*3.3. Chlorophyll a Fluorescence*

The maximum quantum efficiency of PSII photochemistry (Fv/Fm) was comparable in all the red + blue spectral treatments and single red (Figure 7). Monochromatic blue light favored the increase in Fv/Fm. There were variations in the level of relative operating efficiency of PSII, but the differences among the treatments were not significant. Higher effectiveness of the photochemical processes was observed in response to monochromatic blue light and in treatments without red or far-red light (changes of the photochemical electron transport, ETR). Chlorophyll *a* non-photosynthetic quenching (NPQ) was relatively higher in the monochromatic-blue-light treatment. *Plants* **2022**, *11*, x FOR PEER REVIEW 10 of 17

**Figure 7.** (**a**) Maximum quantum efficiency (Fv/Fm) of photosystem II (PSII); (**b**) photochemical electron transport rate (ETR); (**c**) relative PSII operating efficiency (ΦPSII); (**d**) chlorophyll *a* nonphotosynthetic quenching (NPQ) in the leaves of lettuce plants in response to various light treatments. Means ± standard error (SE); means followed by the same letter were not different at *p* ≤ 0.05. For light treatments legend see Figure 4. **Figure 7.** (**a**) Maximum quantum efficiency (Fv/Fm) of photosystem II (PSII); (**b**) photochemical electron transport rate (ETR); (**c**) relative PSII operating efficiency (ΦPSII); (**d**) chlorophyll *a* nonphotosynthetic quenching (NPQ) in the leaves of lettuce plants in response to various light treatments. Means ± standard error (SE); means followed by the same letter were not different at *p* ≤ 0.05. For light treatments legend see Figure 4.

#### *3.4. Carbon Isotopes Discrimination 3.4. Carbon Isotopes Discrimination*

distinct though not very great.

Sampling of plant material was carried out with 6 h intervals. Different light treatments showed multidirectional effects on the carbon isotope composition of leaf biomass, resulting in isotopic shifts in opposite directions (Figure 8). The strongest effects were observed in plants in response to monochromatic red and blue light as compared to the combined reference spectrum. Isotopic changes occurred in opposite directions. Thus, blue-light treatment resulted in 12C enrichment of the leaf biomass; after 6 h of illumination it was 2.56‰ "lighter" in relation to the biomass in control treatment. Red light, on the contrary, induced 13C enrichment of the leaf biomass; after 6 h of illumination it was 2.34‰ "heavier" in relation to the biomass in control experiment. In all the other treatments with combined spectrum, blue lightpresence in the spectrum resulted in a stable isotopic shift towards the enrichment of biomass with the 12C isotope. Additionally, in a combinedspectrum environment missing blue light, the presence of red light resulted in biomass enrichment with the 13C isotope. Sampling of plant material was carried out with 6 h intervals. Different light treatments showed multidirectional effects on the carbon isotope composition of leaf biomass, resulting in isotopic shifts in opposite directions (Figure 8). The strongest effects were observed in plants in response to monochromatic red and blue light as compared to the combined reference spectrum. Isotopic changes occurred in opposite directions. Thus, blue-light treatment resulted in <sup>12</sup>C enrichment of the leaf biomass; after 6 h of illumination it was 2.56‰ "lighter" in relation to the biomass in control treatment. Red light, on the contrary, induced <sup>13</sup>C enrichment of the leaf biomass; after 6 h of illumination it was 2.34‰ "heavier" in relation to the biomass in control experiment. In all the other treatments with combined spectrum, blue lightpresence in the spectrum resulted in a stable isotopic shift towards the enrichment of biomass with the <sup>12</sup>C isotope. Additionally, in a combined-spectrum environment missing blue light, the presence of red light resulted in biomass enrichment with the <sup>13</sup>C isotope.

The results of the carbon isotopic differences in leaf biomass during the transition from light to dark are of particular interest. One could expect a strong rearrangement of the metabolic fluxes between day and night periods. Indeed, as we can see from Figure 8, during the light period, the leaf biomass became enriched with the 12C isotope as compared to the biomass carbon composition detected at the end of the dark period. These isotopic differences occurred in all the lighting modes. Isotopic differences were quite

daily variations in the carbon isotope composition of the *Ricinus communis* plants and had found similar daily variations not only in the carbon of the plant leaf biomass but also in

the carbon of its water-soluble and water-insoluble fractions and phloem sap.

