**4. Materials and Methods**

*4.1. Light Characterization*

The CoeLux® growth room (University of Insubria) is composed of two standard 1 TEU containers assembled one above the other. The upper container hosts the two CoeLux® 45HC lighting systems, while the lower one is insulated and equipped with an air conditioner for temperature control to function as a growth room. The lighting system is sourced by full-spectrum white LEDs with a color temperature of 6500 K. This light is subsequently filtered to obtain the desired skylight effect [6], modifying both spectra and intensity of the original light. Therefore, both light quality and intensity were characterized within a representative section of the growth room (Figure 7).

The HD 2302.0 Light Meter (Delta Ohm) was used to measure the photosynthetically active radiation (PAR) along the central section of the growth room (566 cm × 256 cm). A custom-made rail was designed to guide the instrument sensor along the selected section and perform measurements exactly every 10 cm across the whole surface. The resulting data were analyzed to obtain a color-scale map (Figure 7). The light radiated by the CoeLux® systems is not uniformly diffused inside the growth room, being concentrated within the sunbeam ray of light with a fixed angle of 45◦ . Within the sunbeam, the highest PAR intensity, measured at 10 cm from the lighting system, was 140 µmol m−<sup>2</sup> s −1 , while at a further distance, it drops rapidly to around 20 µmol m−<sup>2</sup> s −1 . These values are even of a lower magnitude when measured within the shade, ranging from 26 µmol m−<sup>2</sup> s −1 under the blue sky from the system to less than 1 µmol m−<sup>2</sup> s −1 in the most shaded parts of the growth room. Increased light intensity was observed in some shade areas due to light reflection on the walls of the growth room and the frames of the CoeLux® systems skylight (Figure 7).

Spectra measurements every 4 nm in the range between 380 nm and 780 nm were taken on a horizontal white reflector using the SpectraScan PR655 (Photo Research), both inside the CoeLux® growth room and under the HPS lamps that we used as control. Inside the CoeLux® growth room, a total of 23 measurements were performed: 17 of them along the central section of the growth-room at five different heights from the ground floor (0, 50, 100, 150, 200 cm), inside the sunbeam of the CoeLux® system, outside the sunbeam but under the blue panel of the lighting system (sky), and in the deep shade part of the container (Figure 7). The other 6 measurements were taken near the lateral and bottom walls of the growth room to investigate the influence on the light spectra of light reflecting on the grey walls of the growth room. Within the same measurement, the instrument

also provides a light intensity value in the form of luminance (cd/m<sup>2</sup> ), which was used to normalize the spectra measurements. The spectra were divided into color components: blue light is the integral between 400 and 490 nm, green light is the integral between 490 and 560 nm, yellow light is the integral between 560 and 590 nm, red light is the integral between 590 and 700 nm, and far-red light is the integral between 700 and 780 nm. The red-to-far-red ratio (R/FR) and the blue-to-green ratio (B/R) were calculated according to Sellaro et al. [25]. *Plants* **2021**, *10*, x FOR PEER REVIEW 10 of 15

**Figure 7.** The photosynthetically active radiation (PAR) color-scale map of the double container showroom at Insubria University Campus (Varese, IT). The PAR values in the figure are given in μmol m−2s −1. Spectra measurements (M) were taken at positions M1 to M23. **Figure 7.** The photosynthetically active radiation (PAR) color-scale map of the double container showroom at Insubria University Campus (Varese, IT). The PAR values in the figure are given in µmol m−<sup>2</sup> s −1 . Spectra measurements (M) were taken at positions M1 to M23.

Spectra measurements every 4 nm in the range between 380 nm and 780 nm were taken on a horizontal white reflector using the SpectraScan PR655 (Photo Research), both inside the CoeLux® growth room and under the HPS lamps that we used as control. Inside the CoeLux® growth room, a total of 23 measurements were performed: 17 of them along the central section of the growth-room at five different heights from the ground floor (0, 50, 100, 150, 200 cm), inside the sunbeam of the CoeLux® system, outside the sunbeam but under the blue panel of the lighting system (sky), and in the deep shade part of the container (Figure 7). The other 6 measurements were taken near the lateral and bottom walls of the growth room to investigate the influence on the light spectra of light reflecting on the grey walls of the growth room. Within the same measurement, the instrument also provides a light intensity value in the form of luminance (cd/m<sup>2</sup> ), which was used to normalize the spectra measurements. The spectra were divided into color components: blue light is the integral between 400 and 490 nm, green light is the integral between 490 and We observed only small differences between the light spectrum measured within the sunbeam (red lines in Figure 8) and that measured within the shade (light blue lines in Figure 8), independently of the distance from the light source or room walls. In both cases, the spectra covered almost the entire visible wavebands; however, the total irradiance was differently distributed. Within the sunbeam, the spectra presented a broad peak between 490 and 700 nm and a sharp peak of irradiance of comparable height in the blue region (400–490 nm), representing 14% of the entire irradiance. Within the shade, the spectrum had a similar pattern but with a higher peak at 450 nm (representing 26% of the entire irradiance) and lower values in the red component of light between 590 and 700 nm (30%vs. 41%). Thus, at an equal light intensity, plants placed in a shade position received more blue and green light while plants placed inside the sunbeam receive more red and far-red light. In the small frontier positions between sun and shade, we found spectra with an intermediate shape (yellow lines in Figure 8).

