Effects of Elevated CO₂ on Maize Physiological and Biochemical Processes

Abstract

Maize (Zea mays) is a critical global crop, serving as a source of food, livestock feed, and industrial raw materials. Climate changes, driven by rising atmospheric carbon dioxide (CO₂) levels, have substantial effects on maize physiology, growth, and nutrient content. This review investigates the impact of elevated CO₂ on maize, with a particular focus on photosynthesis enhancement as it improves water use efficiency (WUE), which can lead to increased biomass production. Despite this, elevated CO₂ results in a decreased concentration of essential nutrients, including nitrogen, phosphorus, potassium, and folate. The reduction in folate, which is vital for both plant development and human nutrition, poses challenges, especially for population heavily reliant on maize. Additionally, biofortification through traditional breeding and genetic engineering is proposed as a strategy to enhance folate level in maize to mitigate nutritional deficiencies. Elevated CO₂ stimulates lignin production, improving stress resistance and carbon sequestration capacity. However, the increase in guaiacyl-rich lignin may negatively affect biomass degradability and efficiency in biofuel production. The findings emphasize the importance of balancing maize’s stress resilience, nutrient profile, and lignin composition to address future climate challenges. This balance is essential for optimizing maize cultivation for food security, biofuel production, and environmental sustainability.

1. Introduction

Maize is one of the most significant crops globally, valued for its versatility as food, animal feed, and a critical industrial raw material [1,2]. Its adaptability allows it to be cultivated across a wide range of agroecological zones, with varying altitudes, temperatures, and soil types, though yield per hectare may differ greatly depending on these conditions. North America leads global maize production, followed by Asia, particularly East Asia. Due to its relatively high yields compared to other cereal crops, maize is particularly attractive in regions facing land scarcity and high population density [3]. Globally, about 61% of maize is utilized as livestock feed, while only 13% is consumed directly by humans. It plays a crucial role in the diets of millions, especially in Africa, South Asia, and parts of Latin America, where it serves as a staple food. In contrast, East Asia primarily uses maize for livestock feed. Despite large production volumes, significant maize deficits persist in Asia and Africa, making these regions key net importers and critical areas of focus in this review. To address future challenges, maize farming must become more efficient, with an emphasis on breeding high-yield, stress-tolerant varieties to counter adverse climate conditions [4].

In many regions, maize serves as an essential dietary source of carbohydrates, proteins, iron, vitamin A (in yellow and orange varieties), various B vitamins (excluding B12), and essential minerals. While foods rich in vitamin A such as fruits, vegetables, and animal products are often expensive and inaccessible to poor households, maize remains a staple, with many families consuming it multiple times per day. In Asia, maize’s growing prominence, now being the second most important crop after rice, can be attributed to a surge in demand [5]. This demand stems from its wide range of uses, including feed for poultry and cattle, production of high-quality starch, and numerous industrial applications, such as dextrose, maltose, ethanol, and maize oil. Additionally, maize is processed into various food products, including sweet corn, popcorn, baby corn, and other corn-based convenience foods. The supply and demand dynamics in Asia have played a pivotal role in shaping the global maize economy [6]. Although maize’s direct use for human consumption is decreasing, its role in the feed and wet milling industries is expanding at a rate much faster than previously expected [7].

Several factors, such as technological advancements, improved input usage, and better pest management, have driven the increase in maize production [8]. Projections suggest that by 2050, demand for maize in developing countries will double, driven by population growth and evolving dietary preferences [9]. The widespread adoption of high-yielding hybrid maize varieties has led to substantial yield increases in various Asian agroecosystems. The average maize yield in Asia stands at approximately 4.97 tons per hectare (TE 2012), which is slightly below the global average of 5.2 t/ha [10]. In the TE 2013, five countries such as Bangladesh, China, India, Indonesia, and Pakistan produced roughly 252.42 million tons of maize grain, accounting for 87.5% of Asia’s total maize production on 48.84 million hectares of land [1]. The maize sector in Asia has experienced rapid expansion in recent years, with significant developments in technology (especially hybrids), a growing role of the private sector in technology dissemination, and increased demand, particularly from the livestock and poultry industries [10].

This review aims to explore how elevated CO₂ levels influence the growth of maize, with a particular focus on key nutritional and biochemical parameters, folate biosynthesis, and lignin formation. While maize serves as a crucial staple crop worldwide, the ongoing rise in CO₂ concentration poses both challenges and opportunities for its cultivation. Understanding how elevated CO₂ affects maize growth, nutrient quality (including folate contents), and biochemical components like lignin is essential for future agriculture strategies. Folate is a vital nutrient influencing human health, while lignin plays a critical role in plant structure and resilience, impacting both crop yield and quality. This review synthesizes findings on these topics, aiming to provide insight into how elevated CO₂ may alter maize agronomic traits, nutritional profile, and structural components, with implications for food security and crop adaptation strategies.

2. Effects of Elevated CO₂ on Maize Growth and Development

The increasing levels of carbon dioxide (CO₂) in the atmosphere are a major contributor to global warming [11] and are predicted to cause irreversible climate changes for centuries [12]. The rise in atmospheric CO₂ level is primarily driven by the combustion of fossil fuels for energy, deforestation, and numerous agricultural practices [13]. The industrial revolution is known to have greatly influenced the increase in atmospheric CO₂ levels [14]. The expansion of the transportation sector, including the widespread use of automobiles, has significantly increased CO₂ emissions [15].

