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Article

Synergistic Regulation of Growth and Quality in Substrate-Grown Spinach by LED Light Quality and Intensity in PFALs

by
Pengpeng Yu
1,
Chenzhi Wang
1,
Rezwangul Tursun
1,
Xianchao Zeng
1,
Wei Cai
2,
Jinxiu Song
1,* and
Wei Geng
3,*
1
College of Agricultural Engineering, Jiangsu University, Zhenjiang 212013, China
2
College of Water Resources & Civil Engineering, China Agricultural University, Beijing 100083, China
3
Jilin Academy of Vegetable and Flower Sciences, Changchun 130119, China
*
Authors to whom correspondence should be addressed.
Horticulturae 2025, 11(9), 1065; https://doi.org/10.3390/horticulturae11091065
Submission received: 29 July 2025 / Revised: 24 August 2025 / Accepted: 2 September 2025 / Published: 4 September 2025
(This article belongs to the Section Protected Culture)
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Abstract

In this study, a full factorial experiment was conducted to investigate the interactive effects of different red-to-blue light ratios (with R–B ratios of 0.6, 1.2, and 2.4) and photosynthetic photon flux densities (PPFDs of 200, 250, 300, and 350 μmol·m−2·s−1) on the growth, biomass accumulation, and nutritional quality of spinach (Spinacia oleracea L.) in a plant factory using substrate cultivation. The results demonstrated that both LED light quality and light intensity had significant regulatory effects on spinach’s morphological development, pigment biosynthesis, photosynthetic activity, and nutritional quality. The treatment combining an R–B ratio of 1.2 with a PPFD of 300 μmol·m−2·s−1 produced the most favorable outcomes, resulting in the largest leaf area (98.3 cm2), the highest net photosynthetic rate (16.4 μmol·m−2·s−1), and the greatest shoot fresh mass (48.7 g·plant−1). Moreover, this treatment also led to the highest vitamin C content in the leaves and a notable reduction in nitrate accumulation. Correlation analysis revealed significant positive relationships (r ≥ 0.70) between leaf number and shoot fresh mass, chlorophyll content, and vitamin C content. Principal component analysis further indicated that PC1 and PC2 jointly accounted for 83.4% of the total variance, with growth-related and quality-related traits contributing primarily to PC1 and PC2, respectively. Among all treatment combinations, the R–B ratio of 1.2 and 300 μmol·m−2·s−1 condition achieved the highest comprehensive performance score. These findings underscore the critical role of finely tuned LED light environments in optimizing spinach productivity and nutritional quality in a controlled environment. Based on the results, an R–B ratio of 1.2 combined with a PPFD of 300 μmol·m−2·s−1 is recommended as the optimal lighting strategy for spinach cultivation in plant factories.

1. Introduction

With the continuous growth of the global population, accelerating urbanization, and increasing scarcity of arable land, improving the efficiency of high-nutrient vegetable production within limited spaces has become a pressing challenge in modern agriculture [1,2]. Plant factories with artificial lighting (PFALs), as highly intensive agricultural production systems, have attracted worldwide attention due to their precisely controlled environment, high productivity, and year-round supply capabilities [3,4]. By the end of 2024, a total of 493 PFALs were in operation across China. Among the various environmental factors in plant factories, the light environment plays a central role, as it directly affects the photosynthetic efficiency, morphological development, biomass accumulation, and nutritional quality of crops. Consequently, optimizing the light environment has become a core aspect of regulation strategies in plant factory systems [5,6].
In recent years, light-emitting diodes (LEDs) have increasingly replaced traditional fluorescent and high-pressure sodium lamps in PFALs, owing to their advantages of low energy consumption, adjustable spectral output, and extended service life [7,8]. An appropriate LED light intensity can modulate photosynthetic efficiency in leaves, thereby promoting dry matter accumulation and enhancing crop quality [9,10]. Studies have shown that increasing light intensity generally improves plant height, leaf number, biomass accumulation, soluble sugar and protein contents, and vitamin C levels in leafy vegetables [11,12]. However, excessive light intensity may induce photoinhibition, suppressing chlorophyll synthesis and carbon assimilation [13,14], and can even cause physiological disorders such as tipburn in lettuce [15,16]. In contrast, insufficient light intensity often leads to reduced leaf expansion and lower photosynthetic capacity in leafy crops [17]. For instance, a light intensity range of 200–300 μmol·m−2·s−1 has been reported to support optimal growth and morphological balance in various lettuce cultivars [18,19]. Moreover, elevated light intensity has been associated with reduced nitrate accumulation, contributing to improved nutritional safety of vegetables [20].
In addition to light intensity, the spectral composition of LED light—particularly the red-to-blue light (R–B) ratio—plays a crucial role in regulating the growth and quality of leafy vegetables. Red light tends to promote stem elongation and biomass production, whereas increased proportions of blue light enhance chlorophyll and carotenoid biosynthesis, improve antioxidant capacity, and elevate nutritional value [21,22]. It has been reported that combinations such as 91% red + 9% blue and 95% red + 5% blue significantly enhance both fresh and dry mass in various leafy vegetable species [23].
At present, most studies on R–B ratios have focused on the effects of specific fixed ratios on individual crop species, with limited exploration of the interactive effects between R–B ratios and light intensity on the integrated physiological and quality of crops [24,25]. However, coordinated regulation of light quality and intensity is crucial for further optimizing the growth performance of leafy vegetables [14,26]. For instance, red–blue LED lighting under high irradiance has been reported to not only enhance photosynthetic efficiency and yield in lettuce, but also stimulate the accumulation of anthocyanins and carotenoids, thereby improving crop quality [27,28]. These findings highlight that precise manipulation of both light quality and intensity is a promising strategy to improve yield and nutritional quality in leafy vegetables. Selecting appropriate R–B ratios and optimal light intensities, while considering species-specific responses, is essential for achieving efficient and high-quality vegetable production in a controlled environment [23,29]. Previous research has demonstrated that moderate light intensity contributes to biomass accumulation, whereas an appropriate R–B ratio benefits the improvement of nutritional quality in leafy vegetables. When combined, a suitable R–B ratio and moderate light intensity can maximize both growth and quality, which holds considerable importance for the efficient production of leafy vegetables in plant factories.
The present study employed spinach (Spinacia oleracea L.) as a model crop due to its widespread cultivation in PFALs and its recognized nutritional importance. Spinach is rich in essential vitamins, minerals, and bioactive compounds, making it highly relevant for both human consumption and controlled-environment agriculture research [30,31]. Using this species, we systematically investigated the effects of different LED light qualities and intensities on leaf morphology, pigment content, physiological characteristics, biomass accumulation, and nutritional quality. This choice allows the findings to provide practical guidance for optimizing LED lighting strategies in PFALs while addressing both crop productivity and nutritional outcomes. In addition, correlation analysis and principal component analysis (PCA) were performed to clarify the internal relationships between these characteristics. The goal was to elucidate the synergistic effects of light quality and intensity on spinach performance, identify the optimal light environment for high yield and quality, and provide a theoretical and technical foundation for LED lighting strategies in spinach and other leafy vegetable production systems in PFALs.

