Effects of Elevated CO₂ on Grain Yield and Quality in Different Wheat Cultivars

Abstract

While the effects of elevated CO₂ (eCO₂) on crops have been extensively studied, cultivar-specific responses and impacts under higher CO₂ concentrations (>800 μmol/mol) remain unclear. Here, we addressed these two aspects to reveal the effects of eCO₂ (approximately 900 μmol/mol) on the yield and quality of three wheat cultivars, Chinese spring (CS), Chuanmai 44 and Neimai 9. The results indicated the net photosynthetic rate (Pn) and water use efficiency (WUEi) of the three cultivars significantly increased under 900 μmol/mol CO₂ concentration. Elevated CO₂ increased the hundred-grain weight (HGW) of Chuanmai 44 (+32.51%, p < 0.05) and Neimai 9 (+8.47% p < 0.05), but had little effect on HGW of CS. CO₂ elevation significantly increased the N content in the grain of CS (+7.27%, p < 0.05). Elevated CO₂ enhanced amino acid biosynthesis in CS but suppressed it in Chuanmai 44 and Neimai 9. No significant changes in grain mineral concentrations occurred in CS and Chuanmai 44 under eCO₂ conditions. Neimai 9 demonstrated significant decreases in K and Mg, with non-significant reductions in other elements. The effects of eCO₂ on grain yield and quality were closely linked to cultivars. This study will provide insights for understanding effects of CO₂ concentration and cultivar interactions on crop growth and selecting wheat cultivar to cope with future climate change.

1. Introduction

Carbon dioxide (CO₂), a critical atmospheric component accounting for approximately 0.04% of atmospheric composition, serves as the primary substrate for plant photosynthesis. Its major sources include respiration in plants and animals, microbial decomposition of organic matter, and combustion of fossil fuels in industrial processes. It is indisputable that atmospheric CO₂ concentrations have been steadily increasing over time. According to a study by the Intergovernmental Panel on Climate Change (IPCC), CO₂ concentration has reached 413.92 μmol/mol, with further predictions of 936 μmol/mol (RCP8.5) by the year 2100 [1].

Atmospheric CO₂ concentration constitutes a primary environmental regulator for photosynthesis in C3 plants. Elevated CO₂ (eCO₂) levels directly influence crop photosynthetic rates, stomatal conductance, growth development, and ultimately yield [2]. The projected continuous rise in CO₂ concentration will persistently impact global agricultural production in the foreseeable future [3]. As one of the three major cereals in the world, wheat (Triticum aestivum L.) is an important source of starch and energy, providing a large number of nutrients for humans, including protein, free amino acids and mineral elements. Extensive studies have suggested that the growth and grain yield of wheat have been significantly influenced by high atmospheric CO₂ concentration because of its photosynthesis-enhancing effects. Thousand-grain weight (TGW) often increased under eCO₂, indicating positive effects on grain quality in terms of milling quality and hence economic value [4]. Long-term FACE experiments in Arizona in the United States and in Beijing in China have shown that eCO₂ could increase the yield of winter wheat by 25–28%, even under persistent moderate-drought conditions. The yield increase surpassed the reported 11–19% gain under irrigation [5,6]. In experiments using FACE technology under realistic field conditions, aboveground biomass and yield increased by 10–16% (550 versus 380 μmol/mol CO₂) when given ample nutrients and water [7]. When CO₂ levels were further increased to 600 μmol/mol, it still had a promoting effect on wheat yield [8]. However, some studies suggested that the increase of CO₂ concentration may not significantly enhance crop yields. As demonstrated by Tan et al. [9], eCO₂ concentrations up to 600 μmol/mol failed to offset the adverse effects of warming (+2.5~3.0 °C) on the growth and yield of winter wheat. A FACE experiment conducted across five different countries showed that the increase in CO₂ concentration (550 μmol/mol) had no significant effect on the yield of durum wheat and bread wheat, which may be attributed to the adaptation of photosynthetic capacity to high CO₂ concentrations [10], a phenomenon known as “photosynthetic adaptation”. Most studies have found that elevated atmospheric CO₂ concentrations have a negative effect on crop grain quality. When CO₂ levels rise to 550 μmol/mol, the contents of various amino acids tended to decrease overall [4]. The composition of proteinogenic amino acids was also found to change in the study of Högy et al. (2009) [11], but no clear response pattern of different types of amino acids was observed under CO₂ enrichment [11].

Elevated CO₂ concentrations generally reduced mineral element content in wheat [12]. K, Mo and Pb increased, while Mn, Fe, Ca and Si decreased, suggesting that adjustments in agricultural practices may be required to retain current grain quality standards [13]. Högy et al. [4] found that elevated CO₂ concentrations (500 μmol/mol) significantly reduced the concentration of macro-elements in wheat grains except K and P, among which Ca and Mg decreased by 9.7% and 4.8%, respectively. The study also observed declines in micronutrient concentrations, with Fe and Zn decreasing by 18.3% and 13.1%, respectively. Elevated CO₂ concentrations altered the entire stoichiometry of plants, resulting in lower concentrations of most elements [14]. The majority of studies have shown that eCO₂ led to decreased nitrogen concentration and increased carbon concentration in crops [15]. However, recent research has found that when CO₂ rise to 800 μmol/mol, wheat grain carbon concentrations decreased, possibly because higher CO₂ concentrations inhibited carbohydrate transport from leaves to grains, resulting in a decrease in grain carbon concentrations [16].

