Performance Evaluation of Three Peanut Cultivars Grown under Elevated CO₂ Concentrations

Abstract

This study explored the performance and physiological responses of three commercially used peanut cultivars in Australian farming systems under ambient and elevated CO₂ conditions, aiming to identify the most suitable genotype for dual-purpose (grain and graze) cropping experiments. The experiment utilized an open-top chamber (OTC) facility to regulate CO₂ concentrations. The elevated CO₂ (EC) treatment targeted approximately 650 ± 50 µmol mol⁻¹, while both ambient CO₂ (AC) and control plots operated at a concentration of approximately 400 µmol mol⁻¹. Notably, control plots without chambers served as a reference for current CO₂ and environmental conditions. In contrast, despite having the same ambient CO₂ concentration, AC plots were enclosed in chambers, allowing for plant growth comparisons with EC plots with the same environmental conditions aside from CO₂ levels. The analyses revealed significant effects of CO₂ enrichment on peanut plants. In particular, the EC treatment led to enhanced photosynthetic rates (20% in Kairi, 31% in Holt, and 19% in Alloway), alongside reduced stomatal conductance (−55% in Kairi, −32% in Holt, and −40% in Alloway), transpiration, and increased water use efficiency compared to AC conditions. Elevated CO₂ levels positively influenced pod yields in Kairi (+41%) and Alloway (+36%). However, CO₂ enrichment did not significantly alter the protein content, total phenolic content, cupric-reducing antioxidant capacity, and ferric-reducing antioxidant power of peanut plant material. Furthermore, no significant differences were observed in the phytochemical composition among the three cultivars under ambient or elevated CO₂ conditions. On the other hand, analysis of the fibre structure conducted on peanut stover harvested at plant maturity suggested potential declines in feedstock quality. Based on the findings of this research, further investigations and testing, including simulated grazing trials, will be carried out to identify a single breed line suitable for dual-purpose management under future elevated CO₂ conditions.

1. Introduction

The constant increase in the global atmospheric CO₂ concentration poses a potential threat to crops utilizing the C₃ metabolic pathway, potentially altering their nutrient composition [1]. Studies suggest that countries with plant-based diets may see a reduction in protein intake of over 5% due to elevated CO₂ levels, particularly impacting tropical and subtropical regions [2]. However, legumes, with their ability to fix atmospheric nitrogen through symbiosis with nitrogen-fixing bacteria, may respond differently to elevated CO₂ levels. Therefore, promoting legume cultivation in agricultural systems could help alleviate pressure on the existing farming systems to maintain food productivity levels amid increasing demand.

The benefits of elevated CO₂ concentrations on C₃ plants are well documented in certain plant species, including increased biomass, improved resource use efficiency, higher yields, and stimulation of the photosynthetic rate [3,4]. However, this photosynthetic stimulation is often observed only in short-term periods, typically lasting from a few days to two or three weeks [5,6,7].

Elevated CO₂ concentrations have been linked to diverse effects on legumes’ vegetative growth and productivity, influenced by complex interactions between environmental and growth conditions. These interactions include intra- and interspecific variability, nitrogen fixation capability, and nutrient uptake dynamics under elevated CO₂ conditions. While some studies suggest that legumes exposed to elevated CO₂ may perform more efficiently than non-nitrogen-fixing legumes, the impact of elevated CO₂ on yield and biomass production remains unclear due to instances of reduced yield in certain cases [8].

Peanuts, a significant oilseed crop globally, are valued for their oil content, which typically ranges from 32 to 57% w/w, and protein concentration, which varies from 22 to 30% w/w, depending on genotype and growth conditions [9,10]. However, their productivity faces potential threats from climate change, including elevated CO₂ levels. While peanuts, as C₃ plants, may benefit from increased CO₂ concentrations, the outcomes are complex and depend on various environmental factors such as temperature, rainfall patterns, and the prevalence of pests and diseases.

Several studies have explored the impact of elevated CO₂ (EC) on peanut performance. For instance, Stanciel et al. [11] found that peanut plants grown under EC conditions of 800 µmol mol⁻¹ exhibited significantly higher net photosynthetic rates than those grown at ambient concentrations (AC) of 400 µmol mol⁻¹. This reduction in stomatal conductance accompanied this increase in photosynthetic rates, and EC has been shown to positively affect specific morphological parameters of peanuts, such as increased biomass traits (leaf fresh and dry weights) and higher pod fresh weights, resulting in an improved harvest index (HI).

A study conducted by Vaidya et al. [12] similarly documented that CO₂ enrichment enhances leaf net photosynthesis while reducing stomatal conductance, leading to increased water use efficiency. However, the study also revealed significant intraspecific variability in the physiological performance of different peanut genotypes under elevated CO₂ conditions. Additionally, elevated CO₂ positively influenced growth and yield parameters, although this effect exhibited variability between genotypes.

More recent studies have found that EC positively impacts peanut performance, particularly by increasing leaf-level CO₂ assimilation and improvising transpiration efficiency, leading to enhanced vegetative biomass production and pod yield [13,14]. The positive effect of elevated CO₂ is further enhanced when combined with higher temperatures, as elevated CO₂ ameliorates the negative impact of heat on yield and growth traits [15].

However, the response is susceptible to significant inter-specific variability, as reported in a study conducted on leguminous cover crops grown under different CO₂ [16,17]. Additionally, significant intra-specific variability in the response of yield and nutritional traits has been observed in soybean genotypes [18].

Given the observed inter- and intraspecific variability in the response of different crops to CO₂ enrichment, it is increasingly important to understand the metabolic response of crops and plants to this stimulation. This understanding assists in the selection of genotypes with the potential for increased productivity under elevated CO₂ conditions.

This study aims to evaluate the performance of three commercially used peanut cultivars within Australian farming systems. The specific goals are as follows:

• Assess the physiological, growth, and yield responses of the three peanut cultivars under both ambient CO₂ conditions (~400 µmol mol⁻¹) and enriched CO₂ conditions (650 ± 50 µmol mol⁻¹) and analyse variability among cultivars in terms of growth, yield, biomass production, and kernel and forage attributes. We anticipate observing variability in physiological responses, such as differences in photosynthesis rates, water use efficiency, stomatal conductance, and transpiration. Additionally, we expect variations in growth metrics, including biomass accumulation and yield components such as pod number, kernel size, and overall yield. Furthermore, we predict differences in kernel attributes, such as variations in protein content and nutritional quality. This hypothesis also extends to include variations in the nutritional quality of the forage. These expectations are based on the premise that while the three cultivars may share similar potential yield, growth, and maturity characteristics, they will likely demonstrate distinct responses to ambient and enriched CO₂ conditions due to inherent genetic differences.
• Identify the most promising and suitable genotypes for future dual-purpose (grain and graze) cropping experiments. This includes evaluating the potential for yield productivity and stock feed biomass.

By achieving these objectives, we aim to provide insights into the optimal peanut genotype for enhanced productivity and resilience in the context of changing CO₂ levels. This work completes our previous research focused on the implications of elevated CO₂ on the physicochemical traits of peanuts [19].

2. Materials and Methods

2.1. Experiment’s Site and Growth Conditions

The three peanut cultivars were grown at the Central Queensland Innovation Research Precinct (CQIRP), located in Rockhampton, Queensland, Australia (23°19′22″ S; 150°30′53″ E, 43 m elevation).

Plant cultivation and CO₂ enrichment were conducted using an open-top chambers (OTCs) facility, of which the design, fabrication, and validation have been described in detail in our previous work [20].

In our study, we utilized a total of twelve plots, with four replicates for each of the three treatments. Specifically, we had eight plots with OTCs—four allocated for elevated CO₂ enrichment (EC) maintaining a target concentration of 650 µmol mol⁻¹ and four for ambient CO₂ (AC)—and four plots without OTCs serving as controls. Both AC and control plots maintained ambient CO₂ concentrations of approximately 400 µmol mol⁻¹.

Carbon dioxide enrichment began 12 days after sowing (DAS) when the plants had fully emerged and continued during daytime hours (0530–1800) until harvest at 132 DAS. The daily mean [CO₂] within the open-top chambers (OTCs) and the standard deviation during the injection period from 9 February 2022 to 29 June 2022 was 655 ± 42 µmol mol⁻¹—the weather data collected during the experiments are reported in Figure A1. Temperature sensors monitored the chamber temperature at regular intervals (10 min). The daily average temperature in the AC chambers was 25.3 ± 3.5 °C, while in the EC chambers, it was slightly higher at 25.9 ± 3.5 °C. The minimum daily temperature in the control group was 19.8 ± 3.2 °C, and the maximum daily temperature was 29.9 ± 4.3 °C. The total rainfall was recorded using a rain gauge installed near the experimental site, totalling 287 mm during the study period. Water was applied at set intervals with an automated irrigation system to maintain the crop under well-watered conditions throughout the experiment. Soil moisture at 80% above field capacity and automated irrigation was deactivated during rainy days. Soil moisture was measured with plug-in probes (Meter Group, Pullman, WA, USA) installed in the soil at crop root depth (10–20 cm), and data were recorded with ECH2O Em50 dataloggers (Meter Group, Pullman, WA, USA).

2.2. Peanut Material and Planting Pattern

Three high-oleic peanut lines, Holt, Kairi, and Alloway, were sown from seed material provided by the Peanut Company of Australia (PCA). The characteristics of the cultivars used in this study are reported in

Table 1. Description of the main agronomic traits for the cultivars Alloway, Holt, and Kairi adapted from the peanut cultivar sheets of the Peanut Company of Australia.

