*3.1. Growth Responses*

Lettuce plant growth (leaf biomass accumulation) was significantly retarded in the monochromatic blue light (Figures 3 and 4). It was also reduced in the combined-spectrum environment missing blue light. Monochromatic red light was especially favorable for

*Plants* **2022**, *11*, x FOR PEER REVIEW 6 of 17

*Plants* **2022**, *11*, x FOR PEER REVIEW 6 of 17

**3. Results** 

**3. Results** 

*3.1. Growth Responses* 

*3.1. Growth Responses* 

biomass accumulation. Red light's absence was unfavorable for dry biomass accumulation, though it did not affect fresh biomass yield. environment missing blue light. Monochromatic red light was especially favorable for biomass accumulation. Red light's absence was unfavorable for dry biomass accumulation, though it did not affect fresh biomass yield. biomass accumulation. Red light's absence was unfavorable for dry biomass accumulation, though it did not affect fresh biomass yield.

Lettuce plant growth (leaf biomass accumulation) was significantly retarded in the monochromatic blue light (Figures 3 and 4). It was also reduced in the combined-spectrum

Lettuce plant growth (leaf biomass accumulation) was significantly retarded in the monochromatic blue light (Figures 3 and 4). It was also reduced in the combined-spectrum environment missing blue light. Monochromatic red light was especially favorable for

**Figure 3.** Representative photo of plants from each light treatment, 20 days after emergence: (1) "460 + 640 + 660 + 730"—4-peak reference treatment; (2) "460 + 640 + 730"—3-peak treatment missing redlight 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; (6) "450"—monochromatic blue-light B450 region; (7) "659" monochromatic red-light R659 region. **Figure 3.** Representative photo of plants from each light treatment, 20 days after emergence: (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; (6) "450"—monochromatic blue-light B<sup>450</sup> region; (7) "659" monochromatic red-light R<sup>659</sup> region. **Figure 3.** Representative photo of plants from each light treatment, 20 days after emergence: (1) "460 + 640 + 660 + 730"—4-peak reference treatment; (2) "460 + 640 + 730"—3-peak treatment missing redlight 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; (6) "450"—monochromatic blue-light B450 region; (7) "659" monochromatic red-light R659 region.

In the treatment missing blue light, plants demonstrated a tendency towards bolting. The highest bolting resistance was observed in response to single-blue-light treatment or to the combined spectrum missing far-red light. Additionally, leaf size was reduced in the single-blue-light treatment and in response to the combined spectrum missing far-red light. In the blue-light treatment, leaf size reduction resulted in dramatically reduced total leaf area. However, in the combined spectrum missing far-red light, there was no reduction in leaf area due to the increased total leaf number; also, the highest specific leaf weight was observed under these conditions. Blue-light knockout in the combined spectrum resulted in a considerable leaf area increase. In the treatment missing blue light, plants demonstrated a tendency towards bolting. The highest bolting resistance was observed in response to single-blue-light treatment or to the combined spectrum missing far-red light. Additionally, leaf size was reduced in the single-blue-light treatment and in response to the combined spectrum missing far-red light. In the blue-light treatment, leaf size reduction resulted in dramatically reduced total leaf area. However, in the combined spectrum missing far-red light, there was no reduction in leaf area due to the increased total leaf number; also, the highest specific leaf weight was observed under these conditions. Blue-light knockout in the combined spectrum resulted in a considerable leaf area increase. In the treatment missing blue light, plants demonstrated a tendency towards bolting. The highest bolting resistance was observed in response to single-blue-light treatment or to the combined spectrum missing far-red light. Additionally, leaf size was reduced in the single-blue-light treatment and in response to the combined spectrum missing far-red light. In the blue-light treatment, leaf size reduction resulted in dramatically reduced total leaf area. However, in the combined spectrum missing far-red light, there was no reduction in leaf area due to the increased total leaf number; also, the highest specific leaf weight was observed under these conditions. Blue-light knockout in the combined spectrum resulted in a considerable leaf area increase.

**Figure 4.** Growth parameters of lettuce plants in response to various light treatments. Sampling 30 days after emergence. Means ± standard error (SE); means followed by the same letter were not different at *p* ≤ 0.05. (**a**) Total leaf fresh weight; (**b**) total leaf dry weight; (**c**) length of the biggest leaf; (**d**) total leaf number per plant; (**e**) total leaf area; (**f**) specific leaf weight; (**g**) stem length. Light treatments from the bottom of *y*-axis: "460 + 640 + 660 + 730"—4-peak reference treatment; "460 + 640 + 730"—3-peak treatment missing red-light R660 region; "460 + 640 + 660"—3-peak treatment missing far-red-light FR730 region; "640 + 660 + 730"—3-peak treatment missing blue-light B460 region;

The highest net photosynthesis was observed in plants grown in the combinedspectrum light environment missing red or blue light (Figure 5). Interestingly, the net photosynthesis in the reference treatment was lower than in all the other treatments.