Since the industrial revolution, CO₂ levels have significantly increased, particularly in the last 50 years, rising from 320 ppm to 390 ppm [16]. By 2018, these levels had reached 409.23 ppm [17], and projections suggest that CO₂ concentrations may climb to 800 ppm by the year 2100 [12]. Elevated CO₂ levels can influence both the morphology and physiology of crops [18] as climate change impacts plant growth and development, primarily due to changes in photosynthetic carbon uptake [19]. Plants absorb atmospheric CO₂ through the chemical reduction of carbon, a process that both stores chemical energy and supplies the carbon frameworks needed for the synthesis of organic molecules that make up plant structures [20]. In theory, plants could help mitigate climate change by converting atmospheric CO₂ into carbohydrates and other organic compounds through photosynthesis [21]. However, the full scope of these changes, particularly their effects on crop production and productivity, requires further study [19].

Elevated CO₂ levels impact numerous physiological processes in plants, producing a mix of both positive and negative responses depending on the species [17]. The responses of C3 and C4 plants to increased CO₂ differ as this increase affects biological processes at multiple levels of organization [22]. While research on C3 plants under elevated CO₂ conditions is abundant, C4 plants have received less attention due to the assumption that their CO₂-concentrating mechanism makes them less responsive to higher CO₂ levels [19]. However, studies on C4 plants like Saccharum officinarum L. [23,24], and Sorghum bicolor L. [25], have demonstrated increased photosynthetic rates in high CO₂ environments. In Zea mays L., elevated CO₂ has been associated with changes in transpiration rates and the production of metabolites like glucose, mannose, and galactose [21]. Moreover, increased CO₂ may enhance plant vulnerability to pests and diseases, largely due to reductions in phytochemical and phytohormone production, which are essential for plant defense [26,27].

Although C4 plants account for a relatively small percentage of global plant species, they are of great ecological and economic significance [24]. Among these species, maize (Zea mays L.) is the most significant crop worldwide in terms of production [28]. Maize is widely cultivated for food and as a feedstock for ethanol, starch, and oil production [29]. However, climatic fluctuations can negatively impact maize yield, as seen in southern Brazil where climate change has led to an average reduction of 0.8% in productivity levels [30]. Although there is extensive research on the effect of elevated CO₂ on C3 plants, there is still a significant gap in understanding how CO₂ influences C4 plants, specifically in term of changes in physiological processes, crop productivity, and plant defense mechanisms.

3. Positive and Negative Impacts of Elevated CO₂ on Plant Nutrition

Over the last 3 million years, the rise in atmospheric CO₂ levels (from 280 to 415 ppm) has been both rapid and unprecedented in scale [31]. This increase, beyond its major role in driving climate change, has profound implications for plant nutrition due to the dual role of CO₂ as both a substrate and a signal molecule. On the positive side, because current atmospheric CO₂ levels limit C3 photosynthesis, a rise in CO₂ concentration is expected to enhance CO₂ uptake in C3 plants, leading to improved biomass production often referred to as the “CO₂ fertilization effect” [32]. This is a crucial development as enhanced photosynthesis is essential to meet increasing food demands while mitigating CO₂ accumulation [33]. Various experimental setups, such as controlled growth chambers, open-top chambers (OTCs), and free-air CO₂ enrichment (FACE) systems, have been utilized to study this CO₂ fertilization effect under artificially elevated CO₂ conditions [34]. Yields of C3 crops grown at CO₂ levels anticipated for the latter half of this century have frequently shown increases of 20–30%, although these results can vary depending on the species and experimental conditions [35]. These advancements present a vital opportunity to secure global food and feed production. However, recent studies suggest that actual gains in photosynthesis and biomass production are often lower than expected [36]. This is commonly attributed to reduced photosynthetic efficiency in plants exposed to elevated CO₂ compared to ambient levels, a phenomenon known as “photosynthetic acclimation to elevated CO₂”. This adaptation is linked to the buildup of non-structural carbohydrates, a decrease in total leaf protein content (including Rubisco), and a reduction in the activation state of Rubisco [32,34].

On the downside, a surprising result is the harmful effect of elevated CO₂ on the mineral content of C3 plants. Increased CO₂ levels have been shown to reduce the concentrations of key nutrients in plant tissues, a phenomenon observed for over two decades [37]. Nearly all species and essential nutrients (N, P, K, S, Fe, Mg, and Zn) are affected, with nutrient concentrations decreasing by 5–25%, depending on the nutrient, the level of CO₂, and the experimental conditions [38]. This decline in nutrient content is expected to have two significant negative effects. First, it may reduce the nutritional quality of major staple crops, exacerbating global malnutrition and associated health problems. Second, the shift in nutrient concentrations, particularly changes in C/N and C/P ratios, may impact the stability of soil organic matter and alter key biogeochemical processes that regulate nutrient cycling in ecosystems [39]. A recent study has been reviewed that elevated CO₂ reduces plant metabolites and closes stomata partially to reduce water loss [40]. In addition to C3 plants’ responses to elevated CO₂, several other studies have also reported on the impacts of elevated CO₂ on C4 plants. Comparing the C3 plants with C4 plants, a study has shown that elevated CO₂ increases the productivity of C3 plants compared to C4 plants [41], which shows that CO₂ elevation affects the productivity of C4 plants. It has been reported that C3 plants tend to respond more positively to elevated CO₂ levels, with a photosynthesis rate boost of about 58% when CO₂ baubles, whereas C4 species show minimal changes as their photosynthesis is nearly saturated at current CO₂ levels [42]. In C4 plants, the positive effect of elevated CO₂ can significantly enhance photosynthesis under drought stress; however, some recent studies have also observed growth in various C4 grasses with higher CO₂ levels, even under well-watered conditions [43,44,45]. The fertilizer impact of elevated CO₂ on photosynthesis in C4 species remains unclear although evidence suggests that CO₂ enrichment can boost leaf photosynthesis (Pn) and lower transpiration rates, potentially raising leaf temperatures and further enhancing Pn in C4 plants [42].

