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. 2025 Sep 4;48(12):8712–8726. doi: 10.1111/pce.70141

Investigating the Impact of Elevated CO2 on Biomass Accumulation and Mineral Concentration in Foliar and Edible Tissues in Soybeans

Ravneet Kaur 1,2, Mary Durstock 3,4, Stephen A Prior 5, G Brett Runion 5, Elizabeth A Ainsworth 6, Ivan Baxter 7, Alvaro Sanz‐Sáez 3, Courtney P Leisner 1,2,✉
PMCID: PMC12586913  PMID: 40905342

ABSTRACT

Rising atmospheric carbon dioxide (CO₂) levels are expected to enhance biomass and yield in C3 crops. However, these benefits are accompanied by significant reductions in the concentrations of essential nutrients in both foliar and edible tissues, posing potential global nutritional challenges. In this study, we grew three soybean cultivars (Clark, Flyer, and Loda) in ambient ( ~ 438 ppm) and elevated CO₂ ( ~ 650 ppm) conditions using open top chambers and measured changes in leaf‐level physiological responses, biomass accumulation, and nutrient concentrations across developmental stages. Elevated CO₂ increased carbon assimilation and decreased stomatal conductance, which led to an increase in seed yield, while root biomass remained unchanged. Seed nutrient concentrations, particularly iron (Fe), zinc (Zn), manganese (Mn), boron (B), phosphorus (P), potassium (K), and magnesium (Mg), decreased at maturity. We hypothesize that reductions in seed mineral concentration resulted from enhanced carbon assimilation and biomass accumulation without a concomitant response in root biomass and nutrient uptake. This constrained the plant's ability to maintain nutrient status with increased yield at elevated CO₂, and this response was conserved across the cultivars included in this study. Future work is needed to further understand the molecular mechanisms associated with these physiological responses at elevated CO2 in soybean.

Keywords: carbon dioxide, nutrient uptake, photosynthesis, soybean

Summary Statement

  • Elevated CO₂ increased seed yield, reduced seed nutrient content, and did not increase root biomass across soybean (Glycine max L. Merr.) with limited cultivar‐based response variation.

  • We hypothesize increased yield, limited root uptake, and a small effect of reduced transpiration led to reduced seed quality.

1. Introduction

Since the industrial revolution in the late 1800s anthropogenic emissions of carbon dioxide (CO2) have increased (Friedlingstein et al. 2023; Intergovernmental Panel on Climate Change (IPCC) 2023). Consequently, the concentration of CO₂ in the Earth's atmosphere has reached levels unprecedented in human history on a global scale (Friedlingstein et al. 2023). Increased atmospheric CO2 concentrations increases leaf area index, biomass, and yield in C3 plants (Ferris et al. 1999; Dermody et al. 2006; Digrado et al. 2024). This increase in biomass is caused by the enhanced rate of photosynthesis with simultaneous decrease in stomatal conductance which drives an increase in water‐use efficiency (WUE) in C3 plants, resulting in a “fertilization” effect (Drake et al. 1997; Long et al. 2006; Loladze 2014; Myers et al. 2019; Ainsworth and Long 2021). Consequently, the projected rise in atmospheric CO2 concentrations to ~ 650 ppm by 2050 has the potential to positively impact world food production and address the needs of a growing population (Ciais. 2014).

Although there is strong evidence of increased photosynthesis and biomass in C3 plants grown in elevated CO2 (eCO2) conditions, a number of studies have shown this increase in biomass is accompanied by a significant reduction in protein, nitrogen (N) and several other mineral nutrients in plant foliar and edible tissues (Högy and Fangmeier 2009; Fernando et al. 2012; Loladze 2014; Dietterich et al. 2015; Myers et al. 2017; Soares et al. 2019). Recent meta‐analyses have shown C3 grains and legumes grown under eCO2 conditions projected for 2050 exhibit reduced zinc (Zn) and iron (Fe) levels; Zn decreased by 3.3%–9.3% and Fe decreased by 5.1%–5.2% in grasses, while Zn declined by 6.8%–5.11% and Fe declined by ~ 4.1% in legumes (Loladze 2002; Loladze 2014; Myers et al. 2019). This decrease in micronutrients in C3 grains and legumes has the potential to increase essential micronutrient deficiencies in developing and developed nations impacting around 2 billion individuals globally (Tulchinsky 2010; Loladze 2002). Furthermore, previous work has shown that plants grown in eCO2 also have a higher amount of total nonstructural carbohydrates (TNC) in comparison to the plants grown under ambient conditions (Loladze 2014). This in turn, changes in the ratio of TNC:protein and TNC:minerals with a stoichiometric impact of adding a spoonful of carbohydrates (about 5 g of a starch‐and‐sugar mixture) to every 100 g of dry plant tissue (Loladze 2014). The phenomenon where increased carbohydrate content in seeds and other plant parts leads to decreased nutrient content has been deemed mineral dilution (Poorter et al. 1997; Gifford et al. 2000; Taub and Wang 2008; Taub et al. 2008; Chaturvedi et al. 2017). Work in rice demonstrated that even when mineral bioavailability in the soil was high, nutrient translocation to grains under eCO₂ was insufficient to match elevated carbohydrate accumulation and offset mineral dilution (Guo et al. 2015).

In addition to mineral dilution, other physiological mechanisms have been proposed to explain the reduction in nutrient concentration in C3 plants grown in eCO₂ conditions. These include reduced leaf transpiration and associated declines in bulk nutrient flow (Mcgrath and Lobell 2013), as well as changes in root architecture or nutrient transporter activity that affect root mineral absorption and overall nutrient uptake (Beidler et al. 2015; Jauregui et al. 2016). While eCO₂ often leads to a higher root‐to‐shoot ratio and greater fine root production enhancing the plant's capacity for nutrient acquisition (Pritchard and Rogers 2000; Tausz‐Posch et al. 2014), this morphological expansion is not always accompanied by improved nutrient uptake. Some studies suggest root physiological activity may decline under eCO₂ despite increased biomass (BassiriRad et al. 2001), indicating that factors such as root turnover and soil nutrient availability mediate actual nutrient absorption. These mechanisms are not mutually exclusive, but empirical validation remains limited, particularly in important C3 grain and legume crops.

