Performance of the Photosynthetic Apparatus under Glass with a Luminophore Modifying Red-To-Far-Red-Light Ratio—A Case Study

The aim of this study was to examine the effect of the modified light spectrum of glass containing red luminophore on the performance of the photosynthetic apparatus of two types of lettuce cultivated in soil in a greenhouse. Butterhead and iceberg lettuce were cultivated in two types of greenhouses: (1) covered with transparent glass (control) and (2) covered with glass containing red luminophore (red). After 4 weeks of culture, structural and functional changes in the photosynthetic apparatus were examined. The presented study indicated that the red luminophore used changed the sunlight spectrum, providing an adequate blue:red light ratio, while decreasing the red:far-red radiation ratio. In such light conditions, changes in the efficiency parameters of the photosynthetic apparatus, modifications in the chloroplast ultrastructure, and altered proportions of structural proteins forming the photosynthetic apparatus were observed. These changes led to a decrease of CO2 carboxylation efficiency in both examined lettuce types.


Introduction
Plant growth and development, as well as crop productivity in the natural environment are limited by various stress factors. Among these, one of the most-significant is sunlight [1,2]. Light is essential for photochemistry, which drives primary production. It is also the most-important environmental signal that modifies physiological processes and determines the course of plant morphogenesis [3,4]. Light for energy production is absorbed by photosynthetic pigments: chlorophylls-which absorb red and blue light most efficiently, and carotenoids, which absorb blue light [5]. Plants also evolved a sophisticated system of photoreceptors that perceive and assess the intensity, amount, duration, and direction, as well as the spectral composition of light and translate it into information necessary to optimize plant function [6]. Typically, photoreceptors include: phytochromes, which sense mainly red and far-red light, but also light from the blue and green spectrum; cryptochromes and phototropins, receptive to blue, green, and ultraviolet A; and a UV-B photoreceptor, sensing ultraviolet B [6]. Furthermore, plants perceive changes in light of the photosynthetic apparatus of lettuce cultivated in the soil in a greenhouse. Furthermore, such a luminophore is employed to produce glass with integrated photovoltaic panels, which can be used in the future to build greenhouses. With the application of a photoluminescent pigment, part of the absorbed energy through the fiber-optic effect would be transferred to the solar panels and converted into electricity [28]. However, the addition of luminophore to the glass alters its optical properties, and therefore, it is necessary to evaluate the plants' response to the light conditions under such glass. Two types of lettuce were chosen for the study to see if the photosynthetic apparatus response mechanism to the applied conditions would be universal among closely related plants. Our results showed that the performance of the photosynthetic apparatus of both types of lettuce cultivated under glass with red luminophore differed significantly compared to the control conditions. The study indicated that the red luminophore used changed the sunlight spectrum, providing an adequate blue:red light ratio, while a decreased red:far-red radiation ratio. Regarding the lettuce types tested, such light conditions led to a decrease in CO2 carboxylation efficiency, resulting from the disruption of linear electron transport due to a limitation on the acceptor side of PSII and PSI.

Plant Material
The experimental material comprised two types of head lettuce (Lactuca sativa var capitata): butterhead and iceberg. Seeds of the butterhead-type cultivar Melodion were purchased from Enza Zaden Ltd. (Warsaw, Poland), and seeds of the iceberg cultivar from Elenas from Rijk Zwaan Ltd. (Blonie, Poland).

