Plant material and germination environment.

Seeds of ‘Salanova^® Red Oakleaf’ lettuce were double-sown in 128-cell trays (15-mL individual cell volume) using a soilless germination medium (BM2 Germinating Mix; Berger Horticultural Products Ltd., Saint-Modeste, Quebec City, Canada), and moved to a reach-in growth chamber (PG2500; Conviron, Winnipeg, Manitoba, Canada) after sowing. Air temperature and day/night relative humidity in the chamber were set at 21 °C and 55%/65%, respectively. An average extended photosynthetic photon flux density (ePPFD; 400–750 nm) at a canopy height of 200 µmol·m⁻²·s⁻¹ was provided by tunable LED fixtures (tunable fixtures; Elixia; Heliospectra, Gothenburg, Sweden) with individually dimmable B (450 nm), R (660 nm), FR (735 nm), and white (5700 K) channels for a 16-h photoperiod (0600–2200 HR). Trays were misted three to four times daily to ensure germination. Once cotyledons unfolded (4 d after sowing), trays were moved to treatment conditions and grown for 12 d. Trays were thinned to one seedling per cell 1 d after treatment initiation and watered as needed using tap water with added water-soluble fertilizer (Jack’s 13N–0.9P–10.8K Plug LX; J.R. Peters, Inc., Allentown, PA, USA) providing (in mg·L⁻¹) 150 nitrogen (N), 23 phosphorus (P), 150 potassium (K), 69 calcium (Ca), 34 magnesium (Mg), 0.15 boron (B), 0.07 copper (Cu), 0.75 iron (Fe), 0.37 manganese (Mn), 0.07 molybdenum (Mo), and 0.37 zinc (Zn). The pH and electrical conductivity (EC) of the fertilizer solution were confirmed using a handheld meter (GroLine H19814; Hanna Instruments, Woonsocket, RI, USA); the average ± standard deviation (SD) pH and EC were 6.71 ± 0.18 and 1.31 ± 0.02 mS, respectively.

Growth chamber conditions.

Two reach-in growth chambers described were used to create two constant temperature environments. For each experimental replication, one chamber was set at 23 °C, and the second at 18 °C, and the day/night relative humidity was set at 55%/65% in both chambers. Each chamber contained both control and EOD-FR treatment groups to ensure a uniform temperature environment, and white opaque plastic was used to divide the chamber to prevent light pollution. Air temperature was measured using two precision thermistors (ST-100; Apogee Instruments, Inc., Logan, UT, USA) per chamber, with one thermistor on each side of the plastic divider. Leaf temperature was measured with fixed mounted infrared thermocouples with acrylonitrile butadiene styrene plastic housing (OS36–01–T–80F; Omega Engineering Inc., Norwalk, CT, USA). The recorded mean air temperature ± SD averaged across three replications in the 23 °C and 18 °C chambers were 23.16 ± 0.28 °C and 18.18 ± 0.22 °C, respectively, and the recorded mean leaf temperature ± SD averaged across three replications in the 23 °C and 18 °C chambers were 22.97 ± 0.28 °C and 18.17 ± 0.60 °C, respectively.

Seedling trays grown in both the 23 °C and 18 °C chambers received the same ePPFD and light spectral quality throughout the normal photoperiod (16 h; 1700–0900 HR) provided by two tunable fixtures hung approximately 0.9 m above canopy level. The target mean ePPFD and extended DLI (eDLI) of the normal photoperiod were 205 µmol·m⁻²·s⁻¹ and 11.8 mol·m⁻²·d⁻¹, respectively. The target percentages of B, green, R, and FR photon flux densities (PFDs) of the normal photoperiod were 10%, 20%, 55%, and 15%, respectively, and the actual percentages were within 2.2% of the targets, on average, for both the 23 °C and 18 °C chambers (Supplemental Table 1). The actual ePPFD, eDLI, and photon flux densities of B, green, R, and FR light provided during the normal photoperiod in the 23 °C and 18 °C chamber are provided in Table 1. The light intensity and spectral quality of the previously described growth conditions and subsequently described EOD light treatments were measured before the start of each experiment replication using a spectroradiometer (SS-110; Apogee Instruments, Inc., Logan, UT, USA) with no less than 20 scans and 16 scans for normal photoperiod conditions and EOD treatments, respectively.