Although the effects of elevated CO₂ concentration on wheat yield and grain quality have been well studied, most of them focus on a specific wheat variety. In fact, distinct wheat cultivars exhibit differential responses to elevated CO₂ concentrations. For instance, spring wheat cultivar demonstrated more pronounced photosynthetic enhancement under high CO₂ conditions, indicating significant genotype × CO₂ interaction effects on wheat physiology [17]. An intriguing question is whether genotypic variation in leaf morphology and spike architecture mediates differential responses of wheat cultivars to eCO₂, and how such genotype-specific physiological adaptations manifest under eCO₂. Current understanding of these genotype × eCO₂ interactions remain limited. Furthermore, most existing studies on the effect of CO₂ concentration on wheat yield and grain quality have been conducted at concentrations ≤ 800 μmol/mol. Under projected atmospheric CO₂ trajectories, the impacts of higher CO₂ levels (>800 μmol/mol) on wheat productivity and grain quality remain poorly understood, representing a critical knowledge gap in climate change research. We hypothesize that elevated CO₂ concentration does not always lead to yield increase or quality reduction, as its effects are cultivar-dependent, which requires verification.

To this end, the present study employed three wheat cultivars (Chinese spring, Chuanmai 44, and Neimai 9) as experimental materials to investigate how eCO₂ (~900 μmol/mol) influenced yield and quality parameters across different genotypes. The findings will provide critical insights for understanding effects of CO₂ concentration and cultivar interactions on crop growth and selecting suitable wheat cultivar to cope with future climate change, so as to increase wheat yield and improve its grain quality.

2. Materials and Methods

2.1. Experimental Set-Up

The experiment was carried out from January 2022 to June 2022 in a climate-controlled greenhouse at College of Life Sciences, China West Normal University. Three wheat cultivars, namely Chinese spring (CS), Chuanmai 44 and Neimai 9, were selected as materials. Chinese spring was internationally recognized as genetic material. Chuanmai 44 and Neimai 9 were the predominant wheat cultivars extensively grown in the southwestern wheat-growing region of China. Compared to CS, Chuanmai 44 and Neimai 9 exhibited comparatively broad leaves, long spikes and smaller plant heights (~85 cm). They were grown in two climate cells with an ambient CO₂ concentration (aCO₂, 410 μmol/mol) and elevated CO₂ concentration (eCO₂, 900 μmol/mol), respectively. Each CO₂ treatment was repeated three times. Wheat seeds (one seeds per pot) were sown in plastic pots (8.5 cm in height and 10.0 cm in diameter) filled with clay loam soil and surface soil (0–20 cm) of nearby cropland, with a pH (1:5 soil: water) of 5.8–6.3, and contained 1.51% organic carbon (C) and 0.18% total N. To avoid nutrient deficiency, all plants were fertilized with 200 mL standard fertilizer per pot twice from the tillering stage to grain filling stage (stock solution with macronutrient: NPK 14-3-23 + Mg and micronutrient: B 0.23%, Cu 0.14%, Fe 1.32%, Mn 0.5%, Mo 0.05%, Zn 0.18%; diluted 100- fold when applying) [16]. In each greenhouse cell, 90 pots were established and arranged randomly, with thirty replicates for each cultivar. The other climate conditions were set as 24/16 ± 2 °C day/night air temperature, 65% relative humidity (RH) and a 16 h photoperiod with a photosynthetic active radiation (PAR) > 300 μmol m⁻² s⁻¹ supplied by LED lamps.

The CO₂ was supplied from high-pressure cylinders containing pure liquid CO₂. Flexible tubing connected the CO₂ cylinders to PVC pipes inside the growth chambers. A branch rotameter was used to quantify the CO₂ flow rate, and a blower delivered the gas into the growth chambers. The CO₂ concentration inside the chambers was continuously monitored every six seconds using a CO₂ analyzer (GMT222, Vaisala, Helsinki, Finland), and the flow rate from the cylinders was adjusted to maintain the target concentration, with fluctuations kept within ±10%. To avoid excessive heat buildup due to prolonged enclosure and to avoid hypoxia-induced growth inhibition, the lids above the growth chamber were equipped with 45° inward-angled ventilation openings. The greenhouse maintained excellent airflow conditions. CO₂ enrichment was applied daily from 09:00 to 18:00 (accounting for negligible photosynthetic activity at night), and the lid was opened the rest of the time. The elevated CO₂ treatment continued from the tillering stage to the filling stage.