Attribute	Alloway	Holt	Kairi
Type	Runner	Runner	Runner
Growth habit	Semi-erect	Semi-prostrate	Semi-erect
Oil	High oleic	High oleic	High oleic
Plant morphology traits
Canopy density	Dense	Dense	Dense
Branching	Profuse	Profuse	Profuse
Leaf color intensity	Medium	Medium	Dark
Leaflet length	Medium	Medium	Medium
Leaflet shape	Retuse	Retuse	Retuse
Pods and kernels traits
Pod constrictions	Weak	Medium	Weak
Pod reticulation at surface	Medium	Medium	Strong
Number of kernels per pod	Two	Two	Two
100 kernels weight	Medium	Medium	High
Thickness of shell	Thin	Thin	Thin
Plant phenology
Time of maturity	Late	Late	Late
Crop maturity	145–148 DAS	154 DAS	140–150 DAS
Yield
Irrigated	7.5 t h−1	5.5 t h−1	7.5 t h−1
Dry-land	2.5 t h−1	2.2 t h−1	2.5 t h−1
Disease resistance
Pod rot	Medium	Absent	High
Leaf rust	Moderate	Medium	Very high
Leaf spot	High	Moderate	Very high
Net blotch	High	Moderate	Moderate

.

The soil was prepared and fertilized following the recommended rates outlined in the PCA peanut production guidelines [21]. This included 5 g m⁻² of N (as ammonium sulfate), 10 g m⁻² of P (6.6% as insoluble citrate, 5% as soluble citrate), 20 g m⁻² of K (as sulfate of potassium) a, and 100 g m⁻² of Ca (gypsum). Additionally, microelements, including B, Zn Mg, Mn, and Cu, were added to the soil at a rate of 0.4 g m⁻².

The seeds of the three cultivars were pre-treated with a fungicide mix (Captan^®, 0.12%; Quintozene^® 0.12%) and were inoculated with a rhizobia strain selected for peanuts belonging to group type P, strain NC92 (NoduleN^TM, New Edge microbials, Wodonga, VIC, Australia). The inoculant solution was created by blending water with the inoculant powder until a thick slurry formed in a container. Seeds were then gently added to the mixture, ensuring they were uniformly coated.

Seeds were then planted simultaneously in the same plots distributed over three linear meters. Each cultivar was divided into half-lines, and these segments were randomly distributed in a completely randomized design across the plots, ensuring varying line sequences and orientations.

Approximately 20 peanut seeds of each cultivar were sown directly into the soil at a 3–4 cm depth following a Randomised Block Design (RBD). After germination, plants were thinned to 12 individuals for each cultivar per plot. The two plants located near the beds’ wall served as edge plants and were discarded during the measurements to minimize the edge effect.

2.3. Leaf Gas-Exchange Parameters

Leaf gas exchange parameters were measured using a portable infrared gas analyser, the LI-COR 6800 (LI-COR Inc., Lincoln, NE, USA). Measurements were taken from the first to the third youngest, healthy, and fully expanded leaf.

Two types of measurements were performed on different days:

• Instantaneous measurements during reproductive stage R2 using a solar photosynthetic photon flux density (PPFD) value of 1500 µmol mol⁻¹, humidity at 60%, fan speed at 8000 rpm, air flow set at 500 mmol⁻¹, and block cuvette temperature held at 30 °C. Three measurements per plot were taken between 0900 and 1100 AM in the morning and 1300 and 1500 PM. The cuvette [CO₂] was adjusted to either 400 mmol⁻¹ or 650 mmol⁻¹ for each plot, matching the corresponding growth [CO₂] conditions. Carbon net assimilation rates at saturated light (A_net), stomatal conductance (g_sw), transpiration (E), and leaf intercellular [CO₂] (C_i) were measured in peanut plants grown in Control (plots without chamber), AC, and EC plots for three replicates. The intrinsic water use efficiency (_iWUE) was calculated as the ratio between A_net and g_sw.
• Single-leaf long-term measurements and an acclimatization assessment were conducted at the growth stage (R2) using assimilation vs. intercellular CO₂ response curves at different CO₂ levels. These levels included 650, 400, 200, 0, 200, 400, 600, 800, 100, 0, and 650 µmol mol⁻¹ for plants grown under EC conditions. The same set points were used for plots grown under ambient conditions (AC and Control plots) but with initial and final cuvettes [CO₂] of 400 µmol mol⁻¹. The solar photosynthetic photon flux density was set at 1500 µmol m⁻² s⁻¹. A single measurement per cultivar (n = 4 × treatment × cultivar) was carried out in each AC and EC OTC and the Farquhar–von Caemmerer–Berry (FvCB) [22] photosynthesis model was adopted to fit A-Ci curves performed using the plantecophys RStudio (2023.03.0 + 386) package [23]. Subsequently, maximum Rubisco carboxylase activity (V_cmax), maximal electron transport rate (J_max), J_max/V_cmax ratio, and daytime respiration (Rd) were determined.

2.4. Peanuts’ Agronomic Traits

The peanut plants were carefully uprooted from the raised beds, one breed line at a time, digging with a shovel into the ground well below the root system, ensuring that no damage was caused. The length of each plant’s roots and main stems was measured and recorded for individual plants.

Subsequently, the root systems and shoots were separated near the root crown. The above-ground section was then partitioned into leaves and stems, with their respective fresh weight recorded. Plant materials were stored in paper bags and dried in a forced air-drying oven at 65 °C for 72 h or until a constant weight was achieved.

2.4.1. Roots Nodules Score

The nodule presence was assessed using a system described by Corbin et al. [24], designed for visually assessing the nodulation and modified for use with peanut root systems. Briefly, the scores of all plants of a given plot were summed and divided by the number of plants, yielding a mean value. Scores ranging from 0–2 indicated non-existent or poor nodulation, 2–3 denoted moderate nodulation, 3–4 signified good nodulation, and 4–5 represented excellent nodulation.

2.4.2. Pods and Kernels Yield Traits

The peanut pods were separated from the root system, washed, and dried with paper before being counted, weighed, and oven-dried at 30 ± 2 °C until reaching a constant weight. Subsequently, the dried pods and kernels’ dry weights were measured individually using a digital weighing balance (Mettler Toledo PM2000, 0.01 g readability) to determine the average yield per plant for each variety across different treatments.

To assess bulk density, pods and kernels were placed in a 500 mL chondrometer (Graintec Scientific, Toowoomba, QLD, Australia), and their weight (g) was divided by the volume of the container as shown in Equation (1) [25]. This process was replicated three times, and the average values were converted to g:

$$\mathrm{BD} (\mathrm{kg}{\mathrm{m}}^{-3})=\frac{\mathrm{Weight} \mathrm{of} \mathrm{the} \mathrm{sample}}{\mathrm{Volume} \mathrm{of} \mathrm{the} \mathrm{container}}$$

(1)

Sixty healthy pods and kernels from each cultivar in each CO₂ treatment (Control, AC, and EC) were randomly selected for physical characterization. The length (L), width (W), and thickness (T) of pods and kernels were measured in mm using a high-precision digital calliper (Craftright, 0–150 mm range, 0.01 mm). In addition, the weight of individual kernels was measured using a digital weighing balance (Mettler Toledo PM2000, 0.01 g readability).

The following shape and size characteristics were calculated:

The geometric mean diameter (D_g) was determined using the size measurements of length (L), width (W), and thickness (T) following Equation (2) [26]:

$${\mathrm{D}}_{\mathrm{g} }\left(\mathrm{mm}\right)={\left[\left(\mathrm{LWT}\right)\right]}^{1/3}$$

(2)

The sphericity ($\mathsf{\Phi }$) percentage of pods and kernels was calculated as the ratio of the geometric mean diameter (D_g) and the length (L) using Equation (3):

$$\mathsf{\Phi } (\%)=\left(\frac{{\mathrm{D}}_{\mathrm{g}}}{\mathrm{L}}\right) \times 100$$

(3)

The aspect ratio can provide essential insights into kernel or pod properties as it can affect germination and yield. This could help farmers plant optimal quality seeds to ensure good plant health and crop yields.

The aspect ratio (Ra) was calculated as a ratio between the width (W) and the length (L) of the given pod or peanut kernel according to Equation (4):

$$\mathrm{Ra} \left(\%\right)=\left(\frac{\mathrm{W}}{\mathrm{L}}\right) \times 100$$

(4)

The surface area (S) was determined from the geometric mean diameter (D_g) using t Equation (5) [26]:

$$\mathrm{S} \left({\mathrm{mm}}^2\right)=\mathsf{\pi } {{\mathrm{D}}_{\mathrm{g}}}^2$$

(5)

The harvest index for pods and kernels was determined using the Equations (6) and (7):

$$\mathrm{PHI}=\frac{\mathrm{Pods} \mathrm{yield}}{\mathrm{Pods} \mathrm{yield}+\mathrm{Biological} \mathrm{yield}} \times 100$$

(6)

$$\mathrm{KHI}=\frac{\mathrm{Kernels} \mathrm{yield}}{\mathrm{Kernels} \mathrm{yield}+\mathrm{Biological} \mathrm{yield}} \times 100$$

(7)

2.5. Chemical Analysis

2.5.1. Nitrogen, Carbon, Protein Content, and C-to-N Ratio

The samples of peanut stems, leaves, roots, and shells were ground into a fine powder using a rotor mill (unbranded) equipped with a 0.5 mm screen, ensuring uniformity in particle size. Kernel samples were ground into flour using a tube mill (IKA 100 control, Staufen im Breisgau, Germany). Subsequently, a fraction of each powdered sample was collected using the coning and quartering method [27]. The total content of carbon and nitrogen content in kernels and below-ground and above-ground biomass was determined using dry gas combustion with a LECO Tru Mac CN analyser (Leco Corp., St Joseph, MO, USA).