Soybean (Glycine max L. Merr.) is a widely grown C3 crop and a model legume known for its ability to fix atmospheric nitrogen via symbiosis with soil microorganisms (Schmutz et al. 2010). Soybean cultivars show substantial variation in biomass and yield responses to eCO₂ (Bishop et al. 2015; Sanz‐Sáez et al. 2017), and some exhibit reductions in seed mineral content, including Fe and Zn, in eCO₂ conditions (Loladze 2014; Myers et al. 2019). However, the physiological mechanisms underlying this variation remain unclear (Schmutz et al. 2010; Myers et al. 2019; Parvin et al. 2019; Soares et al. 2019).

In this study, we leveraged the phenotypic variation among soybean cultivars to examine potential mechanisms driving reductions in seed nutrient concentration under eCO₂. Using open‐top chambers (OTCs), we grew three cultivars (Clark, Flyer and Loda) with previously documented contrasting responses to eCO₂. Loda, which exhibits increased yield under eCO₂, (Sanz‐Sáez et al. 2017) was used to test the mineral dilution hypothesis. Clark, previously identified as nonresponsive in terms of yield and nutrient concentration (Myers et al. 2019), was used to assess the stability of nutrient uptake. Flyer, which shows significant declines in seed Zn concentration, was included to examine the role of altered transpiration and nutrient transport (Myers et al. 2019). Because Clark and Flyer differ in nutrient content response, we also evaluated whether differences in root biomass contribute to nutrient outcomes under eCO₂. Our findings aim to elucidate physiological drivers of nutrient loss in crops in eCO2 conditions and guide breeding strategies for nutrient‐retentive, climate‐resilient cultivars.

2. Materials and Methods

2.1. Plant Material and Experimental Conditions

Soybeans were grown at the USDA‐ARS National Soil Dynamics Laboratory in Auburn, AL, in OTCs (Figure S1A–D). The OTCs consisted of a cylindrical, aluminum metal frame that was 3 m wide and 2.4 m tall, with the bottom half covered with clear plastic, allowing the sunlight to penetrate and reach the plants (Rogers et al. 1983; Runion et al. 2023). The double‐walled plastic chamber cover included 2.5 cm perforations in the inner plastic wall to facilitate gas distribution within the chamber (Figure S1C). Eight chambers were used in the experiment: four set to ambient CO2 ( ~ 400 μmol mol−1) and four set to elevated CO2 (ambient + 200 μmol mol−1) for 12 h per day (7:00 AM to 7:00 PM CST). The average daily 8‐h CO2 concentration in ambient chambers was 438.33 ± 0.24 μmol mol−1, while in elevated chambers it was 650.31 ± 0.48 μmol mol−1 (Figure S2). Three soybean cultivars (Clark, Flyer, and Loda) were selected based on their differing yield and nutrient accumulation responses to eCO2 (Sanz‐Sáez et al. 2017; Myers et al. 2019). Chambers were arranged in a split plot design (n = 4), with each chamber containing four plants from each of the three cultivars (Figure S1D).

Seeds were inoculated with commercial Bradyrhizobium japonicum (N‐dure, Verdesian Inc. Cary, NC, https://vlsci.com/products/n-dure/) and germinated in a greenhouse on May 6, 2021. On May 10, 2021, seedlings were transplanted into 20‐liter black containers filled with soil from the E.V. Smith Research Station (Shorter, AL). The soil is classified as sandy loam, consisting of 23.6% silt and clay, 76.4% sand, 3.2% clay, and 20.4% silt with a pH of 6.1. Immediately following transplanting, containers were placed in each OTC. Following soil test recommendations from Auburn University Soil Testing Laboratory, 1 gram of potash was applied to each container and 2 grams of Miracle Gro were added to each container to ensure sufficient nutrient availability. Plants were watered daily with a drip tape irrigation system that applied 1.9 liters of water every other day for the first 4 weeks and every day afterwards to avoid drought stress.

2.2. Leaf Gas Exchange, Biomass and Harvest Measurements

Leaf CO2 assimilation (A) and stomatal conductance (g s ) were measured using an infrared gas analyzer (LI‐6800, LI‐COR Biosciences, Lincoln, NE). Measurements were conducted during midday hours (10:00 AM–2:00 PM) on the most recently fully expanded leaf located at the top of the canopy. These measurements were taken 34 days after planting (DAP) on June 9 (V5 vegetative stage) and 91 DAP (full seed, R6 developmental stage; Fehr et al. 1971) on August 5. To ensure accuracy, the light intensity and temperature inside the leaf cuvette were adjusted to match the ambient conditions. The LI‐6800 was used to measure the ambient light intensity. The relative humidity within the leaf cuvette was maintained at 60%–70% and the concentration of CO2 inside the cuvette was set to match that of the ambient or elevated OTC conditions. Gas exchange measurements were averaged from two plants per cultivar per OTC per time point.

Biomass sampling occurred 70 DAP (pod filling, R5 developmental stage) on July 15th and 126 DAP (maturity, R8 developmental stage) on September 9th. At the pod‐filling stage (70 DAP) seed counts were measured immediately at harvest. Both aboveground biomass (shoots, leaves, and seeds) and belowground biomass (roots) were collected at 70 DAP. Root biomass has previously been used as an indicator of root architectural changes to assess how plants modify their belowground structures under eCO₂ conditions and in turn the nutrient uptake (Van Vuuren et al. 1997). The samples were oven‐dried for at least 72 h at 60°C and then weighed. At maturity (126 DAP), aboveground biomass (stems and seeds) was collected. These samples were also dried for at least 72 h at 60°C and then weighed. Biomass measurements were averaged from two plants per cultivar per OTC per time point. Harvest index (HI) was also calculated for both 70 and 126 DAP harvest. HI was calculated as HI = seed yield/(seed yield + aboveground biomass).

