Supplemental red and far-red light in white LED systems regulates growth, phytochemicals, and antioxidant accumulation in indoor-grown lettuce and kale

Introduction

Lettuce and Kale are widely cultivated leafy vegetables valued for their short growth cycles, adaptability to controlled environments, and rich nutritional profiles, serving as important sources of vitamins, minerals, polyphenols, flavonoids, anthocyanins, chlorophylls, and carotenoids essential for healthy diets (Kim et al., 2016). Lettuce is particularly appreciated for its mild flavor and crisp texture, while kale is recognized as a nutrient-dense “superfood” with exceptionally high levels of antioxidants and bioactive compounds (Sikora et al., 2008). Increasing consumer demand for fresh, functional vegetables, together with global challenges such as climate change, soil degradation, and water scarcity, has driven a shift from conventional open-field cultivation to controlled-environment agriculture (CEA). CEA systems, which are fully enclosed facilities with precise control of temperature, humidity, CO₂ concentration, hydroponic nutrients, and artificial lighting, have emerged as an innovative approach for stable, year-round production of high-quality leafy greens (Kozai et al., 2019; Despommier, 2010).

Artificial light-emitting diode (LED) lighting is a cornerstone of modern plant factories because it enables precise control of light intensity, photoperiod, and spectral composition while providing energy-efficient, durable, and wavelength-specific lighting to optimize crop productivity and food quality across different growth stages (Hu et al., 2026; Hogewoning et al., 2010). These advances have transformed indoor horticulture by enabling systematic spectral manipulation to enhance plant growth, pigment accumulation, and secondary metabolism in leafy vegetables. Among different LED spectra, white (W), red (R), and far-red (FR) light play complementary roles in regulating plant growth and secondary metabolism, with white light supporting balanced photosynthesis and morphology, while red light (660 nm) aligns with chlorophyll absorption peaks and efficiently promotes photosynthetic carbon fixation. These lights stimulate leaf expansion, chlorophyll synthesis, and biomass accumulation in both lettuce and kale (Johkan et al., 2010; Dou et al., 2017). FR light (730 nm) is less photosynthetically active, but it influences plant architecture, stomatal behavior, and energy distribution via the phytochrome system. FR light has been shown to increase leaf expansion, modify canopy structure, and enhance light interception in leafy greens (Zhen and Bugbee, 2020; Park and Runkle, 2018). Recent studies report that combining W, R, and FR light can yield synergistic effects on growth and quality in lettuce, kale, spinach, and basil (Hu et al., 2026; Zou et al., 2019; Chen et al., 2017).

The combinations of W and R light often improve photochemical efficiency compared to W light alone, while adding FR can further modify energy partitioning within photosystems (Hogewoning et al., 2010; Zhen and van Iersel, 2017). For kale, supplemental R and FR light promote rapid canopy development, enabling more effective light capture in dense vertical farming setups. FR supplementation has also been linked to increased stomatal conductance in lettuce, improving gas exchange and photosynthetic activity under artificial conditions. In addition, the light spectrum plays a pivotal role in regulating both nitrogen metabolism and secondary metabolism in leafy vegetables. Red (R) light enhances photosynthesis and nitrate reductase activity, promoting nitrate assimilation and amino acid synthesis and thereby reducing nitrate accumulation in leaves, whereas far-red (FR) light lowers the R:FR ratio, suppresses nitrate reductase activity, and often increases nitrate content while stimulating biomass accumulation, with white light generally producing intermediate nitrate and amino acid levels (Lin et al., 2013; Snowden et al., 2016; Lee et al., 2019; Zhang et al., 2018). In addition to nitrogen dynamics, lighting spectra profoundly influence pigment biosynthesis and antioxidant capacity. R and FR light significantly increase chlorophyll a and b content, improving light-harvesting efficiency (Hu et al., 2026). Carotenoids, which play key roles in photoprotection are often accumulated more under R+FR combinations. Anthocyanins are strong antioxidants, responsible for red and purple pigmentation, and respond sensitively to light quality; supplemental R and FR light enhances anthocyanin accumulation in colored lettuce cultivars and kale (Dou et al., 2017; Li and Kubota, 2009). Polyphenols and flavonoids are major contributors to plant antioxidant properties, which are also strongly light-responsive: R light activates phenylalanine ammonia-lyase, promoting phenolic biosynthesis, while FR in combination with W or R light further boosts total phenols and flavonoids (Dou et al., 2017; Zhen and Bugbee, 2020; Li and Kubota, 2009). By fine-tuning the ratios and combinations of W, R, and FR light, growers can control morphology and increase levels of health-promoting compounds such as polyphenols, flavonoids, anthocyanins, and carotenoids without compromising plant growth and yield. This spectral optimization strategy offers a powerful tool in smart plant factories, enabling precise, dynamic light management to boost plant growth and nutritional quality.

