Carbon capture and storage (CCS) technology effectively reduces carbon dioxide (CO₂) emissions from primary pollution sources and is one of the key technologies to achieve global net-zero emission goals (IPCC, 2023; Chen et al., 2024). CCS involves capturing, transporting, and storing CO₂ in geological formations (Bukar and Asif, 2024). However, CO₂ may leak through wells or faults, as stable mineralization rarely occurs (Wu et al., 2024; Xiao et al., 2024), resulting in a persistent risk of leakage (Liu et al., 2022). Thus, assessing leakage risks is essential before large-scale CCS deployment (Zhang et al., 2022). This study focused on the impact of high soil CO₂ concentrations from CCS leakage on plant growth. CO₂ leakage can displace soil oxygen, inhibiting root respiration and reducing biomass (Maček et al., 2005). Natural leaks at Mammoth Mountain caused tree mortality (Farrar et al., 1995), and Mediterranean pastures near vents showed no plant growth within 6 meters, although recovery occurred farther away (Beaubien et al., 2008). As sedimentary basins suitable for CCS frequently overlap farmland, the potential impact of elevated soil CO₂ concentrations on farmland has garnered considerable attention. Projects such as ASGARD (Artificial Soil Gassing and Response Detection), Ginninderra, and ZERT (Zero Emission Research and Technology) simulated leaks to study the responses of crops. ASGARD found that CO₂ injection at a depth of 60 cm inhibited field bean growth and increased mortality due to hypoxia (Al-Traboulsi et al., 2012). Similarly, Zhang et al. (2024) observed reduced chlorophyll levels in wheat under CO₂ leakage conditions, which correlated with the leakage rate. Lake and Lomax (2018) reported that impurities like SO₂ and H₂S in leaks had no significant additional impact compared to pure CO₂. Crops such as maize, clover, and alfalfa show reduced biomass beyond certain CO₂ thresholds (Zhang et al., 2018). However, effects on quality vary: alfalfa showed declines in crude protein and amino acids (Zhang et al., 2022), whereas sunflowers increased in crude protein and linoleic acid under leakage (Yu et al., 2020). Further research is required to assess the effects of CCS CO₂ leakage on crop quality and explore the underlying mechanisms. Plants respond to external stresses through intricate physiological, molecular, and biochemical mechanisms (Bashir et al., 2021), mitigating adverse effects on growth, development, and reproduction (Hasanuzzaman et al., 2013; Ma et al., 2020a). These stress conditions often lead to the accumulation of free radicals and reactive oxygen species (ROS) in plant cells (You and Chan, 2015). To counteract this, synthesizing antioxidants such as vitamins C and E plays a crucial role in scavenging excess ROS, thereby protecting cells from oxidative damage (MacGregor, 2024). Moreover, flavonoids, a class of secondary metabolites, exhibit significant antioxidant properties, further aiding plants in counteracting ROS-induced damage (Macedo et al., 2024). In addition to these biochemical defenses, the cell wall serves a critical function under stress by enhancing its mechanical strength to maintain cellular rigidity and stability (Swaminathan et al., 2022; Zhao et al., 2024). Furthermore, environmental stress can alter the oxalate levels in plants, which may, in turn, be linked to changes in carbon metabolism (Zhou et al., 2019) and antioxidant mechanisms (Shi et al., 2024). Therefore, investigating the effects of soil CO₂ stress on the contents of vitamins, flavonoids, cell walls, and oxalate provides valuable insights into the mechanisms underlying plant responses to CO₂ leakage from CCS.

