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Open Access

Peer-reviewed

Research Article

Influence of day and night temperature and carbon dioxide concentration on growth, yield, and quality of green butterhead and red oakleaf lettuce

Abstract

Production of lettuce (Lactuca sativa) within vertical farms is an expanding segment of controlled environment agriculture—precise manipulation of environmental parameters including mean daily temperature (MDT) and carbon dioxide (CO2) concentration enables year-round production, alongside color, yield, and crop size regulation. Our objectives included 1) quantify how MDT and CO2 interact to influence lettuce growth, development, and quality; 2) model lettuce growth under several MDTs and CO2 concentrations. Green butterhead ‘Rex’ and red oakleaf ‘Rouxaï RZ’ seedlings were transplanted into hydroponic tanks under a photosynthetic photon flux density of 300 μmol·m‒2·s‒1 for 17-h·d‒1. CO2 concentrations of 500, 800, or 1200 μmol·mol−1 and day/night and MDT setpoints of 22/15°C (MDT 20°C), 25/18°C (23°C), or 28/21°C (26°C) were maintained within growth chambers. ‘Rex’ fresh mass increased linearly with MDT, increasing by 18% from 20 to 26°C. ‘Rouxaï RZ’ fresh mass increased quadratically with MDT, with a 32% increase from 20 to 23°C, then a 7% increase from 23 to 26°C. Elevating CO2 concentrations from 500 to 800 μmol·mol–1 increased ‘Rouxaï RZ’ and ‘Rex’ fresh mass by 33 and 16%, respectively, while fresh mass did not increase from 800 to 1200 μmol·mol–1. Both cultivars had the greatest dry mass at 800 μmol·mol–1 CO2 across temperatures. At a high MDT, ‘Rouxaï RZ’ foliage color became more light, vibrant, and green, while a low MDT induced darker, grayer, and redder foliage. Tipburn occurred on ‘Rex’ across treatments, while 25% of ‘Rouxaï RZ’ were afflicted at 500 μmol·mol−1 CO2 and 67% at 1200 μmol·mol−1. At the light intensity studied, we recommend growing ‘Rex’ and ‘Rouxaï RZ’ at an 800 μmol·mol−1 CO2 concentration and MDT of 23°C for greatest biomass and leaf number, and slightly redder foliage in ‘Rouxaï RZ’ than at a 26°C MDT.

Citation: Tarr ST, Lopez RG (2025) Influence of day and night temperature and carbon dioxide concentration on growth, yield, and quality of green butterhead and red oakleaf lettuce. PLoS ONE 20(2): e0313884. https://doi.org/10.1371/journal.pone.0313884

Editor: Elias Kaiser, Wageningen University, NETHERLANDS, KINGDOM OF THE

Received: July 13, 2024; Accepted: November 3, 2024; Published: February 6, 2025

Copyright: © 2025 Tarr, Lopez. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The data underlying the results presented in the study are available at figshare:S1 Dataset. Rex and Rouxai CO2 Temperature Data.xlsx (DOI: 10.6084/m9.figshare.27161685.v2).

Funding: This work was supported by Michigan State University AgBioResearch, the USDA National Institute of Food and Agriculture Hatch project nos. MICL02472 and USDA-NIFA Specialty Crop Research Initiative award no. 2019-51181-30017. There was no additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Lettuce (Lactuca sativa), as a global economic crop, plays a crucial role in the agricultural sector, notably in the United States, where it accounted for a staggering $3.5 billion in wholesale production in 2019 [1]. Although a significant proportion of leaf and romaine lettuce is field grown in California and Arizona, a rising demand for locally grown leafy greens has sparked interest in controlled environments (CE) production. From 2014 to 2019, sales of lettuce grown in CE increased by 28% due to benefits such as consistent production during off-seasons and stable pricing throughout the year [2, 3].

Vertical farms, warehouses, and shipping containers are among the common facilities used in CE food production. They enable the precise control of environmental conditions such as light duration, quantity, and quality; day and night temperatures; air flow; vapor pressure deficit (VPD); and carbon dioxide (CO2) concentration [3–5]. This precision, particularly in controlling factors such as mean daily temperature (MDT) and CO2 concentration, has a profound effect on plant growth, development, flavor, color, and nutrient content. These environmental control capabilities combined with lettuce’s compact growth, short production time, and high market demand make it an ideal candidate for CE production, potentially improving harvest frequency and space efficiency [5, 6].

Temperature plays a pivotal role in plant growth; the rate of development of lettuce increases linearly with increasing temperature, from a species-specific base temperature (Tb) up to an optimum temperature (Topt), after which the developmental rate declines [7]. Excessive temperatures late in the growing cycle can induce bolting in lettuce, reducing overall quality as heads become loose, leaves become bitter, and tipburn risk increases. Additionally, temperature can influence crop quality aspects such color in red-leaf lettuce cultivars. Like many plants, lettuce uses C3 carbon fixation, which is responsive to atmospheric CO2 concentrations. Elevating CO2 availability decreases photorespiration, raising the photosynthetic rate and light saturation point, thus increasing growth until a species-specific CO2 concentration saturation point is reached [8, 9]. There is an initial high impact on growth as CO2 is elevated above ambient levels (~420 μmol·mol‒1) but benefits diminish as the saturation point is approached [8, 9]. CO2 and MDT interact to influence growth and development—at greater CO2 concentrations, the Topt for photosynthesis typically increases [9, 10]. Beyond the Topt, the rate of carboxylation to oxygenation decreases, reducing photosynthetic efficiency due to photorespiration [10].