Several studies have addressed similar or closely related research questions; most have examined the effects on leafy vegetables under optimal growing conditions (Ju et al., 2023; Kelly and Runkle, 2024; Hernández et al., 2025; Jeong et al., 2024). In contrast, this study evaluated the effects of supplemental R and FR light added to a white-light background under relatively low irradiance (100 µmol m^-² s^-¹) and low relative humidity (35-40%), representative of dry environments. We hypothesized that R and FR supplementation would (i) improve growth performance and phenolic accumulation compared with white light alone, (ii) enhance photochemical activity and pigment content, and (iii) produce species-specific responses due to inherent physiological differences between lettuce and kale in low light and low RH conditions.

Materials and methods

Plant materials and growing conditions

Leaf lettuce (Lactuca sativa, cv Salanova@red butter, MTO-OG) and Kale (Brassica oleracea L. cv Winterbor) seeds (Johnny seeds.com, USA) were germinated on moistened 3.0 cm rockwool cubes (Grodan, Roermond, Netherlands). Three weeks after sowing, uniform seedlings were selected and transferred to a deep-flow hydroponic system (a 10-L-capacity plastic container pot) containing a commercial nutrient solution (Humboldt’s Secret Supplies, California, USA). The nutrient solution was applied at 1.6 ml/L of water, according to the manufacturer’s dosage guidelines. Both plant species were grown in an indoor plant factory at 23 ± 2 °C, 35–40% relative humidity, and a 16 h light/8 h dark photoperiod. The hydroponic nutrient solution was maintained at an EC of 1.5–2.0 dS m^-¹ and pH 6.0 to 6.5, adjusted as needed using pH down phosphoric acid (General hydroponics, Rosa, CA, USA). The solution was aerated continuously and replaced every seven days to prevent nutrient imbalance or microbial growth. This experiment was repeated twice; each study collected data from twelve plants per treatment/replication, and plants were grown for 30 days.

Artificial LED light treatments

Three artificial LED light spectra were used: cool white (W; correlated color temperature, CCT 6500 K), red (R; peak wavelength 660 nm), and far-red (FR; peak wavelength 730 nm), delivered using LED panels (LED Panel Grow Light, China). Four spectral treatments were established based on the proportional contribution of each wavelength to the total photosynthetic photon flux density (PPFD): W (control), W+R (3:1), W+FR (3:0.5), and W+R+FR (3:1:0.5). The ratios represent the relative photon flux contributions of each spectral component. The total PPFD was maintained at 100 ± 10 µmol m^-² s^-¹ at canopy level for all treatments, measured using a calibrated quantum sensor (LI-250A, LI-COR Biosciences, USA). Spectral photon distribution and relative wavelength contributions were quantified using a spectroradiometer (LI-180, LI-COR Biosciences, USA). To ensure consistent light exposure throughout the experiment, the LED panel height was adjusted to compensate for plant growth, maintaining uniform PPFD at the canopy level across all treatments. Plants were arranged in a randomized design within the growth area to minimize positional effects.

Photochemical and pigment analysis

The photochemical parameters (maximum quantum yield, F_v/F_m, and performance index, PI), gas exchange parameters (stomatal conductance, g_s), and pigments (chlorophyll a, b, flavonoids, anthocyanins, and nitrogen-flavonoid index) were collected on a fully expanded leaf at 30 days post-transplant. The F_v/F_m and PI were measured using a portable chlorophyll fluorometer (Hansatech Pocket PEA; Hansatech Instruments Ltd., King’s Lynn, Norfolk, UK). Prior to measurement, fully expanded leaves were dark-adapted for 30 min using leaf clips to ensure all PSII reaction centers were fully oxidized (open state). The fluorometer then applied a saturating red-light pulse to induce chlorophyll fluorescence. Minimum fluorescence (F_o) and maximum fluorescence (F_m) were recorded, and F_v/F_m was calculated as (F_m − F_o)/F_m (Maxwell and Johnson, 2000). The PI was derived from the fast fluorescence transient, integrating information on energy absorption, trapping, and electron transport efficiency. Stomatal conductance (g_s) was measured using a SC-1 Leaf Porometer (Meter Group Inc., Pullman, WA, USA). Measurements were taken on the abaxial leaf surface under steady-state conditions. The instrument determines stomatal conductance by quantifying the rate of water vapor diffusion from the leaf to the sensor chamber, which is inversely related to stomatal resistance.

Total chlorophyll, flavonoid, anthocyanin content, and nitrogen balance index (NFI) of fully expanded leaves were assessed non-destructively using an MPM-100 Multiple Wavelength Pigment Meter (Opti-Sciences Inc., Hudson, NH, USA). The device estimates pigment indices based on leaf optical properties (light absorbance and reflectance at specific wavelengths). Chlorophyll is primarily estimated from red and near-infrared absorbance, while flavonoids and anthocyanins are derived from UV and visible light absorption. The NFI was calculated as the ratio of chlorophyll to flavonoid signals, providing an indirect indicator of plant nitrogen status. In each treatment, twelve plants were used for data collection.

Plant morphological measurements

After 30 days of post-transplantation, plants were harvested for morphological and biochemical analyses. The following parameters were recorded including plant height (cm): measured from the base to the apex using ruler; leaf number: total fully expanded leaves per plant; leaf area (cm²): measured using a leaf area meter (LI-3100, LI-COR, USA); root length (cm): measured using ruler; fresh weight (FW) and dry weight (DW) using electric balance. Dry weight was determined after oven-drying samples at 65 °C for 72 h.

