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Article

Physiological and Biochemical Responses of Maize to Elevated CO2 Concentrations: Implications for Growth and Metabolism

by
Pirzada Khan
1,†,
Fardous Mohammad Safiul Azam
1,†,
Tong Lian
1,
Ashraf M. M. Abdelbacki
2,
Mohammed Albaqami
3,
Rahmatullah Jan
4,*,
Kyung-Min Kim
4,* and
Weixuan Wang
1,*
1
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China
2
Deanship of Skills Development, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia
3
Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia
4
Department of Applied Biosciences, Graduate School, Kyungpook National University, Daegu 41566, Republic of Korea
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2024, 14(8), 1751; https://doi.org/10.3390/agronomy14081751
Submission received: 28 June 2024 / Revised: 5 August 2024 / Accepted: 8 August 2024 / Published: 9 August 2024
(This article belongs to the Special Issue Net-Zero Emissions for Sustainable Food Production and Land Management)
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Abstract

:
Rising atmospheric CO2 levels, a significant consequence of anthropogenic activities, profoundly impact global agriculture and food security by altering plant physiological processes. Despite extensive research, a comprehensive understanding of the specific effects of elevated CO2 on maize (Zea mays L.)’s primary and secondary metabolism remains elusive. This study investigated the responses of maize seedlings cultivated in open-top chambers (OTCs) under three CO2 concentrations: ambient (380 ppm), elevated (600 ppm), and high (1800 ppm). Key growth parameters, including plant height, leaf area, and aboveground biomass (leaf and stem), were assessed alongside metabolic profiles encompassing nonstructural and structural carbohydrates, syringyl (S) and guaiacyl lignin, the syringyl-to-guaiacyl (S/G)-lignin ratio, photosynthetic pigments, total soluble protein, and malondialdehyde (MDA) levels. The results demonstrated that exposure to 600 ppm CO2 significantly enhanced plant height, leaf area, and aboveground biomass compared to ambient conditions. Concurrently, there were notable increases in the concentrations of primary metabolites. In contrast, exposure to 1800 ppm CO2 severely inhibited these growth parameters and induced reductions in secondary metabolites, such as chlorophyll and soluble proteins, throughout the growth stages. The findings underscore the intricate responses of maize metabolism to varying CO2 levels, highlighting adaptive strategies in primary and secondary metabolism under changing atmospheric conditions. This research contributes to a nuanced understanding of maize’s physiological adaptations to future climate scenarios characterized by elevated CO2, with implications for sustainable agriculture and food security.

1. Introduction

The escalating atmospheric concentration of carbon dioxide (CO2) since the Industrial Revolution poses a critical global concern, primarily due to its profound impact on climate and agriculture. CO2 levels have risen by approximately 30% since pre-industrial times, primarily due to human activities, such as fossil fuel combustion and deforestation [1,2]. This increase has altered global carbon dynamics, prompting significant concerns about its implications for agricultural production, ecosystem stability, and human livelihoods worldwide.
Of particular concern is the effect of elevated CO2 levels on staple crops like maize (Zea mays L.), which plays a crucial role in global food security and the livestock industry [3,4]. Maize is a versatile crop, serving as a staple food for human consumption, a vital component of animal feed, and a raw material for various industrial products. The global production and consumption of maize have risen steadily in recent decades, driven by its adaptability to diverse climates and its high yield potential [5].
Research examining the influence of elevated CO2 on maize has explored various facets of its growth and metabolic responses. Elevated CO2 generally enhances photosynthesis and biomass production in C3 and C4 plants, including maize [6,7,8]. This phenomenon, known as the CO2 fertilization effect, results from the increased availability of CO2 for photosynthetic carbon fixation, thereby promoting plant growth under optimal conditions [9]. However, alongside these benefits, elevated CO2 concentrations can lead to shifts in nutrient dynamics within plants, impacting their nutritional quality and potentially altering their interactions with pests and pathogens [10,11].
Studies have demonstrated that under elevated-CO2 conditions, maize plants often exhibit decreased concentrations of essential minerals such as calcium, potassium, and magnesium in their tissues. These nutrient reductions have significant implications for both human and animal nutrition, potentially compromising the health and productivity of livestock dependent on maize-based feeds [12,13]. Furthermore, the altered nutrient composition in maize grains may affect food quality for human consumption, highlighting broader implications for food security and public health [14].
The effects of elevated CO2 on maize metabolism are complex and multifaceted, often yielding mixed results in research findings. While some studies suggest that higher CO2 levels enhance maize yields, they also indicate potential trade-offs, such as reduced protein content or altered carbohydrate profiles in plant tissues [6]. These complexities underscore the need for comprehensive studies that consider various environmental factors and management practices influencing maize responses to elevated CO2, especially in diverse agroecosystems across different regions.
Lignocellulosic material from grasses, including corn stover, is a critical resource for both animal fodder and bioethanol production [15]. Although lignin, a major component of lignocellulosic material, has a 30% higher gross energy content compared to cellulose, it significantly reduces the feed value of plant resources [16]. This is because the lignin content in ruminant diets is negatively correlated with digestible energy; higher lignin levels lead to lower digestibility and, thus, lower energy availability for the animals. This presents a challenge for using maize stover as a feed resource, highlighting the need for strategies to improve its degradability and nutritional value. In China, maize cultivation holds significant economic importance, particularly in the livestock sector, where maize plants and their by-products serve as crucial feed resources for cattle, poultry, and swine. Each year, it is estimated that approximately 0.24 billion tons of corn stover, comprising leaves and stems, are produced [17]. The quality and nutritional value of maize, influenced by environmental factors such as CO2 levels, directly impact animal health, growth rates, and reproductive performance [18]. Understanding how elevated CO2 affects maize growth and nutrient composition is therefore essential for optimizing agricultural practices and ensuring sustainable food production in China and other maize-producing regions globally.
This study is designed to evaluate the impact of varying CO2 concentrations on the growth, biomass production, and metabolic responses of maize. While focusing primarily on physiological and metabolic changes, the research also considers the nutritional implications in the broader context of agricultural sustainability and food security. By addressing these dynamics, this research contributes to a comprehensive understanding of how maize responds to future climate scenarios characterized by elevated CO2 levels, informing strategies for resilient and sustainable maize production systems.

