Irradiance levels affect growth parameters and carotenoid pigments in kale and spinach grown in a controlled environment


  • Edited by H. Usuda



Carotenoids play critical roles in both light harvesting and energy dissipation for the protection of photosynthetic structures. However, limited research is available on the impact of irradiance on the production of secondary plant compounds, such as carotenoid pigments. Kale (Brassica oleracea L.) and spinach (Spinacia oleracea L.) are two leafy vegetables high in lutein and β-carotene carotenoids. The objectives of this study were to determine the effects of different irradiance levels on tissue biomass, elemental nutrient concentrations, and lutein β-carotene and chlorophyll (chl) pigment accumulation in the leaves of kale and spinach. ‘Winterbor’ kale and ‘Melody’ spinach were grown in nutrient solution culture in growth chambers at average irradiance levels of 125, 200, 335, 460, and 620 μmol m−2 s−1. Highest tissue lutein β-carotene and chls occurred at 335 μmol m−2 s−1 for kale, and 200 μmol m−2 s−1 for spinach. The accumulations of lutein and β-carotene were significantly different among irradiance levels for kale, but were not significantly different for spinach. However, lutein and β-carotene accumulation was significant for spinach when computed on a dry mass basis. Identifying effects of irradiance on carotenoid accumulation in kale and spinach is important information for growers producing these crops for dry capsule supplements and fresh markets.

Abbreviations – 




chl a

chlorophyll a

chl b

chlorophyll b


dry mass


fresh mass


High Performance Liquid Chromatography




photosynthetically active radiation








Incoming solar radiation is required to provide energy for plant metabolic systems, however, light can be one of the most common environmental stresses. As light strikes the surface of plant leaves, photons are absorbed by antenna pigments which funnel this energy to the photosynthetic reaction centre. In the reaction centre, chlorophyll (chl), pheophytins, and quinones molecules convert light energy into chemical energy (Frank and Cogdell 1996). Carotenoids are bound to pigment–protein complexes within the thylakoid membranes and are utilized as antenna pigments. In higher plants, carotenoid distribution between the two photosystems is unevenly distributed, with pigments of photosystem I having high levels of β-carotene, while L makes up the majority of carotenoid pigments in photosystem II (Demmig-Adams et al. 1996). At high light levels, excess energy must be removed from the photosynthetic system to prevent damage. Carotenoid molecules are in close proximity to the chl molecules and quench the energetic triplet state of the chl molecule to prevent damage to the photosynthetic system (Miki 1991, Frank and Cogdell 1996, Taiz and Zeiger 1998, Tracewell et al. 2001).

Irradiance can affect mineral accumulation and plant secondary compounds, such as L and β-carotene (Havaux et al. 1998). Increases in photosynthesis will increase plant biomass and can result in a dilution effect on elemental concentrations (Mills and Jones 1996). One important class of secondary plant metabolites is the carotenoids. Carotenoids are C40 isoprenoid polyene compounds that form yellow, orange, and red lipid soluble pigments in higher plants, algae and bacteria. In shade leaves, the content of L and β-carotene is less than in sun leaves (Demmig-Adam et al. 1996). Similarly, summer-grown kale has higher L and β-carotene concentrations than kale grown during winter months, when light levels are reduced (Azevedo and Rodriguez-Amaya 2005).

Kale (Brassica oleracea L. var. acephala D.C) ranks highest, and spinach (Spinacia oleracea L.) ranks second among vegetable crops for L and β-carotene concentrations (Holden et al. 1999, USDA 2002). Kale is also an excellent source of Ca, Mg, and K (Mills and Jones 1996). However, kale has low consumption rates, with per capita fresh intake at less than 0.33 kg year−1 in the United States (Lucier and Plummer 2003). Spinach has one of the highest rates of consumption among green-leafy vegetables in the United States, with per capita intakes of 0.73, 0.09, and 0.36 kg year−1 for fresh, canned, and frozen markets, respectively (Lucier and Plummer 2003). Spinach is also high in Fe, Ca, Mg, and K (Zhang et al. 1989, Mills and Jones 1996, USDA 2002).

