*2.1. Photosynthesis*

Fitting net photosynthesis (A) vs. cellular CO<sup>2</sup> concentration (Cc) curves to net photosynthesis over a range of CO<sup>2</sup> concentrations allows for the estimation of parameters that relate to leaf-level photosynthesis and the underlying biochemistry limiting photosynthetic assimilation of CO<sup>2</sup> (Figure 1), with estimates for the potential electron transport rate (J) and maximum RuBP carboxylation rate (Vcmax) in Table 1. A C<sup>c</sup> value of ~270 ppm corresponded to ambient concentrations of CO<sup>2</sup> of 400 ppm under measurement conditions. At that level, photosynthesis was highest in RB and GB (23.9 and 23.6 µmol CO<sup>2</sup> m−<sup>2</sup> s −1 , respectively) followed by RGB, RGB + FR, and B (21.0, 20.5, and 19.6 µmol CO<sup>2</sup> m−<sup>2</sup> s −1 , respectively). Considerably lower photosynthesis values are found for G and RG (11.3 and 10.2 µmol CO<sup>2</sup> m−<sup>2</sup> s −1 , respectively) with R having the lowest photosynthesis of all groups at 5.8 µmol CO<sup>2</sup> m−<sup>2</sup> s −1 .

The estimated maximum rate of Rubisco carboxylation is significantly higher in RB and RGB + FR than all other treatments except GB. The G, R, and RG treatments have significantly lower estimates for Vcmax and J than treatments containing blue light—the B, GB, RB, RGB, and RGB + FR treatments (Table 1).

The photosynthesis measurements under 1000 µmol photons m−<sup>2</sup> s −1 , saturating light differ substantially from the photosynthesis measurements under ambient, treatment light at 170 µmol photons m−<sup>2</sup> s −1 (Figure 2). Under ambient conditions, net photosynthesis was highest in RGB and RG, followed by RB and RGB + FR which had significantly higher net photosynthesis than GB or R. The B and G treatments had the lowest net photosynthesis under ambient conditions.

**Figure 1.** Net photosynthesis (A) vs. cellular CO<sup>2</sup> concentration (Cc) curve fitting for each light treatment. Filled circles represent observed net photosynthesis (A) relative to calculated Cc values. The solid line shows Rubisco limitation, while the dotted line fits RuBP limitation. Triose-phos-**Figure 1.** Net photosynthesis (A) vs. cellular CO<sup>2</sup> concentration (Cc) curve fitting for each light treatment. Filled circles represent observed net photosynthesis (A) relative to calculated Cc values. The solid line shows Rubisco limitation, while the dotted line fits RuBP limitation. Triose-phosphate utilization (TPU) limitation was not apparent.

phate utilization (TPU) limitation was not apparent.



RGB + FR 137.7 ± 2.7 c 101.9 ± 2.1 a

The photosynthesis measurements under 1000 µmol photons m−2 s

GB, RB, RGB, and RGB + FR treatments (Table 1).

light at 170 µmol photons m−2 s

thesis under ambient conditions.

**Figure 2.** Net photosynthesis (A) under ambient treatment lighting. Different lowercase letters indicate significant differences (*p* ≤ 0.05; *n* = 6). Error bars are the standard error. Uppercase letters indicate light treatments. **Figure 2.** Net photosynthesis (A) under ambient treatment lighting. Different lowercase letters indicate significant differences (*p* ≤ 0.05; *n* = 6). Error bars are the standard error. Uppercase letters indicate light treatments.

The estimated maximum rate of Rubisco carboxylation is significantly higher in RB and RGB + FR than all other treatments except GB. The G, R, and RG treatments have significantly lower estimates for Vcmax and J than treatments containing blue light—the B,

differ substantially from the photosynthesis measurements under ambient, treatment

was highest in RGB and RG, followed by RB and RGB + FR which had significantly higher net photosynthesis than GB or R. The B and G treatments had the lowest net photosyn-

−1

−1 (Figure 2). Under ambient conditions, net photosynthesis

, saturating light

Overall, there was no correlation between net photosynthesis under ambient, treatment lighting at 170 µmol photons m−2 s −1 and those observed at the same CO<sup>2</sup> concentration under saturating 90% red, 10% blue light at 1000 µmol photons m−2 s −1 . Overall, there was no correlation between net photosynthesis under ambient, treatment lighting at 170 µmol photons m−<sup>2</sup> s <sup>−</sup><sup>1</sup> and those observed at the same CO<sup>2</sup> concentration under saturating 90% red, 10% blue light at 1000 µmol photons m−<sup>2</sup> s −1 .

