 μmol m−2·s−1, lettuce plants under fluorescent had 65–82 % greater fresh weight than those under G light with 510 nm, 520 nm, or 530 nm peaks; however, at 300 μmol m−2·s−1, lettuce under 510 nm green light had 41–68 % greater fresh weight than all other treatments. On a different study, lettuce (‘Green Oak leaf’) grown under 135 μmol m−2·s−1 (16 h) using white light supplemented with R or B had 63.2 % and 21.7 % greater shoot fresh mass, respectively than lettuce grown in white light alone (Chen et al., 2016). In addition, plants grown with white light supplemented with Fr had 36 % lower shoot fresh mass than in white light alone (Chen et al., 2016). Mickens et al. (2019) grew red pack choi under 25B:75R, white light, and sun-simulated spectrum as well as under white light supplemented with either R, B, or Fr. Red pack choi plants grown under the treatments of 25B:75R or sun-simulated spectrum had greater shoot dry and fresh mass than all other treatments. Although studies have investigated lettuce plant responses to white light supplemented with other colors (UV, B, Y, G, Fr), none have focused on light quality that simulates solar spectrum. Solar radiation has a balanced spectrum on the photosynthetic active radiation range (PAR: 400−700 nm); in addition, it contains UV (UVB:280-320, and UVA 320–380) and far-red (700−800 nm). For example, based on a spectroradiometer scan (Raleigh, NC, 35° 47′ 16.17″ N, 78° 40′ 24.22″ W, June 20th, 13:20, 2016), the solar spectrum contained 5 % UV (UV-B and UV-A), 20 % B (400−500 nm), 26 % G (500−600 nm), 26 % R (600−700 nm), and 23 % Fr (700−800 nm) (solar spectrum can vary based on time of day, season, weather, and geographical location). The spectral composition of solar radiation can elucidate plant responses that may not be expressed under commonly used B:R and white LEDs in VF conditions. For example, UV can affect several plant physiological processes when absorbed different photoreceptors as follows: 1) UV absorption by UVR8 (UV resistance locus 8) can affect pathogen resistance and increase protection to future UV radiation; 2) UV absorption by phytochromes can affect flowering time; 3) UV absorption by cryptochromes can affect leaf anatomy (leaf thickness, stomata number) that can consequently reduce photosynthesis; and 4) UV absorption by phototropins can increase plant compactness and branching (Huché-Thélier et al., 2016). Solar spectrum also contains relatively high levels of far-red; which alters the R:Fr ratio (R:FR of SUN = 1.2) compared to common B:R LEDs (R:FR of 20B:80R = 137). Different R:Fr can elucidate phytochrome related responses such as shade-avoidance (stem and leaf extension) (Kendrick and Kronenberg, 1994), flowering (Craig and Runkle, 2016), and increase overall photosynthetic efficiency when combined with light that overexcites photosystem II (Fr preferentially excites photosystem I) (Zhen et al., 2019). In addition to the presence of UV and far-red light, solar spectrum is a balanced ratio of B, G, R and Fr wavelengths, which can elucidate or regulate responses specific to each independent wavelength. For example, B increases plant compactness (decrease leaf and stem extension); if the B:G ratio decreases (more G) the response rate triggered by B light is reduced (Wang and Folta, 2013). Similarly, phytochemical accumulation such as anthocyanin and carotenoids can be increased by increased B, but can be decreased if the B:FR ratio decreases (more Fr) (Li and Kubota, 2009). The availability of different color LEDs has made it possible to create a spectrum that approaches the percent photon flux ratio for UV, B, G, R, and Fr of the sun spectrum. Therefore, one of the objectives of this study is to assess plant responses elucidated by the unique spectral composition of the sun and compare those to other spectra mainly composed of B and R light. Fixtures mainly containing B and R diodes are commonly used in

2. Materials & methods 2.1. Plant material and growing conditions Pelletized lettuce (Lactuca sativa L.) seeds ‘Green Oakleaf’ and ‘Red Oakleaf’ (Salanova®, Johnny’s Selected Seed Corp., Waterville, ME, USA) were sown in 1 P Fafard (Conrad Fafard Inc., Agawam, MA, USA) potting mix that consisted of 77 % peat, 23 % perlite, and lime, in individual cells. Each cell (3.3 cm upper diameter × 2.8 cm lower diameter × 3.9 cm deep with a volume of 21 ml) was then placed in 98cell trays (711 plants m−2). After sowing, trays were sub-irrigated with water and a thin layer of vermiculite was placed over the seed (2−5 mm thick) with each tray covered in plastic wrap to maintain moisture 2

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nm), and 660 nm (FWHM: 29 nm) for peaks one and two of R, 733 nm for Fr (FWHM: 34 nm), and 5700 K white diodes with peaks at 450 nm (FWHM: 24 nm) and 577 nm (FWHM: 130 nm). Five LED light treatments with different percentages of B and R PFD ratios (B:R) were evaluated: 0B:100R (100R), 20B:80R, 50B:50R, 80B:20R, 100B:0R (100B) (LX601, Heliospectra AB, Göteborg, SE). LED peaks for the B:R treatments were 452 nm for B (FWHM: 23 nm) and 659 nm for R (FWHM: 16 nm). A fluorescent (FL) light treatment with 6500 K was used as the control (Sun Blaze 21-T5 HO, Sunlight Supply Inc., Vancouver, WA, USA). All treatments were set at equal biologically active radiation (300–800 nm) (BAR) and their respective PFD are listed in Table 2. Light measurements were taken at the start and conclusion of each repetition using a spectroradiometer (PS-200, Apogee Instruments, Inc. Logan, UT, USA). BAR is a range of radiation that can impact photosynthesis and photomorphogenesis, whereas photosynthetic active radiation (PAR) does not capture all of this range and the SUN treatment contained radiation outside PAR, thus treatments were set to equal BAR (energy consumption between fixtures more similar when fixtures are set at BAR than PAR). From day 3–9, treatment BAR light intensity was maintained at 100 μmol m−2 s−1 with intensity increased to 200 μmol m−2 s−1 at day 10 and was held at this intensity via lamp height adjustment for the remainder of the experiment (both intensities had 18 h photoperiod). Table 2 details the light intensity specifics of the different treatments in terms of BAR and PAR. Phytochrome stationary state (PSS) and yield photon flux (YPF) were calculated based on Sager and McFarlane (1997). Treated plants were positioned inside a row of border plants to prevent possible edge effects and rotated every two days to equalize light uniformity within the growing area (56 cm × 56 cm).

