**1. Introduction**

Vertical farming refers to hydroponic crop production in buildings with precise environmental control. Vertical farms do not require arable land and can obtain high crop yields from a small land area. Therefore, vertical farms are becoming popular in urban areas. However, the energy costs associated with the required electric lighting and environmental control, especially cooling and dehumidification, in vertical farming are high [1]. The cost of electric lighting in controlled environment agriculture in just the United States has been estimated at ~\$600 million annually [2]. To bring this cost down, research into more efficient production techniques [3] and more energy-efficient crops (more biomass gain per unit of energy use) are required.

Overall, crop growth is a function of the amount of incident light reaching the canopy, which depends on projected canopy size (PCS) [4,5], and light use efficiency (LUE, grams of biomass produced per mol of incident light) [6]. To screen for crops with rapid growth, quantifying PCS development and LUE is essential.

Plants that produce a larger canopy can achieve faster growth by increasing the amount of incident light reaching the canopy compared to plants with a smaller canopy [4,5].

**Citation:** Jayalath, T.C.;

van Iersel, M.W. Canopy Size and Light Use Efficiency Explain Growth Differences between Lettuce and Mizuna in Vertical Farms. *Plants* **2021**, *10*, 704. https://doi.org/10.3390/ plants10040704

Academic Editors: Rosario Muleo and Valeria Cavallaro

Received: 28 February 2021 Accepted: 2 April 2021 Published: 6 April 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

As incident light increases, canopy photosynthesis and biomass accumulation of plants also increase [5], as long as canopy photosynthesis is not light-saturated. This in turn helps plants to produce additional canopy faster than plants with a smaller canopy [4]. Therefore, quantifying the PCS and determining how this affects the amount of incident light, is important. Non-destructive digital imaging has been used in many crops, including tomatoes [7], soybean [8], and lettuce [5,9]. Periodic PCS measurements can be used to estimate the daily PCS [6]. Combining those data with PPFD data allows for estimation of the total incident light over the course of the growing cycle. Projected canopy size is also valuable to make crop growth predictions, since PCS early in the growing cycle may be correlated with the final dry weight of the crop, as we have previously shown in lettuce (*Lactuca sativa*) [6,10] and black-eyed susan (*Rudbeckia fulgida*) [11].

The LUE of a crop describes how efficiently plants use the incident light for growth. The LUE can be calculated by dividing the dry weight of a plant by the total incident light that the plant received throughout the growing period [6]. This provides a physiological measure of how efficiently crops use light, in contrast to calculating LUE based on the amount of light provided to the growing space, which provides insight into production efficiency, but not underlying physiological mechanisms [6]. Factors such as chlorophyll content, the quantum yield of photosystem II (ΦPSII, the fraction of absorbed photons used to drive photochemistry), and CO<sup>2</sup> assimilation are all important in determining the LUE of a crop.

Higher chlorophyll content increases light absorption [12]. The energy of the absorbed photons can then be used in the light reactions of photosynthesis to drive photochemistry (electron transport), while some light energy is dissipated as heat (NPQ, non-photochemical quenching), or re-emitted as fluorescence. Photochemistry competes with NPQ and chlorophyll fluorescence for excitation energy from photons [13,14]. To understand how different lighting strategies affect plant growth, it is important to determine ΦPSII, since this partly determines how efficiently plants use the absorbed light to drive photosynthesis and produce biomass. Previous studies have shown that increasing PPFD decreases ΦPSII, due to the partial closing of photosystem II reaction centers and upregulation of NPQ [15,16]. Although ΦPSII decreases, the electron transport rate (ETR) increases with increasing PPFD [15–17]. The ΦPSII of different species responds differently to increasing PPFDs [16]. In addition, plants acclimated to high light have higher ΦPSII and ETR than those acclimated to low light. Therefore, to increase the production efficiency in plant factories, identifying crops with both high ΦPSII and high ETR is important, because a higher ETR will result in the production of more ATP and NADPH for use in CO<sup>2</sup> assimilation. Both high ΦPSII and high chlorophyll content index (CCI) may increase the ETR, CO<sup>2</sup> assimilation rate, and potentially the growth rate of plants.

A previous indoor study with mizuna (*Brassica rapa* var. *japonica*) and oakleaf lettuce (*Lactuca sativa* 'Green Salad Bowl') found much faster growth of mizuna compared to lettuce [18]. This growth difference must be the result of morphological and/or physiological differences between the two crops, but those underlying reasons were not explored in that study. A better understanding of the underlying reasons for growth differences among crops will facilitate screening for rapid-growing crops and cultivars that are well-suited for vertical farming production systems. It can also enable breeding efforts by providing selection criteria for crops that are well-suited for production in vertical farms. Therefore, our objective was to determine the underlying mechanisms for the growth differences between mizuna and lettuce. We hypothesize that the faster growth of mizuna is the result of a greater PCS, increased canopy incident light, and higher LUE. Since crop growth is affected by PPFD, we also determined how different PPFDs affect the morphological and physiological factors underlying crop growth. Comparing two species, grown at different PPFDs, allowed us to determine how useful PCS and LUE are in explaining crop growth.