2. Materials and Methods

2.1. Experimental Site

The experiment was conducted using open-top chambers (OTCs) at Langfang research farm, located at 39.5380° N, 116.6838° E in Hebei province, China, situated within a cold-and-moderate-temperature zone. The mean annual precipitation is 557 mm, and the mean annual temperature is 12.1 °C. The annual frost-free period is normally between 180 and 200 days, while the rainy season is highly variable, with summer accounting for 75% of precipitation, with rainstorms occurring mostly in July and August.

2.2. Experimental Design and Elevated-CO2 Treatment

During the Kharif seasons of 2018 and 2019, chamber experiments were conducted to examine the impact of elevated CO2 on agronomic and biochemical traits of maize (Zea mays L.). Maize seeds were grown under three different atmospheric CO2 concentrations, 380 ppm, 600 ppm, and 1800 ppm, representing current levels and future projections according to the IPCC (2017) [19] (Table 1).
The experiments were conducted in OTCs with maize (Zea mays L.) planted with a spacing of 20 cm between plants within rows, and rows spaced 60 cm apart. Throughout the growing season, all the seedlings were irrigated by flooding method of irrigation when necessary. CO2 levels within the OTCs were maintained between 8:30 a.m. and 6:00 p.m. using CO2 gas released from cylinders positioned 35 cm above the soil surface along the circumference of the chambers. Air blowers situated near the chamber base circulated air, mixing CO2 with incoming air. The actual CO2 concentration was continuously monitored by CO2 analyzers within the OTCs, and inlet valves were regulated automatically to maintain desired CO2 levels. CO2 supplementation occurred only during daylight hours. Maize crops were fertilized with 120 kg/ha of nitrogen (N), 26 kg/ha of phosphorus (P), and 50 kg/ha of potassium (K). At planting, the total amount of P and K, along with 50% of the N, was applied. The remaining N was split into two equal applications: one at the knee-high stage (25 days after sowing, DAS) and another at the tassel-formation stage (65 DAS) of maize growth. This experimental setup allowed us to explore comprehensively how varying CO2 concentrations affect maize growth and development, alongside an assessment of the biochemical responses of the plants under simulated future atmospheric conditions.

2.3. Open-Top Chamber Design

Four pairs of OTCs were constructed, each standing 3.5 m high with an octagonal ground surface area of 3.73 square meters (Figure 1). These chambers were spaced apart by a 5.5-meter-wide buffer zone to prevent shading between adjacent chambers. Carbon dioxide gas was supplied to the plants within the chambers through a pipe equipped with pinholes, connected to industrial carbon dioxide cylinders containing 99.99% pure liquid carbon dioxide from Anjin Gas Corporation, located outside the chambers. Inside each chamber, a fan facilitated the circulation of the supplied carbon dioxide with fresh air from outside, ensuring thorough mixing throughout the chamber. The gas exited through a top vent, allowing continuous replenishment with fresh air. To monitor CO2 levels continuously, an infrared gas analyzer was employed, providing precise measurements within the chambers throughout the experimental period.

2.4. Growth and Harvesting of Maize

Maize plants (B73) were harvested at three distinct growth stages, vegetative (40 days after sowing, DAS), tasseling (70 DAS), and dent stage (90 DAS), to assess various morphological and biochemical characteristics. At each harvest, measurements were taken for plant height, leaf area, and the fresh and dry weights of leaves and stems. Leaf area for each plant was calculated using the length-width coefficient approach (length × width × 0.75). The total leaf area per plant was determined by summing the areas of all individual leaves. Aboveground biomass was calculated using the dry weights of leaves and stems. Six plants from each treatment were divided into stems and leaves before being dried in a forced-air oven at 60 °C for a minimum of 96 h until fully dried. Subsequently, the dried leaves and stems were weighed separately using an electronic balance.

2.5. Total Soluble Sugar, Starch, and Total Non-Structural Carbohydrate Measurement

The total soluble sugar (TSS) and starch contents were determined using a commercially available chemical kit according to the manufacturer’s instructions (Solarbio Life Sciences, Beijing, China). For analysis of samples, the third youngest leaf from the top was harvested, and it was stored and preserved in liquid nitrogen. While the stem was divided into bottom, middle, and top nodes, depending on the length of each harvested plant, from each part, the healthiest node was collected for the analysis of total soluble sugar and starch. Three leaves from each treatment were collected randomly in liquid nitrogen. Next, 200 mg of leaves were crushed into powder form, in liquid nitrogen, and put into 1.5 mL tube. To each tube, 1 mL of distilled water was added, followed by vortexing and incubation for 10 min. The samples were then centrifuged at room temperature: 3000× g for 10 min for starch and 8000× g for 10 min for sugar. The supernatant was filtered into a 10 mL test tube and diluted to 10 mL with distilled water. Glucose was used as a standard stock solution to prepare a standard curve for quantifying sugar and starch in the samples. For analysis, 200 µL of the sample was mixed with enthrone reagent and 1 mL of 95% sulfuric acid. The mixture was then heated in a water bath at 95 °C for 10 min and subsequently measured at absorbance 620 nm using a UV-1200 spectrophotometer. The total non-structural carbohydrate (TNC) content was calculated as the sum of the total soluble sugar and starch contents determined from the standard curve as described by [20].

2.6. Total Soluble Protein Measurement

Total soluble protein was extracted from 200 mg of leaf tissue using 1 mL of protein-extraction buffer. The samples were homogenized on ice and then centrifuged at 10,000× g at 4 °C for 15 min. The supernatant was assessed using the BCA Protein Assay Kit (Suzhou Keming Biotechnology Co., Ltd., Chefang Town Suzhou, Jiangsu, China), following the manufacturer’s protocols.

2.7. Lignin Contents and Composition Measurements

Lignin extraction was performed using the acetyl bromide method with a commercially available kit following the instructions from Solarbio Life Sciences, Beijing, China. For lignin analysis, leaves and stems of the whole plants were shed, dried, and then, weighing 3 mg each, and having previously passed through a 40-mesh sieve, placed in 1.5 mL EP tubes. The samples were mixed with acetylated perchloric acid and incubated in an 80 °C water bath for 40 min. After cooling, the samples underwent centrifugation at 8000× g for 10 min, and the resulting supernatant was collected. The supernatant was then mixed with glacial acetic acid and the absorbance was measured at 280 nm using a UV-1200 MAPADA spectrophotometer.

