Light intensity and photoperiod: Tools for improving the phytonutrient profile of Brassica rapa ssp. nipposinica for supplementing the space diet

Light intensity and photoperiod: Tools for improving the phytonutrient profile of Brassica rapa ssp. nipposinica for supplementing the space diet | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Light intensity and photoperiod: Tools for improving the phytonutrient profile of Brassica rapa ssp. nipposinica for supplementing the space diet Ethan Darby, Sarah Armstrong, Gioia Massa, Kellie J. Walters This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4338874/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Beyond mere caloric intake, the nutritional quality of food will be key to maintaining astronaut health during exploration-length missions. The production of leafy greens aboard spacecraft can help to provide consistent daily nutrition; however, maximizing the nutritional yield of each plant will be key to sustainable and efficient dietary supplementation. Brasssica rapa ssp. nipposinica ‘Red Hybrid’ was grown under environmental conditions similar to those of the International Space Station and the effect of light intensity and photoperiod on nutritional and biomass yields were evaluated. Four light intensities (200, 400, 600, and 800 µmol·m − 2 ·s − 1 ) applied over a 16- or 24-hr photoperiod were implemented and the resulting concentrations of ascorbic acid, thiamine, phylloquinone, β-carotene, lutein, zeaxanthin, total anthocyanins, calcium, potassium, magnesium, and iron were quantified. Providing 800 µmol·m − 2 ·s − 1 over a 16-hr photoperiod produced the best nutritional profile for supplementing the astronaut diet, offering the following percentages of recommended daily intake per 75 g serving: 100% phylloquinone, 63% ascorbic acid, 2.7% thiamine, and 97% retinol (from precursor β-carotene). However, when evaluated for light use efficiency (yield·MJ − 1 ), the 200 µmol·m − 2 ·s − 1 treatments (16- and 24-hr) performed better, yielding more mass and phytonutrients per MJ of energy utilized. Astronaut mizuna anthocyanins thiamine phylloquinone carotenoids Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The production of fresh vegetables aboard spacecraft has been identified as a key component of successful exploration-length space ventures [ 1 ]. This is due to a need for fresh sources of vitamin C (ascorbic acid), vitamin B 1 (thiamine), and dietary carotenoids (β-carotene, lutein, zeaxanthin), as well as supplemental sources of vitamin K 1 (phylloquinone), potassium, magnesium, calcium, and antioxidants such as anthocyanins [ 2 ]. However, growing fresh produce in space presents many challenges, some of which include limited space aboard craft, limited energy resources, and environmental constraints such as elevated CO 2 concentrations and microgravity [ 3 , 4 ]. Leafy greens are promising candidates for production aboard space craft, as they are more energy- and space-efficient than fruit or root crops. They also provide an advantage over dried supplements, such as pills, as accidental toxicities are harder to incur [ 5 ]. Prior evaluation of leafy greens has shown species within the genus Brassica to be particularly promising from a biomass, organoleptic, and phytonutrient perspective [ 6 ]. More recent research has confirmed that Brassica rapa spp. nipposinica (mizuna) contains high concentrations of desired phytonutrients while still performing well when grown under conditions similar to those of the International Space Station (ISS) [ 7 ]. Additionally, plant tissue concentrations of many phytonutrients respond to environmental stimuli [ 8 , 9 , 10 ], and environmental manipulation may provide methods to increase concentrations of desirable compounds while maintaining or even increasing biomass yield. This is especially true of light, as compounds such as ascorbic acid [ 11 ], secondary carotenoids [ 12 ], and anthocyanins [ 13 ] are involved in light-induced free-radical scavenging and absorption of excess light, while phylloquinone [ 14 ] and primary carotenoids [ 15 ] are directly involved in the photosynthetic machinery. Thus, environmental control of light intensity and photoperiod is a potential tool to improve the quality of leafy greens for consumption as part of the space diet. Light is also used more efficiently by plants at a lower intensity, meaning it can be more energy efficient to apply a lower intensity of light over a longer period of the day to achieve the same total photon exposure, or DLI (Daily Light Integral) [ 16 ]. This study aims to determine which combination of light intensity and photoperiod produces Brassica rapa spp. nipposinica ‘Red Hybrid’ mizuna with the highest phytonutrient concentrations when produced under an environment similar to that of the ISS, while still maintaining a compact morphology and plant vigor. Materials and Methods Experimental Design The experiment was organized in a factorial design with 9 plants grown under each combination of light intensity (4) and photoperiod (2) for a total of 8 treatments. The experiment was conducted twice over time. At harvest, morphological data were collected on all 9 plants per replication. Plant-specific phytonutrient and mineral data were collected on all 9 plants per replication, except for plants grown at a 200 µmol·m − 2 ·s − 1 light intensity and a 16-hr photoperiod which did not provide enough tissue per plant for mineral nutrient extractions. Tissue from multiple plants was pooled to produce mineral concentration data from this treatment. Plant Production Seeds of Brassica rapa spp. nipposinica ‘Red Hybrid’ mizuna (Kitazawa Seed, Oakland, CA) were sown in 162-cell bound coco-peat cubes (Preforma plugs; Jiffy Growing Solutions, Lorain, OH) and placed in a walk-in growth chamber (TC2, Environmental Grow Chambers; Chagrin Falls, OH). Plant production was similar to Darby et al. [ 7 ] with treatment and other select differences. The temperature was 22.7 ± 0.3° C, and the relative humidity was 61.6 ± 7.3%, both measured and logged every 10 minutes by a shielded temperature and relative humidity sensor (Hobo MX2300; OnSet, Bourne, MA). Seedling trays were placed under broad-spectrum light-emitting diodes (Scorpion Diablo, Horticulture Lighting Group; Maynardville, TN) providing 21:36:43 blue:green:red (%) radiation ratios, with a red:far-red ratio of 13:1, and one of four light intensities applied at one of two photoperiods for a total of eight treatments (Table 1 ). Light intensity was monitored by quantum sensors (SQ-500S; Apogee, Logan, UT) for the duration of the experiment. Leaf surface temperature was monitored with ultra-narrow field of view infrared radiometers (SI-131-SS; Apogee, Logan, UT), averaging 21.8 ± 1.1° C across all treatments (Table 1 ). CO 2 (Industrial Grade Carbon Dioxide CD 50S; Airgas, Knoxville, TN) was injected to provide 2,770 ± 180 µmol∙mol − 1 . Dosing was monitored and controlled by an infrared gas analyzer (LI-850; LI-COR Biosciences, Lincoln, NE) connected to a relay switch controlling an electronic regulator. CO 2 concentration was reported to a Raspberry Pi (Raspberry Pi 4 B; Raspberry Pi, Cambridge, England) every 20 seconds where it was recorded in a CSV file. Seeds were irrigated daily with reverse-osmosis water supplemented with 12N-1.8P-13.4K water-soluble fertilizer, providing (mg⋅L − 1 ) 100 nitrogen, 15 phosphorus, 112 potassium, 58 calcium, 17 magnesium, 2 sulfur, 1.4 iron, 0.5 zinc, 0.4 copper and manganese, and 0.1 boron and molybdenum, (RO Hydro FeED; JR Peters, Inc., Allentown, PA) and magnesium sulfate (MgSO 4 ) to provide (mg⋅L − 1 ) 15 magnesium and 20 sulfur. The pH was adjusted to 5.8 with H 2 SO 4 or KCHO 3 . Table 1 Lighting treatments, their intensity, photoperiod, and daily light integral (DLI), as provided to the initial growing surface. Target Light Intensity Actual Light Intensity Photoperiod Actual Daily Light Integral Leaf Temperature (µmol⋅m − 2 ⋅s − 1 ) (µmol⋅m − 2 ⋅s − 1 ) (hrs) (mol⋅m − 2 ⋅d − 1 ) (° C) 200 202 ± 10 16 11.6 ± 0.6 21.6 ± 0.8 400 391 ± 24 16 22.5 ± 1.4 21.4 ± 1.0 600 583 ± 40 16 33.6 ± 2.3 21.9 ± 1.2 800 780 ± 30 16 44.9 ± 1.7 22.1 ± 1.5 200 203 ± 11 24 17.5 ± 1.0 21.5 ± 0.6 400 396 ± 16 24 34.2 ± 1.4 22.1 ± 0.9 600 603 ± 25 24 52.1 ± 2.2 22.0 ± 1.0 800 782 ± 38 24 67.6 ± 3.3 22.4 ± 1.1 Similar to Darby et al. [ 7 ], after thirteen days the seedlings were transplanted into 18-cm-deep by 61-cm-wide by 122-cm-long deep-water culture hydroponic systems (Premium Flood Table, Active Aqua; Petaluma, CA) with 61 by 122 cm sealed-surface foam rafts (36 ct lettuce raft; Beaver Plastics, Acheson, AB, Canada) and grown for 9 days. Plants were harvested 22 days after sowing to prevent over-crowding of the growing space. The environmental conditions after transplant were the same as the seedling stage. Eight systems were filled with 108 L of reverse-osmosis water and supplemented with 12N-1.8P-13.4K water-soluble fertilizer (RO Hydro FeED; JR Peters, Inc.) and MgSO 4 providing twice the concentrations reported during propagation. Electrical conductivity (EC) and pH were monitored (HI9813-6N Portable Waterproof pH/EC/TDS Meter; Hanna Instruments, Woonsocket, RI) and pH was adjusted to 5.8 using H 2 SO 4 or KCHO 3 . Air pumps (Active Aqua 110 L⋅min − 1 commercial air pump; Hydrofarm, Petaluma, CA) and air stones (Active Aqua air stone round 10 cm × 2.5 cm; Hydrofarm, Petaluma, CA) were used to provide dissolved oxygen to the nutrient solution. Harvest, Morphological Data Collection, and Tissue Processing Plants were harvested at the substrate surface and morphological data were collected [ 7 ]. Height from the substrate surface to the tip of the tallest leaf, width at the widest point and perpendicular to the widest point, the number of fully expanded leaves, and fresh mass were collected for 9 plants per replication. After weighing, fresh tissue of 9 plants per treatment was immediately placed in a plastic sample bag and flash frozen in liquid N. Tissue was then stored in a -80°C freezer, freeze dried, and homogenized in liquid N. Once homogenized, the tissue was divided into separate aliquots for future extraction procedures and stored in individual 15 mL centrifuge tubes in the − 80°C freezer. Anthocyanin