### **2. Results** *2.1. Experiment*

**2. Results**

#### *2.1. Experiment* To determine the underlying morphological and/or physiological reasons for growth

*Plants* **2021**, *10*, x 3 of 14

To determine the underlying morphological and/or physiological reasons for growth differences between mizuna and lettuce at different PPFDs, plants were grown at six PPFD levels (~50, 125, 200, 275, 350, and 425 µmol m−<sup>2</sup> s <sup>−</sup><sup>1</sup> at the center of each section) for a 16-hr photoperiod. Projected canopy size was measured twice a week throughout the growing period, and those images were used to estimate the daily PCS and to calculate the total incident light per plant over the growing period. In addition, leaf chlorophyll content index (CCI), anthocyanin content index (ACI), ΦPSII, and net CO<sup>2</sup> assimilation of both crops were measured during the study. Mizuna and lettuce were harvested 27 and 28 days after seeding, respectively. The total leaf area and shoot dry weight were measured. Finally, the LUE was calculated by dividing shoot dry weight by the total incident light over the growing period. differences between mizuna and lettuce at different PPFDs, plants were grown at six PPFD levels (~50, 125, 200, 275, 350, and 425 µmol m−<sup>2</sup> s <sup>−</sup>1 at the center of each section) for a 16-hr photoperiod. Projected canopy size was measured twice a week throughout the growing period, and those images were used to estimate the daily PCS and to calculate the total incident light per plant over the growing period. In addition, leaf chlorophyll content index (CCI), anthocyanin content index (ACI), ΦPSII, and net CO<sup>2</sup> assimilation of both crops were measured during the study. Mizuna and lettuce were harvested 27 and 28 days after seeding, respectively. The total leaf area and shoot dry weight were measured. Finally, the LUE was calculated by dividing shoot dry weight by the total incident light over the growing period. *2.2. Projected Canopy Size*

#### *2.2. Projected Canopy Size* Projected canopy size of both crops increased sigmoidally over time. A PPFD of 50

Projected canopy size of both crops increased sigmoidally over time. A PPFD of 50 µmol m−<sup>2</sup> s −1 resulted in a much lower PCS of both crops compared to PPFD levels <sup>≥</sup> <sup>125</sup> <sup>µ</sup>mol m−<sup>2</sup> s −1 . The PCS of mizuna at the end of the growing cycle was ~340 cm2/plant at PPFDs <sup>≥</sup> <sup>200</sup> <sup>µ</sup>mol m−<sup>2</sup> s −1 (Figures 1 and 2). For lettuce, PCS was similar (~240 cm2/plant) at all PPFDs <sup>≥</sup> <sup>125</sup> <sup>µ</sup>mol m−<sup>2</sup> s −1 (Figures 1 and 2). Mizuna had a larger PCS than lettuce starting from the early growth stages at PPFDs > 50 µmol m−<sup>2</sup> s −1 (Figure 1). The difference in PCS between the two species increased over time, especially at higher PPFDs (<sup>≥</sup> <sup>200</sup> <sup>µ</sup>mol m−<sup>2</sup> s −1 ) (Figure 1). µmol m−<sup>2</sup> s <sup>−</sup><sup>1</sup> resulted in a much lower PCS of both crops compared to PPFD levels ≥ 125 µmol m−<sup>2</sup> s −1 . The PCS of mizuna at the end of the growing cycle was ~340 cm<sup>2</sup> /plant at PPFDs ≥ 200 µmol m−<sup>2</sup> s −1 (Figures 1 and 2). For lettuce, PCS was similar (~240 cm<sup>2</sup> /plant) at all PPFDs ≥ 125 µmol m−<sup>2</sup> s <sup>−</sup>1 (Figures 1 and 2). Mizuna had a larger PCS than lettuce starting from the early growth stages at PPFDs > 50 µmol m−2 s −1 (Figure 1). The difference in PCS between the two species increased over time, especially at higher PPFDs (≥ 200 µmol m−<sup>2</sup> s −1 ) (Figure 1). The PCS of both crops at harvest was low at a PPFD of 50 µmol m−<sup>2</sup> s <sup>−</sup><sup>1</sup> and increased