2.5.2. Biomass Methanolic Extraction for Antioxidant Analysis

The small sample fraction of above- and below-ground biomass and peanut flour were used for methanolic extraction prepared for assessing the total phenolic content (TPC) and the antioxidant capacity using two methods: the ferric-reducing antioxidant power (FRAP) and the cupric ion-reducing antioxidant capacity (CUPRAC) assays. Approximately 0.5 g of fine powder of each sample was weighed in duplicates placed in 10 mL centrifuge tubes and homogenized to 7 mL of 90% v/v aqueous methanol. The extraction was repeated on the solid matrices with an additional 7 mL of 90% methanol to gather any remaining antioxidant compounds. A sequence of vertexing and mixing finalized the procedure. The methanolic extracts were stored in the dark at 4 °C until required for further analyses. Although roots are not directly consumed by humans, they can be integrated into livestock feedstuff along with the above-ground biomass, impacting animal health. Additionally, studying the total phenolic content and the antioxidant activity in roots offers insights into the plant’s antioxidant capacity and defence against oxidative stress, crucial for overall plant health.

2.5.3. Total Phenolic Content

The total phenolic content of the methanolic extracts was determined by performing a modified Folin–Ciocalteu assay as described by Singleton and Rossi [28]. The final absorbance values were measured with a UV-VIS spectrophotometer at 760 nm, and the TPC concentration was calculated as the mean phenolic content expressed as milligrams of gallic acid equivalents per g of dry weight (mg GAE g⁻¹ DW). The absorbance results were then calculated using a calibration curve based on gallic acid standards in the range of 20–120 mg L⁻¹ and with R² = 0.99.

2.5.4. Ferric-Reducing Antioxidant Power

The antioxidant capacity of the methanolic extracts was determined using the FRAP assay developed by Benzie and Strain [29]. The final absorbances were read at 593 nm, and the FRAP was derived as a function of the equivalent absorbance of Trolox in ethanol solution using a calibration curve in the range of 10–175 mg L⁻¹ with R² = 0.99. The results were expressed as milligrams of Trolox equivalents per g of dry weight (mg TXE g⁻¹ DW).

2.5.5. Cupric Ion-Reducing Antioxidant Capacity

The second method for assessing the antioxidant capacity used here as validation was a modified CUPRAC assay developed by Apak et al. [30]. The absorbance values were read at 450 nm using a UV spectrophotometer, and the CUPRAC was derived as a function of the equivalent absorbance of Trolox (TE) in ethanol solution with a calibration curve developed in the range of 50–600 mg L⁻¹ and having R² = 0.99. The results were expressed as milligrams of Trolox equivalents per g dry weight (mg TXE g⁻¹ DW).

2.5.6. Forage Nutritive Fiber Composition

The fibre structure analyses of peanut forage involved calculating the Neutral Detergent Fibre (NDF) and the Acid Detergent Fibre (ADF) using an ANKOM 200 fibre analyser (ANKOM Technology, Macedon, NY, USA) following the method described by ANKOM Technology [31]. The final NDF and ADF percentages were calculated and converted into concentrations (mg g⁻¹ DM).

2.6. Statistical Analysis

Statistical analysis was performed using R programming (R Studio 4.1) to evaluate the interactive effects of two factors, CO₂ concentration ([CO₂]) and cultivar, on plant physiological, morphological, growth, yield, and physiochemical parameters. Data normality was tested using Shapiro–Wilk and Kolmogorov–Smirnov tests. Homoscedasticity, or the equality of variances, was assessed using Levene’s test. A two-way analysis of variance (ANOVA) was then conducted to examine the main effects and interactions between [CO₂] and cultivar. Differences among the means of the output variables were identified using Tukey’s multiple comparison method at a 95% confidence level. The following symbols were used for the different levels of significance: ns = not significant p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001. All data were reported as means ± standard error, and groups with different letters express differences among the treated combinations of [CO₂] and cultivar. Additionally, multivariate analysis was carried out using principal component analysis (PCA) to explain the contribution of the variables. This analysis was conducted to reduce dimensionality and identify the most significant variables contributing to the observed variations.

3. Results

In this section, the results for each parameter or attribute will be compared between the ambient CO₂ (AC) and elevated CO₂ (EC) treatments conducted within controlled OTCs, allowing for direct comparisons. The focus will primarily be on comparing AC and EC treatments, as these represent the main experimental conditions with equal growth parameters (e.g., temperature, light, and air humidity). Comparisons involving the control plots without chambers, which serve as a reference for the current environmental conditions and are not affected by chamber effects, will only be considered when explicitly mentioned for a specific attribute in the relevant section. However, control data will be reported in graphs and tables along with AC and EC data. This approach ensures a clear and focused analysis of the effects of [CO₂] on the parameters under investigation.

3.1. Plant Physiology Traits

3.1.1. Leaf Gas Exchange Parameters

All cultivars grown under elevated CO₂ showed increased photosynthetic rates (20% in Kairi, 31% in Holt, and 19% in Alloway) compared to ambient CO₂ (AC) and control conditions. A two-way ANOVA test revealed significant evidence (p = 0.0036) of A_net increases caused by [CO₂] treatments (AC and EC) (Figure 1a). However, it did not demonstrate significant variation among cultivars or the [CO₂] × cultivars interaction (p > 0.05), indicating that the cultivars in this study exhibited similar responses to e[CO₂] stimulation. The CO₂ treatment was highly significant (p < 0.001) for the other physiological parameters considered in this study, including g_sw, intercellular CO₂ (C_i), transpiration, and _iWUE. However, non-significant differences were recorded in g_sw for genotypes and the [CO₂] × cultivars interaction.

The response in g_sw varied significantly in EC compared to AC plants, with declines observed in Kairi (−55%), Holt (−32%), and Alloway (−40%) (Figure 1b). Additionally, C_i also showed a significant increment in response to e[CO₂], rising by 60, 50, and 17% in Holt, Alloway, and Kairi, respectively (Figure 1c).

Transpiration rates declined in all cultivars, with Kairi showing the most prominent decrease (−48%) (Figure 1d). Elevated CO₂ at 650 µmol mol⁻¹ improved _iWUE in all three cultivars over control and AC conditions, particularly in Kairi, where it increased from 2.68 µmol CO₂ mmol H₂O⁻¹ (the lowest level among the genotypes) to 6.65 µmol CO₂ mmol H₂O⁻¹ (the highest value among the cultivars) (Figure 1e).

3.1.2. Long-Term Measurements and Acclimation Assessment

The analysis revealed no significant effect of CO₂ on V_cmax (p = 0.237), indicating that the differences in V_cmax between AC and EC treatments were not pronounced. Similarly, V_cmax did not differ between the cultivars (p = 0.751), suggesting that the observed variations in V_cmax were not attributed to the different peanut cultivars. Furthermore, the interaction between the [CO₂] and cultivar was also non-significant (p = 0.879). These results suggest that, based on the measured V_cmax values, neither the treatment conditions nor the peanut cultivars significantly influenced the observed photosynthetic capacity (Figure A2a).

The investigation did not exhibit a statistically significant effect of EC on J_max (p = 0.776), suggesting that the observed variations in J_max values between AC and EC treatments were not substantial. Similarly, the cultivar factor did not significantly impact J_max (p = 0.818). Finally, the interaction between [CO₂] and cultivar was also non-significant (p = 0.707) (Figure A2b).

The respiratory parameter R_d, which represents dark respiration rates, significantly differed between AC and EC (p = 0.0225), indicating that the respiration rates differed between AC and EC conditions, with an increase of approximately 35%. Cultivars exhibited a marginally non-significant impact on R_d (p = 0.0824). However, the interaction between [CO₂] and cultivar was not statistically significant (p = 0.2651), implying that the combined effects of treatment conditions and peanut variety did not lead to substantial differences in R_d (Figure A2c).

Tukey’s HSD post-hoc test was conducted to identify specific group differences, indicating that respiration rates were higher in the EC treatment. All three cultivars (Kairi, Alloway, and Holt) shared similar values for the cultivar factor, suggesting no significant intraspecific differences in R_d. Specific combinations (e.g., EC:Kairi, EC:Alloway, and AC:Alloway) shared similar responses in the interaction between factors.

The J_max/V_cmax ratio, a key parameter reflecting the balance between the maximum rates of electron transport and carboxylation in photosynthesis, was highly affected by EC (p = 0.000519) (Figure A2d). The cultivar factor also significantly impacted J_max/V_cmax (p = 0.007862), indicating variability in the ratio among the peanut varieties. Additionally, the interaction between [CO₂] and cultivar was marginally significant (p = 0.045379). Post-hoc analyses using Tukey’s HSD test revealed specific group differences for the Treatment factor, indicating that the J_max/V_cmax ratio was higher under EC. For the cultivar factor, Kairi, Holt, and Alloway demonstrated significant variability in the ratio, while in the interaction, specific combinations showed significant differences in the J_max/V_cmax ratio among these treatment–variety combinations.