2.8. Measurement of Chlorophyll and Malondialdehyde (MDA) Contents

For the determination of MDA content, a commercial chemical kit from Solarbio Life Sciences, Beijing, China was utilized following the manufacturer’s instructions. Initially, 100 mg of leaf sample was homogenized on ice with 1 mL of extraction buffer, followed by centrifugation at 8000× g for 10 min at 4 °C. The supernatant was collected and kept on ice. Subsequently, 200 µL of the sample was mixed with working reagent (600 µL) and reaction buffer, and the mixture was incubated in a water bath at 100 °C for 30 min, followed by rapid cooling in an ice bath. After centrifugation at 10,000× g for 10 min at room temperature, the supernatant was transferred to a 1 mL glass cuvette, and absorbance was measured at 450 nm, 532 nm, and 600 nm using a spectrophotometer.
For measuring total chlorophyll content, 300 mg of fresh leaves were weighed and transferred into a 15 mL Falcon tube containing 80% acetone. The samples were incubated in darkness for 15–30 min, followed by centrifugation at 3000 rpm at 4 °C for 15 min. The supernatant was properly mixed, and the absorbance (A) was measured using a spectrophotometer. Chlorophyll concentrations were calculated using the following formulas, where 80% acetone served as a blank control:
Chlorophyll a (Ca) = [12.7 × A663 − 2.69 × A645] × V/1000 × W
Chlorophyll b (Cb) = [22.9 × A645 − 4.86 × A663] × V/1000 × W
Total Chlorophyll (Ca + b) = [8.02 × A663 + 20.20 × A645] × V/1000 × W
where V represents the volume of the extract in milliliters, and W denotes the weight of fresh leaves in grams.

2.9. Statistical Analysis

Two-way ANOVA was performed to analyze the effects of elevated CO2 on maize using Statistix 10 (Analytical Software, Tallahassee, FL, USA). For the mean comparison, the least significant difference (LSD) was used at 5% level of significance. The GraphPad Prism 7.0 software (GraphPad Software, La Jolla, CA, USA, https://www.graphpad.com/, accessed on 18 February 2024) was used for making figures, while correlational analysis was performed with Excel 2016. The data were expressed as the mean ± standard error (SE) of triplicates.

3. Results

3.1. CO2 Causes Morphological Changes in Maize Plants

The maize plants were grown for up to 13 weeks under ambient CO2 conditions (380 ± 20 ppm CO2) or with varying levels of CO2 enrichment (600 ± 20 ppm and 1800 ± 20 ppm CO2). Throughout each growth stage, the plants subjected to CO2 enrichment exhibited distinct morphological changes compared to those grown under ambient CO2 conditions (Figure 2A–C). Notably, the maize plants grown with CO2 enrichment at 600 ppm were taller than those grown under ambient CO2 conditions, despite having the same number of leaves. In contrast, the plants grown at 1800 ppm CO2 were shorter in height than those grown under ambient conditions. The CO2 enrichment at 600 ppm increased the plant height by 13.47%, 23.61%, and 8.83% at each harvest stage, while at 1800 ppm, the plant height decreased by 65.71%, 30.77%, and 24.26% after 40, 70, and 90 DAS, respectively.
Comparing maize plants grown under ambient (380 ppm) and elevated CO2 levels, there was a significant 37.55% increase in the total biomass at 600 ppm and a notable 30.49% decrease at 1800 ppm by 90 DAS (Table 2). At 600 ppm CO2, the leaf area increased by 10.7%, but it significantly decreased, by 17.66%, at 1800 ppm after 90 days. The increase in total biomass at 600 ppm was primarily due to the enhanced growth of the leaves and shoots. Conversely, the decrease in biomass at 1800 ppm was attributed to reduced leaf and shoot growth, consistent with the shorter stature observed in these plants. The CO2 enrichment also significantly increased the leaf area at 600 ppm by 17.49% at 70 DAS and decreased it by 19.88% at 1800 ppm. By the end of the experiment, the leaf area had increased by 10.7% at 600 ppm and decreased by 17.7% at 1800 ppm. These findings highlight the differential effects of CO2 enrichment on maize growth and biomass accumulation, underscoring its potential impacts on crop productivity under changing atmospheric conditions.
The dry weights of the maize leaves exhibited inconsistent patterns under different CO2 concentrations. This was particularly evident at 600 ppm, where significant increases were observed at specific harvest stages. At 70 DAS, both the leaf and the stem dry weights significantly increased, by 22.48% and 65.34%, respectively, whereas at 90 DAS, only the stem dry biomass showed a significant increase, of 59.48% (Table 2). Conversely, at 1800 ppm CO2, significant decreases were noted in the leaf and stem dry weights at 70 DAS, with reductions of 22.96% and 28.32%, respectively. At 90 DAS, significant reductions in leaf dry weight were observed at both 600 ppm (24.69%) and 1800 ppm (34.48%).
At 40 DAS, although not statistically significant, there was a notable 27.27% increase in stem dry weight at 600 ppm, whereas a 36.36% decrease was observed at 1800 ppm (Table 2).

