AT LEAST ONE LIGHT ABSORBING PIGMENT

FIELD

[0001] The improvements generally relate to photosynthesis and more specifically relate to methods of illuminating plants to increase a biomass yield.

BACKGROUND

[0002] It is generally accepted in the field that, to increase the biomass yield, a given plant should preferably be illuminated with a light beam having optical energy within spectral regions encompassing wavelengths which are specifically directed to absorbing wavelengths of light absorbing pigments of the given plant.

[0003] Although existing plant growing techniques are satisfactory to a certain degree, there remains room for improvement.

SUMMARY

[0004] In accordance with one aspect, there is provided a method of growing a plant having at least one light absorbing pigment, the at least one light absorbing pigment absorbing optical energy at absorbing wavelengths, the method comprising: illuminating the plant with at least one light beam, the at least one light beam having optical energy within at least two spectral regions each encompassing a given wavelength, the given wavelengths being away from the absorbing wavelengths of the at least one light absorbing pigment, the at least two spectral regions including at least both a first spectral region at about 430 nm and a second spectral region at about 595 nm.

[0005] In accordance with another aspect, there is provided a method of growing a plant having at least one light absorbing pigment, the at least one light absorbing pigment absorbing optical energy at absorbing wavelengths, the method comprising: illuminating the plant with at least one light beam, the at least one light beam having optical energy within one or more spectral regions each encompassing a given wavelength being away from the absorbing wavelengths of the at least one light absorbing pigment.

[0006] Many further features and combinations thereof concerning the present improvements will appear to those skilled in the art following a reading of the instant disclosure. DESCRIPTION OF THE FIGURES

[0007] In the figures,

[0008] Fig. 1 is a schematic view of an example of a system for growing a plant having one or more light absorbing pigments by illuminating the plant with a light beam, in accordance with an embodiment;

[0009] Fig. 1A is a graph showing energy as function of wavelength of the light beam of Fig. 1 ;

[0010] Fig. 2 is a graph showing net photosynthetic rate as function of wavelength when tomato leaves and lettuce leaves are illuminated with the light beam of Fig. 1 ;

[001 1] Fig. 3 is a schematic diagram of an example of a LED lighting system showing a power supply, power distribution panel, voltage drivers, voltage regulators and LED assemblies, in accordance with an embodiment;

[0012] Figs. 4A and 4B are graphs showing relative spectral irradiance compositions of LED light treatments with the LED lighting system of Fig. 3, wherein arrow in Fig. 4A indicates the wavelength of the valley using a pc-amber LED assembly with a notch filter;

[0013] Fig. 5 is an image showing a morphology of lettuce ( Lactuca sativa, cv. Breen) plants grown under the 595, 602, 633, and 613-nm light treatments (from left to right) for two weeks, the wavelength values in the figure indicate the peak wavelengths of the light treatments, each grid is 30 mm in size;

[0014] Fig. 6 is an image showing pigmentation on the lettuce ( Lactuca sativa, cv. Breen) leaf grown under the 602-nm light treatments for two weeks;

[0015] Fig. 7 is a graph showing comparative data between the measured whole photosynthetic rates and shoot fresh mass for the lettuce plants ( Lactuca sativa, cv. Breen) under different LED wavelengths;

[0016] Fig. 8 is a graph showing photosynthetic response curve of lettuce ( Lactuca sativa var. Buttercrunch) for three hours (see inset) using a 655-nm LED array at an intensity of 120 pmol m ² sec ¹ ; [0017] Fig. 9 is a graph showing the leaf absorption spectra of lettuce and tomato seedlings grown under a fluorescent light spectrum;

[0018] Fig. 10 is a graph showing the change in light compensation points for lettuce and tomato seedlings at different incident LED light wavelengths;

[0019] Figs. 1 1A and 1 1 B are graphs showing the action spectra of lettuce ( Lactuca sativa) and tomato ( Solarium lycopersicum) seedlings at different light intensities. Error bars for each data point indicates the standard deviation;

[0020] Figs. 12A and 12B are graphs showing the relationship between photosynthetic activity and light compensation point for lettuce ( Lactuca sativa) and tomato ( Solarium lycopersicum) seedlings at LED light wavelengths varying from 400 to 700 nm;

[0021] Fig. 13 is a schematic view a thin film nutrient loop system showing the location of the plants in each channel, LEDs of each treatment were placed directly above each channel;

[0022] Fig. 14 is a graph showing normalized spectral distribution of light from blue, amber and red LEDs in which spectral distribution measurements were performed with a spectroradiometer (PS-300, Apogee instruments Inc., Logan, UT);

[0023] Fig. 15 is a graph showing eigenvectors from the principal component analysis (PCA) for lettuce growth under the red-blue-amber (RBA) treatment in which total, blue (445 nm), amber (600 nm) and red (635 nm) photosynthetic photon flux density (PPFD) were considered in relation to fresh mass (FM) and dry mass (DM);

[0024] Figs. 16A and 16B are graphs showing eigenvectors from the principal component analysis (PCA) for lettuce growth under the RBA treatment for a lower range (39 to 441 pmol m ² sec ¹ ) of photosynthetic photon flux densities (PPFDs) where Fig. 16A is for a higher range (51 1 to 1321 pmol m ² sec ¹ ) of PPFD and in Fig. 16B total, blue (445 nm), amber (600 nm) and red (635 nm) PPFD were considered in relation to fresh mass (FM) and dry mass (DM);

[0025] Fig. 17 is a graph showing the effects photosynthetic photon flux density (PPFD) on lettuce ( Lactuca sativa var. Breen) plant fresh mass (FM) under blue (445 nm), amber (600 nm), red (635 nm) and red-blue-amber (RBA) combination LED light treatments;

[0026] Fig. 18 is a graph showing the effects photosynthetic photon flux density (PPFD) on lettuce ( Lactuca sativa var. Breen) plant dry mass (DM) under blue (445 nm), amber (600 nm), red (635 nm) and red-blue-amber (RBA) combination LED lights;

[0027] Fig. 19 is a graph showing the effects photosynthetic photon flux density (PPFD) on lettuce ( Lactuca sativa var. Breen) plant leaf shape index (LSI) under blue (445 nm), amber (600 nm), red (635 nm) and red-blue-amber (RBA) combination LED lights;

[0028] Figs. 20A and 20B are graphs showing the effects of photosynthetic photon flux density (PPFD) on total leaf area (TLA) of lettuce ( Lactuca sativa var. Breen) plants under blue (445 nm), amber (600 nm), red (635 nm) and red-blue-amber (RBA) combination LED lights., where Fig. 20A shows curves of the relationship between PPFD and TLA and Fig. 20B shows the relationship between TLA and fresh mass FM;

[0029] Fig. 21 is a graph showing the effect of photosynthetic photon flux density (PPFD) on lettuce ( Lactuca sativa var. Breen) leaf SPAD measurements (Chlorophyll) taken 18 days after transplanting under the blue (445 nm), amber (600 nm), red (635 nm), and RBA light treatments;

[0030] Fig. 22 are images showing the effect of the red (635 nm) LED light treatment on lettuce ( Lactuca sativa var. Breen) where the value seen in the lower left corner of each photograph is the photosynthetic photon flux density (PPFD) in pmol m ² sec ¹ ;

[0031] Fig. 23 are images showing the effect of the blue (445 nm) LED light treatment on lettuce ( Lactuca sativa var. Breen) where the value seen in the lower left corner of each photograph is the photosynthetic photon flux density (PPFD) in pmol m ² sec ¹ ;

[0032] Fig. 24 are images showing the effect of the amber (600 nm) LED light treatment on lettuce ( Lactuca sativa var. Breen) where the value seen in the lower left corner of each photograph is the photosynthetic photon flux density (PPFD) in pmol-m ² sec ¹ ; and [0033] Fig. 25 are images showing the effect of the red-blue-amber (RBA) LED light treatment on lettuce ( Lactuca sativa var. Breen) where the value seen in the lower left corner of each photograph is the photosynthetic photon flux density (PPFD) in pmol-m ² sec ¹ and in parenthesis, the respective RBA ratios (red:blue:amber). The RBA ratios varied from 0.4:0.2:1 to 22.7:1.9:1.

DETAILED DESCRIPTION

[0034] Fig. 1 shows an example of a system 10 for growing a plant 12 having one or more light absorbing pigments 14. Examples of such light absorbing pigments 14 can include chlorophyll a, chlorophyll b, carotenoids (e.g., lutein, b-carotene, zeaxanthin and lycopene) and/or any other suitable light absorbing pigments.

[0035] In this example, a given one of the light absorbing pigments 14 of the plant 12 absorbs optical energy at known absorbing wavelengths. As depicted, the system 10 has an illuminator 16 which is configured for illuminating the plant 12 with one or more light beams (hereinafter“the light beam 18”). It was found that unexpected results (e.g., increased net photosynthetic rate, increased biomass yield) can be obtained when the light beam 18 has optical energy within at least two spectral regions each encompassing a given wavelength, where the given wavelengths are away from the absorbing wavelengths of the given light absorbing pigment. More specifically, the spectral regions include at least both a first spectral region 20 at about 430 nm and a second spectral region 22 at about 595 nm.

[0036] In some embodiments, the system 10 can be configured to remove, or otherwise block, optical energy of the light beam 18 which lies within a spectral region encompassing the absorbing wavelengths of the light absorbing pigments 14.

[0037] Fig. 1A is a graph showing optical energy of the light beam 18 as function of wavelength. As shown, the first and second spectral regions 20 and 22 are spectrally spaced from one another. One exemplary spectral absorption band 24 representing the absorbing wavelengths of the given light absorbing pigment 14 is also shown in dashed line in Fig. 1A. As can be seen, the first and spectral regions 20 and 22 of the light beam 18 are substantially spectrally spaced from the absorbing wavelengths of the light absorbing pigment 14 of the plant 12. In some embodiments, the first and second spectral regions 20 and 22 do not overlap with one another. However, in some other embodiments, the first and second spectral regions may have overlapping tails 30, i.e., overlapping spectral regions where optical energy of the first and second spectral regions 20 and 22 has no or insignificant effect on plant growth.

[0038] Examples of the unexpected results are shown in detail in Fig. 2. As depicted, in this example, the light absorbing pigments 14 of the plant 12 have first, second and third spectral absorption bands Dl1 , Dl2 and Dl3. More specifically, the first spectral absorption band Dl1 spans from about 400 nm to about 420 nm, the second spectral absorption band AK2 spans from about 440 nm to about 525 nm and the third spectral absorption band Dl3 spans from about 610 nm to about 700 nm. The skilled reader may appreciate that, in this example, the first absorption band Dl1 spans between the ~400- nm peak of chlorophyll a and the ~425-nm peak of chlorophyll b. Accordingly, in this example, by illuminating the plant 12 with the light beam 18 having the first and second spectral regions 20 and 22 which are each spectrally spaced from the first, second and third spectral absorption bands Dl1 , AK2 and Dl3 of the light absorbing pigments 14, a satisfactory net photosynthetic growth rate can be obtained.

[0039] In some embodiments, the light beam 18 includes a first light beam 18a having optical energy at the first spectral region 20 and a second light beam 18b having optical energy at the second spectral region 22. In these embodiments, the first light beam 18 can be generated using a first light-emitting diode 26a and the second light beam can be generated using a second light-emitting diode 26b. However, in some other embodiments, any other suitable light source can be used such as a lamp, a laser and the like.

[0040] Moreover, the system 10 can have one or more filter elements 28 configured for filtering the first light beam 18a in a manner filtering out optical energy away from the first spectral region 20 and for filtering the second light beam 18b in a manner filtering out optical energy away from the second spectral region 22.

[0041] The first and second spectral regions 20 and 22 can have any suitable bandwidth. In some examples, the first and second spectral regions 20 and 22 are narrow-band. For instance, in some embodiments, the bandwidths of the first and second spectral regions 20 and 22 can be at least about 1 nm, about 5 nm, about 10 nm and the like. [0042] Although not shown in Fig. 1 , the light beam 18 can be focused on an area of the plant 12 in some alternate embodiments. Moreover, in some embodiments, light encompassing a visual region of the electromagnetic spectrum (e.g., about 400 nm to about 700 nm) can be shined onto the plant 12 so as to trigger mechanisms such as flowering of the plant 12 when desired, which would not necessarily be triggered solely using the light beam 18 having the first and second spectral regions 20 and 22.