Isolation and Quantification Total anthocyanins were extracted and analyzed using a method derived from Islam et al. [ 17 ] and previously used in Darby et al. [ 7 ]. Briefly, 50 mg of ground tissue was used per sample, and the procedure occurred under red light. Samples were saturated with 5 mL of 95% ethanol/1.5 N HCl (85:15, v:v), placed on an orbital shaker for 15 minutes, and stored at 4°C for 24 hrs. Samples were then filtered and analyzed with a microplate reader (Agilent Technologies, Santa Clara, CA). Carotenoid Isolation and Quantification The carotenoids β-carotene, lutein, and zeaxanthin were extracted and analyzed using a method derived from Kopsell et al. [ 18 ] and previously described by Darby et al. [ 7 ]. Under red light, 50 mg samples of chilled tissue were extracted with purified water and tetrahydrofuran, and an internal carotenoid standard was used to quantify sample loss during homogenization. Samples were homogenized and tetrahydrofuran was used for a second extraction followed by additional homogenization. The resulting solution was centrifuged, and the eluent was dried down on a stream evaporator. After the addition of acetone, samples were filtered, and an aliquot was stored for subsequent identification and quantification on a 1200 Agilent Series HPLC unit equipped with a diode array detector. A reverse phase C30 column was used with an isocratic mobile phase composed of methyl tert-butyl ether, methanol, and triethylamine (11%, 88.99%, and 0.01%, v/v/v). Fat Soluble Vitamin Isolation and Quantification The fat-soluble vitamin phylloquinone (vitamin K 1 ) was extracted using a method derived from Pokkanta et al. [ 19 ] and previously used in Darby et al. [ 7 ]. Extractions were performed under red light (peak wavelength: 654 nm) and samples were saturated with a series of three solvents (methanol, dichloromethane, and hexane). After being saturated with each solvent, the samples underwent orbital shaking, sonication, and were finally centrifuged. After removing the supernatant, the next solvent in the series was added and the samples again underwent the three previously mentioned procedures. An internal standard was added to the pooled supernatant, and the samples were dried completely, suspended in 5 mL dichloromethane, and filtered. A 1-mL aliquot was then collected for analysis with a 1200 Agilent Series HPLC equipped with a diode array detector, using a reverse phase C18 column and a mobile gradient composed of methanol and water. Phylloquinone was identified at 248 nm. Mineral Isolation and Quantification Nitrogen concentration was quantified via combustion of 0.25 g of tissue and detection with a Thermal Conductivity Sensor (vario MAX cube; Elementar, Ronkonkoma, NY). Other minerals, Ca 2+ , K + , Mg 2+ , and Fe 3+ were extracted and quantified using a method derived from Jones [ 20 ]. A 0.5 g aliquot of tissue was dry ashed in a furnace at 500°C, and then wet ashed by the addition of dilute acid. The resulting solution was then analyzed on an ICP-OES to determine individual mineral concentrations (SPECTROBLUE; Spectro Analytical Instruments Inc., Kleve, Germany). Due to limited tissue production under the 200 µmol⋅m − 2 ⋅s − 1 by 16-hr photoperiod, mineral analysis on this treatment was performed on pooled samples (with two plants contributing tissue to each sample). Water Soluble Vitamin Isolation and Quantification The water-soluble vitamins (WSVs) ascorbic acid (vitamin C) and thiamine (vitamin B 1 ) were extracted using a method derived from Seal and Chaudhuri [ 21 ] and Sun et al. [ 22 ], and previously used in Darby et al. [ 7 ]. Under red light, chilled samples of 200 mg were hydrated with purified water, hydrochloric acid, and a phosphate buffer. Samples were vortexed and supernatant was filtered. The process was repeated, and a 1 mL aliquot was collected for analysis on a 1200 Agilent Series HPLC equipped with a diode array detector. A reverse phase C18 column was used alongside an aqueous formic acid mobile phase. Both vitamins were detected at 255 nm. Statistical Analysis Statistical analysis was performed in R [ 23 ]. Data were organized and compiled using the ‘tidyverse’ package [ 24 ], and an initial two-way analysis of variance was performed with the ‘stats’ package to determine any interaction between the effect of light-intensity and photoperiod on the 14 parameters listed in Table 2 [ 23 ]; when interactions were not present, data were pooled. Linear or quadratic regression were performed with the ‘stats’ package when light intensity or the interaction of light intensity and photoperiod was significantly different [ 23 ]. For final scoring, treatment means of each parameter were calculated, min-max normalized, and multiplied by a weighting value, according to the values given in Table 2 . Weighted means of each parameter were then added together per treatment to produce a final score for comparison of total treatment value. To determine energy use efficiency, the wattage used per treatment per day to power the LED lights was calculated and converted to megajoules. The mean total yield per metric was then divided by the energy used to produce said yield, further described in the following equation: $$mg\bullet {MJ}^{-1} = \frac{\left(mean concentration \bullet mean dry mass yield\right)}{\left(fixture wattage utilized\bullet daily photoperiod hours\right)(0.0036 MJ\bullet {watt hour}^{-1})}$$ The exception to this equation was the calculation for fresh mass energy use efficiency, in which case there was no need to calculate a total yield from the concentration and the dry mass yield. Instead, the mean fresh mass yield was used as the numerator and the end result was described in g∙MJ − 1 . Visualizations were created with ggplot [ 24 ]. Table 2 List of parameters of interest to NASA, their class, and the multiplier to be applied to the normalized mean. Multipliers were based on previous NASA research and reflect the importance of each parameter to astronaut health on future exploration-length missions (Massa et al, 2015). Negative values indicate undesired plant traits, while magnitude of value indicates importance. Metric Category Multiplier Justification Fresh Mass Morphological × 2.5 Total phytonutrient content scales with this metric Dry Matter Morphological × 1.0 Higher dry mass proportion is desirable Plant Volume Morphological × -1.5 Smaller plants are desirable due to limited space Ca Minerals × 1.5 Mitigates bone loss in microgravity [ 25 ] K Minerals × 2.0 Currently deficient in space diet [ 6 ] Mg Minerals × 1.0 Mitigates bone loss in microgravity Fe Minerals × -1.5 Excessive iron can exacerbate bone loss in microgravity [ 6 ] Ascorbic Acid Water-Soluble Vitamins × 1.5 Prevent scurvy; degrades quickly [ 2 , 26 ] Thiamine Water-Soluble Vitamins × 1.5 Prevent beriberi; degrades quickly [ 2 , 26 ] Phylloquinone Fat-Soluble Vitamin × 1.5 Promote proper blood coagulation; deficient in stored space diet [ 6 , 27 ] β-carotene Carotenoids × 1.5 Vitamin A precursor; degrades quickly [ 2 , 28 ] Lutein Carotenoids × 1.5 Preventing ocular photodamage [ 29 ] Zeaxanthin Carotenoids × 1.5 Preventing ocular photodamage [ 29 ] Total Anthocyanin Anthocyanins × 1.0 Potentially beneficial as an antioxidant Results and Discussion Individual Phytonutrient Responses While many of the parameters tested demonstrated an interactive effect between light intensity and photoperiod, ascorbic acid, total anthocyanin, and phylloquinone concentrations did not (Supplementary Table S1 ). Additionally, while total anthocyanin concentrations decreased linearly from 11.9 to 7.2 mg·g − 1 with increasing light intensity (Fig. 1 a, Supplementary Table S2), ascorbic acid and phylloquinone concentrations did not respond significantly to changes in light intensity or photoperiod (Supplementary Table S1 ). Anthocyanins attenuate the negative effects of increasing light concentrations, thus, plants are expected to produce more anthocyanins under high irradiance [ 30 ]. The decrease in concentration seen in this study may be due to a dilution effect, in which primary photosynthetic products (sugars, starches, cellulose, etc.) are produced in higher amounts at high light intensities due to the elevated CO 2 , accounting for a higher percentage of the dry mass than under normal conditions. A similar effect has been seen with the dilution of chlorophyll by increased primary metabolites, both structural and non-structural, in Brassica juncea when grown under elevated CO 2 concentrations [ 31 ]. However, further research would be required to prove this hypothesis against plants grown under atmospheric CO 2 , as all treatments in the current study occurred at elevated concentrations. Ascorbic acid concentrations were approximately 9 mg·g − 1 across all treatments, which was lower than the concentrations found in ' Red Hybrid’ grown at 200 µmol⋅m − 2 ⋅s − 1 applied across a 16-hr photoperiod in a previous study, where ascorbic acid concentrations averaged 17 mg·g − 1 [ 7 ]. The simplest explanation for this difference is that the plants in the previous study were allowed to grow for an additional 8 days, as older leaves have been shown to have higher concentrations of ascorbic acid [ 32 ]. All quantified morphological traits exhibited responses to the interaction of light intensity and photoperiod. Fresh mass (Fig. 2 a, Supplementary Table S2) increased in response to increasing light intensity at both photoperiods, although this increase tapered off after reaching 60 g at 600 µmol⋅m − 2 ⋅s − 1 and decreased to 53 g at 800 µmol⋅m − 2 ⋅s − 1 , an amount similar to the 52 g produced at 400 µmol⋅m − 2 ⋅s − 1 . At each tested light intensity, a 24-hr photoperiod produced higher fresh mass than that of a 16-hr photoperiod, with the exception of the highest light intensity where the 16-hr photoperiod surpassed its 24-hr counterpart. Dry matter percentage increased linearly with increasing light at both photoperiods, although the response was stronger at the 24-hr photoperiod (Fig. 2 b, Supplementary Table S2). Plant volume also increased linearly with increasing light intensity at a 16-hr photoperiod but decreased linearly when grown at a 24-hr photoperiod (Fig. 2 c, Fig. 2 d, Supplementary Table S2). The increase in mass in conjunction