Surface ozone (O₃) is predominantly produced through photochemical reactions involving primary pollutants, such as nitrogen oxides (NO_X) and volatile organic compounds (Monks et al., 2015; Li K, et al., 2018). The increasing levels of NO_X, driven by industrial development and the rapid expansion of vehicle ownership, have led to higher concentrations of O₃ in the atmosphere (Jiang et al., 2012; Feng et al., 2015). Environmental monitoring data reveal that between 2013 and 2019, the daily maximum 8-hour average O₃ concentration in the surface layers of many Chinese cities consistently exceeded 50 ppb, with peak O₃ levels increasing at an annual rate of 3 ppb (Lu et al., 2020). In some areas, such as Jiangsu province, the maximum hourly average concentration has exceeded 100 ppb (Hu et al., 2023; Shao et al., 2023). These increased concentrations of surface O₃ present significant threats to human health (Sicard et al., 2021; Marco et al., 2022), agricultural production (Feng et al., 2022; Ramya et al., 2023), and biodiversity (Ainsworth, 2017).

The herbaceous peony (Paeonia lactiflora) is a well-known traditional flower in China, valued for its ornamental, medicinal, and health-promoting properties. It has emerged as a commercially significant cut flower in both domestic and international markets due to its substantial economic benefits (Ning et al., 2015; Qi et al., 2020). China has become one of the world’s major producers of cut peonies (Yang et al., 2020), with production concentrated in the Beijing, Sichuan, Jiangsu, and Shandong provinces (Du et al., 2018). Extensive research has been conducted on the effects of various abiotic stressors on peony growth, including heavy metals, saline-alkali conditions, drought, waterlogging, temperature extremes, and insufficient light. Lu et al. (2022) reviewed these stressors and their impacts on peony morphology, internal physiological processes, and secondary metabolites. These findings provided valuable insights into the mechanisms by which peonies adapt to stress and improve their tolerance. However, unlike other environmental stressors, O₃ pollution has seasonal variations that make certain crops, including peony, particularly vulnerable during their critical growth phases. Moreover, China’s major peony-producing regions also experience frequent high O₃ concentrations (Feng et al., 2022), particularly during the crucial growth and flowering period in May, which coincides with regional O₃ pollution peaks (Wang et al., 2001; Li et al., 2007; Xu et al., 2008a; Ding et al., 2013; Wang et al., 2017). Despite numerous studies highlighting the harmful effects of O₃ on crops (Feng and Kobayashi, 2009; Peng et al., 2019; Shao et al., 2020; Shang et al., 2024) and woody species (Li et al., 2016; Shang et al., 2018; Dai et al., 2017), minimal research has been conducted on the impacts of O₃ on horticultural plants (Mills et al., 2007; Zhang et al., 2015), particularly on peony, to date.

Furthermore, leaves serve as the primary interface for material and energy exchange between the surface atmosphere and the terrestrial biosphere, and they are also the main organs responsible for sensing O₃ stress responses (Krupa and Manning, 1988). Ozone predominantly enters plants through the stomata. Prolonged exposure to O₃ induces visible leaf injury symptoms in sensitive plants species (Mills et al., 2011; Feng et al., 2014), activates various defense mechanisms, and subsequently leads to progressive chlorophyll degradation, accompanied by a reduction in photosynthetic efficiency (Wittig et al., 2009). These physiological disruptions interfere with carbon assimilation and the distribution of essential mineral nutrients (Shang et al., 2018), ultimately causing growth retardation and premature aging (Matyssek and Sandermann, 2003). Therefore, the plant’s resilience to environmental stress, its ecological fitness, its capacity for carbon sequestration are all compromised (Feng et al., 2022). Significant variability in O₃ tolerance has been observed both among crops (Mills et al., 2007; Paoletti et al., 2009) and within species (Biswas et al., 2008; Krupa et al., 1998; Maggs and Ashmore, 1998; Shi et al., 2009).