2.2. Photosynthetic Index Determination

At the beginning of the filling stage, leaf gas exchange rates were measured on flag leaves in the morning between 9.00 and 11.00 a.m., including the net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr), using a portable photosynthetic system (Li- 6400, Li- Cor, Lincoln, NE, USA). Measurements were performed on one flag leaf per pot under 1450 μmol m⁻² s⁻¹ photon flux density at 25 °C leaf temperature and at [CO₂] of 410 μmol/mol for aCO₂ and 900 μmol/mol for eCO₂ treatment, respectively. Intrinsic water use efficiency (WUEi) was calculated as the Pn:Gs ratio. To avoid edge effects, 15 plants of each cultivar located at the center of the climate cells and showing healthy growth were selected. A total of 45 plants from three replicates were used for analysis, and each plant was measured 3 times.

2.3. Yield-Related Trait Measurement

After maturation, the aboveground parts of the wheat plants were harvested to measure yield components including spike length, spikelet number, grain number on the main spike, tiller number, grain weight per plant, grain length and width, and hundred-grain weight. A total of 15 plants of each cultivar located at the center of the climate cells and showing healthy growth were selected. A total of 45 plants from three replicates were used for trait measurement.

2.4. Determination of Grain Quality Traits

The grain quality parameters, including C, N, amino acid, and mineral concentrations, were analyzed.

The main spikes of 15 healthy plants of each cultivar located at the center of each of the climate cells were selected. Three biological and three technical replicates were performed. The wheat seeds were oven-dried at 105 °C to a constant weight, then grounded using a ball mill (Mixer Mill 400, Retsch, Haan, Germany) before being sieved through a 60-mesh screen. According to the national standard for the determination of amino acids in food (GB5009.124-2016 [18]), the sieved samples were treated with an acid or alkali hydrolysis method, and then the amino acid content was determined by S433D amino acid analyzer (Sykam, Furstenfeldbruck, Germany). The concentrations of carbon and nitrogen were analyzed using the Vario MICRO cube elemental analyzer (Elementar, Langenselbold, Hessen, Germany). The concentrations of macronutrients (Ca, K, Mg) and micronutrients (Cu, Fe, Mn, Zn) in grain were determined by AA-7000 atomic absorption spectrometry (Shimadzu, Kyoto, Japan) after microwave-assisted digestion with 70% HNO₃ and 15% H₂O₂.

2.5. Data Analysis

Two-way analysis of variance (ANOVA) was performed to analyze the effects of [CO₂] and [Cultivar] and their interactions on grain yield and quality parameters using the SPSS 27.0 software. All data were expressed as mean ± standard error. The standard errors were calculated using the ANOVA table’s pooled error term. Treatments were compared using Tukey’s tests at p = 0.05. After the data were standardized, principle component analysis (PCA) was applied to comprehensively evaluate the variation in yield and quality characteristics among wheat cultivars under different CO₂ concentrations. The histograms were made using Origin 2021 software.

3. Results

3.1. Leaf Gas Exchange

Pn and WUE_i were significantly increased, while Tr and Gs decreased significantly under eCO₂ in all three wheat cultivars compared with those under aCO₂. And the magnitude of change in different cultivars varied, indicating an interactive effect of [CO₂] and [Cultivar] (Figure 1).

3.2. Grain Yield Components

Elevated CO₂ (eCO₂) significantly enhanced grain length in both CS and Neimai 9, as well as grain width in Chuanmai 44. Notably, eCO₂ induced a significant elevation in the length-to-width ratio of Neimai 9 grains, resulting in more slender grains. Conversely, Chuanmai 44 exhibited a substantial reduction in grain length-to-width ratio under eCO₂ treatment, leading to a more rounded grain shape (

Table 1. Effects of eCO₂ on grain length and width of three wheat cultivars.

Cultivar-CO2	Grain Length (mm)	Grain Width (mm)	Length/Width
CS-aCO2	6.08 ± 0.16 e	3.15 ± 0.21 b	1.94 ± 0.12 bc
CS-eCO2	6.17 ± 0.28 d	3.16 ± 0.17 b	1.96 ± 0.12 b
CM44-aCO2	6.41 ± 0.22 c	3.10 ± 0.21 b	2.08 ± 0.14 a
CM44-eCO2	6.46 ± 0.15 c	3.38 ± 0.13 a	1.91 ± 0.06 c
NM9-aCO2	6.57 ± 0.23 b	3.40 ± 0.27 a	1.94 ± 0.16 bc
NM9-eCO2	6.76 ± 0.23 a	3.33 ± 0.19 a	2.03 ± 0.11 a
Cultivar	***	***	*
CO2	***	**	ns
Cultivar × CO2	ns	***	***

Note: The data in the table are the mean ± standard error of the means (SE). Values within each column followed by the same letters are not significantly different according to Tukey’s test at p < 0.05. The effects of CO₂ concentration, cultivar and their interactions are presented. *, ** and ***, respectively, represent the significant levels when p < 0.05, p < 0.01 and p < 0.001; “ns” represents non-significant difference. Abbreviations: CS, Chinese spring; CM44, Chuanmai 44; NM9, Neimai 9.