with a decrease of required production space is a favorable combination of factors for production in space; this combination of features is attainable with a high light intensity applied across a 24-hr photoperiod. Apart from magnesium, any trends present in mineral concentration show a linear decrease with increasing light intensity (Fig. 3 ). These trends occurred in calcium, potassium, and iron when light was applied across a 24-hr photoperiod. Linear decreases with increasing light intensity were also present at a 16-hr photoperiod in the concentrations of potassium and iron. This decrease was most apparent in the response of potassium concentration (Fig. 3 b, Supplementary Table S2), which decreased by 39% when light was increased from 200 to 800 µmol⋅m − 2 ⋅s − 1 at a 24-hr photoperiod. Across the same lighting conditions, iron concentrations decreased by 31% (Fig. 3 d, Supplementary Table S2) and calcium decreased by 18% (Fig. 3 a, Supplementary Table S2). The similar trends present in the plants grown under a 16-hr photoperiod were less aggressive when present. Unique among the minerals, magnesium concentrations rose by 26% as light intensity increased from 200 to 800 µmol⋅m − 2 ⋅s − 1 (Fig. 3 c). The general decrease in mineral concentrations with increasing light could be explained by the same dilution effect mentioned regarding anthocyanin pigments [ 31 ]. However, all four minerals quantified in this study are at least partially incorporated into the plant by passive uptake. As passive uptake is driven by transpiration which occurs prominently during the photoperiod, it would be logical to assume that plants grown at a 24-hr photoperiod would contain higher concentrations of passively incorporated minerals. This was not seen (Fig. 3 abcd) and the opposite was true at the highest light intensity, where plants grown under a shorter photoperiod had higher concentrations of all minerals quantified. Previous studies have found that super-elevated CO 2 concentrations can prevent nighttime stomatal closure [ 33 ] and this could partially explain the failure of the 24-hr treatment to produce plants with higher mineral concentrations, although it does not explain why the 16-hr treatment did not experience a similar decline at higher light intensities. Thiamine (vitamin B 1 ) concentrations increased from 0.003 to 0.005 mg·g − 1 as light increased from 200 to 600 µmol⋅m − 2 ⋅s − 1 but decreased back to 0.003 mg·g − 1 as light increased further (Fig. 3 e, Supplementary Table S2). Thiamine helps plants to respond to oxidative stress [ 34 ] and an upregulation in antioxidant compounds is typically seen in response to increasing light intensity. The decrease in thiamine after 600 µmol⋅m − 2 ⋅s − 1 could be due to a variety of factors, including metabolic burnout under high daily light integral and high CO 2 [ 35 ] or dilution due to starch accumulation. Carotenoids are also utilized in plant response to high light stress (secondary function), in addition to participating in the photosynthetic machinery (primary function). β-carotene (Fig. 3 f, Supplementary Table S2) and lutein (Fig. 3 g, Supplementary Table S2) both decreased linearly in response to increasing light and produced lower concentrations at a 24-hr photoperiod. β-carotene [ 15 ] and lutein [ 15 , 36 ] are both required for proper function of the antennae complexes of higher plants, both in gathering light and mitigating the impact of excessive photon exposure, and in leaf tissue are primarily concentrated in PSI and PSII. Zeaxanthin, while also a part of the photosystem, is also found in the lipid bilayer participating in a secondary role as part of the photosynthetic membrane [ 37 ]. This secondary location and function of zeaxanthin could explain its sharp increase at high light at a 24-hr photoperiod (Fig. 3 h, Supplementary Table S2), an increase that did not occur in carotenoids which are primarily tied to the photosystems in leaf tissue. Phytonutrient Profile Evaluation Parameter means were normalized and weighted according to Table 2 and the resulting weighted means were summed by treatment to determine the best conditions for producing optimal ‘Red Hybrid’ plants under ISS-like conditions. We found that plants grown at a light intensity of 800 µmol⋅m − 2 ⋅s − 1 at a 16-hr photoperiod provided the best combination of traits for supplementing the astronaut diet (Fig. 4 ). Producing a 75 g serving of fresh biomass yield of ‘Red Hybrid’ would require on average 1.4 plants when growing at a 800 µmol⋅m − 2 ⋅s − 1 light intensity and a 16-hr photoperiod for 22 days in 0.008 m 3 of production space per plant. This 75 g serving provides all the necessary phylloquinone and 63%, 2.7%, and 97% of ascorbic acid, thiamine, and vitamin A precursor, respectively. Again, mineral nutrients are present but minimal, providing 1.1, 1.3, 0.9, and 4.9% of Ca, K, Mg, and Fe (RDI). Energy Considerations It is important to note that the conditions which provide the highest quality ‘Red Hybrid’ plants may not be the most energy efficient, and energy use is at times more important than crop quality. Once normalized on a yield per MJ of energy spent basis it is apparent that the low light-intensity treatments provide more fresh mass per MJ than any other treatment, regardless of photoperiod (Fig. 5 ). This starkly contrasts the trends presented in Fig. 2 a, in which fresh mass generally increases with increasing light intensity, and those produced by the final scoring, in which score is based on normalized means and weighted multipliers (Fig. 4 ). Digging deeper and looking into the phytonutrient yield per MJ we see a similar trend, where it is almost always more efficient to use the lowest light intensity evaluated (Fig. 6 ). The outstanding exception to this is zeaxanthin, where the gains seen at the 800 µmol⋅m − 2 ⋅s − 1 by 16-hr treatment are large enough to offset the energy expenditure. Little direction emerges in the phylloquinone efficiency, but this is to be expected due to its lack of statistical difference when previously tested. When choosing a lighting protocol for crop production to supplement the astronaut diet, it seems the largest question at play will be one of balancing efficiency vs. peak nutritional output. This question may require more investigation, or it may simply be a shifting target, as different missions and applications will carry different priorities. Regardless, the data presented here can help to inform these decisions, as new lenses could be applied to the dataset as new needs or priorities arise. Declarations Acknowledgements This work was funded by the NASA Space Biology Program grant 80NSSC22K0205. We gratefully thank Dr. John Munafo for his thoughtful review, Lauren Little for plant production assistance, Hannah Schmidt for phytonutrient quantification assistance, Spencer Givens, Kathleen Coffman, and Maggie Whittington for data collection assistance, Horticulture Lighting Group for LED fixtures, and Jiffy Growing Solutions for substrate. We also acknowledge the Mississippi State University Soil Lab for mineral quantification. The use of trade names in this publication does not imply endorsement by the University of Tennessee of products named nor criticism of similar ones not mentioned. Author contributions ED performed the experiment, analyzed the data, and prepared the manuscript. SA aided in phytonutrient quantification and method development. GM and KW provided experimental guidance and reviewed and revised the manuscript. KW conceptualized and designed the study and obtained funding. Data availability statement Data will be made available upon request by the corresponding author. Additional information Research support of LED fixtures and substrate were provided by Horticulture Lighting Group and Jiffy Growing Solutions, respectively. KW has consulted for Vitamin Greens and received compensation. ED owns stock in Plenty. GM and SA declare no conflict of interest. References Perchonok, M. H., Cooper, M. R. & Catauro, P. M. Mission to mars: food production and processing for the final frontier. Annual Review of Food Science and Technology 3 , 311–330 (2012). Cooper, M., Perchonok, M. & Douglas, G. L. Initial assessment of the nutritional quality of the space food system over three years of ambient storage. npj Microgravity 3 , 1–4 (2017). Burgner, S. E. et al. 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R Foundation for Statistical Computing (2023). Wickham, H. et al. Welcome to the Tidyverse. Journal of Open Source Software 4 , 1686 (2019). Flynn, A. The role of dietary calcium in bone health. Proceedings of the Nutrition Society 62 , 851–858 (2003). Jukes, T. H. The prevention and conquest of scurvy, beri-beri, and pellagra. Preventive Medicine 18 , 877–883 (1989). J. Basset, G., Latimer, S., Fatihi, A., Soubeyrand, E. & Block, A. Phylloquinone (vitamin K1): occurrence, biosynthesis and functions. Mini Reviews in Medicinal Chemistry 17 , 1028–1038 (2017). Olson, J. A. & Hayaishi, O. The enzymatic cleavage of beta-carotene into vitamin A by soluble enzymes of rat liver and intestine. Proceedings of the National Academy of Sciences 54 , 1364–1370 (1965). Chylack, L. T. et al. NASA study of cataract in astronauts (NASCA). Report 1: cross-sectional study of the relationship of exposure to space radiation and risk of lens opacity. Radiat Res 172 , 10–20 (2009). Chalker-Scott, L. Environmental significance of anthocyanins in plant stress responses. Photochemistry and Photobiology 70 , 1–9 (1999). Uprety, D. C. & Mahalaxmi, V. Effect of elevated CO 2 and nitrogen nutrition on photosynthesis, growth and carbon-nitrogen balance in Brassica juncea. J Agron Crop Sci 184 , 271–276 (2000). Li, M., Ma, F., Guo, C. & Liu, J. Ascorbic acid formation and profiling of genes expressed in its synthesis and recycling in apple leaves of different ages. Plant Physiology and Biochemistry 48 , 216–224 (2010). Levine, L. H. et al. Physiologic and metabolic responses of wheat seedlings to elevated and super-elevated carbon dioxide. Advances in Space Research 42 , 1917–1928 (2008). Tunc-Ozdemir, M. et al. Thiamin confers enhanced tolerance to oxidative stress in Arabidopsis. Plant Physiology 151 , 421–432 (2009). Reuveni, J. Very high CO 2 reduces photosynthesis, dark