[0043] Example 1 - Manipulating Spectrum Compositions of Red and PC-Amber LEDs by Using Optical Filters to Investigate the Effect on Lettuce ( Lactuca sativa, cv. Breen) Growth

[0044] Deep-red (650-690 nm) light is one of the principal wavelength regions responsible for driving photosynthetic activity and plant development, resulting in the selection of deep-red light emitting diodes (LEDs) being a standard diode in most plant LED lighting systems. However, high pressure sodium (HPS) lamps that peak at ~600 nm are still the industry standard lighting systems, and the impact of amber (590-610 nm) wavelengths on plant development is conflicted in the literature and requires further investigation. There is also sparse information on orange/red (610-630 nm) light due to the limited availability of LED hardware with the corresponding wavelengths, and the limited ability to control LED spectral properties. The objective of this study was to investigate plant performances between 590-630 nm and the effect of 630-nm light using pc-amber and red LEDs. We measured two parameters: photosynthetic rate (minutes) and plant growth (weeks) of lettuce plants ( Lactuca sativa cv. Breen) at an irradiance level of 50 W m ² (243-267 pmol m ² sec ¹ ). To obtain the desired spectra, we manipulated the existing pc-amber (602 nm) and red (633 nm) LEDs with different optical filters. With the use of a shortpass and a notch filter, a narrow spectrum (613 nm) and a double-peak spectrum (595 and 655 nm; identified as 595 nm treatment) without 630-nm light were created. It was found that the highest photosynthetic rate and biomass yield (fresh and dry mass) were observed under the 602-nm light treatments, followed by the 595, 633, and 613-nm light treatments. Photosynthetic rates did not correlate to biomass yield for the plants under the 613 and 632-nm light treatments with relatively high photosynthetic rates, but did result in high biomass for the 595 and 602-nm light treatments. Leaf elongation (poor leaf development) was observed under the 613 and 633-nm light treatments. Shifting and narrowing the peak of the LED wavelengths from 602 to 613 nm and from 633 to 613 nm resulted in a biomass yield decrease by ~50 % and ~80 %, respectively. This may be caused by an interaction effect between the wavelengths, plant architecture, and light interception at leaf level. Comparing the 595 and 602-nm (with a double peak) light treatments, highly reducing 630-nm light resulted in larger leaf areas, expanded plant structures, and the absence of purple coloration (assumed to be anthocyanin accumulation). According to the literature and the present results, we concluded that 630-nm light is necessary to reduce leaf elongation and amber light is beneficial for plant growth.

[0045] Research on plant lighting experiments have shown that red (600-700 nm) light plays a critical role on short term photosynthetic activity and long term plant development in the photosynthetically active radiation (PAR) spectrum (400-700 nm) (Goins et al., 1997; McCree, 1972). From the results shown by early action spectrum and quantum yield using monochromatic light (Inada, 1976; McCree, 1972), red light induces higher photosynthetic activity (~20-40 %) compared to other wavelengths in the PAR spectrum, for typical greenhouse crops. The highest peak found from the early quantum yield data presented by McCree (1972) was at 620 nm with a shoulder at 670 nm in the red wavelength. This measurement resulted in red light emitting diodes (LEDs) being deployed in plant lighting systems (Massa et al., 2008; Morrow, 2008).

[0046] Since the very first results using 660 nm LEDs for plant lighting were obtained by Bula et al. (1991), the effect of deep-red (650-690 nm) LED light has been explored and evaluated for plant development experiments (Brazaityte et al., 2006; Goins et al., 2001 ; Mizuno et al., 201 1). Results from these studies showed that deep-red LED light is beneficial for plant growth in terms of biomass yield. Further studies utilized 640 nm LEDs alone or as supplemental lighting to investigate plant responses (Lefsrud et al., 2008; Mizuno et al., 201 1 ; Olle and Virsile, 2013; Stutte et al., 2009). Positive effects were not observed on plant growth from these studies. However, 640-nm LED light stimulated secondary metabolites and anthocyanin accumulation. Hence, deep-red LEDs are still the basal component in plant LED lighting systems in terms of plant productivity (Massa et al., 2008; Mitchell, 2015). There is, however, a lack of information on the effect of orange/red (610-630 nm) LED light on typical greenhouse crops.

[0047] Amber-biased (~ 590-610 nm) high pressure sodium (HPS) lamps are currently the preferable choice over LEDs in commercial greenhouse facilities, due to varying plant productivity with respect to crop choice and growth stages under LED light (Gomez et al., 2013; Olle and Virsile, 2013). In recent years, experiments that involved comparisons of HPS lamps to blue/red LEDs for plant development have become one of the major subjects for lighting studies (Bergstrand and Schussler, 2013; Dueck et al., 201 1 ; Gajc- Wolska et al., 2013; Gomez et al., 2013; Martineau et al., 2012). These studies indicate that LEDs will become the prominent plant lighting systems compared to HPS lamps, due to the efficient nature of this lighting technology. Yet, according to the same results, plant productivity and physiology showed either no significant differences (Gomez et al., 2013) or were higher under HPS lamp light treatments alone (Brazaityte et al., 2006; Gajc- Wolska et al., 2013; Martineau et al., 2012). Conflicting results were reported on the effect of amber light using HPS lamps (Dougher and Bugbee, 2001 ; Loughrin and Kasperbauer, 2001 ; Vanninen et al., 2010). Suppressed growth on some greenhouse crops such as basil ( Ocimum basilicum L.) and lettuce ( Lactuca sativa, cv. Grand Rapids) under high proportions of amber light was observed (Dougher and Bugbee, 2001 ; Loughrin and Kasperbauer, 2001).

[0048] An advantage of LEDs in a comparison to HPS lamps and other conventional lighting sources is the added ability to select and control specific wavelengths (Morrow, 2008; Singh et al., 2015; Yeh et al., 2010). Unlike conventional lighting sources, LED wavelengths can be selected to target specific plant physiobiological responses (Lefsrud et al., 2008; Massa et al., 2008; Olle and Virsile, 2013). However, users have limited options in terms of wavelength selection from the diode manufacturers. For instance, there is only approximately 10-15 LED nominal wavelength options in the red wavelengths from major diode manufacturers such as Cree and Philips-Lumileds. Furthermore, users have limited control to change optical characteristics of existing LEDs (e.g. peak wavelength, spectral composition, and full width at half maximum (FWHM)) under normal operations. Although manipulating the lighting environment could be achieved by using different colored LEDs, this method is only able to create a mixture of different colored LED light using available LEDs. This method of color mixing has led to some undesired results such as uneven light quality/quantity over plant surfaces and low light outputs in wavelengths of interest (Hogewoning et al., 2010). These limitations make the investigation of specific narrow band wavelengths of LED light quite limited.

[0049] Therefore, the goal of this study was to 1) investigate the effect of 590-630 nm light and 630-nm light on the photosynthetic activity and plant development of lettuce plants (a plant usually used for wavelength testing in the horticultural field) using LEDs at a high irradiance level, and 2) create LED spectra (single and double-peak spectra) that were not obtainable from the major LED manufacturers by using optical filters. The results from this study will provide information on the impact of 590-630 nm light on short term photosynthetic activity measurements and long term plant development. Although photosynthetic photon flux density (PPFD) is considered as a standard unit for plant growth in the horticultural industry, it is usually used to define photosynthetic rates rather than photomorphogenesis. Therefore, the energy unit of irradiance level (W m ^-2 ) used in this study as recommended by Langhans and Tibbitts (1997) and Both et al. (2015), but PPFD (pmol m ² sec ¹ ) was also reported.

[0050] Seeds of lettuce ( Lactuca sativa cv. Breen) were potted in 25 mm rockwool growing cubes (Grodan A/S, Dk-2640, Hedehusene, Denmark) and placed in a growth chamber (TC30, Conviron, Winnipeg, Canada) for germination. Plants were kept in the chamber under cool-white fluorescent bulbs (4200 K, F72T8CW, Osram, Wilmington, MA ) at an average irradiance level of 20 W m ² (equal to ~100 pmol m ² sec ¹ ) with a photoperiod of 16 h. The environmental conditions in the chamber were controlled at 50 % relative humidity and a day/night temperature of 23/21 ± 1 °C with ambient C0 ₂ levels. A full strength Hoagland nutrient solution was provided to the plants every other day as described by Hoagland and Arnon (1950). The young lettuce plants (two weeks after germination) with the emergence of the 4 ^th true leaf were used for whole plant photosynthetic rate measurements, and transferred to a 13-L hydroponic tank (Rubbermaid, Atlanta, GA, USA) with different light treatments provided. Oxygen in the hydroponic tanks was provided using air pumps (Marina 200, Rolf C. Hagen, Baie d’Urfe, QC, Canada).

[0051] Fig. 3 illustrates a LED lighting system used in this study. LED assemblies of pc-amber (LXM2-PL01 , Philips-Lumileds, San Jose, CA, USA) and red (LXM2-PD01 , Philips-Lumileds, San Jose, CA, USA) were the light sources for this study. The pc- amber LED assembly was selected due to its similar spectral composition to HPS lamps. Each LED assembly was connected to a power distribution panel and powered using a DC power supply (DP832, Rigol Tech., Beaverton, OR, USA). An adjustable voltage regulator with a digital voltmeter and a 700-mA dimmable DC voltage driver (A01 1 FlexBlock, LED dynamics, Randolph, VT, USA) were placed between the power distribution panel and the LED assembly, and were used to provide constant current output and to adjust light outputs of the LEDs. All the LED assemblies were mounted on a water jacket (ST-01 1 , Guangzhou Rantion Trading Co., China) and attached with concentrated lenses (25 mm focal length, No. 263, Polymer Optics, Wokingham, Berkshire, UK), to concentrate all the emission from LED assemblies into one single spot (12 mm in diameter). Water was circulated at 15 °C in a water jacket behind the mounted LEDs by using an isotemp bath circulator (4100R20, Fisher Scientific, Hampton, NH, USA). Two types of optical filters were utilized to manipulate the spectral compositions of the pc-amber and red LEDs: a 632.8-nm notch filter (25 mm in diameter, #67-120, Edmund Optics, Barrington, NJ, USA) and a 625-nm short pass filter (25 mm in diameter, #64-604, Edmund Optics, Barrington, NJ, USA). The selection of the shortpass and notch filters was based on the needs to decrease the peak wavelengths of the red LED assemblies and exclude 630-nm light from the pc-amber LED assemblies, respectively. The use of the notch filter eliminated overlapping wavelengths that occur when using two different colored LEDs.

[0052] The whole plant photosynthetic rates of the young lettuce plants under different light treatments were carried out using a LI-6400 photosynthesis system (LI-COR, Lincoln, NE, USA) equipped with a Whole Plant Arabidopsis Chamber (6400-17, LI-COR, Lincoln, NE, USA). The irradiance level was set to 50 W m ² . The relative humidity and temperature of the LI-COR environment was controlled at 50 % and 23 °C, the same as in the germination growth chamber. The C0 ₂ concentration and flowrate in the Arabidopsis chamber were set to 400 ppm and 400 pl rnin ¹ , respectively. The photosynthetic rate measurements were repeated three times with three biological replicates. The leaf area of the plants was measured using the software ImageJ 1.48v (Bethesda, MD, USA). The imagery from the ImageJ software was used to determine the whole plant photosynthetic rate on a per unit leaf area basis.

[0053] After the emergence of the 4 ^th true leaf, the lettuce plants were transplanted to the chamber with the LED lighting system and cultivated under four different light treatments at an irradiance level of 50 W m ² for two weeks: the pc-amber LED assembly (peak wavelength: 602 nm, 256 pmol m ² sec ¹ ), the pc-amber assembly with the notch filter (595 and 655 nm, 250 pmol m ² sec ¹ ), the red LED assembly (633 nm, 267 pmol rn ² sec ¹ ), and the red LED assembly with the shortpass filter (613 nm, 243 pmol m ² sec ¹ ). The peak wavelengths, spectral compositions, and light intensities (irradiance levels and PPFD) of the LED light treatments were measured using a spectroradiometer (PS-300, Apogee, Logan, UT, USA) (Figs. 4A-B and Table 1). With the notch filter, the pc-amber LED spectrum was altered to a narrow 595-nm and a 655-nm spectra (ratio º 3: 1). Here, we named this double-peak spectrum after its main peak (595 nm) throughout this study. In the chamber, the optical filters were secured by using three-screw adjustable ring mounts (#36-605, Edmund Optics, Barrington, NJ, USA) and placed 25 mm below the amber and red LED assemblies. Cardboard covered with black plastic sheets and a hole at the center were placed on the ring mounts, to avoid unfiltered LED spectra from reaching the plants. Between each light environment, black sheets were used as a light barrier. Due to the high irradiance levels and need for uniform light distribution over the plant surface, only a single lettuce plant was placed under each light treatment. The heights between the LED lights and the top of plants were checked every three days and the LED lights were adjusted accordingly to allow the plant canopy to be exposed to the same irradiance level throughout the growth period. The differences of the irradiance levels under each light treatment over the growth period were less than 2 %. Fresh full- strength Hoagland’s solution was provided weekly. The environmental conditions (relative humidity, day/night temperature, C0 ₂ levels, and photoperiod) in the chamber with the LED treatments were the same as the germination growth chamber. The photoperiod was controlled by a timer connected to the DC power supply.

[0054] Table 1. The peak wavelength, full width at half maximum (FWHM), irradiance level, and photosynthetic photon flux density (PPFD) of each LED lighting systems used in this study.