Chemical analysis

Sample preparation

For the analysis of the compounds (phenolics, flavonoids, antioxidants, amino acids, nitrate content), the first set comprised twelve plants from replication 1, and the second set comprised twelve plants from replication 2. Each set of plants was dried and ground into a fine powder using a mortar and pestle, then stored in an airtight Ziploc bag. During analysis, each set was triplicated. In the final calculation, six samples (three from rep 1 and three from rep 2) were averaged and reported as the mean ± SD. Briefly, 0.5 g of dried sample was mixed with 30 mL of 80% methanol in a 50 mL Falcon plastic tube. The mixture was shaken at 300 rpm overnight (12 to16 hr) using an electric shaker. The extract was then filtered through Whatman No. 1 filter paper (0.20 mm, Whatman, 5B Cytiva, Marlborough, MA, USA), and the eluate was stored at 4 °C until further analysis.

Estimation of total phenolic content

The total phenolic (TP) content of lettuce and kale was quantified according to the Folin–Ciocalteu method (Singleton and Rossi, 1965). Briefly, 1 mL of the extract was mixed with 0.2 mL of 1 N Folin–Ciocalteu reagent in a test tube. The mixture volume was then increased by adding 1.8 mL of deionized water, vortexed, and allowed to react for 3 min. Subsequently, 0.4 mL of 10% (v/v) Na₂CO₃ solution was added, and the final volume was adjusted to 4 mL with 0.6 mL of deionized water. After incubation for 1 h at room temperature, the absorbance was measured at 725 nm using a spectrophotometer (UV-1800–240 V, Shimadzu Corporation, Kyoto, Japan). The TP content was calculated from a gallic acid calibration curve and expressed as milligrams of gallic acid equivalents (GAE) per gram of dry weight (dw).

Determination of total flavonoid content

The total flavonoid (TF) content was determined following the method of Ghimeray et al. (2014) with minor modifications. Briefly, 0.5 mL of the sample extract was mixed with 0.1 mL of 10% aluminum nitrate and 0.1 mL of 1 M potassium acetate. The mixture was then diluted to a final volume of 4 mL with 3.3 mL of 80% methanol, vortexed, and incubated for 40 minutes. Absorbance was recorded at 415 nm using a spectrophotometer (UV-1800–240 V, Shimadzu Corporation, Kyoto, Japan). The TF content was expressed as milligrams of coumarin equivalents per gram of dry weight (mg CE g^-¹ dw).

Chlorophyll and carotenoid determination

For the determination of photosynthetic pigments, the fresh leaves (50 mg) from each treatment (three plants/treatment, a total of six samples made from two replications) were extracted (10 mL of 80% acetone) and placed at room temperature for 30 min. The collected extract was transferred into a tube and centrifuged at 4000 rpm for 10 min. The absorbance was measured at 647, 663, and 470 nm using a spectrophotometer (UV-1800, 240 V, Shimadzu Corporation, Kyoto, Japan). Chlorophyll a, Chlorophyll b, and Carotenoid were determined according to the formula proposed by Lichtenthaler (1987) and expressed as mg g⁻¹ fw:

Chl a = 12.25 × A₆₆₃ − 2.79 × A_647.

Chl b = 21.50 × A₆₄₇ − 5.10 × A_663.

Car = (1000 × A₄₇₀ − 1.82 × Chl a − 85.02 × Chl b)/198.

Anthocyanin determination

Total anthocyanins was analyzed according to the modified protocol described by Revilla et al. (1998) Each fresh leaf sample (30 mg) was dissolved in 5 mL of 2% HCl in methanol for 36 h at 4 °C, then centrifuged (1446 × g for 15 min), and the supernatant was filtered through Whatman No. 1 filter paper (0.20 mm, Whatman, 5B Cytiva, Marlborough, MA, USA), and used as a stock solution for anthocyanins analysis. Following that, 400 mL of the collected supernatant (from each sample) was diluted with 2 mL of potassium chloride buffer (0.025 M, pH 1.0) and sodium acetate buffer (0.025 M, pH 1.0). (0.4 M, pH 4.5). After 15 min, each solution was filtered, and absorbance was measured at 515 nm; the maximum absorbance was confirmed in separate scans using a UV–Vis spectrophotometer (UV-1800, 240 V, Shimadzu Corporation, Kyoto, Japan). Total anthocyanins were calculated as the equivalent value of cyaniding-3-glucoside and expressed as mg 100 g⁻¹ fw.

Amino acid analysis

Total amino acid content was determined colorimetrically using the ninhydrin–citrate buffer method by Yemm et al. (1955). A citrate buffer (pH 5.5) was prepared from citric acid monohydrate and trisodium citrate dihydrate, and a fresh ninhydrin reagent was made by dissolving 2 g of ninhydrin in 100 mL of DMSO and diluting to volume with the citrate buffer. For the assay, 1.0 mL of each sample solution was transferred to a clean test tube, followed by the addition of 1.0 mL of ninhydrin reagent. The tubes were mixed thoroughly and heated in a water bath (at 80 °C) for 10 minutes to allow color development. After heating, the tubes were cooled to room temperature for 5 minutes, and 2.0 mL of citrate buffer was added to each tube, bringing the total volume to 4.0 mL. The absorbance of the resulting purple solution was measured at 570 nm against a reagent blank within 30 minutes using a UV–Vis spectrophotometer (UV-1800–240 V, Shimadzu Corporation, Kyoto, Japan). A standard calibration curve was prepared by plotting absorbance against amino acid concentration, and the amino acid content of samples was calculated accordingly.