The impact MDT has on the growth and development of lettuce has been investigated previously [5, 11, 12]. For example, Ouyang et al. (2020) grew ‘Grand Rapids TBR’ at 16, 18, and 20°C under a continuous photosynthetic photon flux density (PPFD) of 210 μmol∙m–2∙s–1 for 30 days after transplant [12]. Lettuce shoot fresh mass (SFM) and height was 38 and 18% (9.9 g and 1.9 cm) greater at 20°C than at 16°C and shoot dry mass (SDM) was 14% (0.5 g) greater at 18 or 20°C than at 16°C [12].

The influence of CO2 concentration on lettuce growth and development in CEs has also been investigated previously [13–16]. SFM of lettuce ‘Blonde of Paris Batavia’ and ‘Oak Leaf’ increased by 55 and 77% (46 and 34 g), respectively, when the CO2 concentration was raised from 400 to 700 μmol·mol–1 under a PPFD of 400 μmol∙m–2∙s–1 at day/night (14 h/10 h) temperatures of 25/18°C [16]. Additionally, the photosynthetic rate, apparent quantum yield, antioxidant capacity, and water-use efficiency increased for both cultivars [16].

Given the strong influence of MDT and CO2 concentration on lettuce growth, development, and quality, identifying the environmental parameters for improved resource-use efficiency and yield in CEs is needed. The objectives of this study were to 1) quantify how MDT and CO2 concentration influence lettuce growth, development, quality, and yield; and 2) create models for predicting growth and development under various MDTs and CO2 concentrations. We hypothesized that increasing CO2 concentration would increase biomass production of lettuce across all temperatures, but there would be less of an effect shifting from the moderate to the highest tested CO2 concentration compared to the shift from the low to medium CO2 concentrations.

Materials and methods

Plant material and propagation conditions

On 28 Apr. 2020, 09 June 2020, 27 July 2020, 16 Sept. 2020, 12 Nov. 2020, and 07 Jan. 2021, and 20 Feb. 2021, seeds of red oakleaf lettuce ‘Rouxaï RZ’ and green butterhead lettuce ‘Rex’ (Rijk Zwaan; Salinas, CA, USA) were sown into 200-cell (2.5 cm × 2.5 cm) rockwool plugs (AO 25/40 Starter Plugs; Gordan, Milton, ON, Canada). The cultivars were selected due to their use in previous indoor production studies and commercial relevance. Plugs were placed in trays and presoaked in deionized water with a pH of 4.4 to 4.5 adjusted using diluted (1:31) 95 to 98% sulfuric acid (J.Y. Baker, Inc.; Phillipsburg, NJ, USA). The trays were covered with translucent plastic domes for 3 d to maintain high humidity during germination. Trays were placed in a 2.4-m-wide, 4.1-m-long, 2.4-m-tall walk-in growth chamber (Hotpack environmental room UWP 2614–3; SP Scientific, Warminster, PA, USA) with an MDT of 22°C, CO2 concentration of 500 μmol·mol‒1, and relative humidity of 60%. Light-emitting diode fixtures (Ray66 Indoor PhysioSpec; Fluence Bioengineering, Austin, TX, USA) provided a total photon flux density (400–800 nm) of 180 μmol∙m–2∙s–1 and a light ratio (%) of 19:39:39:3 blue (400–500 nm): green (500–600 nm): red (600–700 nm): far-red (700–800 nm) radiation for 24 h. After 3 d, the photoperiod was reduced to 20 h until transplant at 11 d. Seedlings were sub-irrigated with deionized water supplemented with water-soluble fertilizer providing (in mg∙L–1): 125 N, 18 P, 138 K, 73 Ca, 47 Mg, 1.56 Fe, 0.52 Mn, 0.36 Zn, 0.21 B, 0.21 Cu, 35 S, and 0.01 Mo (12N–1.8P–13.3K RO Hydro FeED; JR Peters, Inc., Allentown, PA, USA). The pH and electrical conductivity (EC) were adjusted to 5.6 and 1.6 dS·m–1, respectively, as determined with a pH/EC probe (HI 991, 301 pH/TDS/ Temperature Monitor; Hanna Instruments, Smithfield, RI, USA). The pH was adjusted using potassium bicarbonate and sulfuric acid, while the EC was adjusted by adding deionized water and concentrated nutrient solution.