Cultivation Conditions
The experiments were conducted in 2020 at the University of Agriculture in Kraków (Poland) in the high-tech greenhouse of the Faculty of Biotechnology and Horticulture (transplants' production; 50°03′ N, 19°57′ E) and in small, temporary greenhouses located at the vegetable experimental station (main experiment; 50°08′ N, 19°85′ E). Lettuce seeds were germinated in 96-cell multi-pots (60 × 40 cm) filled with Florabalt Seed (Floragard Vertriebs GmbH, Oldenburg, Germany) (pH 5.6; N 140, P2O5 80, and K2O 190 mg•L −1 ) and kept in greenhouse conditions for 5 weeks. Before transplanting, the soil was fertilized with the fertilizer YaraMila Complex (5% N-NO3, 7% N-NH4); P-11% P2O5; K-18% K2O Mg-2.7% MgO; S-20% SO3; B-0.015%; Fe-0.20%, Mn-0.02%; Zn 0.02%) (Yara Poland Our results showed that the performance of the photosynthetic apparatus of both types of lettuce cultivated under glass with red luminophore differed significantly compared to the control conditions. The study indicated that the red luminophore used changed the sunlight spectrum, providing an adequate blue:red light ratio, while a decreased red:far-red radiation ratio. Regarding the lettuce types tested, such light conditions led to a decrease in CO 2 carboxylation efficiency, resulting from the disruption of linear electron transport due to a limitation on the acceptor side of PSII and PSI.

Plant Material
The experimental material comprised two types of head lettuce (Lactuca sativa var. capitata): butterhead and iceberg. Seeds of the butterhead-type cultivar Melodion were purchased from Enza Zaden Ltd. (Warsaw, Poland), and seeds of the iceberg cultivar from Elenas from Rijk Zwaan Ltd. (Blonie, Poland).

Evaluation of Photosynthetic Apparatus Performance
The photosynthetic apparatus performance was evaluated on living plants after four weeks of cultivation.

Photosynthetic Pigment Concentration Assessment
Individual leaves were collected from plants on which chlorophyll a fluorescence and gas exchange were measured. Photosynthetic pigment content analyses were conducted according to the spectrophotometric method of Wellburn [29]. Immediately after collection, leaves were weighted and homogenized with 80% acetone (30 mL) in ice-cold conditions. Samples were centrifuged for 15 min at 4800× g at 4 • C. Diluted extracts were measured at 470, 646, and 663 nm, which correspond to chlorophyll a, chlorophyll b, and carotenoids' absorbance, respectively. The absorbance of samples was measured using the double-beam spectrophotometer U-2900 (Hitachi High-Technologies Corporation, Tokyo, Japan). The content of the photosynthetic pigments were calculated using equations. Moreover, total chlorophylls (Chl a + b) and the ratios of the pigments (Chl a/b) were calculated.

Transmission Electron Microscopy Observation
After 4 weeks of cultivation, fragments of leaves were collected. The material was fixed in 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M cacodyl buffer (pH 7.2) for 2 h. The sections were then washed four times in cacodyl buffer and fixed in a solution of 2% osmium tetroxide in cacodyl buffer for 3 h at 4 • C. After this time, the material was dehydrated through a stepwise ethanol series and replaced with propylene oxide, then embedded in glycidyl ether 100 epoxy resin (SERVA, Heidelberg, Germany). Resin polymerization was carried out at 65 • C for 24 h. Semi-thin sections were prepared with a Jung RM 2065 (Leica, Wetzlar, Germany) microtome, stained with methylene blue and azure B and examined under a light microscope (Olympus-Provis, Tokyo, Japan). Ultra-thin sections were prepared with a Leica Ultracut UCT microtome (Leica, Wetzlar, Germany), collected on formvar-coated grids and stained with uranyl acetate, followed by lead citrate for 1 min. The examination was performed in a transmission electron microscope (Morgagni 268D, Hillsboro, OR, USA). Additionally, on ten randomly chosen chloroplasts of each lettuce type and condition, we measured the chloroplast size-length (using scale bars) and counted the number of grana, starch grains, and plastoglobuli.

Guaiacol Peroxidase Activity Evaluation
The guaiacol peroxidase (POD) activity was assayed according to Lűck [36] as follows: Leaf samples (2 g) were homogenized in an ice bath (4 • C) in 10 mL of 50 mM potassium phosphate buffer (pH 6.2). The mixture was centrifuged at 13.968× g for 15 min at 4 • C. Then, 2 mL of plant extracts diluted five times was mixed with 2 mL of potassium phosphate buffer and 0.2 mL of a 1% solution of p-phenylenediamine. The peroxidase activity was assessed by measuring absorbance at 485 nm on a UV-VIS Helios Beta spectrophotometer (Spectronic Unicam, Cambridge, UK) one minute and two minutes after the addition of 0.2 mL 0.1% H 2 O 2 to each sample. A blind sample was prepared as described above, but without H 2 O 2 addition. A unit of enzyme activity (U) is expressed as an increase in absorbance of 0.1 per minute.