Table 1. The mean ± standard deviation (SD) of the extended photosynthetic photon flux density (ePPFD; 400–750 nm; µmol·m⁻²·s⁻¹), the extended daily light integral (eDLI; 400–750 nm; mol·m⁻²·d⁻¹), and the blue (400–499 nm), green (500–599 nm), red (600–699 nm), and far-red (700–750 nm) photon flux densities (PFDs; µmol·m⁻²·s⁻¹) of the normal photoperiod in the 23 °C and 18 °C chambers averaged over the three experimental replications. Light was provided by tunable light-emitting diode (LED) fixtures (Elixia; Heliospectra, Gothenburg, Sweden) for a 16-h (1700–0900 HR) photoperiod.

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End-of-day light and temperature treatments.

At the end of the normal photoperiod, all seedling trays received one of two 2-h (0900–1100 HR) EOD treatments provided by tunable fixtures in each chamber. Half of the seedling trays received the EOD-FR treatment and the other half received an end-of-day white (EOD-W) treatment that was considered a positive control for this experiment. We selected a positive control because we could not ensure complete blackout conditions within the chambers required for a negative control (no EOD light); therefore, the EOD-W and EOD-FR treatments ran concurrently at EOD to limit the effects of light pollution. The light spectral quality of the EOD-W treatment mimicked the spectral quality of the normal photoperiod; the actual light quality percentages of the EOD-W treatments differed by <1% compared with the normal photoperiod for both the 23 °C and 18 °C chambers (Supplemental Table 1). The EOD-FR treatment contained only R and FR light with a target wide-band R:FR (600–699/700–780 nm) of 0.7. The target ePPFD of both EOD treatments was 25 µmol·m⁻²·s⁻¹, resulting in an eDLI <0.2 mol·m⁻²·d⁻¹. The actual EOD light treatments are summarized for both the 23 °C and 18 °C environments in Table 2. Seedling trays also received one of three air temperature treatments. Air temperature treatments included constant 23 °C, constant 18 °C, or 23 °C for the normal photoperiod; then, they were moved to 18 °C for both the EOD treatment and subsequent dark period, establishing a +DIF treatment. A total of six different EOD light × temperature treatments were created: constant 23 °C + EOD-W; constant 23 °C + EOD-FR; constant 18 °C + EOD-W; constant 18 °C + EOD-FR; +DIF + EOD-W; and +DIF + EOD-FR.

Table 2. The mean ± standard deviation (SD) of the extended photosynthetic photon flux density (ePPFD; 400–750 nm; µmol·m⁻²·s⁻¹), the ratio of red to far-red (R:FR; 600–699 nm/700–780 nm), and the blue (400–499 nm), green (500–599 nm), R (600–699 nm), and FR (700–780 nm) photon flux densities (PFDs; µmol·m⁻²·s⁻¹) of end-of-day FR (EOD-FR) and end-of-day white (EOD-W) treatments in the 23 °C and 18 °C chambers averaged over the three experimental replications. Light was provided by tunable light-emitting diode (LED) fixtures (Elixia; Heliospectra, Gothenburg, Sweden) for 2 h at the end of the normal photoperiod (0900–1100 HR).

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Data collection.

Seedling data were collected 12 d after treatment initiation, and eight seedlings from each treatment were randomly selected for measurement and analysis. Roots of selected seedlings were thoroughly washed and the following measurements were performed: stem length (cm), measured from the base of the hypocotyl to the shoot apical meristem; stem diameter (mm), measured directly under and perpendicular to cotyledons using a digital caliper (Fisherbrand™ Traceable™; Thermo Fisher Scientific, Waltham, WA, USA); and relative chlorophyll content (RCC), measured on the longest leaf using a soil plant analysis development (SPAD) chlorophyll meter (Chlorophyll Meter SPAD-502Plus; Konica Minolta, Inc., Chiyoda City, Tokyo, Japan).

