*2.1. Plant Material and Experimental Design*

Tomato (*Solanum lycopersicum*) seedlings cv. 'Trovanzo' were grafted onto 'Emperator' (TE) or onto 'Kaiser' (TK) in a double-stemmed (twin-head) system. 'Emperator' has been observed to promote vegetative growth (or a vigorous rootstock) whereas 'Kaiser' has been shown to promote generative growth. In this way, we could observe the homeostatic balance between two different rootstock types. Twin-head transplants (5 weeks old) raised by a commercial propagator were placed into rockwool slabs on top of a raised growing trough (30 cm high) in a large glass greenhouse (200 m<sup>2</sup> growing area) at the Harrow Research and Development Centre, Agriculture and Agri-Food Canada, Harrow, Ontario, Canada (42.03◦ N, 82.9◦ W) on 11 November 2018 at a plant density of 2 plants m−<sup>2</sup> (4 stems m−<sup>2</sup> ). The plants were planted into 6 rows, with the 2 outside rows serving as guard rows. The two stems/heads of each plant in the row were trained upward along the vertical strings into a 'V' system as in commercial production. The strings were hung onto the top wires (3.5 m high). Once the plant head reached the overhead wires, a few bottom leaves were removed and the plants were lowered twice every week. The plants were drip-irrigated using a complete nutrient solution [22]. The electrical conductivity and pH were set at 2.8 dS m−<sup>1</sup> and 5.8, respectively. Plants were grown at an enriched CO<sup>2</sup> concentration of 800 µL L−<sup>1</sup> when the greenhouse was not ventilated. The average daytime temperature was held between 21 and 24 ◦C during the months of November, December, and January. Average daytime temperatures during the months of February and March were between 21 and 25 ◦C. Average daytime temperatures during the months of April and May were between 22 and 27 ◦C depending on the ambient solar radiation. Nighttime temperature was maintained at 20 ± 1 ◦C throughout the production period. Relative humidity of 70 ± 10% was maintained during both daytime and nighttime periods.

The 4 middle rows were divided into 16 plots via white curtains, which were impenetrable to light. There were 24 twin-head plants (48 stems) in each plot; 12 plants for each of the 2 rootstocks were planted. Four supplemental overhead lighting treatments were applied to the 16 plots in a Latin square design with 4 replications (one lighting treatment in each row or column of the 16 plots): 100% red from 23:00 to 16:00 (Red 17 h), 100% red from 17:00 to 16:00 (Red 23 h), mixed light from 23:00 to 16:00 (Mix 17 h), and mixed light from 17:00 to 16:00 (Red 23 h; Figure 1). The mixed light had 75% red (600–700 nm), 20% blue (400–499 nm), and 5% green light (500–599 nm), with green light being introduced via white diodes. The four lighting treatments provided similar DLIs (Table 1). All the lighting treatments were applied using Pro 325e smart LED fixtures from LumiGrow (Emeryville, California, USA). A 23 h monochromatic red photoperiod was utilized because red light tends to preferentially support vegetative growth. Furthermore, red diodes are highly efficient, and thus a pure red spectrum is able to achieve the highest photosynthetic photon efficacy (PPE) [23]. The mixed light spectrum was chosen as a control, which provides close to the recommended amounts of blue light [24] and some green light, which has also been shown to improve tomato growth and yield [25].

Equation (1) [26].

**Supplemental Light Intensity (µmol m-2 s-1)** 

**Lighting Treatment** 

**Table 1.** Photosynthetic photon flux density of supplemental lighting treatments (400–700 nm; 80 cm below the LED fixtures) as determined by using a Li-190R quantum line sensor at night. Red to far-red ratios (R:Fr) were estimated by dividing the total photons of red (600–700 nm) by the total photons of far-red (700–780 nm) during both sunny and cloudy days when the supplementary lighting fixtures were on. Phytochrome photostationary state (PSS) was calculated using

Red 17 h 176 ± 7 3.78 25.56 0.82 0.88 10.79 ± 0.41 Red 23 h 127 ± 4 2.81 17.77 0.80 0.87 10.54 ± 0.36 Mix 17 h 169 ± 4 2.55 15.29 0.79 0.86 10.32 ± 0.27 Mix 23 h 134 ± 5 2.30 12.01 0.78 0.86 11.09 ± 0.42

**R:Fr— Cloudy Day** 

**R:Fr— Sunny Day** 

were determined on both sunny and cloudy days (February 11 and February 14, 2019, respectively) by dividing the total photons of red by the total photons of far-red (700–780 nm). Further to this, the phytochrome photostationary state (PSS) was determined using

are the photochemical cross-section of phytochrome in the red absorbing state and far-red