Therefore, the J_max/V_cmax ratio was significantly influenced by both treatment conditions and peanut cultivars. The elevated [CO₂] treatment resulted in a higher J_max/V_cmax ratio (35%) compared to AC, and there were significant variations in the ratio among the peanut varieties. The interaction effect suggests that the combined influence of treatment and variety played a role in shaping the observed differences in the J_max/V_cmax ratio.

3.2. Peanut Agronomic Traits

3.2.1. Fresh above- and below-ground Biomass Plant Partitioning Traits

The two-way ANOVA analysis highlighted that some of the vegetative growth parameters, specifically shoot length and root-to-shoot ratio and root length, were also highly different for [CO₂] treatments (p < 0.001) (Figure A3). Shoot length and root biomass were highly different for genotypes, and the nodulation score was moderately significantly different (p = 0.0265). On the other hand, the interactions of cultivar × [CO₂] levels showed no significance for all biomass parameters considered in this study. Compared to ambient growth conditions (AC), elevated CO₂ improved shoot length in Holt (4%) and Alloway (10%) with no changes in Kairi. Elevated CO₂ also enhanced the leaf weight and stems fresh weight in all cultivars (

Table A1. Fresh biomass data presented as mean ± SE, n = 4 for four replicates for each treatment including Control, AC, and EC.

Cultivars	[CO2]	SL	RL	RFW	LFW	SFW	TAB	NDS	RSR	LSR
		cm		g Plant−1
Kairi	Control	48.0 ± 1.89 c	13.8 ± 0.49 ab	5.6 ± 0.77 a	65.2 ± 8.75 a	104.0 ± 13.90 a	169.2 ± 22.4 a	3.6 ± 0.24 a	0.30 ± 0.02 a	0.64 ± 0.04 a
	AC	96.2 ± 4.66 a	13.6 ± 1.00 ab	2.7 ± 0.24 c	52.2 ± 7.59 a	83.8 ± 5.39 a	136.0 ± 12.6 a	2.1 ± 0.31 b	0.15 ± 0.01 b	0.61 ± 0.05 a
	EC	95.9 ± 3.94 a	14.4 ± 0.74 ab	3.6 ± 0.34 bc	63.1 ± 9.09 a	104.0 ± 11.2 a	167.1 ± 19.5 a	2.6 ± 0.30 b	0.15 ± 0.01 b	0.59 ± 0.05 a
Holt	Control	43.2 ± 1.94 c	14.7 ± 1.02 ab	5.0 ± 0.49 abc	51.5 ± 8.22 a	82.5 ± 8.82 a	134.0 ± 14.6 a	3.3 ± 0.47 a	0.34 ± 0.03 a	0.64 ± 0.07 a
	AC	81.9 ± 3.84 ab	15.8 ± 0.75 ab	3.5 ± 0.38 bc	58.3 ± 8.04 a	90.7 ± 8.05 a	149.0 ± 15.8 a	2.8 ± 0.39 b	0.17 ± 0.01 b	0.60 ± 0.05 a
	EC	85.2 ± 2.87 ab	13.2 ± 0.88 b	4.5 ± 0.50 abc	73.9 ± 8.44 a	115.0 ± 7.65 a	188.9 ± 15.5 a	3.4 ± 0.34 a	0.19 ± 0.01 b	0.62 ± 0.04 a
Alloway	Control	40.0 ± 1.74 c	13.7 ± 1.08 ab	5.1 ± 0.45 ab	47.7 ± 5.10 a	82.2 ± 8.81 a	131.0 ± 12.9 a	3.3 ± 0.43 a	0.35 ± 0.04 a	0.61 ± 0.05 a
	AC	76.2 ± 3.56 b	12.0 ± 0.55 b	3.7 ± 0.44 bc	52.6 ± 5.56 a	105.0 ± 8.34 a	157.6 ± 12.8 a	2.9 ± 0.38 b	0.18 ± 0.02 b	0.51 ± 0.04 a
	EC	83.8 ± 3.52 ab	14.7 ± 0.78 ab	3.6 ± 0.33 abc	61.8 ± 11.0 a	104.0 ± 11.4 a	165.8 ± 21.4 a	2.6 ± 0.44 b	0.18 ± 0.01 b	0.58 ± 0.05 a
Source of variation	Df	SL	RL	RFW	LFW	SFW	TAB	NDS	RSR	LSR
[CO2]	2	***	**	***	ns	ns	ns	*	***	ns
Cultivars	2	***	ns	ns	ns	ns	ns	ns	*	ns
[CO2] × Cultivars	4	ns	ns	ns	ns	ns	ns	ns	ns	ns
Error		14.0	3.45	2.00	35.33	42.01	73.38	1.50	0.08	0.21

Note. SL: shoot length, RL: root length, RFW: root fresh weight, LFW: leaves fresh weight; SFW: stem fresh weight, TAB: total above-ground biomass, NDS: nodules score, RSR: root-to-shoot ratio, LSR: leaves-to-stems ratio. Columns sharing the same letters display no significant difference between groups. The different levels of significance are reported as ns = not significant p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

), resulting in positive impacts on total above-ground biomass (Figure A3c), which increased by 5%, 23%, and 26% for Alloway, Kairi, and Holt, respectively. Regarding root material, declines in root length (were observed in Holt (−16%), with small increases in Kairi and Alloway under elevated CO₂ conditions. The root fresh biomass (Figure A3d) showed significant variability between the three cultivars: Kairi and Holt accumulated biomass in response to elevated CO₂ concentrations, increasing from 2.7 to 3.6 g plant⁻¹ and 3.5 to 4.5 g plant⁻¹, respectively. The root-to-shoot ratio was significantly different for [CO₂] treatment (p < 0.001) and moderately different between the varieties (p = 0.0216) (Figure A3e). The nodule score was significantly impacted by EC treatment (p = 0.0265), with increments observed in Holt (24%) and Kairi (21%), while a decline was shown in Alloway (−10%) (Figure A3f).

3.2.2. Dry Matter Yield and Physiochemical Properties

The two-factor ANOVA failed to highlight any significant impact of [CO₂] treatment, differences between cultivars, and the interaction cultivar × [CO₂] for the dry matter yield parameters, namely leaves, stem (

Table A2. Dry matter yield traits and nutritional quality. Data presented as mean ± SE, n = 4 for four replicates for each treatment including Control, AC, and EC.

Cultivars	[CO2]	LDW	SDW	DM	ADF	NDF	TPC	FRAP	CUPRAC	N	C	C:N	CP
		g Plant−1			mg g−1 DM		mg GAE g−1 DW	mg TXE g−1 DW		mg g−1 DW			%
Kairi	Control	16.77± 2.64 a	24.55± 3.18 a	41.32± 5.63 a	372± 9 a	389± 9 bc	6.65 ± 0.72 a	7.31± 0.93 ab	26.46± 4.67 b	23.4± 0.77 a	413± 1.74 a	17.7± 0.56 a	14.7± 0.48 a
	AC	13.20± 1.72 a	21.61± 1.82 a	34.81± 3.53 a	347± 7 b	399± 11 bc	6.91 ± 0.57 a	7.98± 0.72 ab	29.64± 2.54 a	25.1± 1.25 a	398± 8.41 a	15.9± 0.53 b	15.7± 0.78 a
	EC	11.10± 0.37 a	19.12± 2.69 a	30.22± 2.73 a	368± 14 a	423± 11 ab	7.17 ± 0.33 a	7.86± 0.44 ab	29.86± 1.31 a	24.8± 0.50 a	406± 4.61 a	16.4± 0.23 a	15.5± 0.32 a
Holt	Control	9.46± 3.13 a	19.71± 5.15 a	24.24± 4.82 a	355± 4 b	379± 3 c	5.8 ± 0.79 a	5.17± 0.08 b	20.9± 0.54 b	23.8± 1.02 a	409± 2.54 a	17.3± 0.66 a	14.9± 0.64 a
	AC	11.57± 3.40 a	22.07± 3.74	33.64± 7.13 a	358± 6 b	413± 8 abc	6.91 ± 0.35 a	9.54± 1.22 a	30.77± 3.96 a	25.1± 0.86 a	400± 6.10 a	16.0± 0.34 a	15.6± 0.54 a
	EC	17.19± 1.88	30.12± 2.51 a	47.31± 4.21 a	386± 10 ab	432± 10 ab	6.74 ± 0.34 a	9.42± 0.26 a	33.17± 2.05 a	24.8± 0.30 a	406± 5.58 a	16.5± 0.35 a	15.4± 0.19 a
Alloway	Control	14.42± 1.58 a	19.77± 2.11 a	34.19± 2.75 a	385± 14 ab	404± 10 abc	6.55 ± 0.23 a	6.31± 0.91 ab	25.13± 3.86 b	22.5± 1.06 a	389± 16.5 a	17.3± 0.61 a	14± 0.67 a
	AC	11.23± 2.30 a	19.54± 4.52 a	30.77± 5.74 a	379± 9 ab	444± 5 a	7.32 ± 0.30 a	8.23± 0.18 ab	27.95± 1.72 a	23.5± 0.55 a	391± 5.36 a	16.7± 0.23 a	14.7± 0.34 a
	EC	14.75± 3.10 a	26.06± 3.84 a	40.81± 6.47 a	425± 10 a	450± 8 a	7.5 ± 0.70 a	7.54± 0.77 ab	27.96± 1.61 a	23.1± 0.51 a	397± 5.20 a	17.2± 0.36 a	14.4± 0.32 a
Source of variation	Df	LDW	SDW	DM	ADF	NDF	TPC	FRAP	CUPRAC	N	C	C:N	CP
[CO2]	2	ns	ns	ns	**	***	ns	**	*	ns	ns	**	ns
Cultivars	2	ns	ns	ns	***	***	ns	ns	ns	ns	ns	ns	ns
[CO2] × Cultivars	4	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns
Error		4.56	5.87	9.55	27.67	25.57	1.00	1.47	6.33	1.59	14.84	0.88	0.99

