*3.1. Chlorophyll Fluorescence and Photosynthesis*

Assessing the maximum efficiency of PSII (Fv/Fm) via chlorophyll fluorescence measurements is often used as a proxy measurement to assess the health of a plant. Here, it was used to assess injury related to photoperiod extension. During the initial stage of growth (23 DIT, 8 December 2018), leaves from TE tomato plants had similar maximum efficiency of PSII (Figure 3A). At 62 DIT (16 January 2019), leaves exposed to both red 23 h and mix 23 h lighting treatments produced lower Fv/F<sup>m</sup> values compared to leaves exposed to red 17 h and mix 17 h treatments (Figure 3B). During the late stage of growth (138 DIT, 2 April 2019), leaves under all lighting treatments again produced similar Fv/F<sup>m</sup> values (Figure 3C). Of note, leaves under both red 23 h and mix 23 h treatments showed an increase in Fv/F<sup>m</sup> values from 62 DIT to 138 DIT to values similar to those at the beginning of the experiment, indicating full recovery. As shown in Figure 4, leaves exposed to red 23 h and mix 23 h lighting displayed photoinhibition patterns characteristic of interveinal chlorosis. These patterns were not apparent on the 5th leaf of any treatments at 138 DIT (Figure 4). Leaves under the mix 23 h lighting treatment tended to have more interveinal chlorosis than leaves under the red 23 h lighting treatment at 62 DIT (Figure 4), which is also seen by a slightly higher Fv/F<sup>m</sup> in Figure 3B.

**Figure 3.** Maximum efficiency of PSII (Fv/Fm) from the 5th leaf of TE tomatoes grown under red 17 h, red 23 h, mix 17 h, or mix 23 h lighting treatments at 23 DIT (December 8, 2018, panel **A**), 62 DIT (January 16, panel **B**), and 138 DIT (April 2, 2019, panel **C**). Error bars represent the standard error of the mean of n = 8. Letter groups (A, B) represent significant differences between the lighting treatments at a specific time point and leaf position at *p* < 0.05. **Figure 3.** Maximum efficiency of PSII (Fv/Fm) from the 5th leaf of TE tomatoes grown under red 17 h, red 23 h, mix 17 h, or mix 23 h lighting treatments at 23 DIT (8 December 2018, panel **A**), 62 DIT (January 16, panel **B**), and 138 DIT (2 April 2019, panel **C**). Error bars represent the standard error of the mean of n = 8. Letter groups (A, B) represent significant differences between the lighting treatments at a specific time point and leaf position at *p* < 0.05. **Figure 3.** Maximum efficiency of PSII (Fv/Fm) from the 5th leaf of TE tomatoes grown under red 17 h, red 23 h, mix 17 h, or mix 23 h lighting treatments at 23 DIT (December 8, 2018, panel **A**), 62 DIT (January 16, panel **B**), and 138 DIT (April 2, 2019, panel **C**). Error bars represent the standard error of the mean of n = 8. Letter groups (A, B) represent significant differences between the lighting treatments at a specific time point and leaf position at *p* < 0.05.

**Figure 4.** Spatial response of Fv/Fm from the 5th leaf of TE tomatoes grown under either red 17 h, red 23 h, mix 17 h, or mix 23 h lighting treatments at 23 DIT (December 8, 2018), 62 DIT(January 16, 2019), and 138 DIT (April 2, 2019). **Figure 4.** Spatial response of Fv/Fm from the 5th leaf of TE tomatoes grown under either red 17 h, red 23 h, mix 17 h, or mix 23 h lighting treatments at 23 DIT (December 8, 2018), 62 DIT(January 16, 2019), and 138 DIT (April 2, 2019). **Figure 4.** Spatial response of Fv/Fm from the 5th leaf of TE tomatoes grown under either red 17 h, red 23 h, mix 17 h, or mix 23 h lighting treatments at 23 DIT (8 December 2018), 62 DIT (16 January 2019), and 138 DIT (2 April 2019).

