*3.2. Biochemical Parameters of Lettuce Plants*

In the phase of active growth on the 25th day of cultivation, for 'Lollo Rossa' leaf lettuce in terms of concentration and ratio of pigments, the options with constant illumination 16/8 and impulse were optimal. The plants grown under these light modes had the highest content of total chlorophyll—1.2 mg per 1 g of fresh mass (Figure 2a). The ratio of total chlorophyll to carotenoids was more than four (Figure S1b) which corresponds to the period of active vegetation. As for the semi-head 'Azart' variety, there were no significant differences in the ratio of chlorophylls to carotenoids (Figure S1a,b), and the highest concentration of pigments was observed in the control variant.

**Figure 2.** The concentration of pigments on the 25th (**a**) and 35th (**b**) day of cultivation. The solid colour indicates the columns of the diagram related to the 'Azart' variety; the shading columns of the diagram indicate data related to the 'Lollo Rossa' variety. Values represent mean SEM (n = 10). Letters indicate significant differences among treatment and control samples (*p* < 0.05). Asterisk sign near letters is used for the 'Azart' variety and apostrophe - for the 'Lollo Rossa' variety. One sign is used for Chlorophyll *a*, two signs − for Chlorophyll *b* and three signs − for Carotenoids.

(**b**)

On the 35th day of cultivation, the 'Lollo Rossa' leaf lettuce was distinguished by a more intense accumulation of pigments (Figure 2b). This may be due to the screening effect of anthocyanins [31]. It also accumulated more carotenoids in relation to the total content of chlorophylls in the variants with Constant 24 and Scanning irradiation (Figure S1d), what indicates increased stress level since carotenoids protect the photosynthetic apparatus from negative light exposure [32]. Plants of the 'Azart' variety in the variant with the use of impulse irradiation were distinguished by a reduced concentration of photosynthetic pigments. The ratio of chlorophyll a to chlorophyll b (Figure S1c) in the half-head 'Azart' variety in all variants, except for the Constant 24, was below three, which indicates a slight lack of illumination, and the total chlorophyll content ratio to the carotenoid content was the highest under Impulse and Scanning irradiation modes. Thus, judging by the pigment composition, both constant 16/8 and impulse irradiation modes are suitable for 'Lollo Rossa' leaf lettuce and Constant 24 for the 'Azart' variety.

It is known that the prolongation of the photoperiod can increase the fresh weight and chlorophyll content, meanwhile, a decrease in the photoperiod helps to reduce the content of nitrates in lettuce [33]. In this regard, we checked the content of nitrates in plants at the time of green mass harvesting. The concentration of nitrates was minimal in the control variant and the variant with the use of impulse irradiation (Figure 3). When using the scanning and constant 24 modes, the concentration of nitrates was slightly higher but did not exceed the maximum allowable concentrations for green products. For these lighting options, the absence of a night period contributes to a greater accumulation of nitrates in the leaves of crops. The observed results are a consequence of the influence of the light environment on plant metabolism and carbon and nitrogen exchange processes. The data presented in this article correlates with previous studies [33,34].

#### *3.3. Chlorophyll Fluorescence Parameters*

At the next stage of the study, the influence of the illumination mode on the magnitude of the photosynthetic electron flux through photosystem II (ETR) and non-photochemical fluorescence quenching (NPQ) was studied.

It was shown (Figure 4a) that the lighting regime did not cause significant changes in the 'Azart' lettuce variety; at the same time, there was a trend towards an increase in ETR with increasing actinic light intensity in plants cultivated under continuous light conditions (control). In the 'Lollo Rossa' variety, this increase in the ETR of plants grown under continuous illumination was significant (Figure 4b). NPQ values at intermediate light intensities were reduced in both 'Azart' leaves (Figure 4c) and 'Lollo Rossa' leaves (Figure 4d); under conditions of low or high intensity of actinic light, the effect was absent. At the same time, there were practically no differences between the studied photosynthetic parameters in plants grown under other lighting conditions that were used in the work. These results are consistent with data obtained from red leaf lettuce, where there was no significant difference in the total rate of leaf photosynthesis under impulse illumination with Kf = 75% compared to continuous illumination with LEDs [2], and on green lettuce, where impulse irradiation did not significantly affect the concentration of chlorophyll and fluorescence parameters, except for the lowest frequency (0.2 Hz) [16].