Hydroponic systems

Eleven days after sowing, 14 seedlings of each cultivar were transplanted 20-cm-apart into six 250 L, 0.9-m-wide by 1.8-m-long deep-flow hydroponic systems (Active Aqua premium high-rise flood table; Hydrofarm, Petaluma, CA, USA) distributed within walk-in growth chambers described previously. Each hydroponic system contained a 4-cm-thick extruded polystyrene foam sheet to float on the nutrient solution. Plastic net baskets were placed into 4-cm-diameter holes in the polystyrene foam, and seedlings were placed in the baskets, so the rockwool was in contact with the nutrient solution. Deionized water supplemented with water-soluble fertilizer providing (in mg·L–1) 150 N, 22 P, 166 K, 87 Ca, 25 Mg, 1.9 Fe, 0.62 Mn, 0.44 Zn, 0.25 B, 0.25 Cu, and 0.01 Mo (12N–1.8P–13.3K RO Hydro FeED; JR Peters, Inc.), and 0.31 g·L–1 magnesium sulfate (Pennington Epsom salt; Madison, GA, USA). The EC and pH were adjusted daily to maintain an EC of 1.7 dS·m–1 and pH of 5.6, as described previously. Air pumps (Active Aqua 70 L·min–1 commercial air pump; Hydrofarm) connected to air stones (Active Aqua air stone round 10.2 cm × 2.5 cm; Hydrofarm) were used to increase the dissolved oxygen concentration.

Growth chamber environmental conditions

The air day/night (17 h/7 h) and MDT set points in each growth chamber were 22/15 (20°C), 25/18 (23°C), or 28/21 (26°C), measured every 5 s by a resistance temperature detector (Platinum RTD RBBJL-GW05A-00-M 36B; SensorTec, Inc., Fort Wayne, IN, USA) and logged by a C6 controller (Environmental Growth Chambers, Chagrin Falls, OH, USA). CO2 was maintained at 500, 800, or 1200 μmol·mol–1 in each chamber with compressed CO2 injection, measured with a CO2 sensor (GM86P; Vaisala, Helsinki, Finland) and logged by a C6 Controller (Environmental Growth Chambers) every 5 s. A positive fixed difference between air and night temperatures (DIF) of 7°C was used for each temperature treatment. Relative humidity had a target setpoint of 60%. PPFDs of 300 μmol∙m –2∙s–1 were provided for 17 h∙d–1 by LED fixtures (Ray66; Fluence Bioengineering), providing a DLI of 18.4 mol∙m –2∙d–1, averaged over several measurements (Table 1). The LEDs were mounted 95 cm above the crop canopy. Every 30 s, water temperature, leaf temperature, and PPFD were measured using a thermistor (ST-100; Apogee Instruments, Logan, UT, USA), infrared thermocouple (OS36-01-T-80F; Omega Engineering, INC. Norwalk, CT, USA), and quantum sensor (LI-190R; LI-COR Biosciences, Lincoln, NE, USA), respectively, with means logged every hour by a CR-1000 datalogger (Campbell Scientific, Logan, UT, USA). The average horizontal air speed was approximately 0.37 m⋅s−1 over the growing canopy measured with an anemometer (HHF803; Omega Engineering, Norwalk, CN, USA). Vertical airflow and inter-canopy airflow was not provided or assessed.

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Table 1. Mean (± SD) day and night air, canopy, and water temperatures; carbon dioxide (CO2) concentrations; photosynthetic photon flux density (PPFD); and vapor pressure deficit (VPD) during 36 or 37 days of indoor deep flow hydroponic production for butterhead lettuce ‘Rex’ and red oakleaf lettuce ‘Rouxaï RZ’, respectively.

https://doi.org/10.1371/journal.pone.0313884.t001

Growth data collection and analysis

Parameters assessed for lettuce quality included the foliage coloration of ‘Rouxaï RZ’, relative chlorophyll concentration (RCC), the maximum photosystem II quantum yields (Fv/Fm), and the dry mass. The foliage coloration of ten ‘Rouxaï RZ’ plants in each treatment was measured 35 d after sowing with a tristimulus colorimeter (Chroma Meter CR-400; Konica Minolta Sensing, Inc., Chiyoda, Tokyo), reported as International Commission on Illumination (CIE) L*a*b* color space values, which were then converted to hue angle (h°) and chroma (C*). The RCC of the most recent fully expanded leaf of ten plants of each cultivar in each treatment was then estimated with a SPAD meter (MC-100 Chlorophyll Meter; Apogee Instruments, Logan, UT, USA). One leaf of ten plants per treatment was then dark acclimated for >15 min using three of the manufacturer-supplied clips and then exposed to 3500 μmol·m–2·s–1 of red radiation (peak wavelength 650 nm) to saturate photosystem II and the fluorescence was measured, averaged, and reported as Fv/Fm by a portable chlorophyll fluorescence meter (Handy Plant Efficiency Analyzer; Hansatech Instruments Ltd., Norfolk, UK).

‘Rouxaï RZ’ and ‘Rex’ were harvested 36 and 37 d after sowing, respectively. SFM (g), length and width (cm) of the sixth fully expanded leaf, and leaf number (when >5 cm) were measured on ten plants of each cultivar per treatment. Plant height from the roots to the highest point of the foliage and the foliage diameter at the widest point and perpendicular to the widest point was measured with a ruler and recorded. Incidence, but not severity, of tipburn was recorded. Plants were observed for bolting and no bolting was observed. To provide an integrated measurement of plant size, the growth index (GI) was calculated using the crop height and two foliage diameters mentioned previously (GI = {plant height + [(diameter 1 + diameter 2)/2]}/2). The plant material was placed in a forced-air drier maintained at 75°C for at least 3 d, weighed, and the SDM was recorded.