2. Materials and Methods

2.1. Experimental Materials

The spinach cultivar used in this study was ‘Bofeiter,’ which was kindly supplied by the Chinese Academy of Agricultural Sciences. The experiment took place from January to April 2024 in a PFAL situated at Jiangsu University (32°19′ N, 119°14′ E).
Prior to sowing, spinach seeds were subjected to a pre-germination treatment: they were immersed in warm water at 50 °C for 2 h, followed by an additional 24 h of soaking at ambient temperature. Once radicle protrusion was observed, the seeds were transferred into 72-cell plug trays containing a substrate blend (Jiangsu Xingnong Matrix Technology Co., Ltd., Zhenjiang, China) of peat, vermiculite, and perlite mixed in a 3:1:1 (v/v/v) ratio.
The seedlings were cultivated under T5 fluorescent lamps (FL-T5-28 W, Shanghai Dingduo Lighting Co., Ltd., Shanghai, China) at a photosynthetic photon flux density (PPFD) of 200 μmol·m−2·s−1, with a photoperiod of 12 h·d−1 (08:00–20:00). During the light period, the air temperature and relative humidity were maintained at (20 ± 1) °C and (60 ± 5)%, respectively. In the dark period, the air temperature was controlled at (16 ± 1) °C with a relative humidity of (70 ± 5)%. Meanwhile, the CO2 concentration during the light period was regulated at 500 μmol·mol−1.
After cotyledons had completely unfolded, seedlings were supplied with a half-strength Yamazaki nutrient solution for spinach. The solution consisted of the following compounds (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China): Ca(NO3)2·4H2O, 472 mg·L−1; KNO3, 808 mg·L−1; MgSO4·7H2O, 492 mg·L−1; NH4H2PO4, 152 mg·L−1; Na2Fe-EDTA, 30 mg·L−1; H3BO3, 2.86 mg·L−1; MnSO4·4H2O, 2.13 mg·L−1; ZnSO4·7H2O, 0.22 mg·L−1; CuSO4·5H2O, 0.08 mg·L−1; and (NH4)6Mo7O24·4H2O, 0.02 mg·L−1. Once the first true leaf appeared, the seedlings were irrigated with the full-strength Yamazaki nutrient solution. At the stage when two leaves and one heart were present, the seedlings were transferred into plastic troughs (L120 cm × W90 cm × H30 cm) arranged on multilayer vertical shelves in the PFAL. The cultivation substrate was the same 3:1:1 (v/v/v) mixture of peat, vermiculite, and perlite. The plants were positioned 10 cm apart within rows and 15 cm between rows, accommodating 55 seedlings per trough.

2.2. Experimental Design

After transplanting, spinach plants were subjected to controlled light treatments using LED intelligent lighting modules (RWB/L/TK4, USHIO Lighting Co., Ltd., Kyoto, Japan), with a photoperiod of 14 h·d−1 (06:00–20:00). The LED light sources were mounted 55 cm above the cultivation troughs using fixed brackets. Red-to-blue (R–B) light ratios were adjusted to 0.6, 1.2, and 2.4, designated as L0.6, L1.2, and L2.4, respectively (where L represents light quality, and the number denotes the R–B ratio). Light intensity was measured 15 cm below the LED light source using a handheld quantum sensor (RS-13L, ESPEC Corp., Osaka, Japan). Illumination levels were set at 200, 250, 300, and 350 μmol·m−2·s−1 by adjusting the power settings of the LED fixtures, referred to as P200, P250, P300, and P350, respectively (where P represents light intensity, and the data indicate the numerical values of the light intensity). A full-factorial experimental design was applied with two factors: light quality (R–B) and light intensity, resulting in a total of 12 treatments. Each treatment included three cultivation troughs as biological replicates. During the experiment, the environmental conditions were maintained as follows: the light period temperature at (20 ± 1) °C and the dark period temperature at (16 ± 1) °C; the relative humidity at (60 ± 5)% during the light period and (70 ± 5)% during the dark period. The CO2 concentration during the light period was maintained at 500 μmol·mol−1. Plants were irrigated every two days with full-strength Yamazaki spinach nutrient solution.

2.3. Measurement Methods

2.3.1. Measurement of Morphological Growth

At 18 days post-transplantation, eight spinach plants exhibiting uniform growth were randomly selected from each cultivation trough for morphological assessment. The parameters recorded included leaf number, leaf length, leaf width, and total leaf area. Leaf number was defined as the count of fully expanded leaves per plant, with partially expanded leaves assigned a value of 0.5. Leaf length and width were determined using a ruler, measured on the largest fully expanded leaf. The total leaf area was quantified by scanning all leaves with a flatbed scanner (Lide-110, Canon Marketing Vietnam Company Ltd., Ho Chi Minh City, Vietnam), followed by digital analysis in Adobe Photoshop 2023, applying the method outlined by Song et al. [32].