respiration and yield in wheat. Annals of Botany 80 , 539–546 (1997). Dall’Osto, L. et al. Lutein is needed for efficient chlorophyll triplet quenching in the major LHCII antenna complex of higher plants and effective photoprotection in vivo under strong light. BMC Plant Biology 6 , 32 (2006). Havaux, M. & García-Plazaola, J. I. Beyond non-photochemical fluorescence quenching: the overlapping antioxidant functions of zeaxanthin and tocopherols. in Non-photochemical quenching and energy dissipation in plants, algae and cyanobacteria (eds. Demmig-Adams, B., Garab, G., Adams III, W., & Govindjee) 583–603 (Springer Netherlands, Dordrecht, 2014). Additional Declarations Competing interest reported. Research support of LED fixtures and substrate were provided by Horticulture Lighting Group and Jiffy Growing Solutions, respectively. KW has consulted for Vitamin Greens and received compensation. ED owns stock in Plenty. GM and SA declare no conflict of interest. Cite Share Download PDF Status: Published Journal Publication published 29 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 26 Aug, 2024 Reviews received at journal 22 Aug, 2024 Reviews received at journal 22 Aug, 2024 Reviewers agreed at journal 09 Aug, 2024 Reviewers agreed at journal 24 Jul, 2024 Reviewers agreed at journal 02 Jul, 2024 Reviewers invited by journal 03 May, 2024 Editor assigned by journal 03 May, 2024 Editor invited by journal 02 May, 2024 Submission checks completed at journal 02 May, 2024 First submitted to journal 28 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4338874","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":299598610,"identity":"ed2f44f7-df72-47da-a305-818adc384863","order_by":0,"name":"Ethan Darby","email":"","orcid":"","institution":"University of Tennessee","correspondingAuthor":false,"prefix":"","firstName":"Ethan","middleName":"","lastName":"Darby","suffix":""},{"id":299598611,"identity":"3f87f27b-4616-43b9-8403-2f189d74c258","order_by":1,"name":"Sarah Armstrong","email":"","orcid":"","institution":"University of Tennessee","correspondingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"","lastName":"Armstrong","suffix":""},{"id":299598612,"identity":"2fd95ddd-e581-4400-9520-de598b9e3cbf","order_by":2,"name":"Gioia Massa","email":"","orcid":"","institution":"NASA Kennedy Space Center","correspondingAuthor":false,"prefix":"","firstName":"Gioia","middleName":"","lastName":"Massa","suffix":""},{"id":299598613,"identity":"4a31d757-0fdc-4ad2-91c5-3a789f084baa","order_by":3,"name":"Kellie J. Walters","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvklEQVRIiWNgGAWjYFAC5gYQacAPZDFAMEHA2AjSYyDZwEyqFoMDxGoxOH6w/cHHPXXGxjfyDxswVFgnNhDUciaxsXHGs8NmZjeSmRMYzqQT1mJ2ILGxmefAARuQlgOMbYeJ0HL+YWPznwN1NsYzQFr+EaPlBtAWhgPMZgYSQIcxNhChxf7Gw8aZPQcOG0uceWxskHAs3ZigFsn+5AMffhyoM+xvT3ws8aHGWpagFlSQQJryUTAKRsEoGAW4AAChokQPTbcLsAAAAABJRU5ErkJggg==","orcid":"","institution":"University of Tennessee","correspondingAuthor":true,"prefix":"","firstName":"Kellie","middleName":"J.","lastName":"Walters","suffix":""}],"badges":[],"createdAt":"2024-04-28 17:25:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4338874/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4338874/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-11662-y","type":"published","date":"2025-07-29T16:21:16+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":56086319,"identity":"53ee2ca4-3113-4b10-8100-a78e93f642a2","added_by":"auto","created_at":"2024-05-08 11:07:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":66333,"visible":true,"origin":"","legend":"\u003cp\u003eLight intensity effect on \u003cem\u003eBrassica rapa \u003c/em\u003espp. \u003cem\u003enipposinica \u003c/em\u003e‘Red Hybrid’ mizuna total anthocyanin concentration. Means are represented by black points and a vertical line represents the standard error. Trend line represents regression prediction with a grey confidence interval, with the coefficients presented in Supplementary Table S2.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4338874/v1/90f805748c4773535554cdf0.png"},{"id":56086321,"identity":"07c3ff33-31bc-4420-8f4e-6405cd132dab","added_by":"auto","created_at":"2024-05-08 11:07:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":222127,"visible":true,"origin":"","legend":"\u003cp\u003eLight intensity and photoperiod effect on\u003cstrong\u003e \u003c/strong\u003emorphological characteristics of \u003cem\u003eBrassica rapa \u003c/em\u003espp. \u003cem\u003enipposinica \u003c/em\u003e‘Red Hybrid’ mizuna. Plots a) fresh mass, b) dry matter percentage, and c) plant volume display means represented by triangles (24-hr) or dots (16-hr) and a vertical line represents the standard error. Trend lines represent regression predictions with a shaded confidence interval, with coefficients presented in Supplemental Table 2. In d) images representative of plant morphology from each treatment combination are displayed.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4338874/v1/48e6406113a43affb9376864.png"},{"id":56086318,"identity":"88aa99a4-9bb2-4726-bf4b-4a82f66e56df","added_by":"auto","created_at":"2024-05-08 11:07:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":270340,"visible":true,"origin":"","legend":"\u003cp\u003eLight intensity and photoperiod effect on\u003cstrong\u003e \u003c/strong\u003emineral and phytonutrient concentrations in \u003cem\u003eBrassica rapa \u003c/em\u003espp. \u003cem\u003enipposinica \u003c/em\u003e‘Red Hybrid’ mizuna including a) calcium, b) potassium, c) magnesium, d) iron, e) thiamine, f) β-carotene, g) lutein, and h) zeaxanthin. Means are represented by triangles (24-hr) or dots (16-hr) and a vertical line represents the standard error. Trend lines represent regression predictions with a shaded confidence interval, with coefficients presented in Supplementary Table S2.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4338874/v1/b58b2b66f979d36327ca3df2.png"},{"id":56086323,"identity":"382af796-dbb1-470a-9c14-0994b29f77b5","added_by":"auto","created_at":"2024-05-08 11:07:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":91557,"visible":true,"origin":"","legend":"\u003cp\u003eFinal scores for each treatment combination, calculated by normalizing the means of each parameter of interest for \u003cem\u003eBrassica rapa \u003c/em\u003espp. \u003cem\u003enipposinica \u003c/em\u003e‘Red Hybrid’, multiplying by the weighting factor in Table 2, and summing the results from all 14-parameters into a total score. Components of the score visualized under the 0-line are negatively weighted while those above the line are positive. The black dot represents the total score, or the negative components subtracted from the positive.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4338874/v1/66af695c81218ac261234fbb.png"},{"id":56086711,"identity":"a9988c70-312e-4451-b65a-380764a53db0","added_by":"auto","created_at":"2024-05-08 11:15:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":46350,"visible":true,"origin":"","legend":"\u003cp\u003eFresh mass yield per MJ of electricity spent powering lighting fixtures. Grams per MJ produced by the 16-hr photoperiod are represented by light blue bars, while the efficiency of the 24-hr photoperiod treatments are in dark blue.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4338874/v1/a3d079616f4ed40bc7a531f4.png"},{"id":56086713,"identity":"f873a77a-7e01-4422-a235-9e75ad5b7f32","added_by":"auto","created_at":"2024-05-08 11:15:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":132720,"visible":true,"origin":"","legend":"\u003cp\u003ePhytonutrient yields per MJ of electricity spent powering lighting fixtures. Milligrams per MJ of anthocyanins, ascorbic acid, phylloquinone, zeaxanthin, lutein, and β-carotene are represented. Light blue bars represent 16-hr photoperiod efficiencies, while dark blue bars represent 24-hr photoperiod efficiencies.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4338874/v1/753415181a111ef49c4990c7.png"},{"id":88268247,"identity":"882a5401-4db7-4651-8f7f-48e8fbe8a5ad","added_by":"auto","created_at":"2025-08-04 16:50:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1676355,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4338874/v1/3dd2f477-9d4d-4d94-a10c-8cea8852bcdb.pdf"}],"financialInterests":"Competing interest reported. Research support of LED fixtures and substrate were provided by Horticulture Lighting Group and Jiffy Growing Solutions, respectively. KW has consulted for Vitamin Greens and received compensation. ED owns stock in Plenty. GM and SA declare no conflict of interest.","formattedTitle":"Light intensity and photoperiod: Tools for improving the phytonutrient profile of Brassica rapa ssp. nipposinica for supplementing the space diet","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe production of fresh vegetables aboard spacecraft has been identified as a key component of successful exploration-length space ventures [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This is due to a need for fresh sources of vitamin C (ascorbic acid), vitamin B\u003csub\u003e1\u003c/sub\u003e (thiamine), and dietary carotenoids (β-carotene, lutein, zeaxanthin), as well as supplemental sources of vitamin K\u003csub\u003e1\u003c/sub\u003e (phylloquinone), potassium, magnesium, calcium, and antioxidants such as anthocyanins [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, growing fresh produce in space presents many challenges, some of which include limited space aboard craft, limited energy resources, and environmental constraints such as elevated CO\u003csub\u003e2\u003c/sub\u003e concentrations and microgravity [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLeafy greens are promising candidates for production aboard space craft, as they are more energy- and space-efficient than fruit or root crops. They also provide an advantage over dried supplements, such as pills, as accidental toxicities are harder to incur [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Prior evaluation of leafy greens has shown species within the genus \u003cem\u003eBrassica\u003c/em\u003e to be particularly promising from a biomass, organoleptic, and phytonutrient perspective [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. More recent research has confirmed that \u003cem\u003eBrassica rapa\u003c/em\u003e spp. \u003cem\u003enipposinica\u003c/em\u003e (mizuna) contains high concentrations of desired phytonutrients while still performing well when grown under conditions similar to those of the International Space Station (ISS) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdditionally, plant tissue concentrations of many phytonutrients respond to environmental stimuli [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and environmental manipulation may provide methods to increase concentrations of desirable compounds while maintaining or even increasing biomass yield. This is especially true of light, as compounds such as ascorbic acid [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], secondary carotenoids [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and anthocyanins [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] are involved in light-induced free-radical scavenging and absorption of excess light, while phylloquinone [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and primary carotenoids [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] are directly involved in the photosynthetic machinery. Thus, environmental control of light intensity and photoperiod is a potential tool to improve the quality of leafy greens for consumption as part of the space diet.