2.3. Mineral Concentration Analysis

Nutrient analysis was also completed at 70 and 126 DAP. Aboveground (leaf, stem, and pod) and belowground (root) samples were analyzed at 70 DAP, and aboveground (stem and pods) samples were analyzed at 126 DAP. Dried tissue samples were weighed and ground. The samples were sent to Waters Agricultural Laboratory Inc. in Camilla, GA, for nutrient concentration analysis. Macronutrient concentrations (%) of N, phosphorus (P), potassium (K), magnesium (Mg), sulfur (S), calcium (Ca), and micronutrient concentrations (ppm) of boron (B), Fe, Zn, manganese (Mn) and copper (Cu) were determined using inductively coupled plasma mass spectroscopy (ICP‐MS). Samples were averaged from two plants per cultivar per OTC at both time points. Nutrient concentration of aboveground biomass samples was calculated as the sum of leaf, stem and seed tissues. Nutrient uptake for macronutrients (g/total biomass per tissue per plant) and micronutrients (mg/total biomass per tissue per plant) was calculated from nutrient concentration and biomass data. The percent change in measurements for elevated versus ambient CO2 was calculated as ((elevated‐ambient)/elevated) *100.

2.4. Statistical Analysis and Data Availability

Statistical analysis of the physiological data (gas exchange, biomass, and nutrient analysis) was conducted using R v4.3.1 (R Core Team 2024) and RStudio v2024.04.0 (RStudio Team 2015). A split‐plot design was implemented, where CO₂ treatment was assigned to main plots and cultivar was assigned to subplots within blocks. Cultivar and CO₂ treatment were treated as fixed effects, while block and its nested structure with treatment were treated as random effects to account for variability among experimental units. A linear mixed‐effects model was fitted using the lme4 package, with the general model structure: Response Factor ∼ Treatment × Cultivar + (1 | Block/Treatment).

Model assumptions, including normality, were assessed using histograms, Q‐Q plots, and the Shapiro‐Wilk test. Analysis of variance was performed using the lmerTest package with Kenward‐Roger degrees of freedom approximation (Kuznetsova et al. 2017). Post‐hoc comparisons were conducted using estimated marginal means (emmeans) with Tukey's adjustment for multiple comparisons (Searle et al. 1980). Compact letter displays (CLDs) were generated using the multcompView package (Graves et al. 2015). Data visualization and CLD generation were facilitated by the car and ggplot2 packages (Fox and Weisberg 2019; Wickham. 2016). Pearson correlation analysis was performed to examine relationships between measurement parameters. The correlation matrices were visualized using the corrplot package. Bonferroni corrections were applied to control for multiple comparisons, ensuring that only statistically robust correlations were highlighted. Significant differences were indicated as adjusted p < 0.05, with significance levels indicated as *p < 0.01, **p < 0.001, ***p < 0.0001. Raw data for all figures and tables presented in this study can be found in Tables S1–S4 and Data set 1.

3. Results

3.1. Gas Exchange Parameters Were Impacted by the eCO2 Treatment

At 34 DAP, midday stomatal conductance (g s ) (mol H2O m‐2 s‐1) significantly decreased in plants grown in eCO2 (Figure 1A). Midday stomatal conductance (g s ) decreased by from 73.5% to 82.4% across cultivars with no significant cultivar effect (Figure 1A). At 34 DAP midday carbon assimilation (A) (µmol CO2 m‐2 s‐1) significantly increased in plants grown in eCO2 with no significant cultivar effect (Figure 1B). At 91 DAP there was no significant impact of CO2 treatment or cultivar on g s (Figure 1C). At 91 DAP, A increased by 6.4% to 19%, with a significant main effect of both CO₂ and cultivar, as well as a significant CO₂ × cultivar interaction (Figure 1D). Flyer and Loda showed a significant increase in A with eCO2 while Clark did not (Figure 1D).

Figure 1.

Figure 1

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The effect of eCO2 on stomatal conductance (g s ) and carbon assimilation (A) rate during 34 DAP (A, B) and 91 DAP (C, D). Different letters indicate significant differences across treatments based on Tukey's post‐hoc test following a linear mixed‐effects model. Brackets with asterisks represent significant (p < 0.05) main effects of CO₂, as determined from the mixed‐effects model and are included for clarity. NS represents no significance. Results of main effect statistics are placed in the upper corners.

3.2. Biomass of Aboveground but Not Belowground Plant Tissues Increased in eCO₂ Conditions

At 70 DAP, aboveground biomass (leaves, stems and seeds) was significantly increased under eCO₂ conditions with no significant cultivar effect (Figure 2A; Table S1A). Similarly, leaf biomass was significantly influenced by both CO₂ treatment and cultivar (Figure 2C; Table S1A), with eCO₂ increasing leaf biomass across cultivars. Additionally, significant differences were observed among cultivars, with Clark and Flyer producing greater leaf biomass than Loda under both CO₂ conditions. In contrast, root biomass was unaffected by eCO₂ but varied significantly among cultivars with Loda exhibiting lower root biomass compared to Clark and Flyer (Figure 2D; Table S1A).

Figure 2.

