**1. Introduction**

The application of light-emitting diodes (LED) in horticultural lighting systems provides new possibilities for light intensity and light spectrum fine regulation along with a significant reduction in energy consumption [1–3]. A breathtaking possibility to modulate the LED lighting spectrum can also help in promoting the accumulation of important plant metabolites, which are often associated with nutraceutical properties, as has been shown in various crops, including lettuce [4]. The set-up of plant-specific light protocols for their cultivation is a critical phase in improving the sustainability of indoor growing systems [2].

Besides photosynthesis, plants are capable of perceiving and processing information with light signals from their biotic and abiotic surroundings for optimal growth and development [5]. Reviews of studies on light quality effects on plant growth and development can be found elsewhere [6–8]. Red and blue are generally recognized as the most important light regions necessary for plant development and growth [3]. However, other wavelengths

**Citation:** Tarakanov, I.G.; Tovstyko, D.A.; Lomakin, M.P.; Shmakov, A.S.; Sleptsov, N.N.; Shmarev, A.N.; Litvinskiy, V.A.; Ivlev, A.A. Effects of Light Spectral Quality on Photosynthetic Activity, Biomass Production, and Carbon Isotope Fractionation in Lettuce, *Lactuca sativa* L., Plants. *Plants* **2022**, *11*, 441. https://doi.org/10.3390/plants11030441

Academic Editors: Valeria Cavallaro and Rosario Muleo

Received: 23 December 2021 Accepted: 3 February 2022 Published: 5 February 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

(such as those corresponding to yellow or green colors) could also have a role in affecting the quality of crops [6]. Blue light is involved in a wide range of plant processes such as phototropism, photomorphogenesis, stomatal opening, and leaf photosynthetic functioning [9]. Most studies assessing the effects of blue light (blue LEDs) on the leaf or whole plants have either compared their response to a broadband light source with the response to blue-deficient light [10] or plants grown under red light alone [11,12]. On the other hand, red LEDs emit a narrow spectrum of light (660 nm) that is close to the maximum absorbance for both chlorophyll and phytochromes. The absorption of blue and red light (LEDs) by plants has been measured as 90% [13], which indicates that plant development and physiology is strongly influenced by blue and red light [6]. The effects of green light tend to reverse the processes established by red and/or blue light. In this way, green light may be functioning in a manner similar to far-red light, informing the plant of photosynthetically unfavorable conditions and triggering adaptative responses [14]. Many studies have been reported on several crops grown under deficiency/efficiency or using a combination of red and blue light at different wavelengths [15,16] to investigate their effects on plant growth and development. While red light promotes biomass accumulation, growth, and photosynthesis in lettuce, blue LED light is effective in stimulating photomorphogenesis and adaptive phenomena such as the stomata-opening/closing-regulation mechanism, as well as biomass accumulation and chlorophyll and anthocyanin biosynthesis [3,17]. A positive growth response to the combination of blue and red light was confirmed in Batavia lettuce plants [18]. Green LED light regulates leaf expansion, stem stretching, and stomatal conductance. Moreover, it has been shown that green LED light addition leads to greater dry mass accumulation and growth stimulation [19].

The plant perceives light environment signals by means of photosynthetic apparatus (PSA) and specific photoreceptors sensitive to different light spectral regions. Blue and red light are not equal in their effects on photoreceptors: red light is perceived in addition to PSA by phytochromes only, and blue light is absorbed by both phytochromes and blue-light receptors (cryptochromes, phototropins) [20]. Blue light influences a greater number of photoreceptors and is functionally more versatile. It is most effective in stimulating the transcription of photosynthesis-related genes (via cryptochromes and phytochromes) [21]. Interestingly, barley plants grown with monochromatic red light demonstrated specific organization of chloroplast membranes (shaggy-formed grana) and light-harvesting complexes (increased energy transfer to PSI, possibly due to spillover promoted by this particular granum structure) [20]. These specific responses can be related to contradictory information from the photoreceptors; the signals from the phytochromes and photosynthetic apparatus indicate the incidence of light, while the lack of a signal from the blue-light receptors can be misinterpreted as darkness [20]. Most of the negative monochromatic red-light effects can be avoided by the addition of blue light [22–24]. Furthermore, a combination of red and blue light in certain cases can result in synergetic effects in biomass accumulation [25,26] Plant photosynthesis and growth, directly or indirectly, can also be mediated by the photoreceptor response. Additionally, chloroplasts play an important role in photoreceptor-mediated control of photomorphogenic responses [27]. The main obstacle in the transition to LED lighting in crop production is that it involves a complex system change beyond lighting (e.g., plant light recipes, which are species- and often cultivardependent), resulting in serious associated costs [28]. Lighting systems using specific wavelengths are capable of target compound biosynthesis fortification; however, special attention has to be paid to the stress the artificial light may cause in the photosynthesis and biomass accumulation [29]. To explore the action mode of different light spectrum regions, various experimental approaches are used. Thus, in the studies on the blue-light effects, plant responses to a broadband light source with a response to blue-region-deficient light were compared [10] with plants grown under red light alone [11]. So, the experimental set up can include studies on the effects of monochromatic irradiation. Additionally, plant responses to photosynthetically active radiation (PAR) missing distinct spectrum regions can be investigated [30].