ଷ଼ ሻ

ଷ଼ + ∑ ி ଼

Spectral readings for these calculations were taken between 12:00 and 14:00 using a Li-COR Li-180 spectrometer on both sunny and cloudy days under their respective lighting treatments. The sunny day was one with no observable clouds in the sky and the cloudy day was fully overcast. Throughout the experiment, supplemental lighting remained on regardless of ambient light levels to ensure that all treatments received similar total DLIs (Figure 2). The curtains were closed during cloudy days to prevent light contamination between treatments. During sunny days, the curtains were opened to prevent shading of natural light. On days which were partly cloudy/sunny, the forecast was used to determine the majority (i.e., sun or cloudy) and then curtains were opened or closed as appropriate. Because there was sunlight between 16:00 and 17:00 (i.e., during natural sunset), the actual photoperiods (including sunlight) were 18 h and 24 h (CL) for the 17 h and 23 h lighting, respectively. In commercial greenhouses, bumble bees are used as pollinators for fruit setting. The stopping of supplemental lighting between 16:00 and 17:00 was to facilitate the return of bees to their hives under natural dusk/sunset conditions; otherwise, the bees may get lost and significantly increase the number of bees (and associated cost) needed for pollination [27]. Dusk/sunset varied throughout the course of the experiment from 16:52 to 20:22, which was sufficient to allow for bees to return to their hives.

> **PSS— Sunny Day**

**PSS— Cloudy Day**  **Supplemental DLI (mol m-2 d-1)** 

*r* and *Fr*

ଷ଼ <sup>ሻ</sup> (1)

Equation (1) from Sager et al. [26], where *N* is the photon flux (mol m-2 s-1) and

= ሺ∑ ଼

ሺ∑ ଼

absorbing state, respectively.

**Figure 1.** Normalized photon flux density (380–780 nm) from red and mixed LED lighting treatments during both sunny and cloudy days. The spectra were measured using a Li-180 Spectrometer (Li-COR Inc., Lincoln, NE, USA) at a distance of 80 cm from the light fixtures. Measurements for sunny days were done on 11 February 2019 and cloudy days on 14 February 2019 between 12:00 and 14:00. Panel (**A**) represents spectra from both red light treatments during sunny and cloudy days. Panel (**B**) represents spectra from both mixed light treatments during sunny and cloudy days.

**Table 1.** Photosynthetic photon flux density of supplemental lighting treatments (400–700 nm; 80 cm below the LED fixtures) as determined by using a Li-190R quantum line sensor at night. Red to far-red ratios (R:Fr) were estimated by dividing the total photons of red (600–700 nm) by the total photons of far-red (700–780 nm) during both sunny and cloudy days when the supplementary lighting fixtures were on. Phytochrome photostationary state (PSS) was calculated using Equation (1) [26].


Application of the supplemental lighting treatments began on 16 November 2018. Supplemental light intensities as shown in Table 1 were determined at three different positions within a treatment at 80 cm from the light fixtures (just above the heads of the plants) using a Li-COR 190R (Li-COR Biosciences Inc., Lincoln, NE, USA) quantum line sensor during the nighttime period to exclude natural solar radiation. Red:far-red (R:Fr) were determined on both sunny and cloudy days (11 February and 14 February 2019, respectively) by dividing the total photons of red by the total photons of far-red (700–780 nm). Further to this, the phytochrome photostationary state (PSS) was determined using Equation (1) from Sager et al. [26], where *N* is the photon flux (mol m−<sup>2</sup> s −1 ) and *σ<sup>r</sup>* and *σFr* are the photochemical cross-section of phytochrome in the red absorbing state and far-red absorbing state, respectively.

$$PSS = \frac{\left(\sum\_{380}^{780} N \sigma\_r\right)}{\left(\sum\_{380}^{780} N \sigma\_r + \sum\_{380}^{780} N \sigma\_{Fr}\right)} \tag{1}$$

Spectral readings for these calculations were taken between 12:00 and 14:00 using a Li-COR Li-180 spectrometer on both sunny and cloudy days under their respective lighting treatments. The sunny day was one with no observable clouds in the sky and the cloudy day was fully overcast. Throughout the experiment, supplemental lighting remained on regardless of ambient light levels to ensure that all treatments received similar total DLIs