The PCS of both crops at harvest was low at a PPFD of 50 µmol m−<sup>2</sup> s <sup>−</sup><sup>1</sup> and increased asymptotically with increasing PPFD (*p* < 0.0001, Figure 2). The PCS of both crops was similar at PPFDs of 50 and 125 µmol m−<sup>2</sup> s −1 . However, the PCS of lettuce did not increase further at PPFDs <sup>≥</sup> <sup>125</sup> <sup>µ</sup>mol m−<sup>2</sup> s <sup>−</sup>1, while mizuna PCS was similar at PPFDs <sup>≥</sup> <sup>200</sup> <sup>µ</sup>mol m−<sup>2</sup> s −1 . At PPFDs <sup>≥</sup> <sup>200</sup> <sup>µ</sup>mol m−<sup>2</sup> s −1 , mizuna had a greater PCS than lettuce (Figure 2). asymptotically with increasing PPFD (*P* < 0.0001, Figure 2). The PCS of both crops was similar at PPFDs of 50 and 125 µmol m−<sup>2</sup> s −1 . However, the PCS of lettuce did not increase further at PPFDs ≥ 125 µmol m−<sup>2</sup> s <sup>−</sup>1, while mizuna PCS was similar at PPFDs ≥ 200 µmol m−<sup>2</sup> s −1 . At PPFDs ≥ 200 µmol m−<sup>2</sup> s −1 , mizuna had a greater PCS than lettuce (Figure 2).

**Figure 1.** Sigmoidal regression curves fitted through the projected canopy size (PCS) data of mizuna (*Brassica rapa* var. *japonica*) and lettuce (*Lactuca sativa* 'Green Salad Bowl') over the course of the growing cycle for plants grown at six different photosynthetic photon flux densities (PPFD, upper left corner of each graph) (R<sup>2</sup> ≥ 0.99 for all curves). Inserts show the PCS during the first eight days of the study. **Figure 1.** Sigmoidal regression curves fitted through the projected canopy size (PCS) data of mizuna (*Brassica rapa* var. *japonica*) and lettuce (*Lactuca sativa* 'Green Salad Bowl') over the course of the growing cycle for plants grown at six different photosynthetic photon flux densities (PPFD, upper left corner of each graph) (R<sup>2</sup> <sup>≥</sup> 0.99 for all curves). Inserts show the PCS during the first eight days of the study.

*Plants* **2021**, *10*, x 4 of 14

*Plants* **2021**, *10*, x 4 of 14

**Figure 2.** The projected canopy size (PCS) of mizuna (*Brassica rapa* var. *japonica*) and lettuce (*Lactuca sativa* 'Green Salad Bowl') plants grown at six different photosynthetic photon flux densities (PPFDs). Data were collected at the end of the growing cycle (27 and 28 days for mizuna and lettuce, respectively). Each data point represents the mean of nine plants. *2.3. Incident Light* **Figure 2.** The projected canopy size (PCS) of mizuna (*Brassica rapa* var. *japonica*) and lettuce (*Lactuca sativa* 'Green Salad Bowl') plants grown at six different photosynthetic photon flux densities (PPFDs). Data were collected at the end of the growing cycle (27 and 28 days for mizuna and lettuce, respectively). Each data point represents the mean of nine plants. **Figure 2.** The projected canopy size (PCS) of mizuna (*Brassica rapa* var. *japonica*) and lettuce (*Lactuca sativa* 'Green Salad Bowl') plants grown at six different photosynthetic photon flux densities (PPFDs). Data were collected at the end of the growing cycle (27 and 28 days for mizuna and lettuce, respectively). Each data point represents the mean of nine plants. *2.3. Incident Light*

#### The incident light integrated over the entire crop cycle increased with higher PPFD *2.3. Incident Light* The incident light integrated over the entire crop cycle increased with higher PPFD

levels for both crops, but this increase was more pronounced for mizuna than for lettuce (Figure 3, *P* < 0.0001). The incident light integrated over the entire crop cycle increased with higher PPFD levels for both crops, but this increase was more pronounced for mizuna than for lettuce (Figure 3, *p* < 0.0001). levels for both crops, but this increase was more pronounced for mizuna than for lettuce (Figure 3, *P* < 0.0001).

**Figure 3.** The total incident light on the plant canopy throughout the growing period of mizuna (*Brassica rapa* var. *japonica*) and lettuce (*Lactuca sativa* 'Green Salad Bowl') plants grown at six different photosynthetic photon flux densities (PPFDs) for 27 and 28 days, respectively. Lines show the results from multiple regression analysis (*R<sup>2</sup>* = 0.98), which indicated a significant species × PPFD interaction (*P* < 0.0001). Each data point represents the mean of nine plants. *2.4. Chlorophyll Content Index and Anthocyanin Content Index* **Figure 3.** The total incident light on the plant canopy throughout the growing period of mizuna (*Brassica rapa* var. *japonica*) and lettuce (*Lactuca sativa* 'Green Salad Bowl') plants grown at six different photosynthetic photon flux densities (PPFDs) for 27 and 28 days, respectively. Lines show the results from multiple regression analysis (*R<sup>2</sup>* = 0.98), which indicated a significant species × PPFD interaction (*P* < 0.0001). Each data point represents the mean of nine plants. *2.4. Chlorophyll Content Index and Anthocyanin Content Index* **Figure 3.** The total incident light on the plant canopy throughout the growing period of mizuna (*Brassica rapa* var. *japonica*) and lettuce (*Lactuca sativa* 'Green Salad Bowl') plants grown at six different photosynthetic photon flux densities (PPFDs) for 27 and 28 days, respectively. Lines show the results from multiple regression analysis (*R <sup>2</sup>* = 0.98), which indicated a significant species <sup>×</sup> PPFD interaction (*p* < 0.0001). Each data point represents the mean of nine plants.