content. Seeds were germinated at 24 °C under continuous 5000 K fluorescent lighting (F17-T8-850, TCP International Holdings Ltd., Cham, CH) at 80 μmol m−2 s−1 photosynthetic photon flux density (PPFD) for 48 h (day 0–2) in a germination chamber. At this point (day 2) cotyledons emerged and plants were moved to the experimental chamber. On day 18, lettuce was transplanted into larger pots (7.4 cm × 7.4 cm × 6.3 cm deep with a volume of 173 ml) and placed at a density of 189 plants m−2. On day 33, plants were spaced at a final density of 125 plants m−2 and kept to day 42 (D42) for final plant measurements. From day 2–9 plants were sub-fertigated as needed with hydroponic solution containing (mg L-1) 45 N, 24 P, 72 K, 72 Ca, 30 Mg, 58 S, 45 Cl, 0.34 B, 0.55 Mn, 0.05 Cu, 0.05 Mo, 0.33 Zn, 2 Fe. From day 10–42 the same hydroponic nutrient solution was used but with an added (mg L-1) 50 N, 103 K, 36 Ca.

2.2. Chamber parameters & LED light treatments The experimental growth chamber consisted of seven compartments for individual light treatments. Each compartment (100 cm wide × 84.5 cm high × 61 cm deep) contained both cultivars. Environmental conditions on the compartments were measured and recorded (CR1000, Campbell Scientific Inc., Logan, UT, USA) to provide growing zone air temperature and substrate temperature (thermocouples, 0.005 gauge, T-type, Omega Inc. Stamford, CT, USA), chamber CO2 concentration (GMT222, Viasala Inc., Helsinki, FI), and chamber relative humidity (CS-215, Campbell Scientific Inc., Logan, UT, USA) (Table 1). CO2 enrichment was provided using liquid CO2 controlled by the CO2 sensor. All data was measured every five seconds, averaged and recorded every minute. For growing zone air temperature, thermocouples were placed in the center of the growing zone and located 1 cm above the top of the canopy. Treatment air velocity (Table 1) was measured at the start of the experiment, light quality and intensity (Table 2 and Fig. 1) was measured at the start and end of the experiment while nutrient solution pH and electrical conductivity (EC) were measured at the time of each irrigation (HI 9813-6, Hannah Instrument Inc, Woonsocket, RI, USA) (Table 1). Treatment air velocity (laminar air flow) was provided by four equally spaced fans in the growing plane and measured at nine different points on a grid in the growing zone. Ten measurements every 30 s were taken for each point and averaged. Light treatment conditions are shown in Table 2 and Fig. 1. A sun simulated light treatment (SUN) was created by matching the photon flux density of an outdoor spectral scan (Raleigh, NC, 35° 47′ 16.17″ N, 78° 40′ 24.22″ W, June 20th, 13:20, 2016) at the different 100 nm increment wavelengths of UV (300 nm–399 nm), B (400 nm–499 nm), G (500 nm–599 nm), R (600 nm–699 nm), FR (700 nm–800 nm) using a multi-diode LED fixture (RX30, Heliospectra AB, Göteborg, SE) (Fig. 1). Six LED diodes were used for the SUN treatment at the following wavelengths: 388 nm for UV (full width at half maximum (FWHM): 11 nm), 407 nm (FWHM: 12 nm) and 424 nm (FWHM: 15 nm) for peaks one and two of B, 524 nm for G (FWHM: 30 nm), 625 nm (FWHM: 17

2.3. Measurements and data collection Morphology and growth responses The experiment consisted of two replicates over time. Transplant stage was harvested on day 17 (D17) and 15 plants were randomly selected for repetition one while 10 plants were randomly selected for the second repetition. On day 33 (D33) (baby leaf stage), three random plants per repetition from both cultivars were selected. On harvest day 42 (D42) (head stage), five plants per repetiton were measured from both cultivars. Measurements on harvested plants included leaf count, leaf area, fresh mass, and dry mass (dried for 72 h at 65 °C). A threshold of 1 cm in leaf length qualified leaves for leaf count. Both fresh and dry mass were measured using an electronic scale. Leaves were scanned and then processed with ImageJ 1.51g software (Schneider et al., 2012) (D17 and D33) or 2) and with a LI-3100 Area Meter (LI-COR Inc., Lincoln, NE, USA) (D42). In addition to these morphological responses, stem length, stem fresh mass, stem dry mass, marketable leaf count, marketable leaf mass (both fresh and dry), and marketable leaf area were measured on

Table 1 Environmental parameters (mean ± SD) for each light treatment. R represents percent PFD of red light, B represents percent PFD of blue light; SUN represents the sun-simulated white light spectrum composed of different ultraviolet, blue, green, red, and far-red percent PFD ratios (5.1UV, 20.0B, 26.1 G, 26.3R, 22.6Fr); FL represents cool white fluorescent (6500 K) white light. Parameter

Treatments (percent photon flux density ratio) 100R

Air temperature (°C) Substrate temperature(°C) Air Velocity (m s−1) CO2 (μmol mol−1) RH (%) pH EC (dS m−1)

