*2.2. Supplemental Intra-Canopy Illumination*

The supplemental LED illumination was assembled from Crops IP67 tubes (Bioled Eco Light Systems Ltd., Tzova, Israel), providing cool-white (CW; 5700K) light at 32 W/m. CW was chosen as it was found to be preferable for bell pepper in our earlier study [12]. For simplicity, we refer to the LED tubes as 'Bioled' in the text. Two LED tubes affixed back-toback were installed between the two adjacent rows of the beds (Figure 1). Two illumination

regimes of 12 h were provided: daytime ('LED-D', 6:00 to 18:00) and edge-of-daytime ('LED-N', 4:00–10:00 and 16:00–22:00). The experimental setup encompassed four replicate sections (5.4-m-long each) for each of the two intra-canopy light treatments and for the non-illuminated control (Figure 2). The illumination period began 70 days after planting (28 October 2019), when the canopy height was ~1.5 m. Fixtures were installed at a height of 70–80 cm aboveground at the start of the illumination period, raised to 90–100 cm in the middle of December 2019, and raised again to 110–120 cm in the middle of March 2020. nation regimes of 12 h were provided: daytime ('LED-D', 6:00 to 18:00) and edge-of-daytime ('LED-N', 4:00–10:00 and 16:00–22:00). The experimental setup encompassed four replicate sections (5.4-m-long each) for each of the two intra-canopy light treatments and for the non-illuminated control (Figure 2). The illumination period began 70 days after planting (28 October 2019), when the canopy height was ~1.5 m. Fixtures were installed at a height of 70–80 cm aboveground at the start of the illumination period, raised to 90–100 cm in the middle of December 2019, and raised again to 110–120 cm in the middle of Mar. 2020.

was chosen as it was found to be preferable for bell pepper in our earlier study [12]. For simplicity, we refer to the LED tubes as 'Bioled' in the text. Two LED tubes affixed backto-back were installed between the two adjacent rows of the beds (Figure 1). Two illumi-

*Plants* **2022**, *11*, 424 4 of 17

**Figure 1. Intra-canopy supplemental illumination.** (**A**) Installation of the 'Bioled' light fixtures at the center of the beds (September 2019, prior to start of illumination treatments). (**B**) Side-view of intra-canopy back-to-back LED illumination (picture acquired in March 2020). **Figure 1. Intra-canopy supplemental illumination.** (**A**) Installation of the 'Bioled' light fixtures at the center of the beds (September 2019, prior to start of illumination treatments). (**B**) Side-view of intra-canopy back-to-back LED illumination (picture acquired in March 2020).

Spectra and photosynthetic photon flux densities (PPFD) were recorded using a portable spectroradiometer (EPP2000C, StellarNet, Inc., USA) with a cosine-corrected head (Apogee Instruments Inc., USA) and an LI-250A quantum sensor (LI-COR, USA), respectively. Air temperature within the canopy was recorded using HOBO temperature data loggers (Onset Computer Corporation, USA) hung in proximity (10–15 cm) to the LED fixtures or at the same height in control sections. Spectra and photosynthetic photon flux densities (PPFD) were recorded using a portable spectroradiometer (EPP2000C, StellarNet, Inc., Tampa, FL, USA) with a cosinecorrected head (Apogee Instruments Inc., Logan, UT, USA) and an LI-250A quantum sensor (LI-COR, Lincoln, NE, USA), respectively. Air temperature within the canopy was recorded using HOBO temperature data loggers (Onset Computer Corporation, Bourne, MA, USA) hung in proximity (10–15 cm) to the LED fixtures or at the same height in control sections.