Recent research has further validated these predictions from elevated CO₂ experiments. On a global scale, it now appears that the CO₂ fertilization effect is real as increases in CO₂ have enhanced photosynthetic carbon capture and boosted primary vegetation production. This phenomenon likely explains why the Earth’s vegetation has become greener in recent decades [46], contributing to a rise in the terrestrial carbon sink, which has helped to mitigate global warming [46,47]. Long-term research on forests has revealed a decline in the mineral content of tree leaves over the past few decades, reflecting a broader trend in plant nutrient status [48,49]. Analysis of preserved plant samples has confirmed that modern plants have lower nutrient levels compared to plants from over a century ago [50]. Research has also shown that plants growing in natural CO₂-rich environments, such as volcanic springs, have lower leaf nitrogen content than plants growing in ambient CO₂ conditions nearby [51]. Together, these findings indicate that the ongoing rise in CO₂ levels has already impacted plant nutritional status. This study reflects both the negative and positive effects of elevated CO₂ on plants as shown in Figure 1. While the “CO₂ fertilization effect” is well documented in C3 plants, there is a critical research gap regarding the long-term impact of elevated CO₂ on the nutritional quality of crops, particularly the decline in essential nutrient concentrations.

4. Genetic Responses of Maize to Elevated CO₂

As atmospheric CO₂ levels continue to rise due to anthropogenic activities, understanding the genetic responses of maize (Zea mays) to elevated CO₂ becomes critical. Research indicates that elevated CO₂ can significantly influence maize physiology, growth, and development, particularly through its effects on stress responses [52]. Maize exhibits a range of genetic adaptations that allow it to cope with increased CO₂ levels. One of the key areas of adaptation involves genes associated with stress responses, including those related to drought, heat, and nutrient deficiency. Under elevated CO₂ conditions, maize can improve its water use efficiency, largely, as a result of reduced stomatal conductance [53]. This physiological change is mediated by genetic factors that regulate stomatal movement, such as the genes coding for ion transporters and signaling molecules involved in drought responses. Moreover, the genetic diversity within maize allows for varying responses to elevated CO₂. Studies have shown that different maize genotypes exhibit distinct levels of biomass accumulation and photosynthetic efficiency under high CO₂ [54]. For instance, certain inbred lines possess alleles that enhance the synthesis of key enzymes involved in carbon fixation and stress resilience [55]. Increased CO₂ predominantly suppressed the expression of genes associated with photosynthesis and sugar metabolism pathways. A recent study has shown that elevated CO₂ was found to downregulate genes such as psbY, psaK, petF, PRK, LHCB1, rbcS, GST30, glgC, ppdK, WAXY, pfA, PDHB, GST15, ALDO, BX4, MDH1, DLD, GLUL, MDH2, P5CS, POP2, and thrC, which are involved in pathways like glycolysis, glyoxylate, and dicarboxylate metabolism, as well as fructose and mannose metabolism [56]. Elevated CO₂ often enhances photosynthetic carbon assimilation, increasing fructose, glucose, and sucrose in many plants due to its positive effects on photosynthesis and sugar metabolism [54,57]. However, a recent report demonstrated that elevated CO₂ downregulated the sugar metabolism-related genes, including LDH, ALDO, glgC, and WAXY [56]. Research also highlights the role of transcription factors in maize’s genetic response to elevated CO₂. Genes encoding transcription factors, such as those in the DREB (dehydration-responsive element-binding) family, are activated under stress conditions, facilitating the expression of downstream stress-responsive genes [58]. This genetic network enables maize to maintain growth and development despite the challenges posed by a changing climate. While research has identified specific genetic adaptations in maize to elevated CO₂, there are still studies needed to understand the full extent of genotype-specific responses and the molecular mechanisms underlying these adaptations. This study aims to explore these genetic variations to improve the breeding of maize varieties optimized for future elevated CO₂ conditions.

