Photosynthetic Responses of Swiss Chard, Kale, and Spinach Cultivars to Irradiance and Carbon Dioxide Concentration

HORTSCIENCE 52(5):706–712. 2017. doi: 10.21273/HORTSCI11799-17 of reduced or increased irradiance or CO2 on yield. We also desired to determine Photosynthetic Responses of Swiss whether leafy green varieties differed in the Pn responses to irradiance and CO2 and whether some varieties were more suited to Chard, Kale, and Spinach Cultivars supplemental, or reduced, irradiance and CO2 levels. to Irradiance and Carbon Dioxide We acknowledge that translating instan- taneous Pn measurements on a per-unit-area basis to whole-plant photosynthesis has lim- Concentration itations (see Discussion). Yet, an initial com- John Erwin1,3 and Esther Gesick2 parative study exploring the variation in the Department of Horticultural Science, University of Minnesota, 305 Alderman instantaneous Pn on a per-unit-area basis is valuable in that it provides some insight into Hall, 1970 Folwell Avenue, St. Paul, MN 55108 the degree of variation among species and vari- eties. These data also provide some guidance on Additional index words. Brassica, Beta, Spinacea, light saturation point, CO2 saturation point, respiration irradiance and CO2 levels that maximize the Pn on the uppermost leaves, especially when plants Abstract. The impact of irradiance (0–1200 mmol·mL2·sL1) and carbon dioxide concen- are young and interior leaf shading is limited. tration (CO2; 50–1200 ppm) on kale (Brassica oleracea and B. napus pabularia; three cultivars), Swiss chard (chard, Beta vulgaris; four cultivars), and spinach (Spinacea Materials and Methods oleracea; three cultivars) photosynthetic rate (Pn; per area basis) was determined to facilitate maximizing yield in controlled environment production. Spinach, chard, and Chard, [‘Rhubarb’, ‘Fordhook Giant’, L2 L1 kale maximum Pn were 23.8, 20.3, and 18.2 mmol CO2·m ·s fixed, respectively, across ‘Bright Yellow’, and ‘Bright Lights’ (red)], varieties (400 ppm CO2). Spinach and kale had the highest and lowest light compensation kale [‘Toscano’, ‘Winterbor’, and ‘Red points [LCPs (73 and 13 mmol·mL2·sL1, respectively)] across varieties. The light Russian’ (B. napus pabularia)], and spinach saturation points (LSPs) for chard and kale were similar at 884–978 mmol·mL2·sL1, (‘Melody’, ‘Harmony’, and ‘Bloomsdale but for spinach, the LSP was higher at 1238 mmol·mL2·sL1. Dark respiration was lowest LS’) seeds were sown in 10.5-cm-diameter L2 L1 on kale and highest on spinach (L0.83 and L5.00 mmol CO2·m ·s , respectively). The plastic pots (5 seeds/pot) in premoistened spinach CO2 compensation point (CCP) was lower (56 ppm) than the chard or kale CCP LC-8 soilless growing media (Sun Gro Horti- (64–65 ppm). Among varieties, ‘Red Russian’ kale Pn saturated at the lowest CO2 culture, Bellevue, WA) and placed in a green- concentration (858 ppm), and ‘Bright Lights’ chard saturated at the highest (1266 ppm; house (24 ± 2 C day and 16 ± 2 C night L2 L1 300 mmol·m ·s ). Spinach Pn was more responsive to increasing irradiance than to temperatures; St. Paul, MN). Kale and chard CO2. Kale Pn was more responsive to increasing CO2 than to irradiance, and chard Pn seeds were obtained from Johnny’s Selected was equally responsive to increasing CO2 or irradiance. Implications and limitations of Seeds (Winslow, ME), and spinach seeds this work when ‘‘upscaling’’ to whole-plant responses are discussed. were obtained from W. Atlee Burpee & Co. (Warminster, PA). Seeds germinated in 4–7 d. Greenhouse daylight (0800–1400 HR) was Leafy green vegetable options are in- photosynthesis increases (Bjorkman,€ 1981; supplemented with 75 mmol·m–2·s–1 high- creasing as communities become more eth- Chagvardieff et al., 1994; Dorais, 2003). The pressure sodium lighting when daylight (at nically or racially diverse or both, as the primary inputs into the photosynthetic pro- plant level) was below 200 mmol·m–2·s–1. health and nutritional benefits of greens cess are light (irradiance), CO2, and water After 7 d, the three most uniform (similar consumption are reported (Bertoia et al., (Bjorkman,€ 1981). Therefore, maximizing size) seedlings were left to grow, whereas the 2015; Hu and Rimm, 2015), and as interest photosynthesis in leafy greens to maximize others were removed. As kale and chard flower after they unfold a specific leaf num- in year-round locally produced foods in- yield would require that irradiance, CO2, creases (Feldmann and Hamm, 2015). Three or water not be limited (Fu et al., 2017; ber, after a cool temperature exposure, or increasingly popular leafy vegetables are kale Gaudreau et al., 1994; Gent, 2016). both, daylength was extended with 75 m –2 –1 (Brassica oleracea and B. napus pabularia), In northern climates, year-round leafy mol·m ·s from 1600–2200 HR as flower- spinach (Spinacea oleracea), and Swiss chard green production requires protected cultiva- ing was not a concern [16 h photoperiod; (chard, Beta vulgaris). tion during the late fall, winter, and early mean daily light integral (DLI) = 12.4 · –2· –1 Kale, spinach, and chard leaves are har- spring when temperatures drop below mol m d ] to simulate a typical production vested and sold on a fresh-weight basis. The freezing. Irradiance and CO in protected environment to maximize yield (J. Erwin, 2 personal observation). In contrast, as long ability of plants to increase fresh weight, cultivation often vary, intentionally and days can promote flowering on spinach early or mass, is associated with photosynthesis unintentionally, depending on covering type, in development, spinach seedlings were where plant mass generally increases as plant spacing, degree of ventilation, whether grown under short days (8 h photoperiod; air is