Figure 2

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The effect of eCO2 on aboveground biomass at 70 DAP and 126 DAP (A, B) leaf dry weight and root dry weight at 70 DAP (C, D). Different letters indicate significant differences across treatments based on Tukey's post‐hoc test following a linear mixed‐effects model. Brackets with asterisks represent significant (p < 0.05) main effects of CO₂, as determined from the mixed‐effects model and are included for clarity. Results of main effect statistics are placed in the upper corners. NS represents no significance. [Color figure can be viewed at wileyonlinelibrary.com]

At 70 DAP, seed yield showed a significant increase with eCO₂ treatment across cultivars (Figure 3A; Table S1A). A significant effect of cultivar on seed yield was also observed, with Loda producing significantly higher seed yield than Clark and Flyer, irrespective of CO₂ treatment (Figure 3A). We then analyzed changes in per seed dry weight and total seed number at 70 DAP. Per seed dry weight varied only by cultivar, with Loda having a significantly higher per seed dry weight (Figure 3C). Total seed number was significantly impacted by both eCO2 and cultivar; total seed number was significantly higher with the eCO₂ treatment, and Flyer produced significantly more seeds than Clark and Loda regardless of treatment (Figure S3A; Table S1A). Harvest index (HI) at 70 DAP varied only by cultivar, with Loda having a significantly higher HI compared to the other cultivars, regardless of CO₂ treatment (Figure S4A; Table S1A).

Figure 3.

Figure 3

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The effect of eCO2 on seed yield (A, B) and per seed dry weight (C, D) at 70 and 126 DAP. Different letters indicate significant differences across treatments based on Tukey's post‐hoc test following a linear mixed‐effects model. Brackets with asterisks represent significant (p < 0.05) main effects of CO₂, as determined from the mixed‐effects model and are included for clarity. Results of main effect statistics are placed in the upper corners. NS represents no significance. [Color figure can be viewed at wileyonlinelibrary.com]

At 126 DAP, aboveground biomass was significantly increased with eCO₂ with no significant differences among cultivars (Figure 2B, Table S1B). Plants grown in eCO2 also had significantly higher seed yield, with an increase of ~ 14.46% when averaged across cultivars (Figure 3B, Table S1B). Per seed dry weight varied only by cultivar at 126DAP; Clark and Loda exhibited significantly higher per seed dry weights compared to Flyer (Figure 3D; Table S1B). Additionally, total seed number varied significantly with eCO2 treatment and by cultivar, with a significant CO₂ × cultivar interaction (Figure S3B, Table S1B). The eCO2 treatment increased total seed number by 27% across cultivars, while Flyer produced a significantly higher number of seeds than both Clark and Loda in the eCO2 treatment. Finally, HI decreased significantly with eCO₂ at 126 DAP and varied significantly by cultivar (Figure S4B, Table S1B).

3.3. Macronutrient Concentrations Declined Under eCO₂, While Uptake Increased Across Tissues

The concentration of macronutrients (N, P, K, Mg, Ca, and S) was evaluated at 70 and 126 DAP. At 70 DAP, nutrient concentrations were measured in roots, seeds, and aboveground biomass (leaf, seed, and stem combined). At 126 DAP, only stem and seed concentrations were analyzed.

At 70 DAP, seed macronutrient concentrations varied significantly with cultivar, except for Ca (Figure 4A; Table S2). Both N and S also varied with eCO₂ treatment. Additionally, there was a significant cultivar × eCO₂ interaction for N concentration (Figure 4A; Table S2). Leaf macronutrient concentrations remained unaffected by eCO₂ or cultivar (Table S2). In roots, Mg increased significantly (5.7%–13.4%), while S (5%–21%) and K (4.6%‐36%) decreased significantly under eCO₂ across cultivars, and P was influenced by cultivar as significant main effect (Table S2). For aboveground biomass, P concentrations decreased significantly under eCO₂ and varied significantly with cultivar, whereas K concentrations were decreased significantly with eCO₂ (Table S2).

Figure 4.

Figure 4

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Percent change (%) at eCO2 versus ambient CO2 of seed macronutrient concentration across cultivars. Values are presented for three cultivars: Clark (C), Flyer (F) and Loda (L). The percent change in macronutrient (N, P, K, Mg, Ca, and S) concentration in seed at (A) 70 DAP and (B) 126 DAP. Percent change was calculated as ((elevated‐ambient)/elevated) *100. Asterisks indicate significant differences (p < 0.05) between elevated and ambient measured/absolute values (for values see Table S2,S3). p values are derived from linear mixed‐effects models assessing treatment and cultivar effects. Results of main effect statistics are placed in the table below the figure. [Color figure can be viewed at wileyonlinelibrary.com]

At 126 DAP, seed macronutrient concentrations varied significantly by cultivar for all nutrients (Figure 4B; Table S3). Additionally, eCO₂ caused a significant decrease in P, K and Mg in seeds; these nutrients decreased by 0.7%–10.6% (Figure 4B). In aboveground tissue, concentrations of all macronutrients except Mg varied significantly with cultivar (Table S3). Additionally, K and Mg concentrations decreased significantly with eCO2 treatment, and P decreased marginally (Table S3).

In addition to concentration, macronutrient uptake was also evaluated. At 70 DAP macronutrient uptake increased significantly in leaves with eCO₂ and varied significantly by cultivar, with uptake increasing from 6.3% to 38.4% (Figure S5A; Table S4). There was also a significant CO2 × cultivar interaction for Ca uptake in leaves, with Flyer and Loda having significantly higher uptake than Clark (Figure S5A; Table S4). In roots at 70 DAP nutrient uptake varied significantly by cultivar for all macronutrients, and uptake of Mg and Ca also increased significantly with eCO2 (Figure S5B; Table S4). At 126 DAP seed uptake of all macronutrients increased significantly with eCO₂, with marginal increases observed for S (p = 0.072) (Figure 5; Table S4). Seed macronutrient uptake under eCO₂ increased by 5.05%–17.53% across cultivars (Figure 5; Table S4). Mg, Ca and S uptake in seed also varied significantly by cultivar (Figure 5; Table S4).

Figure 5.

