1. Introduction
In naturally shaded environments, irradiance/photon fluxes in the blue (400 to 500 nm) and red (600 to 700 nm) regions are relatively reduced while the fluxes of photons in the green (500 to 600 nm) and far-red (700 to 750 nm) regions are relatively enriched. In the photobiology literature, the term light is often used to refer to the photon flux, as in blue light, but this terminology does not describe the discrete nature of photons, which drive photobiological reactions. Additionally, light is closely connected to brightness in human perception of photons, thus photon is a preferable term. Here, the terms blue, green, red and far-red photons refer to photons in the regions that induce blue, green, red or far-red color perception.
Plant developmental responses to a relative increase in far-red have been well studied [
1,
2], with species specific increases in leaf area or stem length empirically described as shade tolerance or shade avoidance. It should be noted that shade tolerance does not often specify an increase in leaf area; instead, morphological changes are discussed in terms of an increase in specific leaf area, which is leaf area divided by leaf mass [
3].
Elevated far-red commonly increases leaf area in controlled environments and this response is beneficial because it increases photon capture [
4]. Increases in stem length in controlled environments are typically considered detrimental. In contrast to the well-characterized responses to far-red, shade-responses to green photons are less well studied.
Early light-emitting diode (LED) fixtures for horticultural applications supplied only blue and red photons. One of the first studies that investigated the effects of adding green to this type of spectrum found that increasing the fraction of green photons from zero to 24% increased leaf area in
Lactuca sativa cv. “Waldmann’s Green” by 31% and increased shoot dry mass by 47% [
5]. This early finding created a sustained interest in considering green photons to horticultural fixtures in order to promote growth [
6,
7]. However, more recent studies have shown contradictory results to Kim et al. [
5] (e.g., [
8]), suggesting the need for a reanalysis of the beneficial effects of green photons on plant growth – especially with continued emerging evidence that green photons act antagonistically against blue photons through the photoreceptor cryptochrome.
Cryptochromes are one of the two most well-studied families of blue photon receptors, and they primarily modulate plant growth through the control of gene expression. The other well studied family of blue photon receptors are phototropins, which primarily modulate plant growth through interactions with membranes [
9,
10]. Sunlight has a relatively high fraction of blue photons, which cause reduced stem and leaf elongation. Studies investigating hypocotyl elongation in
Arabidopsis thaliana mutants have indicated that cryptochromes are the primary photoreceptor influencing the decrease in stem length [
11,
12]. Longer-term studies in pea have corroborated this finding as greenhouse grown plants lacking cryptochrome were 20 to 40% longer than the wild-type plants [
13]. Phototropins play an early role in reducing hypocotyl elongation at the onset of blue photons [
14], but this rapid response does not appear to have a prolonged effect [
12]. The role of these photoreceptors in leaf expansion in mature plants is less well studied.
Studies have typically found that increasing the fraction of blue photons decreases leaf area/plant diameter in the horticultural crops lettuce [
8,
15,
16,
17,
18,
19,
20,
21,
22,
23,
24], cucumber [
25,
26,
27,
28] and tomato [
26,
29], but these effects are not always statistically significant [
17,
18,
26,
28,
30,
31,
32,
33,
34] and occasionally go in the opposite direction [
18]. Leaf area is generally highly correlated with dry mass (yield). Thus, increasing the fraction of blue photons also typically result in decreased dry mass [
8,
15,
16,
20,
21,
22,
24,
25,
26,
28,
35,
36], although this is not always the case [
17,
18,
19,
25,
26,
27,
28,
29,
30,
31,
32,
34,
36,
37,
38,
39,
40,
41,
42]. In addition to reducing leaf area and yield, blue photons have also been shown to reduce stem and petiole length in cucumber and tomato [
25,
26,
28,
32,
33], indicating that the effects of blue photons on manipulating cryptochrome activity in
Arabidopsis extend to these horticultural species. We review 29 studies spanning 19 years on the effects of blue photon fraction in lettuce, cucumber and tomato in
Table S1. Comparisons are complex because the studies were conducted at multiple temperatures, study durations, photon fluxes, photoperiods and cultivars.