20B:80R

20.1 ± 1.3 20.0 ± 1.2 19.7 ± 1.4 19.8 ± 1.6 1.00 ± 0.18 1.12 ± 0.1 704.2 ± 34.0 for all treatments 80.4 ± 2.8 for all treatments 6.5 ± 0.1 for all treatments 1.44 ± 0.08 for all treatments

50B:50R

80B:20R

100B

SUN

FL

19.8 ± 1.3 19.6 ± 1.5 0.97 ± 0.1

19.9 ± 1.2 19.8 ± 1.5 1.07 ± 0.2

20.0 ± 1.0 19.7 ± 1.4 0.95 ± 0.1

19.9 ± 1.3 20.1 ± 1.4 0.96 ± 0.3

20.3 ± 1.1 20.2 ± 1.3 1.25 ± 0.1

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Table 2 Spectral characterization of experimental light treatments (mean ± SD). Percent color is provided for biologically active radiation (BAR) for the simulated solar spectrum (SUN) and fluorescence (FL).* Light Parameter

Treatments (percent photon flux density) 100R

BAR** PPFD*** YPFD† Cumulative YPF†† PSS††† R:Fr†††† Photoperiod

20B:80R

200.9 ± 1.3 200.8 ± 1.6 199.3 ± 1.2 199.1 ± 0.5 186.2 ± 1.2 178.6 ± 0.6 463.9 445.27 0.88 0.88 133.0 137.2 18 h for all treatments

50B:50R

80B:20R

100B

SUN (5.1UV, 20.0B, 26.1 G, 26.3R, 22.6Fr)

FL (1.9UV, 29.9B, 39.1 G, 24.1R, 5.0Fr)

200.0 ± 0.4 198.8 ± 0.4 166.4 ± 0.3 415.07 0.86 123.6

201.1 ± 1.7 199.5 ± 1.0 155.2 ± 0.9 386.8 0.79 54.2

199.8 ± 1.3 198.9 ± 1.5 146.7 ± 1.1 366.7 0.49 1.0

201.5 ± 0.2 145.8 ± 0.4 138.7 ± 0.4 345.9 0.69 1.2

199.6 ± 2.5 185.8 ± 6.3 160.0 ± 5.0 400.1 0.81 4.8

* R represents percent PFD of red light, B represents percent PFD of blue light; SUN represents the sun-simulated white light spectrum composed of different ultraviolet, blue, green, red, and far-red percent PFD ratios; FL represents cool white fluorescent (6500 K) white light. ** Biologically active radiation (BAR) is 300−800 nm in μmol m−2 s−1. *** Photosynthetic photon flux density (PPFD) is 400−700 nm in μmol m−2 s−1. † Yield photon flux density (YPFD) values in μmol m−2 s−1 calculated from Sager and McFarlane (1997). †† Cumulative YPF (mol m−2) received per treatment for the entire growing period harvest day (D42). ††† Phytochrome Photoequilibrium (PSS). †††† R = 600−700 nm and Fr = 700−800 nm was used to calculate ratio R:Fr.

on the first harvest day (D17) (Table 3). At the second harvest (D33), lettuce fresh mass differed for plants in FL and 100R, which had 33 % and 30 % greater fresh mass, respectively, than those in 80B:20R (Table 3) while all other treatments were similar. At the final harvest (D42), plants in FL had 27 %, 37 % and 38 % greater fresh mass than in 20B:80R, 50B:50R, and 80B:20R, respectively, but were not different from plants in 100B, 100R, and SUN treatments (Table 3). Lettuce plant responses to B:R treatments varies in the literature, and appears to be cultivar and/or genotype dependent. Green leaf lettuce genotypes ‘Korea’ and ‘Grand Rapid TBR’ gained 18–33 % and 39 % more fresh mass under high red light treatments (Son and Oh, 2013; Kui et al., 2018). Similarly, the red leaf lettuce ‘Sunmang’ had a 70 % gain in fresh mass under 13B:87R than in 24B:76R and 34B:66R light treatments (Son and Oh, 2015) while 0B:100R was the best light quality for fresh mass accumulation for both red leaf (‘Sunmang’) and green leaf (‘Grand Rapid TBR’) lettuce when compared to other B:R treatments (13B:87R, 26B:74R, 35B:65R, 47B:53R, 59B:41R) (Son and Oh, 2013). Increased B PFD (47B:53R) decreased fresh mass accumulation in the red leaf lettuce (‘Sunmang’), but increased green leaf (‘Grand Rapid TBR’) lettuce mass by 46–135 % (Son and Oh, 2013). A general trend is seen in the literature, where good fresh mass accumulation occurs when lettuce is grown under a light treatment containing 30 % B PFD or less (Son and Oh, 2013, 2015; Amoozgar et al., 2017, Kui et al., 2018, Spalholz and Hernández, 2018). However, Amoozgar et al. (2017) grew green lettuce (‘Grizzly’) under 100B, 100R, 30B:70R and white light and found that lettuce shoot dry mass was 5.8 times greater in 100B, white, 30B:70R than in 100R since plants in 100R had severe leaf curling. Despite the 36 % greater PPFD in B:R treatments compared to SUN (Table 2), no gain in lettuce fresh mass was seen compared to plants from the SUN treatment. In fact, lettuce from the SUN treatment had a greater fresh mass to dry mass ratio than the B:R treatments at all harvest dates. These unexpected results suggest that lettuce plants in SUN hold more water per plant dry mass (DM) (Fig. 2). One possible explanation is the presence of far-red in the SUN spectrum (Table 2), which triggers shade-avoidance-responses such as an increase in extensibility of the cell wall by greater turgor pressure (more water in the cell) (Sasidharan et al., 2008). This is also evidenced by the greater leaf length in the SUN treatment plants which was 61–98 % longer than those in B:R treatments (data not shown). A similar response in lettuce fresh mass was seen when far-red light was used to supplement white, Li and Kubota (2009) showed a 28 % increase in lettuce (‘Red cross’). Since lettuce plants are typically sold by fresh weight, this information could be used to design a commercialized spectrum to increase plant