(Figure 2). The curtains were closed during cloudy days to prevent light contamination between treatments. During sunny days, the curtains were opened to prevent shading of natural light. On days which were partly cloudy/sunny, the forecast was used to determine the majority (i.e., sun or cloudy) and then curtains were opened or closed as appropriate. Because there was sunlight between 16:00 and 17:00 (i.e., during natural sunset), the actual photoperiods (including sunlight) were 18 h and 24 h (CL) for the 17 h and 23 h lighting, respectively. In commercial greenhouses, bumble bees are used as pollinators for fruit setting. The stopping of supplemental lighting between 16:00 and 17:00 was to facilitate the return of bees to their hives under natural dusk/sunset conditions; otherwise, the bees may get lost and significantly increase the number of bees (and associated cost) needed for pollination [27]. Dusk/sunset varied throughout the course of the experiment from 16:52 to 20:22, which was sufficient to allow for bees to return to their hives. *Plants* **2021**, *10*, x FOR PEER REVIEW 5 of 23 **Figure 1.** Normalized photon flux density (380–780 nm) from red and mixed LED lighting treatments during both sunny and cloudy days. The spectra were measured using a Li-180 Spectrometer (Li-COR Inc., Lincoln, NE, USA) at a distance of 80 cm from the light fixtures. Measurements for sunny days were done on February 11, 2019 and cloudy days on February 14, 2019 between 12:00 and 14:00. Panel (**A**) represents spectra from both red light treatments during sunny and cloudy days. Panel (**B**) represents spectra from both mixed light treatments during sunny and cloudy days.

**Figure 2.** Daily average solar radiation as measured from November 16, 2018 to May 22, 2019 using a Li-COR LI-200R pyranometer converted from W m-2 to µmol m-2 s-1 using the conversion value of 2.1. Readings were taken above the greenhouse and then corrected for an approximate 50% transmissivity to account for shading from the greenhouse structure, lighting fixtures, and shade curtains. Measurements were taken every 2 h, beginning at 08:00 and concluding at 16:00, between the wavelengths of 400 and 1100 nm. Measurements during this period were averaged to provide an average solar radiation for each day (line plot). The bar graph indicates the average daily solar radiation throughout the month. Breaks in the line plot indicate periods of time which were not documented due to a technical malfunction. **Figure 2.** Daily average solar radiation as measured from 16 November 2018 to 22 May 2019 using a Li-COR LI-200R pyranometer converted from W m−<sup>2</sup> to µmol m−<sup>2</sup> s <sup>−</sup><sup>1</sup> using the conversion value of 2.1. Readings were taken above the greenhouse and then corrected for an approximate 50% transmissivity to account for shading from the greenhouse structure, lighting fixtures, and shade curtains. Measurements were taken every 2 h, beginning at 08:00 and concluding at 16:00, between the wavelengths of 400 and 1100 nm. Measurements during this period were averaged to provide an average solar radiation for each day (line plot). The bar graph indicates the average daily solar radiation throughout the month. Breaks in the line plot indicate periods of time which were not documented due to a technical malfunction.

#### *2.2. Growth Measurements 2.2. Growth Measurements*

from [28].

Growth measurements were performed on 6 randomly selected plants from TE and TK at 31, 60, and 139 days into the treatment (DIT), corresponding with December 16th, 2018, January 14, 2019, and April 3, 2019, respectively. Growth measurements included leaf length, leaf width, and chlorophyll content of the 5th, 10th, and 15th leaf when applicable. Leaf chlorophyll was measured using a SPAD meter (model 502, Konica Minota, Osaka, Japan) and values were converted to chlorophyll content using correction equations generated by spectrophotometric pigment analysis. Chlorophyll correction curves were generated by extracting leaf punches in 95% ethanol at 78 °C for approximately 3 h until the tissue was cleared. Samples were then analyzed at 664.2 nm, 648.6 nm, and 470 nm wavelengths using a spectrophotometer (Beckman DU-640 UV–Vis, Indianapolis, IN, USA). Concentrations of *chlorophyll a*, *b*, and carotenoids were determined via equations Growth measurements were performed on 6 randomly selected plants from TE and TK at 31, 60, and 139 days into the treatment (DIT), corresponding with 16 December 2018, 14 January 2019, and 3 April 2019, respectively. Growth measurements included leaf length, leaf width, and chlorophyll content of the 5th, 10th, and 15th leaf when applicable. Leaf chlorophyll was measured using a SPAD meter (model 502, Konica Minota, Osaka, Japan) and values were converted to chlorophyll content using correction equations generated by spectrophotometric pigment analysis. Chlorophyll correction curves were generated by extracting leaf punches in 95% ethanol at 78 ◦C for approximately 3 h until the tissue was cleared. Samples were then analyzed at 664.2 nm, 648.6 nm, and 470 nm wavelengths using a spectrophotometer (Beckman DU-640 UV–Vis, Indianapolis, IN, USA). Concentrations of *chlorophyll a*, *b*, and carotenoids were determined via equations from [28].

The 5th leaves from TE plants were placed in the chamber of a Li-COR 6400 (Li-COR

temperature was set to 24 °C, with a relative humidity of 55–65% and a CO2 level held at 800 µL L−1. Three leaves from separate plants under each treatment were used at 21 DIT (December 6, 2018) and 55 DIT (January 9, 2019) for both daytime and nighttime measurements. Measurements were taken during the day on cloudy days to maximize the effect

*2.3. Leaf Gas Exchange: Day and Night Measurements* 