Note. LDW: leaves dry weight; SDW: stems dry weight; DM: total above-ground dry matter; ADF: acid detergent fibre; NDF: neutral detergent fibre; TPC: total phenolic content; FRAP: ferric-reducing antioxidant potential: CUPRAC: cupric ion-reducing antioxidant capacity N: shoot [N] DM; C: shoot [C] DM, C:N: C-to-N ratio; CP: crude protein. Columns sharing the same letters display no significant difference between groups. The different levels of significance are reported as ns = not significant p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

), and total dry matter weight (Figure A4a). Although not significant, a positive effect of biomass accumulation was observed in Alloway and Holt, while Kairi’s samples recorded a noticeable decrease. Similarly, no significant variation was determined in N and C content in the above-ground dry matter for all cultivars, albeit a mild decrement of N content was observed alongside a moderate increase in C content (Table A2). However, the C-to-N ratio showed a significant response (p = 0.00866) to the ANOVA, showing increased values under EC conditions. The treatment did not affect the CP content in the dry matter, and no significant differences were observed between cultivars, despite Kairi and Holt sharing similar mean values, slightly higher than Alloway. Both digestibility parameters, ADF and NDF, were significantly impacted by CO₂ treatment, cultivars, and cultivars × [CO₂].

The ADF concentration was significantly impacted by [CO₂] (p = 0.00479), particularly affecting the concentration in Alloway and Holt, which experienced increments of 12 and 8%, respectively, while cultivars responded differently in terms of concentration. Alloway exhibited the highest mean value for all treatments compared to Kairi and Holt, with 385, 379, and 425 mg g⁻¹ DW concentrations, respectively (Figure A4c). Similarly, NDF showed increased mean values from AC to EC treatment, particularly for Kairi and Holt (Figure A4e). Significant differences in responses were noted between cultivars, with Alloway exhibiting the highest concentrations compared to Kairi and Holt (Figure A4d).

Elevated CO₂ moderately enhanced the total phenolic content in shoots of Kairi (4%) and Alloway (2.5%) cultivars, while Holt showed a negative response (−1.6%). The FRAP assay provided a significantly different response among the three cultivars (p < 0.01), with Holt showing the highest values. These values declined from 9.54 in AC conditions to 9.42 mg TXE g⁻¹ DW in EC conditions (Table A2). A positive correlation (p < 0.05) between CUPRAC and [CO₂] was observed in shoot samples, with Holt exhibiting the highest values. Similar to the FRAP assay, Holt recorded the highest values among the three genotypes, which declined in response to enhanced [CO₂]. However, in contrast to FRAP results, Holt’s CUPRAC results showed an increase of 8% for the mentioned cultivar (Table A2).

3.2.3. Dry Root Biomass and Physiochemical Traits

Root dry weight was not significantly impacted by elevated [CO₂] (p > 0.05) (Figure A5a). However, there was significant variability between cultivars, with Kairi exhibiting the lowest values, which declined from 0.99 to 0.77 g plant⁻¹ between AC and EC treatments. Nitrogen and carbon content were not significantly affected by elevated [CO₂], nor was the cultivars × [CO₂] interaction. However, the cultivars showed different responses for N concentration (p = 0.0408) and C concentration (p = 0.0226), with Alloway showing the highest C concentration (426 mg g⁻¹ DM) across all [CO₂] treatments and in Holt under EC conditions (429 mg g⁻¹ DM) (Figure A5c). The C-to-N ratio was not statistically significant for the CO₂ treatment and cultivars, albeit an increment was observed in Kairi and Holt, while Alloway experienced a decrement from AC to EC (Figure A5d). The protein percentage followed a similar trend as N content and was not impacted by CO₂ treatment, with slight differences observed between cultivars, with Holt recording the highest overall concentrations (

Table A3. Root biomass and physiochemical composition data presented as mean ± SE, n = 4 for four replicates for each treatment including Control, AC, and EC.

Cultivars	[CO2]	CP	TPC	FRAP	CUPRAC
		%	mg GAE g−1 DW	mg TXE g−1 DW
Kairi	Control	6.44 ± 0.44 a	3.41 ± 0.13 a	4.03 ± 0.36 a	14.7 ± 2.17 a
	AC	6.95 ± 0.90 a	4.56 ± 0.80 a	4.92 ± 0.96 a	17.5 ± 3.31 a
	EC	6.21 ± 0.45 a	3.98 ± 0.54 a	4.73 ± 0.88 a	14.24 ± 2.75 a
Holt	Control	7.27 ± 0.22 a	4.17 ± 0.44 a	5.04 ± 0.83 a	14.51 ± 0.29 a
	AC	8.24 ± 0.77 a	3.47 ± 0.69 a	4.34 ± 1.10 a	12.05 ± 2.57 a
	EC	7.7 ± 1.77 a	3.63 ± 0.60 a	4.55 ± 1.06 a	14.34 ± 3.01 a
Alloway	Control	6.4 ± 1.02 a	4.85 ± 1.02 a	5.93 ± 1.48 a	20.51 ± 6.13 a
	AC	5.6 ± 0.48 a	3.34 ± 0.48 a	3.94 ± 0.71 a	13.74 ± 1.48 a
	EC	6.7 ± 0.50 a	3.66 ± 0.50 a	4.54 ± 0.97 a	14.22 ± 2.15 a
Source of variation	Df	CP	TPC	FRAP	CUPRAC
[CO2]	2	ns	ns	ns	ns
Cultivars	2	*	ns	ns	ns
[CO2] × Cultivars	4	ns	ns	ns	ns
Error		1.42	1.23	1.90	6.20

Note. CP: crude protein TPC: total phenolic content; FRAP: ferric-reducing antioxidant potential; CUPRAC: cupric ion-reducing antioxidant capacity. Columns sharing the same letters display no significant difference between groups. The different levels of significance are reported as ns = not significant p > 0.05, * p < 0.05.

).

The total phenolic content showed a mild (albeit non-significant) decrease in EC compared to the AC plants in Kairi’s roots, whereas in Holt and Alloway, the CO₂ treatment gave a positive response (Table A3). FRAP increased in Holt and Alloway with EC and decreased in Kairi; however, there was no significant correlation between [CO₂], cultivars, and their interactions (p > 0.05). The statistical analysis performed on CUPRAC of all sample types regarding the interaction between [CO₂] and cultivars showed no significant correlation in root material (Table A3).

3.2.4. Pod Yield

Plants grown under EC conditions showed an increment, albeit not statistically significant, of 49% in Kairi, 24% in Holt, and 16% in Alloway compared to the AC yield. The [CO₂] × cultivars interaction also resulted in statistical significance for the number of pods per plant in the ANOVA (p = 0.0493), as shown in Figure 2a. These increments were reflected in the pods’ fresh weight and dry weight (Figure 2b,c), while no significant difference was observed among cultivars or in the [CO₂] × cultivars interaction. Similarly, hundred-pod weight and pods bulk density were not significant for the [CO₂], cultivars, or their interactions (Figure 2d,e). The ANOVA identified a significant impact caused by [CO₂] treatment on pods’ harvest index, which increased by 34% in Kairi, while in Holt and Alloway, it declined by 24% and 3%, respectively (Figure 2f).

The statistical analysis failed to find any significant change in the pods’ physical properties (

Table 2. Pod shape attribute data presented as mean ± SE, n = 4 for four replicates for each treatment, including Control, AC, and EC.