Irradiance

Peak PPFD

FWHM level

wavelength

(nm)

(nm) _2x (pmol m^sec ¹ )

(W m )

595 48.90 37.5 187.5 pc-amber LEDs+ ¹ ^ ^eak

notch filter ₂ „ _peak

655 41.44 12.5 62.5 pc-amber LEDs 602 74.07 50 256 red LEDs+shortpass filter 613 22.12 50 243 red LEDs 633 20.24 50 267

[0055] After two weeks of growth under the LED light treatments, the plants were harvested and sampled for biomass yield and morphological analysis, which included fresh mass, dry mass, and leaf area. The biomass yield (fresh and dry mass) was determined by a balance (APX-153, Denver Instruments, Bohemia, NY, USA). To determine the dry mass, plant samples were dried at a temperature of 75 °C for no less than 72 h. Five biological replicates were performed under each light treatment

[0056] Statistical analysis was done using JMP 10 (SAS, Cary, NC, USA). Tukey-

Kramer’s HSD was used for the multiple comparisons among spectral treatment means from significant one-way analysis of variance (ANOVA) tests (P< 0.05). [0057] The lettuce plants with the largest size were observed under the 595-nm light treatment, followed by the 602, 630, and 615-nm light treatments (Fig. 5). The leaf- elongated lettuce (poor leaf development) were only observed under the 613 and 633-nm light treatments. Differences in the lettuce leaves in terms of leaf morphology and leaf coloration were observed across the light treatments. The lettuce plants grown under the 595 and 602-nm light treatments had more obvious lateral veins than the 612 and 633- nm light treatments. The lettuce plants grown under the 602-nm light treatment were more compact than those under the 595-nm light treatment as shown in Fig. 5. The plants grown under the 613-nm LED light had longer and thinner leaves compared to the

633-nm LED light treatment. It was observed that the lettuce leaves grown under the 595 and 602-nm light had more curliness near the lateral veins. However, they were relatively smooth when grown under the 613 and 633-nm light. Interestingly, purple pigmentation was only observed on the lettuce leaves grown under the 602-nm light treatment as shown in Fig. 6.

[0058] Table 2 shows the whole plant photosynthetic rates and biomass data for the lettuce plants under the peak LED wavelengths of the 595, 602, 613, and 633 nm. The highest photosynthetic rate was observed at the 602-nm light treatment, followed by the 595, 633, and 613-nm light treatments. The biomass yields (fresh and dry mass) were the highest under the 602-nm light treatment, followed by the 595, 633 and 613-nm light treatments. The same trend was observed for the photosynthetic rate results, but the differences in biomass under different light treatments were significant. The highest biomass yield observed under the 602-nm light treatment was nearly fourfold higher than the lowest biomass yield observed under the 613-nm light treatments, whereas the difference in photosynthetic rate between the 602 and 613-nm light treatments was only ~20 %. Moreover, shifting the wavelength from 633 to 613 nm and from 602 to 613 nm resulted in the biomass yield decreasing by approximately ~50 and ~80 %, respectively. Although the plants with the 602-nm light treatment had the highest photosynthetic rates and fresh/dry mass, the largest leaf area were plants grown under the 595-nm light treatments. Fig. 7 highlights the comparative data between the measured whole photosynthetic rates and shoot fresh mass for the lettuce plants under different LED wavelengths. [0059] Table 2. Photosynthetic rates (n=3) and biomass values (n=5) (average ± standard deviation) of the lettuce ( Lactuca sativa, cv. Breen) plants grown under the different LED light treatments.

Photosynthetic

Peak

Shoot fresh Shoot dry _|-ea f _area rate

wavelengths of

LEDs (nm) ^mass O) mass (g) ( ^cm2 )

(pmol m ² sec ¹ )

595 2.66 ± 0.18 ^ab 27.16 ± 5.97 ^c 1.16 ± 0.20 ^e 419.5 ± 230.7 ⁹

602 2.87 ± 0.12 ^a 27.12 ± 5.37 ^c 1.16 ± 0.21 ^e 400.7 ± 132.5 ⁹

613 2.31 ± 0.03 ^b 5.76 ± 3.51 ^d 0.25 ± 0.17 ^f 136.6 ± 44.1 ^h 633 2.34 ± 0.19 ^ab 1 1.99 ± 3.07 ^d 0.50 ± 0.15 ^f 220.3 ± 90.3 ^h

[0060] The higher photosynthetic rates observed in this study under 595 and 602-nm light, compared to 613 and 633-nm light, were not consistent with the findings of action spectra from McCree (1972) and Inada (1976), which both had higher photosynthetic rates at 620 nm than at 600 nm. Comparing the early studies of McCree (1972) and Inada (1976) and the current study, the main differences with respect to light properties were the light intensities and FWHMs used. The light intensity used to construct the action spectra in these early studies was relatively low (Hess than 150 pmol m ² sec ¹ ) (Inada, 1976; McCree, 1972; Nelson and Bugbee, 2014), whereas in this study we used approximately ~250 pmol m ² sec ¹ for all the light treatments. This result indicated that at higher light intensity, photosynthetic activity would not follow the findings of McCree (1972) and Inada (1976). A similar statement was made by Bugbee (2016). It was stated that using these early action spectra data would not be as appropriate as using higher light intensities to predict whole plant photosynthesis. Furthermore, the high photosynthetic rates at 595 and 602 nm were inconsistent with pigment absorption spectra, which are low in the wavelength of 580-600 nm. In this wavelength range, the absorption percentages of the major pigments such as chlorophylls a and b measured in acetone are relatively lower than blue and red wavelengths (Taiz and Zeiger, 2002). Similar studies made using green (500-600 nm) light, which had as low of a pigment absorbance as amber light, showed that supplementing 550-nm light resulted in higher photosynthetic efficiencies than 680-nm light under strong white light provided by a halogen lamp (Terashima et al., 2009), and that 532-nm light had deeper penetration into leaf tissues when compared to 488 or 650-nm light (Brodersen and Vogelmann, 2010). This difference is mostly due to the scattering effects which allow green light to drive photosynthesis in the lower chloroplasts when the green light penetrated leaf tissues. Based on the literature, it is still not clear if the amber light presents a similar effect within the leaf tissue as the 532 and 550-nm light (Brodersen and Vogelmann, 2010; Terashima et al., 2009), but our results indicated that the amber light across 500-800 nm can induce higher whole plant photosynthetic rates than the narrow orange/red light. This may be caused by deeper penetration within leaves and by a wavelength interaction effect among green, amber, and red light, which is similar to the interaction effect of the 550-nm and white light reported by Terashima et al. (2009). Nevertheless, further investigation into the light absorption profile on leaf internal profile using amber light would be of interest.

[0061] Higher fresh mass, dry mass and leaf areas were found under the 595 and 602- nm light treatments compared to the 613 and 633-nm light treatments. For the amber wavelengths, a similar plant productivity was found using HPS lamps with/without sunlight for greenhouse grown tomato (‘Komeett’ FT and‘Starbuck’ FT) (Gajc-Wolska et al., 2013) and lettuce ( Lactuca sativa var. capitata) (Martineau et al., 2012). However, suppressed growth on lettuce ( Lactuca sativa, cv. Grand Rapids) under HPS lamps was reported due to a high portion of amber light (Dougher and Bugbee, 2001). As we compared the light intensities used in these studies, we can see that a maximum irradiance level threshold strongly influenced plant growth under amber light. Suppression of lettuce growth occurred with the presence of a high portion of amber light when using higher light intensity of HPS lamps (200 and 500 pmol m ² sec ¹ ) (Dougher and Bugbee, 2001). On the other hand, the studies that reported a positive result on plant growth occurred with lower light intensities of HPS lamps (80-170 pmol m ² sec ¹ ) (Gajc- Wolska et al., 2013; Martineau et al., 2012). The light intensity used in this study fell between the light intensities reported in the studies mentioned above, which was approximately ~250 pmol m ² sec ¹ . Therefore, when comparing the results from the published studies and the current study, there is indication that within the amber wavelength, plants respond differently according to the irradiance levels of amber light used. Low irradiance levels of amber light will result in higher plant productivity, as presented in our results and the results from Gajc-Wolska et al. (2013) and Martineau et al. (2012), whereas high irradiance levels of amber light will lead to suppression of plant growth and to defense or interference of primary metabolism (Dougher and Bugbee, 2001 ; Loughrin and Kasperbauer, 2001 ; Vanninen et al., 2010).

[0062] Unlike the results of the 595 and 602-nm light treatments, leaf elongation of the lettuce plants with poor leaf development was observed under the 613 and 633-nm light treatment. This result suggests that the 613 and 633-nm light has a similar effect on plant morphology as the deep-red light (Heo et al., 2002; Johkan et al., 2010). Furthermore, although the 613 and 633-nm light induced photosynthetic rates approximately 20 % less than the 595 and 602-nm light treatments, the biomass yield decreased by nearly ~50 and ~80 % when shifting the LED wavelength from 633 to 613 and 602 to 613 nm, respectively. This result indicates that the lettuce growth was strongly influenced by the peak wavelengths of the LED light treatments at high irradiance levels. This result is not consistent with the conclusions made by Cope et al. (2014) and Johkan et al. (2012), who found that wavelength has a much smaller effect on plant growth rates than light intensity. The lack of accordance with our findings might be due to the differences in the levels of PPFD and wavelengths used in these studies. We studied the wavelengths and light intensities between 595-633 nm and 240-260 pmol m ² sec ¹ , respectively, whereas Cope et al. (2014) and Johkan et al. (2012) reported results using white and 510-530 nm LED spectrums, between 200-500 pmol m ² sec ¹ and 100-300 pmol m ² sec ¹ , respectively. This result reinforces the statement that under higher irradiance levels, plants respond differently and the predictions in terms of plant growth and development based on the early findings of the quantum yields may be inappropriate (Bugbee, 2016; Inada, 1976; McCree, 1972).

[0063] Blocking 630-nm light resulted in the largest leaf areas (~ 5 %) and different responses of leaf colorations compared between the 595 and 602-nm light treatments. Sole or supplemental 650-660 nm light enhancing fresh/dry mass gain and leaf expansion has been observed in lettuce plants (Johkan et al., 2010; Shimizu et al., 201 1 ; Son and Oh, 2013). However, no study yet has reported the effect of the sole 630-nm light on lettuce growth or leaf expansion, except for pea seedlings ( Pisum sativum L.) (Wu et al., 2007), Protea cynaroides L. (Wu and Lin, 2012), and poinsettia ( Euphorbia pulcherrima Willd. ex Klotzsch) (Islam et al., 2012). Comparing the morphology of lettuce plants grown under the 602 and 633-nm light treatments (Fig. 5), the plants grown under the 602-nm light treatment were considered as normal morphology with a compact architecture, and 613 and 633-nm light treatments prompted stem elongation with poor leaf development. However, eliminating 630-nm light from the pc-amber spectrum (595- nm light treatments), resulted in plants with expanded structures and larger leaf areas compared to the 602-nm light treatments. This result suggests that the presence of 630- nm light impacts plant growth and morphology, unlike 650-nm light alone which should not have any significant morphological impact (Bula et al., 1991). These results show that removing or adding individual wavelengths of light can have positive, negative or neutral effects on plant growth and also impact plant architectures.

Purple coloration was observed on the leaves grown under the 602-nm light treatment but not under the 595-nm treatment that blocked 630-nm light with the use of the notch filter. Red and purple coloration in fruits and leaves is typically due to anthocyanin, a polyphenolic pigment (Cheng et al., 2014; Swain, 1976). It was observed that anthocyanins accumulation in lettuce plants can be induced by supplementing 373, 455, 460, 476, 505, 658 and 660-nm light under different light sources (HPS lamp, solar light, and white fluorescent lamps (Li and Kubota, 2009; Owen and Lopez, 2015; Samuoliene et al., 2012). However, its accumulation mechanisms and interactions with light signal transduction pathways still need to be understood (Samuoliene et al., 2012). In the present study, we observed that 630-nm light may impact purple coloration (assumed to be anthocyanin content) in lettuce leaves. More validation on various greenhouse crops would help in understanding the effect of the 630-nm light on morphology and purple coloration in lettuce plants.

[0064] Although the differences on the whole plant photosynthetic rates and biomass yields among the LED treatments for the lettuce plants were not proportional, both sets of data showed quadratic response curves with respect to the LED wavelengths (Fig. 7). For the whole plant photosynthetic rates, the young plants were measured instead of the plants grown under the LED treatments for two weeks, due to the limitation of the instruments (LI-COR chamber size and LED light). As young plants grow under the LED lights, different photosynthetic rates are induced and their plant architectures (e.g. leaf area) are impacted. In a long-term plant cultivation, this would result in different light interceptions between plants grown under different light treatments over the plant life (Evers et al., 2009). In this study, observed differences (50-80 %) on biomass yields for the lettuce plants were found between the LED treatments, even though a constant irradiance level was provided over the plant canopies. Such large differences may be induced by the interaction effects among the plant architectures, light interception, and wavelength. For instance, as lettuce plants grow under 595 and 613-nm light treatments, the former wavelength induces higher photosynthetic rates and results in faster leaf expansion rates than the latter, which further results in different light interceptions at the same irradiance level over the plant canopies between the light treatments. Over time, the differences on the plant growth would become larger between the plants, due to the total energy received by the leaves. Lastly, the leaf elongation induced by the 613-nm light made the differences on the light interceptions between 595 and 613-nm, which resulted in the observed difference on the biomass yields.