circulated, and whether supplemental opaque cloth pulled over plants from 1400– lighting or CO2 are supplied (Kretchen and · –2· –1 Received for publication 20 Jan. 2017. Accepted 0800 HR daily; mean DLI = 10.7 mol m d ) Howlett, 1970). Little work has been con- to inhibit flowering. After 30 d, plants were for publication 5 Apr. 2017. ducted on the effects of irradiance and CO The authors acknowledge and appreciate the 2 transplanted into 7.6-l plastic pots. Through- financial support of the Minnesota Agriculture on the Pn of leafy greens other than lettuce out, plants were watered as needed with Experiment Station, USDA-ARS Floriculture and (Lactuca sativa; Dorais, 2003; Fu et al., 2017; irrigation water containing 250 ppm N from Nursery Research Initiative (FNRI), National In- Gaudreau et al., 1994) and recent work by 15N–0P–15K fertilizer (Peter’s Dark Weather stitute of Food and Agriculture (NIFA), and mem- Gent (2016) on spinach. An understanding of Feed; The Scotts Co., Marysville, OH). All bers of the University of Minnesota through the how irradiance and CO2 impact the Pn of such plants were watered at the same time to ensure Floriculture Research Alliance including Altman greens would facilitate maximizing the Pn to similar media nutritional status (confirmed Plants, Inc., Rocket Farms, Inc., Smith Gardens, Inc., maximize yield in protected cultivation. The and Green Circle Growers, Inc. with soil tests) among species and varieties. 1Professor. objective of the research presented here was to Photosynthetic rate determination. After 2Research Fellow. determine the Pn responses of spinach, kale, kale plants unfolded seven true leaves (>45 3Corresponding author. E-mail: erwin001@umn. and chard to irradiance and CO2 and to inform angle from the stem), chard plants unfolded edu. producers of the advantages or disadvantages four leaves, and spinach plants unfolded eight

706 HORTSCIENCE VOL. 52(5) MAY 2017 –2 –1 leaves (4 weeks across species), the impact (irradiance when estimated Pn =0mmol·m ·s Results of irradiance and CO2 on instantaneous Pn on CO2 fixed), and ‘‘k’’ was a constant that a per-unit-leaf-area basis was determined on represented the ratio of the quantum yield Photosynthetic responses to irradiance. the second leaf below the uppermost fully (q) to the Pn at the LCP (Marino et al., 2010). Pmax (identified by providing saturating irradi- expanded unfolded leaf on five plants of each Rd (dark respiration) was calculated as ance levels at 400 ppm CO2) differed among species and variety. The Pn was measured the estimated Pn (I) when irradiance was species, varieties within a species, and across using a LI-COR LI6400XT portable photo- 0 mmol·m–2·s–1. all varieties (Table 2). Spinach had the highest  m –2 –1 synthesis meter (LI-COR, Inc., Lincoln, NE) Pmax at 23.8 mol CO2·m ·s fixed, whereas kCðÞC 2 ðÞ 0 m –2 –1 using a cuvette (6 cm ) with a built-in vari- Pn C = Pmax 1 e Eq. [2] for kale it was 20.3 mol CO2·m ·s fixed and m · –2· –1 able LED light source. The Pn at 0, 100, 200, for chard it was 18.2 mol CO2 m s fixed 400, 600, 800, 1000, and 1200 mmol·m–2·s–1 Eq. [2] shows Pn responses [(Pn (C)] to (across varieties; Fig. 1; Table 2). Among kale irradiance was determined. The Pn at 50, 200, CO2. Pn responses to increasing CO2 were varieties, ‘Red Russian’ and ‘Toscano’ had m · –2· –1 400, 600, 800, 1000, and 1200 ppm CO2 was asymptotic; ‘‘Pmax’’ in Eq. [2] estimates the ahigherPmax (21.0–22.3 mol CO2 m s ) m · –2· –1 also determined. The Pn was recorded 5 min asymptote. ‘‘C0’’ is the estimated CCP (CO2 than ‘Winterbor’ (17.4 mol CO2 m s ; m · –2· –1 after a change in irradiance or CO2 after the when Pn =0 mol m s CO2 fixed), and Table 2). Among spinach varieties, ‘Melody’ m · –2· –1 Pn had stabilized. Throughout, cuvette tem- ‘‘k’’ is a constant. The CO2 concentration at had a higher Pmax (26.6 mol CO2 m s ) perature was maintained at 24 C, and the 95% of Pmax approximated the CO2 satura- than ‘Harmony’ or ‘Bloomsdale LS’ (22.3– –1 m –2 –1 atmospheric flow rate was 400 mL·min . tion point (CSP; CO2 when Pn was saturated). 22.4 mol CO2·m ·s ; Table 2). Among chard Cuvette CO2 was 400 ppm (outdoor ambient) Experimental design and data analysis. varieties, ‘Fordhook Giant’ had a higher Pmax m · –2· –1 when determining the Pn responses to irradi- Analysis of variance (ANOVA) was conduct- (21.9 mol CO2 m s ) than ‘Bright Lights’ –2 –1 m –2 –1 ance, and irradiance was 300 mmol·m ·s ed on photosynthetic parameters derived or ‘Yellow’ (16.3–16.6 mol CO2·m ·s ; (typical irradiance in a northern U.S. green- from Eqs. [1] and [2] fit to Pn data from each Table 2). Across all species and varieties, house during the winter; personal observation) leaf on each plant as dependent variables. ‘Melody’ spinach had the highest Pmax,and when measuring the Pn responses to CO2. The experiment was analyzed as a two-stage ‘Yellow’ and ‘Bright Lights’ chard had the Photosynthetic parameter determination. nested design using Type I sum of squares, lowest Pmax (Table 2). The Pn data from each leaf of each species which allows the use of Tukey’s honestly Spinach had the highest LCP (73 m · –2· –1 and variety at varying irradiance or CO2 were significant difference (HSD) for mean sepa- mol m s ), the LCP of chard was 25 fit to the nonlinear Mitscherlich and the ration with species as the first factor and mmol·m–2·s–1, and kale had the lowest LCP at nonrectangular hyperbola functions as both cultivar the second. Estimated k values, r2, 13 mmol·m–2·s–1 (Table 2). Kale and spinach are widely used to estimate the Pn responses and mean square errors (MSEs) derived from variety LCP did not differ, but chard variety to irradiance and CO2 (Aleric and Kirkman, the ANOVA are shown in Table 1. Tukey’s LCP