Unlike spectral distributions that lack either blue or red photons [
25,
26,
43], growing plants in the absence of green photons does not necessarily induce abnormal morphology [
25,
26]. Studies from the past two decades have suggested that green photons act antagonistically against blue photons to modulate the action of the photoreceptor cryptochrome in a similar manner to the red and far-red antagonism in the photoreceptor phytochrome. In cryptochrome, the flavin adenine dinucleotide (FAD) chromophore has three potential states. The oxidized form, FADox, is abundant in the dark, and upon photon absorbance it converts into the semi-reduced radical state (flavosemiquinone, FADH
o), which is the active form. Photon absorbance by FADH
o induces conversion into the fully reduced (FADH
–) state, which is inactive [
44,
45]. The absorbance spectra of FADox shows a sensitivity to blue photons at about 450 nm, with little absorbance beyond 500 nm, while comparatively the absorbance spectrum of FADH
o shows relative lower absorbance in the blue region and higher absorbance in the green region [
46]. The fully reduced form has a unique absorbance spectrum [
47,
48], but there are no models of cryptochrome activity that suggest a molecular change in FADH
– by photon absorbance [
44,
49]. This model indicates that green photons ought to partially inhibit/reverse blue induced decreases in stem elongation. If cryptochrome affects leaf expansion, then green photons may increase leaf area and yield.
This hypothesis has been evaluated in multiple studies. Although increasing the green photon flux can induce shade morphology (e.g., increased petiole / total leaf length and decreased leaf angle) in
Arabidopsis thaliana [
50,
51], the effect of green photons in horticultural crops is inconsistent. For example, although some studies in cucumber and tomato showed an increase in stem elongation in response to increasing green fraction [
26] many others have shown no response of stem or petiole length to increasing the fraction of green photons [
25,
26,
27,
29,
33], and occasionally studies show a decrease in stem length [
28], which is in the opposite direction than expected.
Additionally, replacing red photons with green photons under a constant fraction of blue has increased leaf area and dry mass in some studies [
5,
20,
22,
26,
39,
41,
52], but most studies show no response, and several show an opposite response [
8,
20,
23,
25,
26,
27,
28,
29,
33,
36,
38,
40] (also see
Table S2). Overall, the expected morphological (and subsequent growth) responses to blue and green photons do no always occur in the horticultural crops lettuce, cucumber and tomato.
The photosynthetic photon flux density (PPFD), or intensity, affects morphology. One notable example is that leaf thickness typically increases with increasing photon flux. Thick and thin leaves are referred to as sun and shade leaves. This response has recently been partially explained by the involvement of both cryptochromes and phototropins [
53]. In some studies, the blue fraction has been found to be better a predictor of stem elongation and leaf expansion, while in other species and other studies, absolute blue intensity has been found to be a better predictor of morphological responses [
26,
30,
54,
55]. The extent of interactions between photon quality and quantity is not well studied.
Previous studies have investigated some of the following interactions: (1) the effect of blue photons between 10 and 30% blue [
8,
26], (2) interactions with green photons at multiple levels of blue [
8,
20], and (3) interactions with intensity [
26]. We sought to investigate all three parameters and their interactions. We hypothesized that (1) increasing the fraction of blue photons would reduce plant size (e.g., leaf area, dry mass and stem length), while increasing the fraction of green photons would increase plant size; (2) the effect of blue photons would be more significant at lower intensities (as this would cause photoreceptors to be under-saturated); and (3) the effect of green photons would be more significant at a lower fraction of blue photons.