Lutein [(3R,3′R,6′R)-β,?-carotene-3,3′diol] and β-carotene (β,β-carotene) carotenoids possess important human health properties, but cannot be synthesized in mammals. Plants are a primary dietary source of carotenoids, and foods rich in L and β-carotene has been associated with reduced risk of lung cancer and chronic eye diseases, such as cataracts and age-related macular degeneration (Le Marchand et al. 1993, Ames et al. 1995, Landrum and Bone 2001, Semba and Dagnelie 2003)

The amount of light is critical for plant growth and development and can be modified by growing crops at different times of the year, or under various shade environments. What remains unclear, however, is the effect of irradiance on the production of carotenoid pigments. Therefore, the goal of this study was to investigate the effects of different irradiance levels on plant biomass, elemental concentrations, and accumulation of L, β-carotene, and chl pigments in kale and spinach.

Materials and methods

Plant culture

‘Winterbor’ kale and ‘Melody’ spinach (Johnny's Selected Seed, Winslow, ME) were seeded into rockwool growing cubes (Grodan A/S, Dk-2640, Hedehusene, Denmark) and germinated in a greenhouse (22°C day/14°C night) under natural lighting conditions (Lat. 43° 09′-N, Durham, NH). The three replications of kale were seeded on February 3, September 3 and October 27, 2003, and three replications of spinach were seeded on September 25 and December 20, 2002 and September 19, 2003. Peter's 20N-6.9P-16.6K water-soluble fertilizer (Scotts, Marysville, OH) was applied at a rate of 200 mg l−1 every 5 days. After 2 weeks for the kale and 3 weeks for the spinach, the plants were transferred to 38 l plastic containers (Rubbermaid Inc., Wooster, OH). Eight plants of one species were placed into 2-cm round holes set at 10.6 × 9.5 cm spacing on a container lid. Four containers were placed in a growth chamber (E15; Conviron, Winnipeg, Manitoba, Canada) with the air temperature set point for the experiment at 20°C. The plants were grown in 30 l of nutrient solution (Hoagland and Arnon 1950), with elemental concentrations of (mg l−1): N (105), P (15.3), K (117.3), Ca (80.2), Mg (24.6), S (32.0), Fe (0.5), B (0.25), Mo (0.005), Cu (0.01), Mn (0.25), and Zn (0.025). The electrical conductivity of the starting nutrient solution was 0.7 mS cm−1 and pH was measured at 5.6. Solutions were aerated with an air blower (25E133W222; Spencer, Winsor, CT) connected to air stones. Deionized water was added daily to maintain 30 l in each container and the complete nutrient solution was replaced every week throughout the experiment to refresh the solution to the initial nutrient concentrations.

Plants were grown under different irradiance levels within the growth chambers using both fluorescent and incandescent bulbs. Photosynthetically active radiation (PAR) was measured (QSO-ELEC; Apogee Instruments, Logan, UT) at six locations, without plants, on top of each container at the four corner plant holes and between the two side middle plant holes and averaged. Six containers were blocked together for an average irradiance treatment level (PAR) of 125 ± 30, 200 ± 50, 335 ± 60, 460 ± 40, and 620 ± 100 µmol m−2 s−1. The irradiance treatment daily integral was 7.2, 11.5, 19.3, 26.5, and 35.7 mol m−2. The daily photoperiod was16 h, this correlates to the maximum possible field photoperiod in the region where the experiment was located. Irradiance levels were measured at the beginning and confirmed at the end of each replication.

The kale plants were grown for 3 weeks, while the spinach plants were grown for 4 weeks for each experimental replication. At harvest, shoot and root tissues were separated and shoot tissue was weighted. A fully developed, non-shaded leaf from each of the eight plants was randomly selected and a 4 cm2 piece of the leaf was removed. This treatment sample was stored at −20°C prior to lyophilization. The remaining shoot material was dried at 60°C for 72 h, at which time shoot dry mass was determined.

Elemental composition

Dried plant tissues were ground in a sample mill grinder (1093; Cyclotec-Tector, Höganäs, Sweden) to pass a 0.5 mm screen. A 0.300 g tissue sample was mixed with 10.0 ml of 70% nitric acid (HNO3) and digested in a microwave accelerated reaction system (MARS5; CEM Corp., Matthews, NC). The digested solution was cooled to room temperature and deionized water was added to result in a final volume of 40.0 ml. Elemental analysis was determined by Inductively Coupled Argon Plasma – Atomic Emission Spectrometry (Vista AX; Varian, Inc., Palo Alto, CA).