2.3.2. Measurement of Photosynthetic Characteristics

For each treatment, eight spinach leaves of a similar developmental stage and position were randomly selected. After rinsing with distilled water and gently blotting with absorbent paper, the leaves were ground and extracted in 80% acetone (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) to obtain pigment extracts for analysis. The absorbance of the extract was measured at 663 nm, 645 nm, and 470 nm using a UV–visible spectrophotometer (UV2800, Unico Instrument Co., Ltd., Shanghai, China). Based on the absorbance values, the concentrations of chlorophyll a, chlorophyll b, carotenoids, and total chlorophyll, and the chlorophyll a/b ratio were calculated. The specific measurement procedures followed the method described by Lakhiar et al. [33].
Eight spinach plants were randomly selected from each treatment to measure gas exchange parameters. The net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci), and transpiration rate (Tr) were measured on the largest fully expanded leaf using a portable photosynthesis system (LI-6400XT, LI-COR Inc., Lincoln, NE, USA) equipped with a 6400-02B standard light source leaf chamber. Measurements were conducted between 09:00 and 11:00 a.m. The measurement conditions within the leaf chamber were set as follows: the light intensity at 350 μmol·m−2·s−1, the reference CO2 concentration at 500 μmol·mol−1, the flow rate at 500 μmol·s−1, and the chamber temperature at 20 °C. The measurement procedure followed the method described by Tunio et al. [34].

2.3.3. Measurement of Biological Accumulation

Six intact spinach plants were randomly selected from each treatment. The shoots and roots were rinsed separately with clean water, and residual moisture was gently removed using absorbent paper. The shoot fresh mass and root fresh mass of each plant were then measured using an electronic balance (ME204E, Mettler Toledo Technology Co., Ltd., Greifensee, Switzerland). Subsequently, the shoot and root portions were placed in separate kraft paper envelopes and subjected to enzyme deactivation at 105 °C for 2 h in a drying oven (DHG-9053A, Shanghai Yiheng Scientific Instrument Co., Ltd., Shanghai, China), followed by drying at 85 °C to a constant weight. After cooling to room temperature in a desiccator, the shoot dry mass and root dry mass were measured using the same electronic balance. The total fresh mass and total dry mass were calculated accordingly, following the method described by Dou et al. [35].

2.3.4. Measurement of Nutritional Quality

Six spinach leaves of a similar developmental stage were randomly selected from different plants under each treatment. After rinsing with distilled water and gently blotting to remove surface moisture, the leaves were immediately ground into a homogeneous slurry in liquid nitrogen for further biochemical analysis. The vitamin C content in the spinach leaves was determined using the 2,6-dichlorophenolindophenol (DCPIP) titration method, as described by Shyamala et al. [36]. The leaf iron content was measured by the o-phenanthroline spectrophotometric method [37]. The soluble protein content was quantified using the Coomassie Brilliant Blue G-250 colorimetric method, according to Tunio et al. [38]. The oxalic acid content was assessed through potassium permanganate titration, following the procedure outlined in [39]. All assays were conducted in strict accordance with the referenced protocols, and concentrations were calculated based on standard calibration curves. All the reagents used in the above experiments were purchased from Sinopharm Chemical Reagent Co., Ltd. in Shanghai, China.

2.4. Data Processing and Statistical Analysis

Data processing and preliminary figure generation were performed using OriginPro 2024 and Microsoft Office 2019. Statistical analyses were conducted using IBM SPSS Statistics 26.0. One-way analysis of variance (ANOVA) was performed to assess differences between treatment means. Correlation analysis and principal component analysis (PCA) were carried out at a 0.05 confidence level using OriginPro 2024 for both data analysis and graphical presentation.

3. Results

3.1. Effects of LED Light Quality and Intensity on Morphological Characteristics of Spinach

As shown in Table 1, both the LED light quality and light intensity significantly influenced the morphological characteristics of spinach leaves. When the red-to-blue (R–B) ratio was 0.6 or 1.2, the leaf number, leaf length, leaf width, and leaf area of spinach exhibited an initial increase followed by a subsequent decline with increasing light intensity. Notably, the maximum leaf area was observed at a light intensity of 300 μmol·m−2·s−1. No significant differences were found in leaf number, leaf length, or leaf width between the light intensities of 250 and 300 μmol·m−2·s−1 under the treatment with an R–B ratio of 1.2. However, at an R–B ratio of 2.4, the light intensity had no significant effect on the leaf number, leaf length, or leaf width. Under a light intensity of 200 μmol·m−2·s−1, the leaf number, leaf length, leaf width, and leaf area increased progressively with an increasing R–B ratio. In contrast, at light intensities of 250, 300, and 350 μmol·m−2·s−1, these leaf morphological characteristics initially increased and then declined as the R–B ratio rose.