Glutathione Content Evaluation
The reduced form of glutathione (GSH) was assayed using the method described by Guri [37], with some modifications. For this, 2 g of fresh leaves were chopped and homogenized with 10.0 mL of 0.5 mM EDTA and 3% trichloroacetic acid (TCA) in an ice bath (4 • C). The extract was centrifuged at 13.968× g, for 10 min, at 4 • C. The supernatant (2 mL) was mixed with 5 mL K-phosphate buffer (pH = 7.0) to bring the solution pH to the value of ca. 7.0. Next, 1 mL of K-phosphate buffer and 0.1 mL Ellman's reagent (5,5dithiobis-2-nitrobenzoic acid (DTNB)) (Merck KGaA, Darmstadt, Germany) were added to 2 mL of this mixture. The content of reduced glutathione was assessed by measuring absorbance at 412 nm on a UV-VIS Helios Beta spectrophotometer, against a blind sample, prepared as described above, but with 1.1 mL K-phosphate buffer and without Ellman's reagent. The GSH content was calculated based on the calibration curve of GSH and expressed in mg per 1 g fresh weight (FW).

Statistical Analyses
STATISTICA 12.0 (StatSoft Inc., Tulsa, OK, USA) was used to perform the statistical analyses. The results, within each parameter and lettuce type, were subjected to one-way analysis of variance (ANOVA). The Duncan post hoc test at p ≤ 0.05 was used to determine the significance of the differences between the means. All of the spectrophotometric determinations were made in five replications. Chl a fluorescence measurements were performed in ten replications. Gas exchange measurements and electrophoresis were performed in three replications.

Photosynthetic Pigment Concentration
To evaluate changes in the antennae of the lettuce photosynthetic apparatus cultivated in the red glasshouse, the content and ratio of the photosynthetic pigments were analyzed. After 4 weeks of cultivation, there were no changes in the concentration of the chlorophylls (Chl a, Chl b, Chl a + b) and carotenoids (Car) in iceberg lettuce leaves in the red glasshouse in comparison to the control (Table 1). Similarly, no change in the Chl a/b ratio was found (Table 1), whereas, in the leaves of butterhead lettuce, a significant increase of the chlorophylls' and no change in the carotenoids' concentration were observed in the red glasshouse compared to the control. However, the ratio of Chl a/b increased in the leaves of plants grown in the red glasshouse (Table 1).

Chl a Fluorescence
Chl a fluorescence was measured to describe the efficiency of PSII photochemistry. The values of the measured and calculated PSII parameters were normalized against the control (set as 1) and presented on radar charts (Figure 2a,b). Raw values of these parameters are presented in the Supplementary Materials: Table S1. The fluorescence parameters (minimum (F 0 ), maximum (Fm), and variable (Fv) fluorescence) decreased significantly in both types of lettuce examined grown in the red glasshouse compared to the control (Figure 2a,b, Table S1). The maximum quantum yield of PSII (Fv/Fm) and the activity of the water-splitting complex (Fv/F 0 ) did not change in the red glasshouse in both types of lettuce (Figure 2a,b, Table S1). The relative variable fluorescence at 2 ms (V J ) and relative variable fluorescence at 30 ms (V I ) increased significantly in both types of lettuce examined cultivated in the red glasshouse (Figure 2a,b, Table S1). The reduced plastoquinone pool (Area) and total electron carriers per reaction center (RC) (Sm) decreased in both lettuce types in the red glasshouse (Figure 2a,b, Table S1). The parameters describing yield or flux ratios (ϕPo, ϕEo, ϕRo, δRo, ρRo, and ψEo) decreased significantly in the tested plants cultivated in the red glasshouse (Figure 2a,b, Table S1). Specific fluxes or activities per RC (ABS/RC, TRo/RC, ETo/RC, and DIo/RC) increased significantly in butterhead and iceberg lettuce (Figure 2a,b, Table S1). The trapped energy flux per cross-section (CS) (TRo/CSo) and electron transport flux per CS (ETo/CSo) decreased, while the dissipated energy flux per CS (DIo/CSo) increased in both butterhead and iceberg lettuce (Figure 2a,b, Table S1), whereas, the amount of active PSII RCs per CS (RC/CSo) decreased significantly also in both examined lettuce types cultivated in the red glasshouse in comparison to the control glasshouse (Figure 2a,b, Table S1).