	[CO2]	L	W	T	Dg	S	ϕ	Ra
		mm				mm2	%
Kairi	Control	36.80 ± 3.27 ab	15.00 ± 1.37 a	14.53 ± 1.36 ab	20.39 ± 1.46 ab	1313.08 ± 102.45 ab	55.82 ± 3.52 d	43.85 ± 4.38 c
	AC	35.57 ± 5.40 ab	15.52 ± 2.00 ab	14.21 ± 1.62 ab	19.81 ± 2.13 abc	1246.04 ± 88.39 abc	56.26 ± 5.07 d	44.15 ± 7.04 bc
	EC	38.23 ± 4.63 a	15.84 ± 1.06 ab	14.23 ± 0.91 ab	20.47 ± 1.43 a	1322.91 ± 88.88 a	53.87 ± 3.21 d	41.80 ± 3.46 c
Holt	Control	33.07 ± 2.37 bc	15.27 ± 0.89 ab	13.62 ± 0.85 b	19.00 ± 0.99 bcd	1137.63 ± 43.80 bcd	57.59 ± 2.75 bcd	46.32 ± 3.44 bc
	AC	33.50 ± 3.18 bc	14.94 ± 1.07 ab	13.69 ± 0.63 ab	18.98 ± 1.19 bcd	1135.89 ± 117.73 cd	56.84 ± 2.43 d	44.75 ± 3.03 bc
	EC	33.20 ± 2.24 bc	15.86 ± 0.93 ab	13.74 ± 0.83 ab	18.99 ± 0.72 bcd	1135.07 ± 24.58 cd	57.40 ± 3.37 cd	45.60 ± 4.50 bc
Alloway	Control	30.13 ± 2.52 cd	15.33 ± 1.17 ab	14.23 ± 1.29 ab	18.71 ± 1.19 cd	1103.38 ± 64.74 cd	62.29 ± 3.91 ab	51.10 ± 5.57 ab
	AC	29.07 ± 4.60 d	14.75 ± 1.00 b	14.50 ± 0.92 ab	18.33 ± 1.51 d	1062.04 ± 66.39 d	64.44 ± 9.99 a	52.38 ± 15.4 a
	EC	31.10 ± 2.79 cd	15.30 ± 1.37 ab	15.25 ± 3.70 a	19.27 ± 1.82 abcd	1176.54 ± 109.47 abcd	62.08 ± 4.25 abc	49.41 ± 9.92 a
Source of variation	Df	L	W	T	Dg	S	ϕ	Ra
[CO2]	2	ns	ns	ns	ns	ns	ns	ns
Cultivars	2	***	**	**	***	***	***	***
[CO2] × Cultivars	4	ns	ns	ns	ns	ns	ns	ns
Error		3.61	1.25	1.61	1.44	177.19	4.79	7.36

Note. L: length; W: width; T: thickness; Dg: geometric mean diameter; S surface area; ϕ: sphericity; Ra: aspect ratio. Different letters show differences between populations. Columns sharing the same letters display no significant difference between groups. The different levels of significance are reported as: ns = not significant p > 0.05, ** p < 0.01, *** p < 0.001.

) considered in this study, namely length, width, thickness, geometric mean diameter, sphericity, surface area, and aspect ratio for the [CO₂] treatment and the [CO₂] × cultivars interaction. However, ANOVA provided highly significant differences in variability between cultivars for length, geometric mean diameter, sphericity, surface area, and aspect ratio output variables, while the response for width (p = 0.0038) and thickness (p = 0.0039) was significant. Overall, Kairi showed the higher mean values under EC conditions in length (38.23 mm), while Holt recorded the highest mean values for width (15.86 mm) and Alloway for thickness (15.25 mm). These differences in size affected the geometric mean diameter, with the highest mean values recorded in Kairi (20.47 mm) under EC conditions and the pod sphericity and aspect ratio, with the highest percentage reported in Alloway for all CO₂ levels. The total surface areas had the highest mean values in Kairi pods for all CO₂ levels, followed by Alloway under EC conditions.

3.3. Kernel Yield and Physiochemical Traits

The elevated CO₂ concentration significantly impacted the kernel dry weight per plant in Holt and Kairi, which recorded increments from AC to EC of 38 and 26%, respectively, while Alloway remained unchanged (Figure 3a). There was no statistical correlation between hundred-kernel weight and kernel bulk density and [CO₂] cultivars and the [CO₂] × cultivars interaction (Figure 3b,c). The kernel harvest index was highly significant for [CO₂] treatment (p < 0.01), with particular emphasis on Kairi, where an increment of 33% was recorded compared to the AC treatment (Figure 3d). The treatment did not impact nitrogen content and no difference was found between cultivars or in the interaction between the two factors (

Table A4. Kernel physiochemical compositions presented as mean ± SE, n = 4 for four replicates for each treatment including Control, AC, and EC.

Cultivars	[CO2]	N	C	C:N	CP
		mg g−1 DM			%
Kairi	Control	46.5 ± 15.10 a	603 ± 4.58 ab	13.6 ± 1.93 a	25.4 ± 2.78 a
	AC	53.6 ± 0.98 a	592 ± 1.04 d	11.0 ± 0.20 a	29.3 ± 0.54 a
	EC	53.2 ± 0.76 a	592 ± 1.18 cd	11.1 ± 0.14 a	29.0 ± 0.42 a
Holt	Control	48.5 ± 1.28 a	602 ± 0.93 abc	12.4 ± 0.34 a	26.50 ± 0.70 a
	AC	50.8 ± 1.21 a	598 ± 0.29 bcd	11.8 ± 0.32 a	27.70 ± 0.66 a
	EC	52.7 ± 0.86 a	598 ± 1.51 bcd	11.3 ± 0.17 a	28.80 ± 0.47 a
Alloway	Control	45.9 ± 1.22 a	611 ± 2.31 a	13.3 ± 0.40 a	25.0 ± 0.66 a
	AC	46.3 ± 0.65 a	610 ± 1.10 a	13.2 ± 0.19 a	25.30 ± 0.35 a
	EC	49.3 ± 0.89 a	606 ± 1.58 ab	12.3 ± 0.24 a	26.90 ± 0.49 a
Source of variation	Df	N	C	C:N	CP
[CO2]	2	ns	***	*	ns
Cultivars	2	ns	***	ns	ns
[CO2] × Cultivars	4	ns	ns	ns	ns
Error		3.90	4.16	1.38	2.14

Note. N: kernel [N] DM; C: kernel [C] DM, C:N: C-to-N ratio; CP: crude protein. Columns sharing the same letters display no significant difference between groups. The different levels of significance are reported as ns = not significant p > 0.05, * p < 0.05, *** p < 0.001.

). This trend also reflected the crude protein content, where no noticeable difference was observed, with Kairi showing the highest protein content (29%) under EC conditions, followed by Holt (28.8%) and Alloway (27.7%). Alloway recorded the highest C values for all treatments, varying from 611 in the control plants to 610 in AC to 606 mg g⁻¹ DW in EC. The C-to-N ratio was significantly impacted by CO₂ (p = 0.0304), particularly in Alloway, where the ratio showed a decrease (−7%). Similarly, the TPC was moderately impacted by CO₂ (p = 0.0185), with a mild decline observed in Kairi (−10%) and Holt (−3%). The FRAP and CUPRAC measured in kernel samples highlighted no significant differences between cultivars and CO₂ treatments (Figure A6).

The statistical analysis performed on kernels’ physical properties is shown in

Table 3. Kernels’ physical properties data reported as mean ± SE, n = 5 for four replicates for each treatment, including Control, AC, and EC.

Cultivars	[CO2]	KS	L	W	T	Dg	S	ϕ	Ra
		g kernel−1	mm				mm2	%
Kairi	Control	1.01 ± 0.04 ab	18.72 ± 0.34 a	10.18 ± 0.25 bc	8.89 ± 0.19 bcd	11.89 ± 0.10 a	445.87 ± 12.00 a	63.74 ± 1.02 de	54.56 ± 1.43 d
	AC	1.02 ± 0.04 ab	18.50 ± 0.36 a	9.94 ± 0.21 bc	8.28 ± 0.17 d	11.49 ± 0.18 a	416.70 ± 12.91 a	62.22 ± 0.65 e	53.80 ± 0.88 d
	EC	1.03 ± 0.02 a	18.05 ± 0.33 ab	9.85 ± 0.23 bc	8.59 ± 0.13 cd	11.50 ± 0.18 a	417.42 ± 12.50 a	63.84 ± 0.80 de	54.69 ± 1.26 d
Holt	Control	0.90 ± 0.05 ab	16.47 ± 0.30 c	10.36 ± 0.19 bc	9.29 ± 0.19 bc	11.64 ± 0.15 a	426.94 ± 10.63 a	70.96 ± 1.03 bc	63.21 ± 1.32 bc
	AC	1.03 ± 0.05 a	15.63 ± 0.40 cd	10.29 ± 0.22 abc	9.18 ± 0.22 bc	11.43 ± 0.21 a	412.91 ± 15.74 a	73.48 ± 1.14 b	66.08 ± 1.33 b
	EC	0.94 ± 0.03 ab	16.73 ± 0.31 bc	9.70 ± 0.22 c	8.96 ± 0.22 cd	11.31 ± 0.18 a	403.80 ± 12.82 a	67.72 ± 0.75 cd	57.95 ± 1.14 cd
Alloway	Control	1.00 ± 0.04 ab	14.44 ± 0.31 d	11.35 ± 0.19 a	10.36 ± 0.19 a	11.92 ± 0.19 a	448.19 ± 14.12 a	82.78 ± 1.00 a	78.95 ± 1.47 a
	AC	0.85 ± 0.05 b	14.44 ± 0.22 d	10.77 ± 0.21 ab	9.60 ± 0.20 ab	11.42 ± 0.17 a	411.26 ± 12.34 a	79.17 ± 0.92 a	74.73 ± 1.43 a
	EC	0.95 ± 0.03 ab	14.36 ± 0.37 d	10.73 ± 0.22 ab	9.29 ± 0.20 bc	11.24 ± 0.17 a	398.52 ± 12.11 a	78.84 ± 1.44 a	75.48 ± 2.15 a
Source of variation	Df	KS	L	W	T	Dg	S	ϕ	Ra
[CO2]	2	ns	ns	**	***	**	**	*	*
Cultivars	4	*	***	***	***	ns	ns	***	***
[CO2] × Cultivars	4	**	ns	ns	ns	ns	ns	***	**
Error		017	1.46	0.97	0.96	0.80	57.48	4.72	6.32

Note. KS: kernel size; L: length; W: width; T: thickness; Dg: geometric mean diameter; S: surface area; ϕ: sphericity; Ra: aspect ratio. The different levels of significance are reported as: ns = not significant p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001. Columns sharing the same letters display no significant difference between groups.

