Greenhouse Photoluminescent PMMA Panels Improve the Agronomical and Physiological Performances of Lettuce (Lactuca sativa L.)

Abstract

Supplementary lighting of specific wavelengths can be used for inducing morphological and physiological responses in different crops, ultimately improving yield and quality. Based on this approach, new greenhouse covering materials are being developed in order to improve the use of sunlight in horticulture. These new-generation greenhouse coverings may incorporate light spectrum modulation agents or fluorescent additives which convert solar UV radiation into visible light. In this work, we tested the agronomical and physiological response of lettuce grown under a greenhouse covered with poly-methyl-methacrylate (PPMA) panels doped with a blend of the rare-earth inorganic material with a photo-luminescent effect. The doped greenhouse elicited a 36% increase in lettuce yield compared to the undoped greenhouse. Chlorophyll and carotenoid content, as well as antioxidant activity and ascorbic acid content, were not affected by greenhouse cover, but the doped panels induced a 22% reduction in total phenolics and a 14% increase in nitrate content in leaves. The greenhouse covering materials also affected the photochemistry of photosynthesis, as the daily fluctuations in both the effective quantum yield (ΦPSII) and the electron transport rate (ETR) were attenuated under the doped greenhouse. Non-photochemical quenching (NPQ) was closely related to the light environment in all experimental conditions, with the highest values at 14:00 h. Our results showed that the red-supplemented light spectrum under the doped greenhouse cover contributed to increased plant growth and yield, with a corresponding effect on the physiology of photosynthesis.

1. Introduction

Greenhouse crop production plays an important role in agriculture through sustainable crop intensification, involving water-use efficiency optimization and a better control of product quality and safety. This farming system allows for off-season production of vegetables which, in addition to supplying local markets, benefits the foreign trade balance of numerous national economies in the Mediterranean region [1]. Moreover, cultivation under a protected environment, along with the accurate management of cultivation practices, makes it feasible to attain a higher yield than in open field conditions [2].

Each material used to cover greenhouses/tunnels has its own specific optical properties, which modify the light environment (i.e., light intensity and spectral composition), ultimately affecting crop physiology, yield and nutritional quality of the products [3]. In fact, light not only fuels photosynthesis but it affects plant physiology, growth, and productivity in many ways [4,5]. For instance, supplementary lighting with specific wavelengths has been employed for inducing morphological and physiological responses in different crops such as lettuce [6], strawberry [7] or tomato [8,9]. From this perspective, various light sources have been used in greenhouses including incandescent, fluorescent, high-pressure mercury, sodium, or metal-halide lamps. Light-emitting diodes (LEDs) have gained popularity in the last decade due to their numerous advanced features, namely, low power consumption, long life span and an emission spectra which can be tuned to match the light requirement of the plant species [10]. Currently, the widespread use of LEDs is mainly limited by the high upfront costs. Therefore, this technology remains restricted to specific applications such as pilot greenhouses, compensation for low light levels in photovoltaic greenhouses, and high-tech greenhouses for vertical and indoor farming.

Agricultural films for greenhouses are increasingly used to control the light environment and tune it to meet specific light requirements of fruits and vegetables [11]. These films are obtained by adding light conversion agents such as fluorescent materials able to convert UV light into blue–violet or red–orange light. Three different light conversion agents are known: the green-to-red, ultraviolet-to-red, and ultraviolet-to-blue agents. Based on the properties of the material, these agents can be classified into fluorescent dyes, organic rare-earth complexes, and inorganic rare-earth complexes [11]. Therefore, agricultural films doped with light conversion agents can be used to modulate light spectrum inside greenhouses by altering the proportion of blue, red, and far-red light. This ratio is an important determinant of the photomorphogenic responses of crops, ultimately controlling plant growth [12]. For instance, films with far-red light-absorbing dyes were efficient as chemical growth regulators or CuSO₄ filters in controlling the height of bell pepper plants and chrysanthemums [13].