#### *2.2. Chlorophyll Fluorescence 2.2. Chlorophyll Fluorescence*

The relative operating efficiency of PSII was highest in the GB treatment and lowest in the R treatment (Table 2). The R and RG treatments had significantly higher ΦPSII values than the R treatment, but significantly lower than all treatments containing blue light. The maximum quantum efficiency of PSII photochemistry (Fv/Fm) was slightly under 0.83—indicating mild stress—for all treatments with no significant differences observed for any treatments (Table 2). Both light-induced and non-light-induced nonphotochemical quenching were higher in treatments lacking blue light (R, G, and RG) compared to treatments containing blue light (B, RB, GB, RGB, and RGB + FR). The relative operating efficiency of PSII was highest in the GB treatment and lowest in the R treatment (Table 2). The R and RG treatments had significantly higher ΦPSII values than the R treatment, but significantly lower than all treatments containing blue light. The maximum quantum efficiency of PSII photochemistry (Fv/Fm) was slightly under 0.83—indicating mild stress—for all treatments with no significant differences observed for any treatments (Table 2). Both light-induced and non-light-induced nonphotochemical quenching were higher in treatments lacking blue light (R, G, and RG) compared to treatments containing blue light (B, RB, GB, RGB, and RGB + FR).

Under light-saturating conditions, net photosynthesis was significantly lower in the G, R, and RG treatments than all other treatments (Figure 1). However, this had no apparent effect on photosynthesis under ambient conditions (Figure 2). While ambient photosynthesis was lowest in the G treatment, net photosynthesis in R was comparable with GB, and significantly higher than B or G. Finally, ambient photosynthesis in RG was sig-**Table 2.** Maximum quantum efficiency of PSII (Fv/Fm), relative PSII operating efficiency (ΦPSII), coefficient of photochemical quenching (qp), the quantum yield of non-light-induced nonphotochemical quenching (ΦNPQ), the quantum yield of light-induced nonphotochemical quenching (ΦNO), and the fraction of oxidized plastoquinone (qL) calculated using measurements under saturating (1000 µmol photons m−<sup>2</sup> s −1 ) 90% red, 10% blue light. Different letters indicate significant differences (*p* ≤ 0.05; *n* = 3 or 4).


Under light-saturating conditions, net photosynthesis was significantly lower in the G, R, and RG treatments than all other treatments (Figure 1). However, this had no apparent effect on photosynthesis under ambient conditions (Figure 2). While ambient photosynthesis was lowest in the G treatment, net photosynthesis in R was comparable

with GB, and significantly higher than B or G. Finally, ambient photosynthesis in RG was significantly higher than all other treatments save RGB (Figure 2). RGB 0.27 ± 0.01 b 0.82 ± 0.00 a 0.43 ± 0.01 e 0.29 ± 0.00 d 0.42 ± 0.01 b 0.20 ± 0.01 c RGB + FR 0.27 ± 0.01 b 0.81 ± 0.01 a 0.45 ± 0.01 de 0.28 ± 0.00 e 0.43 ± 0.01 b 0.23 ± 0.01 b

−1) 90% red, 10% blue light. Different letters indicate sig-

#### *2.3. Shoot Characteristics 2.3. Shoot Characteristics*

using measurements under saturating (1000 µmol photons m−2 s

nificant differences (*p* ≤ 0.05; *n* = 3 or 4).

Qualitative differences between treatments can be seen in Figure 3, which shows exemplar plants (those closest to treatment average in height and mass) from the replication experiment for each treatment. Shoot dry weight showed no clear trends, except that farred light increased shoot dry weight, with an average of 2.53 g per plant for the RGB + FR treatment and only 1.63 g for the RGB treatment (Figure 4). The RGB + FR and B treatments had significantly higher dry weight than the GB, R, and RB treatments, while the RGB + FR treatment also had significantly higher dry weight than the RG and RGB treatments (Figure 4). Qualitative differences between treatments can be seen in Figure 3, which shows exemplar plants (those closest to treatment average in height and mass) from the replication experiment for each treatment. Shoot dry weight showed no clear trends, except that farred light increased shoot dry weight, with an average of 2.53 g per plant for the RGB + FR treatment and only 1.63 g for the RGB treatment (Figure 4). The RGB + FR and B treatments had significantly higher dry weight than the GB, R, and RB treatments, while the RGB + FR treatment also had significantly higher dry weight than the RG and RGB treatments (Figure 4).