differed from 16 (Bright Lights) to 41 2005; Goudrian, 1979; Johnson et al., 2010; HSD (a < 0.05) was employed for mean mmol·m–2·s–1 (Rhubarb; Table 2). Chard and Laitat and Boussard, 1995; Marino et al., separation except in Table 4 where least kale LSP (884–978 mmol·m–2·s–1) did not 2010; Peek et al., 2002; Potvin et al., 1990). significant difference (LSD; a < 0.05) differ, but spinach LSP was higher (1238 The Mitscherlich equations (Eqs. [1] and [2]) was employed as Tukey’s HSD cannot be mmol·m–2·s–1; Table 2). LSP did not differ fit data best here and provided realistic non- employed with a repeated measures test. A among varieties within any species studied linear parameter values (Table 1; personal Tukey’s test conducted when sphericity is here (Table 2). observation). More complex biochemical violated (often the case with collected re- Photosynthetic responses to CO2 concentra models estimating the leaf Pn [such as used peated measures tests) will have a vastly tion. Pmax (identified by providing saturating m –2 –1 by Farquhar et al. (1980)] were not used, as inflated Type I error rate; SPSS (see below) CO2 concentrations at 300 mol·m ·s )dif- we quantified the Pn responses to CO2 at presumably does not include Tukey’s test as fered among some species, among varieties irradiance levels typical in northern green- an option under repeated measures analysis within some species, and across all varieties houses here and not saturating levels typi- to prevent this error. The best test to compare (Table 3). Chard and kale Pmax did not differ m –2 –1 cally used with biochemical models. multiple comparisons in a repeated measures (17.2–17.6 mol CO2·m ·s fixed), but spin-  design is Bonferroni’s; for our data, Bonfer- ach P was higher at 19.8 mmol CO ·m–2·s–1 ðÞ max 2 ðÞ kI I0 Pn I = Pmax 1 e Eq. [1] roni’s gave the same results as LSD. Aside fixed (across varieties; Fig. 1; Table 3). Pmax from this case, Tukey’s HSD was used when did not differ among spinach varieties. Eq. [1] shows Pn responses [Pn (I)] to possible as it is more statistically rigorous Among kale varieties, ‘Winterbor’ had a irradiance (I). Pn responses to increasing than LSD. Throughout, the SPSS statistical lower Pmax than ‘Red Russian’ (Table 3). –2 –1 irradiance were asymptotic here; ‘‘Pmax’’ in software package (IBM SPSS Statistics, Chard Pmax varied from 16.6 mmol CO2·m ·s –2 –1 Eq. [1] estimates the asymptote. LSP was the Version 23; IBM Corp., Armonk, NY) was for ‘Rhubarb’ to 19.2 mmol CO2·m ·s for irradiance at 95% of Pmax.‘‘I0’’ was the LCP used for statistical analysis. ‘Bright Lights’ (Table 3). Across varieties,

Table 1. Statistics associated with goodness of fit of raw data to the nonlinear Mitscherlich function used to describe responses of kale (Brassica oleracea and B. napus pabularia; three cultivars), spinach (Spinacea oleracea; three cultivars), and Swiss chard (Beta vulgaris; four cultivars) to irradiance and carbon dioxide concentration. The r2, mean square error (MSE), and ‘‘k’’ values (Mitscherlich function) fit to data are shown. Irradiance Carbon dioxide concn Plant type kr2 MSE kr2 MSE Kale 0.0031 0.996 0.157 0.0033 0.998 0.122 ‘Red Russian’ 0.0030 0.999 0.059 0.0038 0.998 0.149 ‘Winterbor’ 0.0034 0.991 0.234 0.0028 0.999 0.048 ‘Toscano’ 0.0030 0.998 0.180 0.0032 0.998 0.168 Spinach 0.0027 0.999 0.038 0.0032 0.999 0.068 ‘Melody’ 0.0026 0.999 0.058 0.0034 0.999 0.056 ‘Harmony’ 0.0028 1.000 0.027 0.0032 0.999 0.064 ‘Bloomsdale LS’ 0.0026 0.999 0.030 0.0030 0.999 0.083 Swiss chard 0.0036 0.998 0.099 0.0030 0.998 0.177 ‘Yellow’ 0.0040 0.999 0.051 0.0030 0.993 0.524 ‘Rhubarb’ 0.0034 0.997 0.165 0.0035 1.000 0.016 ‘Fordhook Giant’ 0.0030 0.999 0.088 0.0030 1.000 0.023 ‘Bright Lights’ 0.0040 0.998 0.093 0.0026 0.999 0.112

HORTSCIENCE VOL. 52(5) MAY 2017 707 Table 2. Variation in predicted maximum photosynthetic rate (Pmax), the light compensation point (irradiance at Pn = 0), the light saturation point (irradiance at 95% of Pmax), predicted dark respiration rate (CO2 evolution in dark at 24 C), and quantum efficiency among three varieties of kale (Brassica oleracea and B. napus pabularia), three varieties of spinach (Spinacea oleracea), and four varieties of Swiss chard (Beta vulgaris) as determined using a fitted Mitscherlich model for each plant of each species and variety. Capital letters denote mean separation [Tukey’s HSD(0.05)] across species, and small letters denote mean separation across varieties. Maximum photosynthetic Light saturation point Light respiration Dark efficiency –2 –1 –2 –1 –2 –1 –2 –1 Plant species/variety compensation rate (mmol CO2·m ·s ) (mmol CO2·m ·s ) point (mmol CO2·m ·s ) rate (mmol CO2·m ·s ) Quantum Kale 20.3 B 13 A 978 A –0.83 C 0.07 A ‘Red Russian’ 22.3 ± 0.5 d 16 ± 2 ab 1,015 ± 2 –1.12 ± 0.12 c 0.07 ± 0.002 ‘Toscano’ 21.0 ± 1.1 bcd 15 ± 2 ab 1,014 ± 2 –0.97 ± 0.11 c 0.06 ± 0.004 ‘Winterbor’ 17.4 ± 0.7 ab 6 ± 1 a 905 ± 61 –0.39 ± 0.10 c 0.06 ± 0004 Spinach 23.8 C 73 C 1,238 B –5.00 A 0.08 B ‘Melody’ 26.6 ± 0.7 e 68 ± 3 e 1,266 ± 123 –5.05 ± 0.44 a 0.08 ± 0.007 ‘Harmony’ 22.4 ± 1.2 d 79 ± 3 e 1,177 ± 102 –5.51 ± 0.43 a 0.08 ± 0.006 ‘Bloomsdale LS’ 22.3 ± 0.8 d 72 ± 4 e 1,270 ± 126 –4.46 ± 0.23 a 0.07 ± 0.007 Swiss chard 18.2 A 25 B 884 A –1.64 B 0.07 AB ‘Bright Lights’ 16.6 ± 0.8 a 16 ± 2 ab 784 ± 64 –10.6 ± 0.13 c 0.07 ± 0.003 ‘Fordhook Giant’ 21.9 ± 0.7 cd 22 ± 2 b 1,021 ± 17 –1.49 ± 0.11 bc 0.07 ± 0.002 ‘Rhubarb’ 18.0 ± 0.8 abc 41 ± 4 c 939 ± 65 –2.60 ± 0.20 b 0.07 ± 0.004 ‘Yellow’ 16.3 ± 0.9 a 21 ± 4 b 790 ± 65 –1.43 ± 0.31 bc 0.07 ± 0.004 Analysis of Variance Species ***z *** *** *** * Variety *** *** NS *** NS HSD = honestly significant difference; NS = nonsignificant. zDenotes significance at the a < 0.05 (*), < 0.01, (**), < 0.001 (***) levels.