Recent research focused on the luminescent properties of some rare-earth incorporating materials [12,14,15,16], which can be used to convert UV, visible or IR light into red and/or blue photons. Ming et al. [17] evolved a highly efficient converter of ultraviolet, visible, and infrared photons into red photons by implementing a luminescent material with the compositions of P₂O₅–Li₂O–Al₂O₃–Sb₂O₃–MnO–Eu₂O₃–Er₂O₃–Yb₂O₃ on phosphate glass. The properties of several glass-based or organic polymer-based materials doped with different rare-earth elements have been reported. Their potential use in agriculture was also suggested [18,19] in view of the effects of light-spectrum modulation on plants [6].

Plant growth and physiology are strongly affected by the environmental light spectrum and red light is known to have a key role in regulating plant development as well as being highly effective in driving photosynthesis [20]. Since photosynthesis is the primary physiological factor driving plant productivity, the effect of treatments was also investigated at the physiological level, focusing on photosynthesis by means of Chlorophyll a (Chl a) fluorescence analysis. Chl a fluorescence emission is widely recognised as a very sensitive tool for detecting even small changes in the structure and functioning of the photosynthetic apparatus [21,22]. This method is based on quick non-destructive measurements and it can be conveniently used in vivo in the field [23].

The principle underlying chlorophyll fluorescence analysis is based on Kautsky’s original observations in 1931 [21]. In the case of healthy unstressed plants, most of the light energy absorbed by chlorophyll molecules in a leaf is used to drive the photochemical reactions of photosynthesis, while excess energy can be dissipated as heat (thermal dissipation) or it can be re-emitted as light (chlorophyll fluorescence). These processes occur in competition with each other, such that any increase in the efficiency of one will result in a decrease in the yield of the other two. Therefore, although the total amount of Chl a fluorescence is very small (about 3–5% of total light absorbed), its measurements provide detailed information about the status and efficiency of the photosynthetic apparatus [24]. When a leaf is exposed to light, photosynthetic metabolism reaches a stationary level known as the light adapted state (LAS), with Chl a fluorescence emission at a low steady-state level (F_t). In this light-adapted state, illumination with a saturating flash of light induces a peak of fluorescence emission known as F’_m. Fluorescence measurements in the LAS allow for the calculation of the ΦPSII parameter (the effective quantum yield of PSII photochemistry), which is related to the “working” efficiency of CO₂ assimilation [25].

The photosynthetic performance of a plant typically follows a diurnal course with two peaks: the first one in the morning, followed by a midday depression around noon and a second peak in the late afternoon. The midday depression is mostly dependent on high irradiance and high temperature, and it corresponds to the energy lost to non-photochemical quenching (NPQ). NPQ is interpreted as a protective system which limits the input of light energy into the photochemical pathway when photosynthesis becomes light saturated at high light intensities, and for this reason NPQ is often referred to as photoinhibition. The regulation of light energy input relies on the dissipation of ‘excess’ light energy harmlessly as heat. This pathway limits the build up of highly reducing electron transfer intermediates that could lead to the formation of damaging reactive oxygen species, ultimately resulting in irreversible damage to the photosynthetic apparatus [26,27,28,29]. From the physiological point of view, NPQ results from the activation of multiple metabolic systems which co-operate for the protection of the plant photosynthetic apparatus. The so-called “quenching analysis” is based on the identification of the separate components of NPQ based on the timing of their relaxation after moving the plant from light to dark [30]. One form of NPQ is ‘energy-dependent exciton quenching’ or qE, which is activated by the acidification of the thylakoid lumen, and it rapidly relaxes within 2–3 min with the depletion of the proton gradient. Another form of NPQ results from photoinhibition or photodamage to PSII reaction centres: it is termed qI and it can be distinguished based on its long recovery time, in the many minutes to hours timescale. Lastly, another short-lived quenching component should be mentioned: this is qT, resulting from a process called state transitions, where a fraction of the light-harvesting antennae dissociate from PSII and migrate to photosystem I (PSI), decreasing the excitation pressure on PSII. This component recovers over intermediate times, in the minutes timescale. However, qT is reported to be small in higher plants and it is often ignored [30].

In this work, we investigated the application of an innovative greenhouse covering material based on poly-methyl methacrylate (PMMA) panels doped with a blend of rare-earth inorganic complexes, with a photo-luminescent effect in the red region of the PAR spectrum. We studied the quantitative and qualitative traits of lettuce production as well as the physiological effects on the structure and functioning of the photosynthetic apparatus through the analysis of chlorophyll fluorescence parameters over the course of the day.