3.2. Elevated CO2 Leads to More Accumulation of Sugar and Starch in Maize Plants

The CO2 treatments had a profound impact on the chemical composition of the maize leaves in this experiment, particularly affecting the carbohydrate contents. The plants exposed to elevated CO2 accumulated varying levels of total soluble sugars, starch, and total nonstructural carbohydrates (TNC) in their leaves and stems compared to those grown under ambient CO2 conditions (Figure 3). Notably, the highest concentrations of soluble sugars, starch, and TNC were recorded at 600 ppm CO2, while the lowest values were observed at 1800 ppm CO2.
The soluble sugars exhibited a decrease with plant age (Figure 3A). At 40 and 70 DAS, the soluble sugars increased slightly at 600 ppm (7.73% and 7.76%, respectively). Conversely, at 70 DAS, the plants exposed to 1800 ppm CO2 showed a drastic decrease in soluble sugar content (25.22%). At 90 DAS, the soluble sugars were significantly reduced at both 600 ppm (16.33%) and 1800 ppm (47%) (Figure 3A). In the stems, the soluble sugars significantly increased at 600 ppm at 40 and 70 DAS (19.47% and 19.78%, respectively) but decreased significantly at 1800 ppm at 70 and 90 DAS (11.59% and 30.15%, respectively) (Figure 3B).
The starch content in the leaves significantly increased at 600 ppm across all the growth stages (66.89%, 20.25%, and 14.17% at 40, 70, and 90 DAS, respectively) but decreased significantly at 1800 ppm at 70 and 90 DAS (15.41% and 23.94%, respectively) (Figure 3C). Similarly, in the stems, the starch contents significantly increased at 600 ppm at 40 and 70 DAS (77.63% and 31.02%, respectively) but significantly decreased at 1800 ppm at 90 DAS (25.53%) (Figure 3D).

3.3. High CO2 Reduces Protein Accumulation in Maize Plant

The impact of elevated CO2 concentrations on the total soluble protein content of the maize was notable across all the growth stages examined in this study (Figure 4). Elevated CO2 levels consistently resulted in decreased protein contents compared to ambient CO2 conditions. Specifically, at 40 days after sowing (DAS), the protein content decreased by 9.3% at 1800 ppm CO2, and at 70 DAS, it decreased by 7.5% at the same CO2 concentration. At 90 DAS, elevated CO2 significantly reduced the soluble protein content; this was particularly evident at 1800 ppm. This phenomenon can be attributed to increased carbohydrate synthesis at the expense of proteins, a concept known as the carbon dilution effect. Moreover, the findings suggest that elevated CO2 may contribute to nitrogen immobilization in various vegetative tissues, considering nitrogen’s pivotal role in protein chemistry (Figure 4). Overall, these observations underscore the complex metabolic adjustments that maize undergoes in response to elevated CO2 levels, impacting its nitrogen utilization and protein synthesis dynamics throughout different growth stages.

3.4. Lignin Accumulation and Composition Is Significantly Affected by CO2 Treatment

The maize plants grown under varying CO2 concentrations exhibited significant effects on the lignin contents in both the leaves and the stems (Figure 5). Across different growth stages, there was considerable variability observed in the maize leaf and stem lignin levels under the different elevated-CO2 treatments. Initially, at 40 DAS, the lignin accumulation increased in both the leaves and the stems, albeit not significantly across all the treatments. Notably, the leaves accumulated significantly more lignin at 1800 ppm CO2 (18.53%) compared to ambient levels. At 90 DAS, the leaves showed significantly higher lignin contents under elevated-CO2 conditions (29.45% at 600 ppm and 18.53% at 1800 ppm). Similarly, the stems exhibited significantly higher lignin accumulation at 600 ppm across all the growth stages (24.28%, 29.42%, and 19.41%). However, higher CO2 levels, particularly at 1800 ppm at 90 DAS, had a negative impact on the lignin accumulation in both the leaves and the stems, resulting in significantly lower lignin contents compared to control levels. This variation in lignin content may be linked to changes in TNC. This is supported by the strong correlation coefficients observed in the leaves and stems across different growth stages.
Interestingly, the total soluble sugars showed a higher correlation coefficient (R2 = 0.69, 0.96; p ≤ 0.05) than the total starch at 40 DAS, suggesting that sugar accumulation might play a more significant role in altering lignin accumulation than starch during early growth stages. Conversely, at 70 and 90 DAS, starch exhibited stronger correlation coefficients than sugar, indicating a shift in the relationship and dynamics between plant primary and secondary metabolites as the plants mature. Additionally, the monomeric composition of lignin was influenced by the different elevated-CO2 treatments. The guaiacyl (G) monomer predominated, initially in the leaves and, at 40 DAS, in the stems, followed by syringyl (S) and p-hydroxyphenyl (H). However, the S monomer became predominant in the stems at 70 and 90 DAS, contributing to stem stiffness and strength. Additionally, the S/G ratio of the lignin decreased under elevated-CO2 conditions, primarily due to lower S lignin values (Table 3 and Table 4). These findings highlight the complex interplay between elevated CO2 levels and the biochemical composition of maize, particularly in terms of lignin content and composition, which are crucial for plants’ structural integrity and responses to environmental stimuli throughout different growth stages.

3.5. Biosynthesis of Chlorophylls, Carotenoids, and MDA in CO2-Treated Maize Plants

Elevated CO2 levels significantly decreased the contents of chlorophyll a (Chl a), chlorophyll b (Chl b), total chlorophyll (Chl a + b), and carotenoids in the maize leaves, with a consistent trend observed across all the growth stages (Table 5). At each harvest, the smallest decrease in pigment content was generally observed at 600 ppm CO2. Specifically, at 40 DAS, the Chl a decreased by 17.58% and 28.04% at elevated CO2 levels, while the Chl b, Chl a + b, and carotenoids decreased by 19.08%, 54.57%, 18.08%, 39.13%, and 32.57%, 51.17%, respectively. Similarly, at 70 DAS, the Chl a decreased by 6.68% and 17.02%, the Chl b decreased by 38.73% and 61.45%, the Chl a + b decreased by 19.04% and 34.15%, and the carotenoids decreased by 28.13% and 46.72% at 600 ppm and 1800 ppm CO2 levels, respectively. By 90 DAS, the Chl a decreased by 6.68% and 17.02%, the Chl b decreased by 38.73% and 61.45%, the Chl a + b decreased by 19.04% and 34.15%, and the carotenoids decreased by 28.13% and 46.72% at the respective CO2 levels.
Consistently, across all the harvests, the maize leaves exposed to the highest CO2 concentration of 1800 ppm exhibited the most pronounced reductions in chlorophyll contents, followed by those at 600 ppm and those at ambient CO2 levels.
MDA is a product of membrane lipid peroxidation and serves as a crucial indicator of cellular membrane damage under stress conditions. In this study, as the CO2 levels increased across different treatments and growth stages, the MDA content in the maize exhibited a significant decrease (Figure 6). Specifically, at each harvest, the MDA content consistently decreased with increasing CO2 concentrations. At 40 DAS, the MDA contents decreased by 22.8% and 32.8% at 600 ppm and 1800 ppm CO2, respectively. At 70 DAS, the MDA contents decreased by 24.5% and 47.9% under the same respective treatments. Similarly, at 90 DAS, the MDA contents decreased by 16.6% and 54.5% at 600 ppm and 1800 ppm CO2 levels, respectively.