Carotenoid and chlorophyll content – tissue extraction

Frozen kale and spinach samples were lyophilized for 72 h (6 l FreeZone, LabConCo, Kansas City, MO). The dried tissue samples were ground with dry ice in a kitchen grinder (Handy Chopper Plus, HC 3000; Household Products Inc, Shelton, CT). Samples were extracted and separated according to the method of Kopsell et al. (2004), which is based on the method of Khachik et al. (1986). A 0.100 g subsample was placed into a Potter–Elvehjem tissue grinder tube (Kontes, Vineland, NJ) and hydrated with 0.800 ml of deionized water. The sample was placed in a 40°C water bath for 20 min. After hydration, 0.800 ml of the internal standard, ethyl-β-8-apo-carotenoate (Sigma Chemical Co., St. Louis, MO) and 2.50 ml of THF stabilized with 25 p.p.m. BHT were added. The sample was homogenized in the tube with approximately 25 insertions with the tissue grinder pestle attached to a drill press (Craftsman 15 inch Drill Press; Sears, Roebuck and Co., Hoffman Estates, IL) set at 540 r.p.m. The sample tube was kept immersed in ice during the grinding process. The tube was placed into a clinical centrifuge for 3 min at 500 gn. The supernate was removed with a Pasteur pipet, placed into a conical 15 ml test tube, capped and held on ice. The sediment was re-suspended in 2.00 ml THF and homogenized with approximately 25 insertions of the grinding pestle. The tube was centrifuged for 3 min at 500 gn and the supernate was collected and combined with the first extracted supernate. The extraction procedure was repeated twice more until the supernate was colourless. The sediment was discarded and the combined four supernates were placed in a 40°C water bath and reduced to 0.50 ml using nitrogen gas (N-EVAP 111; Organomatic Inc., Berlin, MA). Added to the 0.50 ml reduced sample was 2.50 ml of MeOH and 2.00 ml of THF, and then the combined sample solution was vortexed. The sample was filtered through a 0.2 μm PTFE filter (Econofilter PTFE 25/20; Agilent Technologies, Wilmington, DE) using a 5 ml syringe (Becton, Dickinson and Company, Franklin Lakes, NJ).

Carotenoid and chlorophyll content – HPLC analysis

A HPLC unit with photo diode array detector (Agilent 1100; Agilent Technologies, Palo Alto, CA) was used for pigment separation. All samples were analysed for carotenoid compounds using a Vydac RP-C18 5.0 µm 250 × 4.6 mm column (201TP54; Phenomenex, Torrance, CA) fitted with a 4 × 3.0 mm, 7.0 µm guard column compartment. The column was maintained at 16°C using a thermostatic column compartment. Eluents were A: 75% acetronitrile, 20% methanol, 5% hexane, 0.05% BHT, and 0.013% triethyamine (TEA) (v/v) and B: 50% acetonitrile, 25% THF, 25% hexane and 0.013% TEA (v/v). The flow rate was 0.70 ml min−1 and the gradient was 100% eluent A for 30 min; 50% A and 50% B for 2 min; 100% B for 2 min; and 50% A and 50% B for 2 min. The eluent was returned to 100% A for 10 min to re-equlibrate the column prior to the next injection. Eluted compounds from a 20.0 µl injection were detected at 452 nm (carotenoids and internal standard), 652 nm for chl a, and 665 nm for chl b, with data collected and integrated using 1100 HPLC ChemStation Software (Agilent Technologies, Palo Alto, CA). Peak assignment was performed by comparing retention times and absorption spectra obtained from the photodiode array detection of authentic standards (lutein from Carotenature, Lupsingen, Switzerland; β-carotene, chl a, and chl b from Sigma Chemical Co.). Recovery rates of ethyl-β-apo-carotenoate during extraction were above 90%.

Statistical analysis

Data were analysed according to a one-way anova using SAS (Cary, NC). The anova determined the significance of the main effects of the irradiance treatments. LSD was used to determination post hoc multiple comparison tests. The relationship between experimental dependent variables and irradiance treatments were determined by regression analysis using SAS.


