Environmental and Experimental Botany 199 (2022) 104895

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Environmental and Experimental Botany

journal homepage: www.elsevier.com/locate/envexpbot

Phenotypic plasticity and nutritional quality of three kale cultivars

(Brassica oleracea L. var. acephala) under field, greenhouse, and growth
chamber environments
Eyosias L. Ashenafi a, Marianne C. Nyman a, *, Jake M. Holley b, Neil S. Mattson b,
Anusuya Rangarajan b
a
Department of Civil and Environmental Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, United States
b
School of Integrative Plant Science, Cornell University, Ithaca, NY 14583, United States

A R T I C L E I N F O A B S T R A C T

Key words: Comparative analysis of the physiological and biochemical characteristics of three kale cultivars (‘Toscano’,
Kale (Brassica oleracea L.) ‘Redbor’, and ‘Winterbor’) in different agricultural systems was performed. High biomass yield was observed in
Carotenoids the plants grown in the field and greenhouse systems likely due to the higher light intensity (sunlight) and lower
Chlorophyll
planting density during growth. The highest relative growth rate was observed in the field for ‘Redbor’ (104 mg
Growth rate
g-1 d-1) and ‘Winterbor’ kale (115 mg g-1 d-1), while the highest growth rate for ‘Toscano’ kale was found in the
Phenotypic plasticity
Controlled environment greenhouse system (109 mg g-1 d-1). For all three cultivars, the smallest growth rate (72 – 78 mg g-1 d-1) and
leaves with the highest specific-leaf area (295 – 378 cm2 g-1) were observed in the growth chamber environment.
However, the highest concentration of phytochemicals (lutein, violaxanthin, chlorophyll a, and chlorophyll b)
was detected in kale leaves from the growth chamber. The macular pigment, zeaxanthin, was detected in leaf
samples harvested from the field and greenhouse grown kale primarily during high light conditions (PPFD >
1000 μmol m-2 s-1). Based on interaction study, cultivar type (genotype), growth stage at harvest, and farming
system were identified as primary factors that determine nutritional quality in kale.

1. Introduction
Urbanization and emergence of large middle class around the globe
will lead to increased demand for agricultural produce (Tilman et al.,
2011). Concurrently, climatic conditions around the world are increas­
ingly becoming dynamic and extreme in intensity with global warming
(Arnold et al., 2019; Zhu et al., 2018). Agronomists and policy-makers
face uncertainty in predicting crop productivity and variability in
growth cycle, particularly the interaction of high global CO2, high
temperature, and location-dependent changes in rainfall (Hatfield et al.,
2011; Olesen et al., 2011). Such developments pose challenges to global
food security since most crop cultivation is currently based on open-field
farming, which is susceptible to climate impacts and supply chain dis­
ruptions (Nicholson et al., 2020). Hence, it is necessary to investigate the
growth and development of different crops under alternative food pro­
duction systems.
Genetic and environmental factors (e.g., light, temperature, water,
and nutrient availability) influence crop phenotype, rate of
photosynthesis, and leaf biochemistry (Arnold et al., 2019; Hüner et al.,
2012; Stotz et al., 2021). Under dynamic light environment in an
open-field, plants develop large photosynthetic apparatus to drive
higher rate of photosynthesis, while at the same time possess higher
concentration of carotenoids, necessary for photoprotection from
damaging incident radiation (Demmig-Adams et al., 2017; Niinemets
et al., 2003).
Light-dependent xanthophyll cycle, specifically de-epoxidation of
violaxanthin to zeaxanthin is a well-known biochemical response
(Yamamoto et al., 1962). In high light conditions (above saturating
point), violaxanthin is reversibly converted to antheraxanthin and sub­
sequently to zeaxanthin (Peguero-Pina et al., 2013). Zeaxanthin con­
tributes to the safe dissipation of excess photon energy and prevents the
formation of damaging radical species in plant tissues (Demmig-Adams,
1990). From a human health perspective, good nutritional quality (high
zeaxanthin and total phenolic content) in kale and other leafy greens can
be obtained by harvesting plants at high light conditions and low tem­
perature conditions (Cohu et al., 2014; Colonna et al., 2016).

* Corresponding author.
E-mail address: nymanm@rpi.edu (M.C. Nyman).

