Potential Implications of Elevated CO₂ on Physiochemical Parameters in Peanut (Arachis hypogaea L.) Genotypes ^†

Abstract

Three peanut genotypes were cultivated under ambient and elevated CO₂ conditions. Elevated CO₂ treatment was associated with higher total phenolic content (TPC) values in the shoots and roots of all genotypes. Seeds showed lower levels of TPC compared to the other plant tissue, showing a decline in all three genotypes, and no differences between the CO₂ treatments were observed. The Crude Protein (CP) content in seeds varied significantly among the genotypes, and elevated CO₂ treatment was associated with mild increases (not significant) in CP of 4 and 6% in Holt and Alloway cultivars, respectively. It is worth mentioning that Kairi contained the highest CP content under elevated CO₂ (29%).

1. Introduction

Elevated atmospheric carbon dioxide concentrations ([CO₂]) have been linked to declines in the nutritional quality of grains [1,2]. Decreases in the nutritional quality of grains are especially troubling for populations where grains are the primary protein source [2]. Despite these detrimental effects, much of the physiological processes by which reduced grain and forage protein is induced by elevated [CO₂] remains unexamined, and the role of both nitrogen derived from the atmosphere and nitrogen taken up from the soil in grain quality has yet to be fully elucidated [3].

Previous studies have documented increases in phenolic compound concentrations and antioxidative capacity as a response to high [CO₂] exposure, with different magnitudes reported in various crops [4], and these increases have often been associated with increased foliar nitrogen (N) concentrations in crops such as Brassica rapa [5] and arboreal species [6]. However, other studies have reported that elevated [CO₂] has a neutral effect on plants’ N content or, worse, a detrimental impact, suggesting that the responses are subject to species-specific variability.

Thus, it is increasingly important to better understand the metabolic response of crops and plants to this stimulation. This understanding can aid in the selection of genotypes that have the potential for increased productivity under elevated CO₂ conditions.

For this study, we aimed to evaluate the performance of three peanut genotypes, focusing on changes in their physiochemical composition when grown under both ambient (400 ± 50 µmol mol⁻¹) and enriched CO₂ (650 ± 50 µmol mol⁻¹) conditions to identify the most promising genotype for dual-purpose (grain and graze) cropping under elevated CO₂.

2. Materials and Methods

2.1. Site Description

The study was conducted at the Central Queensland Innovation Research Precinct in Rockhampton, Queensland, Australia. The climate in the region is classified as humid subtropical, according to the Köppen climate classification system, and is represented by the code Cfa/Cwa. The project design consisted of eight plots equipped with octagonal-shaped Open-Top Chambers (OTCs), four of which were assigned to ambient carbon dioxide concentration (AC) at ~400 µmol mol⁻¹, while the other four were allocated for elevated carbon dioxide concentration (EC) at 650 µmol mol⁻¹ ± 50 µmol mol⁻¹.

2.2. Planting Pattern and Crop Management

Three types of high-oleic runner peanut varieties (Kairi, Holt and Alloway) were grown in clay loamy soil from seed material provided by The Peanut Company of Australia. The sowing density was ~190,000 seeds h⁻¹. The seeds of the three varieties were pre-treated with a fungicide (Captan^®, 0.12%; Quintozene^® 0.12%) and inoculated with rhizobia group P and planted simultaneously in the same plots distributed in three linear meters. Before planting, the clay loamy soil was fertilized with 20 g m⁻² of phosphorus (P as water-soluble and citrate-soluble) and 20 g m⁻² of potassium (K as sulphate of potassium). Additionally, microelements, including boron (B), zinc (Zn), magnesium (Mg), manganese (Mn), and copper (Cu), were added to the soil at a rate of 0.4 g m^-2. Water was applied through a drip irrigation system twice a day (0600 AM and 1600 PM) for 20 minutes, modifying the application rates during the season according to the crop growing stage and rainfall events and maintaining soil moisture >70% field capacity. The temperature in the OTC chambers was recorded using temperature data loggers. The OTCs’ glasshouse plastic had a light transmissibility of 91%. The [CO₂] inside the OTC was maintained at 650 ± 50 µmol mol⁻¹ for the EC treatment and ~400 µmol mol⁻¹ for the AC treatment. The CO₂ enrichment started 12 days after sowing (DAS), when plants had completely emerged, and it was continuously operated during daytime (0530–1800) until harvest conducted at 132 DAS.