Tissue biomass accumulation

Shoot tissue FM was significantly influenced by irradiance treatment for both ‘Winterbor’ kale (F = 26.5, P = 0.001) and ‘Melody’ spinach (F = 14.4, P = 0.001). Average kale FM increased from 15.3 to 74.3 g per plant as irradiance treatment increased from 125 to 620 µmol m−2 s−1 (Table 1). Kale FM responded linearly to increases in irradiance treatment levels [FM = 8.8 + 0.1 (PAR), r2 = 0.75]. Spinach FM increased from 59.2 to 174.1 g per plant as the irradiance increased from 125 to 620 mol m−2 s−1 (Table 1). A linear increase in FM was also found for spinach [FM = 25.5 + 0.3 (PAR), r2 = 0.55] as the irradiance levels increased.

Table 1.  Mean fresh mass (FM) and dry mass (DM) production in leaf tissues of ‘Winterbor’ kale (Brassica oleracea L.) and ‘Melody’ spinach (Spinacia oleracea L.) grown under increasing irradiance levels in nutrient solution culture. Mean composition of sampled leaf tissue of six replications, eight plants each ±sd. anova results: ***significance at P ≤ 0.001 level.
 Biomass (g)
Irradiance (µmol m−2 s−1)FMDM
Winterbor kale
 12515.3 ± 1.21.2 ± 0.1
 20034.6 ± 6.02.5 ± 0.4
 33554.4 ± 4.14.3 ± 0.4
 46062.5 ± 2.95.4 ± 0.4
 62074.3 ± 6.46.9 ± 0.7
Melody spinach
 12559.2 ± 10.12.5 ± 0.3
 200121.2 ± 13.44.9 ± 0.3
 335122.3 ± 13.35.5 ± 0.6
 460148.2 ± 6.75.7 ± 0.3
 620174.1 ± 11.56.5 ± 0.8

Shoot tissue DM was significantly influenced by irradiance treatment for both ‘Winterbor’ kale (F = 26.6, P = 0.001) and ‘Melody’ spinach (F = 9.9, P = 0.001). Average kale DM increased from 1.2 to 6.9 g per plant as irradiance treatment increased from 125 to 620 µmol m−2 s−1 (Table 1). Kale DM responded linearly to increasing irradiance treatment levels [DM = 0.12 + 0.01 (PAR), r2 = 0.80]. Spinach increased in DM from 2.5 to 6.5 g plant−1 as the irradiance increased from 125 to 620 µmol m−2 s−1 (Table 1). A significant linear increase in DM was found for the spinach [DM = 2.67 + 0.01 (PAR), r2 = 0.46] as the irradiance levels increased.

Macro- and micronutrient accumulation

Changes in irradiance levels accounted for small but significant variation for elemental accumulation in kale. Those mineral elements affected were: P (F = 3.2, P = 0.030), K (F = 3.1, P = 0.035), Ca (F = 2.9, P = 0.041), Cu (F = 5.4, P = 0.003), and Mn (F = 3.5, P = 0.022; Tables 2 and 3). Phosphorous in the leaf tissues of kale increased, then decreased [P = 0.79 + 0.001 (PAR) – 0.000001 (PAR)2, r2 = 0.30] in response to increased irradiance. Kale leaf tissue K decreased [K = 5.0 − 0.002 (PAR), r2 = 0.32] in response to increases in irradiance levels. Calcium accumulation in kale followed a linear decrease [Ca = 4.7 − 0.001 (PAR), r2 = 0.23] due to increasing irradiance level. Kale Cu accumulation also followed a linear decrease [Cu = 7.0 − 0.01 (PAR), r2 = 0.43] as irradiance levels increased. Manganese accumulation in kale decreased linearly [Mn = 1.7 − 0.001 (PAR), r2 = 0.34] with increasing irradiance levels. Spinach leaf tissue Ca (F = 2.9, P = 0.043) and Fe (F = 3.8, P = 0.014) accumulation were both significantly affected by irradiance level (Tables 2,3). Spinach Ca followed a quadratic [Ca = 1.11 − 0.0005 (PAR) + 0.000002 (PAR)2, r2 = 0.21] trend due to increasing irradiance levels. Spinach Fe decreased, then increased [Fe = 440.8 − 1.71 (PAR) + 0.003 (PAR)2, r2 = 0.24] in response to increasing irradiance levels.