https://doi.org/10.1016/j.envexpbot.2022.104895
Received 8 February 2022; Received in revised form 19 April 2022; Accepted 23 April 2022
Available online 29 April 2022
0098-8472/© 2022 Elsevier B.V. All rights reserved.
E.L. Ashenafi et al.
Increase in biomass and size during plant growth and development
can be approximated with an exponential function (Blackman, 1919).
Plants undergo three distinct growth stages: initial lag phase, logarith­
mic (exponential) phase, and finally steady state (decreasing) growth
phase. The rate of accumulation of plant biomass between two time
points can be determined by calculating mass-based relative growth rate
(RGR). RGR of kale grown in the three environmental systems was
calculated using Eq. 1, based on the dry weight (DW) of harvested
samples (Hoffmann and Poorter, 2002). Higher RGR values are reported
in plants grown under optimal conditions.
ln (DW 2 ) − ln (DW)1
RGR =
Δt
Where RGR is the relative growth rate, ln (DW)1 and ln (DW)2 are the
mean of the natural-log transformed dry weights (g) of leaf samples at
time points 1 and 2, respectively, and Δt represents change in time (d).
Other physiological parameters used to measure plant growth are
leaf area ratio (LAR) and specific leaf area (SLA). LAR is the ratio of
change in total leaf surface area (TLA) (photosynthetic apparatus) to
change in plant dry biomass between two growth stages (Eq. 2). SLA is
the ratio of total surface area to DW of leaves at a specific growth stage
(Eq. 3). Leaves from low-light environment exhibit high SLA values
which is ideal for increased light interception (Evans and Poorter, 2001).
TLA2 − TLA1
LAR =
DW 2 − DW 1
Where LAR is leaf area ratio (cm2 g-1). TLA1/TLA2 and DW1/DW2
represent the total leaf area (cm2) and total dry weight (g) of plant leaves
at time points 1 and 2, respectively.
TLA
SLA =
DW
Where SLA is specific leaf area (cm2 g-1), TLA is total leaf area (cm2), and
DW is the total dry weight of leaf samples (g).
Kale is a cold-tolerant, non-heading leafy green that belongs to the
Brassicaceae family. Due to the high concentration of health-promoting
Fig. 1. : Photograph of three kale cultivars (‘Toscano’, ‘Redbor’, and ‘Winterbor’) growing in the field, greenhouse, and growth chambers.
Environmental and Experimental Botany 199 (2022) 104895
compounds, it has been dubbed as “superfood” (Šamec et al., 2019).
From a carotenoid database of 120 fruits and vegetables, the highest
concentration of lutein and zeaxanthin was reported in kale (14.7 –
39.6 mg per 100 g of fresh weight (FW)) (Mangels et al., 1993). In
humans, reduced risk of age-related macular degeneration (AMD) and
cataract disease is associated with increased intake of lutein and zeax­
anthin (Bernstein et al., 2016; Feng et al., 2019). In addition, high
antioxidant content (flavonoids, hydroxycinnamic acids, and glucosi­
nolates) in kale protects against inflammation and cancer (Ferioli et al.,
2013; Olsen et al., 2009).
Locally grown produce is gaining popularity, due to increased con­
sumer awareness of health benefits and impact of food supply chain on
(1)
the environment (Nicholson et al., 2020). To address this demand,
controlled environment agriculture or CEA (plant production in green­
houses and warehouse farms) is being adopted in metropolitan areas in
many countries (Saraswat and Jain, 2021; Wong et al., 2020). CEA
systems can produce fresh, nutrient dense fruits and vegetables, such as
tomato and leafy greens consistently year-round in climate-controlled
environment with significantly less resources (water, fertilizer etc.)
Highly automated vertical farms can provide cultivar-dependent light
treatment, mineral concentrate, and air-conditioning control for opti­
mum production (Eaton et al., 2021).
However, there is limited scientific literature that compares the crop
yield and nutritional quality of leafy greens from CEA systems and from
field-based farming. The objective of this work was to evaluate the
(2)
phenotypic plasticity and phytochemical content of three kale cultivars
from three agricultural systems – open field, semi-controlled green­
house, and controlled growth chamber (see Fig. 1). It was hypothesized
that the large fluctuations in environmental conditions like sunlight and
temperature in the field would produce plants with significantly
different morphology and biochemistry compared to greenhouses and
(3) growth chambers. Specifically, the environmental fluctuation in the
field is expected to result in higher nutritional content in leaves due to
known role of carotenoids in plant stress responses.
2
E.L. Ashenafi et al.
2. Materials and methods
2.1. Planting materials and growing conditions
Seeds for the three kale cultivars (Brassica oleracea cv. ‘Toscano’,
‘Redbor’, and ‘Winterbor’) were obtained from Johnny’s Selected Seeds
(Winslow, ME). The market class of ‘Toscano’, ‘Redbor’, and ‘Winterbor’
kale was dinosaur (flat with dark-green leaves), curly (red), and curly
(green), respectively (Reda et al., 2021).
Field trials were conducted at the H.C. Thompson Vegetable
Research Farm (Freeville, NY) at Cornell University. Plants were grown
in raised beds and fertigated with 13–13–13 N-P-K fertilizer solution
using drip irrigation system. Herbicide and pesticides were applied at
different times. The soil was characterized as slightly acidic (pH = 6.6)
and topsoil with good organic carbon (OC) content (4.4% w/w). Ran­
domized block design was utilized. At each planting cycle, there were
total of 144 plant samples per cultivar at a planting density of 27.6
plants per m2 at the beginning. Seeding and transplanting dates were
May 13/June 7 (planting #1), June 12/July 10 (planting #2), and July
11/August 9 (planting #3), respectively. All field trials were conducted
during the summer and fall of 2019.
Greenhouse trials were conducted at the Kenneth Post Laboratory
(Cornell University, Ithaca, NY), approximately 9 miles from the
Thompson Vegetable Research Farm. Seeds of the three kale cultivars
were sown in a Grodan AX rockwool plugs (Grodan, Netherlands). At
transplanting, they were established in net pots with Hydroton®
expanded clay substrate in a solution culture hydroponic system (Aer­
oFlo2 Aeroponic System, General Hydroponics, Santa Rosa, CA). A
complete hydroponic nutrient solution was provided (Jack’s 5–12–26
Hydroponics Part A, J.R. Peter’s Inc., Allentown, PA and YaraLiva Cal­