**Figure 8.** Carbon isotope composition of the leaves in lettuce plants grown in various light environments during 24 h cycle. Carbon isotope composition is given in PDBV δ13C units. Means ± standard error (SE); means followed by the same letter were not different at *p* ≤ 0.05. (**a**) After 6 h of illumination; (**b**) after 12 h of illumination; (**c**) after 18 h of illumination; (**d**) at the end of night after 6 h of darkness. For light treatments legend see Figure 4. **Figure 8.** Carbon isotope composition of the leaves in lettuce plants grown in various light environments during 24 h cycle. Carbon isotope composition is given in PDBV δ <sup>13</sup>C units. Means ± standard error (SE); means followed by the same letter were not different at *p* ≤ 0.05. (**a**) After 6 h of illumination; (**b**) after 12 h of illumination; (**c**) after 18 h of illumination; (**d**) at the end of night after 6 h of darkness. For light treatments legend see Figure 4.

**4. Discussion**  Studies on the light action in plants using various applications of LED techniques provide new insights into plant photobiology. The plant photosynthesis action spectrum matches with blue and red regions of photosynthetically active radiation in a natural environment [40–42]. In an artificial-light environment, the joint application of red and blue light usually results in increased plant photosynthesis and productivity [11,12,22]. Additionally, blue light is thought to participate in the acclimation of leaf photosynthesis to irradiance during growth [10,43]. These two spectral regions were the basic variables The results of the carbon isotopic differences in leaf biomass during the transition from light to dark are of particular interest. One could expect a strong rearrangement of the metabolic fluxes between day and night periods. Indeed, as we can see from Figure 8, during the light period, the leaf biomass became enriched with the <sup>12</sup>C isotope as compared to the biomass carbon composition detected at the end of the dark period. These isotopic differences occurred in all the lighting modes. Isotopic differences were quite distinct though not very great.

in our photobiological studies. In our experimental set-up, we applied combined-spectrum treatments within two ranges of red light (R640, R660) trying to separate direct light effects on the PSA and lightinduced photomorphogenetic responses controlled by the phytochromes. Indeed, far-red-The data obtained are consistent with the results of Gessler et al. [39], who studied daily variations in the carbon isotope composition of the *Ricinus communis* plants and had found similar daily variations not only in the carbon of the plant leaf biomass but also in the carbon of its water-soluble and water-insoluble fractions and phloem sap.

light absence in the combined spectrum resulted in axial organ growth inhibition as

#### compared to the treatments with far-red light (Figure 4g). Leaf blade elongation was also **4. Discussion**

retarded (Figure 4c) due to the blocking of phytochrome-mediated shade-avoidance syndrome. Interestingly, the total leaf number increased significantly in this treatment, providing the growth of the light-harvesting leaf area of the plant. It is still unclear whether this response was observed due to the decreased plastochrone in the FR-deficient treatment or if other more sophisticated compensation mechanisms were involved. Similar results with stem and leaf growth inhibition were observed in response to monochromatic blue light (Figure 4c,g). Actually, the most serious inhibition of leaf blade growth in comparison with the other treatments was found in response to blue light. A reduction in leaf growth in response to blue light decreased plant biomass accumulation Studies on the light action in plants using various applications of LED techniques provide new insights into plant photobiology. The plant photosynthesis action spectrum matches with blue and red regions of photosynthetically active radiation in a natural environment [40–42]. In an artificial-light environment, the joint application of red and blue light usually results in increased plant photosynthesis and productivity [11,12,22]. Additionally, blue light is thought to participate in the acclimation of leaf photosynthesis to irradiance during growth [10,43]. These two spectral regions were the basic variables in our photobiological studies.