2. Materials and Methods
2.1. Plant Material and Cultural Conditions
Lettuce (Lactuca sativa, var. Red Salad Bowl), tomato (Solanum lycopersicum, cv. Early Girl) and cucumber (Cucumis sativa, var. Boston Pickling) seeds were direct seeded then thinned for uniformity after emergence leaving four plants per module. Planted root modules were randomly placed into the 16 treatment chambers. Each chamber had dimensions of 20 × 23 × 30 (L × W × H, 13800 cm3) with gloss white walls. Fans provided an air velocity of 0.5 m s−1 at the top of the canopy. The root modules measured 20 × 18 × 13 (4680 cm3) and contained a 1:1 ratio of peat and vermiculite by volume with five grams of uniformly mixed Nutricote ® slow-release fertilizer (16-2.6-11.2, N-P-K, type 100). Root modules were watered to 10% excess as needed with dilute fertilizer solution (0.01N-0.001P-0.008K; Scotts ® Peat-lite, 21-5-20; EC = 1 mS cm−1), and were allowed to passively drain. Type-E Thermocouples connected to a data logger (CR1000, Campbell Scientific, Logan, UT, USA) continuously monitored ambient air temperature at the top of the plant canopy. Day/night temperature was 23/20 °C, with less than 1 °C variation over time and 1 °C variation among chambers. CO2 concentration was continuously monitored and was identical for all treatments and varied over time between 450 and 500 ppm.
2.2. Treatments
The system included 16 chambers with eight unique spectral outputs at two intensities for a 16 h photoperiod (PPFD: 200 µmol m
−2 s
−1, DLI: 11.5 mol m
−2 d
−1; and PPFD: 500 µmol m
−2 s
−1 DLI: 28.8 mol m
−2 d
−1). Treatments were developed using LEDs (Luxeon Rebel Tri-Star LEDs; Quadica Developments Inc., Ontario, Canada) to output three white (cool, neutral and warm), three red/blue (RB) combinations, and two red/blue/green (RBG) combinations. The RB combination had about 10, 20 and 30% blue, and the RBG treatments contained about 10 and 20% B with 20 or 10% G, respectively. The spectral distributions of the treatments were measured before each replicate study with a spectroradiometer (model PS-200; Apogee Instruments, Logan, UT, USA) and are shown in
Figure 1. Blue, green and red as a percentage of the PPFD were calculated for each species at the higher and lower PPFD. These are averaged together in
Table 1. PPFD was measured with a full-spectrum quantum sensor (MQ-200, Apogee Instruments, Logan, UT, USA) at the top of the plant canopy, and each chamber was adjusted to maintain PPFD at ± 5%.
2.3. Plant Measurements
All species were harvested after canopy closure – when the leaves of the four plants in one of the treatments began to touch. This occurred 21 days after emergence in lettuce, 12, 13 and 20 days after emergence in tomato, and 11 or 13 days after emergence in cucumber. At harvest, stem and longest petiole length of the each of the four plants per chamber were measured in tomato and cucumber. Leaf area was measured using a leaf area meter (LI-3000; LI-COR, Lincoln, NE, USA). Leaf area index (LAI, m2leaf m−2ground) was calculated by dividing total leaf area per chamber by the ground area of the chamber. Shoot dry mass (DM) was measured after the tissue was dried at 80 °C for 48 h. Dry mass per unit area (g DM m−2ground) was calculated by dividing total dry mass by the chamber area. Specific leaf mass (SLM, kg DMleaf m−2leaf) was calculated by dividing the total leaf dry mass of the four plants by the total leaf area of the four plants. The average stem and longest petiole length from each chamber were used for statistical analysis (four measurements averaged together).
2.4. Statistical Analysis
The study was replicated three times (each with four plants per replicate in time). All data were analyzed using R statistical software (R Foundation for Statistical Computing; Vienna, Austria). Blue, green and PPFD effects on the growth parameters in lettuce, cucumber and tomato were determined using lmer and Anova functions with an F statistic. We present significance at p < 0.05 (marked with a *). In a mixed effects linear model, percent blue and percent green were treated as continuous variables while intensity was treated as a fixed effect. Replicates were treated as random factors. Interaction terms between these three factors were included in the linear model. The three-way interaction was insignificant for all parameters and was therefore pooled into the error term.
In order to understand the interactions, the effect of blue photons was also analyzed by separating the data by intensity (200 and 500 µmol m−2 s−1). This separation was also done in the analysis of the effects of green photons, and because green photons have been implicated in the reversal of blue photon effects, the data were further separated for 10 or 20% blue photons. The RB30 treatment was not included in this analysis, and both the cool and neutral white LEDs were considered about 20% blue.