D42. Three derived responses were calculated as follows: (1) leaf mass area (LMA) the dried shoot mass divided by leaf area (g m−2) for D17 and D33 and dried leaf mass divided by leaf area (g m−2) for D42 (the stem wws not included on D42), (2) ratio of fresh to dry shoot mass (FM:DM) (FM g/ DM g), (3) biomass accumulation on a mole of YPFD basis (DM/mol YPF, respectively) (mg mol-1). Phytochemical Concentrations Leaf chlorophyll concentration was perfomed based on Moran and Porath (1980) from all three-harvest plant stages (sample size n = 3). Two leaf discs, each of 56.55 mm2 area, from a single plant were taken 1 cm from the leaf apex, avoiding the main leaf rib and margin. Total anthocyanin and phenolic content of shoots was measured from three plants of the D42 harvest. These samples were held at −80 °C until analysis. Plant shoots were individually pulverized at −80 °C using liquid N and a genogrinder (SPEX, Metuchen, NJ) and extracted using acidified methanol (formic acid:methanol:deionized water, 60:37:3, v/v/v). The dilution factor of pulverized lettuce sample to extract solvent was 0.6 g to 12 ml. Samples were vortexed and centrifuged (14,000 rpm for 5−10 min at 5 °C). Supernatants were collected for respective anthocyanin and phenolic content analysis. Anthocyanin samples were assayed using the pH differential method (Giusti and Wrolstad, 1999) and phenolics by the method of Singleton et al. (1999). Absorbance was measured using a microplate spectrophotometer (Power Wave XS-BioTek Instruments Inc., Winooski, VT, USA). 2.4. Statistical analysis Statistical analysis for comparing the different treatments was done using ANOVA (p < 0.05) with JMP software Version Pro 13.2 (SAS Institute, Cary, NC, US). Tukey HSD analysis was used for multiple mean comparisons between the different light treatments. When no cultivar x treatment interaction was found, results of the two cultivars ‘Red Oakleaf’ and ‘Green Oakleaf’ lettuce were combined. Where cultivar × treatment interaction was found, the results are presented individually (i.e. dry mass). No repetition and treatment interaction was found. 3. Results and discussion 3.1. Plant fresh mass In this study, light quality had minimal effect on plant fresh mass in all harvest stages. Lettuce fresh mass did not differ among treatments 4

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Fig. 1. Spectral distribution of light-emitting diode (LED) and fluorescent light treatments. R and B represent percent PFD of red light and blue light respectively. FL indicates cool white fluorescent (6500 K) white light and SUN indicates the sun-simulated white light spectrum composed of different ultraviolet, blue, green, red, and far-red percent PFD ratios (5.1UV, 20.0B, 26.1 G, 26.3R, 22.6Fr). The spectra were recorded from a five point light field and averaged together. Each graph is the average reading for both replications.

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Table 3 Fresh mass (combined both cultivars) and dry mass for ‘Green Oakleaf’ and ‘Red Oakleaf’ lettuce cultivars harvested at D17 (transplant), D33 (baby leaf), and D42 (mature heads) after growth under various light treatments.* Mass (g)

Fresh mass

Dry mass ‘Green Oak Leaf’

Dry mass ‘Red Oak Leaf’

Harvest day

17 33 42 17 33 42 17 33 42

Treatments (percent photon flux density ratio)** 100R

20B:80R

50B:50R

80B:20R

100B

SUN

FL

0.57 ± 0.11 a 10.64 ± 0.63 a 21.51 ± 0.92 ab 0.03 ± 0.01 abc 0.58 ± 0.09 a 1.21 ± 0.14 abc 0.03 ± 0.01 ab 0.53 ± 0.05 ab 1.35 ± 0.08 a

0.51 ± 0.13 a 9.82 ± 0.39ab 20.14 ± 1.57b 0.04 ± 0.02 a 0.63 ± 0.03 a 1.40 ± 0.07 ab 0.04 ± 0.01 a 0.56 ± 0.04 a 1.26 ± 0.10 ab

0.43 ± 0.09 a 8.47 ± 0.36 ab 18.65 ± 1.58 b 0.04 ± 0.01 abc 0.55 ± 0.03 a 1.29 ± 0.02 abc 0.03 ± 0.01 ab 0.45 ± 0.02 abc 1.10 ± 0.01 abc

0.40 ± 0.10 a 7.42 ± 0.66 b 18.55 ± 1.21b 0.03 ± 0.01 abc 0.50 ± 0.06 a 1.21 ± 0.05 bc 0.03 ± 0.01 ab 0.39 ± 0.05 bc 0.97 ± 0.07 c

0.42 ± 0.10 a 9.17 ± 1.16 ab 22.33 ± 0.74 ab 0.03 ± 0.01 bc 0.53 ± 0.10 a 1.28 ± 0.08 abc 0.02 ± 0.01 b 0.41 ± 0.08 abc 1.16 ± 0.08 abc

0.47 ± 0.13 a 8.88 ± 1.28 ab 22.83 ± 1.36 ab 0.02 ± 0.01 c 0.46 ± 0.00 a 1.17 ± 0.07 c 0.02 ± 0.01 b 0.34 ± 0.10 c 1.04 ± 0.14 bc

0.55 ± 0.17 a 11.01 ± 1.34 a 25.59 ± 1.91 a 0.04 ± 0.02 ab 0.62 ± 0.05 a 1.45 ± 0.04 a 0.03 ± 0.01 ab 0.44 ± 0.07 abc 1.17 ± 0.14 abc