‘Winterbor’ kale had the lowest Pmax differ from whole-plant responses when a) to irradiance was associated with differences –2 –1 (14.7 mmol CO2·m ·s ),and‘RedRussian’ multiple inputs are changed at once, b) in Rubsico activation and stomatal conduc- and ‘Toscano’ kale had the highest Pmax (17.8 after plants acclimate to altered irradi- tance (gS) (Kaiser et al., 2016). –2 –1 and 19.0 mmol CO2·m ·s ,respectively; ance, CO2, or both, and c) when leaf aging Also, the transferability of our conclu- Table 3). and whole-plant leaf area/shading are taken sions to whole-plant responses is associated Spinach CCP was lower (56 ppm) than into account. with the size (or age) of a plant and leaf area. chard or kale CCPs (64–65 ppm) across One environmental parameter (irradiance High irradiance can result in reduced leaf life varieties (Table 3). CCP did not differ among or CO2) was changed while the other was or smaller leaf area which can result in an spinach or chard varieties, but differed held constant in our research here. Increasing overestimating whole-plant Pn if reduced leaf among kale varieties where ‘Red Russian’ irradiance and CO2 simultaneously may pro- area, more rapid leaf senescence, or both is had the lowest CCP (59 ppm) and ‘Winter- duce different conclusions, likely increasing not taken into account (Austin, 1989). Also, bor’ the highest (72 ppm; Table 3). CSP did LSP, CSP, or both more than reported here. although Pn on the uppermost leaf may be not vary among species or among varieties For instance, Chagvardieff et al. (1994) saturated, whole-plant Pn is likely not satu- within a species, but differed when all vari- observed increasing CO2 and irradiance si- rated as lower leaves are shaded in a canopy eties were compared; ‘Red Russian’ kale had multaneously increased lettuce dry weight as a plant grows and unfolds leaves. Such the lowest CSP (858 ppm), and ‘Bright 69% more than dry weight gains observed shading results in Pn rates lower than the Pmax Lights’ chard had the highest CSP (1266 from increasing CO2 and irradiance sepa- on lower leaves even when irradiance on the ppm; Table 3). rately, i.e., there was a synergy between these uppermost leaves is at the LSP. Irradiance in Dark respiration. Calculated Rd (24 C) factors. Also, other environmental parame- a canopy decreases exponentially from the differed among species. Kale Rd was the ters can interact with irradiance, CO2, or both top to the bottom of a plant following the –2 –1 –kL lowest (–0.83 mmol CO2·m ·s ), chard Rd to impact Pn. Changes in humidity (Kaiser general equation I = Ioe [I = irradiance –2 –1 was –1.64 mmol CO2·m ·s , and spinach Rd et al., 2015) or temperature (Dahal et al., below the canopy; Io = irradiance at the top of –2 –1 was the highest (–5.00 mmol CO2·m ·s ; 2012) can result in markedly different re- the canopy; k = the extinction coefficient across varieties; Table 2). Rd did not differ sponses in Pn to irradiance, CO2, or both. (generally >0.5 for nonvertically oriented among kale and spinach varieties, but dif- Nonenvironmental cultural factors can also leaves); and L = leaf area index (Lambers fered among chard varieties where ‘Rhubarb’ impact Pn and assumptions made here. For et al., 2008)]. Therefore, increasing irradi- and ‘Bright Lights’ chard Rd was –2.60 instance, high irradiance promotion of Pn was ance at the top of the plant above the LSP will –2 –1 and –10.06 mmol CO2·m ·s , respectively most obvious when lettuce was grown under likely increase whole-plant Pn if the leaf area (Table 2). low nitrogen levels only (7 mmol·L–1;Fu index is high. et al., 2017). Although these limitations when trans- Discussion Photosynthetic rate can acclimate to al- lating instantaneous Pn data to crop responses € tered irradiance or CO2 over time (Bjorkman, exist, we believe Pn responses are still in- Limitations of generalizing instantaneous 1981; Lambers et al., 2008; Pons, 2012). formative. Specifically, instantaneous Pn data Pn data on a per-unit-area basis to whole- Therefore, caution should be exercised when on a per-unit-area basis provide insight into plant Pn. Data here provide a framework for extrapolating instantaneous Pn responses to variation in responses among species and determining irradiance and CO2 impacts on whole-plant Pn over time. Pn at high irradi- varieties that is of value and provide some kale, spinach, and chard Pn to facilitate pro- ance or CO2 may be overpredicted, and Pn at insight into which species or varieties may be duction in controlled environment facilities low irradiance or CO2 may be underpredicted more responsive to increases in irradiance or to maximize yield. Pn is often associated with over time (Bunce and Ziska, 2000). The basis CO2 concentration. dry weight gain, fresh weight gain, and yield for Pn acclimation to altered irradiance or Responses to irradiance. Kale and chard –2 –1 in vegetables (Heuvelink and Dorais, 2003). CO2 is not clear. Acclimation of Pn to high Pn saturated at lower (600–800 mmol·m ·s ) There are limitations when extrapolating irradiance or CO2 was more correlated with irradiance levels than spinach (1000–1200 –2 –1 changes in instantaneous Pn measurements soluble saccharides than with day to day mmol·m ·s ; 400 ppm CO2; Fig. 1; Table 2). on a per-unit-area basis to whole-plant Pn variation in CO2 or irradiance alone (Bunce Pmax reported here are consistent with pre- and conclusions drawn from that data. For and Sicher, 2003). In contrast, variation vious data on spinach (Boese and Huner, instance, instantaneous Pn responses can among Arabidopsis varieties in Pn over time 1990; Yamori et al., 2005) and kale

708 HORTSCIENCE VOL. 52(5) MAY 2017 Fig. 1. Effect of increasing irradiance (A) or carbon dioxide (CO2) concentration (B) on spinach (Spinacea oleracea), kale (Brassica oleracea and B. napus pabularia), and Swiss chard (Beta vulgaris) photosynthetic rate across cultivars. Bars represent the ± mean square error as identified through analysis of variance (a < 0.05).