In our studies with lettuce plants, we have used both screens mentioned above, emphasizing research on light spectral quality effects on the carbon isotope composition of plant biomass (Section 2.3). It is known that plant cells are able to fractionate carbon isotopes in the light and in the dark [31,32]. The carbon isotope composition of plant leaf biomass is mainly related to the light processes, CO<sup>2</sup> assimilation, and photorespiration [32,33]. The <sup>12</sup>C enrichment of plant biomass during CO<sup>2</sup> assimilation occurs at Calvin cycle entry during RuBP carboxylation. During photorespiration, carbon isotope fractionation occurs with the opposite sign, thus reducing the effect of CO<sup>2</sup> assimilation and enriching biomass with <sup>13</sup>C. The isotope effect of photosynthetic assimilation and photorespiration are coupled by a key photosynthetic enzyme, Rubisco, that oscillates from CO<sup>2</sup> assimilation to photorespiration and back [34,35]. The effects of monochromatic light and other unique artificial light treatments on the carbon isotope fractioning have not been investigated until now. In our studies with lettuce plants, we have used both screens mentioned above, emphasizing research on light spectral quality effects on the carbon isotope composition of plant biomass (Section 2.3). It is known that plant cells are able to fractionate carbon isotopes in the light and in the dark [31,32]. The carbon isotope composition of plant leaf biomass is mainly related to the light processes, CO2 assimilation, and photorespiration [32,33]. The 12C enrichment of plant biomass during CO2 assimilation occurs at Calvin cycle entry during RuBP carboxylation. During photorespiration, carbon isotope fractionation occurs with the opposite sign, thus reducing the effect of CO2 assimilation and enriching biomass with 13C. The isotope effect of photosynthetic assimilation and photorespiration are coupled by a key photosynthetic enzyme, Rubisco, that oscillates from CO2 assimilation to photorespiration and back [34,35]. The effects of monochromatic light and other unique artificial light treatments on the carbon isotope fractioning have not been investigated until now.

blue-light effects, plant responses to a broadband light source with a response to blueregion-deficient light were compared [10] with plants grown under red light alone [11]. So, the experimental set up can include studies on the effects of monochromatic irradiation. Additionally, plant responses to photosynthetically active radiation (PAR) missing

*Plants* **2022**, *11*, x FOR PEER REVIEW 3 of 17

distinct spectrum regions can be investigated [30].

#### **2. Materials and Methods 2. Materials and Methods**

#### *2.1. Plant Material 2.1. Plant Material*

Lettuce, *Lactuca sativa* L., plants of the Aficion RZ cultivar were used in our studies. This is Batavia-type lettuce, leaves with strongly wavy edge, light green. Batavia lettuce is highly appreciated in the market due to the variability in shape, color, texture, and taste. As for the nutritional value, it is a source of vitamin A, niacin, riboflavin, thiamine, Ca, Fe, K, Mn, Se, and β-carotene [36]. Aficion cultivar is widely grown in greenhouses and vertical farms with artificial lighting. Lettuce, *Lactuca sativa* L., plants of the Aficion RZ cultivar were used in our studies. This is Batavia-type lettuce, leaves with strongly wavy edge, light green. Batavia lettuce is highly appreciated in the market due to the variability in shape, color, texture, and taste. As for the nutritional value, it is a source of vitamin A, niacin, riboflavin, thiamine, Ca, Fe, K, Mn, Se, and β-carotene [36]. Aficion cultivar is widely grown in greenhouses and vertical farms with artificial lighting.