). These findings demonstrated genotype-specific modulation of grain morphology by eCO₂, with distinct effects on dimensional parameters across different wheat cultivars.

Under eCO₂ conditions, the main spike length of CS showed a modest increase, while tiller number was markedly enhanced. However, significant reductions were observed in main panicle spikelet number, fertile spikelet number per main spike, and grain number per spike. Notably, yield-related parameters, including grain weight per panicle and hundred-grain weight (HGW), remained statistically unchanged. Except for the significant increase in HGW, the other indexes of Chuanmai 44 decreased significantly. Neimai 9 exhibited a marked increase in HGW alongside significant decreases in main panicle spikelet number, fertile spikelet number, and total grains per panicle. Remaining agronomic traits including spike length, tiller number, and grain weight per panicle exhibited no statistically significant alterations (

Table 2. Effects of eCO₂ on yield-related traits of three wheat cultivars.

Cultivar-CO2	SL (cm)	SN	FSN	GN	TN	GW (g)	HGW (g)
CS-aCO2	6.32 ± 0.29 d	18.48 ± 1.11 d	13.90 ± 1.93 bc	37.20 ± 6.07 a	2.18 ± 1.05 b	3.50 ± 1.47 a	3.26 ± 0.00 a
CS-eCO2	6.43 ± 0.56 cd	17.76 ± 1.41 e	11.81 ± 3.00 d	29.19 ± 6.35 c	2.95 ± 1.15 a	3.10 ± 1.25 a	3.26 ± 0.00 a
CM44-aCO2	6.62 ± 0.32 bc	21.43 ± 1.45 a	16.46 ± 1.42 a	34.74 ± 6.83 ab	2.41 ± 0.50 b	3.44 ± 1.67 a	3.23 ± 0.00 e
CM44-eCO2	6.17 ± 0.37 d	20.65 ± 1.26 b	12.86 ± 2.34 cd	23.67 ± 7.15 d	1.68 ± 0.72 c	2.59 ± 1.25 b	4.28 ± 0.00 a
NM9-aCO2	6.97 ± 0.65 a	19.42 ± 1.73 c	13.92 ± 2.49 b	32.60 ± 4.05 b	2.14 ± 0.67 b	2.72 ± 0.75 a	3.66 ± 0.00 c
NM9-eCO2	6.73 ± 0.15 ab	18.71 ± 1.24 d	11.94 ± 2.79 d	27.90 ± 7.99 c	2.18 ± 0.81 b	2.52 ± 0.98 a	3.97 ± 0.00 b
Cultivar	***	***	***	**	***	ns	***
CO2	*	***	***	***	ns	ns	***
Cultivar × CO2	*	ns	ns	*	***	ns	***

Note: The data in the table are the mean ± standard error of the means (SE). Within each column values followed by the same letters are not significantly different according to Tukey’s test at p < 0.05. The effects of CO₂ concentration, cultivar and their interactions are presented. *, ** and ***, respectively, represent the significant levels when p < 0.05, p < 0.01 and p < 0.001; “ns” represents non-significant difference. Abbreviations: CS, Chinese spring; CM44, Chuanmai 44; NM9, Neimai 9; SL, main spike length; SN, main panicle spikelet number; FSN, fertile spikelet number on the main spike; GN, grain number on the main spike; TN, tillers number; GW, grain weight per panicle; HGW, hundred-grain weight.

). The PCA plot illustrate distinct clustering patterns among the three wheat cultivars (Figure 2A) and the separation of yield indicators under two CO₂ concentrations (Figure 2B). Figure 2A demonstrate clear spatial segregation of the three cultivars, consistent with ANOVA results, indicating divergent responses of yield component traits to eCO₂ across genotypes. In Figure 2B, all yield indicators except HGW exhibited vector orientations aligned with or proximal to the aCO₂ treatment, while HGW uniquely associated with the eCO₂ treatment. The differential clustering patterns and vector alignments collectively underscored genotype-specific plasticity in balancing grain weight enhancement against reductions in other yield-related parameters under CO₂ enrichment.

3.3. C and N Concentrations in Grain

Elevated CO₂ (~900 μmol/mol) showed differential effects on carbon content, nitrogen content, and C:N ratio in CS, Chuanmai 44, and Neimai 9. Grain carbon content remained statistically unchanged across all three cultivars (p > 0.05). Nitrogen content exhibited an increasing trend, with a significant elevation (7.27%) observed exclusively in CS (p < 0.05). The C:N ratio showed significant reductions in all cultivars (p < 0.05), with CS displaying the most pronounced decrease (7.10%) (

Table 3. Effects of eCO₂ on carbon and nitrogen content in grains of three wheat cultivars.