4. Discussion

Increased CO2 levels in the atmosphere have a significant impact on plant growth, development, and stress response [21]. In the present study, various levels of CO2 concentrations affected primary and secondary metabolites in maize plants. Plant height is a critical factor in crop morphogenesis and grain yield. Proper plant height is associated with higher yield index and higher crop quality [22,23]. At 600 ppm CO2 (Figure 2A–D), the maize plants exhibited significantly increased plant heights compared to those raised at ambient levels (380 ppm). This finding aligns with previous studies highlighting the CO2 fertilization effect, where enhanced carbon availability promotes photosynthetic efficiency and biomass accumulation [8].
Conversely, exposure to 1800 ppm CO2 resulted in a marked inhibition of the maize plants’ heights compared to both ambient and 600 ppm CO2 levels. This inhibitory effect at supra-optimal CO2 concentrations reflects potential metabolic constraints or feedback mechanisms that limit growth beyond a certain threshold [11,13]. High CO2 levels can lead to physiological imbalances, including impaired stomatal conductance and altered nutrient uptake, which may negatively impact overall plant growth and development [8]. The significant height increase at 600 ppm CO2 supports the notion that moderate CO2 enrichment can potentially benefit maize productivity in terms of biomass accumulation, although the long-term implications for grain yield and nutrient content require further investigation [24].
Leaf area is an important factor in many physiological and agronomic processes, especially in insignificant abiotic factor, with significant fertilization effects on crops. As presented in Figure 2E, various levels of elevated CO2 significantly affected the leaf area at different growth stages. At 40 DAS, the leaf area increased in all the elevated-CO2 treatments, though the difference was not statistically significant when compared to the ambient CO2 level. Previous research found that increased CO2 levels significantly increase plants’ growth rate and leaf area [25,26]. Maroco et al. also reported that CO2 enrichment significantly increased the leaf areas of maize plants [27]. Increased plant leaf areas may be due to increased photosynthesis and carbohydrate production in the presence of elevated CO2 concentrations [28]. Increased CO2 availability under 600 ppm CO2 concentrations may allow plants grown under these conditions to maintain higher net C assimilation [27,29], and it was further demonstrated that, at the transcript and metabolite levels, CO2 enrichment stimulates the respiratory breakdown of carbohydrates, providing more energy and biochemical precursors for leaf expansion and growth, thus promoting plant leaf growth under elevated-CO2 conditions. In the current study, the plants had smaller leaf areas at the highest CO2 levels (Figure 2E, Table 2). Therefore, it can be concluded from the present results that plants cultivated at high CO2 levels may be unable to maintain their normal photosynthetic capacity, causing them to fall below the carbon balance threshold. Previous research shows that a loss of carbon balance may hasten senescence and reduce leaf area [30].
The chlorophyll content has been widely used as an indicator of various physiological and agronomic components, particularly in the later stages of growth [31]. It is frequently used to assess chloroplast development and photosynthetic capacity [32]. Various abiotic stresses, as observed in many plant species, accelerate chlorophyll degradation, ultimately reducing photosynthetic capacity [33].
In the present study, elevated CO2 reduced the accumulation of photosynthetic pigments in maize, with reductions in chlorophyll a, b, and total chlorophyll compared to controls (Table 5). The mechanism underlying this reduction is unknown, but it has been suggested that it could be due to a combination of factors, such as carbohydrate dilution, slower N uptake by the roots, and reduced transpiration-driven N flux [13]. It has also been proposed that CO2 inhibits nitrate assimilation [34]. In addition, the reduction in chlorophyll content may be caused by dilution from excessive carbohydrate accumulation, as Wildman hypothesized that starch grain formation may cause disorientation in chloroplasts, resulting in less light interception [35]. Therefore, it can be concluded from the current study that the decrease in chlorophyll contents with increasing elevated CO2 may be due to chloroplast degeneration caused by starch accumulation (Figure 3C, Table 5) [36,37]. Moreover, it was reported that elevated CO2 causes a decrease in the concentration of mineral nutrients in plant leaves, including some essential components of chlorophyll, such as N and Mg [38]. Therefore, a possible explanation for the decrease in chlorophyll here could be a reduction in some necessary mineral nutrients that eventually affects the synthesis of chlorophyll [38]. However, other hypotheses cannot be excluded, e.g., CO2-induced transpiration limitations leading to reduced N uptake, and chlorophyll loss [39]. Numerous studies have found that when CO2 levels are high, the chlorophyll content in C3 and C4 species decreases [27,32,40].
Soluble sugars and starch are the two main types of carbohydrate stored in plant vegetative tissues like stems and leaf sheaths [41]. It is well documented that photosynthesis down-regulation is frequently linked to changes in leaf chemistry, such as insufficient N availability, lower Rubisco concentration activity, and the souce–sink imbalance caused by carbohydrate accumulation in leaves under high CO2 concentrations [42,43]. In the current study, in the maize leaves and stems, the increase in starch content was greater than the increase in soluble sugar concentration (Figure 3). Other researchers observed a higher increase in starch than in soluble sugar production when samples were exposed to elevated CO2, implying that starch production was more up-regulated under elevated CO2 [44]. The increase in primary metabolite production caused by increased CO2 availability could be due to enhanced photosynthesis [45,46]. The accumulation of carbohydrates in CO2-enriched (600 ppm) plants may be attributed to the dilution of plant tissue nitrogen in enhanced plant growth under elevated CO2, especially when nitrogen is limited. It is well understood that nitrogen plays an important role in the regulation of the plant sink strength [47,48]. It is also well known that when exposed to high levels of CO2, the nitrogen content of the plant decreases due to increased growth. This decrease in N may reduce the plant’s sink size, reducing carbohydrates’ translocation to other plant parts [27]. Reduced nitrogen levels may reflect higher nitrogen use efficiency due to the reallocation of protein, an ontogenic drift that accelerates aging due to increased growth rates, or insufficient nitrogen fertilization, uptake, or assimilation [49]. On the other hand, the decreased growth and biomass at 1800 ppm could be attributed to decreased N uptake and reduced demand for nitrogen. Furthermore, the drastic reduction in growth at higher CO2 concentrations could be due to a decreased N assimilation capacity [33]. Similarly, at high CO2 concentrations, a decrease in stomatal conductance may contribute to the down-regulation of the net photosynthetic rate [50,51,52]. Previous research has shown that under elevated-CO2 conditions, there is often a decrease in nitrogen concentrations relative to carbon, resulting in an elevated C/N ratio [8]. This shift can have significant implications for maize growth, nutrient use efficiency, and, ultimately, crop productivity, highlighting the intricate balance between carbon availability and nitrogen utilization in a changing atmospheric environment (Figure 3).
Lignin, a crucial secondary metabolite in plant cells synthesized through the phenylalanine/tyrosine metabolic pathway, plays a key role in plant structure [53]. Increased CO2 levels impact not only primary metabolism, but also the secondary metabolic composition in plant tissues [41,42,54,55]. Contrary to the predictions of source–sink balance theories, experimental studies at elevated CO2 levels reveal no consistent pattern for lignin, an insoluble phenolic polymer and carbon-based structural compound found in cell walls. These levels have primarily been assessed in litter studies. Interestingly, plants exposed to prolonged elevated CO2 have shown higher lignin concentrations [56,57].
With respect to secondary metabolism, it is known that high concentrations of CO2 interfere with the biosynthesis of lignin. However, it has been observed that increasing rates of CO2 can either increase [58], reduce [59], or have no effect on the concentration of lignin [60].
In the present study, the carbohydrates accumulated in the maize at elevated CO2 levels might have been channeled for the production of secondary metabolites i.e., lignin (Figure 5A,B). The up-regulation in lignin could be linked to the balance between carbohydrate source and sink, as the higher the source–sink ratio, the higher the production of secondary metabolites that might occur [43]. The current findings revealed that increased TNC production promoted lignin accumulation in maize under the influence of elevated CO2. Several relevant studies [44,54,61,62,63] have reported changes in the secondary metabolites as CO2 levels rise. On the other hand, a contradiction may occur when elevated CO2-induced N dilution reduces carbohydrate reserves, resulting in a decrease in secondary metabolites [41]. At 70 DAS, a significant decrease in lignin content was observed in the leaves at higher CO2 levels (Figure 5A). The assimilated carbon was most probably incorporated into nonstructural carbohydrates (NSC) at the expense of lignin synthesis. Plants may prefer to invest excess carbon in growth and renewable resources, such as sugar and starch, particularly in foliar tissues, because lignin is a final metabolic product that is not reused [64]. A decrease in lignin content was observed in a number of other studies when CO2 levels were elevated [54,64].
The monomer composition significantly impacts the molecular structure of lignin, influencing aspects like polymer branching and the extent of crosslinking with polysaccharides [53]. Consequently, the types of monomer present determine both the degradability of lignin and the usability of lignocellulosic biomass [53]. In the present study, the monomeric composition of lignin was significantly affected by the elevated CO2 (Table 3 and Table 4). These results are corroborated by those of earlier studies, which revealed that elevated CO2 altered lignin contents and compositions, and that the compositions of monomers have an important influence on the molecular structure of lignin, such as the branching of the polymer and the degree of crosslinking with the polysaccharide [65].
The addition of the enzymatic complex altered the monomeric composition of the lignin in both silages. The guaiacyl (G) monomer was the most abundant, followed by syringyl (S) and p-hydroxyphenyl (H). Notably, the S monomer experienced a greater increase compared to the other monomers, resulting in a higher S/G ratio. This rise in the S/G ratio indicates that the lignin became less condensed after the enzymatic treatment [66], probably making it easier for microorganisms to access the nutritional components.