2. Materials and Methods

2.1. Experimental Design, Plant Material and Crop Management

Red-leaved Lactuca sativa L. cv ‘Canasta’ was cultivated in 0.25 m diameter pots (12 L capacity) at the Department of Agricultural Sciences, University of Naples “Federico II” (Portici, NA, Italy) in the spring season of 2021. Pots were filled with sandy soil (91.0% sand, 4.5% silt, 4.5% clay) characterized by: pH 6.6, organic matter 2.6%, total N 1112 mg kg⁻¹, P₂O₅ 127.2 mg kg⁻¹ and K₂O 471.8 mg kg⁻¹. A plant density of 8 plants m⁻² is considered typical for this cultivar. One plant (growth-stage code 13 of BBCH-scale) per pot was transplanted on April 29, 2021, and the harvest occurred on June 15 (growth-stage code 49 of BBCH-scale). Fertilization was carried out only for nitrogen, added at a dose of 80 kg ha⁻¹ with ammonium nitrate (26%; YaraBela EXTRAN 26, Yara Italia S.p.A, Milano, Italy) and split twice (10 days after the transplant and 20 days after the first fertilization). The water losses by evapotranspiration were calculated with the Hargreaves method and fully restored [31]. The pots were placed under two greenhouses custom built around a steel frame covered with PMMA panels (described in Section 2.2). In the first greenhouse, the panels were doped with rare earth (hereinafter, “doped”), whereas in the second one, they were not doped (hereinafter, “control”). Six pots/replicate were located under each structure. An additional group of six pots was placed in the open air as a reference for the chlorophyll fluorescence measurements.

2.2. Photoluminescent PMMA Panels for Greenhouse Covering

The poly-methyl methacrylate (PMMA) sheets that covered the experimental greenhouses were produced by the cell casting method. In short, liquid MMA monomer was poured between two flat sheets of toughened glass, sealed with a rubber gasket and heated for polymerization. In this experiment, 5% (w/w) of the rare-earth blend was added to PMMA-doped panels. The rare-earth blend constituted two photoluminescent pigments emitting two different wavelengths, red and blue. The red component is due to CaS:Eu (Europium and Dysprosium), while the blue one is due to Sr₄Ca₄Al₂₂O₄₁:Eu, Dy⁺³, Nd⁺³, B₃ (calcium oxide, Strontium oxide, Aluminium Oxide and Europium Oxide) with a weight ratio of 70/30. The resulting PMMA sheets were completely transparent in the case of undoped PMMA or opalescent white in colour, in the case of doped PMMA.

2.3. Light Spectra, Total Irradiance, PAR, and Temperature Measurements

The light spectra inside and outside the greenhouses were measured with an Optics Maya 2000 Pro (Ocean Insight, Oxford, UK) spectrophotometer (spectral range 165–1100 nm) set at 14 ms integration time. The total irradiance in the spectral range 400–1050 nm was measured using a HD2102.2 photoradiometer (Delta Ohm, Padua, Italy) equipped with a LP471RAD (Delta Ohm) radiometric probe with a cosine corrector. The photosynthetic photon flux density (PPFD) in the photosynthetically active radiation (PAR) range from 400 to 700 nm was measured with a PSI PAR-FluorPen FP 110/D (Photon Systems Instruments, Drásov, Czech Republic) equipped with a cosine-corrected PAR sensor.

Under the two experimental greenhouses, the air temperature was monitored during the whole growing period with a Vantage Pro2 weather station (Davis Instruments, Hayward, CA, USA). Data were reported as daily mean.

2.4. Plant Growth and Yield Measurements

At harvest, we analysed as growth parameters the head fresh weight, number and mean weight of leaves, height, and diameter of stems. A representative sample of leaves and stems was oven dried at 70 °C until constant weight to measure dry weight and then to calculate the dry-matter percentage. The marketable yield was expressed as kg m⁻².

2.5. Chroma Meter Measurements and Pigments Determinations

The leaf colour space parameters were measured with a CR-300 Chroma Meter (Minolta Camera Co. Ltd., Osaka, Japan). These measurements are based on the spectral reflectance properties of leaves and express the colour of the reflected light providing a digital output of chromaticity (L*, a*, and b* parameters). The above measurements (n = 10 per treatment) were taken on young fully expanded leaves on the middle portion of the adaxial leaf surface between the midrib and the leaf margin.