Figure 5

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Percent change (%) at eCO2 versus ambient CO2 of the seed nutrient uptake for three cultivars. Values are presented for Clark (C), Flyer (F) and Loda (L). The percent change in macro‐ (g/total biomass per tissue per plant) and micro‐ (mg/total biomass per tissue per plant) nutrient uptake in seeds at 126 DAP. Macronutrients: N, P, K, Mg, Ca, S; micronutrients: B, Zn, Mn, Fe and Cu. Percent change was calculated as ((elevated‐ambient)/elevated) *100. Asterisks indicate significant differences between elevated and ambient measured/absolute values (see Table S4). p values are derived from linear mixed‐effects models assessing treatment and cultivar effects. Results of main effect statistics are provided in the table below the figure. [Color figure can be viewed at wileyonlinelibrary.com]

3.4. Micronutrient Concentration Declined Under eCO₂ With Variable Uptake Patterns

Micronutrient concentration (B, Zn, Mn, Fe and Cu) was also analyzed across various tissues at 70 and 126 DAP. At 70 DAP, eCO2 caused a significant decrease in all seed micronutrients, except for Fe (Figure 6A; Table S2). Seed micronutrient concentration also varied significantly by cultivar, and Zn and Cu exhibited significant CO₂ × cultivar interactions. Zn showed a larger decreased in concentration for Clark (15.7%) compared to Flyer (0.2%) and Loda (9.05%) with eCO2 treatment. Cu showed a larger reduction with eCO₂ in Loda (14.8%) compared to Clark (1.6%) whereas Flyer showed a small increase (0.9%) (Figure 6A; Table S2). Aboveground tissue concentrations of Cu decreased marginally (p = 0.051) with eCO₂, while no significant changes were observed in leaf tissue (Table S2). In roots, all micronutrients varied significantly only by cultivar except for B and Mn (Table S2). B did not vary significantly by cultivar or treatment, and Mn showed a significant reduction with eCO2 regardless of cultivar (Table S2).

Figure 6.

Figure 6

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Percent change (%) at eCO2 versus ambient CO2 of the seed micronutrient concentration across three cultivars. Values are presented for Clark (C), Flyer (F) and Loda (L). The percent change in micronutrient (Zn, Fe, B, Mn, and Cu) concentration in seed at (A) 70 DAP and (B) 126 DAP. Percent change was calculated as ((elevated‐ambient)/elevated) *100. Asterisks indicate significant differences (p < 0.05) between elevated and ambient CO₂ treatments based on linear mixed‐effects model analysis of the absolute values (see Table S2,S3). [Color figure can be viewed at wileyonlinelibrary.com]

At 126 DAP, seed concentrations of B, Mn and Cu decreased significantly with eCO₂, and Zn (p = 0.086) and Fe (p = 0.083) showed moderately significant reduction with eCO2 (Figure 6B; Table S3). All seed micronutrient concentrations varied significantly by cultivar at 126 DAP (Figure 6B; Table S3). B also had a significant CO2 x cultivar interaction. Aboveground tissue concentrations of B and Cu decreased significantly under eCO₂, and B, Zn and Cu also varied significantly by cultivar (Table S3).

Micronutrient uptake in leaves at 70 DAP increased significantly with eCO₂ and varied significantly by cultivar for all micronutrients except Fe (Figure S5A; Table S4). B uptake also had a significant CO2 × cultivar interaction (Figure S5A; Table S4). In roots at 70 DAP, micronutrient uptake varied significantly only by cultivar (Figure S5B; Table S4). At 126 DAP, uptake of all micronutrients in seeds increased significantly under eCO₂, except for B, which significantly decreased, and Fe, which showed a marginally significant increase (p = 0.075; Figure 5). B, Zn and Cu uptake also varied significantly by cultivar (Figure 5; Table S3).

3.5. Correlation Analysis Across Cultivars and Response Variables

Pearson correlation analysis was done to compare the relationship between changes in seed nutrient concentrations, photosynthetic parameters (A, g s ), and yield components (seed yield, seed number, per seed dry weight) (Figure S6, Table S5). Consistent patterns were observed across Clark, Flyer, and Loda, indicating that cultivar responses to eCO₂ were similar. Specifically, most seed nutrient concentrations were negatively correlated with A, total seed yield and seed number across all cultivars (Table S5). Additionally, A showed strong positive correlations with yield traits across all cultivars, with significant positive correlations in Loda at 34 DAP (r = 0.87) and 91 DAP (r = 0.58) (Figure S6, Table S5). Midday gs showed weak or nonsignificant associations with both yield and nutrient traits. Notably, the parallel decline in multiple nutrients across cultivars, paired with the consistent negative correlations between yield and nutrient concentrations, underscores the systemic nature of mineral dilution in response to eCO₂.

4. Discussion

The aim of this study was to explore the physiological mechanisms underlying plant nutrient responses to eCO2 levels by evaluating responses in three soybean cultivars with previously observed contrasting responses to growth in eCO2 conditions (Myers et al. 2019; Sanz‐Sáez et al. 2017). These cultivars were specifically selected to test hypotheses related to nutrient dilution, reduced transpiration, and changes in root architecture under eCO₂. Using an OTC system, we analyzed changes in physiological and yield parameters and their impact on nutrient accumulation at the whole‐plant level.