\u003c/p\u003e \u003cp\u003eLight is also used more efficiently by plants at a lower intensity, meaning it can be more energy efficient to apply a lower intensity of light over a longer period of the day to achieve the same total photon exposure, or DLI (Daily Light Integral) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This study aims to determine which combination of light intensity and photoperiod produces \u003cem\u003eBrassica rapa\u003c/em\u003e spp. \u003cem\u003enipposinica\u003c/em\u003e \u0026lsquo;Red Hybrid\u0026rsquo; mizuna with the highest phytonutrient concentrations when produced under an environment similar to that of the ISS, while still maintaining a compact morphology and plant vigor.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental Design\u003c/h2\u003e \u003cp\u003eThe experiment was organized in a factorial design with 9 plants grown under each combination of light intensity (4) and photoperiod (2) for a total of 8 treatments. The experiment was conducted twice over time. At harvest, morphological data were collected on all 9 plants per replication. Plant-specific phytonutrient and mineral data were collected on all 9 plants per replication, except for plants grown at a 200 \u0026micro;mol\u0026middot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026middot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e light intensity and a 16-hr photoperiod which did not provide enough tissue per plant for mineral nutrient extractions. Tissue from multiple plants was pooled to produce mineral concentration data from this treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePlant Production\u003c/h2\u003e \u003cp\u003eSeeds of \u003cem\u003eBrassica rapa\u003c/em\u003e spp. \u003cem\u003enipposinica\u003c/em\u003e \u0026lsquo;Red Hybrid\u0026rsquo; mizuna (Kitazawa Seed, Oakland, CA) were sown in 162-cell bound coco-peat cubes (Preforma plugs; Jiffy Growing Solutions, Lorain, OH) and placed in a walk-in growth chamber (TC2, Environmental Grow Chambers; Chagrin Falls, OH). Plant production was similar to Darby et al. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] with treatment and other select differences. The temperature was 22.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u0026deg; C, and the relative humidity was 61.6\u0026thinsp;\u0026plusmn;\u0026thinsp;7.3%, both measured and logged every 10 minutes by a shielded temperature and relative humidity sensor (Hobo MX2300; OnSet, Bourne, MA). Seedling trays were placed under broad-spectrum light-emitting diodes (Scorpion Diablo, Horticulture Lighting Group; Maynardville, TN) providing 21:36:43 blue:green:red (%) radiation ratios, with a red:far-red ratio of 13:1, and one of four light intensities applied at one of two photoperiods for a total of eight treatments (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Light intensity was monitored by quantum sensors (SQ-500S; Apogee, Logan, UT) for the duration of the experiment. Leaf surface temperature was monitored with ultra-narrow field of view infrared radiometers (SI-131-SS; Apogee, Logan, UT), averaging 21.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u0026deg; C across all treatments (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). CO\u003csub\u003e2\u003c/sub\u003e (Industrial Grade Carbon Dioxide CD 50S; Airgas, Knoxville, TN) was injected to provide 2,770\u0026thinsp;\u0026plusmn;\u0026thinsp;180 \u0026micro;mol∙mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Dosing was monitored and controlled by an infrared gas analyzer (LI-850; LI-COR Biosciences, Lincoln, NE) connected to a relay switch controlling an electronic regulator. CO\u003csub\u003e2\u003c/sub\u003e concentration was reported to a Raspberry Pi (Raspberry Pi 4 B; Raspberry Pi, Cambridge, England) every 20 seconds where it was recorded in a CSV file. Seeds were irrigated daily with reverse-osmosis water supplemented with 12N-1.8P-13.4K water-soluble fertilizer, providing (mg\u0026sdot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) 100 nitrogen, 15 phosphorus, 112 potassium, 58 calcium, 17 magnesium, 2 sulfur, 1.4 iron, 0.5 zinc, 0.4 copper and manganese, and 0.1 boron and molybdenum, (RO Hydro FeED; JR Peters, Inc., Allentown, PA) and magnesium sulfate (MgSO\u003csub\u003e4\u003c/sub\u003e) to provide (mg\u0026sdot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) 15 magnesium and 20 sulfur. The pH was adjusted to 5.8 with H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e or KCHO\u003csub\u003e3\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLighting treatments, their intensity, photoperiod, and daily light integral (DLI), as provided to the initial growing surface.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTarget\u003c/p\u003e \u003cp\u003eLight Intensity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eActual\u003c/p\u003e \u003cp\u003eLight Intensity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePhotoperiod\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eActual Daily Light Integral\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLeaf Temperature\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(\u0026micro;mol\u0026sdot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026sdot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(\u0026micro;mol\u0026sdot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026sdot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(hrs)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(mol\u0026sdot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026sdot;d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(\u0026deg; C)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e202\u0026thinsp;\u0026plusmn;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e391\u0026thinsp;\u0026plusmn;\u0026thinsp;24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e583\u0026thinsp;\u0026plusmn;\u0026thinsp;40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e780\u0026thinsp;\u0026plusmn;\u0026thinsp;30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e44.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e203\u0026thinsp;\u0026plusmn;\u0026thinsp;11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e396\u0026thinsp;\u0026plusmn;\u0026thinsp;16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e603\u0026thinsp;\u0026plusmn;\u0026thinsp;25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e782\u0026thinsp;\u0026plusmn;\u0026thinsp;38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e67.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSimilar to Darby et al. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], after thirteen days the seedlings were transplanted into 18-cm-deep by 61-cm-wide by 122-cm-long deep-water culture hydroponic systems (Premium Flood Table, Active Aqua; Petaluma, CA) with 61 by 122 cm sealed-surface foam rafts (36 ct lettuce raft; Beaver Plastics, Acheson, AB, Canada) and grown for 9 days. Plants were harvested 22 days after sowing to prevent over-crowding of the growing space. The environmental conditions after transplant were the same as the seedling stage. Eight systems were filled with 108 L of reverse-osmosis water and supplemented with 12N-1.8P-13.4K water-soluble fertilizer (RO Hydro FeED; JR Peters, Inc.) and MgSO\u003csub\u003e4\u003c/sub\u003e providing twice the concentrations reported during propagation. Electrical conductivity (EC) and pH were monitored (HI9813-6N Portable Waterproof pH/EC/TDS Meter; Hanna Instruments, Woonsocket, RI) and pH was adjusted to 5.8 using H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e or KCHO\u003csub\u003e3\u003c/sub\u003e. Air pumps (Active Aqua 110 L\u0026sdot;min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e commercial air pump; Hydrofarm, Petaluma, CA) and air stones (Active Aqua air stone round 10 cm \u0026times; 2.5 cm; Hydrofarm, Petaluma, CA) were used to provide dissolved oxygen to the nutrient solution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eHarvest, Morphological Data Collection, and Tissue Processing\u003c/h2\u003e \u003cp\u003ePlants were harvested at the substrate surface and morphological data were collected [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Height from the substrate surface to the tip of the tallest leaf, width at the widest point and perpendicular to the widest point, the number of fully expanded leaves, and fresh mass were collected for 9 plants per replication. After weighing, fresh tissue of 9 plants per treatment was immediately placed in a plastic sample bag and flash frozen in liquid N. Tissue was then stored in a -80\u0026deg;C freezer, freeze dried, and homogenized in liquid N. Once homogenized, the tissue was divided into separate aliquots for future extraction procedures and stored in individual 15 mL centrifuge tubes in the \u0026minus;\u0026thinsp;80\u0026deg;C freezer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAnthocyanin Isolation and Quantification\u003c/h2\u003e \u003cp\u003eTotal anthocyanins were extracted and analyzed using a method derived from Islam et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and previously used in Darby et al. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Briefly, 50 mg of ground tissue was used per sample, and the procedure occurred under red light. Samples were saturated with 5 mL of 95% ethanol/1.5 N HCl (85:15, v:v), placed on an orbital shaker for 15 minutes, and stored at 4\u0026deg;C for 24 hrs. Samples were then filtered and analyzed with a microplate reader (Agilent Technologies, Santa Clara, CA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCarotenoid Isolation and Quantification\u003c/h2\u003e \u003cp\u003eThe carotenoids β-carotene, lutein, and zeaxanthin were extracted and analyzed using a method derived from Kopsell et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and previously described by Darby et al. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Under red light, 50 mg samples of chilled tissue were extracted with purified water and tetrahydrofuran, and an internal carotenoid standard was used to quantify sample loss during homogenization. Samples were homogenized and tetrahydrofuran was used for a second extraction followed by additional homogenization. The resulting solution was centrifuged, and the eluent was dried down on a stream evaporator. After the addition of acetone, samples were filtered, and an aliquot was stored for subsequent identification and quantification on a 1200 Agilent Series HPLC unit equipped with a diode array detector. A reverse phase C30 column was used with an isocratic mobile phase composed of methyl tert-butyl ether, methanol, and triethylamine (11%, 88.99%, and 0.01%, v/v/v).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFat Soluble Vitamin Isolation and Quantification\u003c/h2\u003e \u003cp\u003eThe fat-soluble vitamin phylloquinone (vitamin K\u003csub\u003e1\u003c/sub\u003e) was extracted using a method derived from Pokkanta et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and previously used in Darby et al. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Extractions were performed under red light (peak wavelength: 654 nm) and samples were saturated with a series of three solvents (methanol, dichloromethane, and hexane). After being saturated with each solvent, the samples underwent orbital shaking, sonication, and were finally centrifuged. After removing the supernatant, the next solvent in the series was added and the samples again underwent the three previously mentioned procedures. An internal standard was added to the pooled supernatant, and the samples were dried completely, suspended in 5 mL dichloromethane, and filtered. A 1-mL aliquot was then collected for analysis with a 1200 Agilent Series HPLC equipped with a diode array detector, using a reverse phase C18 column and a mobile gradient composed of methanol and water. Phylloquinone was identified at 248 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMineral Isolation and Quantification\u003c/h2\u003e \u003cp\u003eNitrogen concentration was quantified via combustion of 0.25 g of tissue and detection with a Thermal Conductivity Sensor (vario MAX cube; Elementar, Ronkonkoma, NY). Other minerals, Ca\u003csup\u003e2+\u003c/sup\u003e, K\u003csup\u003e+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, and Fe\u003csup\u003e3+\u003c/sup\u003e were extracted and quantified using a method derived from Jones [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. A 0.5 g aliquot of tissue was dry ashed in a furnace at 500\u0026deg;C, and then wet ashed by the addition of dilute acid. The resulting solution was then analyzed on an ICP-OES to determine individual mineral concentrations (SPECTROBLUE; Spectro Analytical Instruments Inc., Kleve, Germany). Due to limited tissue production under the 200 \u0026micro;mol\u0026sdot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026sdot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e by 16-hr photoperiod, mineral analysis on this treatment was performed on pooled samples (with two plants contributing tissue to each sample).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eWater Soluble Vitamin Isolation and Quantification\u003c/h2\u003e \u003cp\u003eThe water-soluble vitamins (WSVs) ascorbic acid (vitamin C) and thiamine (vitamin B\u003csub\u003e1\u003c/sub\u003e) were extracted using a method derived from Seal and Chaudhuri [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and Sun et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and previously used in Darby et al. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Under red light, chilled samples of 200 mg were hydrated with purified water, hydrochloric acid, and a phosphate buffer. Samples were vortexed and supernatant was filtered. The process was repeated, and a 1 mL aliquot was collected for analysis on a 1200 Agilent Series HPLC equipped with a diode array detector. A reverse phase C18 column was used alongside an aqueous formic acid mobile phase. Both vitamins were detected at 255 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed in R [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Data were organized and compiled using the \u0026lsquo;tidyverse\u0026rsquo; package [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and an initial two-way analysis of variance was performed with the \u0026lsquo;stats\u0026rsquo; package to determine any interaction between the effect of light-intensity and photoperiod on the 14 parameters listed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]; when interactions were not present, data were pooled. Linear or quadratic regression were performed with the \u0026lsquo;stats\u0026rsquo; package when light intensity or the interaction of light intensity and photoperiod was significantly different [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. For final scoring, treatment means of each parameter were calculated, min-max normalized, and multiplied by a weighting value, according to the values given in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Weighted means of each parameter were then added together per treatment to produce a final score for comparison of total treatment value. To determine energy use efficiency, the wattage used per treatment per day to power the LED lights was calculated and converted to megajoules. The mean total yield per metric was then divided by the energy used to produce said yield, further described in the following equation:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$mg\\bullet {MJ}^{-1} = \\frac{\\left(mean concentration \\bullet mean dry mass yield\\right)}{\\left(fixture wattage utilized\\bullet daily photoperiod hours\\right)(0.0036 MJ\\bullet {watt hour}^{-1})}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThe exception to this equation was the calculation for fresh mass energy use efficiency, in which case there was no need to calculate a total yield from the concentration and the dry mass yield. Instead, the mean fresh mass yield was used as the numerator and the end result was described in g∙MJ\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Visualizations were created with ggplot [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList of parameters of interest to NASA, their class, and the multiplier to be applied to the normalized mean. Multipliers were based on previous NASA research and reflect the importance of each parameter to astronaut health on future exploration-length missions (Massa et al, 2015). Negative values indicate undesired plant traits, while magnitude of value indicates importance.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMetric\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCategory\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMultiplier\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJustification\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFresh Mass\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMorphological\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026times; 2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTotal phytonutrient content scales with this metric\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDry Matter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMorphological\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026times; 1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHigher dry mass proportion is desirable\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlant Volume\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMorphological\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026times; -1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSmaller plants are desirable due to limited space\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMinerals\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026times; 1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMitigates bone loss in microgravity [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMinerals\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026times; 2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCurrently deficient in space diet [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMinerals\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026times; 1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMitigates bone loss in microgravity\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFe\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMinerals\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026times; -1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eExcessive iron can exacerbate bone loss in microgravity [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAscorbic Acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWater-Soluble Vitamins\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026times; 1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePrevent scurvy; degrades quickly [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThiamine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWater-Soluble Vitamins\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026times; 