It has been shown in previous studies that blue and red light induce stomatal opening via different pathways [38]. In our experiment, the highest stomatal conductance and transpiration were observed under monochromatic blue light. Red- or far-red-light absence in the combined spectrum decreased these parameters as compared to blue-light treatment, though more significant response was observed in the absence of blue light. Water use efficiency (WUE, photosynthesis/transpiration ratio) was extremely low under blue light (mostly due to the highest transpiration rate) and increased by three times in

"450"—monochromatic blue-light B450 region; "659" monochromatic red-light R659 region.

*3.2. Photosynthesis and Transpiration* 

the treatments with red or blue light. **Figure 4.** *Cont*.

**Figure 4.** Growth parameters of lettuce plants in response to various light treatments. Sampling 30 days after emergence. Means ± standard error (SE); means followed by the same letter were not different at *p* ≤ 0.05. (**a**) Total leaf fresh weight; (**b**) total leaf dry weight; (**c**) length of the biggest leaf; (**d**) total leaf number per plant; (**e**) total leaf area; (**f**) specific leaf weight; (**g**) stem length. Light treatments from the bottom of *y*-axis: "460 + 640 + 660 + 730"—4-peak reference treatment; "460 + 640 + 730"—3-peak treatment missing red-light R660 region; "460 + 640 + 660"—3-peak treatment missing far-red-light FR730 region; "640 + 660 + 730"—3-peak treatment missing blue-light B460 region; "450"—monochromatic blue-light B450 region; "659" monochromatic red-light R659 region. **Figure 4.** Growth parameters of lettuce plants in response to various light treatments. Sampling 30 days after emergence. Means ± standard error (SE); means followed by the same letter were not different at *p* ≤ 0.05. (**a**) Total leaf fresh weight; (**b**) total leaf dry weight; (**c**) length of the biggest leaf; (**d**) total leaf number per plant; (**e**) total leaf area; (**f**) specific leaf weight; (**g**) stem length. Light treatments from the bottom of *y*-axis: "460 + 640 + 660 + 730"—4-peak reference treatment; "460 + 640 + 730"—3-peak treatment missing red-light R<sup>660</sup> region; "460 + 640 + 660"—3-peak treatment missing far-red-light FR<sup>730</sup> region; "640 + 660 + 730"—3-peak treatment missing bluelight B<sup>460</sup> region; "450"—monochromatic blue-light B<sup>450</sup> region; "659" monochromatic red-light R<sup>659</sup> region.

### *3.2. Photosynthesis and Transpiration 3.2. Photosynthesis and Transpiration*

The highest net photosynthesis was observed in plants grown in the combinedspectrum light environment missing red or blue light (Figure 5). Interestingly, the net photosynthesis in the reference treatment was lower than in all the other treatments. It has been shown in previous studies that blue and red light induce stomatal opening The highest net photosynthesis was observed in plants grown in the combinedspectrum light environment missing red or blue light (Figure 5). Interestingly, the net photosynthesis in the reference treatment was lower than in all the other treatments.

via different pathways [38]. In our experiment, the highest stomatal conductance and transpiration were observed under monochromatic blue light. Red- or far-red-light absence in the combined spectrum decreased these parameters as compared to blue-light treatment, though more significant response was observed in the absence of blue light. Water use efficiency (WUE, photosynthesis/transpiration ratio) was extremely low under blue light (mostly due to the highest transpiration rate) and increased by three times in the treatments with red or blue light. It has been shown in previous studies that blue and red light induce stomatal opening via different pathways [38]. In our experiment, the highest stomatal conductance and transpiration were observed under monochromatic blue light. Red- or far-red-light absence in the combined spectrum decreased these parameters as compared to blue-light treatment, though more significant response was observed in the absence of blue light. Water use efficiency (WUE, photosynthesis/transpiration ratio) was extremely low under blue light (mostly due to the highest transpiration rate) and increased by three times in the treatments with red or blue light.

As for the light response curve determination, the lowest photosynthesis intensity at saturating PPFD was observed in response to red light (Figure 6). Here, low light intensity at light response curve saturation was found, as well. This kind of response is typical for plants originating from the shaded habitats. The highest photosynthesis at saturating light intensity was observed in response to blue light and the combined spectrum without red light R660; in part, the absence of the long-wave red light was compensated for here by short-wave red light R640.

**Figure 5.** CO2—H2O leaf exchange in lettuce plants in response to various light treatments. (**a**) Net photosynthesis; (**b**) stomatal conductance; (**c**) transpiration rate. 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 5.** CO2—H2O leaf exchange in lettuce plants in response to various light treatments. (**a**) Net photosynthesis; (**b**) stomatal conductance; (**c**) transpiration rate. Means ± standard error (SE); means followed by the same letter were not different at *p* ≤ 0.05. For light treatments legend see Figure 4. *Plants* **2022**, *11*, x FOR PEER REVIEW 9 of 17

**Figure 6.** Light response curves in lettuce plants in response to various light treatments Means ± standard error (SE). For light treatments legend see Figure 4. **Figure 6.** Light response curves in lettuce plants in response to various light treatments Means ± standard error (SE). For light treatments legend see Figure 4.

relatively higher in the monochromatic-blue-light treatment.

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

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

*3.3. Chlorophyll a Fluorescence* 