Cultivar-CO2	C concentration (%)	N concentration (%)	C/N
CS-aCO2	41.16 ± 0.25 a	2.89 ± 0.02 d	14.23 ± 0.12 a
CS-eCO2	40.97 ± 0.23 a	3.10 ± 0.06 b	13.22 ± 0.19 c
CM44-aCO2	40.59 ± 0.39 a	3.03 ± 0.02 bc	13.40 ± 0.10 b
CM44-eCO2	41.12 ± 0.28 a	3.08 ± 0.03 b	13.34 ± 0.14 bc
NM9-aCO2	41.04 ± 0.10 a	3.30 ± 0.03 a	12.43 ± 0.08 d
NM9-eCO2	40.78 ± 0.28 a	3.35 ± 0.02 a	12.18 ± 0.07 e
Cultivar	ns	***	***
CO2	ns	***	***
Cultivar × CO2	*	***	***

Note: The data in the table are the mean ± standard error of the means (SE). Within each column values followed by the same letters are not significantly different according to Tukey’s test at p < 0.05. The effects of CO₂ concentration, cultivar and their interactions are presented. * and ***, respectively, represent the significant levels when p < 0.05 and p < 0.001; “ns” represents non-significant difference. Abbreviations: CS, Chinese spring; CM44, Chuanmai 44; NM9, Neimai 9.

).

3.4. Grain Amino Acid Content

Analysis of amino acid profiles under eCO₂ conditions revealed distinct genotype-specific patterns (

Table 4. Effects of eCO₂ on amino acid content in grains of three wheat cultivars.

Cultivar-CO2	Asp	Thr	Ser	Glu	Pro	Gly	Ala	Val	Met	Ile	Leu	Tyr	Phe	His	Lys	Arg
CS-aCO2	15.59 ± 2.43 c	11.07 ± 2.34 b	13.33 ± 1.83 d	56.08 ± 3.26 cd	19.37 ± 5.25 a	11.7 ± 1.12 c	11.44 ± 1.91 b	12.33 ± 1.56 c	8.12 ± 2.99 b	12.64 ± 2.41 b	16.95 ± 1.74 d	10.93 ± 2.64 b	12.6 ± 1.26 e	13.64 ± 2.73 b	10.98 ± 1.77 c	16.16 ± 2.76 b
CS-eCO2	19.5 ± 0.76 ab	14.26 ± 0.39 a	16.26 ± 0.71 abc	59.97 ± 4.96 bc	14.63 ± 0.14 b	13.82 ± 0.63 a	14.17 ± 0.38 a	14.76 ± 0.45 ab	11.99 ± 0.72 a	15.92 ± 0.57 a	20.12 ± 1.00 bc	14.5 ± 0.63 a	15.07 ± 0.70 bc	17.51 ± 0.58 a	13.3 ± 0.46 ab	20.44 ± 0.79 a
CM44-aCO2	19.68 ± 0.39 ab	14.61 ± 0.17 a	16.8 ± 0.29 ab	64.55 ± 1.93 ab	14.64 ± 0.24 b	14.16 ± 0.34 a	14.15 ± 0.34 a	15.2 ± 0.18 ab	11.63 ± 0.15 a	16.26 ± 0.27 a	20.97 ± 0.43 ab	14.49 ± 0.29 a	15.66 ± 0.30 ab	17.61 ± 0.30 a	13.4 ± 0.36 ab	20.15 ± 0.43 a
CM44-eCO2	18.01 ± 0.39 b	13.63 ± 0.17 a	15.23 ± 0.27 c	52.78 ± 1.73 e	13.71 ± 0.15 b	12.71 ± 0.25 b	13.32 ± 0.20 a	13.97 ± 0.21 b	11.8 ± 0.35 a	15.08 ± 0.13 a	18.7 ± 0.36 c	13.78 ± 0.36 a	13.78 ± 0.27 d	16.42 ± 0.33 a	12.38 ± 0.23 bc	18.69 ± 0.29 a
NM9-aCO2	20.25 ± 0.16 a	14.77 ± 0.09 a	17.44 ± 0.08 a	68.43 ± 1.06 a	15.7 ± 0.39 ab	14.32 ± 0.11 a	14.35 ± 0.54 a	15.69 ± 0.41 a	12.39 ± 0.52 a	16.86 ± 0.16 a	21.86 ± 0.20 a	15.16 ± 0.02 a	16.26 ± 0.16 a	18.1 ± 0.10 a	13.97 ± 0.11 a	20.69 ± 0.16 a
NM9-eCO2	18.22 ± 0.37 b	13.76 ± 0.25 a	15.6 ± 0.38 bc	55.59 ± 2.22 cd	16.15 ± 0.50 ab	12.74 ± 0.29 b	13.16 ± 0.56 a	14.04 ± 0.58 b	11.78 ± 0.34 a	15.37 ± 0.12 a	19.22 ± 0.40 c	13.52 ± 0.46 a	14.38 ± 0.35 cd	16.98 ± 0.28 a	12.63 ± 0.29 ab	18.88 ± 0.19 a
Cultivar	*	*	*	ns	ns	ns	ns	*	*	*	**	ns	**	*	ns	ns
CO2	ns	ns	ns	***	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns
Cultivar × CO2	***	**	***	***	ns	***	**	***	*	**	***	**	***	**	**	**