Gas Exchange
Measurements of gas exchange using infrared were carried out to determine the photosynthetic efficiency of the examined plants. Net photosynthesis (Pn) decreased significantly in both tested lettuce types in the red glasshouse compared to the control (Figure 3a,c). In turn, transpiration (E) and stomatal conductance (Gs) decreased significantly only in iceberg lettuce (Figure 3b), while the intercellular CO 2 concentration (Ci) did not change either in butterhead or iceberg lettuce (Figure 3a,c). Moreover, the leaf photosynthesis efficiency of iceberg lettuce growing in the red glasshouse was significantly lower in low light intensity (0-50 µmol·m −2 ·s −1 ) and in the range of light intensity between moderate (100 µmol quanta m −2 ·s −1 ) and high (1500 µmol quanta·m −2 ·s −1 ) (Figure 3d), while the leaf photosynthesis efficiency of butterhead lettuce, cultivated in the same conditions, was lower in light intensity between 100 and 1500 µmol quanta·m −2 ·s −1 (Figure 3b).

Structural and Functional Photosynthetic Proteins
The quantitative participation of photosystem I (PsaA, PsaB, Lhca1), photosystem II (PsbB, PsbC, PsbO, PsbQ, and Lhcb1), proteins, RuBisCo (RbcL), and RuBisCo activase (RA) was estimated by SDS-PAGE and immunoblotting in lettuce isolated chloroplasts. The content of PsaA and PsaB, core proteins of photosystem I, increased both in butterhead and iceberg lettuce types growing in the red glasshouse (Figure 4a,b). The Lhca1 content increased in iceberg lettuce chloroplasts, but did not change in butterhead ones (Figure 4a,b). In turn, the content of the core antenna of PSII, PsbB, increased in both lettuce types cultivated in the red glasshouse, whereas PsbC content did not change either in butterhead or iceberg chloroplasts (Figure 4a,b). Similarly, the content of Lhcb1, the LHCII type I chlorophyll a/b-binding protein, did not change in either lettuce type cultivated in the red glasshouse (Figure 4a,b). Moreover, the content of the subunits constituting the oxygen evolving complex (PsbO and PsbQ) did not change in butterhead and iceberg lettuce chloroplasts (Figure 4a,b). In contrast, a decrease in the content of RuBisCo activase (RA) was recorded in both lettuce types' chloroplasts ( Figure 4a,b), while the content of the RuBisCo large subunit (RbcL) decreased only in butterhead lettuce chloroplasts in the red glasshouse (Figure 4a). iceberg lettuce chloroplasts (Figure 4a,b). In contrast, a decrease in the content of RuBisCo activase (RA) was recorded in both lettuce types' chloroplasts ( Figure 4a,b), while the content of the RuBisCo large subunit (RbcL) decreased only in butterhead lettuce chloroplasts in the red glasshouse ( Figure 4a).