The study was conducted using a randomized block design, where the blocks were defined by sequential time periods. Each block corresponded to a two-month cycle, resulting in six blocks (T1 through T6). Within each block, a single CO2 treatment (500, 800, or 1200 μmol·mol–1) was applied to all three growth chambers. The CO2 treatments were assigned to blocks in the following sequence: 500 μmol·mol–1 in T1 and T2, 800 μmol·mol–1 in T3 and T4, and 1200 μmol·mol–1 in T5 and T6. Within each CO2 treatment block, all three temperature treatments were randomized across the chambers. This randomization was replicated, resulting in each CO2 treatment being paired with each temperature treatment twice before advancing to the next CO2 level. The temperature treatments were distributed between each harvest within each CO2 block to ensure randomization.

This design allowed for the examination of the interaction between CO2 concentration and temperature on lettuce growth, with each growth chamber serving as the experimental unit for the temperature treatments within the given CO2 treatment block. Data were collected from 10 randomly selected lettuce plants in each growth chamber at the end of each cycle, providing a total of 20 plants sampled per treatment combination per time period.

Data were analyzed separately by cultivar with SAS (version 9.4; SAS Institute, Cary, NC, USA) mixed model procedure (PROC MIXED) for analysis of variance (ANOVA), tests of normality and homogeneity of variances were performed, and pairwise comparisons were performed with Tukey-Kramer difference test (p ≤ 0.05). SigmaPlot (version 14.5, Systat Software, Inc., San Jose, CA, USA) was used for regression analysis.

Results

Shoot fresh and dry mass

The SFM of ‘Rouxaï RZ’ increased quadratically with MDT; from 20 to 23°C, SFM increased by 32% (41.6 g), then 7% (12.9 g) from 23 to 26°C (Tables 2 and 3; Fig 1A). ‘Rex’ SFM increased linearly by 18% (28.0 g) from 20 to 26°C (Tables 2 and 3; Fig 1C). Both cultivars showed quadratic increases in SFM as CO2 concentration increased (Table 2). Elevating CO2 from 500 to 800 μmol·mol–1 resulted in SFM increasing by 33 and 16% (46.5 and 24.4 g) for ‘Rouxaï RZ’ and ‘Rex’, respectively, without additional biomass accumulation as CO2 increased from 800 to 1200 μmol·mol–1 (Tables 2 and 3; Fig 1B, 1D).

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Fig 1. Effects of mean daily temperature (MDT; 20, 23, and 26°C) and carbon dioxide (CO2 concentration; 500, 800, 1200 μmol·mol–1) on red oakleaf lettuce ‘Rouxaï RZ’ fresh mass (A and B) and growth index (E), and green butterhead lettuce ‘Rex’ fresh mass (C and D).

Model predictions are represented by lines; coefficients are in Table 3; error bars represent standard errors.

https://doi.org/10.1371/journal.pone.0313884.g001

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Table 2. Influence of mean daily temperature (MDT;°C) and carbon dioxide (CO2; μmol·mol–1) on height and width (cm); growth index; leaf length and width (cm) and number; shoot fresh and dry mass (g); hue angle (h°); chroma (C*); International Commission on Illumination (CIE) L* color value of red oakleaf lettuce ‘Rouxaï RZ’ and green butterhead lettuce ‘Rex’.

Data represent the mean of two replications and cultivars with 10 samples. Analyses of variance for the effects of MDT and CO2 and their interaction are included below each cultivar mean. Within-column means of a given treatment with different letters were significantly different according to Tukey’s honestly significant difference test (P < 0.05).

https://doi.org/10.1371/journal.pone.0313884.t002

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Table 3. Equations of regression analysis and r2 or R2 for mean shoot fresh and dry mass (g), growth index, leaf number, and leaf length (cm) in response to mean daily temperature (MDT; 20, 23, and 26°C) and carbon dioxide (CO2 concentration; 500, 800, 1200 μmol·mol–1) of green butterhead lettuce ‘Rex’ and red oakleaf lettuce ‘Rouxaï RZ’.

All models are in the form of: ƒ = y0 + a*MDT + b*CO2 + c*MDT2 + d*CO22 + e*MDT*CO2.

https://doi.org/10.1371/journal.pone.0313884.t003

MDT and CO2 concentration interacted to influence SDM of both cultivars (Tables 2 and 3; Fig 2A and 2B). Regardless of MDT, the greatest SDM (~7.1 and ~7.9 g for ‘Rouxaï RZ’ and ‘Rex’, respectively) was recorded at a CO2 concentration of 800 μmol·mol–1, while the lowest was at 20°C and a CO2 concentration of 500 or 1200 μmol·mol–1 (5.0 and 6.2 g for ‘Rouxaï RZ’ and ‘Rex’, respectively). ‘Rex’ SDM increased from 20 to 23°C at 500 and 1200 μmol·mol–1 CO2.

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Fig 2.

Effects of carbon dioxide concentration (CO2; 500, 800, 1200 μmol·mol–1) and mean daily temperature (MDT; 20, 23, and 26°C) on green butterhead lettuce (Lactuca sativa) ‘Rex’ shoot dry mass (A), leaf number (C), and growth index (E) and red oakleaf lettuce ‘Rouxaï RZ’ shoot dry mass (B), leaf number (D), and leaf width (F). Model predictions are represented by response surfaces; coefficients are in Table 3.

https://doi.org/10.1371/journal.pone.0313884.g002

Morphology

‘Rouxaï RZ’ GI increased linearly with MDT, while the GI of ‘Rex’ was influenced by an interaction of CO2 and MDT (Tables 2 and 3; Figs 1E and 2E). ‘Rouxaï RZ’ GI increased by 19% as MDT increased from 20 to 26°C. GI of ‘Rex’ increased by 20% as MDT and CO2 concentration increased from 20°C and 500 μmol·mol –1 CO2 to 23°C and 1200 μmol·mol ‒ 1 CO2, respectively, or an MDT of 26°C.