Spinach (Spinacia oleracea L.), known for its high sensitivity to environmental stress, is an ideal vegetable for studying stress tolerance and adaptation mechanisms (Badar et al., 2024). Rich in vitamins, carotenoids, folic acid, and minerals, spinach is an important leafy vegetable with high nutritional value (Watanabe and Ayugase, 2015) and is widely consumed globally (Xu et al., 2017). However, spinach is prone to accumulating nitrates and oxalates during growth, and excessive consumption of spinach containing these substances may pose potential health risks (Ghanati et al., 2024; Khaksar et al., 2024). The effects of elevated atmospheric CO₂ on plant growth have been extensively studied (Genthon et al., 1987; Liu et al., 2018). In studies on spinach under high CO₂ conditions, researchers have primarily focused on FACE (Free-Air CO₂ Enrichment) experiments, investigating the impact of elevated atmospheric CO₂ (eCO₂) on spinach morphology, biomass, and quality. Ahmad et al. (2024) found that under eCO₂ conditions, spinach root length and stem length increased significantly by 36.76% and 20.42%, respectively, and the fresh weight, dry weight, leaf number, and leaf area all showed growth. Increased atmospheric CO₂ promoted photosynthetic activity, enhancing crop productivity (Ma et al., 2021). In a meta-analysis of leafy vegetables, Dong et al. (2020) also found that spinach yield increased by 35% under eCO₂ conditions. Giri et al. (2016) evaluated the effects of eCO₂ concentrations on spinach growth and nutritional quality, revealing a significant decrease in leaf stomatal conductance and a reduction in the concentrations of key nutrients such as protein, potassium, and phosphorus, while total phenols and antioxidant capacity remained unchanged, indicating a negative impact of eCO₂ on spinach’s nutritional quality. However, no experimental studies have yet assessed the effects of CCS leakage on spinach’s physiological morphology, biomass, and nutritional quality. However, a study on lettuce, which belongs to the same leafy vegetable category as spinach, showed that under elevated soil CO₂ conditions, both morphological indicators and yield decreased significantly; although unit nutrient content increased, the total nutrient content still declined due to reduced biomass, indicating potential adverse effects of CCS leakage on leafy vegetable crops (Huang et al., 2025).

Plant adaptation mechanisms to environmental stress include avoidance and tolerance, which may lead to changes in metabolites and nutritional quality under stress conditions (Badar et al., 2024). The response of spinach to soil CO₂ leakage stress and its effects on nutritional quality still require further experimental validation. This study hypothesizes that CO₂ leakage will significantly affect spinach leaf morphology, biomass, and nutritional quality during the CCS process, influencing agricultural production. The main objective of this research is to understand spinach’s response to CCS leakage in terms of (1) morphology and yield and (2) nutritional quality, mainly changes in vitamin C, vitamin E, cellulose, and oxalate content. Although the pot-based approach limits extrapolation to field conditions, this well-controlled study provides the first documented evidence of CO₂ leakage effects on spinach biomass and key nutritional components.

Our study investigated the effects of elevated soil CO₂ concentration on spinach’s growth and nutritional quality. The results indicated that soil CO₂ leakage significantly inhibited spinach growth, particularly in plant height, leaf morphology, and aboveground and belowground biomass. However, it also promoted the accumulation of specific nutrients, such as a significant increase in the content of vitamins C and E, and cellulose. These findings revealed that the effects of elevated soil CO₂ on spinach were complex, involving both suppressive impacts and potential activation of plant stress-defense mechanisms.

Nutrient analysis revealed that soil CO₂ leakage significantly increased the contents of vitamin C and E in spinach by 185.47% and 131.45%, respectively (Table 2). This suggests that spinach may activate its stress-defense mechanisms by enhancing the synthesis of antioxidant vitamins under high CO₂ stress. Both vitamin C and vitamin E play important roles in plant disease resistance, with vitamin C serving as a key inducer of disease resistance. The increase in vitamin C content under CO₂ leakage conditions aligns with previous studies on plant resistance mechanisms (Boubakri et al., 2016), further confirming the critical role of vitamins in plant responses to stress. Notably, our findings on vitamin C content differ from those of FACE experiments, in which the vitamin C content of vegetables such as carrots decreased under elevated atmospheric CO₂ (Azam et al., 2013). This discrepancy suggests that soil and atmospheric CO₂ affected vitamin C synthesis through different mechanisms, potentially due to variations in biomass growth and nutrient absorption patterns.

Additionally, the cellulose content in spinach increased significantly by 315.03% (Table 2). Cellulose is a major structural component of the cell wall, and changes in its content often reflect compensatory or integrity responses that impact vegetable quality and stress resistance (Li et al., 2017). The increase in cellulose under CO₂ leakage likely helped reinforce the cell wall, enhancing the plant’s ability to resist stress. This supported the idea that spinach underwent metabolic adjustments to enhance its tolerance to stress under CO₂ stress conditions. As Taiz and Zeiger (2015) suggested, plants often adapt to adverse environments by adjusting their cell wall composition, which aligns with the increase in cellulose content observed in this study.