3.2. Effects of LED Light Quality and Intensity on Photosynthetic Characteristics of Spinach

As presented in Table 2, the LED light quality and light intensity significantly affected the photosynthetic pigment content in spinach leaves. At R–B ratios of 0.6 and 1.2, the contents of chlorophyll a, chlorophyll b, and total chlorophyll, and the chlorophyll a/b ratio initially increased and then decreased with rising light intensity. However, except for the chlorophyll a/b ratio, no significant differences were observed in the chlorophyll a, chlorophyll b, or total chlorophyll contents between the light intensities of 250 and 300 μmol·m−2·s−1. At an R–B ratio of 2.4, the trends in the chlorophyll a and total chlorophyll contents were consistent with those observed at R–B ratios of 0.6 and 1.2, while the chlorophyll b content remained statistically unchanged, and the chlorophyll a/b ratio gradually declined. With the exception of the R–B ratio of 1.2, light intensity did not significantly influence the carotenoid content in spinach leaves.
When the light intensity was below 350 μmol·m−2·s−1, the contents of chlorophyll a and total chlorophyll, the chlorophyll a/b ratio, and the carotenoid content in spinach leaves exhibited an initial increase followed by a decrease with a rising R–B ratio. No significant differences in the chlorophyll a, chlorophyll b, or total chlorophyll contents were observed between the 250 and 300 μmol·m−2·s−1 treatments. At a light intensity of 350 μmol·m−2·s−1, the chlorophyll a, chlorophyll b, and carotenoid contents in spinach leaves did not show significant changes; however, both the total chlorophyll content and the chlorophyll a/b ratio followed a trend of first increasing and then decreasing as the R–B ratio increased.
As shown in Table 3, the effects of the LED light quality and light intensity on the photosynthetic characteristics of spinach leaves closely resembled those observed for the chlorophyll content. Under the same light quality conditions, the net photosynthetic rate, stomatal conductance, and transpiration rate of spinach leaves increased initially and then declined with rising light intensity. However, under an R–B ratio of 2.4, stomatal conductance and the transpiration rate continuously increased with increasing light intensity. The maximum values for the net photosynthetic rate, stomatal conductance, and transpiration rate were observed under the treatment with an R–B ratio of 1.2 and a light intensity of 300 μmol·m−2·s−1, while the intercellular CO2 concentration reached its lowest point. Under identical light intensity conditions, the net photosynthetic rate and transpiration rate showed an initial increase followed by a decline as the R–B ratio increased. At light intensities of 200 and 250 μmol·m−2·s−1, stomatal conductance and the intercellular CO2 concentration were not significantly affected by light quality treatments. However, at a light intensity of 300 μmol·m−2·s−1, stomatal conductance exhibited a trend of first increasing and then decreasing with a rising R–B ratio, whereas the intercellular CO2 concentration followed the opposite pattern.

3.3. Effects of LED Light Quality and Intensity on Biomass Accumulation of Spinach

The effects of the LED light quality and light intensity on the fresh and dry mass of spinach plants showed consistent trends (Figure 1). Under R–B ratios of 0.6 and 1.2, the shoot fresh mass, root fresh mass, total fresh mass, shoot dry mass, root dry mass, and total dry mass of spinach plants exhibited a pattern of initially increasing and then decreasing as the light intensity increased, with the highest biomass accumulation observed at 300 μmol·m−2·s−1. However, under an R–B ratio of 2.4, no significant changes were detected in either fresh or dry mass across different light intensities. At a light intensity of 200 μmol·m−2·s−1, the shoot fresh mass, root fresh mass, total fresh mass, shoot dry mass, root dry mass, and total dry mass did not vary significantly with an increasing R–B ratio. In contrast, at light intensities of 250, 300, and 350 μmol·m−2·s−1, all biomass parameters showed an initial increase followed by a decrease with an increasing R–B ratio, with the highest biomass consistently recorded under an R–B ratio of 1.2.

3.4. Effects of LED Light Quality and Intensity on Nutritional Quality of Spinach

The LED light quality and light intensity significantly affected the nutritional quality of spinach leaves (Figure 2). The vitamin C content in spinach leaves exhibited distinct trends under different light quality treatments. Specifically, under an R–B ratio of 0.6, the vitamin C content increased progressively with rising light intensity. At an R–B ratio of 1.2, the vitamin C content first increased and then declined as the light intensity increased. In contrast, under an R–B ratio of 2.4, the vitamin C content remained largely unchanged across all light intensities. At light intensities of 200 and 350 μmol·m−2·s−1, no significant differences in the vitamin C content were observed, whereas at 250 and 300 μmol·m−2·s−1, the vitamin C content decreased gradually with an increasing R–B ratio.
Under identical light quality conditions, the oxalic acid content in the leaves increased with increasing light intensity; however, no significant differences were found between the 250 and 300 μmol·m−2·s−1 treatments. At a fixed light intensity, the oxalic acid content exhibited a trend of first increasing and then decreasing with a rising R–B ratio.
Except under the treatment with an R–B ratio of 1.2, where changes in light intensity had no significant effect on the nitrate nitrogen content, both the iron content and nitrate nitrogen content showed an initial increase followed by a decrease as the light intensity increased under each light quality condition. The maximum iron content was observed at 300 μmol·m−2·s−1, while the nitrate nitrogen content showed no significant difference between 250 and 300 μmol·m−2·s−1. Under the same light intensity, both the iron content and nitrate nitrogen content decreased with an increasing R–B ratio, although the difference between the treatments with R–B ratios of 1.2 and 2.4 was not statistically significant.

3.5. Correlation Analysis and Principal Component Analysis of Spinach Traits Under Different LED Light Quality and Intensities

The heatmap of correlation analysis among leaf morphology, pigment content, biomass accumulation, and nutritional quality in spinach under different LED light treatments revealed several significant relationships (Figure 3). Leaf number exhibited strong positive correlations with leaf length, leaf width, leaf area, total chlorophyll content, carotenoid content, and shoot biomass (r ≥ 0.70). Similarly, leaf area was positively correlated with leaf length, leaf width, total chlorophyll content, and shoot biomass (r ≥ 0.65). The total chlorophyll content showed a significant positive correlation with carotenoid content and shoot biomass (r ≥ 0.76).
Furthermore, the vitamin C content in the leaves was strongly and positively correlated with leaf number, leaf length, leaf width, leaf area, total chlorophyll content, and shoot biomass (r ≥ 0.73). The leaf iron content was significantly positively correlated with the nitrate nitrogen content (r = 0.91), while exhibiting a negative, though not statistically significant, trend with morphological characteristics and biomass accumulation. The oxalic acid content showed weak and non-significant correlations with other measured parameters. Overall, the correlation analysis indicated a strong synergistic relationship between leaf morphology, photosynthetic pigment content, and biomass accumulation in spinach plants under varying light quality and intensity conditions.
The results of the principal component analysis of plant trait variables under various LED light qualities and intensities are shown in Table 4. The first two principal components, PC1 and PC2, accounted for 65.2% and 18.2% of the total variance, respectively, explaining a combined 83.4% of the overall variation. Most trait variables exhibited high loadings on PC1, particularly leaf number, leaf length, leaf width, leaf area, shoot biomass, and vitamin C content, all of which showed significant positive projections on PC1 (loadings > 0.32), indicating that PC1 primarily represents biomass-related traits. In contrast, nitrate nitrogen content and iron content had higher loadings on PC2 (loadings > 0.65), suggesting that PC2 is more closely associated with quality-related attributes. According to the comprehensive PCA scores across treatments (Table 5), the highest overall score was observed for the P300-L1.2 treatment (score = 1.52), followed by P250-L1.2, P300-L0.6, P350-L1.2, and P250-L0.6, in descending order.