Chloroplast Ultrastructure
The TEM observation revealed the ultrastructure of chloroplasts from the leaves of butterhead and iceberg lettuce cultivated in the control and red glasshouses ( Figure 5, Supplementary Materials: Figures S1 and S2). Significant differences were observed in the ultrastructure of butterhead and iceberg lettuce chloroplasts from plants cultivated in the red glasshouse in comparison to the control (Supplementary Materials: Figure S3). Under control conditions, the chloroplasts had a regular shape, numerous grana ( Figure S3a,b), and a clustered arrangement of thylakoids (Figures 5a,b,e,f, S1a,d and S2a,d). There were no differences in the size (length) of butterhead chloroplasts from plants cultivated in control and red glasshouse conditions ( Figure S3a). However, iceberg chloroplasts of plants from the red glasshouse were significantly larger (longer) than the chloroplasts of plants from the control conditions ( Figure S3b). Moreover, we noted also numerous plastoglobuli (Figures 5a,b,e,f, S1a,d, S2a,d and S3a,b) and, in butterhead lettuce, visible starch grains (Figures 5a,b, S1a,d and S3a). In the red glasshouse conditions, chloroplasts had less numerous grana and plastoglobuli (Figures 5c,d,g,h, S1e,f, S2e,f and S3a,b). In iceberg lettuce chloroplasts, additionally poorly visible grana, a looser arrangement of thylakoids, and practically no starch grains were observed (Figures 5g,h no differences in the size (length) of butterhead chloroplasts from plants cultivated in control and red glasshouse conditions ( Figure S3a). However, iceberg chloroplasts of plants from the red glasshouse were significantly larger (longer) than the chloroplasts of plants from the control conditions ( Figure S3b). Moreover, we noted also numerous plastoglobuli (Figures 5a,b,e,f, S1a,d, S2a,d, and S3a,b) and, in butterhead lettuce, visible starch grains (Figures 5a,b, S1a,d, and S3a). In the red glasshouse conditions, chloroplasts had less numerous grana and plastoglobuli (Figures 5c,d,g,h, S1e,f, S2e,f, and S3a,b). In iceberg lettuce chloroplasts, additionally poorly visible grana, a looser arrangement of thylakoids, and practically no starch grains were observed (Figures 5g,h, S2e,h, and S3b).

Discussion
According to the available literature, an optimally balanced ratio of blue to red light (1:1-1:7) significantly improves the photosynthetic capacity of leaves [38,39]. In the presented study, the ratio of blue to red light (660 nm:450 nm) was approximately 1:1 in the control conditions and 1:2 under glass with red luminophore (Figure 1a,b). Despite the correct ratio of red and blue light, abnormalities in the structure and function of the photosynthetic apparatus were observed in both examined lettuce types cultivated in the red