Leaves were removed from seedlings at the node, and total leaf area (cm²) was determined using a leaf area meter (LI-3100; LI-COR Biosciences). Average leaf area (cm²) was calculated by dividing the total leaf area by the number of counted leaves, and the length of the longest leaf was measured with a ruler (leaf length; cm). Leaves, stems, and roots of each measured seedling were separated and dried at 70 °C for at least 5 d to determine the dry mass of each using an analytical microbalance (Analytical Balance ME54E; Mettler-Toledo, LLC, Columbus, OH, USA). Leaf mass per unit area (LMA; mg/cm²) was calculated by dividing the leaf dry mass by total leaf area.

The total anthocyanin content (TAC) of seedlings was determined using the pH differential method (Lee et al. 2005). First, the leaves of eight seedlings were weighed before freezing for 72 h at −80 °C and lyophilizing for 72 h (Benchtop Freezone Legacy; Labconco Corporation, Kansas City, MO, USA). Dry mass of the samples was recorded after lyophilization, and samples were stored at −80 °C until analysis. Then, anthocyanins were extracted from ground lyophilized leaves with methanol/water/acetic acid (85:15:0.5), as described by Cheng et al. (2014). All extracts were stored at −80 °C until analysis. To measure total monomeric anthocyanins, samples were diluted in each 0.4 M sodium acetate buffer (pH 4.5) and 25 mM potassium chloride buffer (pH 1.0) to a concentration of 1 mg dry mass per mL. The absorbance of each sample was measured at 510 nm and 700 nm using a BioTek Synergy H1 multimode microplate reader (Agilent Technologies, Santa Clara, CA, USA). The total anthocyanin content was expressed as µg cyanidin-3-O-glucoside equivalents per mg dry mass. Extractions and analyses were performed in experimental duplicate.

Experiment design and statistical analysis.

This experiment was a 2 × 3 factorial randomized complete block design with EOD light (two levels) and temperature (three levels) as treatment factors and replication as the block. The experimental unit was one 128-cell tray, and the measurements from all seedlings sampled from one tray were averaged together for analysis; this resulted in three (N = 3) experimental replicates per EOD light × temperature treatment combination. The three replications were conducted from Jan 2023 through Jul 2023, and chamber temperature conditions were switched between each replication to control for any effect of the chamber. Additionally, the position of each tray was randomized every day to limit the effects of any uneven light distribution. The anthocyanin extractions and dry mass data were only completed for the first two replications (N = 2). The main and interactive effects of temperature and EOD treatment on all parameters were compared using a two-way analysis of variance. If an interaction was significant, then the effects of EOD treatment were compared within each level of temperature by pairwise comparison of estimated marginal means averaged over replication using Tukey’s honestly significant difference (HSD) at P < 0.05. All statistical analyses were completed using R statistical software (R Core Team 2024).

End-of-day far-red light promotes an increase in leaf area compared with end-of-day white light.