2.3. Total Phenolic Content and Antioxidant Capacity

Kernels and above- and below-ground biomass samples were oven-dried for a minimum of 72 h at 65 ° C and subsequently ground into powder form. Following the method of Johnson et al. [7], plant matrices were extracted using approximately 0.5 g of fine powder of plant material in 90% methanol. The methanolic extracts were stored in the dark at 4 °C until required for further analyses. In order to investigate the impact of elevated CO₂ as a potential stress mitigator, the total phenolic content (TPC) and antioxidant capacity of the vegetative matter and peanut seed material were analyzed using the Folin–Ciocalteu [8] assay for TPC and the Ferric Reducing Antioxidant Potential (FRAP) and Cupric Ion Reducing Antioxidant Capacity (CUPRAC) assays for antioxidant capacity.

2.4. Nitrogen and Protein Percentage

Nitrogen concentrations of dried ground kernels and biomass (~0.15 g; particles size: 0.84 mm) were analyzed through the combustion method using a LECO TruMac elemental analyzer (LECO Corporation, St. Joseph, MI, USA). The protein percentages of the kernels and the above- and below-ground biomass samples were calculated by multiplying the nitrogen concentration by a nitrogen/protein conversion factor of 6.25 [9], while the kernel concentrations were calculated using a peanut-specific conversion factor (5.46) [10].

3. Results

The EC treatment impacted the vegetative growth parameters (not shown in this publication) by enhancing the total above-ground biomass, with increases of 5, 23, and 26% being recorded for Alloway, Kairi, and Holt, respectively. The exposure to elevated CO₂ concentration also positively impacted yield traits, namely, the number of pods and the pods’ mass plant⁻¹ in all tree genotypes. The plants grown under EC conditions showed increases in pods’ plant⁻¹ of 43, 24, and 16% in Kairi, Holt, and Alloway, respectively.

3.1. Total Phenolic Content

The total phenolic content (TPC) varied depending on genotypes, plant tissue type, and [CO₂] (

Table 1. Mean of the total phenolic content (mg GAE 100 g⁻¹ DW ± SD), ferric reducing antioxidant potential, and cupric reducing antioxidant capacity (reported as mg 100 g⁻¹ DW ± under ambient (AC) and elevated CO₂ (EC) conditions).

Genotype	[CO2]	TPC (mg GAE 100 g−1 DW)			FRAP (mg TE 100 g−1 DW)			CUPRAC (mg TE 100 g−1 DW)
		Seeds	Roots	Shoots	Seeds	Roots	Shoots	Seeds	Roots	Shoots
Kairi	AC	156 ± 33	456 ± 159	691 ± 114	84 ± 17	492 ± 191	798 ± 145	234 ± 108	1750 ± 661	2593 ± 833
	EC	140 ± 3	398 ± 108	717 ± 65	99 ± 22	473 ± 177	786 ± 88	240 ± 94	1424 ± 550	2986 ± 262
Holt	AC	127 ± 12	347 ± 120	685 ± 70	83 ± 9	434 ± 191	954 ± 244	224 ± 86	1205 ± 444	3077 ± 791
	EC	123 ± 10	363 ± 120	674 ± 68	82 ± 5	455 ± 213	942 ± 53	163 ± 16	1434 ± 601	3317 ± 411
Alloway	AC	132 ± 5	334 ± 96	732 ± 73	84 ± 3	394 ± 143	823 ± 37	220 ± 78	1374 ± 297	2795 ± 344
	EC	125 ± 4	366 ± 99	750 ± 140	88 ± 4	454 ± 193	754 ± 155	196 ± 17	1422 ± 429	2796 ± 322

Abbreviations: TPC, total phenolic content; FRAP, ferric reducing antioxidant potential; CUPRAC, cupric reducing antioxidant capacity; DW, dry weight; GAE, gallic acid equivalents; TE, Trolox equivalents; SD, standard deviation.

). However, the ANOVA tests failed to provide significance between genotypes, [CO₂], and the interaction between [CO₂] and the genotypes (p > 0.05). The TPC showed a mild (albeit non-significant) decrease in EC compared to the control plants in all genotypes’ seeds, with decreases of −10% in Kairi, −5% in Alloway, and −3% in Holt. A similar pattern was observed in Kairi’s roots, whereas in Holt and Alloway, the CO₂ treatment resulted in a positive response. Between the three plant tissues investigated in this study, the shoots displayed the highest overall phenolic content out of all plant parts. EC moderately enhanced the total phenolic content in shoots of Kairi (4%) and Alloway (2.5%) genotypes, while Holt showed a mild negative response (−1.6%).