Total nitrate content analysis

Nitrate concentration in the plant extracts was determined using the salicylic acid method as described by Cataldo et al. (1975) with minor modifications. Briefly, 0.5 g of finely ground dried plant samples were extracted with 10 mL of deionized water by shaking for 12 h at room temperature using an electric shaker (300 rpm). The extract was then filtered through Whatman No. 1 filter paper (0.20 mm, Whatman, 5B Cytiva, Marlborough, MA, USA). For the assay, 0.2 mL of the clear extract was mixed with 0.8 mL of 5% (w/v) salicylic acid prepared in concentrated sulfuric acid (H₂SO₄). The mixture was left at room temperature for 20 min to develop color. Subsequently, 19 mL of 2 N sodium hydroxide (NaOH) was carefully added to neutralize the reaction and bring it to completion. After cooling to room temperature, the absorbance was measured at 410 nm using a UV–Vis spectrophotometer (UV-1800–240 V, Shimadzu Corporation, Kyoto, Japan). A standard calibration curve was prepared using potassium nitrate (KNO₃) solutions ranging from 0 to 100 µg mL^-¹. Nitrate concentration in the samples was calculated from the standard curve and expressed as milligrams of nitrate per gram of dry weight (mg NO₃^- g^-¹ dw).

DPPH free radical scavenging capacity

The antioxidant capacity was determined using the scavenging activity of the s Table 1, 2-diphenyl-1-picryl-hydrazine (DPPH) free radical, according to the methods described by Braca et al. (2003). The DPPH solution was prepared (5.914 mg of DPPH powder dissolved in 100 mL of 100% methanol) to maintain an absorbance of 1.1–1.3 at 517 nm using a UV–Vis spectrophotometer (UV-1800, 240 V, Shimadzu Corporation, Kyoto, Japan). Briefly, 1 mL of stock solution was added to 3 mL of DPPH solution. The blank sample was prepared by adding 1 mL of 80% methanol instead of the stock extract to 3 mL of DPPH solution. The mixture was shaken vigorously and left to stand at room temperature in the dark for 30 min. The absorbance was measured at 517 nm using a spectrophotometer (UV-1800–240 V, Shimadzu Corporation, Kyoto, Japan). The percentage inhibition activities of the plant materials were calculated against a blank sample using the following equation:

Table 1. Chemical analysis of pigment content of lettuce and kale grown under added R and FR light under W LED light background.

Mean separation within columns determined by DMRT. Values are presented as mean ± SD (standard deviation). Significant at p ≤ 0.05. Means with the same letter (small letters for lettuce and capital letters for kale) in a column are not significantly different. W, white; R, red; FR, far red.

Inhibition (%) = [(blank sample − extract sample)/blank sample] × 100.

Ferric reducing antioxidant power assay

The reducing power of the samples was estimated according to the FRAP assay as described by Pulido et al. (2000). In brief, 1 mL of the extracted stock solution was mixed with 1 mL of 0.2 M phosphate buffer to maintain a pH of 6.6. The mixture was then incubated at 50 °C for 20 min. After incubation, 1 mL of trichloroacetic acid (TCA) was added to the solution, and the mixture was then centrifuged at 3000 rpm for 10 min. The collected supernatant was diluted with distilled water at a 1:1 ratio. Finally, 0.25 mL of 0.1% ferric chloride was added, and the absorbance was measured at 700 nm by a spectrophotometer UV-1800–240 V, Shimadzu Corporation, Kyoto, Japan).

Statistical analysis

For the growth analysis, 12 plants from each trial/treatment were considered, for a total of 24 plants across two biological replicates. For analysis of pigments and bioactive compounds, six samples/treatments were considered. The collected data were analyzed using one-way analysis of variance (ANOVA) in Minitab (Minitab LLC, State College, PA, USA) to evaluate treatment effects on each plant species individually. Treatment means were compared using Duncan’s Multiple Range Test (DMRT) at a significant level of p ≤ 0.05. Results are presented as the mean ± SD (standard deviation) for tables and mean ± SE (standard error) for all figures. Principal component analysis (PCA) was performed on standardized (z-score normalized) morphological and biochemical variables to examine relationships among treatments and traits. Principal components were selected based on eigenvalues (>1), and interpretation was made using component loadings and biplot visualization.

Result

Plant growth characteristics and photosynthetic activity

It is observed (Figures 1, 2; Table 2) that the fractions of R and FR light in a W light background significantly influenced the growth characteristics of both plant species. In lettuce, the R light fraction notably increased the number of leaves by 34.5%, leaf area by 56.5%, and plant dry weight by 18.8% compared to W light. In contrast, supplemental FR light with W and W+R backgrounds resulted in 56.4% greater plant height and 27.2% higher fresh weight than W light, respectively. The highest root length was observed in the W+R treatment, and the lowest was in the W+FR treatment.