HORTSCIENCE VOL. 52(5) MAY 2017 709 –2 –1 Table 3. Variation in the predicted maximum photosynthetic rate (Pmax; mmol CO2·m ·s ), the CCP (CO2 concentration when Pn = 0), and the CSP (the CO2 concentration at 95% Pmax) among three cultivars of kale (Brassica oleracea and B. napus pabularia), three cultivars of spinach (Spinacea oleracea), and four cultivars of Swiss chard (Beta vulgaris) as determined using fitted Mitscherlich functions fit to each cultivar and pooled under each species. Capital letters denote mean separation [Tukey’s HSD(0.05)] across species, and small letters denote mean separation across cultivars. –2 –1 Plant species/cultivar Pmax (mmol CO2·m ·s ) CCP (ppm) CSP (ppm) Kale 17.2 A 65.0 B 1,014 ‘Red Russian’ 17.8 + 0.5 bcde 58.8 + 1.5 abc 858 + 51 a ‘Toscano’ 19.0 + 0.3 cde 64.4 + 2.1 bcd 1,013 + 51 ab ‘Winterbor’ 14.7 + 0.5 a 71.8 + 3.1 d 1,170 + 101 ab Spinach 19.8 B 56.1 A 1,005 ‘Melody’ 19.8 + 0.2 de 54.8 + 0.8 a 954 + 61 ab ‘Harmony’ 19.6 + 0.3 de 55.6 + 1.0 ab 1,004 + 51 ab ‘Bloomsdale LS’ 20.0 + 0.4 e 58.0 + 1.1 abc 1,057 + 11 ab Swiss chard 17.6 A 64.3 B 1,102 ‘Bright Lights’ 19.2 + 0.6 cde 67.3 + 2.7 cd 1,266 + 124 b ‘Fordhook Giant’ 17.5 + 0.7 bcd 64.1 + 3.1 abcd 1,113 + 125 ab ‘Rhubarb’ 16.6 + 0.9 ab 63.0 + 1.9 abcd 939 + 73 ab ‘Yellow’ 16.9 + 0.6 abc 62.8 + 0.7 abcd 1,061 + 74 ab Analysis of Variance Species ***z *** NS Cultivar *** ** *

Pmax = maximum photosynthetic rate; CCP = CO2 compensation point; CSP = CO2 saturation point; HSD = honestly significant difference; NS = nonsignificant. zDenotes significance at the a < 0.05 (*), < 0.01, (**), < 0.001 (***) levels.

[Brassica; Dahal et al. (2012) (napus) and these assumptions are based on instantaneous from 400 to 800 ppm as would occur when Ruhil et al. (2015)] at slightly lower CO2 data, and plants may acclimate to lower irradi- using a CO2 injection system (Table 4). For concentrations than used here. Given irradi- ance levels over time. instance, increasing CO2 from 200 to 400 ance in northern climates in greenhouses Table 4 shows predicted percent changes ppm increased predicted Pn by 75% to 98% rarely exceeds LSPs reported here (personal in Pn when irradiance was increased from across species whereas increasing CO2 levels observation), supplemental lighting (up to the 300 to 350 mmol·m–2·s–1 on kale, chard, and from 400 ppm to the CSP increased predicted –2 –1 LSPs, at a minimum) would increase Pn and spinach (DLI = +3.24 mol·m ·d for chard Pn by 38% to 68% across species (Table 4). presumably yield. Supplemental lighting in and kale, and +1.44 mol·m–2·d–1 for spinach These data emphasize the importance of northern greenhouse vegetable production as photoperiod differed). Increasing irradi- ventilating to ensure canopy CO2 levels are facilities is commonplace as growers observe ance from 300 to 350 mmol·m–2·s–1 increased at least similar to ambient outdoor levels. Of increased yield when providing supplemen- predicted spinach Pn by 15% and that of kale course, the predicted increases in Pn if CO2 tal light. In greenhouse lettuce production, and chard by 9% to 11% (Table 4). is increased from 400 to 800 ppm may be daylight is often supplemented with 50–100 Responses to CO2. Kale and chard CSP greater if irradiance was simultaneously in- mmol·m–2·s–1 (DLI = 12–13 mol·m–2·d–1) was lower (600–800 ppm) than that of spin- creased to the LSP. during the winter to realize 140% to 270% ach (1000–1200 ppm) (irradiance = 300 As with Pn responses to irradiance, al- –2 –1 increases in yield compared with plants with mmol·m ·s ; Fig. 1; Table 3). Responses though CO2 levels on the uppermost leaves no supplemental lighting in Canada (Gaudreau observed on kale here were similar to those may be at CSP levels, lower leaf Pn is likely et al., 1994). Similarly, Brassicaceae micro- observed by others (700 ppm CO2)onB. not at CSP levels as CO2 is consumed by green (seedling) fresh weight and nutritional oleracea and B. napus under different irradi- photosynthesis in the canopy and replaced value increased when irradiance was increased ance levels (Bunce and Sicher, 2003; Dahal slowly (depending on air circulation and –2 –1 from 0 to 320–440 mmol·m ·s , but not when et al., 2012, respectively). Given all CSPs ventilation). Therefore, increasing CO2 levels irradiance was further increased from 440 to reported here are higher than the ambient to above the CSP often results in increased –2 –1 545 mmol·m ·s (Samuoliene et al., 2013). In CO2 levels, supplementing greenhouses or yields as lower leaf Pn is less CO2 limited. This contrast, Colonna et al. (2016) showed that the growth rooms with CO2 (above ambient) is increasingly important as leaf area index impact of irradiance on the nutritional value of would likely increase the Pn of crops stud- increases in a canopy. Kale and chard had 10 leafy vegetables varied with species. ied here (Table 3). In fact, injecting CO2 lower CSP than spinach; therefore, these crops Daily light integral can be more associated to increase CO2 levels to 800–1000 ppm would perform better in greenhouses with withyieldthanwithirradiance as it represents is commonplace in commercial vegetable poor ventilation or with close plant spacing the cumulative light delivered over 24 h. For production greenhouses to increase yield than spinach (Table 3). instance, spinach dry weight as a ratio of fresh (personal observation; Dorais, 2003). For Respiration. Spinach, kale, and chard weight increased as normalized daily light in- instance, increasing CO2 from 330 to predicted Rd (24 C) observed here are tegral (DLI/leaf area