At 21 DIT (December 6, 2018), leaves under the mix 17 h treatment produced the highest daytime net carbon exchange rate (NCER; Figure 5A). Both red 23 h and mix 23 h as well as the red 17 h treatment produced statistically lower daytime NCER values compared to leaves exposed to mix 17 h. Respiration rates, indicated by a negative NCER, were the highest under both 17 h lighting treatments (Figure 5A). In both 23 h lighting treatments, an increase in NCER was observed as there was light from the LEDs during this subjective nighttime period (Figure 5A). It should be noted that the red 23 h lighting treatment had a higher NCER than the mix 23 h lighting treatment during the subjective nighttime period, which may indicate the first signs of the alleviation of photoperiod-related injury by red light (Figure 5A). At 55 DIT (January 9, 2019), leaves exposed to mix 23 h produced drastically reduced daytime NCER compared to leaves exposed to mix 17 At 21 DIT (December 6, 2018), leaves under the mix 17 h treatment produced the highest daytime net carbon exchange rate (NCER; Figure 5A). Both red 23 h and mix 23 h as well as the red 17 h treatment produced statistically lower daytime NCER values compared to leaves exposed to mix 17 h. Respiration rates, indicated by a negative NCER, were the highest under both 17 h lighting treatments (Figure 5A). In both 23 h lighting treatments, an increase in NCER was observed as there was light from the LEDs during this subjective nighttime period (Figure 5A). It should be noted that the red 23 h lighting treatment had a higher NCER than the mix 23 h lighting treatment during the subjective nighttime period, which may indicate the first signs of the alleviation of photoperiod-related injury by red light (Figure 5A). At 55 DIT (January 9, 2019), leaves exposed to mix 23 h produced drastically reduced daytime NCER compared to leaves exposed to mix 17 At 21 DIT (6 December 2018), leaves under the mix 17 h treatment produced the highest daytime net carbon exchange rate (NCER; Figure 5A). Both red 23 h and mix 23 h as well as the red 17 h treatment produced statistically lower daytime NCER values compared to leaves exposed to mix 17 h. Respiration rates, indicated by a negative NCER, were the highest under both 17 h lighting treatments (Figure 5A). In both 23 h lighting treatments, an increase in NCER was observed as there was light from the LEDs during this subjective nighttime period (Figure 5A). It should be noted that the red 23 h lighting treatment had a higher NCER than the mix 23 h lighting treatment during the subjective nighttime period, which may indicate the first signs of the alleviation of photoperiodrelated injury by red light (Figure 5A). At 55 DIT (9 January 2019), leaves exposed to mix 23 h produced drastically reduced daytime NCER compared to leaves exposed to mix 17 h

lighting treatment (Figure 5B), indicating severe damage caused by the long photoperiod with a mixed light spectrum. However, leaves exposed to red 23 h produced statistically similar daytime NCER values as both 17 h lighting treatments. Leaves exposed to both 17 h lighting treatments produced the highest respiration rates during the nighttime period, as expected (Figure 5B). Surprisingly, leaves under both 23 h lighting treatments also produced negative NCER values (indicating respiration), even during a period with an appreciable amount of supplemental light (Figure 5B). However, it should be noted that the respiratory rate in both 23 h treatments was lower than those in the 17 h treatment. While light was present in the night, it might not have been utilized to the full extent due to the leaf chlorosis (Figures 3B and 4).

Daytime transpiration rates at both 21 DIT and 55 DIT were similar among all lighting treatments (Figure 5C,D). At 21 DIT, nighttime transpiration rates were also similar among all lighting treatments (Figure 5C). However, at 55 DIT, the transpiration rate was the highest in leaves exposed to the mix 23 h lighting treatment and the lowest under the mix 17 h treatment (Figure 5D). Water use efficiency (WUE) indicates the rate of CO<sup>2</sup> and H2O exchange through stomata, with a positive rate indicating photosynthesis and a negative rate indicating respiration. At 21 DIT and 55 DIT, the daytime WUE of leaves exposed to the mix 17 h lighting treatment was higher than leaves exposed to the mix 23 h lighting treatment (Figure 5E,F). At 55 DIT, WUE from leaves exposed to the red 17 h treatment was also higher than leaves under the mix 23 h treatment. However, WUE was not different between 17 h and 23 h with red light. Therefore, light spectral compositions did affect the response of WUE to the long photoperiod (23 h). Nighttime WUE at 21 DIT was the lowest under the red 17 h treatment and the highest under both red 23 h and mix 17 h (Figure 5E). At 55 DIT, nighttime WUE was the lowest in leaves exposed to the mix 17 h lighting treatment but the highest under the mix 23 h lighting treatment (Figure 5F).