4.1. Nutrient Dilution Likely Played a Role in Decreased Seed Nutrient Concentration in Response to eCO₂

Plants grown in eCO₂ exhibited enhanced A and reduced g s during early development, leading to increased WUE (Figure 1). Across all cultivars, A increased significantly at both 34 and 91 DAP, with Loda showing the greatest enhancement (Figure 1D). At maturity (126 DAP), eCO2 increased biomass and increased seed yield across cultivars (Figure 2B; Figure 3B). Additionally, seed number increased without changes in individual seed weight. Despite yield gains, seed concentrations of macronutrients (P, K, Mg) and micronutrients (Fe, B, Mn, Zn, Cu) were significantly reduced under eCO₂, with Fe and Zn showing marginal reductions at 126 DAP (Figures 4, 5, 6; Table S2–S3). Taken together, this study indicates that enhanced carbon assimilation and increased seed production in eCO2 conditions may be leading to increased seed carbohydrate content (Saha et al. 2015; Jiang et al. 2022) and decreased nutrient content in edible seeds, or mineral dilution. The reductions in seed nutrient concentrations observed in this study aligns with previous reports that increased yield under eCO₂ often leads to lower concentrations of key nutrients (P, K, Mg, Fe, B, Zn, Cu) (Monasterio and Graham 2000; Mcgrath and Lobell 2013; Myers et al. 2019). This yield‐driven dilution phenomenon is consistent with previous findings in other C₃ crops, including wheat, rice, and soybeans (Poorter et al. 1997; Loladze 2002; Broberg et al. 2017). While we did not directly measure TNC in seeds, seed yield has previously been shown as an indicator for carbon accumulation, as greater sink strength often promotes the remobilization of stored carbohydrates during grain filling (Jiang et al. 2022). Future work is needed to further evaluate the source‐sink demand, including analysis of seed TNC and protein levels, to further support this hypothesis.

4.2. Limited Influence of Transpiration on Nutrient Allocation in eCO₂ Conditions

Reductions in gs at 34 DAP (Figure 1A) support the widely observed increase in WUE in plants grown in eCO₂, a hallmark of the “CO₂ fertilization” effect in C₃ species (Drake et al. 1997; Long et al. 2006; Loladze 2014; Myers et al. 2019; Ainsworth and Long 2021). The observed 73.5%–82.4% decrease in gs across cultivars under eCO₂ at 34 DAP suggests reduced transpiration water loss, which may have contributed to early vegetative biomass gains. However, gs was no longer significantly different at 91 DAP (Figure 1C), indicating that the reductions in transpiration was not sustained during reproductive stages when seed development was underway.

The transpiration‐stream hypothesis suggests that reduced transpirational flow under eCO₂ limits the mass flow of mobile nutrients like N, Fe, and Mg to shoots and developing seeds (Mcgrath and Lobell 2013). While our findings align with this hypothesis to some extent, especially given reduced P and K concentrations in aboveground biomass and seeds at 70 and 126 DAP (Table S2, S3) the lack of strong positive correlation between seed nutrient concentrations and gs (Figure S6, Table S5) suggests that dilution from increased carbon assimilation may have played a more dominant role.

Additionally, our nutrient uptake data support the idea that transpiration did not markedly limit nutrient acquisition. Macronutrient uptake in leaves significantly increased under eCO₂ at 70 DAP, with consistent increases across cultivars for S, Ca, Mg, and K (Figure S5A). This enhanced uptake occurred due to the increase in aboveground and seed biomass at elevated CO2 and despite reduced gs , indicating that plants maintained or even improved nutrient acquisition efficiency potentially through compensatory mechanisms such as increased root absorption or altered transporter activity. Similarly, aboveground uptake of most micronutrients increased significantly with eCO₂ (Figure S5A), further weakening the argument for persistent transpiration‐driven nutrient limitations. A similar conclusion was drawn by Houshmandfar et al. (2018), who found that although nutrient uptake in wheat was correlated with transpiration under eCO₂, nutrient uptake per unit of water transpired was higher under eCO₂, suggesting improved nutrient use efficiency despite reduced transpiration.

4.3. Lack of Response in Root Biomass and Implications for Nutrient Concentration in Seeds

The spatial pattern of soil nutrient exploitation is largely influenced by root architectural traits, such as lateral branching, root hair density, and overall root biomass, which determine a plant's ability to acquire nutrients efficiently (Kong et al. 2013). However, despite notable enhancements in aboveground biomass and seed yield (Figure 2; Table S1), our study revealed no significant increase in root biomass under eCO₂ at 70 DAP, suggesting a bottleneck in nutrient transport to developing seeds (Figure S5A, Table S4). Root architecture is particularly critical during the seed‐filling phase (R5), where nutrient remobilization peaks (Bender et al. 2015). While N, P, S, and Cu rely on redistribution from vegetative tissues, elements like Ca and Mg depend on continuous uptake from the soil (Bender et al. 2013; Bender et al. 2015; Gaspar et al. 2018). Although previous studies in soybean, wheat and canola have shown that eCO₂ can stimulate root growth, leading to increased length, branching, and deeper rooting for enhanced nutrient and water acquisition (Rogers. 1992; Nie et al. 2013; Uddin et al. 2018; Ainsworth et al. 2025), similar root enhancements were not observed in soybean in our study. This aligns with previous findings that eCO₂ can delay root development during reproductive stages, potentially limiting nutrient uptake and translocation to seeds (Van Vuuren et al. 1997).

Although eCO₂ can stimulate root growth, it may still reduce the efficiency of nutrient transport to aboveground tissues. Prior studies report reductions in stele and xylem area under eCO₂, impairing water and nutrient flow to aerial tissues (Cohen et al. 2018, 2019). Additionally, downregulation of key nutrient transport genes, particularly those involved in N uptake, may further restrict root‐to‐shoot nutrient translocation, exacerbating nutrient dilution effects in seeds (Jauregui et al. 2016; Gojon et al. 2023). Species‐specific differences in root proliferation patterns under eCO₂ further influence nutrient acquisition. For instance, wheat and canola exhibit deeper root systems under eCO₂, allowing them to access subsoil nutrient and water reserves during drought stress (Franzaring et al. 2011; Uddin et al. 2018). Soybeans, however, tend to develop more lateral roots in upper soil layers, potentially limiting their ability to tap into deeper nutrient pools during seed‐filling stages (Chaudhuri et al. 1990).

In our study, while large pots were used to mitigate spatial constraints, it remains possible that root depth and lateral expansion were still somewhat restricted compared to field conditions, potentially affecting nutrient foraging. Although nutrient uptake increased in vegetative tissues like leaves under eCO₂ (Figure S5A), this did not translate into higher seed nutrient concentration. This reinforces speculation that nutrient partitioning and increased carbon assimilation along with lack of comparable increase in root nutrient uptake were the primary factor driving reduction in seed nutrient concentration. Enhancing root lateral branching, hair density, and overall nutrient uptake efficiency may represent a key strategy to sustain crop nutrition in future atmospheric conditions. Future studies should examine how root system responses differ under field conditions, where soil depth and nutrient heterogeneity play a more significant role in plant development.