#### Increasing the PPFD increased both CCI and anthocyanin content index (ACI) of both Increasing the PPFD increased both CCI and anthocyanin content index (ACI) of both *2.4. Chlorophyll Content Index and Anthocyanin Content Index*

crops (*P ≤* 0.003) (Figure 4). This was more pronounced in mizuna than in lettuce; CCI of mizuna increased by 0.08 for each µmol m−<sup>2</sup> s −1 increase in PPFD, compared to an increase crops (*P ≤* 0.003) (Figure 4). This was more pronounced in mizuna than in lettuce; CCI of mizuna increased by 0.08 for each µmol m−<sup>2</sup> s −1 increase in PPFD, compared to an increase Increasing the PPFD increased both CCI and anthocyanin content index (ACI) of both crops (*p* ≤ 0.003) (Figure 4). This was more pronounced in mizuna than in lettuce; CCI of

in lettuce. As the PPFD increased from 50 to 400 µmol m−<sup>2</sup> s

in lettuce. As the PPFD increased from 50 to 400 µmol m−<sup>2</sup> s

−1 , the

−1 , the

−1

−1

of 0.01 per µmol m−<sup>2</sup> s

of 0.01 per µmol m−<sup>2</sup> s

mizuna increased by 0.08 for each µmol m−<sup>2</sup> s −1 increase in PPFD, compared to an increase of 0.01 per µmol m−<sup>2</sup> s −1 in lettuce. As the PPFD increased from 50 to 400 µmol m−<sup>2</sup> s −1 , the CCI of mizuna increased from ~7 to ~40, while that of lettuce increased from ~2 to about ~10 (Figure 4A). For each 1 µmol m−<sup>2</sup> s increase in PPFD, the ACI of mizuna increased by 0.015 and that of lettuce by 0.004. As the PPFD increased from 50 to 400 µmol m−<sup>2</sup> s −1 , the ACI of mizuna increased from ~3 to ~8, while that of lettuce only increased from ~2 to about ~3.

CCI of mizuna increased from ~7 to ~40, while that of lettuce increased from ~2 to about

The ACI showed a similar pattern as CCI; increasing PPFD increased ACI in both crops (*P* ≤ 0.003) (Figure 4B). Mizuna had a higher ACI than lettuce at all PPFD levels (*P* < 0.0001) and mizuna ACI increased more rapidly with increasing PPFD than that of lettuce.

*Plants* **2021**, *10*, x 5 of 14

−1

~10 (Figure 4A).

**Figure 4.** (**A**) Chlorophyll content index (CCI) and (**B**) anthocyanin content index (ACI) of mizuna (*Brassica rapa* var. *japonica*) and lettuce (*Lactuca sativa* 'Green Salad Bowl') plants grown at six different photosynthetic photon flux densities (PPFD). Data were collected a day before the harvesting (26 and 27 days for mizuna and lettuce, respectively). Lines show the results from multiple regression analysis, which indicated a significant species × PPFD interaction for both CCI (*R<sup>2</sup>* = 0.82, interaction *P* < 0.0001) and ACI (*R<sup>2</sup>* = 0.88, interaction *P* < 0.0001). **Figure 4.** (**A**) Chlorophyll content index (CCI) and (**B**) anthocyanin content index (ACI) of mizuna (*Brassica rapa* var. *japonica*) and lettuce (*Lactuca sativa* 'Green Salad Bowl') plants grown at six different photosynthetic photon flux densities (PPFD). Data were collected a day before the harvesting (26 and 27 days for mizuna and lettuce, respectively). Lines show the results from multiple regression analysis, which indicated a significant species × PPFD interaction for both CCI (*R <sup>2</sup>* = 0.82, interaction *p* < 0.0001) and ACI (*R <sup>2</sup>* = 0.88, interaction *p* < 0.0001).