Light use efficiency (LUE) is the calculation of how much CO<sup>2</sup> is fixed per incoming unit of photons. In this way, it provides a metric which allows for the assessment of light capture and carbon fixation. All leaves at 21 DIT produced similar LUE values (Figure 5G). At 55 DIT, leaves grown under both 17 h lighting treatments produced higher LUE values than leaves under the mix 23 h lighting treatment (Figure 5H). At both time periods, nighttime LUE for the 17 h treatments was non-resultant due to a light intensity of 0 µmol m−<sup>2</sup> s −1 (Figure 5G,H). At 21 DIT, the nighttime LUE was higher under the red 23 h treatment than the mix 23 h lighting treatments (Figure 5G). Similar results were obtained during measurements at 55 DIT (Figure 5H). This indicates that leaves grown under an extended red photoperiod were better able to utilize the light during the subjective nighttime period than those under the mixed spectrum, likely due to less photoperiod-related injury.

At 20 DIT (5 December 2018), photosynthetic light response curves were generated for the fifth leaf of TE tomatoes (Figure 6). All photosynthetic parameters (i.e., respiration rate, light compensation point (LCP), quantum yield (QY), and maximum photosynthetic rate (Pnmax)) were similar among all treatments (Table 2). At 54 DIT (8 January 2019), leaves exposed to the mix 17 h lighting treatment produced the lowest respiration rate and leaves exposed to the mix 23 h lighting treatment produced the highest (Table 2). Both 23 h lighting treatments produced drastically higher LCP than leaves exposed to the 17 h treatments, showing an inability to utilize light well (Figure 6F; Table 2). At 54 DIT, leaves exposed to the mix 17 h treatment produced the highest QY out of all lighting treatments. Furthermore, leaves exposed to the red 17 h treatment produced higher QY than leaves exposed to either red 23 h or mix 23 h treatments (Table 2). At 54 DIT, Pnmax was greatly reduced in both 23 h lighting treatments compared to both red 17 h and mix 17 h lighting treatments (Table 2). At 134 DIT (29 March 2019), respiration rate, LCP, and QY were similar between all lighting treatments. However, Pnmax was higher in leaves exposed to the mix 23 h lighting treatment than the red 17 h lighting treatment (Table 2).

**Figure 5.** Net carbon exchange rate (NCER; panel **A**,**B**), transpiration (panel **C,D**), water use efficiency (panel **E**,**F**), light use efficiency (panel **G**,**H**) of the 5th leaf from TE tomato plants grown under either red 17 h, red 23 h, mix 17 h, or mix 23 h lighting treatments at 21 DIT (December 6, 2018, panels **A**,**C**,**E**,**G**) or 55 DIT (January 9, 2019, panels **B**,**D**,**F**,**H**) during the daytime and nighttime. Measurements were performed using a Li-COR 6400 fitted with a clear-top chamber on a cloudy day or night and thus represent the NCER driven mostly by the supplemental lighting. Error bars represent the standard **Figure 5.** Net carbon exchange rate (NCER; panel **A**,**B**), transpiration (panel **C,D**), water use efficiency (panel **E**,**F**), light use efficiency (panel **G**,**H**) of the 5th leaf from TE tomato plants grown under either red 17 h, red 23 h, mix 17 h, or mix 23 h lighting treatments at 21 DIT (6 December 2018, panels **A**,**C**,**E**,**G**) or 55 DIT (9 January 2019, panels **B**,**D**,**F**,**H**) during the daytime and nighttime. Measurements were performed using a Li-COR 6400 fitted with a clear-top chamber on a cloudy day or night and thus represent the NCER driven mostly by the supplemental lighting. Error bars represent the standard error of the mean of n = 3. Letter groups (A, B, C) represent significant differences within a panel between the lighting treatments at a specific data collection period at *p* < 0.05.

treatments at a specific data collection period at *p* < 0.05.

error of the mean of n = 3. Letter groups (A, B, C) represent significant differences within a panel between the lighting