**Figure 4.** Dependence of the rate of electron transport (**a**,**b**) and non-photochemical (**c**,**d**) quenching of chlorophyll fluorescence on PPFD of lettuce plants on the 35th day of cultivation. Values represent mean SEM (n = 6).

The obtained results suggest that it is the high ETR value and low NPQ that can be the reason for the high productivity of lettuce plants under continuous illumination (Table 1); however, they do not explain the high efficiency of impulse lighting, since the photosynthetic parameters when grown under such conditions do not differ from the options with scanning lighting or simulated daylight hours (l/d = 16/8 h).

#### *3.4. Study of the Influence of the Light Regime during Cultivation on Leaf Reflectance Indices*

Analysis of the studied reflectance indices (NDVI, ZMI, GM1, GM2, CRI1 and CRI2) (Figure S2a,b) showed that their values were somewhat lower in the leaves of the 'Lollo Rossa' lettuce variety compared to the leaves of the 'Azart' variety. This result was in good agreement with the lower values of the chlorophyll index and the net productivity of photosynthesis in the 'Lollo Rossa' variety (Table 1 and Figure 2) since the studied indices correlate with the content of photosynthetic pigments.

Comparison of NDVI, ZMI, GM1, GM2, CRI1 and CRI2 indices in lettuce leaves of the same variety grown under different lighting conditions did not show statistically significant differences between the studied groups. At the same time, with the use of impulse and scanning irradiation, the 'Azart' variety showed a tendency to decrease in several indices sensitive to the concentration of chlorophyll in plants (NDVI, ZMI, GM1 and GM2), which is consistent with a decrease in the content of chlorophyll (Figure 2). Similar trends were also observed for carotenoid-sensitive indices CRI1 and CRI2 (Figure S2b), which correlate with changes in carotenoid concentration (Figure 2).

Even though the changes in the studied reflectance indices were not statistically significant, the observed trends corresponded well to changes in the content of photosynthetic pigments under different lighting conditions: a decrease in the content of chlorophyll a was accompanied by a decrease in the reflectance indices NDVI [25], ZMI [26], GM1 and GM2 [27], while a decrease in the content of carotenoids led to a decrease in CRI1 and CRI2 [28]. At the same time, small values and low significance of changes showed a relatively weak effect of the lighting regime on the reflectance spectrum of the sheet; Given the more pronounced differences in indices between the studied lettuce varieties (Figure S2a,b), the variety factor seems to be more significant for reflectance indices than the light regime during cultivation. On the other hand, Table 1 and Figure 2 show that under different growing regimes, differences in morphometric parameters of plants were observed, which suggests that such a difference may be associated with a change in the anatomical parameters of the leaf. Such features can significantly depend on the illumination parameters [35,36] and modify the reflectivity of the sheet [37] by decreasing or increasing changes in the reflectance indices.

For the "Lollo Rossa" lettuce variety, which has a high content of anthocyanins, the anthocyanin content index (ARI) was additionally measured (Figure 5). It was shown that the impulse lighting mode led to the increase in this index and has a positive effect on the anthocyanin concentration in lettuce leaves.

**Figure 5.** Anthocyanin content index (ARI) of lettuce plants on the 35th day of cultivation. Values represent mean SEM (n = 10). Letters indicate significant differences among treatment and control samples (*p* < 0.05).

#### *3.5. Raman Spectroscopy of Lettuce Leaves*

Usually, as a plant culture in Raman spectroscopy, *Lactuca sativa* is used to determine toxicants, plastics and heavy metals [38–40]. To identify the optimum points for collecting the Raman spectrum for both varieties, spectral data were collected from the inner space of the cell, the centre of the open stomata, and its valve. Based on the data obtained, the most informative were the spectra of the intracellular space (Figure 6).

Most often, peaks associated with pigments, especially with carotenoids and chlorophylls, can be detected in the spectra of plant samples. The main pigment peaks are observed in the range of 1500–1550 and 1150–1170 cm<sup>−</sup>1, this part of the range is associated with in-phase stretching oscillations C=C and C-C of the polyene chain. In addition, swing patterns in the plane of CH3 groups attached to the polyene chain can be identified as peaks of average intensity in the range of 1000–1020 cm [41]. Assessing the average spectra of 'Azart' and 'Lola Rossa' lettuce varieties, it should be noted that the overall colour and content of pigments significantly affected the data: the leaves of plants of the 'Lola Rossa' variety showed a higher and noisier spectrum, with isolated peaks characteristic of photosynthetic pigments and organic acids (Figure S3). In general, the intensity of the red-leaf 'Lola Rossa' lettuce variety detected by CR was more than two times higher than that of the green-leaf 'Azart' variety, which is associated with an increased content

of carotenoids and anthocyanins (Figure 2), which have the properties to raise the overall intensity of the spectrum. A change in the illumination mode had little effect on the cell biochemistry reflected in the spectral data. This was more pronounced in the leaves of 'Lola Rossa' variety lettuce, which is explained by a change in the concentration of anthocyanins with a change in daylight hours (Figure 7).