Table 2. Growth characteristics of lettuce and kale grown under supplemental R and FR light under W LED light background.

Mean separation within columns determined by DMRT test. Values are presented as mean ± SD (standard deviation). Significant at p ≤ 0.05. Means with the same letter (small letters for lettuce and capital letters for kale) in a column are not significantly different. W, white; R, red; FR, far red.

In kale, plant growth varied with different light treatments (Figure 2). The W+R+FR treatment increased plant height by 54.7%, leaf number by 17.3%, leaf area by 43.9%, and fresh weight by 23.4% compared to W light. This also indicated that R and FR light alone improved growth compared to W light alone. However, no significant difference was observed for root length, although the lowest root length was found in the W+R+FR treatment in kale.

The maximum quantum yield (F_v/F_m) increased by 10.6% for lettuce and 16.7% for kale under the W+R+FR light treatment and the differences from other treatments were statistically significant (Figure 3). At the same time, the lowest yield was at W light (0.66) for both plant species.

Furthermore, 62.5% increased stomatal conductance was observed under the FR light treatment compared with W light in lettuce. In addition, in kale, stomatal conductance was 46.2% higher under the W+R+FR treatment than under W light (Figure 4).

Pigments, phenolic compounds, and antioxidant capacity

The pigments, including chlorophyll a, chlorophyll b, carotenoids, and anthocyanin, were measured for lettuce and kale (Table 1). In lettuce, FR light significantly increased chlorophyll a by 35% and chlorophyll b by 36.5% compared to W light. Furthermore, the combination of R and FR light treatment enhanced carotenoid levels by 54% and anthocyanin content by 30.9% compared to W light.

In kale, pigment profiles showed a similar trend across different light treatments (Table 1). Chlorophyll a and b content increased by 66.5% and 43.2% under W+R compared to W light. Carotenoids were also increased by 9.7% in W+R and anthocyanins were increased by 572% under W+R+FR treatment compared to W.

The non-destructive analysis also showed the variations in pigment content in lettuce and kale. The overall trends followed the chemical analysis with some exceptions (Table 3). It is observed that addition of FR and R+FR treatment increases the total chlorophyll content in lettuce. Total flavonoids were higher in R+FR, and total anthocyanins in R and R+FR; however, the nitrogen-flavonoid index decreased in the R treatment for lettuce. On the other hand, for kale, higher chlorophyll and flavonoid content was achieved in W+R treatment, no differences for anthocyanin, and a lower nitrogen-flavonoid index were observed in W+R+FR.

Table 3. Non-destructive pigment analysis (SPAD value) of lettuce and kale grown under supplemental R and FR light under W LED light background.

Mean separation within columns determined by DMRT. Values are presented as mean ± SD (standard deviation). Significant at p ≤ 0.05. Means with the same letter (small letters for lettuce and capital letters for kale) in a column are not significantly different. W, white; R, red; FR, far red.

For anthocyanin content, the addition of R and FR with W background increased levels in both chemical and non-destructive analyses for lettuce, but kale showed no difference in non-destructive analysis. The total phenol content was increased by 11.1% under W+R+FR for lettuce and by 10.7% under W+R for kale. In the same way, flavonoids increased by 30.8% under W+R+FR for lettuce and by 18.1% under W+R for kale (Table 4).

Table 4. Total phenols and total flavonoid analysis of lettuce under added R and FR light under W LED light background.

Mean separation within columns determined by DMRT. Values are presented as mean ± SD (standard deviation). Significant at p ≤ 0.05. Means with the same letter (small letters for lettuce and capital letters for kale) in a column are not significantly different. W, white; R, red; FR, far red.

Regarding antioxidant analysis, the antioxidant capacity of kale was significantly increased under W+R and W+FR light treatments, as determined by the DPPH and FRAP methods (Figures 5, 6). The antioxidant capacity increased by 4.3% under W+R+FR for lettuce and by 6.2% under W+R for kale compared to W. The FRAP method shows that antioxidant capacity increased by 14.1% under W+R+ FR in lettuce and by 56.9% under W+R and W+FR in kale.

It was observed that the W+FR treatment significantly increased the total amino acid content in lettuce by 21.5% compared with other treatments. However, total amino acid content did not differ significantly among the treatments in kale (Figure 7).

In addition, total nitrate content increased significantly across treatments in lettuce and kale (Figure 8). The nitrate accumulation increased by 53.6% under W+R+FR and 39.8% under W+R for lettuce. And for kale, the nitrate content increased by 64.8% and 69.1% under W+FR and W+R+FR, respectively.

Discussion

Morphophysiological and photochemical responses

Results showed that supplemental R and/or FR light to a W background enhances growth characteristics and photochemical attributes of both plants, lettuce and kale: Red (R) light increased leaf number, leaf area, and dry mass compared with white (W) light alone, whereas far-red (FR) light promoted stem elongation and enhanced fresh mass, likely due to FR-induced shade-avoidance responses and improved whole-canopy light interception that stimulate plant growth (Li et al., 2024; Carotti et al., 2024). Because R light is efficiently absorbed by chlorophyll and strongly drives photosynthetic electron transport, while FR preferentially excites photosystem I, the combined application of R and FR light can improve overall photosynthetic efficiency compared with either spectrum alone (McCree, 1971). Far-red (FR) light also regulates the phytochrome system, promoting leaf expansion and improving light capture (Smith, 2000), while its deeper canopy penetration enhances whole-plant photosynthesis and biomass accumulation (Zhen and Bugbee, 2020); therefore, the combined application of R and FR light increases leaf area, improves photon-use efficiency, and accelerates plant growth.