5. Impact on Water Use Efficiency and Drought Resistance

Elevated atmospheric CO₂ levels have significant implications for maize (Zea mays), particularly regarding its water use efficiency (WUE) and drought resistance [59]. Understanding these impacts is crucial as maize is a staple crop that faces increasing challenges from climate change, including drought conditions. Research indicates that elevated CO₂ enhances maize’s water use efficiency by promoting physiological changes that reduce water loss through transpiration. Higher CO₂ concentrations lead to decreased stomatal conductance, which minimizes water loss while maintaining photosynthetic rates [60]. This adaptation allows maize to utilize water more efficiently, thereby improving its overall productivity in water-limited environments [61]. Furthermore, elevated CO₂ can enhance the drought resistance of maize. Studies have shown that plants grown under high CO₂ conditions exhibit increased root biomass and altered root architecture, which facilitates better water uptake during periods of drought stress [62]. The enhancement of root systems is essential for accessing deeper soil moisture, thereby increasing the plant’s resilience to prolonged droughts. Physiologically, elevated CO₂ influences the expression of genes associated with stress response and drought tolerance. For instance, the upregulation of certain aquaporins, which are water channel proteins, has been observed in maize exposed to higher CO₂ levels [63]. This genetic response enhances the plant’s ability to maintain cellular turgor and metabolic functions under drought conditions. Additionally, elevated CO₂ can interact with other environmental factors, potentially complicating maize’s response to drought. While increased CO₂ improves WUE, it may also alter the balance of nutrient availability, which is critical for maintaining plant health and productivity under stress [64]. Therefore, understanding these interactions is vital for developing strategies to enhance maize’s resilience to future climate scenarios. Although elevated CO₂ is known to enhance water use efficiency and drought resistance in maize, there is research needed to evaluate the interaction between increased CO₂ and nutrient availability under drought conditions.

6. Folate Role in Maize Growth and Development Under Stress Condition

6.1. Importance and Significance

Folates are essential compounds involved in metabolism across all living organisms [65]. They have a dual function as donors and acceptors of one-carbon units in transfer reactions, which are crucial for the synthesis of several key biomolecules, including nucleic acids, amino acids, and pantothenate (vitamin B5). In the methylation cycle of folate, the methyl group plays a key role in increasing the function of folate in terms of gene regulation, lipid, protein, chlorophyll, and lignin biosynthesis. Many staple crops, such as corn, rice, potato, and cassava, contain relatively low levels of folates, which leads to widespread folate deficiencies, particularly in regions where these foods are the chief source of energy [66].

Inadequate folate intake has been associated with various developmental problems and health conditions, such as anemia and neural tube defects. Furthermore, folate scarcity has been linked to high risks of cardiovascular disease, dementia, and some cancers. To mitigate these health risks, increasing folate intake has become a priority, with biofortification of staple crops emerging as a cost-effective and sustainable solution. Although advances have been made in enhancing the folate content of certain plants, such as rice, lettuce, and tomato [67,68], challenges remain. For example, the outcomes of biofortification can differ across plant species, likely due to variations in the regulation of the folate biosynthetic pathway. Another significant issue is the inherent instability of folates, which can result in considerable folate loss during post-harvest processing and storage [69]. Additionally, the potential impacts of excessive folate production on overall plant metabolism require a thorough examination to avoid interference with critical metabolic processes.

Folates are also critical for plant health and development. Studies have shown that proper folate metabolism is essential for plant growth [70], influencing signaling pathways [71], as well as carbon and nitrogen metabolism [6]. Folate supplementation has been found to enhance a plant’s resistance to biotic stress [72]. Furthermore, folate metabolism responds differently under various abiotic stress conditions, underscoring its role in stress adaptation and its potential for fine-tuning in response to environmental challenges [73,74]. These insights suggest that understanding the physiological roles and regulatory mechanisms of folate metabolism is crucial in the development of crops with improved productivity and stress resilience.

6.2. Folate Biosynthesis in Plants

Folate refers collectively to tetrahydrofolate (THF) and its derivatives. In plants, folate is synthesized primarily from three components: pterin, p-aminobenzoate (PABA), and glutamate, as shown in Figure 2.

This synthesis occurs in three different cellular compartments. Pterin is synthesized in the cytosol, PABA in plastids, and THF in mitochondria (Figure 3) [75]. Unlike animals, plants can synthesize their own folate. The folate biosynthesis process begins in the cytosol where guanosine triphosphate (GTP) is converted into dihydroneopterin triphosphate by GTP cyclohydrolase I. This is followed by dephosphorylation and side chain removal to produce 6-hydroxymethyldihydropterin (HMDHP) [76]. In the plastids, PABA is synthesized from chorismate through two sequential steps [77]. Chorismate is first converted into aminodeoxychorismate and then into PABA, with both steps occurring in the plastids. The final step in pterin synthesis, catalyzed by dihydroneopterin aldolase (DHNA), involves the removal of the dihydroneopterin side chain to produce glycolaldehyde and HMDHP [78]. In mitochondria, HMDHP undergoes pyrophosphorylation by HMDHP pyrophosphokinase, forming HMDHP-P2. This compound combines with PABA through the action of dihydropteroate synthase to form dihydropteroate (DHP). The biosynthesis of THF is completed by attaching a glutamate moiety via dihydrofolate synthase (DHFS) and reducing it via dihydrofolate reductase (DHFR). THF may then be further modified by folylpolyglutamate synthase (FPGS) or transported to various subcellular compartments [76].

6.3. Effects of Elevated CO₂ on Folates

Plants are essential to human health and well-being, forming the cornerstone of our diet. Of the approximately 50,000 edible plant species, just three staple grains—rice, maize, and wheat—account for more than 60% of the global caloric intake (FAO, undated). Plant biomass and nutritional value are derived from atmospheric CO₂, along with water, nitrogen, and micronutrients absorbed from the soil. Consequently, plant composition and nutritional quality are influenced by environmental conditions and soil health. With the rapid rise of atmospheric CO₂, significant changes in plant stoichiometry are expected, which could negatively impact their nutritional profiles. These changes pose potential risks to the nutritional integrity of ecosystems, including human diets.