Chlorophyll a, chlorophyll b, and carotenoids were determined according to Lichtenthaler and Buschmann [32]. Briefly, frozen leaf samples were extracted in pure acetone and, after centrifugation at 3000× g for 5 min, the absorbance of the supernatants was measured at 662, 645, and 470 nm, respectively, via a Hach DR 2000 spectrophotometer (Hach Co., Loveland, CO, USA). Total chlorophylls was calculated as the sum of chlorophyll a and b.

2.6. Hydrophilic and ABTS Antioxidant Activities, Total Ascorbic Acid, Total Phenols and Nitrate Content

After harvest, leaf samples were rapidly frozen in liquid nitrogen and then lyophilized prior to storage at −80 °C for the subsequent analyses. Hydrophilic and ABTS antioxidant activity (HAA and ABTS, respectively), and total phenolic content were determined on freeze-dried leaf samples, with total ascorbic acid (TAA) on fresh samples, using previously described procedures [33].

Nitrate content was determined on dried leaf samples by Foss FIAstar 5000 spectrophotometer (FOSS Italia S.r.l., Padua, Italy) continuous flow analyzer. Briefly, 0.5 g of plant material was added to 50 mL of bi-distilled water in Falcon tubes. Tubes were shaken on a Universal Table Shaker 709 (Bicasa, Bernareggio, MB, Italy) for 60 min at 250 rpm. Tube were then centrifuged for 5 min at 5000 rpm and samples filtered using LLG-medium/fast qualitative filter paper 150 mm (LLG labware, Meckenheim, Germany). The resulting supernatant was used for the determination of nitrates using a wavelength of 540 nm. Results were expressed in fresh weight based on each sample dry-matter percentage.

2.7. Chlorophyll Fluorescence Measurements

Non-destructive chlorophyll a fluorescence measurements were performed in the field at the end of the cultural cycle, on leaves randomly sampled among the young fully expanded leaves of lettuce plants, using a PAR-FluorPen FP 110/D portable fluorimeter (Photon Systems Instruments, Drásov, Czech Republic) equipped with detachable leaf clips. Ten replicate measurements for each experimental treatment were taken between 07:30–08:30 (Central European Summer Time) for the morning (h 08), between 13:30–14:30 for the midday (h 14) and between 18:30–19:30 for the late afternoon (h 19) measurements. Fluorescence measurements were carried out according to the following procedure: a first measurement was recorded immediately after clipping the fluorimeter onto the leaf in its light-adapted state (LAS), then the leaf clip was closed, and the leaf was dark-adapted for 30 min. Afterwards, a second measurement in the dark-adapted state (DAS) was recorded using the same fluorimeter settings. Fluorescence data were then acquired using the FluorPen software ver. 1.1 (Photon Systems Instruments). The ΦPSII parameter (effective quantum yield of PSII photochemistry) was calculated according to Genty et al. [25], while the NPQ_(T) and related quenching parameters qE_(T) and qI_(T) were calculated according to Tietz et al. [30]. All of the above calculated fluorescence parameters are dimensionless ratios. The linear electron transport rate or ETR was calculated according to Kalaji et al. [34] as ETR = ΦPSII × PPFD × 0.85 × 0.5 and expressed as µmol electrons m⁻² s⁻¹, where PPFD is the photosynthetic photon flux density incident on the leaf, 0.85 is the leaf absorptivity coefficient and 0.5 is a correction factor for PPFD assuming that half of the photons are absorbed by PSII and the other half by PSI.

2.8. Statistical Analysis

All experimental data were subjected to the statistical analysis of variance (ANOVA) using the SPSS software (SPSS version 22, Chicago, IL, USA). Mean separation of physiological parameters was performed with post-hoc Tukey’s HSD test (p ≤ 0.05).

3. Results

3.1. Air Temperature and Light Parameters

Under both greenhouses, the mean air temperature was higher than that recorded in open air, with a 22.0%, and 7.0% increase for the control and doped greenhouses, respectively (Figure 1). The minimum temperatures did not exhibit notable differences between the three growth conditions: the mean values were 13.5, 14.0, and 14.0 °C for the doped greenhouse, control greenhouse, and open-air conditions, respectively. Instead, differences in maximum temperatures were higher with the two greenhouses that elicited a 57.9% and 29.6% increase over external conditions.