cium Nitrate, Yala International ASA, Oslo, Norway). The nutrient
composition (in ppm) was N (200), P (52), K (216), Ca (185), Mg (63), Fe
(3.07), B and Mn (0.51), Zn and Cu (0.15), and Mo (0.11). The pH and
electrical conductivity (EC) were maintained at 5.8 and 1.8 dS m-1,
respectively, to ensure nutrient availability for plants. There were 36
plants per cultivar, and the planting density was 31.0 plants per m2 at
the start of each planting cycle. Seeding and transplanting dates were
May 16/June 10 (planting #1), July 8/July 25 (planting #2), and
August 30/September 14 (planting #3), respectively. All greenhouse
trials were conducted during summer and fall of 2019. Natural sunlight
was the sole lighting source inside glass greenhouse (~ 70% trans­
mittance). Measured PPFD ranged from 0 to 1885 μmol m-2 s-1.
For growth chamber trials, kale seeds were sown in Grodan AX
rockwool plugs (Grodan, Netherlands) and germinated under low light
(~ 50 µmol m-2 s-1) inside two large growth chambers (Conviron®
Model A2000, Winnipeg, Canada). Upon germination, the plants were
supplied with Jack’s 5–12–26 N-P-K Hydroponics Part A and 15–0–0
Cal-Tarte LX (J.R. Peter’s Inc., Allentown, PA). The nutrient composition
(in ppm) was N (200), P (50), K (206), S (78.4), Ca (182.7), Mg (60.3), Fe
(3.63), B (0.63), Mn (0.86), Zn (0.52), Cu (0.22), and Mo (0.17). For
each planting cycle (replicate), there were a total of 39 plants per
cultivar with initial planting density of 335.8 plants per m2.
PPFD was maintained at 200 ± 10% µmol m-2 s-1 in the PAR range
(400–700 nm). Lighting was provided by T5 cool-white fluorescent
(CWF) lamps (Philips, Amsterdam, Netherlands). The light schedule was
16-hr photoperiod followed by 8-hr of darkness. Spectral power distri­
bution of the lamp was measured using PS-300 spectroradiometer
(Apogee Instruments, Logan, UT). Temperature and relative humidity
(RH) were set at 24 ◦ C and 60–80% inside the growth chambers,
respectively. Block design, harvest schedule, and environmental data of
each farming system are presented in Supplementary Materials.
Three developmental stages were identified based on number of
leaves from growth kinetics experiment (see Supplementary Materials).
These were seedling or stationary phase (3–4 leaves), juvenile or mid-log
phase (22–27 leaves), and mature or steady state phase (> 40 leaves).
These stages were used as benchmarks to compare biomass yield and
3
Environmental and Experimental Botany 199 (2022) 104895
pigment density of kale cultivars from the different production systems.
There were three biological replicates (replicated crop cycles) in
each environmental system. Ten randomly selected plant samples were
harvested for morphology analysis from the field at three developmental
stages (seedling, juvenile, and mature). For greenhouse and growth
chamber trials, four plant samples were harvested per cultivar at seed­
ling and juvenile stages for morphology analysis. Furthermore, leaves
from identical number of plants (n = 10 for field and n = 4 for green­
house and growth chamber environments) were harvested from each
agricultural system for nutrient analysis at each developmental stage.
2.2. Morphological measurements
For biomass and morphological assessment, number of leaves, FW,
DW, and TLA were measured at each growth stage. DW was determined
by drying leaf samples at 70 ◦ C to constant weight inside an oven (WTC
Binder, Germany). TLA was determined by measuring surface area of
true leaves from each plant using portable CI-202 laser leaf area meter
(CID-Bioscience, Camas, WA) for growth chamber trials and LI-3100
area meter (LI-COR Biosciences, Lincoln, NE) for field and greenhouse
trials. LAR and SLA of leaf samples were found using Eqs. 2 and 3.
2.3. Extraction of leaf pigments
Across the three environmental treatments, leaf samples for nutrient
analysis were harvested between 10 AM and 12 PM to minimize circa­
dian rhythm effects. At harvest time, instantaneous PPFD was measured
with LI-190R quantum sensor (LI-COR Biosciences, Lincoln, NE). The
fourth leaf from the growing tip was flash frozen in liquid nitrogen and
stored in labelled aluminum envelopes. For field and greenhouse sam­
ples, the envelopes were transported in Styrofoam containers with dry
ice and stored in a − 80 ◦ C freezer until analysis.
During nutrient analysis, frozen leaf samples were homogenized
under liquid nitrogen using a mortar and pestle after removal of midribs.
Approximately 50 mg (mass known) of leaf powder was placed in
1.5 mL polypropylene microcentrifuge tubes (VWR International, Rad­
nor, PA). One mL of 80:20 (v/v) acetone: deionized water solution was
added as an extraction solvent. The tubes were vortexed at 2800 rpm
several times and centrifuged at 14,800 rpm for 5 min at 4 ◦ C (Eppen­
dorf Centrifuge 5403, Enfield, CA). Next, two-hundred microliter of
supernatant solution was drawn with glass micropipettes and mixed
with 1300 µL of the same extraction solvent. Particles from the mix
solution were filtered with 0.45 µm nylon syringe filters (VWR Inter­
national, Radnor, PA), and final samples were stored in amber HPLC
vials. Pigment extraction was conducted at room temperature under dim
light to minimize photodegradation and photoisomerization of the
desired analytes.
2.4. HPLC analysis
Each extract solution was analyzed using Prominence-i high perfor­
mance liquid chromatograph LC-2030 3D with photodiode array (PDA)
detector (Shimadzu Scientific Instruments, Kyoto, Japan). The station­
ary phase was polymeric YMC C-30 Carotenoid column (4.6 ×250 mm x
5 µm) (YMC America, Allentown, PA). Isocratic mobile phase of 81:15:4
(v/v/v) methanol: MTBE: deionized water was used for analysis of plant
pigments. Total run time was 36 min. Flow rate was 1 mL min-1 and
10 µL injection volume was analyzed. Duplicate injections were per­
formed for each sample. Autosampler and column oven temperatures
were maintained at 4 ◦ C and 23 ◦ C, respectively.
Compounds were identified based on retention time of standards and
absorption spectra. Pigment concentration was determined from a
calibration curve of external standards. Chromatogram of sample kale
extract solution and external calibration curves can be found in Sup­
plemental Materials.
High purity standards of chlorophyll a (≥ 85%), chlorophyll b (≥
E.L. Ashenafi et al. Environmental and Experimental Botany 199 (2022) 104895