#### *2.2. Cultivation Conditions 2.2. Cultivation Conditions*

Plants were grown in growth chambers (Urbangrower 150, China; Figure 1) with various light treatments according to experimental layout described in Section 2.3. Each chamber had dimensions of 1.50 <sup>×</sup> 0.90 <sup>×</sup> 2.00, 2.7 m<sup>3</sup> , with gloss white walls. Chambers were supplied with fans; day/night temperature was 20/18 ◦C, with less than 1 ◦C variation over time and 1 ◦C variation among chambers. Plants were grown in growth chambers (Urbangrower 150, China; Figure 1) with various light treatments according to experimental layout described in Section 2.3. Each chamber had dimensions of 1.50 × 0.90 × 2.00, 2.7 m3, with gloss white walls. Chambers were supplied with fans; day/night temperature was 20/18 °C, with less than 1 °C variation over time and 1 °C variation among chambers.

**Figure 1. Figure 1.**  Plant-growing chambers with various light environments. Plant-growing chambers with various light environments.

Plants were grown in 2 L vegetational vessels (3 plants in each container). Seeds were sown directly into the commercial neutralized peat-based substrate "Agrobalt-C" (Pindstrup, Pskov region, Russia) with pH 6.0–6.5 and complete macro- and micronutrient supply including 150 mg L−<sup>1</sup> [NH<sup>4</sup> <sup>+</sup> andNO<sup>3</sup> <sup>−</sup>], 270 mg L−<sup>1</sup> P2O5, and 300 mg L−<sup>1</sup>

K2O. Substrate humidity was maintained at 70% of full water capacity, watering up to calculated weight. PPFD at ±5%. To provide uniform PPFD, plant pots were moved and rotated within the marked uniform light platform every second day.

Plants were grown in 2 L vegetational vessels (3 plants in each container). Seeds were sown directly into the commercial neutralized peat-based substrate "Agrobalt-C" (Pindstrup, Pskov region, Russia) with pH 6.0–6.5 and complete macro- and micronutrient supply including 150 mg L−1 [NH4+ andNO3−], 270 mg L−1 P2O5, and 300 mg L−1 K2O. Substrate humidity was maintained at 70% of full water capacity, watering up to calculated

Plant chambers were illuminated with lamps consisting of various light-emitting diode (LED) bars specifically designed to provide a custom spectrum in each chamber. Fixtures consisted of light modules with tunable light-emitting diodes varying in wavelength

Four types of high-performance narrow-band 3-Watt LEDs (Estar Technology, Changchun, China) were used: short-wave red (∆λ0.5 = 623 ÷ 641 nm, λmax = 632 nm), longwave red (∆λ0.5 = 646 ÷ 674 nm, λmax = 660 nm), far-red (∆λ0.5 = 727 ÷ 751 nm, λmax = 739 nm), and blue (∆λ0.5 = 452 ÷ 477 nm, λmax = 465 nm). The control light treatment included all 4 types of LEDs, and in each of the other regimes one of them was excluded (except shortwave red) in order to elucidate the wavelength that affected distinct crop physiological processes. Short-wave red was used as an additional background spectral region to provide chlorophyll *a* excitation in the absence of long-wave red light. The same daily light integral (DLI) of 9.72 mol m−2 d−1 was maintained in all the treatments with photosynthetic photon flux density (PPFD) 150 µmol m−2 s−1 , photoperiod 18 h. Spectra of the resulting lamp systems were measured with a spectrometer UPRtek PG100N (Taiwan). To measure the PPFD in the PAR region, an LI-191R quantum sensor with an LI-250A data logger (LI-COR Biosciences, NE, USA) was used. It was measured at the top of the plant canopy (the distance from the light source was ≥50 cm), and each chamber was adjusted to maintain

and spectral composition of the emitted light over wide ranges (Figure 2).

#### *2.3. Light Treatments* In the experiment on the red and blue monochromatic light effects, two types of tunable LEDs (Cree, USA) were used: red (∆λ0.5 = 647 ÷ 671 nm, λmax = 659 nm) and blue (∆λ0.5

weight.