4. Discussion

4.1. Leaf Morphological Characteristics

Among environmental factors, light remains one of the most influential drivers of plant growth and morphological formation [40,41]. Low light intensity restricts biomass production by limiting photosynthetic energy input, whereas excessive intensities can damage photosynthetic tissues and limit further growth [17]. For instance, 240–300 μmol·m−2·s−1 has been identified as the optimal range for growth and morphological balance in various lettuce cultivars [18]. Both red and blue wavelengths significantly influence plant development, yet each can reduce photosynthetic efficiency when used in isolation, and their effects are modulated by light intensity [42,43]. This study systematically examined how different LED light qualities (R–B ratios) and light intensities affect growth, physiology, and quality in substrate-cultivated spinach. The results showed that both light factors significantly regulate shape formation, pigment accumulation, photosynthetic capacity, and nutrient quality, with clear interaction effects between R–B ratios and light intensity.
Specifically, in treatments with R–B ratios of 0.6 and 1.2, an increase in light intensity led to a rise and then a decline in leaf number, length, width, and area, peaking at 300 μmol·m−2·s−1. This underscores that moderate light intensity combined with balanced red–blue spectra supports optimal leaf expansion and architectural development. Although Nguyen et al. [44] and Thuy et al. [45] found optimal growth under 190–240 μmol·m−2·s−1, that discrepancy may arise because the present research measured irradiance 15 cm below the LED source, not at the canopy level, and vertical light attenuation was steep. Other researchers have noted beneficial effects at irradiances up to 400 μmol·m−2·s−1 for leafy crops like canola and alfalfa [46,47], which aligns with findings by Gao et al. [48] and Lin et al. [49] showing optimal R–B ratios of around 1.0–1.2. Conversely, at an R–B ratio of 2.4, morphological responses to light intensity flattened, indicating that excessive red light may suppress blue-light regulatory pathways and reduce organ development efficiency [50,51].
It is worth noting that the flattened morphological responses observed at an R–B ratio of 2.4 likely result from the excessive proportion of red light, which can reduce photosystem II efficiency and alter photoreceptor-mediated signaling pathways that regulate leaf expansion [52]. High red-light ratios may also affect the balance between light harvesting and photoprotection, limiting the plant’s ability to dissipate excess energy effectively [53]. As a result, leaf elongation and biomass accumulation are constrained, leading to the observed plateau in growth responses [29]. These physiological mechanisms provide a plausible explanation for the diminished morphological changes under high red-light conditions and highlight the importance of optimizing the red-to-blue ratio to maintain both growth and photoprotective efficiency.

4.2. Photosynthetic Characteristics

The chlorophyll a, b, and total chlorophyll contents were highest under an intermediate R–B ratio (1.2) and moderate PPFD (250–300 μmol·m−2·s−1), consistent with Wang et al. [54], underscoring the role of appropriate blue light in stimulating pigment synthesis and light capture efficiency [23]. Within this intensity range, the pigment content increased with the R–B ratio up to a point, and then declined, suggesting that moderate red light improves chlorophyll stability and photosynthetic potential, while excessive red light can disrupt pigment metabolism [54,55]. Mechanistically, this pattern may result from the regulation of chlorophyll biosynthesis enzymes, such as glutamyl-tRNA reductase and chlorophyll synthase, which are sensitive to light quality [56]. Blue light can enhance the expression of genes involved in chlorophyll synthesis and maintain photosystem stability [57], whereas excessive red light may shift the balance toward chlorophyll degradation pathways, reducing pigment accumulation [58].
Similar trends were observed in carotenoid accumulation, reflecting their tight association with chlorophyll pathways [59]. The observed variability in carotenoid responses, especially at an R–B ratio of 1.2, likely reflects the balance between light harvesting and photoprotection. Specific red-to-blue ratios may alter the excitation pressure on photosystems, inducing differential carotenoid accumulation to dissipate excess energy [59]. Carotenoids play a key role in non-photochemical quenching, protecting the photosynthetic apparatus from light-induced stress [55]. These mechanisms help explain the treatment-dependent differences in carotenoid content observed in this study.
The photophysiological parameters mirrored the pigment trends: with an R–B ratio of 1.2 and PPFD of 300 μmol·m−2·s−1, the net photosynthesis (Pn), stomatal conductance (Gs), and transpiration rate (Tr) reached their maximum, while the intercellular CO2 concentration (Ci) was lowest, indicating a highly efficient photosynthetic regime with effective stomatal regulation. This is comparable to the findings of Le et al. [29], who reported elevated Pn at an R–B ratio of 1.0. Matsuda et al. [60] noted that even at 500 μmol·m−2·s−1, spinach maintained high photosynthesis under white LEDs, likely due to reduced photoinhibition relative to monochromatic LEDs. In contrast, treatments with an R–B ratio of 2.4 showed weaker photosynthetic responses: Gs increased but Pn did not, possibly due to lower energy utilization efficiency or enhanced photoinhibitory stress [61], aligning with the observations of Gao et al. [48].