Research into the impact of elevated CO₂ on plants is still in its early stages, particularly regarding its implications for human health and global food security. The ongoing increase in atmospheric CO₂ levels is predicted to have numerous detrimental effects on both global food production and nutrient adequacy [80]. One notable example is the documented decline in nutrient concentrations in food crops grown under elevated CO₂ conditions. Controlled experiments over the past several decades have consistently demonstrated significant reductions in essential nutrients across multiple food crops, including elements critical for human health [81,82]. Although some early studies produced inconclusive results when replicated in open-field free-air carbon enrichment (FACE) experiments [83], more recent FACE experiments have confirmed nutrient losses, especially in iron, zinc, and protein content, across key staple crops such as rice, wheat, barley, and, to a lesser extent, maize, soybeans, and potatoes [20,84].

Modeling studies suggest that the resulting nutrient deficiencies could place 138–175 million people at risk of zinc deficiency [85], and an additional 122–148 million people may face protein deficiency by 2050 due to CO₂-induced nutrient losses [86]. Furthermore, over a billion people already suffering from zinc and protein deficiencies may see these issues worsen. While the risk of iron deficiency could not be estimated with the same precision, it is expected to disproportionately affect women of childbearing age and children under five, putting them at heightened risk [85].

Collectively, these findings underscore the significant threat posed by CO₂-induced nutrient losses to global nutritional sufficiency. Recent research has expanded on these insights by examining the effects of elevated CO₂ on both mineral and vitamin content in rice [87]. This study not only confirmed the reductions in iron, zinc, and protein but also revealed declines in several B vitamins, including thiamin, riboflavin, pantothenic acid, and folate, with average losses ranging from 13 to 30%. Although the study focused solely on rice, it highlights a critical vulnerability in the global food system linked to rising anthropogenic CO₂ emissions.

6.4. Folate Stability and Degradation Under Environmental Stresses

Folate, or vitamin B9, is an essential nutrient that plays a crucial role in plant development and human health. In maize (Zea mays), the stability and degradation of folate can be significantly affected by various environmental stresses, including elevated temperatures, drought, and increased atmospheric CO₂ levels [88]. Understanding these impacts is vital for improving the nutritional quality of maize in the face of climate change. Elevated temperatures are known to adversely affect folate stability in maize. High temperatures can lead to the degradation of folate through increased enzymatic activity and oxidative stress, resulting in reduced folate concentrations in plant tissues [88]. Studies indicate that folate biosynthesis is temperature-sensitive, with elevated temperatures inhibiting the expression of key genes involved in folate metabolism, leading to diminished folate levels [89]. Drought stress also plays a significant role in folate degradation. Under water-limited conditions, maize plants exhibit physiological stress responses that can affect folate synthesis and stability. Drought can induce oxidative stress, which further contributes to the breakdown of folate compounds [90]. Moreover, drought conditions may alter the availability of essential nutrients required for folate synthesis, exacerbating folate degradation [76]. Increased atmospheric CO₂ levels present a complex interaction with folate stability in maize. While elevated CO₂ can enhance plant growth and productivity, it may also influence folate biosynthesis pathways [91]. Research suggests that CO₂ enrichment can modify the expression of genes related to folate metabolism although the exact effects can vary based on other environmental factors [88]. Furthermore, enhanced growth under high CO₂ conditions may lead to dilution effects where increased biomass does not proportionally increase folate concentration, potentially impacting nutritional quality [92].

6.5. Biofortification Strategies for Folate Enhancement in Crops

Biofortification is a promising approach to improving the nutritional quality of staple crops, including maize (Zea mays), particularly in the context of increasing global food insecurity and changing environmental conditions. Recent advances in biofortification strategies aim to enhance folate content, which is crucial for human health [66]. One of the key strategies for folate biofortification involves traditional breeding methods [93]. Researchers have identified maize genotypes with naturally higher folate levels, which serve as breeding stock for developing new varieties [94]. For instance, a study by Palacios-Rojas et al. (2020) demonstrated that specific landraces of maize possess elevated folate concentrations due to variations in metabolic pathways related to folate biosynthesis [95]. By utilizing these genotypes in breeding programs, it is possible to increase the folate content of modern maize varieties. Genetic engineering techniques also hold significant promise for enhancing folate levels in crops. The introduction of genes associated with folate biosynthesis, such as encoding enzymes like GTP cyclohydrolase I (GCH1), and 4-dihydroxy-2,3,3′,4′-tetrahydropteroate synthase (DHPS), has shown potential in model systems and some crops [96]. For example, transgenic approaches have successfully increased folate levels in rice, which could be adapted for maize and other staple crops [93]. In addition to genetic approaches, agronomic practices can also play a role in folate biofortification. Studies have indicated that soil nutrient management, including the application of micronutrients and organic fertilizers, can enhance folate content in crops [88]. For instance, the use of foliar fertilizers containing micronutrients has been linked to increased folate concentrations in maize and wheat under field conditions [97]. Moreover, as environmental stresses like drought and elevated temperatures become more prevalent due to climate change, integrating stress resilience into biofortification strategies is crucial. Research has shown that maintaining optimal growing conditions through irrigation and nutrient management can help sustain folate levels in crops under stress [98]. This multifaceted approach ensures that biofortified crops remain nutritionally adequate even in challenging environments. Despite the above information, there is still research needed to evaluate how elevated CO₂ affects folate stability in maize, especially under combined abiotic stresses. There is also a gap in understanding genotype-specific responses to elevated CO₂, the interaction between folate and lignin synthesis, and the impact on maize nutrient quality.