90%), and violaxanthin (≥ 95%) were obtained from Sigma-Aldrich (St. Table 1
Louis, MO). Primary grade standards of lutein and zeaxanthin (98.6%) Morphology measurements of kale cultivars grown under field, greenhouse, and
were purchased from ChromaDex (Irvine, CA). Methyl tert-butyl ether growth chamber environments at different growth stages.
(MTBE) was sourced from Acros Organics (Fair Lawn, NJ). HPLC-grade A. ‘Toscano’
methanol and acetone were purchased from Fisher Scientific (Waltham, Environment Seedling Stage Juvenile Stage
MA). Liquid nitrogen and dry ice were purchased from Airgas Store
FW DW TLA FW DW TLA
(Albany, NY). DI water was obtained with Thermo Fisher Barnstead™
GenPure™ system (Waltham, MA). Chemicals were used as received. Field 6.8 0.9 157 326 37 4352
Stock solution of pigment standards was prepared individually in 100% ± 0.5a ± 0.06a ± 12ab ± 35a ± 5a ± 457a
Greenhouse 8.8 0.8 211 224 20 3440
acetone solution and stored at – 80 ◦ C in amber vials. ± 1a ± 0.1a ± 29a ± 25a ± 2b ± 325a
Growth 3.2 0.4 106 42 4 947.2
2.5. Statistical analysis Chamber ± 0.3b ± 0.03b ± 9b ± 9b ± 1b ± 162b