5. Conclusions

This study revealed that elevated CO2 levels influence maize plants’ growth and alter both primary and secondary metabolites. Lignin, a crucial component for biomass resistance against enzymatic digestion by ruminants, showed significant changes under elevated CO2. Higher CO2 levels increased the total nonstructural carbohydrates, which enhanced the lignin production initially. However, the highest CO2 concentration negatively impacted the plant growth, leading to a reduced accumulation of nonstructural carbohydrates and, subsequently, of lignin. These effects can be understood through the carbon–nutrient balance hypothesis, which posits that excess carbon under high CO2 conditions is diverted from primary metabolism to secondary-metabolite biosynthesis. This shift favors carbon-based secondary metabolites like lignin over nitrogen-based compounds, thereby decreasing chlorophyll and soluble protein levels in plants. Increased lignin accumulation enhances biomass resistance but lowers forage quality by reducing digestibility and animal performance. Conversely, lower lignin content improves digestibility but increases stems’ susceptibility to lodging under stress. To enhance biomass and forage crop quality, further research into the mechanisms regulating lignin accumulation under elevated-CO2 conditions is crucial.

Author Contributions

Conceptualization: P.K., T.L., R.J. and W.W.; methodology: P.K., T.L. and F.M.S.A.; statistical analysis: P.K., F.M.S.A. and A.M.M.A.; writing and original draft preparation: P.K., W.W. and M.A.; review and editing, K.-M.K., R.J. and A.M.M.A.; supervision: K.-M.K., W.W., R.J. and M.A.; funding acquisition K.-M.K., W.W., A.M.M.A. and M.A. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by Central Public-interest Scientific Institution Basal Research Fund (Y2020PT23) China, and “Cooperative Research Program for Agriculture Science and Technology Development (Project No. RS-2024-00348677)” Rural Development Administration, Republic of Korea. The authors extend their appreciation to the Researchers Supporting Project number (RSPD2024R978), King Saud University, Riyadh, Saudi Arabia.