4.3. Biomass Accumulation

The fresh and dry mass of both shoots and roots were significantly influenced by the R–B ratio and light intensity. The combination with an R–B ratio of 1.2 and PPFD of 300 μmol·m−2·s−1 produced the highest yields, consistent with previous studies [16,62]. The superior performance observed at an R–B ratio of 1.2 may be attributed to the Emerson enhancement effect, where the simultaneous absorption of red and blue light enhances the overall photosynthetic efficiency beyond the sum of individual wavelengths. Biomass distribution patterns corresponded strongly with leaf morphology, pigment concentration, and photosynthetic performance, reinforcing that biomass formation depends on the coordinated development of structure and photosynthetic capacity [63,64]. Low light intensity (200 μmol·m−2·s−1) or high red ratios (an R–B ratio of 2.4) notably constrained biomass, likely due to insufficient electron transport under low light intensity [29], or reduced blue light leading to impaired stomatal regulation, diminished chlorophyll synthesis, and lower energy use efficiency [65].
Correlation heat map analysis revealed strong positive relationships (r ≥ 0.76) between shoot fresh mass and leaf number, leaf width, light length, leaf area, and total chlorophyll content. Leaf area, in particular, correlated with biomass at r ≥ 0.91, highlighting its critical role in light interception. The total chlorophyll content correlated with biomass at r ≥ 0.76, indicating that optimized light regimes can enhance photosynthetic efficiency and thereby yield [23]. Similarly, the carotenoid content was positively correlated with biomass (r ≥ 0.73), suggesting its role in photoprotection and stabilization of PS II under stronger light [59].

4.4. Nutritional Quality

Nutritional quality responses under varying LED light qualities and intensities reflect spinach’s physiological and metabolic adjustments [66]. Under the treatment with an R–B ratio of 1.2 and PPFD of 300 μmol·m−2·s−1, the leaf vitamin C content was significantly elevated compared with other treatments, consistent with Meas et al. [67]. Strong positive correlations (r ≥ 0.73) between vitamin C content, total chlorophyll content, biomass, and leaf area suggest that pigment synthesis and antioxidant accumulation may follow a coordinated pathway [68], likely driven by the blue light activation of antioxidant enzyme systems [69]. The vitamin C content loaded heavily on PC1 in the PCA, indicating its dual role in quality and growth regulation [68].
The leaf iron content and nitrate content showed similar trends: nitrate peaked under the treatment with an R–B of 1.2 at a PPFD of 250–300 μmol·m−2·s−1 and then declined at a higher light or R–B ratio of 2.4, aligning with Gao et al. [48]. Both features were predominantly associated with PC2 (the nutrient quality axis), with strong loadings (>0.66), and exhibited a high positive correlation (r = 0.91), supporting the notion that nitrate availability may facilitate Fe2+ uptake [70]. Despite weak negative correlations with morphology and biomass, these traits appear linked more to nutritional metabolism than to growth per se [71].
The oxalic acid content displayed a rising-then-declining pattern with an increasing R–B ratio and was lowest under the treatment with an R–B ratio of 2.4, echoing the findings of Alrifai et al. [72], where red escalation further reduced oxalate levels [73]. Though oxalate generally increased with light intensity, this varied across light quality, possibly due to interactions between light and organic acid metabolism enzymes [74]. No significant correlations were found between the oxalate concentration and other physiological traits, suggesting its accumulation is more influenced by genotype, stress, or maturity than by controlled light variables [75].

5. Conclusions

Using a factorial design of LED red–blue light ratios and light intensities, this study elucidated the synergistic impacts of light quality and light intensity on spinach growth, biomass accumulation, and nutritional quality under plant factory conditions. The combination of an R–B ratio of 1.2 and a PPFD of 300 μmol·m−2·s−1 emerged as the most favorable for both yield and quality, especially for this spinach species (cv. Bofeiter). Correlation and PCA analyses confirmed close linkages between leaf morphology, pigment content, biomass, and nutritional traits, and identified the optimal lighting regime through comprehensive scoring. In summary, moderate blue light and optimized PPFD significantly enhance both the productivity and nutritional quality of substrate-cultivated spinach. Therefore, an LED configuration of an R–B ratio of 1.2 with a PPFD of 300 μmol·m−2·s−1 irradiance is recommended for spinach (cv. Bofeiter) production in plant factories.
While our study provides clear evidence that an intermediate R–B ratio (1.2) combined with a moderate PPFD (250–300 μmol·m−2·s−1) optimizes growth and quality in substrate-grown spinach, several limitations should be acknowledged. The experiments were conducted on a single cultivar under specific PFAL conditions, and responses may differ in other cultivars or with inclusion of additional light qualities, such as green or far-red. Furthermore, although the identified optimum yields the best physiological performance, higher light intensities entail increased energy consumption, highlighting the need to consider economic and energy-efficiency aspects. Future research should explore multi-cultivar responses, long-term growth and nutritional performance, and underlying molecular and metabolic mechanisms. Such studies will not only refine LED lighting strategies for spinach but also provide broader insights for optimizing the growth and quality of other leafy vegetables in PFALs.

Author Contributions

Conceptualization, J.S. and W.G.; methodology, J.S. and W.G.; validation, R.T., W.C. and X.Z.; formal analysis, P.Y. and C.W.; investigation, P.Y. and C.W.; data curation, P.Y. and C.W.; writing—original draft preparation, P.Y. and C.W.; writing—review and editing, J.S. and W.G.; visualization, R.T., W.C. and X.Z.; supervision, R.T., W.C. and X.Z.; funding acquisition, J.S. and W.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (one thousand and four hundred dollars), grant number PAPD-2023-87, and the Key Laboratory of Desert—Oasis Crop Physiology, Ecology, and Cultivation, MOARA (five thousand dollars), grant number xjnkywdzc-2025002-01-03.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Acknowledgments

The authors gratefully acknowledge the support from the Undergraduate Innovation and Entrepreneurship Training Program and the Scientific Research Funding Program of Jiangsu University.

Conflicts of Interest

The authors declare no conflicts of interest.