* (Mean ± SE) Values represent mean +/- SE. Differences within row and across light treatments are indicated by different letters, (Tukey HSD, p < 0.05). ** R represents percent PFD of red light, B represents percent PFD of blue light; SUN represents the sun-simulated white light spectrum composed of different ultraviolet, blue, green, red, and far-red percent PFD ratios; FL represents cool white fluorescent (6500 K) white light.

had 39 % more dry mass than SUN (Table 3). By the heading stage (D42), ‘Green Oakleaf’ plants in FL treatment had 24 % more dry mass than those from the SUN treatment, and dry mass was 38 % higher in 100R than in 80B:20R for ‘Red Oakleaf’ plants (Table 3). In the present study, the same PFD in BAR range (300−800 nm) was used to grow lettuce plants. The greater dry mass for plants in 20B:80R than plants grown in SUN would be expected as plants in the 20B:80R were irradiated with higher photon flux density for PPF and YPF (Table 2), both known to drive photosynthesis (Sager and McFarlane, 1997). This would indicatate that for increased plant growth (dry mass), the electrical energy should be invested into the production of photons that drive photosynthesis, rather than in photons such as far-red light that mainly control photomorphogenesis. Additionally, when comparing total dry mass accumulation to cumulative YPF, plants in SUN had the same ability to accumulate dry mass per YPF input (mg mol−1) than in 20B:80R treatment (Table 4). In other studies with cucumber seedlings (Hernandez & Kubota, 2016) and lettuce (Son and Oh, 2013), a reduction of dry mass with the reduction of YPF was also found. In addition to using YPF to predict growth, light capture, measured by leaf area, provides plant growth (Lambers and Poorter, 1992). In the present study, plants in SUN and 100B:0R had greater leaf area than plants in the 20B:80R treatment. The increased leaf area did not translate into greater plant dry mass (compared to 20B:80R), possibly due to the high plant density in this study (i.e. 125 plants m-2 for D42, or 3 times higher than commercial density). A higher plant density will consequently reduce the time for canopy closure between plants and reduce the benefits from rapid leaf expansion (increase leaf area). Although plants in the SUN treatment had lower dry mass accumulation due to the lower YPF, there was greater leaf area and higher specific leaf area (1/LMA), both of which are positively correlated with relative growth rate (RGR) (Lambers and Poorter, 1992). This information should be considered in future light studies designed to increase light intersection at specific lettuce growth stages. For example, far-red supplementation can be applied during early growth stages to increase leaf extension and light capture, and removed after canopy

Fig. 2. Fresh Mass to Dry mass (FM:DM) ratio, averaged for cultivars under different light treatments. Different color bars represent the three harvests of transplant (D17), loose-leaf (D33) and head lettuce (D42). Different letters represent light treatment differences that correlate with each respective harvest period only (Tukey HSD, p < 0.05) lower case for D17, lower case underline for D33 and upper case for D42. See Table 2 for light treatment specifications.

weight. 3.2. Plant dry mass For transplants (D17),’ Green Oakleaf’ lettuce grown under 20B:80R had 15 % and 28 % greater dry mass than those grown in 100B and SUN, respectively (Table 3). Similarly, ‘Red Oakleaf’ grown under 20B:80R had 27 % and 39 % greater dry mass than in 100B and SUN, respectively (Table 3). At the baby leaf stage (D33), no differences in dry mass were found for ‘Green Oakleaf’ lettuce grown under any of the light treatments while the‘Red Oakleaf’ plants grown under 20B:80R

Table 4 Dry mass accumulation relative to cumulative moles of YPF (mg/mol) under different light treatments for lettuce plants at 42 days. Different letters represent light treatment differences (in-row) (Tukey HSD, p < 0.05). Cultivar*

‘Green Oakleaf’ ‘Red Oakleaf’

Treatment (photon flux ratios)** 100R

20B:80R

50B:50R

80B:20R

100B

SUN

FL

2.7 ± 0.2 b 2.9 ± 0.2 a

3.1 ± 0.0 ab 2.8 ± 0.23 a

3.1 ± 1.0 ab 2.7 ± 0.0 a

3.1 ± 0.1 ab 2.5 ± 0.2 a

3.5 ± 0.2 a 3.2 ± 0.2 a

3.4 ± 0.2 a 3.0 ± 0.4 a

3.6 ± 0.0 a 2.9 ± 0.3 a

* Interaction of cultivar with light treatment was significant, p < 0.05. ** See Table 2 for light treatment specifications. 6

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of B PFD decreased leaf area and increased LMA in 0B:100R, 20B;80R, 50:50R and 80B:20R. However, higher B:R ratio by itself cannot explain the greater leaf area and lower LMA in 100B, SUN, and FL which contain 100B, 20B, and 30B, respectively. In the case of FL, light quality has a high red to far-red ratio and adequate amount of B-light (Table 2), which should not induce typical shade avoidance responses (longer and thinner leaves). The higher leaf area of plants in FL can be explained by green-light-mediated shade avoidance responses. Similar results were observed on Arabidopsis seedlings grown under fluorescent light (Wang and Folta, 2013; Zhang et al., 2011) and authors concluded that the addition of green light can induce shade avoidance response which are co-regulated by cryptochrome receptors and potentially by an unknown green light receptor. Other studies have reported an increase of lettuce leaf area and decreased LMA by the addition of G light. For example, Kim et al. (2004) grew lettuce under 16B:84R, 10B:86G:4R, 15B:24G:61R, and a coolwhite-fluorescence treatment (CWF:19B:52G:30R) and found 31–70 % increase in lettuce leaf area in 15B:24G:61R and CWF when compared to 16B:84R; and a decrease in LMA (1/specific leaf area) in 10B:86G:4R when compared to the other treatments. With lettuce in the SUN treatment, the inclusion of G PFD could have also contributed to the increase of lettuce leaf area and plants were also exposed to higher far-red PFD. Far-red light or low red to farred ratio (R:Fr) elucidates a phytochrome induced shade avoidance responses (Kendrick and Kronenberg, 1994) and the decrease of R:FR in SUN treatment also contributed to the higher leaf area and lower LMA compared to the B:R treatments. Plants grown under 100B had been reported to have increased leaf area and decreased LMA (Hernández and Kubota, 2016; Jishi et al., 2016; Wang et al., 2015). Cucumber grown under 100B had 15–49 % greater leaf area than plants in 0B:100R, 50B:50R, 75B:25R (Hernández and Kubota, 2016). Jishi et al. (2016) used time intervals under 100B and 100R radiation and found that ‘Cos’ lettuce had greater leaf area and thinner leaves as the B time interval increased. Cucumber plants grown under 100B had 61 % and 16 % greater leaf area than plants in 100R and W spectrum, respectively (Wang et al., 2015). Cucumber chloroplasts from plants grown in 100B had more grama lamella, bigger stacks of thylakoids, and less starch granulates than those grow in W or 100R. Wang et al. (2015) suggested that plants under 100B had a strong adaptability to a low light environment (50 μmol m−2 s−1) since daytime starch accumulation in the chloroplast was depleted at night to potentially increase leaf and stem extension. The physiological mechanisms that drive increased leaf area and stem extension under 100B are still unknown.