index) increased from 3 to 900 ppm increased tomato yield by 21% comparable with those measured by others 27 mol·m–2·d–1 (Gent, 2016). However, it is (Dorais, 2003). although temperatures differed somewhat important to note that a high correlation between Pn in unventilated greenhouses or en- (Dahal et al., 2012; Yamori et al., 2005). DLI and plant dry weight will occur only if irra- closed rooms can be limited by declining We observed variation in Rd among species diance is below the LSP; irradiance levels above CO2 levels as plants use CO2 for photosyn- and among varieties of some species. Al- the LSP would not result in an increase in Pn. thesis. It is not uncommon for CO2 levels in though spinach variety Rd was high and Based on the instantaneous Pn data here, a canopy to drop to 200–250 ppm during the similar, kale and chard variety Rd varied with kale and chard may be better suited for day in an unvented greenhouse or growth some varieties having a 4-fold higher Rd than production in naturally low irradiance loca- room (J. Erwin and J. Frantz, personal obser- with other varieties (Table 2). Variation in Rd tions or facilities than spinach as their LSP vations). Therefore, ventilating production among varieties of other vegetable species were lower than that of spinach (Table 2). environments with outdoor air (400 ppm (such as asparagus) has also been observed Also, as spinach had a higher LCP (60–75 CO2) will increase CO2 concentration inside (Kitazawa et al., 2011). Such variation in –2 –1 –2 –1 mmol·m ·s ) than kale (10–15 mmol·m ·s ), thus increasing Pn. Our data predict ventilat- Rd among species and varieties here are kale may be grown under lower irradiance ing greenhouses to increase CO2 levels from especially important to quantify as Rd can conditions than spinach and still have a net 200 to 400 ppm can result in a greater in- be negatively correlated with postharvest increase in mass. Again, we emphasize that crease in Pn than that from increasing CO2 performance (Kitazawa et al., 2011). For

710 HORTSCIENCE VOL. 52(5) MAY 2017 –2 –1 –2 –1 Table 4. Predicted instantaneous photosynthetic rates (Pn per unit area; mmol CO2·m ·s ) at low irradiance (typical cloudy day in winter; 200 mmol·m ·s ), –2 –1 ambient irradiance (typical 300 mmol·m ·s ), and at 95% of predicted Pmax (Pn saturated) of kale (Brassica oleracea and B. napus pabularia), Swiss chard (Beta vulgaris), and spinach (Spinacea oleracea) varieties. Predicted Pn at low CO2 levels (200 ppm; depleted enclosed environment), at ambient CO2 levels (400 ppm, current outdoor), and at 95% of predicted Pmax of kale, Swiss chard, and spinach varieties. Percentage in parenthesis is the percent increase in Pn when increasing from the rate one line up and to the left, to the level above the number in parentheses. Letters denote mean separation [Tukey’s HSD (a < 0.05)] within a variety to increasing irradiance or increasing carbon dioxide concentration. Irradiance (mmol·m–2·s–1) Carbon dioxide (ppm)

Species and cultivar Low 200 Ambient 300 +50 350 Pn Saturated 600–1,000 Deficient 200 Ambient 400 Pn Saturated 800–1,000 Kale ‘Red Russian’ 9.46 b 12.80 c (+35%) 14.13 d (+10%) 22.34 f (+75%) 7.37 a 12.87 c (+75%) 17.81 e (+38%) ‘Toscano’ 8.96 b 12.10 c (+35%) 13.34 d (+10%) 21.04 e (+74%) 6.66 a 12.44 cd (+87%) 18.98 e (+53%) ‘Winterbor’ 8.33 b 10.90 c (+31%) 11.89 d (+9%) 17.41 f (+60%) 4.42 a 8.77 b (+98%) 14.72 e (+68%) Spinach ‘Melody’ 7.66 a 11.89 b (+55%) 13.63 c (+15%) 26.58 e (+124%) 7.70 a 13.61 bc (+77%) 19.83 d (+46%) ‘Harmony’ 6.37 a 10.22 b (+60%) 11.78 c (+15%) 22.45 d (+120%) 7.24 a 13.04 c (+80%) 19.59 d (+50%) ‘Bloomsdale LS’ 6.27 a 9.85 b (+57%) 11.31 c (+15%) 22.27 e (+126%) 6.94 a 12.84 c (+85%) 20.00 d (+56%) Chard ‘Yellow’ 8.21 b 10.81 c (+32%) 11.77 d (+9%) 16.35 e (+53%) 5.70 a 10.75 c (+88%) 16.89 e (+63%) ‘Rhubarb’ 7.55 a 10.52 b (+39%) 11.67 c (+11%) 17.87 e (+70%) 6.23 a 11.33 bc (+82%) 16.55 d (+46%) ‘Fordhook Giant’ 9.05 b 12.37 c (+37%) 13.69 d (+11%) 21.86 f (+77%) 5.76 a 10.87 c (+89%) 17.50 e (+61%) ‘Bright Lights’ 8.54 b 11.12 cd (+30%) 12.08 d (+9%) 16.62 fe (+50%) 5.53 a 10.92 c (+97%) 19.17 f (+76%) HSD = honestly significant difference. instance, data here suggest that spinach may parameters (Tables 1 and 2). This was not (60–75 mmol·m–2·s–1) than kale (10– have a shorter postharvest life than kale and unexpected as vegetable crops are often in- 15 mmol·m–2·s–1), kale may be grown that among kale varieties and among chard terspecific hybrids, and varieties can vary under lower irradiance conditions varieties, ‘Bright Lights’ may have a shorter greatly genetically. Similar variation in Pmax than spinach and still have an increase postharvest life than ‘Rhubarb’ (Table 2). among lettuce varieties was observed by in mass. Irradiance versus CO2. The question Behr and Wiebe (1992). Gu et al. (2012) 2. Kale and chard CSP were lower arises as to whether a spinach, kale, or chard observed variation in Pmax (17% to 25% (600–800 ppm) than that of spinach producer should increase irradiance or CO2 variation) in rice (13 lines; Oryza sativa L.) (1000–1200 ppm). Given all CSPs to increase yield most. Low CO2 conditions at ambient CO2 levels (380 ppm) and that reported here are higher than ambi- (200 ppm) reduced predicted Pn more than variation was associated with stomatal and ent CO2 levels, supplementing CO2 –2 –1 low irradiance (200 mmol·m ·s ) conditions mesophyll conductance. Yu et al. (2016) would increase the Pn of crops stud- on both kale and chard here (Table 4). There- found variation in Cucumis Pn varieties to ied