Table 2.  Mean macronutrient accumulation in the leaf tissues of ‘Winterbor’ kale (Brassica oleracea L.) and ‘Melody’ spinach (Spinacia oleracea L.) grown under increasing irradiance levels in nutrient solution culture. Mean composition of sampled leaf tissue of six replications, eight plants each ±sd. anova results: ns, *, non-significant or significance at P ≤ 0.05, level, respectively.
 Macronutrients (% DM)
Irradiance (µmol m−2 s−1)PKCaMgS
Winterbor kale
 1250.95 ± 0.034.85 ± 0.194.62 ± 0.180.73 ± 0.020.72 ± 0.03
 2000.97 ± 0.044.66 ± 0.104.28 ± 0.120.72 ± 0.020.68 ± 0.02
 3351.12 ± 0.044.51 ± 0.144.36 ± 0.100.75 ± 0.020.69 ± 0.01
 4601.09 ± 0.054.40 ± 0.234.33 ± 0.120.77 ± 0.030.70 ± 0.02
 6201.09 ± 0.054.02 ± 0.203.88 ± 0.210.72 ± 0.020.72 ± 0.03
Melody spinach
 1251.83 ± 0.0712.3 ± 0.41.02 ± 0.071.15 ± 0.080.27 ± 0.01
 2001.96 ± 0.1310.2 ± 0.41.21 ± 0.161.36 ± 0.160.40 ± 0.06
 3351.74 ± 0.1310.9 ± 0.80.98 ± 0.031.17 ± 0.060.29 ± 0.03
 4601.77 ± 0.1310.2 ± 0.21.31 ± 0.081.23 ± 0.040.34 ± 0.03
 6201.86 ± 0.1410.3 ± 0.91.42 ± 0.151.26 ± 0.050.33 ± 0.03
Table 3.  Mean micronutrient accumulation in the leaf tissues of ‘Winterbor’ kale (Brassica oleracea L.) and ‘Melody’ spinach (Spinacia oleracea L.) grown under increasing irradiance levels in nutrient solution culture. Mean composition of sampled leaf tissue of six replications, eight plants each ±sd. anova results: ns, *, **, non-significant or significance at P ≤ 0.05, 0.01 level, respectively.
 Micronutrients (mg kg−1 DM)
Irradiance (µmol m−2 s−1)BCuFeMnMoZn
Winterbor kale
 12535.8 ± 1.75.91 ± 0.6384.4 ± 11.6161.7 ± 4.80.73 ± 0.0765.5 ± 21.7
 20037.3 ± 2.46.08 ± 0.9380.1 ± 26.5150.9 ± 9.60.57 ± 0.1287.5 ± 18.1
 33537.2 ± 2.34.08 ± 0.4256.1 ± 10.2150.3 ± 4.90.63 ± 0.1356.5 ± 19.2
 46040.6 ± 1.93.29 ± 0.4862.1 ± 6.8130.6 ± 9.60.54 ± 0.1038.0 ± 3.0
 62038.9 ± 2.52.69 ± 0.6957.3 ± 11.8120.3 ± 13.20.67 ± 0.1251.6 ± 4.2
Melody spinach
 12531.5 ± 1.816.7 ± 5.5187.4 ± 16.1330.8 ± 41.80.57 ± 0.08246.9 ± 29.1
 20043.9 ± 6.125.5 ± 6.8349.7 ± 88.5317.9 ± 35.70.54 ± 0.2381.2 ± 24.7
 33531.2 ± 2.39.4 ± 3.6153.7 ± 29.9288.8 ± 18.10.55 ± 0.1395.3 ± 16.4
 46036.9 ± 3.017.7 ± 6.1205.9 ± 43.1337.9 ± 24.60.61 ± 0.05130.8 ± 47.5
 62034.9 ± 2.519.7 ± 5.9489.4 ± 121.7361.6 ± 47.10.59 ± 0.14178.5 ± 68.5