On the other hand, the oxalate content in spinach decreased significantly by 43.08% (Table 2). As an antinutrient, oxalate accumulates in spinach and can interfere with human calcium and zinc absorption (Petropoulos et al., 2016). Environmental stressors influence the accumulation of oxalate in plants (Ding et al., 2007; Jiang et al., 2008). Yu et al. (2012) demonstrated that elevated atmospheric CO₂ levels under heat stress resulted in a 68% reduction in oxalate abundance in cool-season grasses. Similarly, Zhuang et al. (2019) reported that salt stress combined with increased CO₂ altered metabolite accumulation patterns in the leaves and roots of Kentucky bluegrass, leading to a decrease in oxalate content.

Spinach leaf morphology showed a high sensitivity to elevated soil CO₂ levels (Figures 2A, B), significantly reducing plant height, leaf size, number, and leaf area compared with CK. Notably, plant height rapidly responded, with a significant change observed by the fifth day after CO₂ leakage. Moreover, the maximum leaf length and width decreased by 67.97% and 76.76%, respectively, while the leaf area decreased by 92.76% (Table 2). These results were opposite to those of eCO₂ experiments, where spinach exposed to high atmospheric CO₂ for 80 days showed a substantial increase in leaf area (Jain et al., 2007). However, the findings aligned with CCS-simulated soil CO₂ leakage studies, demonstrating a significant inhibitory effect on plant morphology and biomass (Lakkaraju et al., 2010; Ma et al., 2020b). Our results suggested that elevated soil CO₂ concentrations had a potent suppressive effect on spinach growth and caused a continuous deterioration in leaf number and health. These morphological changes may be attributed to reduced oxygen availability in high CO₂ soil environments (Zhang et al., 2016), which affects root respiration and the uptake of water and nutrients.

SPAD showed a significant decline in spinach under CO₂ leakage conditions (Figure 3E). While no noticeable changes were observed early on, SPAD significantly decreased by 18.38% at harvest compared with CK (Table 2). This suggested that the inhibitory effect of high CO₂ on chlorophyll content in spinach intensifies over time, especially under prolonged exposure. This fluctuating decline may be due to disruptions in the chlorophyll synthesis process or accelerated chlorophyll degradation under high CO₂ conditions (Kumari et al., 2013; Muthusamy et al., 2019). Although the reduced SPAD and chlorosis phenomena suggest a possible decline in photosynthetic performance, no direct measurements of gas exchange, chlorophyll fluorescence, or root respiration were conducted to confirm physiological impairment (Zhang et al., 2020). Therefore, this interpretation remains tentative and should be verified by future studies combining physiological and biochemical assays. Further analysis of chlorophyll a and b content revealed a more significant decline in chlorophyll b (35.48%, Table 2), which may lead to a weakened efficiency of the auxiliary light-capturing function of chlorophyll b (Guo et al., 2006). Previous studies on the impact of CCS on leaf photosynthetic capacity support this hypothesis, with findings indicating that high soil CO₂ concentrations suppress photosynthesis in pasture grass, alfalfa, and soybean (Xue et al., 2021), as evidenced by reductions in photosynthetic parameters such as Pn, Tr, and Gs (Zhang et al., 2015). We expected elevated soil CO₂ concentrations to weaken spinach photosynthesis, which hindered biomass accumulation.

The results also indicated that soil CO₂ leakage had a significant negative impact on the aboveground and belowground biomass of spinach (Figures 4C, D). Under CO₂ leakage conditions, the fresh and dry weights of aboveground biomass decreased by 93.46% and 84.46%, respectively (Table 2), indicating severe growth inhibition. Furthermore, the aboveground water content decreased by 17.38% (Table 2). The high CO₂ concentration in the soil impaired the growth vigor of the aboveground portion, resulting in a significant decrease in biomass and compromised water regulation of spinach. Below the ground, the root biomass also decreased significantly, with the fresh root weight of G1500 reduced by 95.83% compared with CK (Table 2). This result was consistent with findings in alfalfa, where soil CO₂ leakage at a rate of 1500 g·m^-2·d^-1 led to reductions of 43.55% and 66.06% in aboveground and belowground fresh weight, respectively, and a decline in the root-shoot ratio from 1.04 to 0.63 (Zhang et al., 2022). In our study, the reduction in aboveground fresh weight was less than that of the belowground biomass, indicating that CO₂ leakage had a more pronounced inhibitory effect on the root system.