Note: The data in the table are the mean ± standard error of the means (SE). Within each column values followed by the same letters are not significantly different according to Tukey’s test at p < 0.05. The effects of CO₂ concentration, cultivar and their interactions are presented. *, ** and ***, respectively, represent the significant levels when p < 0.05, p < 0.01 and p < 0.001; “ns” represents non-significant difference. Abbreviations: CS, Chinese spring; CM44, Chuanmai 44; NM9, Neimai 9.

). In cultivar CS, 14 amino acids showed significant increases (p < 0.05), except proline and glutamic acid. Contrastingly, Chuanmai 44 exhibited declining trends in 15 amino acids, with significant reductions (p < 0.05) in tryptophan, glutamic acid, glycine, leucine, and phenylalanine, despite marginal elevation in methionine. Neimai 9 demonstrated reduced levels in 14 amino acids, with significant decreases (p < 0.05) in aspartic acid, serine, glutamic acid, glycine, valine, leucine, and phenylalanine, while proline showed slight accumulation. Collectively, eCO₂ enhanced amino acid biosynthesis in CS but suppressed it in Chuanmai 44 and Neimai 9.

PCA (Figure 3) further elucidated these responses. Figure 3A displays extensive overlap in amino acid clustering across cultivar, paralleled by CO₂-concentration-dependent intermixing in Figure 3B. This spatial convergence aligns with Table 4 findings, collectively demonstrating significant CO₂ × genotype interactive effects (p < 0.05) on amino acid metabolism.

3.5. Grain Mineral Concentration

The effects of eCO₂ (~900 μmol/mol) on grain mineral composition across cultivars CS, Chuanmai 44, and Neimai 9 are summarized in

Table 5. Effects of eCO₂ on the content of mineral elements in grains of three wheat cultivars.

Cultivar-CO2	Ca (mg/g)	K (mg/g)	Mg (mg/g)	Cu (mg/g)	Mn (mg/g)	Zn (mg/g)	Fe (mg/g)
CS-aCO2	0.670 ± 0.113 ab	3.810 ± 0.510 bc	0.403 ± 0.130 ab	0.047 ± 0.031 a	0.039 ± 0.005 a	0.021 ± 0.006 b	0.049 ± 0.013 a
CS-eCO2	0.524 ± 0.156 ab	4.110 ± 0.109 bc	0.423 ± 0.035 ab	0.024 ± 0.002 a	0.040 ± 0.003 a	0.038 ± 0.007 ab	0.050 ± 0.003 a
CM44-aCO2	0.713 ± 0.085 a	4.686 ± 0.355 ab	0.520 ± 0.031 ab	0.030 ± 0.015 a	0.043 ± 0.003 a	0.025 ± 0.013 ab	0.038 ± 0.015 a
CM44-eCO2	0.709 ± 0.162 a	4.109 ± 0.558 bc	0.338 ± 0.053 b	0.028 ± 0.003 a	0.037 ± 0.012 a	0.029 ± 0.003 ab	0.052 ± 0.002 a
NM9-aCO2	0.622 ± 0.081 ab	5.195 ± 0.439 a	0.617 ± 0.096 a	0.030 ± 0.006 a	0.045 ± 0.014 a	0.041 ± 0.005 a	0.046 ± 0.012 a
NM9-eCO2	0.477 ± 0.111 b	3.583 ± 0.787 c	0.417 ± 0.198 b	0.020 ± 0.006 a	0.042 ± 0.016 a	0.028 ± 0.013 ab	0.045 ± 0.004 a
Cultivar	*	ns	ns	ns	ns	ns	ns
CO2	ns	*	*	ns	ns	ns	ns
Cultivar × CO2	ns	*	ns	ns	ns	*	ns

Note: The data in the table are the mean ± standard error of the means (SE). Within each column values followed by the same letters are not significantly different according to Tukey’s test at p < 0.05. The effects of CO₂ concentration, cultivar and their interactions are presented. * represent the significant levels when p < 0.05; “ns” represents non-significant difference. Abbreviations: CS, Chinese spring; CM44, Chuanmai 44; NM9, Neimai 9.

. No statistically significant changes in Ca, Cu, K, Mg, Zn, Fe, or Mn concentrations were detected in CS and Chuanmai 44 under eCO₂ conditions. Neimai 9 demonstrated significant decreases in K and Mg (p < 0.05), with non-significant reductions in other elements. PCA ordination revealed high varietal dependency in mineral responses (Figure 4A). The intermixed clustering among cultivars and CO₂ treatments, coupled with divergent parameter vector orientations, confirmed strong varietal effects and significant genotype × CO₂ interactions. Figure 4B further illustrates bidirectional regulation: overlapping clusters between CO₂ treatments with parameter vectors distributed across both aCO₂ and eCO₂ axes, quantitatively demonstrating cultivar-specific mineral reallocation patterns. These findings established that eCO₂ differentially modulated grain mineral homeostasis.