Leaf number was influenced by the interaction of MDT and CO2 concentration for both cultivars (Tables 2 and 3; Fig 2C and 2D). The leaf number of ‘Rex’ was primarily influenced by the MDT, and as MDT increased from 20 to 23°C and 23 to 26°C, an average of 5 and 8 more leaves unfolded, respectively. The fewest number of leaves (22 leaves) was observed for ‘Rex’ at a CO2 concentration of 500 or 1200 μmol·mol‒1 at an MDT of 20°C, while the greatest number of leaves (43) was at a CO2 concentration of 1200 μmol·mol‒1 and MDT of 26°C. However, ‘Rouxaï RZ’ unfolded 21 leaves at an MDT of 20°C and an additional 8 leaves at 23°C, regardless of CO2 concentration. Only 4 additional leaves unfolded as the MDT increased from 23 to 26°C at a CO2 concentration of 1200 μmol·mol‒1.

Leaf length of both cultivars and leaf width of ‘Rouxaï RZ’ were influenced by the interaction of MDT and CO2, while leaf width of ‘Rex’ was influenced only by MDT (Tables 2 and 3; Fig 2F). Leaves of ‘Rouxaï RZ’ were 22% (2.7 cm) longer and 15% (2.9 cm) wider at an MDT of 26°C and CO2 concentration of 1200 μmol·mol‒1 than those grown at 20°C and 500 μmol·mol‒1 CO2. At 500 μmol·mol‒1 CO2, ‘Rex’ leaves were 10% longer at an MDT of 26°C than at 20°C, and were 4% wider at 20°C than at 26°C.

Tipburn incidence, recorded as the percentage of plants affected, was 25% and 67% at CO2 concentrations of 500 and 1200 μmol·mol‒1 for ‘Rouxaï RZ’. Tipburn was present on all of ‘Rex’ irrespective of MDT or CO2 concentration.

Fv/Fm, relative chlorophyll concentration, and pigmentation

The maximum photosystem II quantum yields (Fv/Fm) ranged from 0.813 to 0.861, suggesting minimal impacts of stress on photosynthetic reactions. For ‘Rex’, the relative chlorophyll concentrations were 7% lower at 23°C (30.6) than at 20 or 26°C (32.6)

Foliage pigmentation of ‘Rouxaï RZ’ was influenced by MDT (Table 2). As MDT increased from 20 to 26°C, h° increased from 71.4 to 89.2, C* from 6.6 to 9.1, and L* from 29.9 to 32.5. These greater h°, C*, and L* values correspond to slightly lighter and more vibrant green foliage than the darker-gray, more yellow/red foliage with lower values.

Discussion

Lettuce sales and yield are primarily determined by marketable fresh mass, with time to harvest and quality parameters such as color, plant size, and tipburn incidence being of particular importance to growers. Lettuce biomass accumulation varies by cultivar [17–19] and depends on stage of growth and time to harvest [20, 21], and environmental conditions, including PPFD [17, 19, 22], MDT [17, 20, 23], and CO2 concentration [13, 21, 24]. In the present study, SFM of ‘Rouxaï RZ’ and ‘Rex’ were influenced by CO2 and MDT independently, while SDM was influenced by the interaction of CO2 and MDT. SFM of both cultivars increased as MDT, with a 6°C positive difference in day and night temperature, increased from 20 to 26°C, with ‘Rex’ and ‘Rouxaï RZ’ following linear and quadratic responses, respectively (Table 2; Figs 1A, 1C; 2A and 2B). The quadratic response of ‘Rouxaï RZ’, with reduced SFM gain from the 23 to 26°C MDTs, may indicate the optimal temperature was being reached, after which a plateau and decline will occur until growth ceases and the maximum temperature is reached. Given this reduced SFM benefit at the higher MDT, growers should consider if the yield increase at 26°C is worthwhile compared to the energy cost of maintaining the higher temperature. Overall, the temperature response we saw aligns with other studies observing increased growth rate and biomass accumulation under heightened MDTs in lettuce [5, 20]. The recommended MDT for lettuce varies by variety and cultivar, but Choi et al. (2000) suggested a 22–26°C day and 15–20°C night temperature during the early and middle growth stages, followed by a 20–24°C and 15–20°C during the later stages of growth for butterhead ‘Omega’ and leaf lettuce ‘Grand Rapids’. In that study, the rate of photosynthesis and transpiration 15, 25, and 35 days after treatment of day/night temperatures of 10/7, 20/15, and 30/25°C, the high temperature treatment of 30/25°C had the greatest photosynthetic and transpiration rates at 25 days, while 20/15°C had the greatest at 35 days after treatment [20]. Altering the temperature based upon growth stages may provide opportunities for further optimization within plant factories if divisions within the facilities allow for temperature control between planting dates of crops.