Discussion
According to the available literature, an optimally balanced ratio of blue to red light (1:1-1:7) significantly improves the photosynthetic capacity of leaves [38,39]. In the pre-sented study, the ratio of blue to red light (660 nm:450 nm) was approximately 1:1 in the control conditions and 1:2 under glass with red luminophore (Figure 1a,b). Despite the correct ratio of red and blue light, abnormalities in the structure and function of the photosynthetic apparatus were observed in both examined lettuce types cultivated in the red glasshouse. These differences may be explained by the influence of other factors, including the other aspects of the spectral composition of the light used. The relevance of a reasonably high blue:red light ratio is the subject of numerous studies [4,19,21,22,40,41]. In contrast, the contribution and impact of far-red radiation and its ratio to red light is very often neglected. Most studies dealing with this topic focus on the far-red and redlight-induced photomorphogenic response associated with phytochrome induction [24]. Among studies that focus on the structure and function of the photosynthetic apparatus, there is a considerable discrepancy in the description of plant responses to the ratio of red to far-red [24,42,43]. In our study, the red:far-red (660 nm:720 nm) ratio in red glasshouse was approximately 1:0.6 in comparison to 1:0.8 in the control glasshouse. Few studies indicate that a low red:far-red ratio leads to a reduction in the number of grana thylakoids and their stacking degree in the ultrastructure of chloroplasts [24]. Our results showed that the butterhead lettuce chloroplast ultrastructure was less susceptible to a changed spectral composition than iceberg lettuce, which were significantly longer than the control ones ( Figure S3). In contrast, more far-red light, in tobacco studies, caused chloroplast elongation [44]. However, chloroplasts of both lettuce types in the red glasshouse had significantly less grana, starch grains, and plastoglobuli ( Figure S3). Meanwhile, studies of other authors indicated a very different response of the chloroplast ultrastructure (decrease or increase of grana thylakoids) to excessive or deficient far-red light [24,44,45].
Chloroplast ultrastructural changes arise from alterations in the structure of the photosynthetic apparatus, of which protein complexes (e.g., PSII, PSI, LHCII, LHCI) are located in the membranes, building the grana and stroma thylakoids [11,46]. In this study, under enhanced red radiation, an increase in chlorophyll pigments was observed in butterhead lettuce accompanied by an increase in the content of the cortical subunits of PSI, i.e., PsaA and PsaB (Figure 4a), which are responsible for light harvesting, charge separation, and electron transport [47]. Indeed, only Chl a is found in photosystems [11], so an increase in the Chl a/b ratio (Table 1) is associated with an increased proportion of photosystem proteins relative to antenna proteins, as observed in butterhead lettuce (Figure 4a). In contrast, iceberg lettuce showed no change in pigment content, but an increase in PsaA, PsaB, and Lhca1-a chlorophyll a/b-binding protein of LHC of PSI (Figure 4b). Since PSI is localized in the stroma thylakoids, an increase in the content of these subunits is associated with a change in chloroplast structure, where the proportion of stroma thylakoids increases and the stacking of grana thylakoids decreases [11].
The structural modifications described above were accompanied by changes in the function and efficiency of the photosynthetic apparatus. Although in plants grown in the red greenhouse, no electron limitation was observed on the donor side of PSII-no change in OEC capacity and structure, as evidenced by unchanged PsbO and PsbQ content ( Figure 4) and Fv/F 0 ( Figure 2)-there was a reduction in the number of open reaction centers (RC) of PSII among all active reaction centers (V J increase; Figure 2). A decrease in the efficiency of electron transporters (Area decrease; Figure 2) was also observed. There was a reduction in the content of the membrane PQ pool (Sm decrease; Figure 2) and, within this pool, a decrease in its rapidly reducing fraction (V I increase; Figure 2), as well as the reserve PQ pool accumulated in plastoglobuli, the number of which decreased ( Figure S3). Moreover, in both types of lettuce in the red greenhouse, a limitation in electron transport was observed both on the acceptor side of PSII, between Q A and Q B (ϕEo decrease; Figure 2) and on the acceptor side of PSI (ρRo, δRo, ϕRo decrease; Figure 2). The limitation on the acceptor side of PSI may result from the efficiency of the Calvin-Benson cycle, especially the amount and activity of RuBisCo [33]. A high proportion of far-red light activates RuBisCo [48]. On the other hand, the amount of RuBisCo also depends on light intensity, not only on light quality [49]. In addition, RuBisCo activity is regulated by the stroma pH, which is too low when electron transport is not efficient [50]. In our study, we observed both a decrease in activity (RA decrease; Figure 4) and in the amount of RuBisCo (RbcL decrease; Figure 4). All this led to a decrease in CO 2 carboxylation efficiency (Pn decrease; Figure 3). The limitation on the acceptor side of PSII and PSI in the absence of a limitation on the donor side of PSII suggested the elevated generation of reactive oxygen species around PSII, as evidenced by the catalase content increase ( Figure 6) and, in iceberg lettuce, also by the higher glutathione content ( Figure 6). Regarding the functioning of the photosynthetic apparatus, some researchers point to an increased linear electron transport and photosynthetic intensity in a reduced red:farred ratio [51]. The meaning of far-red light is related to the enhanced oxidation of photosystem I, which, in combination with an efficiently functioning NADH thioredoxin reductase (NTRC), leads to the oxidation of plastocyanin and ferrodoxin. Furthermore, far-red radiation induces cyclic electron transport around PSI via both the PGR5 and PGRL pathways and NDH [52]. As a result, it enables efficient linear electron transport, as well as cyclic transport around PSI [52]. In our study, the light conditions in the red glasshouse were characterized by increased in the red:far-red ratio relative to the control, which resulted in the aforementioned changes in chloroplast ultrastructure and also the described limitation on the acceptor side of PSI, resulting from the lack of efficient PSI oxidation, consequently leading to the disruption of linear electron transport.