Carotenoid and chlorophyll pigment accumulation

A small but significant amount of variation for kale leaf tissue L concentrations (F = 3.08, P = 0.03) resulted from changes in irradiance levels. Kale leaf tissue L accumulation ranged from 9.1 mg 100 g−1 at 125 µmol m−2 s−1, to as high as 15.1 mg 100 g−1 at 335 µmol m−2 s−1 (Table 4). The trend in kale leaf tissue L accumulation was an increasing then decreasing quadratic trend [L = 5.36 + 0.04 (PAR) – 0.00005 (PAR)2, r2 = 0.21] in response to increasing irradiance levels. Similarly, β-carotene accumulation in the kale leaf tissues responded significantly to irradiance treatments (F = 4.77, P = 0.005). Kale leaf tissue β-carotene accumulation ranged from 5.7 mg 100 g−1 at 125 µmol m−2 s−1, to as high as 11.1 mg 100 g−1 at 335 µmol m−2 s−1 (Table 4). Kale leaf tissue β-carotene increased, then decreased [β-carotene = 2.29 + 0.036 (PAR) – 0.00004 (PAR)2, r2 = 0.30] in response to irradiance treatments. Overall, there tended to be a dilution of fresh mass carotenoid concentrations in both kale and spinach with increasing total fresh mass production.

Table 4.  Mean pigment accumulation as expressed on a fresh mass (FM) basis in the leaf tissues of ‘Winterbor’ kale (Brassica oleracea L.) and ‘Melody’ spinach (Spinacia oleracea L.) grown under increasing irradiance levels in nutrient solution culture. Mean composition of sampled leaf tissue of six replications, eight plants each ±sd. anova results: ns, *, **, non-significant or significance at P ≤ 0.05, 0.01 level, respectively. Post hoc LSD test for multiple comparison: a, b.
 Pigment concentration (mg 100 g−1 FM)
Irradiance (µmol m−2 s−1)Luteinβ-caroteneChl aChl b
Winterbor kale
 1259.1 ± 1.4 a5.7 ± 0.9 a145.0 ± 23.1 a35.1 ± 6.4 a
 20012.0 ± 1.6 ab8.1 ± 1.1 ab184.4 ± 26.0 ab47.2 ± 6.7 ab
 33515.1 ± 1.4 ab11.1 ± 1.2 ab247.3 ± 10.0 ab59.0 ± 2.2 b
 46012.0 ± 0.6 ab8.6 ± 0.5 ab211.4 ± 13.0 ab55.1 ± 1.5 b
 62012.7 ± 0.5 b9.3 ± 0.4 b216.3 ± 18.2 b55.2 ± 4.2 b
Melody spinach
 1257.9 ± 0.76.4 ± 0.6127.2 ± 10.834.1 ± 2.6
 20011.1 ± 1.59.2 ± 1.3179.2 ± 24.746.1 ± 6.1
 3358.7 ± 0.67.2 ± 0.6143.1 ± 11.535.7 ± 3.0
 4608.7 ± 1.27.4 ± 1.1142.7 ± 19.832.5 ± 4.4
 6207.1 ± 0.96.2 ± 0.9119.2 ± 17.129.1 ± 3.7

Chlorophyll pigments concentrations in the leaf tissues of kale and spinach were much higher when compared to carotenoid concentrations. Concentrations of chl a (F = 4.1, P = 0.011) and chl b (F = 4.1, P = 0.011) pigments in kale leaf tissues differed among irradiance treatments. Similar to the carotenoid pigments, maximum chl pigment accumulation in kale leaf tissues occurred at 335 µmol m−2 s−1, with chl a at 247.3 mg 100 g−1 and chl b at 59.0 mg 100 g−1 (Table 4). Kale leaf tissue chl a and chl b accumulation both followed a quadratic trend [chl a = 68.3 + 0.75 (PAR) – 0.0008 (PAR)2, r2 = 0.32; chl b = 16.6 + 0.19 (PAR) – 0.0002 (PAR)2, r2 = 0.36] in response to increases in irradiance levels.

The largest accumulation of carotenoid and chl pigments in spinach leaf tissues occurred at the irradiance level of 200 µmol m−2 s−1, with L levels at 11.1 mg 100 g−1, β-carotene levels at 9.2 mg 100 g−1, chl a at 179.2 mg 100 g−1, and chl b at 46.1 mg 100 g−1 (Table 4). However, spinach leaf tissue carotenoid and chl FM concentrations were not statistically affected by irradiance treatment.