In the present study, SFM of both cultivars only increased as the CO2 concentration was raised from 500 to 800 μmol·mol–1, with no further increases from 800 to 1200 μmol·mol–1 (Table 2). Caplan (2018) reported a similar response: butterhead lettuce ‘Fairly’ grown at an MDT of 22°C under PPFDs ranging from 156 to 330 μmol·m−2·s−1 and CO2 concentrations from 400 to 1300 μmol·mol–1 had the greatest SFM and SDM at 850 μmol·mol−1 CO2, while yields decreased as CO2 concentration increased [25]. Conversely, the SFM of lettuce ‘Partavousi’ increased by 6 and 55% as CO2 concentration increased from 400 to 800 and 800 to 1200 μmol·mol−1, respectively, under a PPFD of 300 μmol·m−2·s−1 and at an MDT of 25°C for 40 d, without additional biomass accumulation at CO2 concentrations of 1200 to 1600 μmol·mol−1 [21]. The reduced impact on growth at higher CO2 concentrations in the present study may be due to the CO2 saturation point being reached for the tested cultivars, after this saturation point the photosynthetic rate would no longer be limited by CO2 availability, but instead factors such as light, nutrients, or enzyme capacity. Additionally, there may have been other factors limiting lettuce growth once CO2 was elevated, such as the onset of tipburn due to the lack of vertical or inter-canopy airflow while under rapid growth conditions.

SDM was influenced by the interaction of CO2 and MDT for both cultivars (Tables 2 and 3; Fig 2A and 2B). Regardless of MDT, the greatest SDM occurred at a CO2 concentration of 800 μmol·mol−1, while at higher or lower CO2 concentrations, SDM was primarily influenced by MDT. Esmaili et al. observed that the SDM of ‘Partavousi’ increased by 31 and 147% as CO2 concentration increased from 400 to 800 and 800 to 1200 μmol·mol−1, respectively, with similar SDM at 1200 and 1600 μmol·mol−1 [21]. Within the conditions of our study, the 800 μmol·mol−1 CO2 concentration appears to be optimal for biomass accumulation for both SFM and SDM across temperature treatments.

The GI, a measure of plant size that integrates plant height and width, was greatest at an MDT of 26°C for both cultivars, while ‘Rex’ was marginally influenced by the interaction of CO2 and MDT (Tables 2 and 3; Figs 1E and 2E). Tarr et al. reported MDT only influencing the GI of ‘Rouxaï RZ’ as it increased from 20 to 23°C [17]. The size of plants can impact recommended planting density and packaging of heads of lettuce into clamshells; understanding that size increases within MDTs of 20 to 26°C enables growers to adjust conditions based upon market preferences. Leaf size was influenced by the interaction of MDT and CO2, but primarily by MDT (Table 2). MDT has been suggested to have a greater impact on leaf mass area (leaf dry mass per leaf area) than CO2 concentration [26]. Both cultivars had the shortest leaves at 20°C, while ‘Rouxaï RZ’ leaves were narrowest and ‘Rex’ leaves were widest at 20°C.

The interaction of CO2 concentration and MDT impacted the leaf unfolding rate for both cultivars (Tables 2 and 3; Fig 2C and 2D). Leaf unfolding increased from 20 to 26°C when CO2 concentrations were pooled, as expected with developmental rates increasing up to the Topt [27]. The influence of CO2 and MDT on the leaf unfolding rate of lettuce is not well documented. Lettuce ‘Grand Rapids’ grown at an MDT of 16.7°C and ~500 μmol·mol−1 CO2 unfolded 3 more leaves than at 18.3°C and at CO2 concentrations of 200–400 μmol·mol−1 [14]. In CO2-limited conditions, the leaf unfolding rate and photosynthetic rate may be restricted as photorespiration occurs [8].

A major concern for CE lettuce producers is tipburn, the necrosis on a leaf margin induced by calcium deficiency [5, 22]. Lettuce undergoing rapid growth with limited transpiration at the growing point is susceptible to tipburn. In the current study, tipburn incidence of ‘Rouxaï RZ’ was greatest under a PPFD of 300 μmol∙m‒2∙s‒1 at a CO2 concentration of 1200 μmol·mol−1 (Table 2), while all ‘Rex’ treatments had signs of tipburn. We did not assess severity of tipburn, so distinctions between beginning signs of tipburn development on inner leaves at harvest and more severe tipburn occurrence was not made. When grown at an MDT of 22°C and under a PPFD of 330 μmol·m−2·s−1, tipburn occurrence in butterhead lettuce ‘Fairly’ was not observed at CO2 concentrations of 400 and 550 μmol·mol−1, but 10, 10, 25, and 33% of plants had tipburn at 700, 850, 1000, and 1300 μmol·mol−1 CO2, respectively [25]. The increased incidence of tipburn at elevated CO2 concentrations is likely due to a reduction in stomatal conductance [8, 25] as stomata close in elevated CO2 concentrations, reducing transpiration and, consequently, calcium movement to the growing point. MDT did not influence tipburn occurrence, aligning with Tarr et al. [17]. There are confounding reports in the literature about the influence of MDT on lettuce tipburn, as results varied by cultivar and environmental conditions [23, 28, 29]. The VPD may have influenced tipburn incidence in our study. We maintained a constant 60% relative humidity at 20, 23, and 26°C, which translates to VPDs of ~0.9, 1.1, and 1.3 kPa, respectively (Table 1). Transpiration rate can increase with VPD, potentially increasing calcium access at the growing point and reducing tipburn incidence, but this response varies depending on VPD exposure, as stomata can be closed to prevent waterloss [5, 28]. Tipburn occurrence may have been suppressed at the higher MDTs due to greater VPDs, and therefore affected how MDT may influence tipburn. However, the most likely cause of our high tipburn incidence is a lack of sufficient vertical airflow over the lettuce canopy under rapid growth conditions.