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. View of open-top chambers at Lang Fang (Hebei province), China.
Figure 1. View of open-top chambers at Lang Fang (Hebei province), China.
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Figure 2. Effects of ambient and elevated CO2 on plant height (A) and leaf area (B) at different growth stages (40, 70, and 90 DAS). (C) Shows pictorial representation of plant height, (D) shows graphical representation of plant height, and (E) shows graphical representation of leaf area. Different letters indicate significant differences at p < 0.05.
Figure 2. Effects of ambient and elevated CO2 on plant height (A) and leaf area (B) at different growth stages (40, 70, and 90 DAS). (C) Shows pictorial representation of plant height, (D) shows graphical representation of plant height, and (E) shows graphical representation of leaf area. Different letters indicate significant differences at p < 0.05.
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Figure 3. Effects of ambient and elevated CO2 on soluble sugar in leaf (A), stem (B), starch in leaf (C), stem (D), TNC in leaf (E), and stem (F) at different growth stages (40, 70, and 90 DAS). Different letters indicate significant differences at p < 0.05.
Figure 3. Effects of ambient and elevated CO2 on soluble sugar in leaf (A), stem (B), starch in leaf (C), stem (D), TNC in leaf (E), and stem (F) at different growth stages (40, 70, and 90 DAS). Different letters indicate significant differences at p < 0.05.
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Figure 4. Effects of ambient and elevated CO2 on total soluble protein at different growth stages (40, 70, and 90 DAS). Different letters indicate significant differences at p < 0.05.
Figure 4. Effects of ambient and elevated CO2 on total soluble protein at different growth stages (40, 70, and 90 DAS). Different letters indicate significant differences at p < 0.05.
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Figure 5. Effects of ambient and elevated CO2 at different growth stages (40, 70, and 90 DAS) on lignin accumulation in maize leaf (A) and stem (B), Different letters indicate significant differences at p < 0.05.
Figure 5. Effects of ambient and elevated CO2 at different growth stages (40, 70, and 90 DAS) on lignin accumulation in maize leaf (A) and stem (B), Different letters indicate significant differences at p < 0.05.
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Figure 6. Effects of ambient and elevated CO2 on MDA contents in maize leaf. Different letters indicate significant differences at p < 0.05.
Figure 6. Effects of ambient and elevated CO2 on MDA contents in maize leaf. Different letters indicate significant differences at p < 0.05.
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Table 1. Experimental treatment descriptions. C0 shows carbon level zero (ambient), C1 shows carbon level 1 (600 ppm), and C2 shows carbon level 2 (1800 ppm).
Table 1. Experimental treatment descriptions. C0 shows carbon level zero (ambient), C1 shows carbon level 1 (600 ppm), and C2 shows carbon level 2 (1800 ppm).
Treatment No.NameDescription
1C0Ambient (380 ± 20 ppm)
2C1Elevated CO2 (600 ± 20 ppm)
3C2Elevated CO2 (1800 ± 20 ppm)
Table 2. Dry biomass accumulation of maize plants grown under ambient (380 ± 20 ppm) and elevated CO2 (600 and 1800 ppm). Plants were harvested at 40, 70, and 90 DAS. Values are means and SE (in parentheses); different letters indicate significant difference (p < 0.05).
Table 2. Dry biomass accumulation of maize plants grown under ambient (380 ± 20 ppm) and elevated CO2 (600 and 1800 ppm). Plants were harvested at 40, 70, and 90 DAS. Values are means and SE (in parentheses); different letters indicate significant difference (p < 0.05).
Growth StagesPlant OrgansCO2 Concentration
Ambient600 ppm1800 ppm
40 DASLeaf (g)4.43 (0.17) d4.93 (.084) d3.53 (0.18) d
Stem (g)1.833 (0.40) f2.33 (0.33) f1.16 (0.16) f
Total6.263 (0.57) g7.26 (1.17) g4.69 (0.34) g
70 DASLeaf (g)11.61 (0.68) b14.22 (0.66) a8.94 (0.85) c
Stem (g)28.83 (2.32) c47.66 (1.64) a20.66 (2.5) d
Total40.44 (3) d61.88 (2.3) a29.6 (3.35) e
90 DASLeaf (g)13.5 (0.88) a14.33 (1.25) a10.16 (0.60) bc
Stem (g)19.33 (1.62) d30.83 (0.90) c12.66 (1.52) e
Total32. (2.5) e45.16 (2.15) c22.82 (2.12) f
Table 3. Effects of ambient and elevated CO2 on lignin monomers in maize leaf. Data are means ± SE (n = 3). Lignin monomers are expressed in µmol/mg. Third leaf (from top) of maize was collected at three different growth stages, i.e., 40, 70, and 90 DAS. Means in the same category followed by different letters indicate significant differences at p < 0.05 using the least significant difference (LSD) test.
Table 3. Effects of ambient and elevated CO2 on lignin monomers in maize leaf. Data are means ± SE (n = 3). Lignin monomers are expressed in µmol/mg. Third leaf (from top) of maize was collected at three different growth stages, i.e., 40, 70, and 90 DAS. Means in the same category followed by different letters indicate significant differences at p < 0.05 using the least significant difference (LSD) test.
Harvest TimeLignin MonomersCO2 Treatment
Ambient600 ppm1800 ppm
40 DASp-Hydroxyphenyl (H)27.34 ± 0.55 c27.43 ± 0.48 c30.66 ± 0.62 b
Guaiacyl (G)94.75 ± 10.32 c160.48 ± 7.35 b73.90 ± 1.62 c
Syringyl (S)25.88 ± 3.44 de37.52 ± 7.55 bc17.84 ± 1.10 e
S/G0.27 ± 0.007 bc0.23 ± 0.039 cd0.24 ± 0.009 cd
70 DASp-Hydroxyphenyl (H)14.69 ± 0.53 e27.15 ± 0.34 c2.81 ± 0.29 h
Guaiacyl (G)186.93 ± 10.65 b221.03 ± 8.80 a67.94 ± 3.58 c
Syringyl (S)36.79 ± 1.43 bcd42.66 ± 0.83 b18.72 ± 0.61 e
S/G0.19 ± 0.007 de0.19 ± 0.07 de0.27 ± 0.08 abc
90 DASp-Hydroxyphenyl (H)15.89 ± 0.16 de13.46 ± 0.59 ef9.81 ± 0.58 g
Guaiacyl (G)182.32 ± 1.34 b182.13 ± 10.01 b70.07 ± 3.78 c
Syringyl (S)28.65 ± 6.09 cde60.98 ± 2.036 a22.67 ± 1.55 e
S/G0.15 ± 0.15 e0.33 ± 0.01 a0.32 ± 0.32 cde
Table 4. Effects of ambient and elevated CO2 on lignin monomers in maize leaf. Data are means ± SE (n = 3). Lignin monomers are expressed in µmol/mg. Stems of maize (Bottom, mid, and top) were collected at three different growth stages, i.e., 40, 70, and 90 DAS. Means in the same category followed by different letters indicate significant differences at p < 0.05 using the least significant difference (LSD) test.
Table 4. Effects of ambient and elevated CO2 on lignin monomers in maize leaf. Data are means ± SE (n = 3). Lignin monomers are expressed in µmol/mg. Stems of maize (Bottom, mid, and top) were collected at three different growth stages, i.e., 40, 70, and 90 DAS. Means in the same category followed by different letters indicate significant differences at p < 0.05 using the least significant difference (LSD) test.
Harvest TimeLignin MonomersCO2 Treatment
Ambient600 ppm1800 ppm
40 DASp-Hydroxyphenyl (H)9.02 ± 0.80 e25.08 ± 1.89 a6.81 ± 0.24 e
Guaiacil (G)143.56 ± 6.86 g207.47 ± 14.28 ef155.33 ± 12.23 g
Syringe (S)144.75 ± 15.55 e148.4 ± 11.77 e127.79 ± 11 e
S/G1.021 ± 0.15 cd0.71 ± e 0.0070.82 ± de 0.005
70 DASp-Hydroxyphenyl (H)21.78 ± 1.81 bc25.01 ± 0.47 ab13.59 ± 2.54 d
Guaiacil (G)372.77 ± 20.42 d420.03 ± 5.31 c158.76 ± 9.22 g
Syringe (S)251.53 ± 19.97 d425.39 ± 6.28 c231.18 ± 19.28 d
S/G0.67 ± 0.05 e1.012 ± 0.002 cd1.45 ± 0.03 b
90 DASp-Hydroxyphenyl (H)14.06 ± 0.38 d14.66 ± 0.50 d18.99 ± 0.48 c
Guaiacil (G)785.18 ± 12.27 a454.3 ± 14.56 b238.64 ± 13.04 e
Syringe (S)827.64 ± 12.23 a458.12 ± 14.94 b546.24 ± 8.71 c
S/G1.05 ± 0.03 c1.01 ± 0.04 cd2.29 ± 0.08 a
Table 5. Effects of ambient and elevated CO2 on chlorophyll a (mg/g FW), chlorophyll b (mg/g FW) chlorophyll a + b (mg/g FW), and carotenoid contents (mg/g FW) in maize leaf. Means in the same category followed by different letters indicate significant differences at p < 0.05 using the least significant difference (LSD) test. The data represent the means of three replications ± standard error.
Table 5. Effects of ambient and elevated CO2 on chlorophyll a (mg/g FW), chlorophyll b (mg/g FW) chlorophyll a + b (mg/g FW), and carotenoid contents (mg/g FW) in maize leaf. Means in the same category followed by different letters indicate significant differences at p < 0.05 using the least significant difference (LSD) test. The data represent the means of three replications ± standard error.
HarvestCO2 LevelsChlorophyll
aba + bCarotenoid
IAmbient4.62 ± 0.10 ab2.31 ± 0.17 b7.01 ± 0.03 b2.2 ± 0.17 ab
600 ppm3.81 ± 0.65 bcd1.87 ± 0.64 bcd5.74 ± 0.29 bcd1.54 ± 0.64 cd
1800 ppm3.32 ± 0.98 cde1.05 ± 0.35 fgh4.26 ± 0.92 ef1.12 ± 0.35 def
IIAmbient5.22 ± 0.18 a3.31 ± 0.12 a8.61 ± 0.59 a2.56 ± 0.12 a
600 ppm4.87 ± 0.20 ab2.03 ± 0.02 bc6.97 ± 0.27 b1.84 ± 0.02 bc
1800 ppm4.33 ± 0.43 abcd1.27 ± 0.12 efg5.67 ± 0.68 cd1.3 ± 0.12 cde
IIIAmbient4.51 ± 0.05 abc1.74 ± 0.03 bcde6.32 ± 0.09 bc1.70 ± 0.03 bcd
600 ppm4.18 ± 0.14 abcd1.50 ± 0.07 cdef5.74 ± 0.25 bcd1.54 ± 0.07 cd
1800 ppm2.35 ± 0.13 e0.45± 0 02 gh2.88 0.17 f0.77 ± 0.02 f
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MDPI and ACS Style