Compared with EOD-W, EOD-FR generally promoted an increase in leaf area, whereas growth at 18 °C or +DIF reduced leaf area compared with plants grown at 23 °C (Table 3). The significant interaction between temperature and EOD treatment is likely attributable to a difference in magnitude rather than direction of response because EOD-FR promoted 4.4-cm² (P < 0.0001), 1.7-cm² (P = 0.0173), and 1.2-cm² (P = 0.0649) increases in leaf area compared with EOD-W at 23 °C, 18 °C, and +DIF conditions, respectively (Table 3). The difference in leaf area at each level of temperature is primarily caused by individual leaf expansion because leaf number was not significantly affected by EOD treatments, while average leaf area was greater under EOD-FR compared with EOD-W (Table 3). Additionally, the length of the longest leaf was significantly affected by EOD treatment, temperature, as well as an interaction between the two (Table 3). On average, the longest leaf was 11.5% (P = 0.0004), 7.7% (P = 0.0650), and 2.5% (P = 0.3761) greater under EOD-FR compared with that under EOD-W for the 23 °C, 18 °C, and +DIF conditions, respectively, suggesting that cooler temperatures inhibit leaf elongation (Table 3). However, because leaf width was not collected and only one leaf per plant was measured for length, it is difficult to completely attribute changes in leaf area to this difference. Notably, the total leaf area under EOD-FR was 13.2% and 12.6% greater than that under EOD-W at 23 °C and 18 °C, respectively, but only 4.2% greater under the +DIF treatment (Table 3). For some rosette species, leaf expansion in response to FR light has been thought to be dependent in part on resource competition between the stem and the leaves (Casal et al. 1987; Cogliatti and Sanchez 1983). Stem length and diameter were not affected by EOD treatment in the present study (Table 3), suggesting there may not be significant resource competition between the stems and leaves under the present experimental conditions. Collectively, these results indicate that EOD-FR in this experiment had a promotive effect on individual leaf expansion when plants were grown at a constant temperature, but that a shift to cooler temperatures for the EOD treatment and subsequent dark period (+DIF) reduced the effectiveness of the EOD-FR treatment.

Table 3. The mean ± standard error of the total leaf area, average leaf area, leaf length, leaf number, stem length, and stem diameter averaged over three experiment replicates (N = 3). The main and interactive effects of temperature and end-of-day (EOD) light treatment were compared by a two-way analysis of variance (ANOVA). If a significant interaction was detected, then the effects of EOD treatment (white, far-red) were compared at each level of temperature and means sharing a letter within a temperature treatment row (23 °C, 18 °C, DIF) are not statistically different according to Tukey’s honestly significant difference test at P < 0.05. If only main effects were significant, then means sharing a letter within the “temperature mean” column or “EOD mean” row are not statistically different according to Tukey’s honestly significant difference test at P < 0.05.

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Changes in leaf expansion in response to DIF vary by species (Erwin and Heins 1995; Kim et al. 2023). For example, Kim et al. (2023) found that leaf area for cucumber seedlings was greater under +DIF (25/15 °C) compared with neutral DIF (0DIF; 20/20 °C) and −DIF (15/20 °C); however, for tomato seedlings, the greatest leaf area was observed under 0DIF compared with +DIF and −DIF. It is important to note the effects of +DIF in the present experiment result from a reduced NT or increased DT when compared with the technically 0DIF 23 and 18 °C treatments, respectively. This is in contrast to previous research because many DIF experiments use a +DIF, −DIF, and 0DIF with the same average daily temperature (e.g., 26/18 °C, 18/26 °C, and 22/22 °C) (Kim et al. 2023; Thingnaes et al. 2008) or only +DIF and −DIF treatments with the same average daily temperature (Ohtaka et al. 2020; Xiong et al. 2002). Warmer night temperatures have been found to increase leaf area for Galega officinalis and Medicago sativa (Patterson 1993), while Arabidopsis leaf length increased by 37% as night temperature decreased from 27 °C to 12 °C (Thingnaes et al. 2003). In the present experiment, we found that cooler temperatures at night (23 °C vs. +DIF) as well as cooler temperatures during the day (18 °C vs. +DIF) reduced the total and average leaf area, length of the longest leaf, as well as leaf number for both EOD-FR and EOD-W treated plants (Table 3); these results suggest that differences in leaf area between temperature treatments were caused by both individual leaf expansion and the number of leaves. Because leaf growth occurs during both the day and night (Pantin et al. 2011), cooler temperatures during the day and night or just the night may have inhibited leaf expansion; this is also supported by biomass data because leaf dry mass was lower under +DIF compared with 23 °C, and it was lower under 18 °C compared with both +DIF and 23 °C (Table 4).