#### *2.3. Chlorophyll Content and Fluorescence 2.3. Chlorophyll Content and Fluorescence*

Measurements were conducted non-destructively on attached leaves of the inner canopy. Chlorophyll (Chl) content was assayed using an MC-100 Chl measurement system (Apogee, USA). Chl-*a* fluorescence emission was measured using a portable pulse-amplitude-modulated fluorometer (PAM-2000, Heinz Walz GmbH, Germany) at its default setting designed to determine Fv/Fm ('Da-2000' program). In brief, leaves were subjected to dark adaptation for 20 min using dark leaf clips (DLC-8, Walz), and then initial Chl-*a* fluorescence (F0) and maximum Chl-*a* fluorescence in dark (Fm) were recorded after applying a saturating light pulse for 0.8 s. The Fv/Fm = [(Fm-F0)/Fm] values were calculated by the program and recorded. Measurements were conducted non-destructively on attached leaves of the inner canopy. Chlorophyll (Chl) content was assayed using an MC-100 Chl measurement system (Apogee, Chesapeake, VA, USA). Chl-*a* fluorescence emission was measured using a portable pulse-amplitude-modulated fluorometer (PAM-2000, Heinz Walz GmbH, Pfullingen, Germany) at its default setting designed to determine Fv/Fm ('Da-2000' program). In brief, leaves were subjected to dark adaptation for 20 min using dark leaf clips (DLC-8, Walz), and then initial Chl-*a* fluorescence (F0) and maximum Chl-*a* fluorescence in dark (Fm) were recorded after applying a saturating light pulse for 0.8 s. The Fv/Fm = [(Fm − F0)/Fm] values were calculated by the program and recorded.

**Figure 2. Schematic map of the experimental tunnel.** The experiment was carried out in the three central double-row beds (2, 3, and 4) of the tunnel. Intra-canopy lighting was applied at the center of the beds between the two rows (Figure 1A), along 5 m-long sections. The illumination was applied either during daytime ('LED-D') or the two edges of daylight period ('LED-N'), with nonilluminated sections of the same length as controls ('CR'). Each treatment had four replicates. A spacing of at least 2 m was kept between sections. 'E' and 'W' denote the eastern- and westernfacing rows, with regard to the results presented in further Figures and Tables. Note that beds and experimental sections are not drawn to scale. **Figure 2. Schematic map of the experimental tunnel.** The experiment was carried out in the three central double-row beds (2, 3, and 4) of the tunnel. Intra-canopy lighting was applied at the center of the beds between the two rows (Figure 1A), along 5 m-long sections. The illumination was applied either during daytime ('LED-D') or the two edges of daylight period ('LED-N'), with non-illuminated sections of the same length as controls ('CR'). Each treatment had four replicates. A spacing of at least 2 m was kept between sections. 'E' and 'W' denote the eastern- and western-facing rows, with regard to the results presented in further Figures and Tables. Note that beds and experimental sections are not drawn to scale.

#### *2.4. Gas-Exchange Measurements 2.4. Gas-Exchange Measurements*

Gas-exchange measurements were conducted on attached leaves of the inner or outer canopy using a portable LCi photosynthesis system (ADC BioScientific Ltd., UK) with a clear top chamber at ambient conditions. For the inner canopy, leaves were sampled 10– 15 cm above the upper LED fixtures, at a height of 110–120 cm above the ground, and at the same height in non-illuminated plots. For outer canopy measurements, leaves from the eastern- and western-facing canopy were probed in the morning or afternoon during peak photosynthetically active radiation (PAR) intensities on each side. Positioning of the LCi chamber was adjusted according to the leaf being measured (to keep the leaf attached) and to allow natural sunlight or light from the LEDs to reach the leaf. Gas-exchange measurements were conducted on attached leaves of the inner or outer canopy using a portable LCi photosynthesis system (ADC BioScientific Ltd., Hoddesdon, UK) with a clear top chamber at ambient conditions. For the inner canopy, leaves were sampled 10–15 cm above the upper LED fixtures, at a height of 110–120 cm above the ground, and at the same height in non-illuminated plots. For outer canopy measurements, leaves from the eastern- and western-facing canopy were probed in the morning or afternoon during peak photosynthetically active radiation (PAR) intensities on each side. Positioning of the LCi chamber was adjusted according to the leaf being measured (to keep the leaf attached) and to allow natural sunlight or light from the LEDs to reach the leaf.