here. fore, ventilating enclosed production spaces changes in irradiance was associated with 3. Ventilating greenhouses to increase (where CO2 may have dropped to 200 ppm) differences in leaf cholorophyll content. In CO2 from 200 to 400 ppm may result to ensure CO2 levels are at ambient levels another work, differences in Arabidopsis in a greater increase in Pn than that (400 ppm) may increase Pn and likely yield variety Pn responses to changing irradiance from increasing CO2 from 400 to on kale and chard more than on spinach. In was associated with differences in Rubsico 800 ppm. contrast, in nearly all cases (except ‘Toscano’ activation and gS (Kaiser et al., 2016). What- 4. Although spinach variety Rd was sim- kale, and ‘Yellow’ and ‘Bright Lights’ ever the basis, our data infer genetic diversity ilar, kale and chard variety Rd varied chard), increasing irradiance from ambient (based on Pn responses) of kale and chard with some varieties having a 4-fold –2 –1 irradiance (300 mmol·m ·s ) to the LSP may be greater than that of the spinach higher Rd than others. increased predicted Pn more than increasing varieties studied here. 5. Low CO2 reduced kale and chard Pn CO2 from ambient (400 ppm) to the CSP Combining both Pn and Rd. It cannot also more than low irradiance. Therefore, (Table 4). This suggests that irradiance may be assumed that higher Pn will result in ventilating production spaces to ensure be more limiting than CO2 with the crops increased fresh or dry weight and yield as CO2 levels are at 400 ppm may increase studied here. yield is associated with carbon loss or Rd.As yield more on kale and chard than on Future work must examine the synergy Rd occurs during both day and night, it can spinach. between irradiance and CO2 on kale, chard, have a significant negative impact on net 6. In nearly all cases (except ‘Toscano’ and spinach. Increasing CO2 from 400 to 800 daily carbon gain. When predicted daily kale, and ‘Yellow’ and ‘Bright Lights’ ppm and increasing irradiance from 400 to carbon gain was calculated by taking both chard), increasing irradiance from 300 –2 –1 –2 –1 800 mmol·m ·s significantly increased let- Pn (Rd already quantified in direct Pn read- mmol·m ·s to the LSP increased the tuce dry weight by 25% (1.5 g/plant) and ings) and Rd (at night only) into account {net Pn more than increasing CO2 from –1 19%, (1.15 g/plant), respectively (Chagvardieff carbon gain = [(Pmax · 18 h·d (kale) or ambient to the CSP. This suggests –1 –1 et al., 1994). However, increasing CO2 concen- 8h·d (spinach)] – [(Rd · 8h·d (kale) or irradiance may be more limiting than –1 tration and irradiance simultaneously acted 16 h·d (spinach)]}, we observed spinach CO2 on these crops. synergistically [2.65 g/plant when benefits carbon gain/d (380.8–80.0 = 300.8 mmol 7. When predicted daily carbon gain was –2 –1 added individually vs. 4.21 g/plant (+69%) CO2·m ·s ) was lower than that of kale calculated by taking both Pn and Rd into –2 –1 when increased together] when conducted (365.4–6.6 = 358.8 mmol CO2·m ·s )even account, spinach carbon gain per day 23 to 40 d after sowing (Chagvardieff et al., though Pmax was greater (Table 2). was lower than that of kale even though 1994). Take home messages. the Pmax was greater. Variety differences. Varieties (of some species) differed in response to increasing 1. Kale and chard Pn saturated at lower –2 –1 irradiance or CO2 suggesting different ge- (600–800 mmol·m ·s ) irradiance Literature Cited netic backgrounds. There was little variation levels than spinach Pn (1000–1200 Aleric, K.M. and K.L. Kirkman. 2005. Growth and m –2 –1 among spinach varieties in Pmax, LCP, CCP, mol·m ·s ), and kale and chard photosynthetic responses of the federally LSP, and CSP (Tables 1 and 2). However, may be better suited for production in endangered shrub, Lindera melissifolia (Laur- there were substantial differences among low-irradiance facilities than spinach. aceae), to varied light environments. Amer. J. kale and chard varieties for these same Also, as spinach had a higher LCP Bot. 92:682–689.

HORTSCIENCE VOL. 52(5) MAY 2017 711 Austin, R.B. 1989. Genetic variation in photosyn- cultural practices. Can. Greenhouse Conf. temperature on the postharvest quality of three thesis. J. Agr. Sci. Camb. 112:287–294. 2003, 1–8. asparagus cultivars harvested in spring. J. Jpn. Behr, U. and H.J. Wiebe. 1992. Relations between Farquhar, G.D., S. von Caemmerer, and J.A. Soc. Hort. Sci. 80(1):76–81. photosynthesis and nitrate content of lettuce Berry. 1980. A biochemical model of CO2 Kretchen, D.W. and F.S. Howlett. 1970. CO2 cultivars. Sci. Hort. 49:175–179. assimilation in leaves of C3 species. Planta enrichment for vegetable production. Trans- Bertoia, M.L., K.J. Mukamal, L.E. Cahill, T. Hou, 149:78–90. actions of the ASAE13:252–256. D.S.Ludwig,D.Mozaffarian,W.C.Willett, Feldmann, C. and U. Hamm. 2015. Consumers’ Laitat, E. and H. Boussard. 1995. Comparative F.B. Boese, and N.P.A. Huner. 1990. Effect perceptions and preferences for local food: A response on gas exchange of Picea spp. ex- of growth temperature and temperature shifts review. Food Qual. Prefer. 40:152–164. posed to increased atmospheric CO2 in open on spinach leaf morphology and photosynthesis. Fu, Y., H. Li, J. Yu, H. Liu, Z. Cao, N.S. Manukovsky, top chambers at two test sites, p. 241–248. Plant Physiol. 