Marketability of crops is influenced by foliage color, with green foliage being undesirable in red-leaf cultivars [30]. Most red, blue, and purple coloration of foliage is primarily caused by anthocyanins [31]. At low temperatures, anthocyanins can accumulate in leaves, inducing darker, more pigmented foliage. The color of ‘Rouxaï RZ’ foliage at an MDT of 26°C was a lighter, more vibrant green than the darker, grayer yellow/red foliage at 20°C, which is consistent with other studies on foliage coloration [17, 32, 33].

Future studies comparing lettuce growth responses to CO2 concentrations and temperatures applied at different growth stages are needed to identify when supplemental inputs are most valuable. Esmaili et al. reported lettuce growth responses 10, 20, 30, and 40 d after sowing, with the greatest growth rate change occurring after 30 d [21]. However, this was using constant environmental conditions over the growth cycle, rather than comparing CO2 supplementation at different growth stages. Response to MDT may also vary by growth stage; relative growth rate of butterhead lettuce ‘Omega’ 25 d after transplant was greater at 30/25°C than 20/15°C, but by 35 d after transplant, relative growth rate was lowest at 30/25°C [20]. Identifying the growth stage that specific MDT and CO2 supplementation is most beneficial enables strategic applications for efficient input use.

In conclusion, under a PPFD of 300 μmol∙m‒2∙s‒1 and with a 6°C difference in day and night temperature, we recommend growing ‘Rex’ and ‘Rouxaï RZ’ at a CO2 concentration of 800 μmol·mol−1 and MDT of 23°C as this provided the greatest biomass and leaf number, kept plants moderately compact, and, for ‘Rouxaï RZ’, induced redder foliage than growth at 26°C. Increasing to 1200 a CO2 concentration of 800 μmol·mol−1 did not provide additional benefits for growth or quality parameters while increasing tipburn occurrence in ‘Rouxaï RZ’.