#### *2.5. Fruit Set Quantification 2.5. Fruit Set Quantification*

In each of the experimental replicate sections (four sections for each treatment) shown in Figure 2, ten plants were selected for the analysis: five in the eastern-facing row and five in the western-facing row of the same bed. Fruitlets were counted, labelled, and In each of the experimental replicate sections (four sections for each treatment) shown in Figure 2, ten plants were selected for the analysis: five in the eastern-facing row and five in the western-facing row of the same bed. Fruitlets were counted, labelled, and

screened for survival on eleven dates throughout the season. Survival of fruit labelled on one date were assayed on the next fruitlet labelling date. On the last day of the experiment (7 May 2020), fruitlets remaining on the plants were counted. screened for survival on eleven dates throughout the season. Survival of fruit labelled on one date were assayed on the next fruitlet labelling date. On the last day of the experiment (May 7, 2020), fruitlets remaining on the plants were counted.

#### *2.6. Daily Light Integral (DLI) Recording 2.6. Daily Light Integral (DLI) Recording*

PAR (400–700 nm) was recorded using LI-190SB-L quantum sensors (LI-COR, Lincoln, NE, USA) installed at the eastern- and western-facing canopy (at a height of 1 m above ground), as well as above the canopy (height of 3 m) at 90 degrees. Data were recorded every ten minutes and logged by a Campbell system (CR10X—Campbell Scientific, Logan, UT, USA). PAR (400–700 nm) was recorded using LI-190SB-L quantum sensors (LI-COR, USA) installed at the eastern- and western-facing canopy (at a height of 1 m above ground), as well as above the canopy (height of 3 m) at 90 degrees. Data were recorded every ten minutes and logged by a Campbell system (CR10X—Campbell Scientific, USA).

#### *2.7. Statistical Analysis 2.7. Statistical Analysis*

Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Tukey's multiple comparisons as a post hoc test. For comparisons of two groups, the Student's *t*-test was used. The level of significance is provided in the figure legends/table. Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Tukey's multiple comparisons as a post hoc test. For comparisons of two groups, the Student's *t*-test was used. The level of significance is provided in the figure legends/table.

#### **3. Results 3. Results**

#### *3.1. Intra-Canopy Illumination 3.1. Intra-Canopy Illumination*

The 'Bioled' light fixtures utilized as intra-canopy illumination provided cool-white (CW) light (Figure 3A). The effect of the added illumination on the light intensity between the double-row beds was assessed when the canopy height was ~2 m. In control ('CR') non-illuminated sections, the light intensity of the inner canopy below 1.5 m was generally <50 µmol photons m−<sup>2</sup> s −1 , and for the most part even <20 µmol photons m−<sup>2</sup> s −1 (Figure 3B). In the illuminated sections, a region of almost 1 m in height, from 30 to 120 cm aboveground, exhibited significantly higher light intensities, reaching an average intensity of 225 µmol photons m−<sup>2</sup> s −1 in proximity to the fixtures (Figure 3B). At a canopy height of 1.5 to 1.8 m, the light intensities in the CR and illuminated sections were similar, and the considerable higher intensity at 1.8 m is due to sunlight penetration at this height. The 'Bioled' light fixtures utilized as intra-canopy illumination provided cool-white (CW) light (Figure 3A). The effect of the added illumination on the light intensity between the double-row beds was assessed when the canopy height was ~2 m. In control ('CR') non-illuminated sections, the light intensity of the inner canopy below 1.5 m was generally < 50 µmol photons m−2 s−1, and for the most part even <20 µmol photons m−2 s−1 (Figure 3B). In the illuminated sections, a region of almost 1 m in height, from 30 to 120 cm aboveground, exhibited significantly higher light intensities, reaching an average intensity of 225 µmol photons m−2 s−1 in proximity to the fixtures (Figure 3B). At a canopy height of 1.5 to 1.8 m, the light intensities in the CR and illuminated sections were similar, and the considerable higher intensity at 1.8 m is due to sunlight penetration at this height.