Leaf tissue percentage DM was influenced by irradiance levels for both kale (F = 8.1, P ≤ 0.001) and spinach (F = 4.3, P = 0.009). Average kale percentage DM increased from 9.7 to 15.1% as irradiance levels increased from 125 to 620 µmol m−2 s−1 (Table 5). Spinach leaf tissue percentage DM increased from 6.8 to 7.7% as the irradiance increased from 125 to 620 µmol m−2 s−1 (Table 5). The trend of kale leaf tissue percentage DM followed a quadratic trend [% DM = 7.7 + 0.0001 (PAR) – 0.000004 (PAR)2, r2 = 0.54] in response to increasing irradiance levels. No trend was reported for spinach.

Table 5.  Mean percentage dry mass (DM) and pigment concentration as expressed on a dry mass basis in the leaf tissues of ‘Winterbor’ kale (Brassica oleracea L.) and ‘Melody’ spinach (Spinacia oleracea L.) grown under increasing irradiance levels in nutrient solution culture. Mean composition of sampled leaf tissue of six replications, eight plants each ±sd. anova results: ns, *, **, ***, non-significant or significance at P ≤ 0.05, 0.01, 0.001 level, respectively. Post hoc LSD test for multiple comparison: a, b, and c.
  Concentration (mg g−1 DM)
Irradiance (µmol m−2 s−1)% DMLuteinβ-carotene
Winterbor kale
 1259.7 ± 0.3 a0.93 ± 0.130.56 ± 0.08
 2009.7 ± 1.0 a1.20 ± 0.080.81 ± 0.06
 33512.7 ± 0.7 a1.19 ± 0.110.88 ± 0.09
 46013.1 ± 0.7 ab0.94 ± 0.090.67 ± 0.08
 62015.1 ± 1.1 b0.86 ± 0.050.63 ± 0.05
Melody spinach
 1256.8 ± 0.5 a1.17 ± 0.09 a0.94 ± 0.06 a
 2007.7 ± 0.6 ab1.41 ± 0.1 ab1.17 ± 0.09 ab
 3357.4 ± 0.6 ab1.19 ± 0.04 abc0.98 ± 0.04 ab
 4607.5 ± 0.6 b1.14 ± 0.09 bc0.96 ± 0.08 a
 6207.7 ± 0.4 b0.91 ± 0.08 c0.79 ± 0.08 ab

The concentration of spinach leaf tissue L (F = 4.6, P = 0.007) and β-carotene (F = 3.4, P = 0.023) measure on a DM basis responded to increases in irradiance levels (Table 5). Spinach L DM accumulation ranged from 0.9 mg g−1 at 620 µmol m−2 s−1, to as high as 1.41 mg g−1 at 200 µmol m−2 s−1. Spinach β-carotene DM accumulation ranged from 0.79 mg g−1 at 620 µmol m−2 s−1, to as high as 1.17 mg g−1 at 200 µmol m−2 s−1. The trend in L accumulation in the spinach as a function of dry mass was quadratic [L DM = 1.13 + 0.001 (PAR) – 0.00001 (PAR)2, r2 = 0.33] in response to increasing irradiance levels. The trend in spinach β-carotene DM accumulation was also quadratic [β-carotene DM = 0.90 + 0.001 (PAR) – 0.00001 (PAR)2, r2 = 0.23] in response to increasing irradiance levels.


Irradiance level directly influences the photosynthetic rate of plants, resulting in increased production of carbohydrates and total biomass (Mills and Jones 1996, Taiz and Zeiger 1998). Results from the current study showed linear increases in both FM and DM as the light irradiance levels increased from 125 to 620 µmol m−2 s−1. Research by Weng (1992) also showed a linear increase in FM for B. oleracea as the irradiance level was increased to 750 µmol m−2 s−1. However, as light levels increase, the photosynthetic efficiency decreases when a light saturation point is reached. The level of the light saturation point can be affected by a number of factors including plant nutrition, species, variety, and specific genetic factors (Weng 1992). The light saturation point for members of B. oleracea is estimated to be between 750 and 1000 µmol m−2 s−1, but has been reported to be as high as 1600 µmol m−2 s−1 for one variety of rapeseed (Weng 1992). The light saturation point can explain why both FM and DM for spinach and kale increased steadily and then levelled as irradiance treatments increased in the current study. From regression analysis of the data the potential light saturation point based on FM would be 650 and 775 µmol m−2 s−1 for the kale and spinach, respectively.