The PMMA covering panels also modified the spectra of transmitted light inside of the greenhouse, compared with the outside solar light spectrum (Figure 2A). Most of the solar UVA radiation below 400 nm was effectively screened by both control and doped PMMA panels. Moreover, the solar UV radiation also excited the photoluminescence emission of Europium and Dysprosium particles embedded within the doped PMMA panels. This resulted in the peaks at 617 nm; 626 nm; 704 nm; and 706 nm enriching the orange—far-red region of the light spectrum inside the doped greenhouse (Figure 2B).

The PPFD in the PAR region (total photosynthetically active radiation between 400 and 700 nm) measured at different times during the day (on 4 June 2021) highlighted a shading effect of the doped PMMA panels compared with undoped panels (Figure 3). At h 14, PAR intensity in the doped greenhouse was 529 µmol m⁻² s⁻¹, about 36.3% lower than inside the Control greenhouse (1459 µmol m⁻² s⁻¹); at the same time, PAR intensity was 2042 µmol m⁻² s⁻¹ under direct sunlight. PAR measurements were taken concomitantly with the Chl a fluorescence measurements.

3.2. Lettuce Yield, and Growth Parameters

The lettuce marketable yield was significantly affected by the type of greenhouse cover material, with a 36% increase over the control plants (Figure 4). This was due to the increase in the fresh weight of lettuce heads (

Table 1. Lettuce growth parameters (head fresh weight (fw), number and mean weight of leaf, leaves dry matter (dm), and fresh weight, height and diameter of stem).

Treatment	Head	Leaf		Stem
	fw	Number	dm	fw	Height	Diameter
	g Head−1	n	%	g	cm	cm
Control	215.0	30.5	5.91	18.16	4.38	2.10
Doped	296.7	28.5	4.94	13.50	4.05	1.95
	*	ns	*	ns	*	ns

* = Significant at p ≤ 0.05; ns = not significant.

). As regards the growth parameters, significant differences were found for the dry matter of the leaves; it was lower in lettuce grown under the doped greenhouse (about −16.4%) (Table 1). In addition, stem height was significantly higher in the control (Table 1).

3.3. Color Parameters, Pigments and Nitrate Content of Lettuce Leaves

The greenhouse cover material significantly affected the CIELAB colour space parameters. For all three parameters, the higher values were recorded in lettuce grown under the doped greenhouse, with a 7.5%, 74.1%, and 47.8% increase, for brightness (L*), green (a*) and yellow (b*) intensity, respectively (

Table 2. Leaf colour parameters of the lettuce leaves. Lightness (L*) ranges from 0 (black, no reflection) to 100 (white, perfect diffuse reflection); a* ranges from green (–60) to red (+60); b* ranges from blue (−60) to yellow (+60).

Treatment	L*	a*	b*
Control	41.6	−4.64	15.27
Doped	44.7	−8.08	22.57
	**	**	**

** = Significant at p ≤ 0.01.

).

A significant effect of the greenhouse cover material was not observed for chlorophyll a, b, total chlorophylls, and carotenoids (

Table 3. Chlorophyll a, b, and total, carotenoids, and nitrate content of the lettuce leaves in greenhouse as affected by the cover material.

Treatment	Chlorophyll a	Chlorophyll b	Total Chlorophylls	Carotenoids	Nitrates
	mg g−1 fw	mg g−1 fw	mg g−1 fw	µg g−1 fw	mg kg−1 fw
Control	0.756	0.313	1.069	394.0	3065.4
Doped	0.894	0.411	1.304	394.7	3492.1
	ns	ns	ns	ns	*

* = Significant at p ≤ 0.05; ns = not significant.

). The nitrate content was higher in leaves of lettuces grown under doped greenhouse, with an increase of about 13.9% over the control lettuce plants (Table 3).