4.4. Limited Cultivar‐Specific Responses to eCO2 Were Observed

Despite inherent genetic differences, all cultivars exhibited a broadly similar response to eCO₂. At the physiological level, enhanced A and reduced gs at 34 DAP improved WUE, contributing to biomass accumulation at 70 and 126 DAP (Figure 2A–B). This pattern was consistent across cultivars, with increased yield under eCO₂ accompanied by reductions in seed nutrient concentrations. While CO₂ × cultivar interactions were limited, nutrient concentrations did differ significantly among cultivars (Table S1), indicating underlying genetic variation in baseline nutrient profiles. All cultivars showed declines in macro‐ and micronutrient concentrations, with Loda expected to show the highest yield displaying the most pronounced and consistent nutrient reductions across all elements except for N (Figures 4, 5, 6). Clark and Flyer followed similar trends, though Flyer exhibited a slightly lower decline in Zn concentration at 126 DAP compared to the others (Figure 6). The correlation matrices (Figire S6) further support these findings, showing similar trends across all cultivars under eCO₂ for nutrient concentration, A, and g s . These cultivar‐level trends suggest a common physiological response mechanism, rather than cultivar‐specific trade‐offs, further highlighting the widespread impact of eCO₂ on nutrient distribution in seeds.

While previous research has highlighted cultivar‐specific responses to eCO₂, particularly in yield and nutrient accumulation (Bishop et al. 2015; Sanz‐Sáez et al. 2017; Digrado et al. 2024), the uniformity observed in this study suggests that environmental conditions—such as OTCs versus field settings may influence the degree of variation (Ainsworth et al. 2002). In contrast to Free‐Air CO₂ Enrichment (FACE) studies that reported significant genotype × environment interactions affecting yield and nutrient concentration (Köhler et al. 2019; Bishop et al. 2015), our results indicate a more uniform response across cultivars. These findings emphasize the importance of considering environmental settings when interpreting cultivar‐specific responses to eCO₂ and highlight the necessity of breeding strategies that optimize both yield and nutritional retention under future atmospheric conditions.

4.5. Alternative Hypotheses Not Tested by This Study

While this study primarily tested hypotheses related to nutrient dilution, reduced transpiration, and root responses, it is important to acknowledge that other mechanisms have also been proposed to explain reductions in nutrient concentrations under eCO₂. These include decreased photorespiration, downregulation of photosynthetic proteins, changes in nitrate assimilation, and alterations in root transporter activity and architecture (Rachmilevitch et al. 2004; Bloom et al. 2010; Bloom 2015; Gojon et al. 2023). Many of these mechanisms are not mutually exclusive and may interact, with processes like reduced photorespiration potentially lowering the reducing power needed for nitrate assimilation, while altered nutrient transporter activity could limit nutrient uptake and translocation (Ujiie et al. 2019). Additionally, a reduction in physiological demand for certain minerals under elevated CO₂ has also been suggested. For example, lower demand for Mg due to decreased synthesis of Rubisco or chlorophyll may reduce Mg uptake (Mcgrath and Lobell 2013). This altered mineral requirement could influence both uptake and partitioning patterns, especially for micronutrients. Recent work has also indicated that changes in the binding affinity of Mg²⁺ and Mn²⁺ to Rubisco may affect the balance between carboxylation and photorespiration, further impacting nutrient dynamics (Bloom and Lancaster 2018).

Our findings highlight that mineral dilution, driven by enhanced carbon assimilation, is the likely mechanism by which nutrient reduction occurs in seeds under eCO₂ for the soybean cultivars we tested. Reduced transpiration occurred but is likely playing less of a role because it did not show any significant reduction under eCO2 around the seed filling stage. Additionally, no significant reduction in seed nutrient uptake was observed at maturity. Our data support that root biomass and nutrient uptake were limited under eCO2 conditions, which could create a mismatch between nutrient demand and carbon assimilation in leaves possibility leading to mineral dilution in seeds. Future research is needed to disentangle the timing, extent, and interactions of these processes to better understand how eCO₂ impacts nutrient concentrations in crops like soybeans.

5. Conclusion

This study reveals that for the three cultivars we examined reduced nutrient concentrations in soybean seeds under elevated eCO₂ was primarily driven by a combination of mineral dilution and limitations in root architecture and nutrient uptake (Figure 7). While increased photosynthesis and biomass enhance seed yield, we hypothesize there was disproportionate allocation of resources to carbohydrate production which diluted seed nutrients as described before in the literature. We also hypothesize that root constraints during reproductive stages further limit nutrient uptake and translocation, failing to match the increased aboveground demand under eCO₂. Reduced transpiration plays a minor but nuanced role in nutrient transport. Overall, these findings suggest that maintaining seed nutritional quality under future CO₂ conditions will require targeted breeding strategies to improve root development, nutrient uptake efficiency, and nutrient remobilization.

Figure 7.

Figure 7

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Conceptual model summarizing the physiological responses of soybean (cultivars Clark, Flyer and Loda) to elevated atmospheric CO₂ (eCO₂). Similar physiological trends were observed across all cultivars examined. Arrows indicate directionality of processes, solid arrows indicate significant changes, while dashed arrows denote inferred or nonsignificant changes in physiological processes under eCO2. [Color figure can be viewed at wileyonlinelibrary.com]

Conflicts of Interest

The authors declare no conflicts of interest. Dr. Lisa Ainsworth is an Editorial Board member of Plant, Cell, and Environment and a coauthor of this article. To minimize bias, they were excluded from all editorial decision‐making related to acceptance of this article for publication.