560 nm, yellow light is the integral between 560 and 590 nm, red light is the integral between 590 and 700 nm, and far-red light is the integral between 700 and 780 nm. The redto-far-red ratio (R/FR) and the blue-to-green ratio (B/R) were calculated according to Sellaro et al. [25]. We observed only small differences between the light spectrum measured within the High-pressure sodium (HPS) lamps (Philips MasterColour CDM-T MW eco 230W/842) were used to provide a control light type in our study. To characterize this light spectrum, a total of 12 spectra measurements were performed at different positions in the range between 120 and 20 µmol m−<sup>2</sup> s −1 . Data were normalized on luminance to compare the spectra generated by the CoeLux® systems within the sunbeam with those of HPS lamps

sunbeam (red lines in Figure 8) and that measured within the shade (light blue lines in Figure 8), independently of the distance from the light source or room walls. In both cases,

differently distributed. Within the sunbeam, the spectra presented a broad peak between 490 and 700 nm and a sharp peak of irradiance of comparable height in the blue region (400–490 nm), representing 14% of the entire irradiance. Within the shade, the spectrum had a similar pattern but with a higher peak at 450 nm (representing 26% of the entire irradiance) and lower values in the red component of light between 590 and 700 nm (30%

(Figure 9). The HPS light type were shown to have a higher blue component (24% vs. 14%), while the CoeLux® light type had more yellow (15% vs. 11%) and red (41% vs. 35%) components. The green light component was almost identical even if a statistically significant difference was detected, while the far-red component showed no significant difference between the two, as it represented 6% of the total radiation for both types of light (Figure 9). vs. 41%). Thus, at an equal light intensity, plants placed in a shade position received more blue and green light while plants placed inside the sunbeam receive more red and far-red light. In the small frontier positions between sun and shade, we found spectra with an intermediate shape (yellow lines in Figure 8).

*Plants* **2021**, *10*, x FOR PEER REVIEW 11 of 15

**Figure 8.** Light spectra measured within the sunbeam (red lines), in shade areas (light blue lines), and in frontier positions between sun and shade (yellow lines). Measurements (M) are reported in order of appearance in the graph. Color components mean values are reported and statistically significant differences are marked with an asterisk (\*). R/FR: red/far-red ratio; B/G: blue/green ratio. **Figure 8.** Light spectra measured within the sunbeam (red lines), in shade areas (light blue lines), and in frontier positions between sun and shade (yellow lines). Measurements (M) are reported in order of appearance in the graph. Color components mean values are reported and statistically significant differences are marked with an asterisk (\*). R/FR: red/far-red ratio; B/G: blue/green ratio. *Plants* **2021**, *10*, x FOR PEER REVIEW 12 of 15

**Figure 9.** Mean light spectra curves collected within the CoeLux® sunbeam (blue) and under HPS light (green). Color components mean values are reported and statistically significant differences are marked with an asterisk (\*). R/FR: redto-far-red ratio; B/G: blue-to-green ratio. **Figure 9.** Mean light spectra curves collected within the CoeLux® sunbeam (blue) and under HPS light (green). Color components mean values are reported and statistically significant differences are marked with an asterisk (\*). R/FR: red-to-far-red ratio; B/G: blue-to-green ratio.

*Arabidopsis thaliana* wild-type (WT) seeds were stratified at 4 °C for 5 days on 1% agar gel and subsequently transferred to pot flats (Araflats; Arasystem; Ghent/Belgium) composed of 51 individual pot cavities with a 5 cm diameter, filled with sterilized commercial soil-less substrate. Plants were grown at a temperature as close as possible to 22 °C, with

cm water layer was maintained in the tray and 1mL liquid fertilizer (NPK 7.5–3−6) was supplied weekly. In the CoeLux® growth room, full pot flats were located at four different positions at progressive distances from the light source (20, 85, 205, 365 cm) inside the system's sunbeam, each corresponding to a different value of light intensity, respectively

for plant growth with the highest light intensity achievable. In a separate growth room, with the same environmental parameters of the CoeLux® growth room, plants were illuminated with HPS lamps as reference light (control), recreating the same light intensity of

light.

Phenological analysis [35] was performed through the recording of different developmental stages for a period of 100 days after sowing, considered as day 0 (Table 1). A

facility, 120 μmol m−2s

−1 is the position suitable

*4.2. Plant Material and Growth Conditions*

each of the four positions under the CoeLux®

**Stage Description**

Germination Plants with fully expanded cotyledons

Bolting and flowering Plants with a floral stalk taller than 1cm Silique formation Plants with at least one fully developed silique

120, 70, 30, 20 μmol m−2s

*4.3. Plant Analysis*

−1. In our CoeLux®

total of 10 plants were monitored for each growth condition.

**Table 1.** *A. thaliana* growth stages recorded in this work.

4-leaf stage Plants with the first two rosette leaves bigger than the cotyledons 6-leaf stage Plants with the second couple of rosette leaves bigger than the first one

Ripening and senescence Plants with at least one silique totally brown or open with ripe seeds