3.2. Ferric Reducing Antioxidant Potential

In the root material, the ferric antioxidant potential (FRAP) increased in Holt and Alloway with EC and decreased in Kairi; however, there was no significant correlation between [CO₂], genotypes, and the interaction between [CO₂] and the genotypes (p > 0.05). Similarly, the FRAP measured in the seed samples showed no significant differences between genotypes and CO₂ treatments. The shoots’ responses significantly differed among the three genotypes (p < 0.01), with Holt showing the highest values, which declined from 954 in AC conditions to 942 mg TE 100 g⁻¹ DW in EC conditions.

3.3. Cupric Reducing Antioxidant Capacity

Statistical analysis performed on the cupric reducing antioxidant capacity (CUPRAC) of all sample types in combination with the [CO₂] and genotypes showed no significant correlation in the roots and seeds. However, a positive correlation (p < 0.05) between CUPRAC and genotypes was observed in the shoot samples, with Holt having the highest value (3317 mg TE 100 g⁻¹ DW, under EC conditions) as illustrated in

Table 2. Summary of total phenolic content (mg GAE 100 g⁻¹ DW ± SD), ferric reducing antioxidant potential (mg TE 100 g⁻¹ DW ± SD), and cupric reducing antioxidant capacity (mg TE 100 g⁻¹ DW ± SD) under ambient (AC) and elevated CO₂ (EC) conditions.

Plant Material	Parameter	AC	EC	p Value	Alloway	Holt	Kairi	p Value	[CO2] × Genotype Interaction
Root	CUPRAC	1465 ± 503	1427 ± 481	NS	1615 ± 767	1371 ± 422	1548 ± 525	NS	NS
	FRAP	441 ± 162	461 ± 176	NS	480 ± 217	463 ± 169	456 ± 147	NS	NS
	TPC	382 ± 129	376 ± 100	NS	395 ± 145	374 ± 101	399 ± 113	NS	NS
Shoot	CUPRAC	2945 ± 537	3033 ± 379	*	2717 ± 486	2895 ± 720	2865 ± 594	NS	NS
	FRAP	859 ± 166	828 ± 129	**	480 ± 151	463 ± 242	456 ± 135	NS	NS
	TPC	703 ± 83	714 ± 94	NS	713 ± 96	652 ± 94	691 ± 104	NS	NS
Seed	CUPRAC	226 ± 83	200 ± 60	NS	236 ± 72	247 ± 130	233 ± 103	NS	NS
	FRAP	84 ± 10	89 ± 14	NS	87 ± 4	90 ± 22	90 ± 17	NS	NS
	TPC	139 ± 24	134 ± 19	NS	138 ± 17	136 ± 20	155 ± 38	NS	NS

Note. NS = not significant (p > 0.05), * p < 0.05, ** p < 0.01. Abbreviations: TPC, total phenolic content; FRAP, ferric reducing antioxidant potential; CUPRAC, cupric reducing antioxidant capacity; DW, dry weight; GAE, gallic acid equivalents; TE, Trolox equivalents; SD, standard deviation.

. As observed in the FRAP assay, Holt recorded the highest values (954 mg TE 100 g⁻¹ DW, under AC conditions) between the three genotypes as a response to enhanced CO₂ in shoot samples. However, in contrast to FRAP for shoot material, Holt’s CUPRAC results showed an increase of 8% with elevated CO₂.

3.4. Nitrogen and Protein Content

The mean values of N and protein contents in the kernels, as well as the above and below-ground biomass of the three peanut genotypes considered in this study, are reported in

Table 3. Mean of nitrogen and protein percentage (based on dry weight) ± SD (standard deviation) in cultivars grown under ambient (AC) and elevated CO₂ (EC) conditions.