[0065] From our results using the optical filters to manipulate the LED spectra, the importance of wavelength on plant growth and development was underlined. Currently, lighting studies on plant performance mainly utilize single wavelength LEDs or create mixed wavelength treatments with the standard LED wavelengths for plant growth (e.g. 460 and 655 nm), which is similar to a bottom-up approach. With this approach, the narrow FWHM of LEDs (~20-30 nm) allows to construct and optimize light recipes for plant growth. However, typical LED spectra can cover a range of over 50 nm and even as great as 100 nm for some“narrow” spectrum LEDs. Our results for the 613 and 633-nm light treatments showed the wavelength had a strong effect on plant biomass yield production and morphology. In this scenario, using these typical LED spectra would still result in acquiring wavelength interaction effects on plant development and consequently lead to wrong (or partially wrong) conclusions if only considering their peak wavelengths. This opens the discussion relating to the need of narrower spectrum lighting systems, such as laser diodes for plant lighting experimentations. With the results obtained using the notch filer in this study, it highlights that eliminating the wavelength that has negative effects on plant growth would promote plant development. This method is similar to a top- down approach, which is not previously reported on plant lighting studies. Optimal light recipes for plant growth can be created by removing certain wavelengths, which a new method that currently has not been used in the LED lighting field. Further applications on plant lighting studies with this approach would be of interest.

[0066] This research investigated lettuce growth and photosynthetic performances under amber and orange/red light treatments. The spectra were manipulated using optical filters at the same resultant irradiance level. Our results found positive plant responses for the photosynthetic activity and biomass yield production under 595 and 602-nm light treatments. With the use of a notch filter, we observed that 630-nm light impacts plant development such as leaf growth, plant architectures, and purple coloration. Unlike the 595 and 602-nm light treatments, leaf-elongated plant were observed under the 613 and 632-nm light treatments. Shifting the wavelengths from 602 to 613 nm and from 632 to 613 nm resulted in a biomass yield decrease by approximately ~50 and ~80 %, respectively. The results in this study suggest that under high irradiance levels, predicting plant growth and development according to early findings of the quantum yield data or photosynthetic rates would be inappropriate. It also highlights the necessity of higher wavelength resolution for plant performance investigations. Our results showed that amber (590-610-nm) and orange/red (610-630- nm) lights strongly affected plant growth and development, however, more subsequent validations are required to understand the effects of these wavelengths. [0067] Bergstrand, K.-J., and H. Schussler. 2013. Growth, development and photosynthesis of some horticultural plants as affected by different supplementary lighting technologies. European Journal of Horticultural Science: 1 19-125.

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[001 14] Example 2 - Measurement of the Light Compensation Point and Action Spectra of Lettuce and Tomato Using Light Emitting Diodes

[001 15] The light compensation point and action spectra of intact seedlings of lettuce (Lactuca sativa) and tomato ( Solanum lycopersicum) were measured under 14 different light emitting diode (LED) wavelengths (405 nm to 700 nm) at four different light intensities (0, 30, 60 and 120 pmol m ² sec ¹ ) each. This was done by germinating the plant seedlings in a growth chamber and transferring them into a portable photosynthesis measurement machine where they were subjected to the light treatments and their photosynthetic rates were monitored. Through regression analysis, the average light compensation points over the measured wavelengths (405 to 700 nm) of lettuce and tomato were determined to be 39.56 ± 12.33 and 83.83 ± 15.62 pmol m ² sec ¹ , respectively, which corresponded to the characterization of each species as a sun or shade plant. The action spectra for photosynthetic rates using the LEDs resembled previous studies on blue wavelength responses, which showed localized blue peaks within the range of 430 to 449 nm. However, a peak at 595 nm was observed at all tested light intensities for both plant species, which was not consistent with previous observations. Results from this study can be applied to optimize the output spectrum of electrical lighting systems used in the horticultural industry, and to extend the postharvest storage period of plants.

[001 16] Historically, light emitting diodes (LEDs) have been used as low wattage indicator lights found in electronic devices until technological developments made them much more powerful, compact and efficient (Morrow, 2008). LEDs are slowly replacing incandescent, fluorescent, halogen and high-intensity discharge (HID) lamps in many lighting applications (Tsao et al., 2010). Unlike conventional light sources, LEDs are small and durable, operate with low and variable power, and emit narrow bandwidth emissions that allow significant flexibility in design and enhance spectral control (Massa et al., 2008; Van leperen and Trouwborst, 2007). In commercial greenhouse plant growth, LEDs provide spectral advantages over popular greenhouse lights such as high pressure sodium (HPS) lamps, which have peak emissions in the amber waveband (~590 nm), where photosynthetic utilization efficiency is low when compared to the red and blue portions of the visible spectrum (Lister et al., 2004; McCree, 1972). Manipulating the output spectrum of HPS lighting, particularly in the red portion of the visible spectrum, cannot be readily achieved and significant improvements in HPS spectral efficiency with respect to photosynthetic activity, fruiting and flowering are not expected (Deram et al., 2014; Tamulaitis et al., 2005). In the horticultural industry, unlike HPS lamps, LEDs can be selected based on their emitted wavelengths through knowledge of plant specific data. For instance, specific diodes can be chosen to optimize the rate of photosynthesis, or to induce hormonal and morphological changes (Menard et al., 2005; Mitchell, 2012; Olle and Virsile, 2013). By using the narrow bandwidth spectrum emitted by LEDs, plants can be provided with light that corresponds to the peaks of the photosynthetically active radiation (PAR) curve, where maximum photosynthetic activity occurs at red (660 nm) and blue (450 nm) wavelengths (Bulley et al., 1969; Inada, 1976; McCree, 1972). [001 17] As the leaves of a plant are subject to light after a period of respiration, the photosynthetic carbon dioxide (C0 ₂ ) assimilation eventually reaches a point at which C0 ₂ uptake exactly balances C0 ₂ release. This is the light compensation point, and the rates of photosynthesis and respiration are equal, yielding a net zero gas exchange. Plant specific data on light compensation can be obtained from light response curves (Ogren and Evans, 1993; Taiz and Zeiger, 2010). Light response curves provide data on the plant action spectrum, which is the photosynthetic utilization efficiency across the PAR range, from 400-700 nm. Proper application of the light compensation point and action spectrum can allow for optimization of the wavelengths found in sole-source or supplemental horticultural lighting. Other potential applications can include LEDs being used to extend the post-harvest storage time for plant biomass to maintain quality and suppress growth during shipping and storage (Kubota and Kozai, 1995; Tamulaitis et al., 2005).

[001 18] The light compensation point provides insight on the light limiting step of photosynthesis. Plant photosynthetic activity is regulated by three metabolic steps; RuBisCo activity, regeneration of RuBP and the metabolism of triose phosphate (Taiz and Zeiger, 2010). At low light intensity, prior to the light compensation point, light is the limiting factor for photosynthesis due to the limited regeneration capacity of RuBP. Increases in light intensity above the light compensation point yields a proportional increase in photosynthesis.

[001 19] The light compensation point varies by cultivar, wavelength and surrounding environmental factors, and is dependent on the properties of the plant that impact respiration and photosynthesis (Ashton and Turner, 1979). For example, higher temperatures result in higher respiration rates, and as a consequence, greater light intensity is required to reach the light compensation point (Bazzaz and Carlson, 1982). Factors such as leaf morphology, optical density, spectral quality of the incident light, leaf temperature, ambient air speed, and ambient C0 ₂ concentration are critical factors that need to be measured during photosynthetic rate measurement (Ashton and Turner, 1979). Notable differences in light compensation point exist between plants grown in full sunlight and those grown in the shade. Under natural sunlight, the compensation point of shade plants can vary between 1 and 5 pmol m ² sec ¹ , whereas that of sun plants can be in the range of 10 and 20 pmol m ² sec ¹ (Taiz and Zeiger, 2010). Broadley et al. (2001) and Tsormpatsidis et al. (2010) found that the average light compensation point of various lettuce species was 35.8 pmol-m ² sec ¹ under 400 W HPS lights, and 14.1 pmol-m ² sec ¹ under natural light. Nederhoff and Vegter (1994) measured the light compensation point of tomato using natural light and varying C0 ₂ levels, and obtained values between 23 and 24 pmol-m ² sec ¹ . Albert et al. (2009) estimated the light compensation point for petunia under natural light supplemented with metal halide lighting and found that it varied from 18 to 47 pmol m ² sec ¹ for plants cultivated under low and high natural light levels, respectively. Lieth and Ashton (1961) reported that weather patterns can impact the light compensation point as the light compensation points of the plants decreased from early spring to the onset of senescence in late spring, but that in early summer, there was an average increase of 6.1 pmol-m ² sec ¹ . This change in light compensation in the plant was attributed to changes in dark respiration of the measured plants. However, changes in light compensation point have exceptions in adverse weather conditions, such as severe frosts, when the compensation point increases greatly without any corresponding increase in the dark respiration rate (Pavletic and Lieth, 1958). This peculiarity can be explained by the fact that in some sensitive plants, frosts and the accompanying wilting can reduce photosynthesis and increase respiration (Levitt, 1956).

[00120] The action spectrum of a plant is influenced by the absorption spectra of photosynthetic pigments, mainly chlorophylls and carotenoids (notably lutein, b-carotene, zeaxanthin and lycopene) (Lokstein and Grimm, 2013). The plant action spectra depend on the concentrations, composition, activity and combined absorption spectrum of their pigments, as well as the water content, internal structure, thickness and surface optical properties of the leaves (McCree, 1972; Taiz and Zeiger, 2010; Vogelmann, 1993). The action spectrum is directly related to light intensity, which is shown by the hyperbolic relationship between light intensity and gross photosynthetic rate, depending on wavelength (McCree, 1972). This relationship suggests that the distribution of the action spectrum is dependent on irradiance, which supports research that indicates the shifting of peak wavelength response is possible as light intensities increase (Koster and Heber, 1982). Peak absorption in plant pigments can shift up to 38 nm depending on a plant’s surrounding environmental conditions, including irradiance (Heber and Shuvalov, 2005).

[00121] The most comprehensive plant action spectrum data set to date was measured by McCree (1972), who observed action spectrum peaks from 450, 500, and 675 nm when the action spectra of 22 plant species were measured and averaged using cut leaves. The 450 and 675 nm peaks corresponded strongly to the absorption of chlorophyll a and b, and to the peaks found in other action spectrum experiments (Avers, 1986; Bulley et al., 1969; Inada, 1976; McCree, 1972). However, these experiments were conducted under either fixed irradiance level (Bulley et al., 1969; Inada, 1976) or at the same photosynthetic rates (McCree, 1972), and therefore the response of action spectrum to varying light intensities is still unknown. Furthermore, the bandwidths of the monochromatic light obtained from the monochromators in these early experiments were 25 nm or greater, which can explain the peak wavelength shift seen in the results from these studies. For instance, in some work, the difference between the measured peaks was between 13 and 17 nm in the blue and red wavelengths, which was within the reported experimental error (Balegh and Biddulph, 1970; Bulley et al., 1969; Hoover, 1937; McCree, 1972).

[00122] The focus of the present research was to create a light response curve for lettuce ( Lactuca sativa) and tomato ( Solarium lycopersicum) by collecting light compensation and plant action spectrum data under multiple different wavelengths and light intensities, using narrow bandwidth LED lights (20 nm). Although light compensation data is available for lettuce and tomato under natural and artificial greenhouse lighting, there is no reported research on light compensation under LED lights. Moreover, plant action spectra data for individual plant species at multiple light intensities and wavelengths has not yet been reported, therefore the effect of light intensity on a plant’s action spectrum is also unknown. This research aims to address these gaps.

[00123] The light treatments were provided with 14 monochromatic LED arrays, each with a distinct peak wavelength. The 14 prototype LED arrays had the following centroid wavelengths: 405 nm (LedEngin, San Jose, CA), 419 nm (Norlux, Stokke, Norway), 430 nm (Marubeni, Japan), 449 nm, 470 nm, 501 nm, 527 nm (Phillips-Lumileds, San Jose, CA), 575 nm (Marubeni, Japan), 595 nm, 624 nm, 633 nm (Phillips-Lumileds, San Jose, CA), 661 nm (LedEngin, San Jose, CA), 680 nm and 700 nm (Marubeni, Japan). Each LED array exhibited similar bandwidths (full width at half maximum) of 20 nm. The test light intensities were 30, 60 and 120 pmol m ² sec ¹ . Due to weak carrier confinement in the semiconductor materials used to manufacture the 575 nm LEDs (Ill-phosphide and Ill-nitride) (Lafont et al., 2012), high intensity (> 30 pmol m ² sec ¹ ) in the 575 nm light treatment was not possible. [00124] The LED arrays were current-controlled using a single channel controller, to produce stable light intensities of each wavelength. The controller had a 24 V DC, 2.0 A maximum unit, with current selected and displayed (0-1.92 A DC), and automatic voltage control. Maximum power output of the LED arrays was at 28 W, with optical energy ranging from 0.4 to 5.5 W.