Supporting information

Dataset 1: Biomass (DW) for Leaf, stem, root and seed (seed yield) was measured, aboveground (AbovG) biomass (shoots, leaves, and seed), macronutrient concentrations (%) of nitrogen (N), phosphorus (P), potassium (K), magnesium (Mg), sulfur (S), calcium (Ca), and micronutrient concentrations (ppm) of boron (B), iron (Fe), zinc (Zn), manganese (Mn) and copper (Cu) and nutrient uptake for macronutrients (g/total biomass per tissue per plant) and micronutrients (mg/total biomass per tissue per plant) was calculated from nutrient concentration and biomass data, were measured at 70 days after planting (DAP).

PCE-48-8712-s001.xlsx (44.3KB, xlsx)

Figure S1: Study site and treatment design of the Open Top Chambers (OTCs) at USDA‐ARS National Soil Dynamics Laboratory. Figure S2: Daily average CO2 concentration in treatment chambers at the Open Top Chambers (OTCs) at USDA‐ARS National Soil Dynamics Laboratory. Figure S3: The effect of eCO2 on seed number at (A) 70 DAP and (B) 126 DAP. Figure S4: The effect of eCO2 on harvest index at (A) 70 DAP and (B) 126 DAP. Figure S5: Percent change (%) at eCO2 versus ambient CO2 of the leaf and root nutrient uptake in Clark (C), Flyer (F) and Loda (L). Figure S6: Pearson correlation matrix of macro‐ and micronutrient concentrations in seeds.

PCE-48-8712-s003.docx (3.5MB, docx)

Table S1: Mean above ground, leaf, seed (seed yield), per seed dry weight, total seed number and harvest index (HI) at (a) 70 DAP and (b) 126 DAP in three cultivars under ambient and eCO2 treatments. Table S2: Seed, leaf, root, and total aboveground biomass macro‐ (%) and micronutrient (ppm) concentration in three cultivars under ambient and eCO2 treatments at 70 DAP. Table S3: Total Aboveground biomass and seed macro‐ (%) and micronutrient (ppm) concentration in three cultivars under ambient and eCO2 treatments at 126 DAP. Table S4: Root and leaf macro (g/total biomass per tissue per plant) and micronutrient (mg/total biomass per tissue per plant) uptake in three cultivars under ambient and eCO2 treatments at 70 DAP and seed mineral uptake at 126 DAP. Table S5: Pearson correlation coefficients (r) and Bonferroni‐adjusted p‐values for pairwise comparisons between nutrient concentrations, photosynthetic parameters (A and gs at 34 and 91 DAP), and seed traits across three soybean cultivars—Clark, Flyer, and Loda.

PCE-48-8712-s002.xlsx (50.8KB, xlsx)

Acknowledgements

We would like to thank Collin Modelski for assisting in various activities of data collection. This study is supported by the Physiology of Agricultural Plants program, project award no. #2022‐67013‐36126, from the U.S. Department of Agriculture's National Institute of Food and Agriculture to C.P.L.

Ravneet Kaur and Mary Durstock indicates co‐first author.

Data Availability Statement

All raw data from this study is presented the supporting tables and Data Set 1.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Dataset 1: Biomass (DW) for Leaf, stem, root and seed (seed yield) was measured, aboveground (AbovG) biomass (shoots, leaves, and seed), macronutrient concentrations (%) of nitrogen (N), phosphorus (P), potassium (K), magnesium (Mg), sulfur (S), calcium (Ca), and micronutrient concentrations (ppm) of boron (B), iron (Fe), zinc (Zn), manganese (Mn) and copper (Cu) and nutrient uptake for macronutrients (g/total biomass per tissue per plant) and micronutrients (mg/total biomass per tissue per plant) was calculated from nutrient concentration and biomass data, were measured at 70 days after planting (DAP).

PCE-48-8712-s001.xlsx (44.3KB, xlsx)

Figure S1: Study site and treatment design of the Open Top Chambers (OTCs) at USDA‐ARS National Soil Dynamics Laboratory. Figure S2: Daily average CO2 concentration in treatment chambers at the Open Top Chambers (OTCs) at USDA‐ARS National Soil Dynamics Laboratory. Figure S3: The effect of eCO2 on seed number at (A) 70 DAP and (B) 126 DAP. Figure S4: The effect of eCO2 on harvest index at (A) 70 DAP and (B) 126 DAP. Figure S5: Percent change (%) at eCO2 versus ambient CO2 of the leaf and root nutrient uptake in Clark (C), Flyer (F) and Loda (L). Figure S6: Pearson correlation matrix of macro‐ and micronutrient concentrations in seeds.

PCE-48-8712-s003.docx (3.5MB, docx)

Table S1: Mean above ground, leaf, seed (seed yield), per seed dry weight, total seed number and harvest index (HI) at (a) 70 DAP and (b) 126 DAP in three cultivars under ambient and eCO2 treatments. Table S2: Seed, leaf, root, and total aboveground biomass macro‐ (%) and micronutrient (ppm) concentration in three cultivars under ambient and eCO2 treatments at 70 DAP. Table S3: Total Aboveground biomass and seed macro‐ (%) and micronutrient (ppm) concentration in three cultivars under ambient and eCO2 treatments at 126 DAP. Table S4: Root and leaf macro (g/total biomass per tissue per plant) and micronutrient (mg/total biomass per tissue per plant) uptake in three cultivars under ambient and eCO2 treatments at 70 DAP and seed mineral uptake at 126 DAP. Table S5: Pearson correlation coefficients (r) and Bonferroni‐adjusted p‐values for pairwise comparisons between nutrient concentrations, photosynthetic parameters (A and gs at 34 and 91 DAP), and seed traits across three soybean cultivars—Clark, Flyer, and Loda.

PCE-48-8712-s002.xlsx (50.8KB, xlsx)

Data Availability Statement

All raw data from this study is presented the supporting tables and Data Set 1.

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