References

  1. 1. U.S. Department of Agriculture (USDA), 2020a. Crop Values 2019 Summary. National Agricultural Statistics Service. Washington DC. 11 December 2020. <https://www.nass.usda.gov/Publications/Todays_Reports/reports/cpvl0220.pdf>
    • 2. U.S. Department of Agriculture (USDA). 2020b. 2019 Census of Horticultural Specialties. U.S. Dept. Of Agr., Washington, D.C. 12 January 2021. <https://www.nass.usda.gov/Publications/AgCensus/2017/Online_Resources/Census_of_Horticulture_Specialties/HORTIC.pdf>
      • 3. Beacham A.M., Vickers L.H. and Monaghan J.M. 2019. Vertical farming: a summary of approaches to growing skywards. The J. of Hort. Sci. and Biotechnol. 94:277–283.
      • 4. Gómez C., Currey C.J., Dickson R.W., Kim H.J., Hernández R., Sabeh N.C., et al. 2019. Controlled environment food production for urban agriculture. HortScience 54, 1448–1458.
      • 5. Ahmed H.A., Yu-Xin T. and Qi-Chang Y. 2020. Optimal control of environmental conditions affecting lettuce plant growth in a controlled environment with artificial lighting: A review. South African J. of Bot. 130:75–89.
      • 6. McCartney L. and Lefsrud M. 2018. Protected agriculture in extreme environments: a review of controlled environment agriculture in tropical, arid, polar, and urban locations. Appl. Eng. in Agr. 34, 455–473.
      • 7. Blanchard M.G., Runkle E.S. and Fisher P.R. 2011. Modeling plant morphology and development of petunia in response to temperature and photosynthetic daily light integral. Scientia Hort. 129:313–320.
      • 8. Dusenge M.E., Duarte A.G. and Way D.A. 2019. Plant carbon metabolism and climate change: elevated CO2 and temperature impacts on photosynthesis, photorespiration and respiration. New Phytologist 221, 32–49.
      • 9. Mattson N. and Holley J. 2021. Carbon dioxide: Building block for plant growth, p. 234–256. In: Beytes C., (ed.) Ball RedBook: Greenhouse Structures, Equipment, and Technology, 19th ed. Ball Publishing, West Chicago, IL.
        • 10. Ainsworth E.A. and Rogers A. 2007. The response of photosynthesis and stomatal conductance to rising [CO2]: mechanisms and environmental interactions. Plant, Cell and Environ. 30, 258–270.
        • 11. Heins R.D., Liu B. and Runkle E.S. 1998. Regulation of crop growth and development based on environmental factors. Acta Hort. 17–28.
        • 12. Ouyang Z., Tian J., Yan X. and Shen H. 2020. Effects of different concentrations of dissolved oxygen or temperatures on the growth, photosynthesis, yield and quality of lettuce. Agr. Water Mgt. 228, 105896.
        • 13. Kitaya Y., Niu G., Kozai T. and Ohashi M. 1998. Photosynthetic photon flux, photoperiod, and CO2 concentration affect growth and morphology of lettuce plug transplants. HortScience 33:988–991.
        • 14. Frantz J.M., 2011. Elevating carbon dioxide in a commercial greenhouse reduced overall fuel carbon consumption and production cost when used in combination with cool temperatures for lettuce production. HortTechnology 21, 647–651.
        • 15. Park Y.G., Park J.E., Hwang S.J. and Jeong B.R. 2012. Light source and CO2 concentration affect growth and anthocyanin content of lettuce under controlled environment. Hortic. Environ. Biotechnol. 53, 460–466.
        • 16. Pérez-López U., Miranda-Apodaca J., Muñoz-Rueda A. and Mena-Petite A. 2015. Interacting effects of high light and elevated CO2 on the nutraceutical quality of two differently pigmented Lactuca sativa cultivars (Blonde of Paris Batavia and Oak Leaf). Scientia Hort. 191, 38–48.
        • 17. Tarr S.T., Valle de Souza S.; Lopez R.G. 2023. Influence of day and night temperature and radiation intensity on growth, quality, and economics of indoor green butterhead and red oakleaf lettuce production. Sustainability. 15(1):829.
        • 18. Fu W., Li P. and Wu Y. 2012. Effects of different light intensities on chlorophyll fluorescence characteristics and yield in lettuce. Scientia Hort. 135:45–51.
        • 19. Kelly N., Choe D., Meng Q. and Runkle E.S. 2020. Promotion of lettuce growth under an increasing daily light integral depends on the combination of the photosynthetic photon flux density and photoperiod. Scientia Hort. 272:109565.
        • 20. Choi K.Y., Paek K.Y. and Lee Y.B. 2000. Effect of air temperature on tipburn incidence of butterhead and leaf lettuce in a plant factory, p. 166–171. In: Kubota C. & Chun C. (eds.). Transplant Production in the 21st Century. Springer Netherlands, Dordrecht.
          • 21. Esmaili M., Aliniaeifard S., Mashal M., Ghorbanzadeh P., Seif M., Gavilan M.U., et al. 2020. CO2 enrichment and increasing light intensity till a threshold level, enhance growth and water use efficiency of lettuce plants in controlled environment. Notulae Botanicae Horti Agrobotanici Cluj-Napoca 48, 2244–2262.
          • 22. Sago Y. 2016. Effects of light intensity and growth rate on tipburn development and leaf calcium concentration in butterhead lettuce. HortScience 51:1087–1091.
          • 23. Lee R.J., Bhandari S.R., Lee G. and Lee J.G. 2019. Optimization of temperature and light, and cultivar selection for the production of high-quality head lettuce in a closed-type plant factory. Hortic. Environ. Biotechnol. 60:207–216.
          • 24. Pérez-López U., Miranda-Apodaca J., Muñoz-Rueda A. and Mena-Petite A. 2013. Lettuce production and antioxidant capacity are differentially modified by salt stress and light intensity under ambient and elevated CO2. J. of Plant Phys. 170:1517–1525.
          • 25. Caplan, B., 2018. Optimizing carbon dioxide concentration and daily light integral combination in a multi-level electrically lighted lettuce production system. The Univ. of Arizona, Tucson, M.S. Thesis.
            • 26. Poorter H., Niinemets Ü., Poorter L., Wright I.J. and Villar R. 2009. Causes and consequences of variation in leaf mass per area (LMA): a meta-analysis. New Phytologist 182:565–588. pmid:19434804
            • 27. Hatfield J.L. and Prueger J.H. 2015. Temperature extremes: Effect on plant growth and development. Weather and Climate Extremes, USDA Res. and Programs on Extreme Events 10:4–10.
            • 28. Lee J.G., Choi C.S., Jang Y.A., Jang S.W., Lee S.G. and Um Y.C. 2013. Effects of air temperature and air flow rate control on the tipburn occurrence of leaf lettuce in a closed-type plant factory system. Hortic. Environ. Biotechnol. 54:303–310.
            • 29. Carotti L., Graamans L., Puksic F., Butturini M., Meinen E., Heuvelink E., et al. 2021. Plant factories are heating up: hunting for the best combination of light intensity, air temperature and root-zone temperature in lettuce production. Front Plant Sci. 11. pmid:33584743
            • 30. Runkle E. 2017. Factors that influence leaf coloration. Greenhouse Prod. News 27:38.
            • 31. Boldt J.K., Meyer M.H. and Erwin J.E. 2014. Foliar anthocyanins: a horticultural review, p. 209–252. In: Horticulture Reviews: Vol. 42. Wiley.
              • 32. Marin A., Ferreres F., Barberá G.G. and Gil M.I. 2015. Weather variability influences color and phenolic content of pigmented baby leaf lettuces throughout the season. J. Agr. Food Chem. 63, 1673–1681. pmid:25650715
              • 33. Walters, K.J. 2020. Controlled environment production impacts hydroponically grown culinary herb physiology, biochemistry, and consumer preference. Michigan State Univ., East Lansing, PhD Diss.