Table 4. The mean ± standard error root dry mass, stem dry mass, leaf dry mass, and leaf mass per unit area averaged over two experiment replicates (N = 2). The main and interactive effects of temperature and end-of-day (EOD) light treatment were compared by a two-way analysis of variance (ANOVA). If a significant interaction was detected, then the effects of EOD treatment (white, far-red) were compared at each level of temperature and means sharing a letter within a temperature treatment row (23 °C, 18 °C, DIF) are not statistically different according to Tukey’s honestly significant difference test at P < 0.05. If only main effects were significant, then means sharing a letter within the “Temperature mean” column or “EOD mean” row are not statistically different according to Tukey’s honestly significant difference test at P < 0.05.

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Although the main effect of temperature on leaf area in the present study is relatively clear, the small inhibitory effect of cooler night temperatures on EOD-FR-induced responses is less so. It is possible that the lower temperatures at night simply reduced the growth rate of leaves, leading to similar leaf area between EOD-FR and EOD-W treatments, but it is interesting that the same response was not observed under the 18 °C treatment (Table 3). The EOD-FR treatment in this study provided a R:FR of 0.7; therefore, the cooler temperatures at night could also slow the reversion of Pfr to Pr in the dark post-EOD treatment, possibly affecting response magnitude, but this would not explain the difference in leaf area observed at 18 °C. Interestingly, stem length, stem dry mass, and stem diameter were similar between +DIF and 23 °C plants, while all three were lower under the 18 °C treatment compared with both the +DIF and 23 °C treatments (Tables 3 and 4). Thus, it is possible that there was a small promotive effect of the +DIF treatment on hypocotyl growth at the expense of leaf growth, resulting in the reduced difference in leaf area in response to EOD-FR under this temperature treatment.

It is important to note that the difference in leaf area in response to EOD-FR in the present experiment was relatively small (12% to 13%) compared with that observed by Zou et al. 2019 (23.8%) and Zou et al. 2021 (24.3%) for lettuce ‘Tiberius’ in response to an EOD-FR treatment. The difference in response magnitude compared with the present study are likely caused by the age of the plants and/or the number of days of EOD-FR treatments. In an FR substitution study, Zhen and Bugbee (2020) found that leaf fractional groundcover of lettuce was greater under the substitution treatments, and that the difference between the substitution treatment and the control increased as the number of days of treatment increased. Zou et al. (2019) initiated EOD-FR treatments after the second true leaf had unfolded and continued for 15 d, while in the current experiment EOD-FR treatments began after both cotyledons had unfolded and continued for 12 d. Thus, plants evaluated by Zou et al. (2019) had more leaves at the start of their experiment that would be affected by EOD-FR treatments as well as more time for the development of new leaves. Future research investigating the timing of EOD-FR treatments on leaf expansion during crop development may contribute to the development of effective EOD lighting strategies for a production setting.

The 14% FR light in the light spectrum during the day could have also limited the effectiveness of the EOD-FR treatment in the present study because EOD-FR treatments and FR substitution treatments reach a saturating dose (Zou et al. 2021) or FR % (Kusuma and Bugbee 2023), respectively. Thus, an EOD-FR treatment may have a smaller effect on PPS if light quality during the day has a higher percentage of FR light. This was supported by Ilias and Rajapakse (2005), who found that EOD-FR treatments increased Petunia ×hybrida height by 67% under a photoselective film that increased the R:FR to 1.51 compared with 7% (nonsignificant) under a neutral density film (R:FR 1.05) in a greenhouse. To our knowledge, no other researchers have explicitly investigated the impact of light quality during the day on responses to EOD-FR light, but it is notable that even a 30-min FR substitution (i.e., increased FR percentage with no significant change in light intensity) at the end of the photoperiod had significant effects on several parameters, including total chlorophyll and anthocyanin content as well as shoot fresh weight and dry weight compared with a negative control (no FR substitution) for ‘Red Butter’ lettuce and ‘Green Butter’ lettuce (Li et al. 2020). Future research should include an examination of this topic because it also has implications for the selection of negative controls for EOD-FR experiments under sole-source lighting. In summary, the measured difference between control and treatment groups could be affected by light quality during the day because of its effects on PPS at EOD.