3.4. Antioxidant Activities, Total Phenols, and Total Ascorbic Acid of Lettuce Leaves

The tested antioxidant activities and total ascorbic acid of lettuce leaves did not exhibit any significant changes between the different greenhouse cover-material treatments. Total phenols were higher (+28.6%) under the control treatment (

Table 4. Hydrophilic antioxidant activity (HAA), ABTS antioxidant activity (ABTS), phenolics, and total ascorbic acid (TAA) of lettuce leaves in greenhouse as affected by the cover material.

Treatment	ABTS	HAA	Total Phenols	TAA
	mM Trolox 100g−1 dw	mM AA 100g−1 dw	mg Gallic Acid g−1 dw	mg g−1 fw
Control	23.18	8.50	3.19	26.99
Doped	21.51	8.49	2.48	24.69
	ns	ns	*	ns

* = Significant at p ≤ 0.05; ns = not significant.

).

3.5. Chlorophyll Fluorescence Parameters

The ΦPSII, or the effective PSII quantum yield, measures the fraction of absorbed light which is used by the Photosystem II to drive the photochemical reactions under steady-state photosynthetic lighting conditions. Therefore, ΦPSII is a measure of the plant photosynthetic “working” efficiency at certain light/temperature conditions. In our study, ΦPSII varied during the day (Figure 5A), following an opposite trend to the daily fluctuation in the solar light intensity. Compared to plants grown in the open air, the fluctuation in ΦPSII was restricted to within a narrower range under the control greenhouse and it was even further restricted under the doped greenhouse.

The electron transport rate (ETR) is a Chl a fluorescence LAS parameter which combines the ΦPSII with the PAR light level, and it is considered to be a proxy for net photosynthetic rate in the field, under the assumption that the photochemical and biochemical processes of photosynthesis are linearly related (i.e., when a plant is under heavy stress, a part of the electron flux is directed towards the dissipative energy process rather than towards carbon fixation, with an overall reduction of photosynthetic efficiency).

As a consequence of the different PAR intensities at h 08, 14 and 19, the ETR values were found to fluctuate significantly during the day, although fluctuations were attenuated under both doped and control greenhouses (Figure 5B).

In primary photochemical reactions of photosynthesis, a fraction of the absorbed light is not used for photosynthetic linear electron transports but is exploited in several protective mechanisms that form non-photochemical quenching (NPQ). The NPQ dissipates excess light energy, thus protecting the photosynthetic apparatus [35]. In this study, we estimated the NPQ(T) parameter and its components (the rapidly relaxing qE and the slow recovery qI) as described by Tietz and coworkers [30].

Non-photochemical quenching was closely related to the light environment in all three experimental conditions: the highest NPQ values were recorded at h 14 and the values were proportional to the incident light level, although the contributions of either qE or qI varied with time among the treatments (Figure 6A–C).

4. Discussion

The application of material able to convert light wavelength in greenhouse covers is a new frontier in agronomic research devoted to improving light use efficiency and productivity. Among the light conversion agents (organic or inorganic rare-earth complexes and fluorescent dyes), the inorganic rare-earth complexes are characterized by low price, ease of preparation and storage, oxidation resistance and high-temperature resistance. Moreover, they have high luminous efficiency, a wide light-wavelength conversion range and an emission spectrum that can match the absorption spectrum of chlorophyll [11]. In the current research, we found that the greenhouse cover doped with rare earth elicited a 36% increase in lettuce marketable yield compared to plants grown under the undoped cover. Nishimura et al. [36] tested a light conversion film able to convert green light to red light on several horticultural crops (lettuce, spinach, celery, and sweet potato). Specifically for lettuce, they reported that fresh and dry weight were higher under light conversion films. Kwon et al. [37] evaluated the effect of three greenhouse covering films with different spectrum conversion properties (red, blue, red + blue) on the growth and quality of lettuce, tomato, and melon. They reported that the red + blue film elicited an increase in leaves number and the fresh weight of lettuce. Wu et al. [38] also reported an increase in both the fresh and dry weight in the case of Chinese flowering cabbage grown under a film cover converting blue-violet to red-orange light. Contrastingly, we observed that the doped greenhouse determined a lower dry-matter percentage of leaves (−16.4%).