In crops, susceptibility to O₃ is typically measured by reductions in yield and biomass. Whereas, the evaluation of O₃ sensitivity in woody and ornamental plants often involves monitoring leaf traits such as specific leaf weight, photosynthetic performance, malondialdehyde (MDA) levels, and antioxidant enzyme activity. Furthermore, high-concentration, short-term O₃ exposure often induces oxidative damage in the form of leaf necrosis, accompanied by increased antioxidant metabolism, as a result of programmed cell death. However, the plant’s antioxidant response to chronic exposure to lower O₃ concentrations is less well understood and remains a subject of debate. Some studies suggest that chronic O₃ stress leads to increased antioxidant activity, as observed in soybeans (Gillespie et al., 2011), while others have observed a reduction in antioxidant pools and key enzymes in wheat leaf tissues under elevated O₃ concentrations (Feng et al., 2010; Wang et al., 2014a). No studies have examined how O₃ affects peony’s antioxidant system or photosynthetic efficiency. Therefore, a systematic study on the effects of O₃ elevation on peony leaf performance is essential to determine its sensitivity to O₃ stress. In this study, O₃ fumigation experiments were conducted under two distinct concentrations, non-filtered ambient O₃ concentration (NF) and NF supplemented with an additional 60 ppb of O₃ (NF60), to evaluate the physiological responses of two widely cultivated cultivars of Paeonia lactiflora. The primary objective was to elucidate how O₃ exposure influences leaf morphological traits, photosynthetic physiology, and oxidative stress responses in the two peony cultivars and to provide an initial evaluation of cultivar sensitivity to O₃ exposure, offering foundational insights for optimizing peony cultivation practices in O₃-polluted regions.

The Herbaceous peony (Paeonia lactiflora Pall.), renowned for its majestic beauty, is often compared to its tree (Paeonia suffruticosa Andr.) and is regarded as the “monarch of herbaceous flowers”. Its large, vibrant, and fragrant flowers have made it a popular choice for urban landscaping, garden cultivation, potted displays, and the premium cut flower market (Holloway and Buchholz, 2013). However, during the introduction and cultivation of the peony, the plant faces numerous abiotic stresses, including fluctuations in temperature, light exposure, water availability, saline-alkali conditions, and heavy metal contamination (Haak et al., 2017). While moderate stress can stimulate growth, excessive stress inhibits development. It causes morphological abnormalities in roots, stems, and leaves, altering internal organic compounds, inorganic ions, and enzymatic activities, which may ultimately lead to plant mortality (Zhang et al., 2018). Among the various abiotic stresses, elevated ground-level O₃ concentrations have emerged as a key stress factor for plant health (Ashmore, 2005). Despite this, research on the impact of O₃ stress in peony remains limited.

The leaf photosynthesis is an essential physiological process that sustains plant life. There are few reports on the effects of O₃ stress on ornamental plants. Qin et al. (2020) conducted a study on O₃ stress in Cleome spinosa, but unfortunately, the study did not measure the response of leaf photosynthetic parameters. The impact of O₃ stress on leaf photosynthesis has been the subject of substantial research, revealing that O₃ exposure significantly reduces photosynthetic rates in various crops and tree species. Studies on wheat (Feng and Kobayashi, 2009; Xu et al., 2025), rice (Ainsworth, 2008; Shang et al., 2024), maize (Yendrek et al., 2017), and trees (Li et al., 2016; Shang et al., 2019) demonstrate that O₃ impairs photosynthetic capacity. The present study showed that NF60 treatment resulted in a 23.0% significant decrease in the photosynthetic rate in peony leaves compared to NF ( Table 1 ), with similar reductions in both cultivars, indicating significant O₃-induced damage. Moreover, the reduction in photosynthetic rate in peony leaves under NF60 treatment was lower compared to the reductions observed in rice (Ainsworth, 2008; Shao et al., 2023), wheat (Feng et al., 2016), maize (Yendrek et al., 2017), and soybeans (Morgan et al., 2003). The observed decrease in photosynthesis can be attributed to both stomatal and non-stomatal limitations (Dai et al., 2017; Shang et al., 2024). Furthermore, under NF60, stomatal conductance was significantly reduced, with DFG showing a greater reduction than HHBT. However, O₃ exposure also reduced intercellular CO₂ concentration by 12.7% on average in DFG, but no significant effect or a slight increase was observed in HHBT, as indicated by a significant O₃ × cultivar interaction ( Table 1 ). This suggests that stomatal limitations play a key role in the decline of photosynthesis in DFG, while both stomatal and non-stomatal factors contribute to the decrease in HHBT. Moreover, significant O₃-induced decreases in leaf ETR were identified, which may represent another non-stomatal mechanism of photosynthetic inhibition (Feng et al., 2016; Schcidegger and Schroeter, 1995).