B. ‘Redbor’
Means from measured and calculated parameters were compared
Environment Seedling Stage Juvenile Stage
using one-way analysis of variance (ANOVA) with post hoc Tukey’s
FW DW TLA FW DW TLA
honestly significant difference (HSD) test with α = 0.05. Type III anal­
ysis of Satterthwaite’s method was used to investigate interaction be­ Field 7.6 0.9 158 294 33 2910
tween variables using ‘lmerTest’ package in R software. More ± 0.5a ± 0.06a ± 10a ± 25a ± 3a ± 195a
Greenhouse 8.9 0.8 207 210 16 2588
specifically, a linear mixed model comparing the variable to the fixed
± 1.6a ± 0.1ab ± 29a ± 32a ± 3b ± 317a
effects of Environment, Cultivar, Age, and PAR at harvest, as well as Growth 6.7 0.6 210 66 5 1443
Environment X Age, Cultivar X Age, and Environment X Age in­ Chamber ± 0.7a ± 0.06b ± 16a ± 10b ± 1b ± 205b
teractions, with random effects of Planting, Block, and specific Plant C. ‘Winterbor’
Number was implemented. Statistical analysis was performed using R
Environment Seedling Stage Juvenile Stage
Studio Ver. 1.4.1103 (Boston, MA).
FW DW TLA FW DW TLA
3. Results Field 7.8 1.0 167 400 48 3468
± 0.6b ± 0.07a ± 15b ± 27a ± 4a ± 196a
Greenhouse 12.4 1.0 269 239 19 3062
3.1. Leaf Biomass, Morphology, and RGR
± 2.4a ± 0.2a ± 46a ± 32b ± 3b ± 337a
Growth 5.2 0.5 173 60 5 1260
Field and greenhouse grown ‘Toscano’ kale yielded significantly Chamber ± 0.5b ± 0.05b ± 14ab ± 8c ± 1b ± 146b
higher FW than growth chamber plants at the seedling stage (Table 1).
Abbreviations: FW = fresh weight (g), DW = dry weight (g), and TLA = total leaf
No statistical difference was observed in fresh biomass for ‘Redbor’ kale
area (cm2).
harvested at seedling stage between the three systems. However, field Sample size per cultivar: n = 10 for field kale and n = 4 for greenhouse and
and greenhouse grown ‘Toscano’ plants yielded significantly higher FW growth chamber kale. Values are reported as mean ± SEM. Significance differ­
than growth chamber plants. For ‘Winterbor’ kale, field and growth ence at p < 0.05 level indicated by different letters within columns, based on
chamber treatments produced higher FW than the greenhouse Tukey’s HSD test with a > b > c.
environment.
At juvenile stage, the FW of ‘Toscano’ and ‘Redbor’ kale from field conditions, respectively. An identical trend was also observed in LAR
and greenhouse treatments was significantly greater than growth values from seedling to juvenile stage. The SLA of mature ‘Toscano’,
chamber plants. For ‘Winterbor’ kale, observed biomass yield followed ‘Redbor’, and ‘Winterbor’ leaves from field trials were 87 ± 7, 71 ± 2,
field, greenhouse, and growth chamber treatments in decreasing order. and 48 ± 1 cm2 g-1, respectively.
Measured DW and TLA across the three environments followed a similar The highest RGR between seeding and juvenile stage was observed in
trend at this stage with lower values found in plants from growth the field and greenhouse treatments (Table 2). For the three kale culti­
chamber environment. vars, calculated RGR in field trials was 104 – 115 mg g-1 d-1, while
At the steady state or mature stage, the FW, DW, and TLA for field similar RGR values were found in greenhouse experiments (101 –
‘Toscano’ kale were 557 ± 38 g, 81 ± 6 g, and 6270 ± 392 cm2, 109 mg g-1 d-1). On the other hand, growth chamber plants had the
respectively. For ‘Redbor’ kale grown in the field, the measured FW, lowest RGR values (72 – 78 mg g-1 d-1). The RGR of ‘Toscano’, ‘Redbor’,
DW, and TLA at mature stage were 426 ± 25 g, 56 ± 3 g, and 3837 and ‘Winterbor’ kale cultivated in the field were 41.3%, 44.4%, and
± 195 cm2, respectively. Finally, the FW, DW, and TLA of mature 47.2% higher than the RGR of the same cultivars cultivated in growth
‘Winterbor’ kale from the field were 759 ± 70 g, 116 ± 10 g, and 5404 chambers, respectively.
± 430 cm2, respectively. Kale plants were not grown to mature stage in In field trials, average LAR and RGR values from juvenile to mature
the greenhouse and growth chamber systems due to growth stagnation growth stages for ‘Toscano’ kale were 44 g cm-2 and 38.5 mg g-1 d-1,
and space limitations. respectively. For ‘Redbor’ kale, the LAR value was 41 g cm-2, while the
Higher leaf area results in higher light interception for growth and RGR was 24 mg g-1 d-1 during the same growth transition. Finally, the
development, which was evident in field and greenhouse trials for each LAR and RGR values for “Winterbor’ kale were 28 g cm-2 and 38.6 mg g-
kale cultivar (Table 1). In field trials, a positive linear relationship be­ 1 -1
d , respectively.
tween cumulative daily light integral (DLI) and kale yield (leaf biomass
and surface area) was observed (Fig. 2). Cumulative DLI in the field was
calculated by accounting for measured solar DLI from transplanting date 3.2. Leaf Phytochemicals (Chlorophylls and Carotenoids)
to final harvest time.
Kale plants grown inside growth chambers had large and thin leaves Overall, the highest pigment concentration was measured in kale
with deep green pigmentation. For all three cultivars, calculated SLA in plants grown inside growth chambers followed by greenhouse and field
growth chamber plants was significantly higher than greenhouse plants environments (see Table 3). In terms of cultivar selection, ‘Toscano’ kale
followed by field plants (Table 2). For instance, at seedling stage, the had higher density of chlorophyll and carotenoid pigments than ‘Red­
SLA of ‘Winterbor’ leaves harvested from growth chambers was 104.1% bor’ and ‘Winterbor’ kale.
and 38.7% larger than the ‘Winterbor’ SLA in field and greenhouse Leaves from field grown ‘Redbor’ kale had purplish-red color (see