Khan, P.; Safiul Azam, F.M.; Lian, T.; Abdelbacki, A.M.M.; Albaqami, M.; Jan, R.; Kim, K.-M.; Wang, W. Physiological and Biochemical Responses of Maize to Elevated CO2 Concentrations: Implications for Growth and Metabolism. Agronomy 2024, 14, 1751. https://doi.org/10.3390/agronomy14081751

AMA Style

Khan P, Safiul Azam FM, Lian T, Abdelbacki AMM, Albaqami M, Jan R, Kim K-M, Wang W. Physiological and Biochemical Responses of Maize to Elevated CO2 Concentrations: Implications for Growth and Metabolism. Agronomy. 2024; 14(8):1751. https://doi.org/10.3390/agronomy14081751

Chicago/Turabian Style

Khan, Pirzada, Fardous Mohammad Safiul Azam, Tong Lian, Ashraf M. M. Abdelbacki, Mohammed Albaqami, Rahmatullah Jan, Kyung-Min Kim, and Weixuan Wang. 2024. "Physiological and Biochemical Responses of Maize to Elevated CO2 Concentrations: Implications for Growth and Metabolism" Agronomy 14, no. 8: 1751. https://doi.org/10.3390/agronomy14081751

APA Style

Khan, P., Safiul Azam, F. M., Lian, T., Abdelbacki, A. M. M., Albaqami, M., Jan, R., Kim, K. -M., & Wang, W. (2024). Physiological and Biochemical Responses of Maize to Elevated CO2 Concentrations: Implications for Growth and Metabolism. Agronomy, 14(8), 1751. https://doi.org/10.3390/agronomy14081751

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