*2.5. Quantum Yield of Photosystem II and CO<sup>2</sup> Assimilation* The quantum yield of photosystem II (ΦPSII) of both crops decreased linearly with increasing PPFD (*P* = 0.0008) (Figure 5A). Increasing PPFD by 1 µmol m−<sup>2</sup> s <sup>−</sup>1 reduced ΦPSII of lettuce and mizuna by 0.0003 mol mol−<sup>1</sup> . Mizuna always had a higher ΦPSII (~0.05 mol mol−1) than lettuce regardless of PPFD (*P* < 0.0001). The net CO<sup>2</sup> assimilation rate of both crops increased with increasing PPFD, but this tended to be more pronounced in mizuna The ACI showed a similar pattern as CCI; increasing PPFD increased ACI in both crops (*p* ≤ 0.003) (Figure 4B). Mizuna had a higher ACI than lettuce at all PPFD levels (*p* < 0.0001) and mizuna ACI increased more rapidly with increasing PPFD than that of lettuce. For each 1 µmol m−<sup>2</sup> s −1 increase in PPFD, the ACI of mizuna increased by 0.015 and that of lettuce by 0.004. As the PPFD increased from 50 to 400 µmol m−<sup>2</sup> s −1 , the ACI of mizuna increased from ~3 to ~8, while that of lettuce only increased from ~2 to about ~3.

−1 at a

#### than in lettuce (*P* = 0.08). Both crops had a CO<sup>2</sup> assimilation rate of ~1 µmol m−2 s *2.5. Quantum Yield of Photosystem II and CO<sup>2</sup> Assimilation*

PPFD of 50 µmol m−2 s −1, but at PPFD of 425 µmol m−2 s −1 mizuna had a CO<sup>2</sup> assimilation rate of ~18 µmol m−2 s −1 while that of lettuce was only ~13 µmol m−2 s −1. The assimilation rate of mizuna and lettuce increased by 0.044 and 0.035 µmol m−<sup>2</sup> s <sup>−</sup><sup>1</sup> per 1 µmol m−<sup>2</sup> s −1 increase in PPFD, respectively. The quantum yield of photosystem II (ΦPSII) of both crops decreased linearly with increasing PPFD (*p* = 0.0008) (Figure 5A). Increasing PPFD by 1 µmol m−<sup>2</sup> s −1 reduced ΦPSII of lettuce and mizuna by 0.0003 mol mol−<sup>1</sup> . Mizuna always had a higher ΦPSII (~0.05 mol mol−<sup>1</sup> ) than lettuce regardless of PPFD (*p* < 0.0001). The net CO<sup>2</sup> assimilation rate of both crops increased with increasing PPFD, but this tended to be more pronounced in mizuna than in lettuce (*p* = 0.08). Both crops had a CO<sup>2</sup> assimilation rate of ~1 µmol m−<sup>2</sup> s <sup>−</sup><sup>1</sup> at a PPFD of 50 µmol m−<sup>2</sup> s −1 , but at PPFD of 425 µmol m−<sup>2</sup> s <sup>−</sup><sup>1</sup> mizuna had a CO<sup>2</sup> assimilation rate of ~18 µmol m−<sup>2</sup> s <sup>−</sup><sup>1</sup> while that of lettuce was only ~13 µmol m−<sup>2</sup> s −1 . The assimilation rate of mizuna and lettuce increased by 0.044 and 0.035 µmol m−<sup>2</sup> s <sup>−</sup><sup>1</sup> per 1 µmol m−<sup>2</sup> s −1 increase in PPFD, respectively.

Quantum yield (mol mol-1

)

1500

*P* < 0.0001

**A B**

*Plants* **2021**, *10*, x 6 of 14

**Figure 5.** (**A**) Quantum yield of photosystem II and (**B**) net CO<sup>2</sup> assimilation rate of mizuna (*Brassica rapa* var. *japonica*) and lettuce (*Lactuca sativa* 'Green Salad Bowl') plants grown and measured at six photosynthetic photon flux densities (PPFD*s*). Data were collected a day before the harvesting (26 and 27 days for mizuna and lettuce, respectively). Lines show the results from multiple regression analysis, which indicated no significant species × PPFD interaction for quantum yield (*R<sup>2</sup>* = 0.72, interaction *P* = 0.62), but significant effects of PPFD and species (both *P* < 0.0001). For CO<sup>2</sup> assimilation rate, there was a weak species × PPFD interaction effect (*R<sup>2</sup>* = 0.91, interaction *P* = 0.08). *2.6. Final Leaf Area and Canopy Overlap Ratio* **Figure 5.** (**A**) Quantum yield of photosystem II and (**B**) net CO<sup>2</sup> assimilation rate of mizuna (*Brassica rapa* var. *japonica*) and lettuce (*Lactuca sativa* 'Green Salad Bowl') plants grown and measured at six photosynthetic photon flux densities (PPFD*s*). Data were collected a day before the harvesting (26 and 27 days for mizuna and lettuce, respectively). Lines show the results from multiple regression analysis, which indicated no significant species × PPFD interaction for quantum yield (*R <sup>2</sup>* = 0.72, interaction *p* = 0.62), but significant effects of PPFD and species (both *p* < 0.0001). For CO<sup>2</sup> assimilation rate, there was a weak species × PPFD interaction effect (*R <sup>2</sup>* = 0.91, interaction *p* = 0.08). **Figure 5.** (**A**) Quantum yield of photosystem II and (**B**) net CO<sup>2</sup> assimilation rate of mizuna (*Brassica rapa* var. *japonica*) and lettuce (*Lactuca sativa* 'Green Salad Bowl') plants grown and measured at six photosynthetic photon flux densities (PPFD*s*). Data were collected a day before the harvesting (26 and 27 days for mizuna and lettuce, respectively). Lines show the results from multiple regression analysis, which indicated no significant species × PPFD interaction for quantum yield (*R<sup>2</sup>* = 0.72, interaction *P* = 0.62), but significant effects of PPFD and species (both *P* < 0.0001). For CO<sup>2</sup> assimilation rate, there was a weak species × PPFD interaction effect (*R<sup>2</sup>* = 0.91, interaction *P* = 0.08).