4
E.L. Ashenafi et al.
Fig. 2. : Linear relationship between cumulative DLI in field plants (from transplanting to harvest) and fresh biomass (a), dry biomass (b), and total leaf area (c) for
the three kale cultivars (p < 0.005 for all plots). Error bars represent mean ± SE (n = 9). Based on hourly solar radiation data at Freeville, NY weather station from
May 13 – Oct. 12, 2019.
Data source: https://newa.cornell.edu; accessed on 12/3/2019.
Fig. 1), indicating high concentration of anthocyanins and other
phenolic compounds due to strong blue and UV light found in sunlight
(Ying et al., 2021; Zietz et al., 2010). In the growth chamber environ­
ment, ‘Redbor’ leaves appeared green, while the petioles were red in
color.
At seedling stage, lutein concentration was found to be 37 – 72%
higher under growth chamber environment in comparison to the field
and greenhouse systems. Similarly, the violaxanthin content in leaves
from the growth chamber harvested at the juvenile stage was 9 – 31%
and 8 – 68% higher than leaves from the greenhouse and field trials,
respectively. Chlorophyll a and b content also followed a similar trend.
Zeaxanthin concentration was dependent on the PPFD level at har­
vest time. The pigment concentration of field kale (in mg zeaxanthin per
100 g FW) was 1.3 – 6.5 (‘Toscano’), 0.7 – 3.1 (‘Redbor’), and 0.7 – 3.8
(‘Winterbor’), when PPFD was greater than 1500 μmol m-2 s-1. In kale
leaves from greenhouse trials, lower concentration of the pigment was
detected (1 – 3.7 in ‘Toscano’, 3.2 – 3.4 mg per 100 g of FW in ‘Redbor’,
and ND in ‘Winterbor kale). Finally, zeaxanthin was not detected in
leaves obtained from growth chamber environment likely due to the low
5
Environmental and Experimental Botany 199 (2022) 104895
indoor PPFD (~ 200 μmol m-2 s-1). The measured PPFD at harvest time
was 76 – 2240 μmol m-2 s-1 in the field and 51 – 1226 μmol m-2 s-1 inside
the greenhouse.
In field grown plants, decrease in nutrient concentration was
observed with age in all kale cultivars with leaves from earlier stages
(seedling and juvenile) containing the highest pigment content (Fig. 3).
An exception was lutein and violaxanthin in ‘Toscano’ kale which had
higher concentration at juvenile stage than at seedling stage.
The chl. a/b ratio increases from seedling to juvenile growth stage
but decreases from juvenile to mature stage, indicating smaller reaction
systems and larger antenna systems at later stages (Fig. 3) (Lichtenthaler
and Buschmann, 2001). Furthermore, high correlation between total
chlorophyll and total carotenoid content was observed (p < 0.005) in all
three cultivars, indicating parallel biosynthesis of photosynthetic pig­
ments (Fig. 4).
3.3. Interaction of Different Factors on Pigment Content
In this study, cultivar type and growth stage at harvest were
E.L. Ashenafi et al. Environmental and Experimental Botany 199 (2022) 104895