**Figure 6.** Photosynthetic light response curves from TE leaves grown under either red 17 h, red 23 h, mix 17 h, or mix 23 h lighting treatments at 20 DIT (December 5, 2018, panel **A**), 54 DIT (January 8, 2019, panel **B**), and 134 DIT (March 29, 2019, panel **C**) as determined using a Li-COR 6400 with a red/blue standard Li-COR light source. Measurements were performed at a CO2 concentration of 800µL L<sup>−</sup>1, leaf temperature of 24 °C, and a relative humidity of 55–65%. Regression lines were fit to y = yo + a(1 − e(−b\*x)) for each lighting treatment. Panels (**D**–**F**) are magnifications of 0–100 µmol m<sup>−</sup>2 s−1 PAR regions fit to the regression line y = mx + b. **Figure 6.** Photosynthetic light response curves from TE leaves grown under either red 17 h, red 23 h, mix 17 h, or mix 23 h lighting treatments at 20 DIT (5 December 2018, panel **A**), 54 DIT (8 January 2019, panel **B**), and 134 DIT (29 March 2019, panel **C**) as determined using a Li-COR 6400 with a red/blue standard Li-COR light source. Measurements were performed at a CO<sup>2</sup> concentration of 800µL L−<sup>1</sup> , leaf temperature of 24 ◦C, and a relative humidity of 55–65%. Regression lines were fit to y = y<sup>o</sup> + a(1 − e (−b∗x)) for each lighting treatment. Panels (**D**–**F**) are magnifications of 0–100 µmol m−<sup>2</sup> s <sup>−</sup><sup>1</sup> PAR regions fit to the regression line y = mx + b.

**Table 2.** Summary of the major physiological traits as determined by leaf light response curves (Figure 6) from tomatoes grown under red 17 h, red 23 h, mix 17 h, and mix 23 h at 20 (December 5, 2018), 54 (January 8, 2019), and 134 DIT (March 29, 2019). Respiration values were the averages of NCER when the light level was 0 µmol m<sup>−</sup>2 s−1. The light compensation point (LCP) and quantum yield (QY) were calculated from a regression line (y = mx + b) fitted to the values between the PAR values of 0–100 µmol m-2 s-1. The photosynthetic maximum (Pnmax) was calculated from y = yo + a(1 − e(−b\*x)). Values ± the standard error of the mean are representative of n = 3. Within each parameter and time of measurement, letter groups (A, B, C, D) represent a statistical difference as determined by a two-way ANOVA with a Tukey–Kramer adjustment (*p* < 0.05). **Table 2.** Summary of the major physiological traits as determined by leaf light response curves (Figure 6) from tomatoes grown under red 17 h, red 23 h, mix 17 h, and mix 23 h at 20 (5 December 2018), 54 (8 January 2019), and 134 DIT (29 March 2019). Respiration values were the averages of NCER when the light level was 0 µmol m−<sup>2</sup> s −1 . The light compensation point (LCP) and quantum yield (QY) were calculated from a regression line (y = mx + b) fitted to the values between the PAR values of 0–100 µmol m−<sup>2</sup> s −1 . The photosynthetic maximum (Pnmax) was calculated from y = y<sup>o</sup> + a(1 − e (−b∗x)). Values ± the standard error of the mean are representative of n = 3. Within each parameter and time of measurement, letter groups (A, B, C, D) represent a statistical difference as determined by a two-way ANOVA with a Tukey–Kramer adjustment (*p* < 0.05).


At 20 DIT (5 December 2018), all parameters related to photosynthetic performance (Vcmax and Jmax) were similar among all lighting treatments (Figure 7; Table 3). At 54 DIT (8 January 2019), leaves exposed to the mix 17 h lighting treatment produced the highest Vcmax, Jmax, and Pnmax compared to the other lighting treatments (Table 3). During the same time period, leaves exposed to the red 17 h lighting treatment produced higher values of Vcmax, Jmax, and Pnmax than leaves exposed to either 23 h lighting treatment (Table 3). At 134 DIT (29 March 2019), Vcmax, Jmax, and Pnmax were similar among all lighting treatments, returning to levels observed at the beginning of the experiment (Table 3). At 20 DIT (December 5, 2018), all parameters related to photosynthetic performance (Vcmax and Jmax) were similar among all lighting treatments (Figure 7; Table 3). At 54 DIT (January 8, 2019), leaves exposed to the mix 17 h lighting treatment produced the highest Vcmax, Jmax, and Pnmax compared to the other lighting treatments (Table 3). During the same time period, leaves exposed to the red 17 h lighting treatment produced higher values of Vcmax, Jmax, and Pnmax than leaves exposed to either 23 h lighting treatment (Table 3). At 134 DIT (March 29, 2019), Vcmax, Jmax, and Pnmax were similar among all lighting treatments, returning to levels observed at the beginning of the experiment (Table 3).