Notably, the doped PMMA panels had a lower light transmittance which resulted in a lower daytime temperature inside the doped greenhouse compared to the undoped PMMA panels. Considering the period of cultivation, this “shading effect” had a beneficial impact on plant growth parameters. On the other hand, several authors reported that the use of a shading net (comparable to the shading effect of the doped greenhouse), reduces temperature and solar radiation intensity, improving microclimate [39], and, therefore, the fresh yield in some cultivars of lettuce [40,41]. The high temperatures usually elicit a stem elongation that is a negative trait of lettuce quality. The doped film prevented this response, allowing a lower value of stem height (−7.5% compared to control plants). The improvement in the growth and yield of lettuce is probably due to the improvement in its photosynthetic activity. On the other hand, it is known that the red-orange (600–700 nm) wavelength is required for plant photosynthesis [11], and our PMMA panels were doped with photo-luminescent pigments emitting two different wavelengths, red and blue.

The measurements of Chl a fluorescence in vivo in the LAS allowed us to assess the efficiency of photochemistry under steady-state photosynthetic lighting conditions non-destructively at three different time points during the day. PAR and temperature conditions inside the greenhouses were substantially affected by the different cover materials. Overall, we observed a close relationship between the amplitude of PAR / Temp fluctuation, the effective photochemical efficiency (ΦPSII), and the electron transport rate (ETR) values. Since these parameters are considered proxies of the photosynthetic carbon fixation, these results suggest that the higher productive performance of plants under doped material corresponds to a minimal daily fluctuation, which is a constant photosynthetic rate throughout the daily cycle. A large four-fold increase in NPQ_(T) was observed at h 14 in the open air grown plants. Since the non-photochemical quenching of chlorophyll fluorescence is regarded as a photoprotection mechanism, the large activation of NPQ could be interpreted as a symptom of high-light stress in these plants [42]. At the same time, lettuce plants grown inside the doped greenhouse did not show any variation in NPQ_(T) compared to the morning measurements, while plants grown under the undoped PPMA greenhouse experienced an intermediate stress level. Overall, a close relationship between the PAR PPFD reaching the plants and the activation of NPQ_(T) was observed, suggesting a positive shading effect of the doped PMMA panels.

Moreover, at h 14, the qI_(T) component of NPQ_(T) was five times higher in open-air grown plants than in plants under doped material, confirming the higher extent of photo-inhibition in open-air grown plants compared to those under doped material. Since qI reflects all mechanisms that result in the light-induced decrease in the PSII quantum yield [43], we suggest that the doped PMMA greenhouse cover reduces the photo-inhibition of PSII in the central hours of the day. Although the qI_(T) is considered a slow relaxing component of non-photochemical quenching, as it recovers in the timescale of hours [44], at h 19 (i.e., five hours after reaching the midday peak), the lowest daily qI_(T) values were recorded for all treatments with no major differences among them. The relaxation to such low qI values indicates that in the evening the higher NPQ of open-air grown plants has to be attributed to qE rather than qI. At h 19, this could still result from a high trans-thylakoidal pH gradient, particularly in open-air grown plants and active violaxanthin de-epoxidase resulting in zeaxanthin formation and high qE values. Plant development and physiology are strongly influenced by the light spectrum of the growth environment. However, the metabolic pathways underlying the effects on the physiological, biochemical and nutritional traits of plants are not fully understood [45,46]. In terms of light quality, both red and blue light (supplied as monochromatic light or at different blue/red light ratios) have been shown to alter plant architectural development as well as its physiology [47]. At the molecular level, chlorophylls and carotenoids are key pigments operating in the photosynthetic pathway and the content of these pigments in plants is very responsive to light spectrum and intensity. Together with other factors (e.g., size and shape of leaves, number, size, and structure of thylakoids), the concentration of photosynthetic pigments affects the amount of light absorbed by leaves [48,49]. In addition, it is also known that chlorophyll biosynthesis requires light [50,51]: reportedly, red light does not seem to promote chlorophyll formation because of the decrease in tetrapyrrole precursor 5-aminolevulinic acid [52,53], while blue light was often reported to increase the accumulation of this pigment [52,54,55].