*2.3. Light Treatments* 

Plant chambers were illuminated with lamps consisting of various light-emitting diode (LED) bars specifically designed to provide a custom spectrum in each chamber. Fixtures consisted of light modules with tunable light-emitting diodes varying in wavelength and spectral composition of the emitted light over wide ranges (Figure 2). = 438 ÷ 462 nm, λmax = 450 nm). To provide easy reading of the figure legends, wavelengths representing figures for combined-spectra regions (blue, short-wave red, long-wave red, and far-red) are "rounded" to 460, 640, 660, and 730, respectively.

*Plants* **2022**, *11*, x FOR PEER REVIEW 4 of 17

**Figure 2.** Light treatments: (**1**) "460 + 640 + 660 + 730"—4-peak reference treatment; (**2**) "460 + 640 + 730"—3-peak treatment missing red-light R660 region; (**3**) "460 + 640 + 660"—3-peak treatment missing far-red-light FR730 region; (**4**) "640 + 660 + 730"—3-peak treatment missing blue-light B460 region; (**5**) "450"—monochromatic blue-light B450 region; (**6**) "659" monochromatic red-light R659 region. **Figure 2.** Light treatments: (**1**) "460 + 640 + 660 + 730"—4-peak reference treatment; (**2**) "460 + 640 + 730" —3-peak treatment missing red-light R<sup>660</sup> region; (**3**) "460 + 640 + 660"—3-peak treatment missing far-red-light FR<sup>730</sup> region; (**4**) "640 + 660 + 730"—3-peak treatment missing blue-light B<sup>460</sup> region; (**5**) "450"—monochromatic blue-light B<sup>450</sup> region; (**6**) "659" monochromatic red-light R<sup>659</sup> region.

*2.4. Plant Growth Parameters Analyses*  Four plants per each treatment were destructively harvested 30 days after emergence. The number of leaves (>1 cm) per plant was counted, and total leaf area was measured using a leaf area meter LI-3000A (LI-COR Biosciences, NE, USA). Shoot fresh weight was measured using an electronic balance. Subsequently, shoots were oven-dried to a constant weight at 70 °C for dry weight determination. Specific leaf weight (SLW) was calculated by dividing leaf weight by leaf area (dry weight per unit leaf area). Four types of high-performance narrow-band 3-Watt LEDs (Estar Technology, Changchun, China) were used: short-wave red (∆λ0.5 = 623 ÷ 641 nm, λmax = 632 nm), long-wave red (∆λ0.5 = 646 ÷ 674 nm, λmax = 660 nm), far-red (∆λ0.5 = 727 ÷ 751 nm, λmax = 739 nm), and blue (∆λ0.5 = 452 ÷ 477 nm, λmax = 465 nm). The control light treatment included all 4 types of LEDs, and in each of the other regimes one of them was excluded (except shortwave red) in order to elucidate the wavelength that affected distinct crop physiological processes. Short-wave red was used as an additional background spectral region to provide chlorophyll *a* excitation in the absence of long-wave red light. The same daily light integral (DLI) of 9.72 mol m−<sup>2</sup> d <sup>−</sup><sup>1</sup> was maintained in all the treatments with photosynthetic photon flux density (PPFD) 150 µmol m−<sup>2</sup> s −1 , photoperiod 18 h. Spectra of the resulting lamp systems were measured with a spectrometer UPRtek PG100N (Taiwan). To measure the PPFD in the PAR region, an LI-191R quantum sensor with an LI-250A data logger (LI-COR Biosciences, NE, USA) was used. It was measured at the top of the plant canopy (the distance from the light source was ≥50 cm), and each chamber was adjusted to maintain PPFD at ±5%. To provide uniform PPFD, plant pots were moved and rotated within the marked uniform light platform every second day.

In the experiment on the red and blue monochromatic light effects, two types of tunable LEDs (Cree, USA) were used: red (∆λ0.5 = 647 ÷ 671 nm, λmax = 659 nm) and blue (∆λ0.5 = 438 ÷ 462 nm, λmax = 450 nm).

To provide easy reading of the figure legends, wavelengths representing figures for combined-spectra regions (blue, short-wave red, long-wave red, and far-red) are "rounded" to 460, 640, 660, and 730, respectively.