Conclusions
Our studies indicated that the ultrastructure and function of the photosynthetic apparatus of both lettuce types studied is more significantly affected by the respective red:far-red ratio than by the blue:red ratio. Despite the high value of the blue:red ratio, a low red:far-red ratio implies a decrease in the intensity of CO2 carboxylation, resulting from the disruption of linear electron transport due to the limitation on the acceptor side of PSII and PSI. The study indicated that the red luminophore used provides an adequate blue:red ratio, while a decreased red:far-red ratio. The disruption of photosynthetic efficiency observed in lettuce in our experiments may not necessarily be observed in other species grown under such light conditions, which requires further research.
Supplementary Materials: The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Table S1. Raw values of structural and functional parameters of photosynthetic apparatus of butterhead and iceberg lettuce types cultivated in transparent (control) and red glasshouses. Figure S1. Chloroplasts ultrastructure of butterhead lettuce type cultivated in trans- Regarding the functioning of the photosynthetic apparatus, some researchers point to an increased linear electron transport and photosynthetic intensity in a reduced red:far-red ratio [51]. The meaning of far-red light is related to the enhanced oxidation of photosystem I, which, in combination with an efficiently functioning NADH thioredoxin reductase (NTRC), leads to the oxidation of plastocyanin and ferrodoxin. Furthermore, far-red radiation induces cyclic electron transport around PSI via both the PGR5 and PGRL pathways and NDH [52]. As a result, it enables efficient linear electron transport, as well as cyclic transport around PSI [52]. In our study, the light conditions in the red glasshouse were characterized by increased in the red:far-red ratio relative to the control, which resulted in the aforementioned changes in chloroplast ultrastructure and also the described limitation on the acceptor side of PSI, resulting from the lack of efficient PSI oxidation, consequently leading to the disruption of linear electron transport.

Conclusions
Our studies indicated that the ultrastructure and function of the photosynthetic apparatus of both lettuce types studied is more significantly affected by the respective red:far-red ratio than by the blue:red ratio. Despite the high value of the blue:red ratio, a low red:far-red ratio implies a decrease in the intensity of CO 2 carboxylation, resulting from the disruption of linear electron transport due to the limitation on the acceptor side of PSII and PSI. The study indicated that the red luminophore used provides an adequate blue:red ratio, while a decreased red:far-red ratio. The disruption of photosynthetic efficiency observed in lettuce in our experiments may not necessarily be observed in other species grown under such light conditions, which requires further research.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/cells12111552/s1, Table S1. Raw values of structural and functional parameters of photosynthetic apparatus of butterhead and iceberg lettuce types cultivated in transparent (control) and red glasshouses. Figure S1. Chloroplasts ultrastructure of butterhead lettuce type cultivated in transparent (control) (a-d) and red (e-f) glasshouses. Scale bars: 1 µm (a-e,g) 0.5 µm (f,h). Figure S2. Chloroplasts ultrastructure of iceberg lettuce type cultivated in transparent (control) (a-d) and red (e-f) glasshouses. Scale bars: 1 µm. Figure S3. Chloroplasts size and parameters (number of grana, starch grains and plastoglobuli) of butterhead (a) and iceberg (b) lettuce type cultivated in transparent (control) and red glasshouses. * statistically significant difference within each parameter at p ≤ 0.05; (n = 10).

Conflicts of Interest:
The funders had no role in the design of the study; in the collection, analyses, or interpretation of the data; in the writing of the manuscript; nor in the decision to publish the results. Fluorescence intensities at 50 µs F100µs