Fig. 3. Leaf area averaged for cultivars under different light treatments. Different color bars represent the three harvests of transplant (D17), loose-leaf (D33) and head lettuce (D42). Different letters represent light treatment differences that correlate with each respective harvest period only (Tukey HSD, p < 0.05). See Table 2 for light treatment specifications.

closure.

3.3. Leaf area Only a few significant differences in leaf area were present in the first two harvest dates. For example, at D17, leaf area in 100R and FL was 84 % and 93 % greater than in 80B:20R plants, respectively (Fig. 3). Similarly, at D33, plants in 100R, 100B, SUN and FL had 56–87 % greater leaf area than in 80B:20R (Fig. 3). However, for D42, plants in 100B, SUN, and FL had 39–78 % greater leaf area than plants in 20B:80R, 50B:50R, and 80B:20R (Fig. 3). Leaf mass area (LMA) can be used to estimate leaf thickness where higher LMA represents greater thickness. For all three harvest dates, plants in 100B, FL, and SUN had lower LMA than in the B:R treatments; for example, at D42, plants in 100B, FL, and SUN had an average of 35 % lower LMA than plants in the B:R treatments (Fig. 4). Cryptochrome photoreceptors are known to inhibit leaf expansion, and maximal activity is in the B wavelength with a peak around 450 nm (Ahmad and Cashmore, 1996; Ahmad et al., 2002). In the present study, the increase

3.4. Leaf number Plants in 100B and SUN had fewer leaves than other treatments for all harvest days. On D42, plants in 100B and SUN had 31 % and 27 % fewer leaves than in 20B:80R (Fig. 5). Although temperature is known to affect leaf initiation and growth (Adams et al., 2001; Hernández and Kubota, 2015; Lieth and Pasian, 1990), temperature was the same in all treatments in this study (Table 1). Other studies have found similar responses in leaf number using 100B. For example, Hernández et al. (2016) grew tomato seedlings under different B:R treatments and found that plants in treatments containing both B and R photons had 21–28 % more leaves than plants in 100B. Similarly, in lettuce, Wang et al. (2016) found that leaf number in 0B:100R, 11B:89R, and 8B:92R was 25–50 % greater than in monochromatic B (100B:0R). In this study, the decreased leaf count in SUN plants could be attributed to the developmental change from vegetative to reproductive stage, as the plants developed inflorescences and started bolting (Fig. 7F). Flowering transition is controlled by the shoot apical meristem (SAM), which is a pool of stem-cells that continuously divide (Fletcher, 2002). During vegetative stage, the SAM produces leaves and after transitioning to the flowering stage, the SAM elongates and

Fig. 4. Leaf mass area averaged for cultivars under different light treatments. Different color bars represent the three harvests of transplant (D17), loose-leaf (D33) and head lettuce (D42). Different letters represent light treatment differences that correlate with each respective harvest period only (Tukey HSD, p < 0.05) lower case for D17, lower case underline for D33 and upper case for D42. See Table 2 for light treatment specifications. 7