Irradiance levels can indirectly affect elemental concentrations in plants by first impacting enzymatic activity and photosynthetic rates. This increases the amount of FM produced by the plant and results in a dilution effect for elemental concentrations (Mills and Jones 1996). Calcium concentrations can increase as a response to lower K concentration at low irradiance levels (Mills and Jones 1996). A study using soybean (Glycine max L. Merr.) revealed that low light resulted in increases in P, K, Cu, Fe, and Mn, however, the form of N can affect which minerals increase at low light levels (Mills and Jones 1996). In our study, the kale concentrations of Ca, Cu, K and Mn all increased at low irradiance while the P concentrations decreased. The spinach mineral concentrations were not significantly different for most irradiance levels, but both Ca and Fe decreased at low light levels. The specific reason for the decrease of P in kale and decrease of Ca in spinach is not known.

Behera and Choudhury (2003) reported increases in wheat (Triticum sativum L.) L (858–1211 µg g−1) and β-carotene (22–92 µg g−1) concentrations as irradiance levels increased from 70 to 210 µmol m−2 s−1. Chlorophyll pigments also responded to changes in irradiance levels, with chl a increasing from 1694 to 2403 µg g−1 and chl b increasing 480–537 µg g−1 for the studied light levels, respectively. When the plants were exposed to 1250 µmol m−2 s−1 of light, the pigment concentrations decreased. Behera and Choudhury (2003) results are very similar to our results, where the carotenoid and chl concentration increased linearly for kale from 125 to 300 µmol m−2 s−1 and for spinach from 125 to 200 µmol m−2 s−1. At irradiance levels above 300 mol m−2 s−1 for the kale and 200 µmol m−2 s−1 for the spinach, the carotenoid and chl levels start to decrease and remain fairly constant after 400 µmol m−2 s−1 for both species. The decrease in carotenoid and chl concentrations could be due to a combined effect of photodegradation of the pigment molecules and dilution of the concentration as the plant grows. The moisture content of the plants changed significantly as the irradiance increased. The kale percentage DM increased from 9.7 to 15.0% and spinach increased from 6.8 to 7.7%. The larger change in percentage DM for kale followed an increasing linear trend while spinach had no change after removing the lowest irradiance level. This change in percentage DM may result in dilution of the carotenoids, which could explain the reason for the significant effect irradiance had on kale but not spinach.

Results from the current study suggest that the irradiance level has no impact on the carotenoid concentrations in spinach expressed on a FM basis. Most spinach is consumed fresh (Lucier and Plummer 2003) and changes in water content in the plant tissue using traditional agriculture based farming practices for growing, handling, storage, or food processing, can results in changes in the nutritional value of the plant (Ezell and Wilcox 1959, Gil et al. 1999). Based on USDA procedures for reporting food carotenoid content on a FM basis, there were no significant differences in L or β-carotene accumulations for the spinach over the irradiance levels tested. However, with the increased interest in dietary antioxidant supplements, such as dried spinach capsules, warrants investigations into DM pigment concentrations (Lefsrud et al. 2005). When spinach carotenoid data are reported on a DM basis, there were significant decreases in both L and β-carotene concentrations. Thus, reporting L and β-carotene carotenoids on both a FM and DM basis may be a more accurate way to express the nutritional value of spinach.

Previous research shows that modification of the growing environment air temperature can influence carotenoid accumulation in both kale and spinach (Lefsrud et al. 2005). Environmental modification of irradiance levels of these cool-season crops resulted in changes in fresh biomass production, and the accumulation of L, β-carotene, and chl b pigments. Carotenoid concentrations in the leaves of kale were maximized at 335 µmol m−2 s−1, while spinach carotenoids concentrations were highest at 200 µmol m−2 s−1. In many parts of the United States, cool-season crops such as kale and spinach are grown as both spring and fall crops. Average field irradiance levels can vary dependent on location, time of year, shading, and atmospheric conditions. Therefore, the influence of irradiance levels on kale and spinach carotenoid concentrations should be considered when selecting appropriate growing conditions for these cool-season crops. Changes in carotenoid concentrations would be expected to influence the nutritional value of kale and spinach.

Acknowledgements –  This paper was funded in part by a grant received by the Cooperative State Research, Education, and Extension Service, U.S. Department Agr., under Agreement no. 2001-52102-11254. The authors also wish to thank Laura Dukach for her technical support during this research.