[00125] Measurement of the PAR, wavelength distribution and leaf transmittance spectra for each LED array was performed using a spectroradiometer (PS-300, Apogee, Logan, UT) equipped with a converging lens and optical fiber aperture to minimize sampling error from critical angle losses. Absorption was calculated by measuring total irradiance and subtracting reflectance and transmittance spectrum between 400 and 700 nm measured with a 45-degree reflectance probe (AS-003, Apogee, Logan, UT) using a xenon bulb and the spectroradiometer, respectively. For each treatment, the absorption of three leaves from different plants was measured.

[00126] Seeds of lettuce (‘Buttercrunch’, lot A1 , OSC, Ontario, Canada) and tomato (‘Beefsteak’, lot A1 , OSC, Ontario, Canada) were sown into rockwool growing cubes (Grodan A/S, Dk-2640, Hedehusene, Denmark) and germinated in a growth chamber (E15, Conviron, Winnipeg, Canada) under cool-white fluorescent light (150 pmol m ² sec ¹ , 4200 K, F72T8CW, Osram, Wilmington, MA). The plants were provided half-strength Hoagland nutrient solution as described in Hoagland and Arnon (1950) and exposed to a day/night temperature of 23 ± 1 °C and 21 ± 1 °C, respectively, and photoperiod of 16 h. Plants used for measurements were selected 14 days after germination and based on the emergence of the second true leaf, to allow for a relatively reproducible and symmetrical plant distribution.

[00127] Whole plant photosynthetic measurements were made using a LI-6400XT photosynthesis system (LI-COR, Lincoln, NE) equipped with a 6400-17 Whole Plant Arabidopsis Chamber. Whole seedlings rooted in wet rockwool were placed in the chamber and parafilm was placed on top of the rockwool cube to ensure moisture retention at the root zone. The LI-6400XT controlled relative humidity (50 %), C0 ₂ concentration (400 ppm), airflow rate (400 mI-min ¹ ), and temperature (23 °C). Once the plants were placed inside the chamber and the chamber environment was stabilized, the plants were kept in the dark for 10 minutes to collect baseline dark respiration rates. The test plants were then exposed to a certain light wavelength and intensity, depending on the treatment. The light from the LED arrays was converged using a Fresnel lens (focal length of 17.8 cm) and reflected perpendicular to the LICOR aperture with a flat circular mirror (diameter of 7 cm) orientated at a 45-degree tilt with respect to the LED array. Light maps over the chamber aperture (external dimensions 7 cm diameter by 2.0 cm height) (LICOR, Lincoln, NE) were made before using each LED array. The irradiance varied from 2 to 4 % (LED array dependent) from the mean light intensity of eight sampling points over the chamber aperture. The order of wavelengths and light intensities tested was randomized to minimize the potential of interaction effects between wavelengths and between light intensities. Photosynthetic rate was recorded for five minutes after photosynthetic rates had stabilized. From our preliminary test on photosynthetic rates under different wavelength and light intensity exposures, the stabilization of photosynthetic rates often occurred after 20 minutes of light exposure and was maintained over a ~35 mins and 3 hours periods, as illustrated in Fig. 8. At least three replicates were obtained per plant species for each wavelength. Leaf areas were determined by taking a digital image of leaves and using software Image J (Bethesda, MD, USA) to determine photosynthetic rates on a per unit leaf area basis.

[00128] The data was analyzed using SAS (Cary, NC, USA) using proc Mixed (p > 0.05). The significance of random effect parameters was determined by running proc Mixed with and without random effect parameters and comparing the Bayesian Information Criterion (BIC). If the BIC criterion for the model without the random effect parameter was less than or equal to the BIC criterion for the model with the random effect parameter, the random effect parameter was considered not statistically significant.

[00129] Regression analysis was conducted using the GLM (generalized linear model) procedure of SPSS (Chicago, ILd). The relationship between experimental dependent variables and treatments was estimated by regression analysis to determine the light compensation points. Orthogonal polynomials were used to study changes associated with treatments by partitioning 64 the sums of squares into components associated with linear and quadratic terms. Standard-error bars are presented and correspond to a confidence of 95 % using the Tukey- Kramer’s method.

[00130] Leaf absorption spectra of lettuce and tomato were shown in Fig. 9. The average absorptions are 74.46 ± 14.30% and 87.39 ± 5.93% for lettuce and tomato, respectively. [00131] Fig. 10 shows comparative data per plant species showing changes in their light compensation points (determined through a linear regression analysis), depending on light wavelength.

[00132] The average light compensation point was 39.6 ± 12.3 and 83.8 ± 15.6 pmol-m ² sec ¹ for lettuce and tomato seedlings, respectively. The resulting R ² of the linear equation for all test wavelength varied between 0.94 and 0.99.

[00133] The action spectra of lettuce showed fluctuating maxima and localized peaks with greater variation between light intensities, in comparison to the action spectra of tomato (Fig. 1 1A-B). At all three light intensities, tomato had peak photosynthetic responses at 430 and 595 nm with shoulders at 470 and 633 nm at certain light intensities. The action spectrum of lettuce at 120 pmol-m ² · sec ¹ showed that the maximum photosynthetic response occurred at 595 nm, with a secondary peak at 430 nm. At 60 pmol m ² sec ¹ , maximum photosynthetic response was between 595 and 624 nm with a secondary peak at 449 nm. At 30 pmol m ² sec ¹ , two maximum photosynthetic responses were at 449 and 595 nm with shoulders at 501 and 633 nm. As irradiance increased from 30 to 120 pmol m ² sec ¹ , the shift in blue peaks was approximately 20 nm, and no obvious shift was observed in red wavelength. There is no data at 575 nm for both plant species due to the limitation of the LED arrays in reaching light levels above 30 pmol m ² sec ¹ . These results compare well with measurements from previous work, however, the peak response shifted from 675 nm to 595 nm in the red spectrum (Bulley et al., 1969; Hoover, 1937; McCree, 1972).

[00134] When the photosynthetic rate data was analyzed in proc Mixed in SAS (Table 1), wavelength and irradiance were both statistically significant for lettuce and tomato. As for the irradiance x wavelength interaction effect, it was statistically significant for both lettuce and tomato. The individual plant or replicate effect was not statistically significant and was removed from the statistical model.

[00135] Table 1. Summary of the statistical analysis of the single and combined effect of wavelength and species on photosynthetic rate in lettuce ( Lactuca sativa) and tomato (. Solarium lycopersicum) using SAS proc Mixed (P > 0.05).

Effect Pr > F Seedlings lettuce tomato species <0.0001 <0.0001 wavelength <0.0268 <0.0001 species x wavelength <0.0001 <0.0268

[00136] A comparison was carried out to identify the relationship between the light compensation point and photosynthetic activity. The light compensation point reached its peak between 501 and 527 nm, where photosynthetic activity was at its lowest value (0.14 pmol m ² sec ¹ at 527 nm) for tomato (Figs. 12A-B). The same relationship was also observed in lettuce (Fig. 12B) where the peaks in photosynthetic activity corresponded to the troughs in light compensation point. The relationship and resulting R ² values between the photosynthetic activity and light compensation points were -0.79 and -0.52 for tomato and lettuce, respectively. Therefore, the relative photosynthetic efficiency (relative to a particular wavelength at a given irradiance) is strongly reflected in increased photosynthetic activity and decreased light compensation point.

[00137] The results highlighted noticeable differences between the plant species. The average light compensation point was 39.6 ± 12.3 and 83.8 ± 15.6 pmol m ² sec ¹ for lettuce and tomato seedlings, respectively. There was significant variation between this data and previous published results (where light compensation points were lower) (Nederhoff and Vegter, 1994; Xiaoying et al., 2012), which may be since older studies were based on equipment with poor resolution, and also the measurements may have been made with a leaf clip, in which case it is not representative of whole plant data. As a shade plant, lettuce had a much lower light compensation point in comparison to tomato (which are usually considered sun plants). This is due to the adaptation of shade plants to survive in light limited environments through lower respiration rates, allowing for the slightest net photosynthesis to bring the net C0 ₂ exchange to zero. As reported in previous studies, the light compensation point is affected by photosynthetic activity (Bazzaz and Carlson, 1982; Taiz and Zeiger, 2010).

[00138] The action spectra produced noticeable similarities between the two plant species. The action spectra were characterized by localized blue and amber peaks within the range of 430 to 449 nm and 595 nm, respectively. The revealed peak ranges in the blue spectrum are consistent with corn and radish (Bulley et al., 1969), red kidney beans (Balegh and Biddulph, 1970), wheat (Hoover, 1937) and the McCree action spectrum (including tomato and lettuce (McCree, 1972)). However, the 595 nm peak was observed in the action spectra at all light intensities for both plant species, which was not consistent with action spectra observations made by McCree (1972), Balegh and Biddulph (1970) and Bulley et al. (1969), who observed a peak at 675 nm. This might be due to the difference in the bandwidths of monochromatic light and the experimental designs between previous measurements and this study. Narrower light spectra for action spectrum and photosynthetic rate measurements lead to more accurate plant specific data and more precise observations of wavelength effects. The 595-nm peak was also inconsistent with the light absorption spectra for these plant species (Fig. 9) as all plants had relatively low absorption at 595 nm when compared to the absorption percentages in the blue and red spectra. Previous research on light compensation points by Bazzaz and Carlson (1982), Taiz and Zeiger (2010), Broadley et al. (2001), Tsormpatsidis et al. (2010) and others, has shown that the light compensation for the same plant species is different under sunlight when compared to electrical light sources. This may be because of the presence of a strong amber waveband in sunlight, which supports the observation of the action spectrum peak at 595 nm. Although there is no action spectra data (short term measurements) supporting our finding, a few studies regarding long term plant cultivation have shown that plant yield under an amber-biased spectrum was the same or better than the under blue and red LED spectra (Bergstrand and Schussler, 2013; Dueck et al., 201 1 ; Gajc-Wolska et al., 2013; Gomez et al., 2013; Martineau et al., 2012). Further investigations on the effect of amber spectrum on plant photosynthetic activity and growth would be of interest.

[00139] As for the blue peaks, a peak shift was observed in the tomato seedlings, but not in the lettuce seedlings. For the tomato seedlings, the blue peaks shifted to the next LED array color (toward a smaller wavelength) as the light intensity increased. Similar shifts were observed in experiments conducted by Heber and Shuvalov (2005) who observed shifts by as much as 38 nm in absorption spectra, however, these were tested in acetone. These shifted blue peaks were not consistent with the action spectrum measured in previous work (Bulley et al., 1969; Inada, 1976; McCree, 1972). The wavelength dependent hyperbolic relationship between irradiance and photosynthetic activity estimated by McCree (1972) predicts a consistent positive or negative shift in action spectra peaks as irradiance increases. This suggests that the 25 nm (or less) shifts in blue and red peaks may be a result of noise, particularly since the bandwidth of the LED light treatments was ~20 nm (full width at half maximum).

[00140] The light compensation point of lettuce and tomato seedlings were different. Not only was the light compensation point different according to the species, but also according to light wavelength and intensity. These results were considerably different than those from previous studies, obtained from wide bandwidth light sources. The measured light compensation points using LED light sources correlated to the categorization of the plant as either a sun or shade species, with lettuce (shade plant) at 39.6 ± 12.3 pmol m ² sec ¹ and tomato (sun plant) at 83.8 ± 15.6 pmol nT ² sec ¹ . It was also shown that light compensation point is negatively correlated to photosynthetic activity in both lettuce and tomato. Localized blue and amber peaks in the action spectra occurred between 430 nm to 449 nm and at 595 nm. Further research should investigate the peak observed at 595 nm.