However, contrasting effects of blue light were reported, depending on the dose and duration of the treatment. For example, blue light was reported to stimulate photosynthesis by inducing stomatal opening [56,57,58], increasing stomatal conductance and intercellular CO₂ concentrations [59], or increases in leaf mass area (LMA), nitrogen and chlorophyll content [46]. Contrastingly, Abidi et al. [47] reported that blue light reduced the net CO₂ assimilation rate of fully expanded leaves in two rose cultivars, despite increasing stomatal conductance and intercellular CO₂ concentrations and the reduction in CO₂ assimilation under blue light being related to a decrease in photosynthetic pigment content, while the chl a/b ratio increases. As first proposed by Wheeler et al. [60], plant developmental response to blue light could be dependent on absolute blue-light levels, rather than the relative amount of blue light in the % of total PPFD. This was recently confirmed by Snowden et al. [61], who found that chlorophyll concentration significantly increased with increasing blue light in tomato, cucumber, radish, and pepper at high light levels, while in lettuce, chlorophyll content was little or not affected by blue-light intensity. In our case, since the light environment in our two experimental conditions (Control and Doped greenhouse) differed in both the light spectrum (Figure 2) and the light intensity (Figure 3), we could not draw any clear conclusion on the direct effect of either of the experimental factors on leaf pigment content without conducting further research.

The CIELAB color space parameters indicated that the leaves of lettuce grown under the Doped greenhouse were of a lighter and brighter green color compared with the Control. On its own, this finding may indicate a higher chlorophyll content and it may be in agreement with Nishimura et al. [36], who found that the SPAD values (an indirect measure of chlorophyll content and, in turn, of colour) of lettuce grown under light conversion films were better. However, we found no significant differences in the spectrophotometrically determined leaf pigment contents (chlorophyll a; chlorophyll b; carotenoids) between the treatments. This may be explained as follows: even though the CIELAB color space parameters may be used to estimate leaf chlorophyll content, to the best of our knowledge, an unequivocal straight correlation between these parameters cannot always be found. This is especially true in the case of colored-leaf plants containing a combination of chlorophylls, carotenoids and anthocyanins, such as the lettuce cv. Canasta, which we chose for our experiment. Recently, Li et al. [62] reported that the difference in leaf color among three cultivars (red, green, and mixed red and green) of A. bettzickiana is due to the combined effects of different chloroplast morphology and chlorophyll-to-anthocyanin ratios. This may also apply to lettuce cv. Canasta, where the differences in leaf color parameters (L*, a*, b*) between plants grown under the Control or the Doped greenhouse are likely to result from a combination of leaf anatomy, morphology and pigment content rather than from differences in chlorophyll content only.

Among the nutritional quality parameters, only total phenols were affected by greenhouse cover with lower values in plants grown under doped films. Finally, one of the most important quality traits of green leafy vegetables is the nitrate content in the leaves. It depends on several factors including fertilization (dose, types of fertilizers, etc. [63,64,65,66]) and climatic factors, above all light, which strongly affects nitrate reductase activity and, consequently, nitrate assimilation and accumulation [67]. The relationship between light and nitrate accumulation is inverse; however, in addition to the light intensity, the type of radiation also affects the nitrate content accumulation [68]. Our findings highlighted a minor increase in nitrate content in leaves, with values not exceeding the 4000 ppm threshold set for lettuce (in a protected environment and harvested between 1 April and 31 October) by the Regulation EU 1258/2011 [69].

5. Conclusions

Our results suggest that the use of greenhouse covering doped with light-modulating agents allows for improved crop performance combined with a low environmental impact. As such, the incorporation of rare-earth inorganic complexes within the greenhouse covering can be regarded as a promising technological innovation aimed at the development of sustainable agricultural practices.

From the physiological point of view, the light environment under the doped greenhouse had significant effects on the functioning of the photosynthetic apparatus. Over the course of the day, the midday depression of photosynthetic efficiency (ΦPSII) was very effectively attenuated, while little activation of the protective systems for energy dissipation at the thylacoidal level (NPQ) was recorded. Overall, the combined effect of these two factors resulted in steady photosynthetic efficiency and minimum light-energy dissipation during the whole light period, compared to both the control greenhouse and to the open-air grown plants. This ultimately resulted in increased plant growth and marketable yield without affecting quality traits.

Therefore, these results may lead to new developments in protected horticulture based on the exploitation of an innovative technology that can increase sustainable practices in agriculture while preserving technical and financial affordability.