significantly. Data on the decreased total leaf fresh weight yield in the treatment combining all four spectral regions in comparison with monochromatic red were unexpected. However, there are other data suggesting that lettuce biomass under monochromatic red was greater than under mixed red and blue light [44]. Comparable responses in other species were In our experimental set-up, we applied combined-spectrum treatments within two ranges of red light (R640, R660) trying to separate direct light effects on the PSA and lightinduced photomorphogenetic responses controlled by the phytochromes. Indeed, farred-light absence in the combined spectrum resulted in axial organ growth inhibition as compared to the treatments with far-red light (Figure 4g). Leaf blade elongation was also retarded (Figure 4c) due to the blocking of phytochrome-mediated shade-avoidance syndrome. Interestingly, the total leaf number increased significantly in this treatment, providing the growth of the light-harvesting leaf area of the plant. It is still unclear whether

this response was observed due to the decreased plastochrone in the FR-deficient treatment or if other more sophisticated compensation mechanisms were involved. Similar results with stem and leaf growth inhibition were observed in response to monochromatic blue light (Figure 4c,g). Actually, the most serious inhibition of leaf blade growth in comparison with the other treatments was found in response to blue light. A reduction in leaf growth in response to blue light decreased plant biomass accumulation significantly.

Data on the decreased total leaf fresh weight yield in the treatment combining all four spectral regions in comparison with monochromatic red were unexpected. However, there are other data suggesting that lettuce biomass under monochromatic red was greater than under mixed red and blue light [44]. Comparable responses in other species were observed by Wollaeger and Runkle [45]. So, the synergetic or antagonistic effects of red and blue light on lettuce are still confused, and more studies need to be conducted [46].

As far as plant growth was inhibited in monochromatic blue light, net photosynthesis was also at a low rate in comparison with the combined-spectra treatments missing distinct spectral regions. The photosynthesis rate in the treatment missing long-wave red light R<sup>660</sup> was one of the highest due to the compensation by short-wave red R640; photosynthesis at saturating light intensity (light response curve) was also very high (Figure 5).

Net photosynthesis in the reference treatment was lower than in all the other treatments. This is most likely because in more stressful environments lacking distinct spectral regions, compensation mechanisms were activated. Additionally, the red-light PPFD share in the combined-spectrum treatment was much lower than PPFD in the monochromaticred-light treatment. On the other hand, we observed plant acclimation to the abnormal light environments as a long-term process (sampling 30 days after emergence). This is most likely because an increased assimilate demand and increased sink capacity were the drivers of photosynthesis in monochromatic red light. We shall try to investigate this phenomenon in the future studies. Blue-light treatment significantly increased stomatal conductance and transpiration rate in plants and decreased their WUE; comparable results were obtained in tomato plants [47].

The main points of carbon isotope fractionation during photosynthesis are located at the crossings of the central metabolic pathways; therefore, the isotopic effects are reflected in the carbon isotope composition of biomass, fractions, and of the overwhelming number of metabolites [35]. The first carbon isotope fractionation point is located at the entry of the pentose phosphate reduction cycle (Calvin cycle) and is associated with the reaction of enzymatic carboxylation of ribulosebisphosphate (RuBP). As a result, the assimilated carbon atoms are enriched in <sup>12</sup>C in relation to the environmental CO2. The enzyme that controls carboxylation, Rubisco, has the properties of oxygenase and is able to simultaneously redirect a part of the carbon flux assimilated in Calvin cycle to glycolate cycle, where it is partly oxidized to CO<sup>2</sup> and released back into the environment, creating so-called photorespiration flux. A probable mechanism of switching the functions of the enzyme is maintained by the changing ratio of CO2/O<sup>2</sup> concentrations in the cell [48]. Due to such organization of photosynthesis, the activities of the Calvin cycle and glycolate cycle are separated in time, and the fluxes of carbon substrates resulting from assimilation and photorespiration become independent and discrete, that is, represented as separate portions [49]. In our experiment, we observed increased stomatal conductance in response to blue-light treatment (Figure 5). As a result, an increased CO<sup>2</sup> supply could enhance Rubisco carboxylating activity and it was followed with leaf tissue <sup>12</sup>C enrichment (Figure 8). These results are consistent with the data of other authors that have shown that δ <sup>13</sup>C correlates negatively with stomatal conductance [50].