**Figure 3. Light spectrum and intensity within the canopy.** (**A**) Spectra of the cool-white 'Bioled' light fixtures. (**B**) Light intensity within the canopy in control vs. illuminated sections, recorded in December 2019. Intensities were measured with the light sensor directly below or above the fixtures at the indicated distances above the ground. Values shown are means ± SD of three control (CR) and three illuminated (LED) plots. **Figure 3. Light spectrum and intensity within the canopy.** (**A**) Spectra of the cool-white 'Bioled' light fixtures. (**B**) Light intensity within the canopy in control vs. illuminated sections, recorded in December 2019. Intensities were measured with the light sensor directly below or above the fixtures at the indicated distances above the ground. Values shown are means ± SD of three control (CR) and three illuminated (LED) plots.

The effects of the supplemental illumination, provided by Bioled fixtures, on the photosynthetic parameters and gas-exchange activity of inner canopy leaves were characterized in LED-D plots (Table 1). The chlorophyll content ('Chl'; measured non-destructively) was somewhat higher (~8%) in the illuminated leaves as compared to control ones. The effects of the supplemental illumination, provided by Bioled fixtures, on the photosynthetic parameters and gas-exchange activity of inner canopy leaves were characterized in LED-D plots (Table 1). The chlorophyll content ('Chl'; measured non-destructively) was somewhat higher (~8%) in the illuminated leaves as compared to control ones. The CO<sup>2</sup>

assimilation rates ('A') in leaves of LED sections were ~3.3-fold higher than non-illuminated leaves. The stomatal conductance ('Gs') and transpiration rate ('E') were, respectively, 5.2 and 3.5-fold higher in illuminated leaves vs. control ones. As the temperature of the leaves probed for the gas-exchange recordings was the same in both control and LED, the higher Gs and E can be attributed to the supplemental light. The average light intensity, recorded during the measurements ('PAR'), was ~3.7-fold higher in LED than in CR.

**Table 1.** Photosynthetic and gas-exchange parameters of the inner canopy † .


† Measurements were recorded non-destructively on inner canopy leaves from control (CR) and illuminated sections (LED). Recordings were made on leaves found 10–20 cm above the LED fixtures, and at the same height in control sections. For chlorophyll (Chl) and Fv/Fm measurements, n = 15 and 18 leaves, respectively. For gas-exchange measurements, n = 12 leaves from LED-D or CR sections. A, CO<sup>2</sup> assimilation rate; Gs, stomatal conductance; E, transpiration rate; Ci, intercellular CO2. Leaf temperature (T) and light intensity (PAR) were recorded during the gas-exchange measurement. Values shown represent means ± SD; distinct letters denote statistical significant differences (*p* < 0.05) between CR and LED.

The effect of the illumination on air temperature within the canopy, in vicinity of the LED fixtures, was recorded along the season. The daily minimal (T-min) and maximal (Tmax) air temperatures in the canopy of the three treatments are shown in Figure S1. Three examples for raw air temperature data on representative days depict how air temperature in the canopy is affected by operation of the illumination in LED-D and LED-N (Figure S2). In CR sections, T-min typically occurs between 04:00 and 07:00, and T-max between 12:00 and 15:00. The timing of T-max depends on the time of year and on whether the day is sunny (Figure S2A) or cloudy (Figure S2B). In LED-D sections, where the illumination operated from 06:00 to 18:00, the air temperature was higher by ~4.5 ◦C than the CR during the operation time. Accordingly, T-max was higher to a similar extent on most days in LED-D (Figure S1). In LED-N sections, higher air temperatures within the canopy were observed at the edges of daytime, in line with the operation times of the illumination for this treatment. As compared to CR, the air temperature within the canopy in LED-N was higher by 3.9 to 5.2 ◦C from 04:00 to 8:00 and by 4.2 to 5.6 ◦C from 16:00 to 22:00 (Figure S2). On some days, T-max in LED-N was higher than in CR and occurred around 10:00, just prior to the end of the first illumination period (Figure S2B,C). This is also evident in the whole season graph for T-max (Figure S1). Although LED-N operated from 04:00 to 10:00, the effect of the illumination on daily T-min was minor (Figures S1 and S2). The increased air temperature observed in LED treatments is mostly limited to the regions surrounding the light fixtures. Even though the increase in air temperature may not necessarily result in considerable increases in foliage temperature, it should still be kept in mind as a factor that can affect the plant physiology.