The F_v/F_m values indicated that both plants maintained functional PSII activity across all spectral treatments, although they were low, suggesting physiologically acceptable growth under the experimental conditions. Although these values were lower than the theoretical optimum for fully dark-adapted leaves (~0.83), they likely reflect mild photoinhibitory acclimation rather than structural damage to PSII (Maxwell and Johnson, 2000; Murchie and Lawson, 2013). Under artificial LED spectra, particularly with a relatively low blue-photon fraction, PSII recovery kinetics and energy dissipation differ from those under natural sunlight, resulting in a moderately reduced F_v/F_m while plants remain physiologically active. The higher stomatal conductance observed under FR supplementation than under W light is also consistent with previous studies showing that FR light can enhance gas exchange by altering energy partitioning, canopy structure, and hormonal regulation (Shmarev et al., 2024; Park and Runkle, 2018).

Light spectral composition profoundly influences the biosynthesis of pigments and phenolic compounds in leafy vegetables such as lettuce, primarily by modulating the photosynthetic and phenylpropanoid pathways. In this study, FR light significantly enhanced chlorophyll a and chlorophyll b contents compared to W light, confirming that FR wavelengths contribute to improved light-harvesting capacity and chloroplast development. The elevated chlorophyll levels observed under W+FR treatment indicate enhanced photo-acclimation and light-use efficiency. Similar responses were reported by Kong and Nemali (2021) and Son et al. (2013), who found that FR light supplementation stimulates chlorophyll synthesis via phytochrome-mediated gene regulation, thereby improving photosynthetic performance and leaf expansion in lettuce.

The W+R+FR treatment promoted synergistic increases in carotenoid and anthocyanin concentrations, comparable to those of FR alone, but with slightly greater enhancement in total antioxidant potential. This trend aligns with the findings of Dou et al. (2017) and Hu et al. (2026), who reported that R and FR combinations stimulate photomorphogenic responses through the phytochrome B and PIF signaling network, leading to higher carotenoid accumulation and anthocyanin biosynthesis. Carotenoids act as photoprotective antioxidants, while anthocyanins provide both photoprotection and enhanced nutraceutical quality; therefore, their upregulation under W+R+FR suggests optimized redox homeostasis under enhanced photon flux at longer wavelengths. The non-destructive pigment indices represent relative optical estimates rather than absolute biochemical concentrations. Data presented in Table 1 and 3 show a slight inconsistency that likely reflects changes in leaf optical properties and morphology under FR light (e.g., leaf thickness and internal light scattering) rather than contradictions in pigment accumulation. Therefore, the chemical extraction data were considered the primary quantitative measurement, while the optical indices were used only as supportive indicators of physiological trends.

The observed increases in total phenolic and flavonoid contents under the W+R+FR treatment suggest that the combined red and far-red spectra effectively stimulated the phenylpropanoid pathway. Red and far-red light are known to enhance phenylalanine ammonia-lyase (PAL) activity, thereby promoting the biosynthesis and accumulation of secondary metabolites, as previously reported in lettuce and basil (Carvalho et al., 2020; Sawatdee et al., 2023). The significant enhancement of antioxidant capacity, as demonstrated by the DPPH and FRAP assays under the W+R+FR treatment, indicates a close association between light-induced phenolic accumulation and improved radical-scavenging activity. Previous studies have also reported that combined R and FR light can strengthen oxidative defense systems by promoting the accumulation of polyphenolic antioxidants and stimulating antioxidant-related enzymes involved in the ascorbate–glutathione cycle (Son et al., 2013; Yorio et al., 2001). Consequently, these results highlight the importance of integrating R and FR spectra to optimize both photosynthetic and secondary metabolic outcomes in leafy greens production in a controlled environment.

Kale exhibited distinct morphological and physiological responses to different LED light spectra, reflecting the species’ strong adaptability to variable light environments. The W+R+FR treatment produced the tallest plants, greatest leaf area, and highest fresh biomass, suggesting a synergistic interaction between red and far-red light in promoting vegetative growth. These responses are consistent with the shade-avoidance mechanism regulated by the phytochrome B–PIF signaling pathway, in which supplemental far-red light lowers the phytochrome photostationary state (PSS), thereby stimulating stem elongation and leaf expansion (Franklin, 2008; Leivar and Quail, 2011). Supplemental far-red light reduces the phytochrome photostationary state (PSS), decreasing the proportion of the active phytochrome form perceived by the plant. Plants interpret this spectral shift as a shade signal, which triggers shade-avoidance responses such as stem elongation and leaf expansion to enhance light capture. Consequently, the addition of far-red light often promotes taller plants with greater leaf area (He et al., 2021; Park and Runkle, 2018).