Variety	Treatment	Nitrogen %			Protein %
		Seeds	Roots	Shoots	Seeds	Roots	Shoots
Kairi	AC	5.36 ± 0.20	1.11 ± 0.29	2.51 ± 0.25	29.3 ± 1.07	6.95 ± 1.79	15.7 ± 1.56
	EC	5.32 ± 0.15	0.99 ± 0.14	2.48 ± 0.10	29.0 ± 0.83	6.21 ± 0.89	15.5 ± 0.63
Holt	AC	5.08 ± 0.24	1.32 ± 0.21	2.50 ± 0.17	27.70 ± 1.32	8.24 ± 1.34	15.6 ± 1.08
	EC	5.27 ± 0.17	1.23 ± 0.33	2.47 ± 0.06	28.80 ± 0.94	7.70 ± 2.09	15.4 ± 0.37
Alloway	AC	4.63 ± 0.13	0.89 ± 0.17	2.35 ± 0.11	25.30 ± 0.71	5.55 ± 1.08	14.7 ± 0.69
	EC	4.93 ± 0.18	1.06 ± 0.19	2.31 ± 0.10	26.90 ± 0.97	6.65 ± 1.19	14.4 ± 0.64

. The results of our statistical analysis showed that the protein contents in all plant materials were not significantly impacted by the elevation in CO₂ (p > 0.05) (

Table 4. Impact of elevated CO₂ conditions on root nitrogen and protein content in three peanut genotypes.

Parameter	AC	EC	p Value	Alloway	Holt	Kairi	p Value	Treatment × Variety Interaction
N	1.08 ± 0.28	1.09 ± 0.25	NS	0.97 ± 0.24	1.25 ± 0.22	1.03 ± 0.21	*	NS
Protein	6.69 ± 1.68	6.85 ± 1.48	NS	6.06 ± 1.43	7.68 ± 1.35	6.55 ± 1.18	*	NS

NS = not significant (p > 0.05), * p < 0.05.

). However, there was significant (p < 0.05) variability between genotypes in the root material for all parameters. Kairi showed the highest protein content (29%) in the seed material under EC conditions, followed by Holt (28%) and Alloway (27%). Holt had the highest protein content in the roots under EC conditions, whereas the protein content in the shoot material was relatively consistent between the genotypes.

4. Discussion

This study found no significant impact of CO₂ treatment, genotypes, or CO₂ × genotype interaction for TPC content in the roots, shoots, and seeds. The shoots had the highest TPC compared to the roots and seeds. Similar results have been reported in other crops [7]. A similar pattern was observed by Gillespie et al. [11] in soybean (Glycine max; another legume), where increasing CO₂ had no impact on total phenolic content. Again, Fernando et al. [12] observed no significant effect in the TPC of wheat (Triticum aestivum) following CO₂ elevation.

Despite this lack of variation seen in TPC, CO₂ elevation did have a significant impact on the antioxidant capacity of the shoot material, as measured by FRAP (p < 0.01) and CUPRAC (p < 0.05). Somewhat surprisingly, the trend was not consistent, increasing under EC conditions for CUPRAC but decreasing for FRAP. Although these assays do have different reaction potentials and measure slightly different aspects of antioxidant activity, further investigation into this trend is required. It is possible that very specific compounds are being up- or down-regulated by the plant’s physiological responses to EC, leading to an increase in CUPRAC (+8% average increase) but not in FRAP (−3.6% average decline). Both Fernando et al. [12] and Gillespie et al. [11] reported an increase in antioxidant capacity following CO₂ elevation. There were no significant differences between genotypes regarding antioxidant capacity for any plant part. However, there was a significant genotypic difference in the root protein content—where Holt showed the highest protein content (7.7%), while Alloway showed the lowest (6.1%).

5. Conclusions

Atmospheric CO₂ represents the primary substrate for photosynthesis; thus, in high concentrations, it is known to boost plants’ growth and biomass accumulation by increasing plants’ carbohydrate contents. However, this process, known as the fertilization effect, could lead to a mismatch with N content in plant tissues, as well as a consequent reduction in N-based compounds such as proteins. Importantly, in this study, no significant effects of increasing the CO₂ level were observed on the protein content, TPC, CUPRAC, or FRAP of the peanut seed material, suggesting that peanuts grown under EC conditions should have a similar level of nutritional and health benefits (in terms of protein, phenolics, and antioxidants) to those grown under current ambient CO₂ levels.

Additionally, the three peanut genotypes explored in this study did not vary significantly from one another in their TPC or antioxidant capacity (for any plant part) when grown under either AC or EC levels. This suggests that, aside from their root composition, they are quite comparable in terms of their phytochemical composition.

In our future work, we will explore the impact of EC conditions on the morphology and physiology of these three peanut genotypes, allowing for a definitive selection of the best genotype(s) for commercial production under future EC scenarios. This will enable peanut producers to be better prepared for potentially altered environmental conditions.