Table 2 4. Discussion
Comparative analysis of plant morphology and growth rate.
A. ‘Toscano’ The large variation in plant density (competition) and total light
Environment Seedling SLA Juvenile SLA LAR (S-J) RGR (S-J) irradiance between the environmental systems likely contributed to the
Field 173 ± 7c 134 ± 9c 118 104 differences in growth rates. From a meta-analysis of 20,000 plant
Greenhouse 257 ± 10b 183 ± 8b 171 109 research papers, indoor grown plants were observed to have higher SLA
Growth Chamber 295 ± 9a 289 ± 24a 238 74 and lower rate of photosynthesis than field plants (Poorter et al., 2016).
B. ’Redbor’ Furthermore, the authors found a significant decrease in RGR and total
vegetative and generative mass with a doubling of plant density. In
Environment Seedling SLA Juvenile SLA LAR (S-J) RGR (S-J)
regards to light intensity effects, Tan and co-workers (2020) observed
Field 182 ± 8c 97 ± 5c 85 104
higher RGR in choi sum (Brassica rapa var. parachinensis) grown under
Greenhouse 280 ± 14b 181 ± 12b 156 102
Growth Chamber 378 ± 20a 291 ± 17a 255 72
160 μmol m-2 s-1 compared to 80 μmol m-2 s-1 PPFD light intensity.
In the present study, plant density in growth chambers was
C. ’Winterbor’
approximately ten times that of field and greenhouse trails. In addition,
Environment Seedling SLA Juvenile SLA LAR (S-J) RGR (S-J) leaves growing inside chambers were not exposed to saturating light
Field 168 ± 7c 81 ± 4c 71 115 intensity (Asat) for photosynthesis, unlike field and greenhouse plants
Greenhouse 248 ± 16b 173 ± 8b 158 101 which experience fluctuating, dynamic light levels throughout the day
Growth Chamber 344 ± 12a 250 ± 15a 223 78 (PPFD > 2000 μmol m-2 s-1 at noon on a clear summer day). Finally,
Abbreviations: S-J = growth from seedling to juvenile stage; SLA = specific leaf significantly lower growth rates were observed in the field at later
area (cm2 g-1); LAR = leaf-area ratio (g cm-2); and RGR = relative growth rate developmental stages (juvenile to mature) due to senescence (Wer­
(mg g-1 d-1). SLA values are reported as mean ± SEM. Significance difference at aduwage et al., 2015).
p < 0.05 level indicated by different letters within columns, based on Tukey’s In field plants, measured FW, DW and TLA were positively correlated
HSD test with a > b > c. to the total amount of sunlight available (r2 > 0.91; Fig. 2). Similarly,
positive relationship between crop yield (biomass and RGR) and irra­
diance level has been reported previously (Lawlor, 1995; Loconsole
Table 3
et al., 2019; Poorter et al., 2016; Poorter and van der Werf, 1998).
Measured pigment density in kale cultivars grown in the field, greenhouse (GH),
and growth chambers (GC) environments at different growth stages. Nutritional quality in leafy greens is dependent on many factors
including light quality, cultivar type, developmental stage, and growth
Cultivar and Pigment density (mg per 100 g of FW)
media (Carvalho and Folta, 2014; Kopsell et al., 2004). Light, in
environment
Lutein Violaxanthin Chlorophyll a Chlorophyll b particular, is an important environmental variable that regulates chlo­
‘Toscano’ – Seedling rophyll and carotenoid biosynthesis in plants (Cazzonelli and Pogson,
Field 23.1 ± 0.8b 10.5 ± 0.8a 227.5 ± 9b 81.5 ± 3.8b 2010). Light-induced conversion (de-epoxidation) of lutein-epoxide to
GH 19.7 ± 1.2b 10.9 ± 0.7a 213.0 ± 13.6b 76.9 ± 3.6b lutein and vice versa is minimal and lutein exists as the dominant form in
GC 31.7 ± 2.2a 13.1 ± 0.5a 324.7 ± 23.0a 118.1 ± 6.1a
‘Toscano’ – Juvenile
leaves (Leonelli et al., 2017; McElroy et al., 2006). However, zeaxanthin
Field 26.6 ± 1.6a 11.9 ± 0.5a 197.8 ± 11.4b 71.3 ± 4.5b concentration is dependent on irradiance level at harvest time and pool
GH 19.5 ± 1.3b 10.3 ± 0.5a 171.9 ± 10.3b 66.3 ± 3.4b of xanthophyll cycle pigments including violaxanthin (Kromdijk et al.,
GC 29.3 ± 1.7a 12.9 ± 0.9a 283.9 ± 12.2a 101.3 ± 4.6a 2016).
‘Redbor’ – Seedling
In terms of cultivar-specific differences, the highest concentration of
Field 12.0 ± 0.3b 6.3 ± 0.3b 113.4 ± 3.9b 38.6 ± 1.4b
GH 10.6 ± 0.3b 6.8 ± 0.6b 122.6 ± 5.9b 43.2 ± 1.3b chlorophyll and carotenoid compounds were found in ‘Toscano’ kale.
GC 16.9 ± 1.2a 9.0 ± 0.6a 194.8 ± 15.5a 72.9 ± 4.8a Similarly, Kopsell and colleagues (2004) reported the largest accumu­
‘Redbor’ – Juvenile lation of lutein and β-carotene pigments in the leaves of ‘Toscano’ from a
Field 8.32 ± 0.4c 4.3 ± 0.3b 51.8 ± 2.4c 16.7 ± 0.78c study of 23 different kale cultivars. Furthermore, inter-species variation
GH 10.8 ± 0.6b 5.8 ± 0.2a 82.8 ± 7.5b 31.4 ± 2.6b
in FW, minerals, and secondary metabolites has been previously re­
GC 14.1 ± 1.1a 6.3 ± 0.6a 152.9 ± 15.0a 53.5 ± 4.7a
‘Winterbor’ - Seedling ported, highlighting the importance of cultivar selection (Nassar et al.,
Field 12.9 ± 0.4b 5.8 ± 0.4b 119.7 ± 5.2b 38.5 ± 1.6c 2015; Yoder and Davis, 2020).
GH 12.1 ± 0.4b 6.2 ± 0.8b 138.0 ± 9.4b 46.1 ± 2.6b Growth stage at harvest time was another important variable that
GC 20.8 ± 0.7a 9.3 ± 0.4a 239.5 ± 10.2a 82.8 ± 2.5a
affects nutrient content. Young kale leaves harvested at seedling stage
‘Winterbor’ – Juvenile
Field 8.50 ± 0.5b 3.8 ± 0.2c 68.4 ± 3.3c 20.4 ± 1.1c
had higher pigment density than older leaves (Fig. 3). Lefsrud et al.
GH 9.52 ± 0.5b 4.9 ± 0.2b 96.4 ± 6.2b 30.9 ± 1.3b (2007) also found higher concentration of lutein, β-carotene, chloro­
GC 14.5 ± 1.1a 6.4 ± 0.4a 166.2 ± 11.8a 53.1 ± 3.7a phyll a and b in young leaves (1 – 3 weeks old after seeding) than fully
Values are reported as mean ± SEM (n = 30 for field plants and n = 12 for GH developed, mature leaves (> 4 leaves) of ‘Winterbor’ kale. However, De
and GC trials from three planting cycles). Different lower-case letters in super­ Azevedo and Rodriguez-Amaya (2005) reported higher violaxanthin but
script represent significant differences based on Tukey’s HSD test. lower lutein and β-carotene content in young kale leaves, in comparison
to mature leaves.
identified as significant factors for all pigment molecules (all with This study adds to the relatively small existing body of work
p ≤ 0.001, except age on lutein, p ≤ 0.01) (Table 4). Environmental comparing the nutritional quality of leafy greens from different growth
production system had significant effect on lutein (p ≤ 0.01), chloro­ media. In soil media, plants invest extensively in their root system to
phyll a (p ≤ 0.001), and chlorophyll b (p ≤ 0.001), whereas no signifi­ locate essential minerals in subsurface, while in a hydroponic system, a
cant effect was found on violaxanthin levels. Instantaneous PAR at formulated mineral concentrate is dissolved in liquid solution and
harvest time had a significant effect on lutein and violaxanthin levels readily available for root uptake (Mattson and Lieth, 2019). Higher
(p ≤ 0.001), as expected due to the xanthophyll cycle. However, no concentration of bioactive compounds (ascorbic acid and α-tocopherol)
significant effect of PAR on chlorophyll a and b levels was observed. have been previously reported in lettuce and strawberries that were
Results from two-way ANOVA showed significant impact of “environ­ grown with hydroponic system, in comparison to soil grown varieties
ment x cultivar” on lutein (p ≤ 0.001) and chlorophyll a and b (p ≤ 0.01) (Buchanan and Omaye, 2013; Treftz et al., 2015). However, in another
content. study, signficantly lower carotenoid content was found in
hydroponically-grown lettuce than conventional field-grown lettuce