In kale, the photosynthetic efficiency (F_v/F_m) showed incremental improvement with the addition of R and FR light, peaking under W+R+FR (0.77) followed by W+FR (0.74). These enhancements suggest improved photochemical efficiency of photosystem II (PSII) and better energy utilization under extended-spectrum lighting. Similar increases in F_v/F_m and PI under combined R and FR irradiation have been reported in kale and spinach, where FR enrichment improved the distribution of light energy between PSI and PSII, leading to enhanced photochemical quenching and photosynthetic assimilation (Park and Runkle, 2017; Son et al., 2013). Furthermore, stomatal conductance increased significantly with R and FR supplementation compared to W light in kale. Red and FR wavelengths play important roles in regulating stomatal behavior through phytochrome- and cryptochrome-mediated signaling pathways, thereby promoting stomatal opening, enhancing CO₂ uptake, and improving transpiration efficiency. These spectral effects ultimately contribute to greater photosynthetic performance and carbon assimilation under controlled-environment conditions (Savvides et al., 2012; Hogewoning et al., 2010).

Pigment and secondary metabolite dynamics

The pigment composition of lettuce and kale was markedly influenced by light spectrum, particularly with respect to chlorophyll a, chlorophyll b, carotenoids, and anthocyanins. The enhanced pigment accumulation under combined R and FR treatments suggests that R and FR photons act synergistically to stimulate chlorophyll biosynthesis. This interaction likely enhances the activity of protochlorophyllide oxidoreductase (POR), the key light-dependent enzyme that converts protochlorophyllide to chlorophyllide during chlorophyll formation. Increased chlorophyll synthesis under R and FR supplementation may improve light-harvesting efficiency and photosynthetic capacity, thereby contributing to greater biomass accumulation and physiological performance (Reinbothe et al., 1996; Tanaka and Tanaka, 2007). Activation of POR also supports the function of other enzymes in the tetrapyrrole pathway, the metabolic route that produces chlorophyll. As a result, plants exposed to R and FR light show higher chlorophyll accumulation because the entire chlorophyll-forming pathway is more efficiently activated (He et al., 2021; Zhen and Bugbee, 2020).

Carotenoid content increased modestly under both R and FR treatments, thereby enhancing photoprotection and regulating oxidative stress. The marked rise in anthocyanin concentration under W+R and W+R+FR indicates activation of the flavonoid and anthocyanin biosynthetic genes (e.g., CHS, DFR, UFGT) by R-enriched light, consistent with reports by Carvalho et al. (2020). R light acts as a potent inducer of anthocyanin accumulation via both phytochrome and sucrose signaling pathways, thereby enhancing pigmentation and antioxidant defense in plants (Ma et al., 2021). The total phenolic and total flavonoid contents also showed strong dependence on R spectral enrichment, reaching their maximum under W+R. These increases are consistent with the upregulation of phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) under R photon flux, as observed in lettuce and spinach (Hogewoning et al., 2010). The slightly reduced phenolic levels under W+FR may reflect a shift in carbon allocation toward primary metabolism and growth rather than secondary defense synthesis (Johkan et al., 2010).

Antioxidant capacity and functional enhancement

The antioxidant assays (DPPH and FRAP) demonstrated that the W+R treatment in kale and the W+R+FR treatment in lettuce significantly enhanced radical-scavenging activity compared with W light alone. The increased antioxidant capacity was closely associated with higher phenolic and flavonoid contents, indicating that these secondary metabolites play a major role in oxidative defense by scavenging reactive oxygen species (ROS) and protecting cellular structures from oxidative damage. The enhanced antioxidant responses under R- and FR-containing spectra suggest that these wavelengths stimulated phenylpropanoid metabolism and antioxidant-related pathways more effectively than white light alone. Red light likely promoted carbon assimilation and metabolite biosynthesis, whereas FR light contributed to phytochrome-mediated acclimation responses associated with stress tolerance and antioxidant production. Similar findings have been reported by Hu et al. (2026); Son and Oh (2013), Zhang et al. (2018), and Zhen and Bugbee (2020), who showed that supplemental R and FR light enhanced antioxidant enzyme activity, phenolic accumulation, and non-enzymatic antioxidants, thereby improving antioxidant capacity and nutritional quality in leafy vegetables.

Total amino acid and nitrogen assimilation

In contrast to the relatively stable amino acid content, nitrate accumulation was strongly influenced by FR supplementation, indicating that nitrogen metabolism was more sensitive to spectral quality than total amino acid biosynthesis. Both lettuce and kale exhibited significantly higher nitrate concentrations under W+R+FR and W+FR treatments compared with white light alone. This response suggests that FR-enriched spectra may stimulate nitrate uptake and accumulation more rapidly than nitrate assimilation capacity. Although FR supplementation promoted biomass production, chlorophyll accumulation, and photosynthetic performance, the concurrent increase in nitrate concentration represents an important physiological and practical trade-off, particularly for fresh leafy vegetables. Excessive nitrate accumulation in edible tissues is undesirable because high dietary nitrate intake has been associated with potential human health concerns and is regulated in many vegetable crops, especially leafy greens (Santamaria, 2006).