. This study found no significant impact of elevated [CO₂] on kernel size, although it decreased in Holt and Kairi (−9% and −1%) and increased in Alloway (12%). However, ANOVA found significant variability between cultivars (p = 0.01475) and a significant [CO₂] × cultivars interaction.

Cultivars showed a highly significant correlation between kernels’ thickness and [CO₂] treatment and the [CO₂] × cultivars interaction, with a decreasing trend seen in all cultivars, consisting of −4, −3.2, and −2.4% in Kairi, Alloway, and Holt, respectively. Similarly, width had a significant correlation with CO₂ level and cultivars (p = 0.0098). Kernel length was not affected by CO₂ level but showed highly significant variability between cultivars, with Kairi recording the highest mean values, as seen for pod length. The geometric mean diameter (p = 0.00384) and the aspect ratio (p = 0.0381) showed a significant response to [CO₂]; however, the post-hoc test determined no significant variability between the AC and EC means. Kernel aspect ratio variability was also highly significant for cultivars and showed a significant [CO₂] × cultivars interaction (p = 0.00478). The sphericity parameter resulted as moderately significant in the ANOVA (p = 0.015) for the [CO₂] treatment, particularly for Holt, whose percentage decreased by 8%, and significant for the cultivars and the [CO₂] × cultivars interaction.

3.4. Cultivar Performance

Eighteen key parameters based on qualitative and productivity criteria were considered to understand the interactive effects between three varieties, namely Kairi, Holt, and Alloway, and three distinct CO₂ treatments: Control, AC, and EC. The traits included NP (number of pods), PFW (pod fresh weight), PDW (pod dry weight), KDW (kernel dry weight), LW (leaf weight), SW (seed weight), TAGB (total above-ground biomass), RW (root weight), PHI (pod harvest index), KHI (kernel harvest index), KS (kernel size), protein content (protein), carbon-to-nitrogen ratio (C:N), nitrogen concentration (N), carbon concentration (C), total phenolics content (TPC), FRAP (ferric-reducing antioxidant power), and CUPRAC (cupric-reducing antioxidant capacity).

A representation of the interaction between cultivars and the selected traits under different growth conditions is reported in Figure 4. The biplot analysis unveiled the patterns within the Control treatment, showcasing Kairi’s better performance across all traits, including phytochemical content, N, antioxidants, and biomass. Conversely, Alloway and Holt exhibited comparable but less pronounced performances, with higher harvest indices for kernels and pods compared to Kairi. The AC treatment revealed a heterogeneous trend among the cultivars, with Alloway demonstrating better yield traits and Holt producing more biomass. Under EC treatment conditions, Kairi displayed superior overall performance, particularly regarding TPC, CUPRAC, protein content, pod harvest index, and kernel size. Alloway demonstrated a slight advantage in yield traits, such as the number of pods and pod weight, while Holt experienced a penalty across all parameters. A noteworthy observation was made when comparing AC to EC treatments, highlighting Kairi as the cultivar experiencing the most significant benefits under elevated CO₂ conditions, particularly regarding biomass productivity and kernel physiochemical traits. This comprehensive assessment provided a broader overview of the interactions between cultivars and CO₂ levels, allowing us to plan strategies to optimize crop performance and yield.

4. Discussion

4.1. Summary of the Cultivars’ Performance

This experiment aimed to assess the performance of three peanut cultivars under EC conditions, focusing on their physiological, phenological, and physiochemical responses, with particular attention to productivity and quality traits. Additionally, the study aimed to assess the magnitude of intraspecific variability between cultivars and identify potential candidates for simulated grazing trials under EC stimulation, laying the foundation for our future work.

Overall, CO₂ enrichment improved photosynthetic rates, reduced stomatal conductance, and decreased transpiration, leading to enhanced water use efficiency. In this respect, the Kairi cultivar showed a more significant reduction in stomatal conductance and enhanced _iWUE compared to Holt and Alloway, making it particularly advantageous in environments with limited water availability. While EC had a fertilization effect on certain biomass traits such as shoot and root length, as well as dry matter yield, no significant impact was observed on kernel morphology. However, EC improved pod and kernel yield to some extent across all cultivars.

Importantly, there were no significant effects of the CO₂ treatment on N concentration—and therefore CP content—TPC, CUPRAC, or FRAP of the peanut seed material. This suggests that peanuts grown under EC should have similar nutritional and health benefits to those grown under current ambient CO₂ levels, while Kairi showed the highest kernel protein content among cultivars.

Additionally, the three peanut cultivars did not significantly differ in their TPC or antioxidant capacity when grown under either AC or EC levels, indicating comparable phytochemical composition. This suggests that aside from their root composition, where there was high intra-specific variability, they are quite comparable in terms of their phytochemical composition.

However, there were potential threats to the fibre structure and digestibility of peanut stover, as evidenced by increased ADF and NDF values, which could compromise feedstock quality.

4.2. Elevated CO₂ Increased Photosynthesis and Reduced Stomatal Conductance

It has been demonstrated that elevated CO₂ affects plant growth by enhancing the carboxylation process in photosynthetic metabolisms in C₃ plants [3,4].

The physiological performance was assessed in three peanut cultivars grown under Control, AC, and EC conditions during the late flowering stage and the beginning of seed pegging. The response in A_net to e[CO₂] stimulation was significant for all cultivars, with an average increment of 22%, and Holt showed the highest photosynthetic rates under EC. Similar results have been reported in CO₂ enrichment experiments conducted on peanuts [12]. All cultivars showed a similar decreasing trend in stomatal conductance as a consequence of prolonged exposure to EC, with reductions of −49% in Kairi, −40% in Alloway, and −25% in Holt. The three cultivars responded to the stimulation by closing stomata and limiting gas exchange, possibly due to changes in morphometric parameters impacted by e[CO₂], such as a significant reduction in leaf adaxial and abaxial stomatal density reported in previous research on peanuts [32]. Moreover, changes in the mechanisms involved in the control and inhibition of the guard cells might affect cell aperture and closure through K⁺ decreases, leading to consequently decreased cell turgor [33]. Holt showed the highest level of intercellular CO₂ under EC conditions, supporting higher photosynthetic rates through increased carbon availability for the carboxylation process. Transpiration rates were positively correlated with elevated [CO₂], exhibiting a decreasing trend for all cultivars (−40 in Kairi, −33 in Alloway, and −22% in Holt). Elevated CO₂ considerably improved _iWUE with a highly significant increase for all cultivars. Kairi emerged as the most efficient and promising cultivar in terms of physiological performance, particularly under regimes with limited water content.

The absence of significant changes in V_cmax under elevated CO₂ conditions during stage R2 suggests that the photosynthetic capacity of peanut cultivars considered in this study was not substantially influenced by increased atmospheric CO₂. While the CO₂ treatment did not significantly impact the cultivars, they experienced different photosynthetic responses. Alloway showed a decreased V_cmax, reflecting results reported for peanuts [13] and the general decreasing trend reported for legumes [4]. Kairi, on the other hand, experienced a slight increase due to CO₂ enrichment, which typically implies that the maximum rate at which a plant converts carbon dioxide into organic compounds during photosynthesis has increased, leading to increased plant growth and productivity as the plant has more resources (i.e., CO₂) available for energy production.

However, our study showed a non-significant increase in J_max for all cultivars except Alloway. An increase in J_max due to CO₂ enrichment indicates a higher maximum rate at which a plant can transport electrons during photosynthesis, contributing to increased growth and productivity by facilitating energy production from solar radiation. In this study, J_max increased in Kairi and Holt, consistent with findings from a study on pigeon peas [34]. Several factors could explain this moderate response and variability. Firstly, it is possible that the plants were already operating at or near their maximum photosynthetic capacity under ambient CO₂ conditions, reaching a saturation point where further increases in CO₂ did not significantly enhance carboxylation rates. Additionally, the duration of CO₂ exposure and the specific genetic characteristics of the plant likely influenced its responsiveness.

Although not statistically significant, the slight increases observed in J_max could indicate a potential acclimation response that may involve adjustments in other physiological processes rather than changes in J_max itself. The relationship between V_cmax and J_max is integral to photosynthetic efficiency. If the two parameters are tightly coordinated, the lack of significant changes in V_cmax might constrain any substantial increase in J_max. The plant may optimize its photosynthetic performance within a certain physiological balance, ensuring that carboxylation and electron transport rates remain coordinated. These results contrast with findings from other research where no impact on V_Cmax/V_Cmax was reported [5]. This study found an increased R_d rate at higher CO₂ concentrations due to the stimulation of energy production by the plant and a subsequent reduction in stomatal conductance and transpiration. Furthermore, elevated CO₂ levels might cause a shift in carbon allocation within the plant, directing more carbon towards respiration rather than growth, resulting in an increase in R_d.