**Figure 6.** Measurement and selection of points on a lettuce leaf of the 'Azart' variety on the 35th day of cultivation, where red is the intracellular space, purple is the centre of the stomata, and green is the stomatal valve.

**Figure 7.** Varieties and variations in average intensity of significant peaks of the 'Azart' variety and 'Lola Rossa' on the 35th day of cultivation. Values represent mean SEM (n = 5). Letters indicate significant differences among treatment and control samples of 'Azart' variety, letters with asterisk sign–of 'Lollo Rossa' variety (*p* < 0.05).

As a result of the analysis of various types of lighting, it was found that the most pronounced spectra are plants grown under the scanning and constant type of lighting, which indicates a greater overall biochemical activity, expressed in an increase in the concentrations of pigments, sugars and organic acids in plant cells. The spectral data for both varieties of lettuce correlate with each other (Figure 7).

#### **4. Conclusions**

For the semi-head 'Azart' lettuce variety, the largest accumulation of the fresh mass of the plant was recorded on the stand with constant irradiation (24 h), and the smallest under scanning illumination. For the red-leaved 'Lollo Rossa' lettuce variety, the accumulation of fresh mass under constant (24 h), impulse and constant (16/8 h) were approximately the same values with a slight advantage in impulse irradiation, and the smallest value was also recorded for the scanning variant. From the point of view of qualitative analysis, the highest concentrations of carotenoids in the 'Lollo Rossa' lettuce variety were found for constant (24 h) and scanning irradiation, since carotenoids protect the photosynthetic apparatus from negative and intense light exposure. The highest value of chlorophyll content was found for plant variants under constant (16/8 h) and impulse irradiation. It was shown that impulse lighting during plant cultivation led to a statistically significant increase in the anthocyanin content index (ARI), which indicates a positive effect of impulse lighting on the concentration of anthocyanins in lettuce leaves. For the 'Azart' lettuce variety, the minimum value of the pigment concentration was found for impulse irradiation, for constant (24 h), scanning and constant (16/8) irradiation, the obtained values did not differ significantly. Thus, judging by the accumulation of fresh mass and the pigment composition, constant (16/8 h) and impulse irradiation is suitable for the 'Lollo Rossa' lettuce variety.

For the semi-head 'Azart' variety, the options with constant (24 h) and impulse irradiation are optimal. Further research will be aimed at researching and evaluating the modes and methods for introducing impulse irradiation for growing leafy crops in greenhouses.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/agriculture12121988/s1, Figure S1: The ratio of the concentrations of chlorophyll a to chlorophyll b on the 25th day (a) and the 35th day (c) of 'Azart' and 'Lollo Rossa' lettuce variety cultivation; the ratio of the concentrations of total chlorophyll to carotenoids on the 25th day (b) and the 35th day of cultivation (d) using different LED irradiation time modes; Figure S2: Vegetation indices of lettuce leaves NDVI, ZMI, GM1, GM2 (a) and CRI1, CRI2 (b) of 'Azart' and 'Lollo Rossa' varieties on the 35th day of cultivation using different LED irradiation time modes. Values represent mean SEM (n = 10); Figure S3: Comparison of normalized Raman spectra of 'Azart' (a) and 'Lola Rossa' (b).

**Author Contributions:** Conceptualization, N.O.C., A.A.S. and N.A.S.; methodology, A.A.S., A.S.D. and N.O.C.; formal analysis, Y.A.P. and V.S.; investigation, A.A.S., N.A.S. and M.M.G.; resources, A.A.S., A.S.D. and N.O.C.; writing—original draft preparation, A.A.S. and N.A.S.; writing—review and editing, N.O.C., V.V. and V.S.; visualization, Y.A.P.; supervision, V.P.; project administration, A.S.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This investigation was funded by the Ministry of Science and Higher Education of the Russian Federation for large scientific projects in priority areas of scientific and technological development, contract no. 075-15-2020-774.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available upon request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

#### **References**