Effects of EOD treatment and temperature on dry mass.

A significant interaction between temperature × EOD treatment was not found for any dry mass parameter (Table 4). Leaf dry mass and LMA were not significantly affected by EOD treatment, but leaf dry mass was generally higher and LMA was lower under EOD-FR treatments compared with EOD-W (Table 4). The small difference in dry mass may reflect the small increase in leaf area because overall fresh and dry mass have been found to increase in response to EOD-FR treatments for lettuce (Zou et al. 2019, 2021). A decrease in LMA (or increase in specific leaf area) is a typical response to a low R:FR in both shade-avoiding and shade-tolerant species and is adaptative to optimize light interception in low-light conditions (Gommers et al. 2013). However, LMA is also dependent on eDLI; therefore, the similar LMA between EOD-FR and EOD-W could be caused by the equal eDLI across EOD treatments (Poorter et al. 2009, 2019). Leaf dry mass and LMA were significantly affected by temperature (Table 4). Leaf dry mass was lower under 18 °C compared with +DIF and 23 °C, and it was lower under +DIF compared with 23 °C; this is likely attributable to slower growth at colder temperatures because leaf growth occurs during the day and night (Pantin et al. 2011). Leaf mass per unit area was greater under 18 °C compared with 23 °C and +DIF, which is a common response for plants grown at colder temperatures (Poorter et al. 2009). Specific leaf area has been found to decrease in response to a low night temperature for some species (Jing et al. 2016), but a night temperature of 18 °C was not low enough to affect LMA for lettuce in the current study. In a previous experiment, we similarly found that a shift to a NT of 16 °C from a DT of 21 °C did not significantly affect LMA for Petunia ×hybrida ‘Dreams Midnight’ (Percival and Craver 2024). Stem dry mass was similar between EOD-W and EOD-FR treatments regardless of temperature treatment, but it was greater at 23 °C and +DIF compared with 18 °C, which is likely caused by the slower growth at 18 °C. As previously discussed, the +DIF treatment may also have had a slight promotive effect on hypocotyl elongation, potentially resulting in similar stem dry mass and morphology between the +DIF and 23 °C plants.

Effects of EOD treatment and temperature on total anthocyanin content and relative chlorophyll content.

No interaction was observed between EOD treatment and temperature for TAC or for RCC; therefore, we tested the main effects on both parameters. The EOD treatment did not significantly affect TAC, but TAC was slightly higher on average for EOD-W compared with EOD-FR treatments (Table 5). As discussed, EOD-FR treatments have been shown to reduce (Li et al. 2020) or minimally affect anthocyanin content for lettuce (Zou et al. 2023). The variation in the observed effects of EOD-FR treatments on lettuce leaf anthocyanin content among the current study and those by Li et al. (2020) and Zou et al. (2023) may be attributable to cultivar-specific responses to FR light. Specifically, Zou et al. (2023) found that neither a supplemental FR nor EOD-FR treatment significantly impacted anthocyanin content for the green leaf lettuce ‘Tiberius’. In contrast, Li et al. (2020) found that lettuce ‘Red Butter’ and ‘Green Butter’ had 67% and 83% lower anthocyanin contents, respectively, under EOD-FR. The lack of a clear effect of EOD treatment on TAC for lettuce ‘Salanova^® Red Oakleaf’ in the present study could be cultivar-specific and would align with the relatively small effect of EOD treatment on other parameters, but the differences in light conditions during the day and at EOD may also explain variability in responses across experiments, as discussed previously. Supplemental FR treatments can reduce anthocyanin content for lettuce (Li and Kubota 2009; Liu and van Iersel 2022), and supplemental FR treatments or growth under a low R:FR throughout the day has a greater effect on responses compared with EOD-FR treatments (Franklin 2008; Sellaro et al. 2012; Zou et al. 2019). The FR light comprised 14% of the daily light spectrum in our experiment and may have limited the effects of the EOD-FR. In contrast, FR light comprised 2% of the daily light spectrum in the study by Li et al. (2020); therefore, the EOD-FR treatment may have been more effective.