**Red 23 h** −1.45 ± 0.24 A 25.44 ± 4.78 A 0.055 ± 0.002 A 25.93 ± 1.03 AB **Mix 17 h** −2.31 ± 0.49 A 39.00 ± 9.12 A 0.058 ± 0.002 A 28.15 ± 2.36 AB **Mix 23 h** −1.83 ± 0.24 A 31.08 ± 3.30 A 0.058 ± 0.002 A 35.00 ± 1.36 A

*Plants* **2021**, *10*, x FOR PEER REVIEW 12 of 23

**Figure 7.** Photosynthetic CO2 response curve from leaves grown under red 17 h, red 23 h, mix 17 h, and mix 23 h lighting treatments at 20 DIT (December 5, 2018, panel **A**), 54 DIT (January 8, 2019, panel **B**), and 134 DIT (March 29, 2019, panel **C**). As determined using a Li-COR 6400 with a red/blue standard Li-COR light source. Measurements were performed at 300 µmol m<sup>−</sup>2 s−1 PAR, a temperature of 24 °C, and relative humidity of 55–65%. Rubisco- and RuBP-limited fit lines were determined using temperature corrections from [31,32]. **Figure 7.** Photosynthetic CO<sup>2</sup> response curve from leaves grown under red 17 h, red 23 h, mix 17 h, and mix 23 h lighting treatments at 20 DIT (5 December 2018, panel **A**), 54 DIT (8 January 2019, panel **B**), and 134 DIT (29 March 2019, panel **C**). As determined using a Li-COR 6400 with a red/blue standard Li-COR light source. Measurements were performed at 300 µmol m−<sup>2</sup> s <sup>−</sup><sup>1</sup> PAR, a temperature of 24 ◦C, and relative humidity of 55–65%. Rubisco- and RuBP-limited fit lines were determined using temperature corrections from [31,32].

**Table 3.** Summary of the major physiological traits as determined by leaf CO2 response curves (Figure 7) from tomatoes grown under red 17 h, red 23 h, mix 17 h, and mix 23 h at 20 (December 5, 2018), 54 (January 8, 2019), and 134 DIT (Mar. 29, 2019). The maximum rate of Rubisco carboxylation (Vcmax) and the maximum rate of electron transport (Jmax) were determined using equations from [31,32]. Pnmax was calculated from y = yo + a(1−e(−b\*x)) and indicates the maximum rate of photosynthesis at a light level of 300 µmol m-2 s-1 at a saturating CO2 level. Values ± the standard error of the mean are representative of n = 3. Within each parameter and time of measurement, letter groups (A, B, C) represent a statistical difference as determined by a two-way ANOVA with a Tukey–Kramer adjustment (*p* < 0.05). **Lighting Treat-Vcmax (µmol CO2 m-2 s-1) Jmax (µmol e− m-2 s-1) Pnmax (µmol CO2 m-2 s-1) Table 3.** Summary of the major physiological traits as determined by leaf CO<sup>2</sup> response curves (Figure 7) from tomatoes grown under red 17 h, red 23 h, mix 17 h, and mix 23 h at 20 (5 December 2018), 54 (8 January 2019), and 134 DIT (29 March 2019). The maximum rate of Rubisco carboxylation (Vcmax) and the maximum rate of electron transport (Jmax) were determined using equations from [31,32]. Pnmax was calculated from y = y<sup>o</sup> + a(1−e (−b∗x)) and indicates the maximum rate of photosynthesis at a light level of 300 µmol m−<sup>2</sup> s <sup>−</sup><sup>1</sup> at a saturating CO<sup>2</sup> level. Values ± the standard error of the mean are representative of n = 3. Within each parameter and time of measurement, letter groups (A, B, C) represent a statistical difference as determined by a two-way ANOVA with a Tukey–Kramer adjustment (*p* < 0.05).