#### The final leaf area of both mizuna and lettuce increased asymptotically with increas-*2.6. Final Leaf Area and Canopy Overlap Ratio 2.6. Final Leaf Area and Canopy Overlap Ratio*

ing PPFD (*P* < 0.0001) (Figure 6A). Both crops had the highest leaf area at PPFDs ≥ 275 µmol m−<sup>2</sup> s −1 , but lettuce leaf area increased faster with increasing PPFD than mizuna leaf area (*P* < 0.0001). At a PPFD of ≥ 275 µmol m−<sup>2</sup> s −1 lettuce had a leaf area of ~1200 cm<sup>2</sup> per plant, while that of mizuna was only ~800 cm<sup>2</sup> . However, lettuce had a lower PCS at harvest compared to mizuna at PPFDs ≥ 200 µmol m−<sup>2</sup> s <sup>−</sup><sup>1</sup> (Figure 2). This apparent contradiction between a greater leaf area and a lower PCS of lettuce can be explained by the canopy overlap ratio (leaf area/PCS); lettuce had a much higher canopy overlap ratio than mizuna at PPFDs ≥ 125 µmol m−<sup>2</sup> s <sup>−</sup><sup>1</sup> (Figure 6B). The canopy overlap ratio of lettuce increased more rapidly, from 1.2 to 5.2 with increasing PPFD compared to that of mizuna (increasing from 1.1 to 2.3) (*P* < 0.0001). 6 The final leaf area of both mizuna and lettuce increased asymptotically with increasing PPFD (*p* < 0.0001) (Figure 6A). Both crops had the highest leaf area at PPFDs ≥ 275 µmol m−<sup>2</sup> s −1 , but lettuce leaf area increased faster with increasing PPFD than mizuna leaf area (*<sup>p</sup>* < 0.0001). At a PPFD of <sup>≥</sup> <sup>275</sup> <sup>µ</sup>mol m−<sup>2</sup> s −1 lettuce had a leaf area of ~1200 cm<sup>2</sup> per plant, while that of mizuna was only ~800 cm<sup>2</sup> . However, lettuce had a lower PCS at harvest compared to mizuna at PPFDs <sup>≥</sup> <sup>200</sup> <sup>µ</sup>mol m−<sup>2</sup> s −1 (Figure 2). This apparent contradiction between a greater leaf area and a lower PCS of lettuce can be explained by the canopy overlap ratio (leaf area/PCS); lettuce had a much higher canopy overlap ratio than mizuna at PPFDs <sup>≥</sup> <sup>125</sup> <sup>µ</sup>mol m−<sup>2</sup> s −1 (Figure 6B). The canopy overlap ratio of lettuce increased more rapidly, from 1.2 to 5.2 with increasing PPFD compared to that of mizuna (increasing from 1.1 to 2.3) (*p* < 0.0001). The final leaf area of both mizuna and lettuce increased asymptotically with increasing PPFD (*P* < 0.0001) (Figure 6A). Both crops had the highest leaf area at PPFDs ≥ 275 µmol m−<sup>2</sup> s −1 , but lettuce leaf area increased faster with increasing PPFD than mizuna leaf area (*P* < 0.0001). At a PPFD of ≥ 275 µmol m−<sup>2</sup> s −1 lettuce had a leaf area of ~1200 cm<sup>2</sup> per plant, while that of mizuna was only ~800 cm<sup>2</sup> . However, lettuce had a lower PCS at harvest compared to mizuna at PPFDs ≥ 200 µmol m−<sup>2</sup> s −1 (Figure 2). This apparent contradiction between a greater leaf area and a lower PCS of lettuce can be explained by the canopy overlap ratio (leaf area/PCS); lettuce had a much higher canopy overlap ratio than mizuna at PPFDs ≥ 125 µmol m−<sup>2</sup> s −1 (Figure 6B). The canopy overlap ratio of lettuce increased more rapidly, from 1.2 to 5.2 with increasing PPFD compared to that of mizuna (increasing from 1.1 to 2.3) (*P* < 0.0001).