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[00181] Example 3 - Growth of Lettuce under Monochromatic and Combination Blue, Amber, and Red Light-emitting Diodes at High Photosynthetic Photon Flux Densities

[00182] Light-emitting diodes (LEDs) offer many advantages over traditional forms of horticultural lighting. Notably, LEDs offer the ability to vary light intensity and select specific wavelengths for targeted plant responses. The combined effects of varying light wavelengths and intensities on plant growth remain misinterpreted due to limited light configurations. Most previous work involves the use of relatively low photosynthetic photo flux densities (PPFDs) (< 800 pmol m ² sec ¹ ). The objective of this study was to investigate the effects of monochromatic and combined red (635 nm), blue (445 nm), and phosphor-converted (PC) amber (595 nm) light-emitting diode (LED) light on lettuce growth ( Lactuca sativa, cv. Breen) up to a PPFD of ~1300 pmol m ² sec ¹ . Two-week old lettuce plants were subjected to four light treatments lasting 18 days and consisting of 28 different PPFDs: red from 39 to 1333 pmol m ² sec ¹ , blue from 33 to 1 194 pmol nT ² sec ¹ , amber from 59 to 1392 pmol m ² sec ¹ , and red-blue-amber RBA from 39 to 1321 pmol m ² sec ¹ . The plants were grown hydroponically under a 16-h photoperiod. The largest fresh mass (FM) was 39.6 g under the amber light at 695 pmol m ² sec ¹ with similar yield occurring at PPFDs ranging from 500 to 800 pmol m ² sec ¹ . The second largest yield was 29.7 g FM under the red light at 660 pmol m ² sec ¹ , followed by the RBA light at 1063 pmol m ² sec ¹ (22.6 g FM) and blue light (21.0 g FM) at 1 194 pmol rn ² sec ¹ . The plants grown using the amber light resulted in 33.3 % more yield than the next closest wavelength (red) at the same PPFD, but under both red and amber light alone, high PPFDs (> ~700 pmol m ² sec ¹ ) led to growth suppression. Blue light was the least productive of the treatments, but showed no suppression at high intensities and provided red pigmentation. The treatment consisting of the combination of red, blue and amber (RBA) light resulted in slightly greater yield in comparison to the blue light treatment and showed no growth suppression under high intensities. Various degrees of pigmentation occurred under each light treatments, but bleaching of pigments were only observed under the amber light at PPFDs above 1000 pmol m ² sec ¹ . [00183] Light properties (wavelength and intensity) are the primary factors contributing to plant growth and development due to their fundamental impact on photosynthetic performances (Fan et al., 2013; Hernandez and Kubota, 2013; Long et al., 1994). Hence, the use of supplemental lighting is widespread in the horticultural industry to ensure optimal plant productivity (Liu, 2012; Singh et al., 2015). The most common supplemental lighting systems are high pressure sodium (HPS) lamps that have an output peak in the amber range of wavelengths (590-620 nm) but lack in the blue and red spectra (Mitchell et al., 2015; Nelson and Bugbee, 2014), and more recently, light emitting diodes (LEDs) that offer monochromatic blue (400 to 500 nm) or red (600 to 700 nm) light (Gomez et al., 2013; Goto, 2003; Wheeler, 2008).

[00184] HPS lamps are commonly used in the greenhouse industry for supplemental and sole-source lighting (Bergstrand and Schussler, 2013; Moe, 1994; Van leperen and Trouwborst, 2007). HPS lamps offer sufficient light intensity over large plant-growing areas and increase greenhouse temperature through their high heat emission, which is favorable in colder climates (Brault et al., 1989; Gomez et al., 2013; Nelson and Bugbee, 2014). Moreover, they are economically viable because they are a mass-produced light source used in many other industries (Gomez et al., 2013). HPS lamps have some critical drawbacks: high heat emission [> 200 °C, (Brault et al., 1989)] limits lamp proximity to the plants, thus limiting high light intensity growth conditions, and the nature of the lamps prevents control of light intensity via current or voltage. Greenhouse growers have therefore started to use red and blue LED lighting systems, due to their controllability of light intensity and promising developments in terms of low heat emission and high efficiency (Massa et al., 2008; Morrow, 2008).

[00185] The use of LED lighting has shown positive impacts on plant growth (Mitchell, 2015; Olle and Virsile, 2013; Singh et al., 2015; Yeh and Chung, 2009). Some of the first LEDs to be deployed in horticulture research were in the red spectrum (600-660 nm) (Bula et al., 1991 ; Mitchell et al., 2015), and therefore numerous early studies reported the effects of red to far-red (700 to 800 nm) LED light, in conjunction with low intensity blue (400 to 500 nm) and green light (500 to 600 nm), on plant growth (Brown et al., 1995; Goins et al., 1997; Kim et al., 2004; Morrow, 2008). Subsequently, LEDs became available in a variety of wavebands ranging from the ultraviolet-C (~250 nm) to the near- infrared range (~1000 nm), including many useful wavelengths to the horticulture industry (Mitchell et al., 2015; Olle and Virsile, 2013). With ongoing and rapidly progressing improvements in terms of LED efficiencies and availabilities, users now have a greater amount of control on the light intensity and wavelengths offered to their crops (Bula et al., 1991 ; Gomez et al., 2013; Haitz and Tsao, 201 1 ; Morrow, 2008; Tennessen et al., 1994). To date, one of the focal points of horticultural LED research has been the investigation of the effect of the red to blue LED ratio on plant growth (Deram et al., 2014; Fan et al., 2013; Martineau et al., 2012; Son and Oh, 2013). Generally, the most electrically efficient LED colors are blue (49%), red (32%), and cool white LEDs (33%) (Nelson and Bugbee, 2014).

[00186] Although studies have shown the positive impacts of monochromatic and combined LED light on different vegetables and flowering plants (Goins et al., 1997; Hernandez and Kubota, 2015; Hogewoning et al., 2010; Massa et al., 2015), HPS lamps are still the preferred choice in the horticultural industry. The effects of particular LED wavelengths and light intensities on plant growth are influenced by plant species and growth stages (Olle and Virsile, 2013). Additionally, most existing studies have involved treatments of red and blue LED ratios, conducted at fixed intensities, or otherwise called photosynthetic photon flux densities (PPFDs) (Son and Oh, 2013; Wang et al., 2016; Yang et al., 201 1). Other studies have considered fixed wavelength ratios under various PPFDs, but rarely investigate treatments that surpass 550 pmol m ² sec ¹ (Avercheva et al., 2014; Fan et al., 2013). Light intensities from HPS, LEDs or a combination thereof, beyond a PPFD of 1000 pmol m ² sec ¹ , have seldom been reported in the literature for plant growth experimentation. Under these various scenarios, the optimal light configurations observed only represent partial light optimization, in terms of either ratios or PPFD.

[00187] The objective of the present study was to evaluate the effects of monochromatic red (635 nm), blue (445 nm) and phosphor-converted (PC) amber (595 nm), and a combination thereof LED light on lettuce growth ( Lactuca sativa, cv. Breen), with a customized LED system capable of supplying a maximum of ~1300 pmol m ² sec ¹ under two light configurations: (1) single wavelengths at a variety of PPFDs, and (2) multiple combined wavelengths, or red-blue-amber (RBA), at a variety of ratios and PPFDs. Biomass yield (fresh and dry mass), morphology (total leaf area, leaf shape, and leaf colour), and SPAD values were obtained for all treatments. [00188] All experiments were conducted at the Macdonald Campus of McGill University in the Phytorium facilities (Ste-Anne-de-Bellevue, QC, Canada). Seeds of lettuce (L sativa, cv. Breen; pelleted MT0 OG, Johnny’s Selected Seeds, Winslow, ME) were germinated in rockwool cubes (25 by 25 by 30 mm, Grodan, Etobicoke, ON, Canada) in germination trays (0.28 by 0.54 m, Mondi Products, Vancouver, BC, Canada) filled with 2 L half strength Hoagland’s solution (Hoagland and Arnon, 1950). The seeds were germinated and grown in a growth chamber (TC30, Conviron Controlled Environment Ltd, Winnipeg, Canada) under a PPFD of 70 pmol m ² sec ¹ supplied by cool white fluorescent lamps (4200 K, F72T8CW OSRAM Sylvania Inc., Wilmington, MA), with a photoperiod of 16 h. After 2 weeks, when the lettuce plants developed their first two true leaves (4 to 6 mm in length) they were transplanted into an adjacent growth chamber (TC30, Conviron Controlled Environment Ltd., Winnipeg, Canada) equipped with a nutrient loop systems and the experimental LED lighting systems. The thin film nutrient loop system consisted of four hydroponic channels installed 0.10 m apart (Fig. 13), and each channel provided room for seven plants, spaced 0.14 m from each other. Twenty- eight lettuce plants were grown under the LED light for 18 d after transplanting. A fresh full-strength Hoagland’s solution was provided and replaced every 9 d. The pH (Oakton waterproof pH tester 30, EuTech Instruments, Vernon Hills, IL) and electrical conductivity (EC) (combo EC meter, Hanna instruments, Ann Arbor, Ml) of the nutrient solutions were maintained at 5.94 ± 0.4 and 1.38 ± 0.09 dS m ¹ , respectively. An air pump (Marina 200, Rolf C. Hagen, Baie d’Urfe, QC, Canada) provided oxygenation to the nutrient solution. The environmental conditions in both growth chambers were set to 50 % relative humidity, 20 °C, and ambient carbon dioxide levels.

[00189] The LED light system was comprised of a frame (1.27 by 0.75 by 0.60 m in length, width and height, respectively) that allowed us to secure and adjust the positions of four LED bars, to obtain the desired PPFD within the treatments. Each LED bar (LED Innovation Design, Laval, QC, Canada) was equipped with 200 diodes with nominal wavelengths of 447, 590, and 627 nm, for the blue, PC amber and red light respectively, as reported by the manufacturer (Phillips-Lumileds, San Jose, CA). Spectral distribution measurements were performed using a spectroradiometer (PS-300, Apogee instruments Inc., Logan, UT) and revealed that the centroid wavelengths were in fact 445, 595 and 635 nm (Fig. 14). Centroid wavelength measurements were completed twice throughout the experiment. It was revealed that the PC amber diode provided a wide output spectrum in comparison to the red and blue diodes, and showed a skewed output towards the red spectrum (Fig. 14). A water cooling circulation system (M4100C, Isotemp cooling circulator, Thermo Fisher Scientifics, Waltham, MA) was used to regulate the temperature of the LED bars to 10 °C with a mixture of water and glycol (1 :3 ratio). The LED bars were able to provide a PPFD up to ~1300 pmol m ² sec ¹ in each of the three wavelengths alone, or in combination to the RBA ratios, at a distance of 0.30 m from the diodes. The selection of the specific red and blue wavelengths was made based on typical wavelengths currently used for LED lighting in horticulture. The PC amber LEDs were selected for their ability to supply high PPFD in the amber spectrum as HPS lighting does. The RBA treatment was included in the experiment in an attempt to emulate the spectrum of HPS lighting with an LED system.

[00190] The PPFD delivered by each LED bar was controlled by a current control box using the software Tera-Term pro 2.3 (Ayera Technologies Inc., Modesto, CA). The LED bars were spaced 0.10 m apart, and placed 0.25 m above the hydroponic channels. The PPFD measurements at each plant location were performed using a light meter (LI-250A; LI-COR Inc., Lincoln, NE) with an underwater quantum sensor (LI-192, LI-COR Inc.) (Deram et al., 2014). Prior to the PPFD measurements, the LED bars and the cooling circulator were switched on and stabilized for 5 min. The PPFD measurements were taken at the beginning and end of each treatment replication to confirm a constant PPFD (Table 1). Each light treatment was repeated three times.

[00191] Table 1. Summary of the PPFD measurements for each LED lighting system. Three wavelengths were used and the ratios were measured but not controlled. The average PPFDs from three replications of monochromatic blue, amber and red, and red- blue-amber combination (RBA) are presented.

Full width half

Wavelength (nm) PPFD range (pmol m ² sec ¹ ) Ratio

maximum (nm)

445 (blue) 33.3 ± 0.6 to 1 194.0 ± 3.6 21.5

600 (amber) 58.8 ± 0.1 to 1392.0 ± 2.9 76.3

635 (red) 38.93 ± 0.6 to 1333.3 ± 0.6 22.35

RBA 0.4:0.2:1 to

39.1 ± 0.6 to 1320.8 ± 2.7

(red:blue:amber) 22.7:1.9:1

[00192] After harvesting, the plant fresh mass (FM) and dry mass (DM) were measured using a balance (APX-153, Denver Instruments, Bohemia, NY). To obtain DM, plants were stored at 4 °C for 12 h and then transferred to a freeze drier (Christ Gamma 1-16 LSC, Shrophire, UK) for 48 h. To verify dryness, plants were weighed after 48 h and again after another 12 h, ensuring less than a 5% change. The leaf shape response to the light treatments was measured using the leaves harvested on day 18 from individual plants. The leaf length (LL) and width (LW) were determined using a digital Vernier caliper (Mitutoyo 500, Mitutoyo Canada Inc. Mississauga, ON, Canada). The leaf shape indices (LSI) were calculated by LL divided by LW for each light treatment (Son and Oh, 2013). The average LSI of each plant was plotted against the PPFD received by the individual plant. Leaves from individual plants for each replication were carefully separated on day 18 and measured for total leaf area (TLA) (LI-COR 3100 Leaf Area Meter; Li COR, Lincoln, NE, USA). Chlorophyll content measurements occurred at the center, off the mid vein of the second fully expanded leaf of each plant using a SPAD meter (Chlorophyll Meter SPAD-502Plus, Konica Minolta, Sakai, Osaka, Japan). Individual plants grown under each different light treatment and PPFD were photographed to observe color and morphology (Canon t4i, Tokyo, Japan).