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Horticulturae 11 01065 g001
Figure 1. Effects of LED light quality and intensity on the biomass accumulation of spinach (Spinacia oleracea L. cv. Bofeiter). In the treatment names, P represents light intensity, with the number following P indicating the value of light intensity; L represents light quality, with the number following L indicating the R–B ratio. The treatments with different letters are significantly different at p ≤ 0.05.
Figure 1. Effects of LED light quality and intensity on the biomass accumulation of spinach (Spinacia oleracea L. cv. Bofeiter). In the treatment names, P represents light intensity, with the number following P indicating the value of light intensity; L represents light quality, with the number following L indicating the R–B ratio. The treatments with different letters are significantly different at p ≤ 0.05.
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Figure 2. Effects of LED light quality and intensity on nutritional quality of spinach (Spinacia oleracea L. cv. Bofeiter). The treatments with different letters are significantly different at p ≤ 0.05.
Figure 2. Effects of LED light quality and intensity on nutritional quality of spinach (Spinacia oleracea L. cv. Bofeiter). The treatments with different letters are significantly different at p ≤ 0.05.
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Figure 3. Correlation analysis between leaf morphology, photosynthetic pigments, bioaccumulation, and nutritional quality of spinach plants. The shape and color of the ellipses in the figure represent the strength and direction of the correlation between variables. Red indicates a positive correlation, while blue indicates a negative correlation. Deeper color and more elongated ellipses denote stronger correlations. Asterisks indicate statistically significant correlations at the 0.05 level. The numbers represent Pearson correlation coefficients, ranging from −1 (perfect negative correlation) to +1 (perfect positive correlation).
Figure 3. Correlation analysis between leaf morphology, photosynthetic pigments, bioaccumulation, and nutritional quality of spinach plants. The shape and color of the ellipses in the figure represent the strength and direction of the correlation between variables. Red indicates a positive correlation, while blue indicates a negative correlation. Deeper color and more elongated ellipses denote stronger correlations. Asterisks indicate statistically significant correlations at the 0.05 level. The numbers represent Pearson correlation coefficients, ranging from −1 (perfect negative correlation) to +1 (perfect positive correlation).
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Table 1. Effects of LED light quality and intensity on morphological characteristics of spinach (Spinacia oleracea L. cv. Bofeiter).
Table 1. Effects of LED light quality and intensity on morphological characteristics of spinach (Spinacia oleracea L. cv. Bofeiter).
TreatmentLeaf Number
Piece·Plant−1		Leaf Length
cm		Leaf Width
cm		Leaf Area
cm2·Plant−1
P200-L0.67.3 ± 1.0 d9.7 ± 1.0 d6.0 ± 0.6 d37.5 ± 5.1 f
P250-L0.68.2 ± 0.4 cd11.5 ± 1.1 bc6.5 ± 0.7 cd53.6 ± 3.8 d
P300-L0.69.2 ± 1.0 b12.8 ± 2.1 b7.2 ± 0.2 b69.5 ± 3.5 b
P350-L0.67.9 ± 0.6 cd11.0 ± 0.9 c6.2 ± 0.6 cd44.9 ± 1.6 cd
P200-L1.28.4 ± 0.5 c12.3 ± 0.7 bc6.4 ± 0.5 cd53.5 ± 2.6 d
P250-L1.210.8 ± 0.5 a13.5 ± 0.6 ab7.9 ± 0.5 ab64.7 ± 2.9 c
P300-L1.211.0 ± 1.0 a14.7 ± 1.9 a8.7 ± 1.4 a98.3 ± 5.6 a
P350-L1.29.3 ± 0.6 b12.7 ± 1.4 b6.7 ± 0.7 c68.5 ± 4.4 b
P200-L2.48.7 ± 0.5 bc12.2 ± 1.2 bc7.0 ± 0.8 bc63.0 ± 3.8 c
P250-L2.48.8 ± 1.3 bc11.5 ± 1.5 bc7.0 ± 0.7 bc52.0 ± 4.8 de
P300-L2.49.3 ± 0.5 b11.5 ± 1.6 bc6.8 ± 0.8 bc50.9 ± 2.2 de
P350-L2.48.8 ± 0.4 bc10.7 ± 0.6 cd6.6 ± 0.6 c51.4 ± 4.4 de
Note: In the treatment names, P represents light intensity, with the number following P indicating the value of light intensity; L represents light quality, with the number following L indicating the R–B ratio. The results are expressed as means ± standard deviations (SDs; n = 8), and treatments with different letters are significantly different at p ≤ 0.05.
Table 2. Effects of LED light quality and intensity on chlorophyll content of spinach (Spinacia oleracea L. cv. Bofeiter).
Table 2. Effects of LED light quality and intensity on chlorophyll content of spinach (Spinacia oleracea L. cv. Bofeiter).
TreatmentChlorophyll a Content
mg·g−1		Chlorophyll b Content
mg·g−1		Carotenoid Content
mg·g−1		Total Chlorophyll Content
mg·g−1		Chlorophyll a/b
P200-L0.61.45 ± 0.21 cd0.47 ± 0.06 c2.19 ± 0.27 c3.05 ± 0.15 d0.27 ± 0.04 bc
P250-L0.61.66 ± 0.15 bc0.56 ± 0.07 b2.48 ± 0.33 bc2.97 ± 0.17 de0.26 ± 0.05 bc