4. Discussion

4.1. Elevated CO₂ Levels Changed Plant Gas Exchange and Improved Water Use Efficiency

The physiological impacts of eCO₂ concentrations on angiosperms are well-documented, particularly regarding enhanced Pn and WUEi, though species-specific response patterns exist [17,19,20]. In this study, under eCO₂ concentrations (~900 μmol/mol), all three wheat cultivars displayed significant Pn enhancement (p < 0.05), but this varied among cultivars (CS: +28.95%, Chuanmai 44: +10.60%, Neimai 9: +24.80%), suggesting that there may be differential stomatal regulation strategies under eCO₂ [21]. Another relatively consistent response to eCO₂ was an increase in WUEi. Our data showed the WUEi of three cultivars was enhanced by eCO₂ in the order of Chuanmai 44 (+279.20%) > Neimai 9 (+148.63%) > CS (+97.82%). These differences in physiological response to eCO₂ among different wheat cultivars revealed that the underlying biochemical and molecular mechanisms of their response to CO₂ enrichment may be different, which warrants further exploration.

4.2. Elevated CO₂ Levels Increased the Hundred-Grain Weight (HGW) but Decreased Individual Plant Yield

Theoretically, synergistic increases in Pn and WUEi should drive biomass accumulation and yield enhancement. Both open-top chamber (OTC) and Free-Air CO₂ Enrichment (FACE) experiments confirmed eCO₂-mediated yield increased in crops [22,23]. In the present study, under ~900 μmol/mol CO₂, Chuanmai 44 and Neimai 9 exhibited significant hundred-grain weight (HGW) increases (+32.51% and +8.47%, p < 0.05), which was consistent with previous studies [24,25]. The increase in HGW with eCO₂ may be due to increased grain length and/or grain width [26]. Our research found that eCO₂ significantly elongated the grain in Neimai 9 (+2.89%, p < 0.05) or broadened the grain in Chuanmai 44 (+9.03%, p < 0.05), whereas that of CS remained relatively stable (Table 1). This partially accounted for the observed increase in HGW in Neimai 9 and Chuanmai 44, in contrast to the unaltered HGW in CS. Additionally, it could also be related to the higher starch content in grains [27], as the density of starch was significantly greater than that of structural polysaccharides and proteins [28]. Of course, this needs further verification.

However, for Chuanmai 44 and Neimai 9, critical yield components displayed contrasting trends: significant reductions (p < 0.05) occurred in main spike length (Chuanmai 44: −6.80%, Neimai 9: −3.44%), spikelets per spike (−3.64% and −3.64%), fertile spikelets (−21.87% and −14.22%), grains per spike (−31.87% and −14.42%), and single-plant spike weight (−24.71% and −7.35%). Chuanmai 44 additionally showed decreased tiller number (−30.29%, p < 0.05), whereas Neimai 9 maintained stable tillering. These results are inconsistent with many previous studies [29]. These physiological trade-offs between grain filling and architectural parameters ultimately constrain whole-plant productivity, revealing a complex balance between eCO₂-driven photosynthetic gains and source–sink regulation [22,30,31].

This study was conducted under controlled greenhouse conditions with two applications of standard fertilizer during the tillering initiation phase and heading stage. All environmental parameters, including temperature and water supply, were maintained consistent across treatments, eliminating confounding stressors such as high-temperature or drought conditions. While eCO₂ would theoretically enhance wheat yield through CO₂ fertilization effects, the three investigated cultivars—particularly Chuanmai 44 and Neimai 9—exhibited significant reductions in key yield components, including spikelet per main spike, fertile spikelet per main spike, and grains per main spike (Table 2). This paradoxical response could be attributed to two primary factors. First, the responsiveness to eCO₂ varies significantly across plant genotypes [32,33]. The plant type and structure were supposed to have an important effect on source–sink relations, and an ideal plant type characterized by more vertical upper leaves and more horizontal lower leaves has been proposed [34,35]. Compared to CS, Chuanmai 44 and Neimai 9 have relatively larger leaves, which often droop vertically under eCO₂ conditions later in their growth period. This somewhat affected the source of individual stems, thereby affecting the source–sink relationship. Second, the concentration-dependent nature of CO₂ fertilization benefits was only effective within a specific threshold range [36]. From the jointing to grain-filling stages, three cultivars were continuously exposed to hyper-elevated CO₂ concentrations. We have also observed that such supra-optimal CO₂ levels accelerated plant growth and developmental processes, inducing premature phenological transitions. The entire phenological periods of Chuanmai 44 and Neimai 9 were shortened by an average of 5~6 days, while no significant effect was observed on CS. This temporal compression reduced the duration for dry matter accumulation and truncated critical reproductive phases, including grain filling and pollination, thereby directly compromising yield formation [37]. These findings demonstrate that CO₂ fertilization effects are constrained by both genetic determinants and concentration thresholds, with hyper-elevated CO₂ (>900 μmol/mol) overriding potential benefits through maladaptive phenological shifts.