*Plants* **2021**, *10*, x FOR PEER REVIEW 5 of 19

**Table 2.** Maximum quantum efficiency of PSII (Fv/Fm), relative PSII operating efficiency (ΦPSII), coefficient of photochemical quenching (qp), the quantum yield of non-light-induced nonphotochemical quenching (ΦNPQ), the quantum yield of light-induced nonphotochemical quenching (ΦNO), and the fraction of oxidized plastoquinone (qL) calculated

**Treatment ΦPSII Fv/F<sup>m</sup> ΦNPQ ΦNO q<sup>P</sup> q<sup>L</sup>**

B 0.26 ± 0.01 b 0.81 ± 0.01 a 0.48 ± 0.01 c 0.26 ± 0.00 f 0.43 ± 0.01 b 0.23 ± 0.01 ab G 0.14 ± 0.00 c 0.80 ± 0.00 a 0.54 ± 0.00 a 0.32 ± 0.00 c 0.24 ± 0.01 c 0.12 ± 0.00 d GB 0.29 ± 0.01 a 0.82 ± 0.00 a 0.44 ± 0.01 e 0.26 ± 0.00 f 0.46 ± 0.01 a 0.24 ± 0.01 a

RB 0.26 ± 0.01 b 0.82 ± 0.01 a 0.47 ± 0.01 cd 0.26 ± 0.00 f 0.42 ± 0.01 b 0.22 ± 0.01 bc RG 0.12 ± 0.00 d 0.80 ± 0.01 a 0.51 ± 0.00 b 0.37 ± 0.00 b 0.19 ± 0.01 d 0.08 ± 0.00 e

Far-red light also increased plant height, with the RGB + FR treatment being significantly taller than the RGB treatment (Figure 4). Conversely, supplemental blue light decreased plant height, with shorter plants in RB than R, GB than G, and RGB than RG. However, plants grown in the B treatment were taller than all other treatments except RGB + FR. The RGB + FR and B treatments, in addition to being the tallest, also had the lowest leaf dry weight fraction (leaf dry weight divided by shoot dry weight) (Figure 4).

There were no clear trends for stem diameter, except that far-red light enhanced stem diameter, with plants in the RGB + FR treatment having significantly greater stem diameter than plants in the RGB treatment (Figure 4).

The RGB + FR treatment also resulted in significantly greater leaf area than the RGB treatment (Figure 4). Due to high within groups variability, there were no other significant trends in leaf area, although with a larger sample size, a trend of decreasing leaf area with supplemental blue light may be observed.

Far-red light also increased plant height, with the RGB + FR treatment being significantly taller than the RGB treatment (Figure 4). Conversely, supplemental blue light decreased plant height, with shorter plants in RB than R, GB than G, and RGB than RG. However, plants grown in the B treatment were taller than all other treatments except RGB + FR. The RGB + FR and B treatments, in addition to being the tallest, also had the Specific leaf weight (SLW), the dry weight of a leaf divided by its area, does show a clear trend with blue light significantly increasing SLW. Higher specific leaf weights were observed in the GB treatment relative to G, RB relative to R, and RGB relative to RG (Figure 4). Far-red light decreased SLW, with the RGB + FR treatment having significantly lower SLW than the RGB treatment.

#### lowest leaf dry weight fraction (leaf dry weight divided by shoot dry weight) (Figure 4). There were no clear trends for stem diameter, except that far-red light enhanced stem *2.4. Stomatal Characteristics*

diameter, with plants in the RGB + FR treatment having significantly greater stem diameter than plants in the RGB treatment (Figure 4). The RGB + FR treatment also resulted in significantly greater leaf area than the RGB treatment (Figure 4). Due to high within groups variability, there were no other significant Stomatal conductance under ambient lighting was significantly higher in B (0.24 mol m−<sup>2</sup> s −1 ) relative to R (0.09 mol m−<sup>2</sup> s −1 ) or G (0.09 mol m−<sup>2</sup> s −1 ) and significantly higher in GB (0.28 mol m−<sup>2</sup> s −1 ) relative to G, in RB (0.19 mol m−<sup>2</sup> s −1 ) relative to R, and in RGB (0.27 mol m−<sup>2</sup> s −1 ) relative to RG (0.09 mol m−<sup>2</sup> s −1 ), demonstrating a blue light-mediated increase in stomatal conductance (Figure 5A).

trends in leaf area, although with a larger sample size, a trend of decreasing leaf area with

Specific leaf weight (SLW), the dry weight of a leaf divided by its area, does show a clear trend with blue light significantly increasing SLW. Higher specific leaf weights were observed in the GB treatment relative to G, RB relative to R, and RGB relative to RG (Figure 4). Far-red light decreased SLW, with the RGB + FR treatment having significantly

Stomatal conductance under ambient lighting was significantly higher in B (0.24 mol

−1) and significantly higher in GB

−1) relative to R, and in RGB (0.27 mol

−1), demonstrating a blue light-mediated increase in

−1) or G (0.09 mol m−2 s

−1) relative to G, in RB (0.19 mol m−2 s

supplemental blue light may be observed.

lower SLW than the RGB treatment.