4.3. Elevated CO₂ Increased Biomass Traits but Slightly Reduced Forage Fiber Digestibility

This research found significant increases in shoot length and root length in Alloway at plant maturity, while root fresh dry weight increased in Holt and Kairi in response to EC over AC conditions, as similarly seen in a peanut study conducted by Bagudam et al. [35]. The fresh above-ground biomass parameters, namely leaf fresh weight, stem fresh weight, and the total above-ground biomass, showed increments in all cultivars, although these increases were not statistically significant. Similar findings were reported in peanuts grown under EC [36], with increased stem weight by 19–38%, values comparable to those calculated in Kairi (25%) and Holt (28%). The root-to-shoot ratio remained unaffected by CO₂, while the nodules score was impacted by the treatment, showing increases in EC plants over AC, although these increases were not statistically significant according to ANOVA. Both parameters showed a clear chamber effect, with Control values significantly higher than those in the OTCs. No significant interaction was noticed in the leaf-to-stem ratio, suggesting that the partitioning of the extra carbon assimilated through enhanced photosynthetic rates did not significantly affect a specific plant organ in the above-ground section. Overall, it can be concluded that the e[CO₂] treatment had a moderate impact, visible only in some of the traits investigated in this study, while cultivars exhibited similar performance in most traits.

The analysis conducted on the dry matter data suggests that the CO₂ treatment did not significantly affect the dry matter yield of the three cultivars, aligning with results reported for peanut total dry matter yield [35]. Similarly, our study showed no consistent results regarding peanut cultivars’ biomass yield [37]. This variation could stem from the distinct intrinsic growth characteristics of each cultivar, which influence their response to elevated CO₂. For instance, the Holt cultivar may have shown a positive response due to its better adaptation to utilize higher CO₂ levels, while the Kairi cultivar may have exhibited a negative response due to its slower growth rate. Additionally, the interaction between the specific characteristics of each cultivar and the elevated CO₂ treatment could have contributed to the non-significant results. Interestingly, the nitrogen content and, consequently, the protein percentage were not significantly impacted by elevated CO₂, although a slight reduction was observed across all cultivars. This suggests that the dilution effect was minimal in this study, likely due to the unchanged biomass accumulation. In terms of forage quality and digestibility, this study found significant increases in ADF in Holt and Alloway and NDF in Kairi and Holt, comparing EC over AC samples showing, to some extent, a heterogeneous response between cultivars. There is a lack of evidence in the literature of NDF changes induced by elevated CO₂ in peanuts. Other works conducted in alfalfa reported increases or decreases in the NDF measurements under EC conditions, which were mainly dependent on the rhizobia strain [8]. An increase in ADF and the consequent decline in forage quality was also reported by Bagudam et al. [35] in peanuts grown under 550 µmol mol⁻¹ but not at 700 µmol mol⁻¹. Increases were observed in wheat forage [38], where ADF increased with EC conditions and plant maturity and in C₄ metabolism species, including prairie grasses [39,40].

While the TPC was not significantly impacted by CO₂ elevation, similar to findings reported by Booker et al. [41], the antioxidant capacity of the shoot material, as measured by FRAP (p < 0.01) and CUPRAC (p < 0.05), provided a significant response. Interestingly, the trend was not consistent, with antioxidant activity increasing under EC for CUPRAC but decreasing for FRAP. Although these assays have different reaction potentials and measure slightly different aspects of antioxidant activity, further investigation is needed to understand this trend. It is possible that specific compounds are being differentially regulated by the plant’s physiological responses to EC, resulting in an average increase of 8% in CUPRAC but a 3.6% average decline in FRAP. These results align with findings reported by Fernando et al. [42] and Gillespie et al. [43] who observed an increase in antioxidant capacity under elevated CO₂.

The investigation on root traits highlighted that [CO₂] and the cultivars × [CO₂] interaction did not significantly affect peanut plants overall. Root dry weight did increase in two cultivars, albeit this increment was not significant. More significant increases have been reported in previous research on peanuts [11,35,44], attributed to enhanced C partitioning in the root systems. Holt and Alloway showed increases in dry weight under EC over AC. However, statistically significant variability was observed for the cultivars factor, with these cultivars also exhibiting the highest below-ground biomass values per plant. Root length was significantly impacted by elevated CO₂ but with different trends between cultivars. Root length increased in Kairi and Alloway, consistent with findings from a previous study conducted by Manjula et al. [45] on peanuts, while Holt experienced a consistent decrease. The C and N stoichiometry varied between cultivars, with a general reduction in N combined with an increase in C content in Kari and Holt, affecting the C-to-N ratio. Similarly, the protein content decreased in Kari and Holt, but the same cultivars showed the highest overall C content compared to Alloway. There were no significant differences between cultivars in the antioxidant capacity for the root material analysed in this study. However, a significant genotypic difference was observed in the root protein content, with Holt showing the highest protein percentage (7.7%) and Alloway the lowest (6.1%) under EC.

4.4. Elevated CO₂ Moderately Improved Yield with No Impact on Kernel Quality Traits

The pod yield parameters, significantly and positively influenced by an elevated CO₂ concentration, included the number of pods per plant, which also exhibited high variability between cultivars, as well as pods’ fresh and dry weights. These traits also demonstrated a chamber effect, with higher values in control plots. However, the positive treatment effect was more visible in plants under EC than in AC treatment. Holt and Alloway showed a positive impact of CO₂ on the pod harvest index, while Kairi, in contrast, showed increased productivity and a reduced harvest index, consistent with the findings reported by Laza et al. [13]. This study found no impact of EC on hundred-pod weight and pod bulk density, suggesting that the improvement was due to enhanced yield rather than intrinsic changes in pod physical composition. Supporting this hypothesis, ANOVA found no significant impact of EC on pod physical properties parameters, which were affected solely by intra-specific variability.

The elevated CO₂ treatment was associated with improved kernel dry weight per plant compared to AC treatment for all cultivars except Alloway, which showed no difference. Similarly, the kernel harvest index was enhanced under EC in Kairi and Holt, while Alloway exhibited a negative trend. As observed with pod physical properties, there was no significant impact of the CO₂ treatment on hundred-kernel weight and kernel bulk density, with no significant variability emerging between cultivars.

This study found that EC had no significant effect on crude protein levels, consistent with the findings of Burkey et al. [46]. Similarly, the total phenolic content (TPC), as well as the CUPRAC and FRAP assays, showed no variation in peanut kernels under EC. This suggests that peanuts grown in elevated CO₂ environments retain the same nutritional and health benefits, such as protein, phenolics, and antioxidants, as those grown in current ambient CO₂ conditions. Furthermore, there was no significant effect of CO₂ treatment, cultivar, or the interaction between CO₂ and cultivar on the TPC content in kernels. These results could be due to the regulation of enzyme activity involved in these pathways, maintaining a balance despite increased photosynthetic carbon assimilation.

This research investigated the kernel size with the assumption that additional carbon-based compounds would be allocated to seeds as sink tissue. The literature reports heterogeneous trends regarding seed size under EC: several studies highlighted that EC might alter seed size [47,48], while others found a negative effect [49] or no change at all [50].

In the present work, seed size varied among cultivars but was not impacted by EC, in accordance with the results reported by Prasad et al. [51]. However, the cultivar Alloway showed a mild increase of 12%, while Kairi remained unchanged. These findings suggest that kernels may not be the primary sink organs for the surplus of compounds produced through enhanced carboxylation. Despite higher C and C:N values observed under EC, the additional assimilated carbon might be preferentially allocated to other plant organs or metabolic processes rather than contributing significantly to kernel size. This indicates that kernels might have a limited capacity to utilize the excess carbon, leading to negligible changes in seed size under elevated CO₂ conditions.

There was a significant negative correlation between kernel thickness, width, and the [CO₂] treatment, as well as the [CO₂] × cultivars interaction, with no effect on kernels’ length. Overall, Kairi recorded the highest mean values. These results showed an impact on peanut physical attributes, similar to findings for rice width [52,53], but in contrast with other research on chickpeas, where seed properties were not significantly different between CO₂ treatments [54], and wheat, which showed significant increases in thickness and width [55].

5. Conclusions

In conclusion, this study successfully evaluated the performance of three peanut cultivars under elevated CO₂ conditions and assessed intraspecific variability between cultivars. The experiment focused on physiological, phenological, and physiochemical responses, with particular attention to productivity and quality traits of both kernel and feedstock.

The fertilization effect of elevated CO₂ significantly enhances plant growth and biomass accumulation through increased carbohydrate production, potentially leading to a mismatch with nitrogen content in plant tissues and a reduction in nitrogen-based compounds like proteins. However, this study identified specific cultivars that positively responded to elevated CO₂, showing improved vegetative growth, physiological efficiency, and kernel yield. Notably, Kairi demonstrated superior performance in water use efficiency compared to Holt and Alloway, making it particularly suitable for environments with limited water availability. While certain biomass traits responded favourably to elevated CO₂, no significant effects were observed on kernel morphology. However, pod and kernel yield improved across all cultivars.

Importantly, peanut kernels’ nutritional and antioxidant properties remained largely unaffected by elevated CO₂, suggesting similar benefits to those grown under ambient conditions. Despite variations in root composition, the cultivars exhibited comparable phytochemical compositions, highlighting their resilience to changing CO₂ levels.

However, concerns were raised regarding the potential decline in the fibre structure and digestibility of peanut stover under elevated CO₂, which could compromise feedstock quality.

This study identified Kairi as the most promising cultivar for future experiments under elevated CO₂ conditions and simulated grazing scenarios. Our future research will explore the combined effects of elevated CO₂ and biomass removal by simulating grazing impacts on peanut cultivars grown as dual-purpose crops. These efforts aim to enhance the productivity and resilience of peanut genotypes within Australian farming systems amidst rising CO₂ levels.