7. Lignin’s Role in Maize Growth and Development Under Stress Condition

7.1. Importance and Significance of Lignin

Lignin, a complex polymer derived from phenylpropanoid compounds and a major component of plant cell walls, is the second most abundant organic compound in plants, representing about 30% of the organic carbon in the biosphere [99]. The process of lignification is believed to have emerged around 430 million years ago, facilitating plant adaptation to terrestrial environments [100]. The functional roles of lignin primarily include providing mechanical support that enables plants to maintain an upright structure, facilitating water transport in xylem vessels, and serving as a defense mechanism against pests and pathogens [100]. The first two functions are linked to lignin’s interactions with other cell wall components, which enhance the structural integrity of the plant. Lignin, while vital for plant growth, presents challenges for human use, such as increasing costs and environmental harm in paper production and hindering biofuel production by blocking cellulose breakdown [101]. Lignin, essential for plant survival but problematic for human applications and animal forage digestibility, is the subject of extensive research aimed at engineering its content and composition in trees and forage crops to better serve human needs.

7.2. Lignin Biosynthesis in Grasses

Lignin is a complex phenolic polymer made up of three main units: p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S). These units originated from hydroxycinnamic alcohols, known as monolignols, specifically p-coumaryl alcohol, and sinapyl alcohol. The main difference between these units is their level of methylation. Monolignols are synthesized from phenylalanine or tyrosine (in grasses) through the phenylpropanoid pathway, which serves as a precursor for various specialized metabolites, including flavonoids, tannins, and coumarins [102]. This process also involves specific pathways for monolignol synthesis in the cytosol, culminating in polymerization within the cell wall. Although the processes included in monolignol production are well established [99,103], the mechanisms by which these compounds are deposited from the cytosol into the secondary cell wall remain unclear, and there is a continued debate regarding whether they are transported via passive diffusion or active transport [104].

The monolignol-specific pathway is highly adaptable, exhibiting many interspecific differences in genes with coordinated regulation (Figure 4). This variation plays a role in the intricate structure of the lignin polymer, which can vary greatly between plant species and even among different cell types. In grasses, lignin primarily consists of S- and G-units, with the inclusion of H-units and substantial quantities of ferulic acid (FA) and p-coumaric acid (pCA) [105,106]. These acids are essential for reinforcing the lignocellulosic matrix by facilitating cross-linking, thereby strengthening the cell wall structure. They achieve this by forming covalent bonds or ether linkages with polysaccharides and lignin components [107]. Additionally, tricin, a compound belonging to the flavonoid group, has been recently identified as an initiator of lignin chains within the polymer [108,109]. Tricin is predominantly found in grasses, with smaller amounts found in certain other monocots and trace amounts in alfalfa [108]. The lignin composition is particularly significant for its recalcitrance during bioconversion, especially following thermochemical pretreatment and subsequent enzymatic or acid/alkaline hydrolysis. The structure of lignin, characterized by recalcitrant C-C and C-O-C (ether) bonds, contributes to its resistance to degradation. The coupling of monolignols differs: H- and G-units can form β–5 linkages (through monomer–monomer and monomer–oligomer reactions) and 5–5 C-C linkages (through oligomer–oligomer reactions), while S-units are primarily connected through β–O–4 linkages, which are more amenable to degradation [110]. The S/G ratio, which compares the abundance of S-units to G-units, is often used as an indicator of cell wall digestibility. A high S/G ratio (above 1.0) indicates a greater prevalence of S-units relative to G-units, while a low ratio (below 1.0) suggests the opposite. It is generally believed that a higher S/G ratio enhances digestibility, potentially due to an increase in the abundance of labile β–O–4 bonds, which promote enzymatic degradation. However, a higher concentration of S-units can also result in more linear structure, resulting in uncondensed lignin that interacts more extensively with cellulose fibers, which may reduce enzymatic digestibility. Therefore, the S/G ratio provides only a partial explanation for biomass recalcitrance.

Moreover, the linkage of pCA to S-units through ether bonds may inhibit fermentation processes because of its toxic effects on yeast [111]. Likewise, alterations in FA compounds through the monolignol ferulate transferase (FMT) gene can impact recalcitrance by incorporating ester bonds that are more readily cleaved [112,113]. Therefore, altering lignin composition and content to improve saccharification can be achieved by expressing genes in the monolignol-specific pathway without affecting overall biomass. Consequently, lignin has been a focus of genetic modification efforts over the last few decades and continues to be a subject of significant interest today.

Figure 4. A monolignol biosynthetic pathway in grasses. The monolignols, including p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, are synthesized in the cytosol and subsequently transported to the secondary cell wall. Within the cell wall, they undergo oxidative reactions mediated by cell wall-bound peroxidase (PRX) and laccase (LAC), followed by radical coupling to form the lignin polymer. Mutant lines are indicated in indigo italics, including brown midrib maize (bm), brown midrib sorghum (bmr), orange lemma barley (rob), and gold hull and internode rice (gh). Indigo lines denote mutations that inhibit enzymatic activity within this pathway. The abbreviations for enzymes involved are as follows: phenylalanine ammonia-lyase (PAL), tyrosine ammonia-lyase (TAL), cinnamate 4-hydroxylase (C4H), 4-coumarate coenzyme A ligase (4CL), p-hydroxycinnamoyl-CoA/shikimate hydroxycinnamoyl transferase (HCT), p-coumarate 3-hydroxylase (C3′H), caffeoyl shikimate esterase (CSE), caffeoyl-CoA O-methyltransferase (CCoAOMT), cinnamoyl CoA reductase (CCR), ferulate 5-hydroxylase (F5H), caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT), and cinnamyl alcohol dehydrogenase (CAD). The illustration is adapted from reference [114].