−1) relative to R (0.09 mol m−2 s

stomatal conductance (Figure 5A).

−1) relative to RG (0.09 mol m−2 s

*2.4. Stomatal Characteristics*

m−2 s

m−2 s

(0.28 mol m−2 s

**Figure 5.** (**A**): Stomatal conductance under saturating light by light treatment. Different lowercase letters indicate significant differences (*p* ≤ 0.05; *n* is between 25 and 30 for each treatment). (**B**): Stomatal conductance under ambient, treatment lighting (Stomatal conductanceA) vs. adaxial stomatal density. (**C**): Net photosynthesis under ambient, treatment lighting vs. stomatal conductance under ambient, treatment lighting. (**D**): Water content vs. instantaneous water use efficiency. **Figure 5.** (**A**): Stomatal conductance under saturating light by light treatment. Different lowercase letters indicate significant differences (*p* ≤ 0.05; *n* is between 25 and 30 for each treatment). (**B**): Stomatal conductance under ambient, treatment lighting (Stomatal conductanceA) vs. adaxial stomatal density. (**C**): Net photosynthesis under ambient, treatment lighting vs. stomatal conductance under ambient, treatment lighting. (**D**): Water content vs. instantaneous water use efficiency. (**E**): Stomatal conductance under saturating light vs. stomatal conductance under ambient, treatment lighting. (**F**): Water content vs. abaxial stomatal density. Uppercase letters indicate light treatments.

We found a significant increase in conductance from RB to RGB, but there was no difference in stomatal conductance between G, R, and the RG treatments (Figure 5A). Far-red light decreased stomatal conductance, with conductance significantly lower in RGB + FR (0.18 mol m−<sup>2</sup> s −1 ) compared to RGB. Stomatal conductance under ambient, treatment lighting was highly correlated with adaxial stomatal density (Figure 5B, R <sup>2</sup> = 0.87). However, stomatal conductance was not correlated to net photosynthesis under ambient, treatment lighting (Figure 5C, R<sup>2</sup> = 0.00).

When measuring the A vs. C<sup>c</sup> curves, all plants were subjected to saturating levels of 90% red, 10% blue light at 1000 µmol photons m−<sup>2</sup> s −1 . Despite being illuminated with the same spectrum, conductance trends were similar to those obtained when illuminated by treatment spectra. Overall, average conductance values under saturating light were higher in all treatments compared to ambient lighting conditions except the B treatment, with R<sup>2</sup> = 0.70 (*p* < 0.01) (Figure 5E).

Like conductance, blue light resulted in an increased stomatal density, with abaxial stomatal density higher in B relative to R (although not different from G), and higher abaxial density in GB relative to G, RB relative to R, and RGB relative to RG. The same trends were found for adaxial stomatal density, except that adaxial stomatal density in B was significantly higher than G (Table 3).

**Table 3.** Stomatal density, abaxial (AB) to adaxial (AD) stomatal density ratio, intrinsic water use efficiency, and water content of cucumber.


Abaxial stomatal density was also lower in RGB + FR than RGB, though there was no difference in adaxial stomatal density (Table 3). We observed significantly higher abaxial:adaxial ratios for the G and R treatments relative to all other treatments.

Intrinsic water use efficiency (iWUE) under ambient conditions, which is calculated by dividing the net photosynthesis by stomatal conductance, was highest in RG, followed by G and R, while iWUE was lowest in the B treatment. Like [30], we found no difference in iWUE between RB and RGB; however, [31] did find a significant increase in iWUE in a low R:FR treatment compared to a high R:FR treatment, while we found no difference between the RGB and RGB + FR treatment.

Intrinsic water use efficiency had only a weak correlation with water content at harvest (Figure 5D, R<sup>2</sup> = 0.23). Water content, the percentage of fresh weight from water, was lower in GB, RB, and RGB than all other treatments. Interestingly, while stomatal conductance was best explained by adaxial stomatal density, water content at harvest was best explained by abaxial stomatal density (R<sup>2</sup> = 0.82, Figure 5F).