The experiment also observed significant degradation of spinach root morphology, with the primary roots becoming shorter and thinner and the number, length, and thickness of fine roots decreasing dramatically (Figure 2C). The root weight of G1500 decreased significantly by 95.83% compared with CK (Table 2). High CO₂ concentrations severely inhibited root development, restricting the roots’ ability to absorb water and nutrients. This finding is consistent with previous studies, such as Han et al. (2020), who reported reductions of 44.73%, 34.14%, and 19.16% in root length, surface area, and volume of maize, respectively, under CO₂ leakage conditions of 2000 g·m^-2·d^-1. These findings indicate that elevated soil CO₂ concentrations cause severe physiological damage to spinach roots, hindering their ability to obtain water and nutrients.

Despite the improvements in spinach’s nutritional quality, the significant reduction in aboveground fresh weight (93.46%, Table 2) indicated a substantial decline in overall total nutritional content per plant. The reduction in edible yield adversely affected the nutritional and economic value of spinach. The total content of vitamin C and vitamin E decreased drastically by 79.71% and 84.88%, respectively; similarly, a pronounced reduction was observed for cellulose (72.96%) (Table 3). These findings highlighted the dual impact of CO₂ leakage: while it enriched certain nutrients, it significantly impaired spinach growth and marketability. This suggests two non-mutually exclusive mechanistic explanations: a concentration effect due to biomass suppression, and/or active metabolic upregulation under stress conditions. Under atmospheric CO₂, plants may experience a “dilution effect” (Taub and Wang, 2008), whereby nutrient content per unit biomass decreases as plant size increases (Li et al., 2023; Ahmad et al., 2024). In contrast, soil CO₂ may inhibit spinach growth, thereby passively increasing nutrient concentrations through a “concentration effect”. However, the potential for stress-induced metabolic activation cannot be ruled out. Previous studies have demonstrated that elevated CO₂ can active antioxidant and signaling pathways (Yao et al., 2015), upregulate defense-related genes (Loewus, 1999; Wang et al., 2010), and alter carbon allocation and enzyme regulation (Demmig-Adams and Adams, 1996). Moreover, enhanced activity of NADPH- and glutathione-related pathways under CO₂ stress has been observed in other C₃ species (Flexas and Medrano, 2002), further supporting possible biochemical adjustments in spinach.

However, this study lacks an in-depth investigation of the dominant mechanisms underlying nutritional changes in spinach under elevated soil CO₂. While alterations in vitamin C, vitamin E, oxalate, and cellulose contents were observed, the precise metabolic pathways involved remain unverified. Critical evidence regarding key enzymatic activities, intermediate metabolites, and potential pathway regulations is still lacking. Without further analysis of biosynthetic pathways (such as the L-galactose pathway for vitamin C and photorespiratory pathway for oxalate), we cannot determine whether the observed changes result from active metabolic regulation or from the passive concentration effect. In addition, similar experiments should be systematically conducted on other crop species to compare metabolic responses and assess whether the patterns observed in spinach are species-specific or represent a broader physiological response.

This study examined the growth and quality variations of spinach under high soil CO₂ concentrations resulting from carbon capture and storage (CCS) leakage. Regarding nutritional quality, soil CO₂ leakage promoted an increase in vitamins C and E as well as cellulose content in spinach leaves, altering the plant’s metabolic pathways. However, the reduction in biomass resulted in a corresponding decrease in the total amount of key nutrients per plant. Additionally, the significant reduction in oxalate content presents a potential avenue for research aimed at mitigating oxalate accumulation in spinach. Elevated soil CO₂ significantly reduced spinach leaf area, relative chlorophyll content (SPAD), and aboveground fresh weight, severely impairing plant growth and photosynthetic efficiency, thereby affecting overall physiological functions. The reduction in fresh weight of the belowground biomass was more pronounced than that of the aboveground biomass, indicating that the root system was susceptible to high CO₂ stress.

Our study quantified spinach’s response to soil CO₂ leakage, showing that while CO₂ leakage may enhance specific nutritional components, the damage to biomass reduced overall yield and the total amount of key quality components. These findings highlighted the potential threat posed by changes in soil CO₂ levels to spinach growth and nutritional quality. Our study offers novel insights into how spinach responds to CO₂ leakage, which can assist CCS project stakeholders in more effectively evaluating the safety of CCS technologies and developing appropriate risk management strategies. Future research could further explore the effects of soil CO₂ leakage on the growth and quality of other crops to enhance understanding of its broader implications.