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temperature differential) (Sukprakarn, 1985; Chen et al., 2018), longday photoperiod (Waycott, 1995), and application of exogenous gibberellin (Fukuda et al., 2011) can accelerate or induce lettuce bolting while the molecular mechanisms that regulate the transition from the vegetative to the reproductive state remain largely unknown (Chen et al., 2018). The extent of these stimuli upon bolting is cultivar dependent (Waycott, 1995; Sanchez et al., 1989). In our study, temperature and photoperiod were the same across treatments so the development of excessive stem elongation and sequential lettuce bolting due to light quality was not expected. Lettuce is classified as a quantitative long day plant (Waycott, 1995) and the 18 h photoperiod used in the present study is typical to elicit a long day response (bolting and floral primordia development) (Figs. 6 and 7F). The only plants to bolt in this experiment were grown in the SUN treatment, even though all treatments were grown under the same photoperiod and relative cool temperature (20 C°). Additionally, both SUN and FL are considered white light treatments and contain UV, G, and Fr light, yet plants in FL treatment did not demonstrate a bolting response. One possible explanation for this difference may be that the amount of Fr light is higher in the SUN treatment compared to other light treatments. The amount of Fr, a lower PSS, and lower R:Fr ratio can signal stem elongation in lettuce (Chia and Kubota, 2010) or flower initiation response in other plant species (Runkle, 2013). It is thought that phytochrome-driven flower initiation depends on light conversion from the inactive PR to the active PFR, a process mainly driven by R light. However, several studies in ornamental crops have shown that for some long-day plants, a moderate R:Fr during the photoperiod promotes flowering more effectively than a high R:Fr (van Haeringen et al., 1998; Kim et al., 2002; Runkle and Heins, 2003; Craig and Runkle, 2016). A possible explanation for the bolting under the SUN treatment is that, in lettuce, long day detection occurs when a moderate or threshold amount of Fr is included in the spectrum (in this experiment the PSS was close to 0.67 and R:Fr close to 1:1.2 in the SUN treatment). Responses similar to this experiment were seen in a study using different colored nets. Lettuce plant stem length was 62 % and 42 % when grown under red colored and pearl nets, respectively, than plants grown directly under the sun (Ilic et al., 2017). Also, these plants had a greater bolting incidence (bud appearance) than plants under the sun (no bolting, just initial stem elongation). In addition, both the red colored nets and the pearl colored nets reduced the R:FR ratio (more far-red) inside the growing environment (Ilic et al., 2017). Studies in ornamental crops (van Haeringen et al., 1998; Kim et al., 2002; Runkle and Heins, 2003); for example, Runkle and Heins (2003), showed that pansy plants grown under a Fr reducing filter with a R:Fr of 1.5 had a lower flowering percent than plants grown under a natural filter with a R:Fr of 1.1. Kim et al. (2002) demonstrated that four petunia cultivars took longer to flower when grown under a R:Fr of 1.7 than when grown under R:Fr of 1.1. Another possible explanation for the bolting response may be from the augmented interaction between G and Fr in the SUN treatment, which increase shade induced characteristics more than Fr or G alone (Wang and Folta, 2013). The G and Fr wavelengths in the SUN treatment could have synergistically increased lettuce stem elongation and perhaps also increased flower initiation. More research is needed to determine which spectrum range or color ratios initiate bolting in

Fig. 5. Leaf count averaged for cultivars under different light treatments. Different color bars represent the three harvests of transplant (D17), loose-leaf (D33) and head lettuce (D42). Different letters represent light treatment differences that correlate with each respective harvest period only (Tukey HSD, p < 0.05) lower case for D17, lower case underline for D33 and upper case for D42. See Table 2 for light treatment specifications.

Fig. 6. Stem length averaged for both cultivars under different light treatments. Different letters represent light treatment differences (Tukey HSD, p < 0.05). See Table 2 for light treatment specifications.

produces inflorescences and consequently stops the production of leaves (Chen et al., 2018; Kobayashi et al., 2012).

3.5. Light quality effect on lettuce bolting In mature lettuce plants (D42), plant stem length was 2.1–4.4 times longer and stem fresh mass was 1.7-3.1 times greater in SUN than in all other treatments (Fig. 6, Table 5). Lettuce stem elongation is indicative of bolting, a reproductive trait that precedes floral initiation. Premature bolting is detrimental to growers as it decreases overall lettuce value by decreasing flavor and appearance quality (Chen et al., 2018). Certain stimuli such as temperature (heat stress, lack of day-night

Table 5 Stem fresh mass and dry mass in grams for each treatment (Mean ± SE) for mature lettuce plants (D42). Data is combined for ‘Red Oakleaf’ and ‘Green Oakleaf’ cultivars. Different letters represent light treatment differences (in-row) (Tukey HSD, p < 0.05). Treatment*

100R

20B:80R

50B:50R

80B:20R

100B

SUN

FL

Stem fresh mass Stem dry mass

0.89 ± 0.07 b 0.09 ± 0.01 ab

0.84 ± 0.06 b 0.10 ± 0.02 ab

0.77 ± 0.04 b 0.09 ± 0.01 b

0.72 ± 0.08 b 0.07 ± 0.01 b

1.3 ± 0.05 b 0.09 ± 0.01 ab

2.25 ± 0.39 a 0.12 ± 0.03 a

1.1 ± 0.08 b 0.09 ± 0.01 b

* See Table 2 for light treatment specifications. 8

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Fig. 7. Morphology of ‘Red Oakleaf’ lettuce under different LED treatments. Pictures were taken on D17-transplant top view (A), D33-loose leaf side view (B) and top view (C), D42-head lettuce top view (D) and side view (E), and side view at 8 weeks (F). See Table 2 for light treatment specifications.

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Table 6 Average total phenolic, total anthocyanin, and total chlorophyll content, for each treatment at day 42 (final harvest). Treatments (photon flux ratio)*

Parameter

−1

Total Phenolics (mg kg ) Total Anthocyanin (mg kg−1)** Chlorophyll Total (g m−2)

100R

20B:80R

50B:50R

80B:20R

100B

SUN

FL

91.0 ± 9.9 b 12.4 ± 0.6 cd 0.17 ± 0.01 d

152.3 ± 18.3 a 68.6 ± 22.8 ab 0.26 ± 0.01 bc

175.6 ± 11.5 a 77.1 ± 17.6 a 0.30 ± 0.01 ab

167.8 ± 9.4 a 70.3 ± 1.0 ab 0.33 ± 0.01 a

109.2 ± 5.7 b 19.5 ± 0.5 cd 0.20 ± 0.01 cd

77.1 ± 3.6 b 6.2 ± 0.4 d 0.15 ± 0.00 d

92.8 ± 10.7 b 37.8 ± 9.6 abcd 0.20 ± 0.00 cd

* Values represent mean ± SD and similar letters withing a row indicate no significant differences, HSD (p < 0.05). Refer to Table 1 for description of treatments. ** Total anthocyanin content is for ‘Red Oakleaf’ lettuce only.