94:1830–1836. and H. Liu. 2017. Interaction effects of light Journal of Biogeography, Vol. 22, No. 2/3, Bertoia, M.L., K.J. Mukamal, L.E. Cahill, T. Hou, intensity and nitrogen concentration on growth, Terrestrial Ecosystem Interactions with Global D.S. Ludwig, D. Mozaffarian, W.C. Willett, photosynthetic characteristics and quality of Change, Volume 1 (March–May 1995). F.B. Hu, and E.B. Rimm. 2015. Changes in intake lettuce (Lactuca sativa L. var. youmaicai). Sci. Lambers, H., F.S. Chapin, III, and T.L. Pons. 2008. of fruits and vegetables and weight change in Hort. 214(5):51–57. Photosynthesis, p. 11–99. Plant Physiological United States men and women followed for up Gaudreau, L., J. Charbonneau, L.P. Vezina, and A. ecology. Springer, New York, NY. to 24 years: Analysis from three prospective Gosselin. 1994. Photoperiod and PPF influence Marino, G., M. Aqil, and B. Shipley. 2010. The leaf cohort studies. PLOS Medicine, doi: 10.1371/ growth and quality of greenhouse-grown let- economics spectrum and the prediction of journal.pmed.1001878. tuce. HortScience 29:1285–1289. photosynthetic light–response curves. Funct. Bjorkman,€ O. 1981. Responses of different quan- Gent, M.P.N. 2016. Effect of irradiance and tem- Ecol. 24:263–272. tum flux densities, p. 57–107. In: O.L. Lange, perature on composition of spinach. Hort- Peek, M.S., E. Russek-Cohen, D.A. Wait, and I.N. P.S. Nobel, C.B. Osmond, and H. Ziegler Science 51:133–140. Forseth. 2002. Physiological response curve (eds.). Encyclopedia of plant physiology. vol. Goudrian, J. 1979. A family of saturation type analysis using nonlinear mixed models. Oeco- 12A, Springer-Verlag, Berlin, Germany. curves, especially in relation to photosynthesis. logia 132:175–180. Boese, S.R. and N.P.A. Huner. 1990. Effect of Ann. Bot. 43:783–785. Pons, T.L. 2012. Interaction of temperature and growth temperature and temperature shifts on Gu, J., X. Yin, T. Stomph, H. Wang, and P.C. irradiance effects on photosynthetic acclima- spinach leaf morphology and photosynthesis. Struik. 2012. Physiological basis of genetic tion in two accessions of Arabidopsis thaliana. Plant Physiol. 94:1830–1836. variation in leaf photosynthesis among rice Photosynth. Res. 113:207–219. Bunce, J.A. and R.C. Sicher. 2003. Daily irradiance (Oryza sativa L.) introgression lines under Potvin, C., M.J. Lechowicz, and S. Tardif. 1990. and feedback inhibition of photosynthesis at drought and well-watered conditions. J. Expt. The statistical analysis of eco- physiological elevated carbon dioxide concentration in Bras- Bot. 63(14):5137–5153. response curves obtained from experiments sica oleracea. Photosynthetica 41(4):481–488. Heuvelink, E. and M. Dorais. 2003. Crop growth involving repeated measures. Ecology 71: Bunce, J.A. and L.H. Ziska. 2000. Impact of and yield. In: E. Heuvelink (ed.). Tomato. CAB 1389–1400. measurement irradiance on acclimation of pho- International, Wallingford, Oxon, UK. Ruhil, K., Sheeba, A. Ahmad, M. Iqbal, and B.C. tosynthesis to elevated CO2 concentration in Hu, E. and B. Rimm. 2015. Changes in intake of Tripathy. 2015. Photosynthesis and growth re- several plant species. Photosynthetica 37(4): fruits and vegetables and weight change in sponses of mustard (Brassica juncea L. cv Pusa 509–517. United States men and women followed for Bold) plants to free air carbon dioxide enrich- Chagvardieff, P., T. d’Aletto, and M. Andre. 1994. up to 24 years: Analysis from three prospective ment. Protoplasma 252:935–946. Specific effects of irradiance and CO2 concen- cohort studies. PLOS Medicine 12:e1001878. Samuoliene, G., A. Brazaityte, J. Jankauskiene, tration doublings on productivity and mineral Johnson, I.R., J. Thornley, J.M. Frantz, and B. A. Virsile, R. Sirtautas, A. Novickovas, S. content in lettuce. Adv. Space Res. 14(11): Bugbee. 2010. A model of canopy photosynthe- Sakalauskaite, and P. Duchovskis. 2013. LED 269–275. sis incorporating protein distribution through the irradiance level affects growth and nutritional Colonna, E., Y. Rouphaei, G. Barbieri, and S. De canopy and its acclimation to light, temperature quality of Brassica microgreens. Cent. Eur. J. Pascale. 2016. Nutritional quality of ten leafy and CO2. Ann. Bot. 106:735–749. Biol. 8:1241–1249. vegetables harvested at two light intensities. Kaiser, E., A. Morales, J. Harbinson, J. Kromdijk, E. Yamori, W., K. Noguchi, and I. Terashima. 2005. Food Chem. 199:702–710. Heuvelink, and L.F.M. Marcelis. 2015. Dynamic Temperature acclimation of photosynthesis in Dahal, K., K. Kane, W. Gadapati, E. Webb, L.V. photosynthesis in different environmental con- spinach leaves: Analysis of photosynthetic Savitch, J. Singh, P. Sharma, F. Sarhan, F.J. ditions. J. Expt. Bot. 66(9):2415–2426. components and temperature dependencies of Longstaffe, B. Grodzinski, and N.P.A. Huner.€ Kaiser, E., A. Morales, J. Harbinson, E. Heuvelink, photosynthetic partial reactions. Plant Cell 2012. The effects of phenotypic plasticity on A.E. Prinzenberg, and L.F.M. Marcelis. 2016. Environ. 28:536–547. photosynthetic performance in winter rye, win- Metabolic and diffusional limitations of pho- Yu, X., R. Zhou, X. Wang, K.H. Kjaer, E. Rosenqvist, ter wheat and Brassica napus. Physiol. Plant. tosynthesis in fluctuating irradiance in Arabi- C. Ottosen, and J. Chen. 2016. Evaluation of 144(2):169–188. dopsis thaliana. Sci. Rep. 6:31252. genotypic variation during leaf development Dorais, M. 2003. The use of supplemental lighting Kitazawa, H., S. Motoki, T. Maeda, Y. Ishikawa, in four Cucumis genotypes and their response for vegetable crop production: Light intensity, Y. Hamauzu, K. Matsushima, H. Sakai, T. to high light conditions. Environ. Expt. Bot. crop response, nutrition, crop management, Shiina, and Y. Kyutoku. 2011. Effects of storage 124:100–109.

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