[00193] Analysis of variance and principal component analysis (PCA) were performed on the data using RStudio version 1.1.383 (RStudio, Inc., Boston, MA). Tukey's honest significant difference (HSD) test was used for the multiple comparisons among spectral treatment means from significant one-way analysis of variance (ANOVA) tests (a < 0.05, unless otherwise stated).

[00194] The seeds were germinated and grown in a growth chamber with an average temperature of 20.4 ± 0.5 °C. The mean water temperature of the hydroponic system was stable throughout the experiment at 16.5 ± 0.3 °C.

[00195] An ANOVA clearly revealed that both PPFD and wavelength had significant effects on the FM and DM of the lettuce plants (P<0.001). A follow-up ANOVA revealed that there were significant differences amongst the FM and DM of the four wavelength treatments. The interaction effect of PPFD and wavelength was not considered significant (P = 0.0569). A Tukey HSD test revealed that only amber light provided a significantly different FM and DM in the plants (greater FM and DM). There were no significant differences between the FM and DM measurements of the blue, red and RBA light treatments. [00196] The RBA light treatment, throughout the range of PPFDs (39 to 1321 pmol-m ² -sec ¹ ), had varying ratios of blue, red and amber light. The ratios were normalized to the amber light for analysis. A two-way ANOVA with interaction effect was used to investigate the importance of the red, blue or interaction of both wavelengths on the FM and DM of the lettuce plants. The red ratios in the RBA treatment are associated with significantly different FM and DM (most likely smaller FM and DM, although we cannot confirm). The blue ratios did not significantly alter FM or DM (P = 0.5135 and 0.5018, respectively), and the interaction effect of the red and blue light was not considered significant (P = 0.0896). These results indicated that the relationships between the red ratios and FM or DM do not depend on the blue ratios.

[00197] Through a PCA of the RBA treatment, it was revealed that under the full range of PPFDs (39 to 1321 pmol-m ^sec ¹ ), the FM and DM were both tightly related to total RBA PPFD (regardless of ratio) and to the blue light PPFD (Fig. 15). Although not tightly related in the PCA, red and amber light PPFD had positive but divergent effects on FM and DM. It was revealed visually by a curve fitting analysis that both the red and amber light treatments had suppression effects on FM and DM beyond 660 and 732 pmol-m ² -sec ¹ , respectively. The curve fitting analysis of FM and DM under the blue light did not reveal a suppression of FM and DM, rather it showed a steadily increasing effect beyond 150 pmol m ² sec ¹ . In the RBA treatment, the PPFD supplied by the blue diodes never surpassed 325.1 pmol m ² sec ¹ , while the red and amber diodes supplied well beyond this, and even beyond 1000 pmol m ² sec ¹ at times. While a suppression was observed under high PPFD red and amber light alone, but not under blue light alone, it appears that with the presence of the blue light under the higher RBA PPFDs, no FM or DM suppression occurred.

[00198] Through further PCA of the RBA treatment, where alterations to the range of the data analysed were made, the effects of the components at lower and higher total RBA PPFD were examined. Initially, the PPFD range was limited to 39 to 441 pmol-m ² -sec ¹ (lower range). Under this range, the FM and DM were still tightly related to total RBA PPFD (regardless of ratio), but the blue light PPFD shifted away from the close relationship seen under the full PPFD range, to now show a mutual relationship with the red light PPFD (Fig. 16A). The effect of the amber light remained positive but divergent to both the red and blue light PPFD. Secondly, the PPFD range was limited to 51 1 to 1321 pmol m ² sec ¹ (higher range). Again, the FM and DM were tightly related to total RBA PPFD (regardless of ratio). However, in this higher range, the FM and DM were negatively related to the blue light PPFD (Fig. 16B). It is important to recall however, that this relationship did not have an overall negative effect on FM and DM. The amber and red light PPFDs were again divergent. In the PCA graph, the amber light PPFD eigenvector shifted further away from FM and DM, potentially showing a suppression effect in the higher RBA PPFD range.

[00199] The lettuce plants grown under various PPFDs of the four light treatments showed recognisable differences in FM and DM (Figs. 17 and 18). A significant increase in FM and DM was observed under the amber light, followed by the red then RBA light, and finally by the blue light. No significant differences, however, were observed between the FM and DM of the plants subject to the red, blue and RBA lights. Under the amber light, the largest FM was 39.6 g observed at 695 pmol m ² sec ¹ . The FM and DM responses to the red and amber light treatments over the full PPFD range showed quadratic relationships with PPFD, with R ^{2 =} 0.96 and 0.85, respectively. FM and DM increased rapidly with increasing PPFD, levelled out at mid-range PPFDs, and severely decreased at high PPFDs. The responses to PPFD under the blue and RBA light treatments over the full PPFD range were fitted to a power regression curve, with R ^{2 =} 0.96 and 0.94, respectively. Both FM and DM increased rapidly with increasing PPFD, but were never suppressed in the higher PPFD range (>1000 pmol m ² sec ¹ ).

[00200] Under the red light treatment, FM increased with increasing PPFD, with the largest FM measurements ranging from 19.2 to 29.7 g at PPFDs between 231 to 997 pmol m ² sec ¹ . The largest FM measurement was found at 660 pmol m ² sec ¹ . PPFDs between 39 to 182 pmol m ² sec ¹ and over 1000 pmol m ² sec ¹ resulted in less FM. Under the red light treatments, DM significantly increased under PPFDs ranging from 39 to 997 pmol m ² sec ¹ , but decreased at values over 1000 pmol m ² sec 1 , and the resulting trend was fitted to a quadratic curve (R ² = 0.96) as seen in Fig. 17.

[00201] The plants grown under the blue light treatment showed an initial rapid increase in FM and DM with an increase in PPFD, followed by a subsequent slower and consistent increase at higher PPFDs. The FM under the blue light treatment was the lowest amongst all treatments, with a maximum at 21.0 g. However, the resulting DM under the blue light treatment eventually surpassed the DM results of both the red and RBA light treatments, with a maximum of 1.75 g. The resulting FM and DM data was fitted to a power regression curve (R ² = 0.96). Unlike the plants under the amber and red light treatments, the FM and DM did not decrease at higher PPFDs.

[00202] The plants grown under the amber light showed an increase of FM under PPFDs ranging from 59 to 223 pmol-m ² · sec ¹ , and in DM from 59 to 530 pmol m ² sec ¹ . No observable difference was noticed under the amber light between 281 to 882 pmol-m ² -sec ¹ for FM, and 552 to 732 pmol-m ² · sec ¹ for DM. Decreased FM was observed at PPFDs over 1000 pmol m ² sec ¹ , and decreased DM was observed between 882 to 1050 pmol m ² sec ¹ . The plants grown under the amber light showed little growth at PPFDs above 1 100 pmol m ² sec ¹ . The resulting trends were fitted to quadratic curves (R ² = 0.85 and 0.93, for FM and DM respectively) as seen in Fig. 17. Under the amber light, at PPFDs ranging from 500 to 700 pmol m ² sec ¹ , FM was 33.3% larger than under the red light at the same PPFDs.

[00203] Similar to the plants under the blue light treatment, the plants grown under RBA light treatment showed an initial rapid increase in FM and DM with an increase in PPFD, followed by a subsequent slower and consistent increase at higher PPFDs. The FM and DM measurements were fitted to a power regression curve (R ² = 0.94). The maximum FM under the RBA combination was 22.56 g at a PPFD of 1064 pmol m ² sec ¹ . The plants showed increased FM and DM under PPFDs ranging from 39 to 1064 pmol-m ² sec ¹ and a decrease in DM was observed in plants treated with PPFDs above 1 164 pmol m ² sec ¹ . Although noticeable (Fig. 17), this decrease was not considered significant.

[00204] The LSI of the lettuce plants was significantly influenced by both the PPFD and wavelength of each treatment (P<0.001), but not by the interaction effect of PPFD and wavelength. Only the LSI of the plants from PPFDs ranging from 33 to 100 pmol m ² -sec

¹ differed significantly from the rest of the results. Under the amber and red light treatments, the LSI of the plants decreased as the PPFD increased to ~550 pmol-m

² sec ¹ , and then increased again until reaching >1000 pmol m ² sec ¹ , creating a shoulder in the trend curves (Fig. 19). These results were fitted to polynomial curves (R ² = 0.96 and 0.89, for amber and red, respectively). Under the blue and RBA treatments, the LSI of the plants decreased as the PPFD increased (Fig. 19), and the results were fitted to power regression curves (R ² = 0.85 and 0.96, respectively). It can be seen that the LSI of the plants under the blue light treatments show a shoulder at ~900 pmol-m ² -sec ¹ , but the resulting third order polynomial regression was not as significant as under the power regression. Statistically, the LSI of the plants under the red and amber treatments were comparable and significantly different from the LSI of the plants under the blue and RBA treatments.

[00205] The TLA of the lettuce plants was significantly influenced by both the PPFD and wavelength of each treatment (P<0.001), and by the interaction effect of PPFD and wavelength. The TLA of the plants increased with increasing PPFD under the blue and the RBA light treatments (Fig. 20A). Under the red light treatment, the TLA initially increased as the PPFD increased, but a local maximum and subsequent local valley were observed at 335 and 995 pmol m ² sec ¹ , respectively. Among all the light treatments, the largest TLA was 402.5 cm ² under the amber light at 530 pmol-m ² - sec ¹ . Similar to the FM and DM measurements obtained from the amber light treatment, the overall greatest TLA values were acquired from PPFDs ranging from 281 to 732 pmol-m ² -sec ¹ . Under the blue light treatment, the largest TLA was 353.3 cm ² at 975 pmol-m ² -sec ¹ . Under the RBA treatment, the largest TLA was 258.1 cm ² at the highest PPFD (1321 pmol m ² sec ¹ ). Lastly, under the blue treatment, a rapid increase occurred under lower PPFDs, and very little change in TLA occurred beyond 666 pmol-m ² -sec ¹ . Upon observation of the relationship between TLA and FM (Fig. 20B), it can be seen that under the blue light treatment, the TLA was capped at ~350 cm ² regardless of an ever- increasing FM, and this occurred at PPFDs beyond 700 pmol-m ² -sec ¹ . Similarly, under the amber treatment, the TLA plateaued at ~400 cm ² at PPFDs beyond 250 pmol-m ² -sec ¹ before decreasing dramatically at PPFDs beyond ~1 100 pmol-m ² -sec ¹ .

[00206] The net chlorophyll content (SPAD reading) of the lettuce plants was significantly influenced by both the PPFD and wavelength (P<0.01 and P<0.0001 , respectively), and by the interaction effect of PPFD and wavelength (P<0.01). The measured chlorophyll content under the blue and RBA treatments showed similar trends, and the results were fitted to a power regression curve (R ² = 0.91 and 0.96, respectively). Under these light treatments, the chlorophyll contents increased rapidly as the PPFDs increased from 30 to ~200 pmol-m ² -sec ¹ . Beyond a PPFD of ~200 pmol-m ² -sec ¹ , the increase in chlorophyll content was much slower. The results from both the blue and RBA treatments suggests that chlorophyll content decreased in the PPFD range of 200 to 500 pmol-m ² -sec ¹ , but a power regression analysis was more significant than a third order polynomial analysis. Under the amber light treatment, the chlorophyll content results showed a similar trend to the other light treatment under low PPFDs (< 500 pmol m ² sec ¹ ), but decreased dramatically at higher PPFDs (> 500 pmol m ² sec ¹ ).

[00207] Plants subjected to different wavelength treatments and PPFDs showed significant differences in morphology and leaf color (Figs. 22-25). Under the red light treatments, longer leaves with shorter width were observed between 38 to 127 pmol-m ² sec 1 , compared to higher PPFDs (Fig. 22). Leaf shape (length and width) and thickness changed beyond 137 pmol m ² sec ¹ . A change in the curliness of the leaves was observed at PPFDs between 137 to 336 pmol m ² sec ¹ . A slight change in leaf color, in terms of red pigmentation, was observed in plants grown at PPFDs above 358 pmol-m ² sec ¹ under the red light. The plants grown under the blue light treatment changed in coloration from green to a dark purple (red pigmentation), and this began at a PPFD of 1 16 pmol m ² sec ¹ . This change in coloration was observed without any physiological damage to the plants in all three replications (Fig. 23). Plants grown under the amber light showed a slightly different physiological and morphological appearance, in comparison to the three other treatments. Longer leaves with smaller width were observed in plants grown under 100 pmol-m ² · sec ¹ . As the PPFD increased, the curliness and leaf thickness increased without a change in the leaf color up to 202 pmol m ² sec ¹ . When the PPFD surpassed 208 pmol m ² sec ¹ but remained below 530 pmol m ² sec ¹ , the leaf color changed to green with slight purple colorations. Beyond 468 pmol m ² sec ¹ , the lettuce plants gradually suffered from bleaching, starting from the lower leaves, upwards, until a PPFD of 1 176 pmol m ² sec ¹ and beyond was reached, where the entirety of the plants became pale yellow. At this point (PPFD > 1 176 pmol-m ² sec ¹ ), the plants showed a significant decrease in chlorophyll content (photo- bleaching), growth and development (Fig. 24). No bleaching was observed under the blue and RBA treatments, but minute amounts of bleaching were observed under the red light treatment. Different ratios within the RBA treatment, starting from 0.4:0.2:1 (39 pmol m ² -sec ¹ ) to 22.7:1.9:1 (1321 pmol m ² sec ¹ ) showed significant variations in plant color, shape and growth (Fig. 25), but followed similar trends in terms of leaf color change (green to dark purple) as seen under the blue LED light treatments.