*P* < 0.0001

5

CO2 assimilation (mol m-2

 s-1

)

**Figure 6.** (**A**) Leaf area per plant and (**B**) canopy overlap ratio of mizuna (*Brassica rapa* var. *japonica*) and lettuce (*Lactuca sativa* 'Green Salad Bowl') plants grown at six different photosynthetic photon flux densities (PPFD*s*) for 27 and 28 days, respectively. The canopy overlap ratio is the ratio between the leaf area and the projected canopy size at harvest. Each data point represents the mean of nine plants. **Figure 6.** (**A**) Leaf area per plant and (**B**) canopy overlap ratio of mizuna (*Brassica rapa* var. *japonica*) and lettuce (*Lactuca sativa* 'Green Salad Bowl') plants grown at six different photosynthetic photon flux densities (PPFD*s*) for 27 and 28 days, respectively. The canopy overlap ratio is the ratio between the leaf area and the projected canopy size at harvest. Each data point represents the mean of nine plants.

Dry weight (g/plant)

0

2

4

6

8

#### *2.7. Shoot Dry Weight and Specific Leaf Area Plants* **2021**, *10*, x 7 of 14

The shoot dry weight at 425 µmol m−<sup>2</sup> s <sup>−</sup><sup>1</sup> was ~50 times higher than at 50 µmol m−<sup>2</sup> s −1 for both lettuce and mizuna, although PPFD increased only ~9 × (Figure 7A, Figure S4). Lettuce and mizuna had a dry weight of 0.08 and 0.11 g/plant at a PPFD of 50 µmol m−<sup>2</sup> s −1 and 3.85 and 6.02 g/plant at a PPFD of 425 µmol m−<sup>2</sup> s −1 , respectively. A 1 µmol m−<sup>2</sup> s −1 increase in PPFD increased the dry weight of lettuce by 10.0 mg/plant and that of mizuna by 15.8 mg/plant. With increasing PPFD, the specific leaf area (SLA, leaf area per gram of dry weight) of both crops decreased (*p* < 0.0001) (Figure 7B). However, due to the higher leaf area and lower dry weight of lettuce compared to mizuna, the SLA of lettuce was greater than that of mizuna at all PPFDs. The difference in SLA between the two species decreased with increasing PPFD (Figure 7B). *2.7. Shoot Dry Weight and Specific Leaf Area* The shoot dry weight at 425 µmol m−<sup>2</sup> s <sup>−</sup>1 was ~50 times higher than at 50 µmol m−<sup>2</sup> s for both lettuce and mizuna, although PPFD increased only ~9 × (Figure 7A, Figure S4). Lettuce and mizuna had a dry weight of 0.08 and 0.11 g/plant at a PPFD of 50 µmol m−<sup>2</sup> s <sup>−</sup><sup>1</sup> and 3.85 and 6.02 g/plant at a PPFD of 425 µmol m−<sup>2</sup> s −1 , respectively. A 1 µmol m−<sup>2</sup> s increase in PPFD increased the dry weight of lettuce by 10.0 mg/plant and that of mizuna by 15.8 mg/plant. With increasing PPFD, the specific leaf area (SLA, leaf area per gram of dry weight) of both crops decreased (*P* < 0.0001) (Figure 7B). However, due to the higher leaf area and lower dry weight of lettuce compared to mizuna, the SLA of lettuce was greater than that of mizuna at all PPFDs. The difference in SLA between the two species decreased with increasing PPFD (Figure 7B). *2.7. Shoot Dry Weight and Specific Leaf Area* The shoot dry weight at 425 µmol m−<sup>2</sup> s <sup>−</sup>1 was ~50 times higher than at 50 µmol m−<sup>2</sup> s −1 for both lettuce and mizuna, although PPFD increased only ~9 × (Figure 7A, Figure S4). Lettuce and mizuna had a dry weight of 0.08 and 0.11 g/plant at a PPFD of 50 µmol m−<sup>2</sup> s <sup>−</sup><sup>1</sup> and 3.85 and 6.02 g/plant at a PPFD of 425 µmol m−<sup>2</sup> s −1 , respectively. A 1 µmol m−<sup>2</sup> s −1 increase in PPFD increased the dry weight of lettuce by 10.0 mg/plant and that of mizuna by 15.8 mg/plant. With increasing PPFD, the specific leaf area (SLA, leaf area per gram of dry weight) of both crops decreased (*P* < 0.0001) (Figure 7B). However, due to the higher leaf area and lower dry weight of lettuce compared to mizuna, the SLA of lettuce was