Table 5. The mean ± standard error total anthocyanin content and relative chlorophyll content averaged over two (N = 2) and three (N = 3) experiment replicates, respectively. The main and interactive effects of temperature and end-of-day (EOD) light treatment were compared by a two-way analysis of variance (ANOVA). If a significant interaction was detected, then the effects of EOD treatment (white, far-red) were compared at each level of temperature and means sharing a letter within a temperature treatment row (23 °C, 18 °C, DIF) are not statistically different according to Tukey’s honestly significant difference test at P < 0.05. If only main effects were significant, then means sharing a letter within the “Temperature mean” column or “EOD mean” row are not statistically different according to Tukey’s honestly significant difference test at P < 0.05.

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In contrast to TAC, EOD-FR-treated plants had significantly lower RCC on average compared with EOD-W treated plants, but this difference was relatively small compared with the effect of temperature (Table 5). A reduction in leaf chlorophyll content is a typical shade-avoidance response to a low R:FR (Franklin 2008), and EOD-FR treatments have been found to reduce leaf chlorophyll content for multiple species such as Petunia axillaris (Casal et al. 1987), Nicotiana tabacum (Kasperbauer and Peaslee 1973), lettuce (Zou et al. 2019), and tomato (Kalaitzoglou et al. 2019). Kalaitzoglou et al. (2019) found that EOD-FR only reduced chlorophyll content for tomato compared with a no EOD control if plants were grown under sole-source R-B (95% R/5% B, approximately 150 µmol·m⁻²·s⁻¹) LEDs, but when grown under the same LEDs as well as a small fraction of sunlight (150 µmol·m⁻²·s⁻¹ from LEDs, solar PPFD = 50 µmol·m⁻²·s⁻¹, solar FR PFD = 10 µmol·m⁻²·s⁻¹), the chlorophyll content between control and EOD-FR treatments was similar. Similar to TAC, FR light throughout the day may have partially masked the effects of the EOD-FR treatment on RCC in the present study. It is also possible that greater differences in TAC and RCC would be observed for plants provided an EOD-FR treatment at a later developmental stage or longer duration (>12 d).

Temperature had a significant effect on both TAC and RCC, but the +DIF and 23 °C plants were similar for both pigments (Table 5). Temperature can affect anthocyanin content in leaves for multiple species, including lettuce (Gazula et al. 2005), Ipomoea batatas (Islam et al. 2005), Brassica rapa (He et al. 2020), and Arabidopsis (Kim et al. 2017). For red leaf lettuce ‘Lotto’, ‘Valeria’, and ‘Impuls’, Gazula et al. (2005) found that growth at 30 °C, relative to 20 °C, resulted in lower anthocyanin and chlorophyll B contents. The same study found that a 30 °C/20 °C (DT/NT) resulted in higher anthocyanin and chlorophyll B content than the 30 °C treatment for all cultivars (Gazula et al. 2005). Sakamoto and Suzuki (2017) similarly found that for lettuce ‘Red Wave’ grown at 20 °C during the day, night temperatures of 10 °C and 5 °C increased anthocyanin content while a 30 °C night resulted in similar anthocyanin content compared with that resulting from a 20 °C night temperature. The lack of difference in TAC between +DIF and 23 °C grown plants may indicate that night temperatures of 18 °C are not sufficiently cold to significantly increase anthocyanin biosynthesis for this cultivar (Table 5). Lower night temperatures may also increase the chlorophyll content (Gazula et al. 2005). We measured RCC with a SPAD meter rather than extracting the pigment to evaluate chlorophyll content per unit dry mass, and the higher LMA (and, thus, a thicker leaf) in the 18 °C treatment likely affected SPAD values, as has been observed in other studies (Marenco et al. 2009); this also aligned with the similar LMA and chlorophyll content found for 23 °C and +DIF plants.