The most intensive lettuce leaf tissue enrichment with <sup>13</sup>C was observed in the treatments with monochromatic red light followed by in the combined-spectra environment missing blue light; in the last case this response could be attributed to the contradictory information from the blue- and red-light photoreceptors, as it was mentioned in Section 1. On the contrary, the biomass of plants subjected to blue-light treatment was enriched with the <sup>12</sup>C isotope. We have to stress here that plants were subjected to the long-term

(during the whole growing cycle) light treatment. Therefore, chloroplast genesis could be affected significantly in the absence of blue light, as it was observed earlier [51]. On the contrary, monochromatic blue light was more favorable for chloroplast development and functioning [20,51]. In our studies, monochromatic-blue-light treatment maintained better plant photosynthetic performance, i.e., the highest maximum quantum efficiency (Fv/Fm) and a higher electron transport rate (ETR). Data from the light response curves show that photosynthesis at saturating light intensity in the blue-light-grown plants was four times higher than in the red-light-grown plants. Taking into consideration the facts discussed above, a possible explanation could be based on the variability in plant adaptations to the abnormal light environments during long-term 30-day exposure. That has resulted in the disturbance of PSA but to a lesser extent in the case of blue-light treatment as compared to red-light treatment.

The second point of carbon isotope fractionation is connected with increased photorespiration when observations show that plant biomass becomes enriched with <sup>13</sup>C [52]. This means that photorespiration is accompanied by an isotope effect of opposite sign than photoassimilation. Numerous studies on isotope fractionation in plants and artificial mutants have proved that the glycine decarboxylase reaction of the glycolate cycle was another place where the isotope effect is observed [33,53].

The third point of carbon isotope fractionation relates to post-photosynthetic metabolism and is associated with the end of the glycolytic chain where pyruvate dehydrogenase reaction proceeds. The observed proximity of the carbon isotope composition of the total plant biomass to assimilatory carbon pool suggests that the glycolytic chain and the majority of metabolites (lipids, proteins, lignins, and some carbohydrates), whose synthesis occurs via glycolytic chain, are supplied with the substrates of the assimilatory pool [54]. At the same time, the syntheses of soluble carbohydrates, organic acids, some amino acids, and other metabolite sis mainly bound to the "heavy" photorespiratory carbon pool. Because of the strict temporal and spatial organization in a cell, noticeable mixing of carbon fluxes does not occur, and various isotope distinctions exist [55].

The idea of the Rubisco oscillating mode of action has been analyzed extensively [32,34,35,56] and theoretically it was shown that oscillations can exist under real photosynthetic cell conditions. In the present paper, we returned to this idea. We assumed the presence of an isotopically "light" assimilatory pool and isotopically "heavy" pool of metabolites appear during photosynthesis as a result of dual function of Rubisco.

In our experiment, in all cases, leaf biomass at the end of light period was enriched in <sup>12</sup>C as compared to the leaf biomass at the end of dark period. Isotopic differences were quite distinct though not very great. Possible explanations of these differences could be given from our earlier paper [57] based on the model of oscillatory photosynthesis discussed above. Plant tissues enrichment with <sup>12</sup>C isotope during the light period was due to the fact that at this time lipids, proteins, lignins, and other structural components were synthesized mainly in the leaf. The isotopically "light" assimilatory pool was the substrate source for them. During the dark period, the outflow of assimilates to generative organs and heterotrophic tissues occurred. The outflow of assimilates occurred mainly in the form of sucrose and other water-soluble carbohydrates and metabolites, the isotopeheavy photorespiratory fund being their carbon source. Different sources of substrates for the synthesis of structural units and transport agents induced isotopic differences in daily variations of leaf biomass. Similar isotopic shifts were observed by other researchers while studying the isotopic differences between photosynthetic and heterotrophic organs and tissues [58].

We can conclude that blue light enhanced the assimilation function of the leaf, while red light enhanced the photorespiratory function. The simultaneous presence of blue and red light compensated for their mutual effects, and therefore the effects of light from the other spectral regions on the isotopic shifts became indistinguishable from the control. It was shown that duration of illumination (6, 12, and 18 h) had a weak effect on the isotope composition of biomass.