−1

−1

*Plants* **2021**, *10*, x 7 of 14

greater than that of mizuna at all PPFDs. The difference in SLA between the two species

**Figure 7.** (**A**) Shoot dry weight and (**B**) specific leaf area of mizuna (*Brassica rapa* var. *japonica*) and lettuce (*Lactuca sativa* 'Green Salad Bowl') plants grown at six different photosynthetic photon flux densities (PPFD*s*) for 27 and 28 days, respectively. Specific leaf area is the ratio between leaf area and dry weight. Each data point represents the mean of nine plants. *2.8. Light Use Efficiency* **Figure 7.** (**A**) Shoot dry weight and (**B**) specific leaf area of mizuna (*Brassica rapa* var. *japonica*) and lettuce (*Lactuca sativa* 'Green Salad Bowl') plants grown at six different photosynthetic photon flux densities (PPFD*s*) for 27 and 28 days, respectively. Specific leaf area is the ratio between leaf area and dry weight. Each data point represents the mean of nine plants. PPFD (mol m-2 s-1) PPFD (mol m-2 <sup>s</sup> -1) **Figure 7.** (**A**) Shoot dry weight and (**B**) specific leaf area of mizuna (*Brassica rapa* var. *japonica*) and lettuce (*Lactuca sativa* 'Green Salad Bowl') plants grown at six different photosynthetic photon flux densities (PPFD*s*) for 27 and 28 days, respectively. Specific leaf area is the ratio between leaf area and dry weight. Each data point represents the mean of nine plants.

Mizuna had a higher LUE than lettuce (*P* < 0.0001) (Figure 8). The LUE of mizuna

<sup>−</sup><sup>1</sup> and decreased to ~0.75 g mol−<sup>1</sup> at a PPFD

#### was ~1.1 g mol−<sup>1</sup> at PPFDs up to 200 µmol m−<sup>2</sup> s *2.8. Light Use Efficiency 2.8. Light Use Efficiency*

of 425 µmol m−<sup>2</sup> s −1 . In contrast, lettuce LUE was greatest at PPFDs of 125 to 350 µmol m−<sup>2</sup> s <sup>−</sup><sup>1</sup> (~0.8 g mol−1) and ~0.6 g mol−<sup>1</sup> at PPFDs of 50 and 425 µmol m−<sup>2</sup> s −1 . Mizuna had a higher LUE than lettuce (*p* < 0.0001) (Figure 8). The LUE of mizuna was ~1.1 g mol−<sup>1</sup> at PPFDs up to 200 µmol m−<sup>2</sup> s <sup>−</sup><sup>1</sup> and decreased to ~0.75 g mol−<sup>1</sup> at a PPFD of 425 µmol m−<sup>2</sup> s −1 . In contrast, lettuce LUE was greatest at PPFDs of 125 to 350 µmol m−<sup>2</sup> s −1 (~0.8 g mol−<sup>1</sup> ) and ~0.6 g mol−<sup>1</sup> at PPFDs of 50 and 425 µmol m−<sup>2</sup> s −1 . Mizuna had a higher LUE than lettuce (*P* < 0.0001) (Figure 8). The LUE of mizuna was ~1.1 g mol−<sup>1</sup> at PPFDs up to 200 µmol m−<sup>2</sup> s <sup>−</sup><sup>1</sup> and decreased to ~0.75 g mol−<sup>1</sup> at a PPFD of 425 µmol m−<sup>2</sup> s −1 . In contrast, lettuce LUE was greatest at PPFDs of 125 to 350 µmol m−<sup>2</sup> s <sup>−</sup><sup>1</sup> (~0.8 g mol−1) and ~0.6 g mol−<sup>1</sup> at PPFDs of 50 and 425 µmol m−<sup>2</sup> s −1 .

2.0 Lettuce

(PPFDs) for 27 and 28 days, respectively. Each data point represents the mean of nine plants. **Figure 8.** Light use efficiency (LUE, grams of shoot dry weight per mol of incident light) of mizuna (*Brassica rapa* var. japonica) and lettuce (*Lactuca sativa* 'Green Salad Bowl') plants grown at six different photosynthetic photon flux densities (PPFDs) for 27 and 28 days, respectively. Each data point represents the mean of nine plants. **Figure 8.** Light use efficiency (LUE, grams of shoot dry weight per mol of incident light) of mizuna (*Brassica rapa* var. japonica) and lettuce (*Lactuca sativa* 'Green Salad Bowl') plants grown at six different photosynthetic photon flux densities (PPFDs) for 27 and 28 days, respectively. Each data point represents the mean of nine plants.

japonica) and lettuce (*Lactuca sativa* 'Green Salad Bowl') plants grown at six different photosynthetic photon flux densities