4.3. Elevated CO₂ Concentrations Negatively Affect Wheat Grain Quality, but These Impacts Exhibit Genotype-Dependent Variation

Numerous studies have established that eCO₂ concentrations lead to significant reductions in nitrogen content across crop species [38], though the underlying mechanisms remain incompletely resolved. A prevailing hypothesis attributes this phenomenon to CO₂-induced photosynthetic enhancement, which drives accelerated biomass accumulation while soil nitrogen availability fails to match the amplified nutrient demands of CO₂-stimulated growth, resulting in nitrogen “dilution” within plant tissues [39]. Contrasting with these established patterns, our study revealed divergent responses under ~900 μmol/mol CO₂ exposure: the nitrogen content in CS grains exhibited a significant increase (+7.27%, p < 0.05), while Chuanmai 44 and Neimai 9 maintained stable nitrogen levels (+1.65% and +1.52%, respectively, p > 0.05). This indicated that the eCO₂ effect on the C metabolism and N assimilation occurred in a highly genotype-specific manner. These three wheat cultivars may have a high nitrogen uptake efficiency, and sufficient nitrogen supplementation throughout the growth cycle may counteract CO₂-driven nitrogen depletion through enhanced soil nitrogen uptake efficiency [40].

The content of amino acids and mineral elements in grains serves as a critical parameter for evaluating wheat grain quality [41]. Previous studies have demonstrated that elevated atmospheric CO₂ concentrations reduce grain amino acid content [4,11] and diminish levels of macronutrients and micronutrients such as iron, zinc, magnesium, and calcium [42]. In this study, under CO₂ enrichment at 900 μmol/mol, the total amino acid content in CS grains showed a significant increase, whereas Chuanmai 44 and Neimai 9 exhibited widespread reductions in major amino acids with statistical significance (Table 4). A similar divergence was observed for mineral elements. All seven mineral concentrations in CS and Chuanmai 44 remained stable. For Neimai 9, only K (−31.03%) and Mg (−32.41%) showed a significant decline, while the reductions in other elements were not insignificant. These findings demonstrated substantial genotypic variation in CO₂ responsiveness of amino acid and mineral profiles, challenging the prevailing paradigm that eCO₂ universally degraded crop grain quality [43]. Notably, the HGW of CS remained unchanged under eCO₂, contrasting with significant increases in Chuanmai 44 and Neimai 9. This contrast highlights a typical inverse correlation between yield enhancement and quality maintenance under eCO₂ [44], emphasizing the metabolic trade-offs inherent in carbon–nutrient allocation.

The detrimental effects of eCO₂ on crop quality are now well-documented. The “carbohydrate dilution” hypothesis posited that CO₂-driven biomass accumulation dilutes nutrient concentrations, but the dilution was not selective [45]. Another mechanism implicated that eCO₂ diminished the transpiration rate, thereby weakening the mass flow of transpiration-driven mineral nutrients, resulting in a parallel downward trend for all mineral elements [46,47]. We observed divergent trends across wheat cultivars and inconsistent patterns within the same cultivar—some nutrients declined while others remained stable or even increased. This variability aligned with emerging reports of similar anomalies in other crops [48], collectively underscoring the insufficiency of current models to fully explain CO₂–nutrient dynamics [49]. Further research on the mechanism needs to be carried out, which can not only improve plant physiology, but also provide a theoretical reference for high-quality crop breeding.

5. Conclusions

Our data indicated that the gas exchange rates, yields, and quality of three cultivars vary under the influence of eCO₂. The Pn and WUEi of the three cultivars significantly increased. Elevated CO₂ increased the HGW of Chuanmai 44 and Neimai 9 in contrast to the unaltered HGW in CS, but decreased individual plant yield. CO₂ elevation significantly or slightly increased the N content in the grains of the three cultivars. Elevated CO₂ enhanced amino acid biosynthesis in CS but suppressed it in Chuanmai 44 and Neimai 9. No statistically significant changes in grain mineral concentrations were detected in CS and Chuanmai 44 under eCO₂ conditions. Neimai 9 demonstrated significant decreases in K and Mg, with non-significant reductions in other elements.

Future research should prioritize integrating CO₂ elevation with other climatic factors (e.g., temperature, water availability) to identify adaptive genotypes. We posit that integrating conventional cross-breeding techniques with molecular marker-assisted selection and gene-editing technologies (e.g., genomic selection, CRISPR, variable-rate fertilization) will enable the development of climate-resilient wheat cultivars.