Figure 4. A monolignol biosynthetic pathway in grasses. The monolignols, including p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, are synthesized in the cytosol and subsequently transported to the secondary cell wall. Within the cell wall, they undergo oxidative reactions mediated by cell wall-bound peroxidase (PRX) and laccase (LAC), followed by radical coupling to form the lignin polymer. Mutant lines are indicated in indigo italics, including brown midrib maize (bm), brown midrib sorghum (bmr), orange lemma barley (rob), and gold hull and internode rice (gh). Indigo lines denote mutations that inhibit enzymatic activity within this pathway. The abbreviations for enzymes involved are as follows: phenylalanine ammonia-lyase (PAL), tyrosine ammonia-lyase (TAL), cinnamate 4-hydroxylase (C4H), 4-coumarate coenzyme A ligase (4CL), p-hydroxycinnamoyl-CoA/shikimate hydroxycinnamoyl transferase (HCT), p-coumarate 3-hydroxylase (C3′H), caffeoyl shikimate esterase (CSE), caffeoyl-CoA O-methyltransferase (CCoAOMT), cinnamoyl CoA reductase (CCR), ferulate 5-hydroxylase (F5H), caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT), and cinnamyl alcohol dehydrogenase (CAD). The illustration is adapted from reference [114].

7.3. Effect of Elevated CO₂ on Lignin

Elevated CO₂ is expected to increase the levels of secondary or structural compounds in accordance with the ‘source-sink balance hypothesis’ [115]. Factors such as elevated CO₂ or nutrient stress can lead to a rise in carbon availability, leading to the buildup of carbon-derived secondary or structural compounds in source leaves. While various studies indicate a general trend of increased secondary compound levels, the specific chemical nature of these compounds varies among different plant species or genotypes [116]. For instance, lignin content has been shown to increase in tree leaves under elevated CO₂ conditions [117,118,119] although long-term Free Air CO₂ Enrichment (FACE) studies have reported no such effects [120]. The impact of elevated CO₂ on lignin content appears to be closely linked to nitrogen (N) supply, as demonstrated in Fagus sylvatica; under limited nutrient conditions, leaf lignin content increased with elevated CO₂, while in nutrient-rich conditions, the lignin content remained unchanged or even decreased [121].

Transcriptomic analyses of plants exposed to elevated CO₂ have revealed stimulation of the phenylpropanoid pathway. For example, transcripts related to this pathway have been found to accumulate in birch trees grown for six years under elevated CO₂ levels during August [122]. The authors of this study proposed that this stimulation might lead to the production of phenolic compounds rather than lignin [122]. In Arabidopsis, elevated CO₂ also resulted in increased expression of genes associated with the phenylpropanoid pathway, including phenylalanine ammonia-lyase (PAL1) and laccases (LAC4), in addition to other genes related to the cell wall. Metabolic profiling revealed that the levels of most amino acids reduced under elevated CO₂, with the exception of histidine, tryptophan, and phenylalanine, suggesting a shift toward secondary metabolism [123]. Similarly, soybeans exposed to elevated CO₂ for 40 days also showed increased gene expression related to secondary metabolism, notably within the phenylpropanoid pathway, with upregulation of caffeic acid O-methyltransferase (COMT) and cinnamoyl-CoA reductase (CCR) in the leaves. The overall gene expression profile suggested a greater allocation of resources to secondary metabolism. However, the response can vary by genotype, as shown by Cseke et al. (2009) [124], who analyzed the transcription profiles, physiology, and biochemistry of leaves from CO₂-responsive and unresponsive Populus tremuloides clones during long-term FACE experiments. While biological processes such as photosynthesis, stomatal conductance, and leaf area index were similar across clones, differences in radial growth were noted. Transcriptomic analysis revealed that the CO₂-responsive clone allocated carbon to pathways related to active defense, stress adaptation, carbohydrate production, and overall growth. In contrast, the CO₂-unresponsive clone channeled carbon into pathways associated with passive defense mechanisms, such as the phenylpropanoid pathway and cell wall fortification. To confirm these findings, additional studies evaluating phenolic and lignin levels in the leaves of inbred lines and mutants are needed.

Most investigations into the impact of elevated CO₂ on plants have focused on source organs, especially leaves. However, changes in leaf physiology and biochemistry can also influence sink organs, such as stems, particularly in trees species. A three-year comparison of gene expression in P. tremuloides leaves and stems under elevated CO₂ conditions with high nitrogen availability revealed a higher prevalence of CO₂-responsive genes in stem than in leaves [125]. This indicates a greater adaptation to elevated CO₂ levels in the stem. In leaves, genes involved in shikimate and flavanol pathways were upregulated, while in stem, genes linked to phenylpropanoid and lignin biosynthesis, such p-coumarate 3-hydroxylase (C3H), caffeic acid O-methyltransferase (COMT), and cinnamyl alcohol dehydrogenase (CAD), showed increased expression. Although it was proposed that elevated CO₂ could enhance lignification in stems, data on lignin content were not available.