The elevated nitrate concentration under FR-containing treatments may result from phytochrome-mediated suppression of nitrate reductase activity, as far-red light shifts phytochrome toward the inactive Pr form, thereby reducing the conversion of nitrate into amino acids and proteins (Lillo, 2008). Under these conditions, nitrate uptake may continue while assimilation becomes comparatively slower, resulting in nitrate accumulation within leaf tissues. In addition, FR-induced leaf expansion and rapid biomass production may increase nitrogen demand and nitrate transport to the leaves, thereby further contributing to nitrate storage in vacuoles rather than its metabolic reduction. Previous studies also reported that FR supplementation can enhance vegetative growth while simultaneously increasing nitrate accumulation due to altered nitrogen partitioning and reduced nitrate reductase activity (Bian et al., 2016; Zhen and Bugbee, 2020).

Species-specific responses were also evident, with kale generally accumulating higher nitrate levels than lettuce under FR treatments. This may reflect differences in nitrate uptake capacity, vacuolar storage potential, and nitrogen use efficiency among species. Therefore, although FR supplementation can enhance growth and productivity in leafy greens, its application should be carefully optimized to minimize excessive nitrate accumulation and potential declines in nutritional quality.

Principal component analysis of biochemical responses

Principal component analysis (PCA) was conducted to evaluate the interactive effects of different light spectra on biochemical composition in lettuce and kale. The first two principal components (PC1 and PC2) together explained approximately 79.6% of the total variance, with PC1 accounting for 54.3% and PC2 for 25.3%. The PCA biplot (Figure 9) clearly separated lettuce and kale according to their light spectral treatments and biochemical traits. PC1 was strongly and positively associated with chlorophyll a, carotenoids, total phenolic, and antioxidant content, indicating that these parameters are primary contributors to the differentiation among treatments. Conversely, PC2 showed higher loadings for flavonoids and anthocyanins, indicating their sensitivity to modulation by R and FR light.

In lettuce, plants grown under combined W+FR and W+R+FR light treatments clustered on the positive side of PC1, indicating enhanced pigment accumulation and antioxidant capacity. These findings are consistent with previous studies demonstrating that supplemental R and FR wavelengths can upregulate genes involved in chlorophyll biosynthesis, phenylpropanoid metabolism, and antioxidant activity (Zhen and Bugbee, 2020; Bian et al., 2015). For kale, plants grown under W+R+FR light showed higher levels of amino acids and antioxidants, suggesting that the combined R and FR spectrum enhanced nitrogen assimilation and stress-responsive metabolic pathways. Similar responses were reported by Carvalho et al. (2020), who observed increased synthesis of amino acids and phenolics in Brassica species exposed to mixed R and FR radiation. The nitrate content vector was oriented opposite to phenolic and antioxidant loadings, implying an inverse relationship; as nitrate assimilation into amino acids and phenolics increases under enriched R/FR conditions, the residual nitrate concentration tends to decrease (Lillo, 2008). PCA indicates correlations among morphological and biochemical traits but does not demonstrate biochemical conversion or metabolic flux. The higher nitrate content observed under FR treatments suggests reduced nitrate reduction or assimilation rather than enhanced incorporation into amino acids. The absence of significant increases in total amino acids supports this interpretation.

Integrative metabolic response under different LED spectra

The clustering pattern shows that lettuce treatments were more closely associated with light-driven pigment biosynthesis (chlorophylls, carotenoids, anthocyanins), whereas kale treatments were more closely associated with nitrogen metabolism and antioxidant activity. This species-specific differentiation may result from morphological and physiological differences in leaf structure and pigment–protein complexes, which influence light-capture efficiency and photoreceptor sensitivity (Walters, 2005; Meng et al., 2019).

The PCA highlights fundamental species-specific physiological strategies: kale tends to favor nitrogen-rich metabolic processes and robust antioxidant pathways shaped by its evolutionary history, whereas lettuce responds more strongly through pigment modulation. These intrinsic metabolic differences explain why kale clusters with nitrogen and antioxidant variables, while lettuce clusters with chlorophylls, carotenoids, and other pigment-related traits.

Conclusion

Qualitative comparisons indicate that both plants exhibit distinct responses to different light combinations, demonstrating that LED spectral composition influences growth, photosynthetic efficiency, and secondary metabolism in lettuce and kale. Generally, the combined R and FR spectrum with W (W+R+FR) produced the most significant enhancement in biomass, pigment accumulation, and antioxidant activity for both plant species. Although R and FR supplementation improved chlorophyll biosynthesis and phenolic accumulation, it also increased leaf nitrate levels, particularly in kale. Kale exhibited stronger morphological and nitrate responses to FR light than lettuce. Overall, balanced spectral management can optimize productivity and nutritional quality in controlled-environment agriculture. However, the study used a single light intensity, nutrient regime, and growth stage, the results should be considered physiological responses rather than production recommendations, and further multi-environment studies are needed before establishing practical lighting guidelines.

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Footnotes

Edited by: Stefano Marino, University of Molise, Italy

Reviewed by: Bozidar Benko, University of Zagreb, Croatia

Derek Stewart, The James Hutton Institute, United Kingdom