6
E.L. Ashenafi et al.
Fig. 4. : Relationship between total chlorophylls and total carotenoids in kale cultivars represented by linear regression model (p < 0.005 for all plots). Both axis in
“mg per 100 g of FW” units. Shaded area around best fit line represents 95% confidence interval.
(Kimura and Rodriguez-Amaya, 2003).
In our work, kale cultivars in the field were grown in the soil, while
the same cultivars were germinated and developed hydroponically in
rockwool plugs in the greenhouse and growth chamber experiments.
From pigment analysis, significantly higher leaf phytochemicals were
obtained in cultivars from growth chamber environment than counter­
parts from field and greenhouse systems both at seedling and juvenile
stages (Table 3). Dilution effect may have contributed to this result,
whereas in the field, high leaf biomass, low SLA (high leaf thickness) and
low pigment content was apparent, while in the growth chamber envi­
ronment, plants develop smaller leaf biomass, but contain higher
pigment density. This effect or trade-off has been observed in previous
studies (Johnson et al., 2019; Shekhar et al., 2015).
7
Environmental and Experimental Botany 199 (2022) 104895
Fig. 3. Carotenoid density (left panel) of lutein (•
symbol with solid line) and violaxanthin (▪ symbol
with broken line) in different kale cultivars from
field environment. Chlorophyll density (right panel)
of chlorophyll a (• symbol with solid line) and
chlorophyll b (▪ symbol with broken line) in
different kale cultivars from field environment. The
x-axis represents the growth stages at harvest time.
The average of Chl. a/b ratio is indicated in gray
background above trendlines. Values reported as
mean ± SE (n = 30 from three planting cycles).
5. Conclusion
In this research work, kale seeds from the same lot were used in each
agricultural system. Furthermore, leaf samples were harvested at the
same developmental stages for biomass and phytochemical evaluation.
From our findings, biomass accumulation and growth rate were largely
dependent on total irradiance during growth. As opposed to our hy­
pothesis, we found some cases where CEA systems had greater xantho­
phyll and chlorophyll content than found in the field system.
Environmental conditions (lighting, air temperature, and minerals) can
be highly optimized in CEA systems for high yield and nutritional
quality, which would be beneficial both for farming operations and end
consumers. Future work should look at individual environmental pa­
rameters such as light intensity or spectrum for their ability to increase
E.L. Ashenafi et al. Environmental and Experimental Botany 199 (2022) 104895

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Hoffmann, W.A., Poorter, H., 2002. Avoiding bias in calculations of relative growth rate.
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interests or personal relationships that could have appeared to influence Chloroplast redox imbalance governs phenotypic plasticity: the “grand design of
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Acknowledgements
control environment through surrogate modeling. PLoS ONE 14 (4), 1–16. https://
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by the U.S. National Science Foundation (NSF) Innovations at the Nexus 2016. Improving photosynthesis and crop productivity by accelerating recovery
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