7.4. Lignin’s Role in Carbon Sequestration Under Elevated CO₂

Lignin is an intricate biopolymer present in the cell walls of vascular plants, particularly in grasses, and plays a critical role in carbon sequestration. As atmospheric carbon dioxide (CO₂) levels rise, understanding how increased lignin biosynthesis in grasses under high CO₂ conditions can contribute to long-term carbon storage becomes increasingly important [122]. Research indicates that elevated CO₂ can enhance lignin synthesis in grasses, which, in turn, can improve carbon sequestration capabilities [126]. Increased levels of CO₂ promote photosynthesis, leading to greater biomass production. This boost in plant growth often correlates with elevated lignin content, as plants allocate more carbon to structural components like lignin to strengthen their cell walls [23]. This structural enhancement not only supports plant growth but also facilitates carbon storage in plant tissues. A study demonstrated that grasses grown in elevated CO₂ conditions exhibited a significant increase in lignin content, which contributed to greater soil organic carbon accumulation [127]. The lignin-derived carbon is more resistant to decomposition compared to other organic carbon forms, leading to enhanced carbon storage in soils [128]. This indicates that increased lignin biosynthesis under high CO₂ conditions can potentially sequester carbon for longer periods. Moreover, the environmental conditions influenced by climate change, marked by elevated temperatures and altered rainfall patterns, may further enhance lignin production. Grasses often respond to abiotic stresses with increased lignin biosynthesis, which helps them cope with drought and other stressors [129]. This adaptive mechanism not only contributes to plant resilience but also has implications for carbon dynamics in ecosystems. However, the effectiveness of lignin as a carbon sink depends on several factors, including soil type, microbial activity, and the overall health of the ecosystem. While lignin enhances carbon sequestration, the balance between lignin production and decomposition processes must be understood to assess its long-term impact on carbon storage [130].

7.5. Impacts of Lignin Modifications on Biofuel Production

Lignin, a multifaceted phenolic compound present in plant cell walls, is a significant component of biomass that affects the efficiency of biofuel production. As atmospheric carbon dioxide (CO₂) levels increase, understanding how elevated CO₂-induced changes in lignin composition impact its suitability for biofuel production is crucial for optimizing renewable energy resources [131]. Research indicates that elevated CO₂ can alter the biosynthesis and composition of lignin in plants, particularly in lingo-cellulosic feedstocks like switch grass and maize (Zea mays). Under high CO₂ conditions, plants often exhibit enhanced lignin deposition, which can affect the structure and properties of lingo-cellulosic biomass [132]. While increased lignin can enhance the structural integrity of plants, it can also complicate the deconstruction process necessary for biofuel production. A study demonstrated that changes in lignin composition—specifically, alterations in the ratio of guaiacyl to syringyl lignin units can significantly influence the digestibility of biomass [133]. Higher ratios of syringyl lignin are generally associated with improved enzymatic hydrolysis, leading to higher yields of fermentable sugars, which are crucial for biofuel production [134]. Conversely, increased guaiacyl lignin content under elevated CO₂ can hinder sugar release, thus reducing the efficiency of biofuel conversion processes [135]. Moreover, elevated CO₂ can enhance the regulation of genes associated with lignin biosynthesis, leading to increased lignin content and potential alterations in the chemical structure of lignin itself [100]. These modifications can have downstream effects on the efficiency of pretreatment processes used to break down lignin and cellulose in biomass [136]. For instance, changes in lignin structure may require different pretreatment strategies to achieve optimal biofuel yields [137]. Understanding these impacts is essential for developing strategies to optimize biofuel production from biomass under changing environmental conditions. Research is ongoing to identify specific lignin modification pathways that can be targeted to enhance the suitability of biomass for biofuel applications, such as through genetic engineering or breeding programs [138]. There is a limitation in this study in terms of understanding the integrated effects of elevated CO₂ on folate biofortification and lignin synthesis in maize, particularly under varying environmental stress conditions. Additionally, the impact of elevated CO₂ on lignin composition and its potential implications for carbon sequestration and biofuel production remain underexplored.

8. Future Directions and Prospects

This study highlights the effect of elevated CO₂ on maize growth, nutrition, and biochemistry, particularly its influence on folate biosynthesis and lignin formation. Future research should focus on identifying maize genotypes that are more resilient and productive under high CO₂ conditions, as well as exploring interactions with other environmental stressors like drought and nutrient deficiencies. Future efforts should focus on improving strategies to increase folate levels in maize to address nutritional challenges and adjusting lignin composition to enhance plant stress tolerance while also making it more suitable for industrial uses like biofuel production. Additionally, long-term field studies and modern tools like high-throughput phenotyping and molecular techniques can deepen our understanding and support sustainable cultivation in charging climates.

9. Conclusions

In summary, knowing the effects of elevated atmospheric CO₂ on maize (Zea mays) is vital for ensuring future food security amid climate change. While elevated CO₂ enhances photosynthesis, biomass production, and water use efficiency, it also reduces the nutritional quality of maize, particularly its folate and essential nutrient content. Developing resilient maize varieties through biofortification, traditional breeding, and genetic engineering is essential to mitigating these challenges and improving nutritional value under environmental stresses. Additionally, lignin plays a dual role in maize, offering structural support and carbon sequestration benefits while posing challenges for biofuel production due to its recalcitrance. Future research should focus on optimizing lignin composition and enhancing maize resilience to maintain productivity and sustainability in a changing climate. These integrated strategies are critical for addressing global food security and nutritional deficiencies associated with maize-based diets.