1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePrevent beriberi; degrades quickly [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhylloquinone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFat-Soluble Vitamin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026times; 1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePromote proper blood coagulation; deficient in stored space diet [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ-carotene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCarotenoids\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026times; 1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVitamin A precursor; degrades quickly [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLutein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCarotenoids\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026times; 1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePreventing ocular photodamage [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZeaxanthin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCarotenoids\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026times; 1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePreventing ocular photodamage [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Anthocyanin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAnthocyanins\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026times; 1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePotentially beneficial as an antioxidant\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIndividual Phytonutrient Responses\u003c/h2\u003e \u003cp\u003eWhile many of the parameters tested demonstrated an interactive effect between light intensity and photoperiod, ascorbic acid, total anthocyanin, and phylloquinone concentrations did not (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Additionally, while total anthocyanin concentrations decreased linearly from 11.9 to 7.2 mg\u0026middot;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with increasing light intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, Supplementary Table S2), ascorbic acid and phylloquinone concentrations did not respond significantly to changes in light intensity or photoperiod (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Anthocyanins attenuate the negative effects of increasing light concentrations, thus, plants are expected to produce more anthocyanins under high irradiance [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The decrease in concentration seen in this study may be due to a dilution effect, in which primary photosynthetic products (sugars, starches, cellulose, etc.) are produced in higher amounts at high light intensities due to the elevated CO\u003csub\u003e2\u003c/sub\u003e, accounting for a higher percentage of the dry mass than under normal conditions. A similar effect has been seen with the dilution of chlorophyll by increased primary metabolites, both structural and non-structural, in \u003cem\u003eBrassica juncea\u003c/em\u003e when grown under elevated CO\u003csub\u003e2\u003c/sub\u003e concentrations [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. However, further research would be required to prove this hypothesis against plants grown under atmospheric CO\u003csub\u003e2\u003c/sub\u003e, as all treatments in the current study occurred at elevated concentrations. Ascorbic acid concentrations were approximately 9 mg\u0026middot;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e across all treatments, which was lower than the concentrations found in \u003cem\u003e'\u003c/em\u003eRed Hybrid\u0026rsquo; grown at 200 \u0026micro;mol\u0026sdot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026sdot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e applied across a 16-hr photoperiod in a previous study, where ascorbic acid concentrations averaged 17 mg\u0026middot;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The simplest explanation for this difference is that the plants in the previous study were allowed to grow for an additional 8 days, as older leaves have been shown to have higher concentrations of ascorbic acid [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAll quantified morphological traits exhibited responses to the interaction of light intensity and photoperiod. Fresh mass (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, Supplementary Table S2) increased in response to increasing light intensity at both photoperiods, although this increase tapered off after reaching 60 g at 600 \u0026micro;mol\u0026sdot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026sdot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and decreased to 53 g at 800 \u0026micro;mol\u0026sdot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026sdot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, an amount similar to the 52 g produced at 400 \u0026micro;mol\u0026sdot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026sdot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. At each tested light intensity, a 24-hr photoperiod produced higher fresh mass than that of a 16-hr photoperiod, with the exception of the highest light intensity where the 16-hr photoperiod surpassed its 24-hr counterpart. Dry matter percentage increased linearly with increasing light at both photoperiods, although the response was stronger at the 24-hr photoperiod (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, Supplementary Table S2). Plant volume also increased linearly with increasing light intensity at a 16-hr photoperiod but decreased linearly when grown at a 24-hr photoperiod (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, Supplementary Table S2). The increase in mass in conjunction with a decrease of required production space is a favorable combination of factors for production in space; this combination of features is attainable with a high light intensity applied across a 24-hr photoperiod.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eApart from magnesium, any trends present in mineral concentration show a linear decrease with increasing light intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These trends occurred in calcium, potassium, and iron when light was applied across a 24-hr photoperiod. Linear decreases with increasing light intensity were also present at a 16-hr photoperiod in the concentrations of potassium and iron. This decrease was most apparent in the response of potassium concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, Supplementary Table S2), which decreased by 39% when light was increased from 200 to 800 \u0026micro;mol\u0026sdot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026sdot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at a 24-hr photoperiod. Across the same lighting conditions, iron concentrations decreased by 31% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, Supplementary Table S2) and calcium decreased by 18% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, Supplementary Table S2). The similar trends present in the plants grown under a 16-hr photoperiod were less aggressive when present. Unique among the minerals, magnesium concentrations rose by 26% as light intensity increased from 200 to 800 \u0026micro;mol\u0026sdot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026sdot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). The general decrease in mineral concentrations with increasing light could be explained by the same dilution effect mentioned regarding anthocyanin pigments [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. However, all four minerals quantified in this study are at least partially incorporated into the plant by passive uptake. As passive uptake is driven by transpiration which occurs prominently during the photoperiod, it would be logical to assume that plants grown at a 24-hr photoperiod would contain higher concentrations of passively incorporated minerals. This was not seen (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eabcd) and the opposite was true at the highest light intensity, where plants grown under a shorter photoperiod had higher concentrations of all minerals quantified. Previous studies have found that super-elevated CO\u003csub\u003e2\u003c/sub\u003e concentrations can prevent nighttime stomatal closure [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and this could partially explain the failure of the 24-hr treatment to produce plants with higher mineral concentrations, although it does not explain why the 16-hr treatment did not experience a similar decline at higher light intensities.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThiamine (vitamin B\u003csub\u003e1\u003c/sub\u003e) concentrations increased from 0.003 to 0.005 mg\u0026middot;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e as light increased from 200 to 600 \u0026micro;mol\u0026sdot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026sdot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e but decreased back to 0.003 mg\u0026middot;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e as light increased further (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, Supplementary Table S2). Thiamine helps plants to respond to oxidative stress [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] and an upregulation in antioxidant compounds is typically seen in response to increasing light intensity. The decrease in thiamine after 600 \u0026micro;mol\u0026sdot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026sdot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e could be due to a variety of factors, including metabolic burnout under high daily light integral and high CO\u003csub\u003e2\u003c/sub\u003e [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] or dilution due to starch accumulation.