[00208] Light energy is the driver of photosynthesis and its level of intensity, or PPFD, plays an important role in deciding the quality and morphology of the plants (Bugbee, 2016; Franklin et al., 2004; Hoenecke et al., 1992; Kim et al., 2004; Ouzounis et al., 2016). In the present study, the highest lettuce plant FM and DM were observed under the amber light treatment, followed by the red, RBA and then the blue treatments. Such results in terms of FM and DM indicate that the photosynthetic reaction centers can readily harvest energy from the amber light at PPFDs below 695 pmol m ² sec ¹ more efficiently than from any other light treatments in this study. As the PPFD surpassed 800 pmol m ² sec ¹ however, the FM and DM decreased dramatically. The specific reason for this response to amber light, in lettuce, is not well known and has rarely been reported or discussed in the literature. Different wavelengths have different quantum yields in terms of carbon dioxide fixation, and red light (600 to 640 nm) has the highest quantum yield, whereas blue and green light (400 to 570 nm) are considerably less efficient in driving photosynthesis (Evans, 1987; Inada, 1976; McCree, 1972). The spectrum of the amber light used in this experiment was red-biased, which could help to explain strong growth that we observed, but the peak of the spectral distribution remained below 600 nm. Kim et al. (2004) reported that the addition of 24% green light (500 to 600 nm) to red and blue LEDs (RGB treatment) enhanced plant growth. However, most previous work that considered growth responses to the‘yellow’ spectrum report a suppression of FM and DM. A study published by Dougher and Bugbee (2001) examined lettuce subjected to 580 to 600 nm yellow light, and it was concluded that yellow light indeed suppressed growth. A study by Kong et al. (2015) revealed that the green light (525 to 575 nm) had much higher contributions to FM and DM in Boston lettuce, in comparison to red (625 to 700 nm) and blue (400 to 475 nm) light. The authors categorized wavelengths from 500 to 600 nm as green light and concluded that additional green light promoted plant growth only within the range of 525 to 575 nm, and that the green-yellow range from 575 to 625 nm had relatively very little effect on growth. Red light is known to have maximum absorption in the reaction centers (Hogewoning et al., 2012; McCree, 1972), and a positive effect of red (660 nm) light on growth has been reported in typical greenhouse crops such as tomato ( Solarium lycopersicum), lettuce, and spinach ( Spinacia oleracea) (Heo et al., 2012; Johkan et al., 2012; Son and Oh, 2013). There is little literature on the spectrum around red 640 nm LED light, but our results suggest that 635 nm light promotes plant growth in lettuce at PPFDs below ~600 pmol m ² sec ¹ . Our work also showed that at a PPFD greater than 660 pmol m ² sec ¹ , red light caused a reduction of DM and FM. The reason for the suppression in FM and DM under both amber and red light might be a physical phenomenon caused by excessive light damaging the antenna pigments and reaction centers resulting in photosaturation and ultimately photo-bleaching (Aro et al., 1993; Foyer and Shigeoka, 201 1 ; Murchie and Niyogi, 201 1). Aro et al. (1993) reported that with the destruction of plant pigments due to damage to the photosynthetic system caused by excessive light, the photosynthetic system released highly toxic substances, which ultimately inhibited plant growth. Foyer and Shigeoka (201 1) reported that excessive light energy causes irreversible damage to photosynthetic component by increasing the reactive oxygen species (ROS) to minimize any further damage to the plant.

[00209] Our results showed that the lettuce plants could more readily utilize red and amber light for improving growth and development, when compared to the blue light, at lower PPFDs (40 to 660 pmol m ² sec ¹ ). However, the opposite was true at PPFDs above ~1000 pmol m ² sec ¹ , where the overall growth of the plant was suppressed. The blue and RBA light treatments resulted in the least amount of growth. It is therefore apparent that combining the three wavelengths resulted in roughly an equivalent yield to the blue light treatment, and thus the plant did not benefit from the photosynthetic efficiencies of the amber or red light alone. Growth inhibition of plants grown under blue light was previously observed in cucumber ( Cucumis sativus L. cv. Burpee's Pickier and cv. Lemon), pea ( Pisum sativum L. cv. Hiderma and cv. Mammoth Melting), and zucchini (Cucurbita pepo L. cv. Fordhook) (Cosgrove, 1981). In a more recent study, a reduction in growth was reported in lettuce under high ratios of blue light, at PPFDs of 200 and 500 pmol m ² sec ¹ (Swan and Bugbee, 2017). Li et al. (2017) reported that auxin repression, and other resistant regulated genes are downregulated and microtubule enhancing genes are upregulated in plants grown under blue light and result in the suppression of total plant growth. Some previous studies have shown that a minimal amount of blue light is however necessary to achieve normal photosynthetic operation (Hogewoning et al., 2010; Trouwborst et al., 2010). Our results from the RBA treatment reveal that the presence of blue light counteracted the decrease in FM and DM that would otherwise (no blue light) be brought upon by amber and red light at a high PPFD. This was confirmed through the RBA combination light resulting in a slightly better yield than the blue light alone.

[00210] There exist many photoreceptors that include the red and far-red-absorbing phytochromes and the blue and UV-A-absorbing cryptochromes, phototropins, and ZTL/ADO family of proteins (Devlin et al., 2007; Lariguet and Dunand, 2005). Evidence now strongly predicts the existence of additional, unknown photoreceptors absorbing in other area of the spectrum (Devlin et al., 2007). Based on a general assessment of the FM and DM results, and from the results of the PCA, the photoreceptors of the plant appear to be transferring energy differently depending on the wavelength. More specifically, amber and red light appear to be utilized by the plant through independent photoreceptors from each other. Blue light appears to have its own photoreceptors and becomes dominant under certain light conditions, relative to the amber and red, in an attempt to counteract high PPFD amber and red light suppression effects.

[0021 1] Leaf shape index (LSI) is a morphological characteristic to measure the effects of different wavelengths and PPFDs on plant leaves, and can be a useful qualifier for quality of leafy greens. Our study has shown that the red light treatment enhanced leaf elongation at very low PPFDs (39 to 55 pmol m ² sec ¹ ), followed by the blue, amber and RBA light treatments. Son and Oh (2013) reported that red (655 nm) light enhanced LSI, and supplemental blue (455 nm) light mixed with red (655 nm) light at various ratios decreased LSI, in comparison to the results under control fluorescent and HPS light. Increased blue light PPFD showed higher LSI in our experiment. A reduction in the TLA of the plant decreases the capacity of the plants to intercept light under limited conditions (Hernandez and Kubota, 2016). In our work, plants grown under the amber light initially showed higher TLA than the plants under the blue, red, and RBA lights, as PPFD was increased. However, in a similar pattern to FM and DM, TLA decreased dramatically under the amber light at very high PPFD.

[00212] Overall, these plant responses help to elucidate the effect of light quality on the efficiency of the energy transfer from the photoreceptor antennas to the reaction centers, and the ability to utilize the absorbed photons from different wavelengths (Aro et al., 1993; Foyer and Shigeoka, 201 1 ; Murchie and Niyogi, 201 1).

[00213] Studies have shown a high positive correlation between the amount of chlorophyll content, and plant growth and development (Brougham, 1960; Li et al., 2012; Mizuno et al., 201 1). The absorbance peaks for chlorophyll a and b are observed between 400 to 500 nm and between 620 to 690 nm (Colmano, 1962). Therefore, it was anticipated that the SPAD values in the lettuce plants grown under the blue, red and RBA light treatments would be elevated, in comparison to the amber treatment. As expected, on the 18 ^th day, the largest SPAD values were observed in plants grown under the RBA treatment, followed by the red, blue, and lastly amber light treatments. Exposure to higher intensities of amber light might induce damage to the antennas and the photosynthetic reaction centers causing chronic photoinhibition, resulting in a drastic decrease in SPAD value (Aro et al., 1993; Foyer and Shigeoka, 2011 ; Murchie and Niyogi, 201 1). Schwartzbach (1990) investigated 597 nm green light on the eukaryote Euglena, and it was determined that at saturation, green light (peak at 597 nm) was less effective than blue in the synthesis of chlorophyll.

[00214] The quality, quantity and duration of light are all characteristics that influence the color and pigmentation of plants, and the effects vary amongst cultivars (Anderson et al., 1995). In the present study, differences in leaf morphology and color were observed in plants grown under different wavelengths and PPFDs (Fig. 22-25). When Arabidopsis plants are grown under red light, the leaves curl downwards; in contrast, flat leaves develop under blue light (Inoue et al., 2008; Kozuka et al., 201 1). Our results showed that less leaf curliness was indeed caused by the presence of blue light (Figs. 23 and 25), but amber light also caused curliness at high PPFDs (Fig. 24).

[00215] The pigment anthocyanin is responsible for the red or dark purple appearance of plants (Andersen and Jordheim, 2010). Studies have shown that blue light can promote anthocyanin and phenolic compounds production in different cultivars of lettuce with various intensities and in combination with other light sources and wavelengths (HPS lamps, green light, fluorescent lamps and UV-A) (Giliberto et al., 2005; Li and Kubota, 2009; Ouzounis et al., 2015). Plants grown under the amber light at PPFDs between 59 to 202 pmol m ² sec ¹ showed green coloration, with a minimum of red pigmentation and leaf curliness. The amber light at PPFDs between 208 to 386 pmol rn ² sec ¹ induced a purple coloration on the leaves of the larger plants. A gradual decrease in overall pigmentation (bleaching) was observed in the plants treated with PPFDs between 468 to 1050 pmol m ² sec ¹ . The plant grown under the amber light at PPFDs over 1 176 pmol m ² sec ¹ showed intense bleaching, with a slight yellow hue (very little pigmentation). The pigmentation of the plants grown under the RBA light treatment had a higher level of red to purple coloration across the entire range of PPFDs compared to the plants grown under the monochromatic lights, except for the blue treatment (Figs. 23 and 25). In our work, the addition of the blue light to the amber and red light induced a gradual increase in the red and eventual purple coloration of the plant. [00216] Based on the results from this experiment, future work will be required to investigate the single effects and interaction effects of additional ratios and combined pairs, such as ratios of amber to red and amber to blue light.

[00217] The growth of lettuce plants can be enhanced through the control of light quality (wavelength) and PPFD (intensity). Our results showed that significantly greater plant growth was observed under amber light at PPFDs up to 732 pmol m ² sec ¹ . High PPFDs (> 1000 pmol m ² sec ¹ ) of both amber and red light resulted in a decline in FM and DM. Under blue light and a RBA light, a gradual increase in FM was observed at high PPFDs, up to ~1200 pmol m ² sec ¹ , but the RBA light with different ratios resulted in higher DM than the blue light alone. Overall, the blue light treatment resulted in the lowest plant FM and DM among the treatments. Red pigmentation of the lettuce plants was most prevalent under the RBA and blue lights, with increasing PPFD. PPFDs from 468 to 1392 pmol m ² sec ¹ under the amber light induced bleaching, which was particularly widespread beyond 1050 pmol m ² sec ¹ . Although the results from this study show a promising fate for amber light in horticulture, the mechanism used by the plant to harvest the 595 nm light is not known.

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[00287] It will be understood that the expression“computer” as used herein is not to be interpreted in a limiting manner. It is rather used in a broad sense to generally refer to the combination of some form of one or more processing units and some form of memory system accessible by the processing unit(s). Similarly, the expression 'controller' as used herein is not to be interpreted in a limiting manner but rather in a general sense of a device, or of a system having more than one device, performing the function(s) of controlling one or more device such as an electronic device or an actuator for instance.

[00288] It will be understood that the various functions of a computer or of a controller can be performed by hardware or by a combination of both hardware and software. For example, hardware can include logic gates included as part of a silicon chip of the processor. Software can be in the form of data such as computer-readable instructions stored in the memory system. With respect to a computer, a controller, a processing unit, or a processor chip, the expression“configured to” relates to the presence of hardware or a combination of hardware and software which is operable to perform the associated functions.

[00289] As can be understood, the examples described above and illustrated are intended to be exemplary only. In some embodiments, the light absorbing pigment can be illuminated when it is in a relaxed state as whereas the light absorbing pigment can be illuminated when it is in a transitional state. The term “optical energy” used in this disclosure is defined broadly so as to encompass any type of optical energy such as optical irradiance, optical power, optical luminance, mole of photons, photon flux and the like. The scope is indicated by the appended claims.