accumulation (Drumm and Mohr, 1978; Mohr and Drumm-Herrel, 1981; Oelmüller and Mohr, 1985). Similarly, Neff and Chory (1998) showed that in Arabidopsis, phyA and cry1 are both necessary for anthocyanin production since the mutation of either photoreceptor prevented anthocyanin accumulation. The lower anthocyanin content in lettuce under the 100B treatment may indicate a need for red light in anthocyanin production or accumulation. In accordance with other reports (Zheng et al., 2019), leaf chlorophyll content in the present study, was greatest in B:R treatments with ≥50 % B light (ie 50B:50R and 80B:20R). Total chlorophyll concentration in lettuce under 50B:50R and 80B:20R was 48–125 % greater than in 100B, 100R, SUN, and FL (Table 6). Blue light is known to promote the chlorophyll tetrapyrrole precursor 5-aminolevulinic acid (ALA). Although monochromatic red light reduced ALA in cabbage, addition of blue light restored chlorophyll concentration (Fan et al., 2013). With lettuce, plants in SUN and FL had decreased chlorophyll content despite the presence of both B and R. The reduced chlorophyll may have resulted from the inclusion of G in SUN and FL, which is known to down-regulate the transcription factors for chloroplast formation in multiple plant species (Folta and Maruhnich, 2007). In contrast, the 50B and 80B treatments had higher LMA (thicker leaves) along with higher chlorophyll. As chlorophyll analysis was done on discs rather than per weight basis, it is possible that the higher dry mass in plants grown in 50B and 80B also had greater chlorophyll concentration per area (g m−2). However, this does not explain the reduced chlorophyll concentration in 20B:80R which also had large LMA.

lettuce when grown under cool environmental conditions. To our knowledge, the present research study is the first report to elucidate lettuce flowering under SUN simulated spectrum, under relative low temperature (20 °C), and indoor growing conditions. 3.6. Phytochemical content Light treatment impacted phytochemical content of different compounds (Table 6). Total phenolic content of lettuce was 98–118 % greater in B:R (20B:80R, 50B:50R, 80B:20R) treated plants than plants in SUN or 100R (Table 6). Total anthocyanin concentration of plants in 20B:80R, 50B:50R, and 80B:20R was 10–11 times greater than plants in SUN (Table 6). B light is mediated by cry1 (cryptochome) and induces gene expression that leads to the accumulation of anthocyanin (Wang and Folta, 2013; Ahmad et al., 1995) and could explain the greater anthocyanin concentration in the B:R treatments when compared to the 100R treatment. Although plants in the SUN treatment were grown under sufficient B and UV-A PFD (25 %, Table 2, Fig. 1) to elucidate anthocyanin and phenolic synthesis, anthocyanin and total phenolic content was less than plants in B:R treatments. G light is present in the SUN spectrum, and when combined with B light, anthocyanin concentration is lower than with B light alone (Wang and Folta, 2013; Zhang et al., 2011; Bouly et al., 2007). Studies in Arabidopsis using cry1 mutant suggested that the response is mediated by cryptochrome (Bouly et al., 2007; Wang and Folta, 2013). The presence of Fr light in the SUN treatment could have also contributed to the reduction in flavanoids, as anthocyanin accumulation is linearly correlated with the amount of phytochrome in the Pfr form (Steinitz et al., 1979). Based on the phytochrome photoequilibrium (PSS in Table 2), plants in the SUN treatment had lower amount of Pfr form (PSS:0.69) than in the B:R treatments (PSS: 0.790.88), contributing to the lower anthocyanin accumulation in SUN treatment. Li and Kubota (2009) reported inhibitory effects of Fr light on phytochemical accumulation in baby leaf lettuce (‘Red Cross’). By adding Fr light to a cool white fluorescent light treatment, anthocyanin, xanthophylls and beta-carotene were reduced by 40 %, 12 %, and 16 %, respectively, compared to those plants in cool white fluorescent lamps without Fr. In addition, plants grown under white light supplemented with Fr had 6 % lower total phenolic content than plants supplemented with red light. The reduction of phytochemical accumulation under 100B is not well understood, although several studies have reported similar results. For example; Hernández et al. (2016) grew tomato seedlings under various B:R treatments and found that plants in 30B:70R, 50B:50R and 75B:25R had 2–3 times greater anthocyanin concentration than plants in 100B. Ryu et al. (2012) grew dandelion greens under a B:R treatment (B6:R4 in energy ratio), fluorescent, monochromatic R (100R), and monochromatic blue (100B); plants in 100B, fluorescent, and 100R had lower anthocyanin concentration than plants in the B:R treatment. Plant-photobiology studies have shown that there is interplay or coaction effect of phytochrome and cryptochrome for anthocyanin accumulation in plant tissue. For example, in milo (grain sorghum), anthocyanin accumulation is mainly driven by phytochrome but a blue light stimulus of the cryptochrome is required for anthocyanin

4. Conclusion The objective of the present study was to investigate the response of lettuce at different growth stages to a SUN simulated spectrum, compared to responses to other common B:R spectrums. While plants in 20B:80R had greater dry mass at the transplant stage (D17) than in other treatments, the increase was not sustained through plant maturity. In contrast, leaf area increased in plants in SUN and 100B in mature plants (D42) compared to LED. The SUN treatment elucidated unique responses, including greatest fresh mass to dry mass ratio and greater leaf area and lower LMA than other LED treatments. Even under cool growing temperatures, plants in SUN showed a clear transition from vegetative to reproductive stage (excessive stem extension and flower initiation). Although plants in the SUN treatment had lower dry mass due to the lower YPFD of the treatment (treatments set at equal BAR: 300−800 nm), dry mass accumulation per cumulative YPF was similar to other LED. These results can be used to tailor plant growth for desired response. For instance, using photons in the PAR range would be the most efficient means of increasing plant growth (dry mass) per energy unit. In contrast, far-red or other wavelengths outside PAR that elicit unique photomorphogenic responses could be used as signals to stimulate flowering. Declaration of Competing Interest The authors declare that they have no known competing financial 10

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interests or personal relationships that could have appeared to influence the work reported in this paper.

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