\u003c/p\u003e \u003cp\u003eCarotenoids are also utilized in plant response to high light stress (secondary function), in addition to participating in the photosynthetic machinery (primary function). β-carotene (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, Supplementary Table S2) and lutein (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg, Supplementary Table S2) both decreased linearly in response to increasing light and produced lower concentrations at a 24-hr photoperiod. β-carotene [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and lutein [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] are both required for proper function of the antennae complexes of higher plants, both in gathering light and mitigating the impact of excessive photon exposure, and in leaf tissue are primarily concentrated in PSI and PSII. Zeaxanthin, while also a part of the photosystem, is also found in the lipid bilayer participating in a secondary role as part of the photosynthetic membrane [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. This secondary location and function of zeaxanthin could explain its sharp increase at high light at a 24-hr photoperiod (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh, Supplementary Table S2), an increase that did not occur in carotenoids which are primarily tied to the photosystems in leaf tissue.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePhytonutrient Profile Evaluation\u003c/h2\u003e \u003cp\u003eParameter means were normalized and weighted according to Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and the resulting weighted means were summed by treatment to determine the best conditions for producing optimal \u0026lsquo;Red Hybrid\u0026rsquo; plants under ISS-like conditions. We found that plants grown at a light intensity of 800 \u0026micro;mol\u0026sdot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026sdot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at a 16-hr photoperiod provided the best combination of traits for supplementing the astronaut diet (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Producing a 75 g serving of fresh biomass yield of \u0026lsquo;Red Hybrid\u0026rsquo; would require on average 1.4 plants when growing at a 800 \u0026micro;mol\u0026sdot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026sdot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e light intensity and a 16-hr photoperiod for 22 days in 0.008 m\u003csup\u003e3\u003c/sup\u003e of production space per plant. This 75 g serving provides all the necessary phylloquinone and 63%, 2.7%, and 97% of ascorbic acid, thiamine, and vitamin A precursor, respectively. Again, mineral nutrients are present but minimal, providing 1.1, 1.3, 0.9, and 4.9% of Ca, K, Mg, and Fe (RDI).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEnergy Considerations\u003c/h2\u003e \u003cp\u003eIt is important to note that the conditions which provide the highest quality \u0026lsquo;Red Hybrid\u0026rsquo; plants may not be the most energy efficient, and energy use is at times more important than crop quality. Once normalized on a yield per MJ of energy spent basis it is apparent that the low light-intensity treatments provide more fresh mass per MJ than any other treatment, regardless of photoperiod (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This starkly contrasts the trends presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, in which fresh mass generally increases with increasing light intensity, and those produced by the final scoring, in which score is based on normalized means and weighted multipliers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Digging deeper and looking into the phytonutrient yield per MJ we see a similar trend, where it is almost always more efficient to use the lowest light intensity evaluated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The outstanding exception to this is zeaxanthin, where the gains seen at the 800 \u0026micro;mol\u0026sdot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026sdot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e by 16-hr treatment are large enough to offset the energy expenditure. Little direction emerges in the phylloquinone efficiency, but this is to be expected due to its lack of statistical difference when previously tested.\u003c/p\u003e \u003cp\u003eWhen choosing a lighting protocol for crop production to supplement the astronaut diet, it seems the largest question at play will be one of balancing efficiency vs. peak nutritional output. This question may require more investigation, or it may simply be a shifting target, as different missions and applications will carry different priorities. Regardless, the data presented here can help to inform these decisions, as new lenses could be applied to the dataset as new needs or priorities arise.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the NASA Space Biology Program grant 80NSSC22K0205. We gratefully thank Dr. John Munafo for his thoughtful review, Lauren Little for plant production assistance, Hannah Schmidt for phytonutrient quantification assistance, Spencer Givens, Kathleen Coffman, and Maggie Whittington for data collection assistance, Horticulture Lighting Group for LED fixtures, and Jiffy Growing Solutions for substrate. We also acknowledge the Mississippi State University Soil Lab for mineral quantification. The use of trade names in this publication does not imply endorsement by the University of Tennessee of products named nor criticism of similar ones not mentioned.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eED performed the experiment, analyzed the data, and prepared the manuscript. SA aided in phytonutrient quantification and method development. GM and KW provided experimental guidance and reviewed and revised the manuscript. KW conceptualized and designed the study and obtained funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available upon request by the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResearch support of LED fixtures and substrate were provided by Horticulture Lighting Group and Jiffy Growing Solutions, respectively. KW has consulted for Vitamin Greens and received compensation. ED owns stock in Plenty. GM and SA declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePerchonok, M. H., Cooper, M. R. \u0026amp; Catauro, P. M. 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Demmig-Adams, B., Garab, G., Adams III, W., \u0026amp; Govindjee) 583\u0026ndash;603 (Springer Netherlands, Dordrecht, 2014). \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Astronaut, mizuna, anthocyanins, thiamine, phylloquinone, carotenoids","lastPublishedDoi":"10.21203/rs.3.rs-4338874/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4338874/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBeyond mere caloric intake, the nutritional quality of food will be key to maintaining astronaut health during exploration-length missions. The production of leafy greens aboard spacecraft can help to provide consistent daily nutrition; however, maximizing the nutritional yield of each plant will be key to sustainable and efficient dietary supplementation. \u003cem\u003eBrasssica rapa\u003c/em\u003e ssp. \u003cem\u003enipposinica\u003c/em\u003e \u0026lsquo;Red Hybrid\u0026rsquo; was grown under environmental conditions similar to those of the International Space Station and the effect of light intensity and photoperiod on nutritional and biomass yields were evaluated. Four light intensities (200, 400, 600, and 800 \u0026micro;mol\u0026middot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026middot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) applied over a 16- or 24-hr photoperiod were implemented and the resulting concentrations of ascorbic acid, thiamine, phylloquinone, β-carotene, lutein, zeaxanthin, total anthocyanins, calcium, potassium, magnesium, and iron were quantified. Providing 800 \u0026micro;mol\u0026middot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026middot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e over a 16-hr photoperiod produced the best nutritional profile for supplementing the astronaut diet, offering the following percentages of recommended daily intake per 75 g serving: 100% phylloquinone, 63% ascorbic acid, 2.7% thiamine, and 97% retinol (from precursor β-carotene). However, when evaluated for light use efficiency (yield\u0026middot;MJ \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), the 200 \u0026micro;mol\u0026middot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026middot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e treatments (16- and 24-hr) performed better, yielding more mass and phytonutrients per MJ of energy utilized.\u003c/p\u003e","manuscriptTitle":"Light intensity and photoperiod: Tools for improving the phytonutrient profile of Brassica rapa ssp. nipposinica for supplementing the space diet","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-08 11:07:20","doi":"10.21203/rs.3.rs-4338874/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-26T17:22:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-22T13:12:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-22T09:17:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"313465675620294131311143232658315000448","date":"2024-08-09T13:44:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"31175786824582703705156428977471476301","date":"2024-07-24T13:32:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"58301280042158577521697774265686474678","date":"2024-07-02T10:42:21+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-03T08:34:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-03T08:07:21+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-05-02T19:36:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-02T19:33:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-04-28T17:17:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dfd516a5-d4fe-44c2-a756-4f16cbb60ba1","owner":[],"postedDate":"May 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-04T16:42:21+00:00","versionOfRecord":{"articleIdentity":"rs-4338874","link":"https://doi.org/10.1038/s41598-025-11662-y","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-29 16:21:16","publishedOnDateReadable":"July 29th, 2025"},"versionCreatedAt":"2024-05-08 11:07:20","video":"","vorDoi":"10.1038/s41598-025-11662-y","vorDoiUrl":"https://doi.org/10.1038/s41598-025-11662-y","workflowStages":[]},"version":"v1","identity":"rs-4338874","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4338874","identity":"rs-4338874","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}