P300-L0.61.78 ± 0.13 b0.55 ± 0.03 b2.59 ± 0.16 b3.22 ± 0.14 c0.25 ± 0.03 bc
P350-L0.61.33 ± 0.21 d0.49 ± 0.09 cd2.06 ± 0.30 cd2.71 ± 0.17 e0.23 ± 0.04 c
P200-L1.21.78 ± 0.21 b0.55 ± 0.06 b2.62 ± 0.27 b3.25 ± 0.16 c0.29 ± 0.04 b
P250-L1.22.16 ± 0.20 a0.59 ± 0.08 ab3.12 ± 0.32 a3.65 ± 0.17 a0.37 ± 0.06 a
P300-L1.22.22 ± 0.16 a0.64 ± 0.05 a3.20 ± 0.20 a3.48 ± 0.18 b0.34 ± 0.03 a
P350-L1.21.47 ± 0.26 cd0.42 ± 0.10 cd2.18 ± 0.26 c3.46 ± 0.20 b0.29 ± 0.05 bc
P200-L2.41.28 ± 0.06 d0.41 ± 0.01 d1.91 ± 0.07 d3.11 ± 0.17 cd0.23 ± 0.02 c
P250-L2.41.33 ± 0.26 d0.42 ± 0.07 d1.99 ± 0.27 cd3.20 ± 0.14 cd0.25 ± 0.02 bc
P300-L2.41.50 ± 0.10 c0.45 ± 0.03 cd2.19 ± 0.12 c3.35 ± 0.16 bc0.25 ± 0.02 c
P350-L2.41.25 ± 0.23 d0.42 ± 0.10 cd1.91 ± 0.22 d2.98 ± 0.16 de0.24 ± 0.04 c
Note: Different letters are significantly different at p ≤ 0.05.
Table 3. Effects of LED light quality and intensity on photosynthetic characteristics of spinach (Spinacia oleracea L. cv. Bofeiter).
Table 3. Effects of LED light quality and intensity on photosynthetic characteristics of spinach (Spinacia oleracea L. cv. Bofeiter).
TreatmentNet Photosynthetic Rate
μmol·m−2·s−1		Stomatal Conductance
mol·m−2·s−1		Intercellular CO2 Concentration
μmol·mol−1		Transpiration Rate
mmol·m−2·s−1
P200-L0.610.2 ± 1.3 e0.642 ± 0.080 bc474 ± 27 a1.35 ± 0.20 de
P250-L0.612.4 ± 1.1 cd0.700 ± 0.051 b458 ± 23 a1.52 ± 0.28 cd
P300-L0.612.6 ± 1.2 cd0.738 ± 0.129 b452 ± 19 a1.88 ± 0.22 c
P350-L0.610.8 ± 1.1 e0.630 ± 0.062 c471 ± 26 a1.28 ± 0.20 e
P200-L1.213.0 ± 1.3 cd0.660 ± 0.038 bc457 ± 16 a1.68 ± 0.15 c
P250-L1.214.8 ± 1.2 b0.788 ± 0.093 ab438 ± 28 ab2.29 ± 0.29 b
P300-L1.216.4 ± 0.9 a0.874 ± 0.087 a424 ± 25 b2.55 ± 0.21 a
P350-L1.213.7 ± 1.5 bc0.607 ± 0.061 c461 ± 33 a1.67 ± 0.36 cd
P200-L2.412.0 ± 1.0 d0.588 ± 0.105 c466 ± 19 a1.46 ± 0.17 d
P250-L2.413.4 ± 1.2 c0.642 ± 0.056 bc457 ± 23 a1.77 ± 0.23 c
P300-L2.413.3 ± 1.1 c0.670 ± 0.066 bc453 ± 20 a1.88 ± 0.32 c
P350-L2.411.6 ± 1.4 de0.688 ± 0.041 b467 ± 17 a1.72 ± 0.20 c
Note: Different letters are significantly different at p ≤ 0.05.
Table 4. Loading matrix and contribution percentage of each factor in principal component analysis.
Table 4. Loading matrix and contribution percentage of each factor in principal component analysis.
Trait VariableFirst Principal Component (PC1)Second Principal Component (PC2)
Leaf length0.3344−0.0634
Leaf width0.3310−0.0796
Leaf area0.3282−0.0678
Total chlorophyll content0.30330.1975
Carotenoid content0.2890−0.0266
Shoot fresh mass0.3474−0.1007
Shoot dry mass0.34280.0361
Vitamin C content0.32140.1785
Iron content−0.02420.6610
Nitrate nitrogen content0.05220.6557
Titratable acid content0.20720.0899
Eigenvalue7.82692.1784
Percentage of variance (%)65.22%18.15%
Cumulative (%)65.22%83.37%
Table 5. Composite scores and ranks of principal component analysis of different treatments.
Table 5. Composite scores and ranks of principal component analysis of different treatments.
TreatmentPrincipal ComponentsComprehensive ScoreRank
F1F2
P200-L0.6−1.14970.8138−0.602111
P250-L0.6−0.25251.54260.11535
P300-L0.60.40511.62790.55973
P350-L0.6−0.79410.8801−0.35827
P200-L1.2−0.3287−0.5310−0.31086
P250-L1.21.3995−0.10670.89342
P300-L1.22.3372−0.04491.51631
P350-L1.20.4457−0.81670.14244
P200-L2.4−0.4477−1.2387−0.516910
P250-L2.4−0.4874−0.4147−0.39329
P300-L2.4−0.4668−0.4087−0.37868
P350-L2.4−0.6606−1.3031−0.667412
Note: F1 and F2 represent the scores of principal component 1 (PC1) and principal component 2 (PC2), respectively.
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Yu, P.; Wang, C.; Tursun, R.; Zeng, X.; Cai, W.; Song, J.; Geng, W. Synergistic Regulation of Growth and Quality in Substrate-Grown Spinach by LED Light Quality and Intensity in PFALs. Horticulturae 2025, 11, 1065. https://doi.org/10.3390/horticulturae11091065

AMA Style

Yu P, Wang C, Tursun R, Zeng X, Cai W, Song J, Geng W. Synergistic Regulation of Growth and Quality in Substrate-Grown Spinach by LED Light Quality and Intensity in PFALs. Horticulturae. 2025; 11(9):1065. https://doi.org/10.3390/horticulturae11091065

Chicago/Turabian Style

Yu, Pengpeng, Chenzhi Wang, Rezwangul Tursun, Xianchao Zeng, Wei Cai, Jinxiu Song, and Wei Geng. 2025. "Synergistic Regulation of Growth and Quality in Substrate-Grown Spinach by LED Light Quality and Intensity in PFALs" Horticulturae 11, no. 9: 1065. https://doi.org/10.3390/horticulturae11091065

APA Style

Yu, P., Wang, C., Tursun, R., Zeng, X., Cai, W., Song, J., & Geng, W. (2025). Synergistic Regulation of Growth and Quality in Substrate-Grown Spinach by LED Light Quality and Intensity in PFALs. Horticulturae, 11(9), 1065. https://doi.org/10.3390/horticulturae11091065

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