Chlorophyll, a crucial component in leaf photosynthesis, plays a vital role in absorbing light, transferring energy, and converting it into chemical forms. Fluctuations in chlorophyll levels significantly influence nitrogen fixation and are indicative of leaf senescence (Singh et al., 2015). Consistent with the observed photosynthetic rate patterns, NF60 treatment led to reductions in chlorophyll a+b and its components in peony leaves, with chlorophyll a showing a more considerable decrease than chlorophyll b. While the high O₃ concentration affected DFG’s chlorophyll content to a lesser extent than HHBT, no significant interaction was found between cultivar and O₃ exposure. The changes in chlorophyll content under O₃ stress likely reflect an imbalance in the production and scavenging of reactive oxygen species (ROS). Specifically, O₃ exposure intensified lipid peroxidation in membranes, increasing MDA and H₂O₂ concentrations ( Table 4 ), and allowing excess ROS to infiltrate chloroplasts, where they contributed to the degradation of chlorophyll. This is primarily due to the fact that these excess ROS exacerbate the peroxidation of chloroplast membranes, impairing the synthesis of chlorophyll-protein complexes (Langebartels et al., 2002). This process accelerates chlorophyll depletion and leaf yellowing, as extensively documented in crops such as rice (He et al., 2024) and wheat (Feng et al., 2016).

Leaf morphology and structure, shaped by the evolutionary processes of natural selection, are closely associated with physiological functions and represent key factors in determining a plant’s ability to adapt to its environment (Liu and Liang, 2016). The LMA is recognized as a reflection of plant adaptations and acclimation to prevailing environmental conditions. Species with low LMA display greater responsiveness to environmental stimuli and higher stress sensitivity compared to high-LMA species, which demonstrate enhanced stress tolerance (Bussotti, 2008; Li et al., 2016). The findings of this study indicated that NF60 exposure reduced leaf LMA by 12-13% (P < 0.1) in both peony cultivars, without any significant interaction observed between O₃ exposure and cultivar, which aligns with similar reports in peach trees (Dai et al., 2017), maize (Peng et al., 2019), and rice (Fu et al., 2021). Stomatal traits, including density and size, are known to be highly responsive to environmental variations (Zheng and Shangguan, 2005). Previous research has reported differing responses of stomatal density to elevated O₃, including increases (Wen et al., 2014), decreases (Li P. et al., 2018), or no significant effects (Xu et al., 2008b), which may be attributed to differences in exposure duration, O₃ concentration, plant species, and the developmental stage of leaves (Li P, et al., 2018). Furthermore, no significant effects on stomatal density or size were found in peony leaves under O₃ exposure ( Table 3 ). This may be because the leaves were fully expanded before treatment, suggesting that the stomatal structure and number had already been established, thus minimizing the potential impact of the O₃ exposure. Therefore, further long-term studies are needed to determine the effects of increased O₃ on stomatal development throughout the lifespan of peony leaves.

The leaf antioxidant system plays a crucial role in defending against and repairing the oxidative damage caused by O₃ stress. However, when environmental stress exceeds a critical threshold, it disrupts the plant’s protective defense mechanisms (Xu et al., 2020). Several studies have shown that O₃-resistant species or cultivars typically display higher levels of antioxidant substances (Dai et al., 2017). In this study, although most leaf antioxidant parameters (such as AsA, TAC, total phenols, and the activities of enzymes like SOD, POD, and CAT) did not show statistically significant responses to O₃ stress ( Table 4 ), peony leaves treated with NF60 showed partial positive responses from their antioxidant system. This was accompanied by an increase in H₂O₂ and MDA content, with all antioxidant enzyme activities showing an upward trend. The results were largely consistent with the findings reported by Qin et al. (2020) regarding 80 ppb O₃ stress on Cleome spinose. Antioxidant indicators generally undergo dynamic changes, starting with an initial increase followed by a decrease as stress intensity increases (Wu et al., 2011). Therefore, measurements taken at a single time point may not adequately reflect the complete dynamic response. Future research should conduct multi-year experiments to elucidate the temporal variations of antioxidants in the cellular repair mechanisms of peony leaves.

Research on the effects of abiotic stress in peony has predominantly focused on factors such as temperature, light, water availability, salinity, and heavy metals. However, there have been no studies on the impact of atmospheric O₃ pollution on peony growth. This study used open-top chambers to evaluate the effects of O₃ stress on the leaf morphology and physiology of herbaceous peony. Compared with NF, the NF60 treatment led to a significant reduction in leaf photosynthetic capacity, as reflected by decreases in chlorophyll content and increases in MDA content, which contributed to accelerated leaf senescence in both peony cultivars tested. Under the same O₃ exposure conditions, the two peony cultivars showed relatively minor changes in leaf morphology and physiology compared to crops such as soybean, rice, maize, and wheat, classifying peony as a species moderately sensitive to O₃. Future research should focus on how elevated O₃ concentrations affect medicinal compounds and key quality traits in cut flowers, such as stem length, flower color, and size. This knowledge will improve the comprehension of the physiological adaptation of peony to O₃ stress, which could inform agronomic practices that optimize peony cultivation and commercialization and improve stress tolerance.