Over the past few decades, speedy industrial growth and unrestrained urbanization in developing nations have greatly increased the concentration of primary and secondary pollutants in the atmosphere (Dhevagi et al., 2021). The concentration of phytotoxic secondary pollutant tropospheric ozone (O₃) depends on the levels of certain primary air pollutants such as nitrogen oxides (NO_x), and volatile organic compounds (VOCs). In recent years, tropospheric ozone has shown a severe impact on plants because of its high oxidative potential (Duque et al., 2021). Background O₃ concentration in the troposphere has increased by 36% since pre-industrial times (Mukherjee and Hazra, 2022). Nearly one-quarter of the world is currently at risk of high O₃ and it is predicted that the levels could rise to 20% by 2050 (Meehl et al., 2018). Because of chemical interactions between O₃ precursors, industrial pollutants, and sunlight, surface ozone levels are increasing at a rate of 0.5 to 2.5 percent each year (Wang et al., 2022). Because it has a negative impact on people’s health, plants, and the ecosystem worldwide, the rising concentration of ground-level O₃ has become a global issue (Mills et al., 2013). Even if strict adherence to the air quality regulations of 2000 is maintained, the O₃-induced damage is projected to worsen, according to future emission scenario legislation taking emissions for the year 2030 (CLE-2030) (Van Dingenen et al., 2009). A number of modeling studies have depicted high tropospheric ozone concentration in the future thus making the study of the impact of elevated ozone on plants even more important.

The stomata are the primary entry point for ozone into plant leaves (Stella et al., 2013). Ozone quickly reacts with intracellular components and forms reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂), superoxide radicals (O^2-), and hydroxyl radicals (OH ^· ) inside the leaf (Zhang et al., 2017). These ROS enhance the antioxidant defense response downstream, which mitigates the fatal consequences of accumulating ROS. Contradictorily, when ROS generation surpasses the antioxidant efficiency, plants show ozone vulnerability in the form of foliar injury symptoms, alteration in metabolic processes, yield loss, and other ozone-related effects (Singh et al., 2014; Singh et al., 2018; Hayes et al., 2020). Excessive ROS levels within the cells brought on by O₃ damage the membranes (lipid peroxidation), oxidize proteins, degrade RNA and DNA, enhance the production of secondary metabolites, degrade chlorophyll, and ultimately result in apoptosis (Choudhury et al., 2017). However, a vast variety of antioxidants and ROS-scavenging enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), and ascorbate peroxidase (APX), produced by cells upon O₃ exposure to attenuate the impacts of ROS in the cells (Picchi et al., 2017). In order to maintain steady development in plants and to aid in the damage repair caused by various stressors, nutrient supplements have frequently been utilized (Sahoo and Tiwari, 2021). Nitrogen (N) is the fourth most common element in living things and is theoretically an important component that supports plant development (Sahoo and Tiwari, 2021). Nitrogen amendment not only maintains greater protein levels it can also be used for repair processes and facilitates the remobilization of nutrients to reproductive parts, sustaining better production (Zeng et al., 2017). Nitrogen applications tend to improve the photosynthetic potential of the plant which can be commonly coupled with stomatal conductance (Zhang et al., 2018). The addition of nitrogen may partly counteract the detrimental effects of O₃ on a few plants’ morphological characteristics (Yendrek et al., 2013). Plants can either strengthen their antioxidant defense against an O₃-induced ROS surge, fix more carbon to use for O₃-induced injury, or direct more biomass into reproductive organs to sustain greater yields in response to N amendments (Podda et al., 2019).

A significant portion of India’s protein needs is fulfilled by pulses, one of the country’s essential crops, which are grown on an area of 28.3 million hectares and produced approximately 25.7 million metric tonnes in the fiscal year 2020–21 (Statista, 2021). In Asia, where the mean ambient ozone concentration ranged between 35 and 75 ppb during the growing season of the crop, legumes had a substantial production loss of 10 to 66 percent (Emberson et al., 2009). Dolichos lablab L. is a versatile crop, several plant components may be consumed as food, animal feed, and green manure (Davari et al., 2018; Rana et al., 2021). It is a low-cost source of protein and micronutrients when compared to other legumes, highlighting the importance of its study in view of food security and nutrition in near future (Letting et al., 2021). These micronutrients, which play a significant role in the diets of resource-limited households in rural areas, include phosphorus, fiber, niacin, and thiamine (Letting et al., 2021). The most recent study shows how efficient lablab bean extracts are at preventing the spread of viral illnesses like influenza and SARS-CoV-2, which has been called a global pandemic (Liu et al., 2020). To the best of our knowledge, the sensitivity of Dolichos lablab L. varieties towards tropospheric ozone has not been defined. The present work is the first effort to characterize the sensitivity of two varieties of Dolichos lablab L. towards O₃ stress. In addition, it also exemplifies the efficiency of nitrogen amendments in the management of O₃ stress and the mechanistic approach adopted therein. Through our work, we hypothesize that the ameliorative effect of N amendments in O₃-stressed plants is mostly credited to the antioxidant response rather than the biophysical parameters like stomatal conductance.

Sicard et al. (2017) predicted a rise in O₃ concentration under all climate change scenarios and identified South Asia as one of the primary O₃ hotspot regions. Intense irradiance, elevated temperature, and low moisture are the ideal circumstances for O₃ generation in the Indo-Gangetic plains, where high episodes of O₃ are common occurrences (Sarkar and Agrawal, 2010). In monitoring studies conducted at the current experimental site between 2002 - 2012, the concentration of O₃ was clearly on the rise (Tiwari and Agrawal, 2018). The results of the present experiment evidently revealed the presence of high ozone episodes during the reproductive phase of both varieties of Dolichos lablab L. The increased O₃ levels during the reproductive phase can be reasoned by the high temperature in the months of January, February, and March as compared to November and December months. High O₃ concentrations have also been recorded in earlier experiments conducted at the current experimental location during the same time period (Singh et al., 2018; Yadav et al., 2019).

Out of the two varieties of Dolichos lablab L. exposed to ambient and elevated O₃ concentrations, Kashi Sheetal had higher endogenously generated H₂O₂ in its control leaves as compared to Kashi Harittima, as evident through the histochemical assay of H₂O₂ localization ( Figure 4 ). Related findings have reported that cultivars with higher levels of endogenous H₂O₂ are more tolerant than cultivars with lower levels of H₂O₂ (Caregnato et al., 2013; Yadav et al., 2019; Gupta and Tiwari, 2020). Caregnato et al. (2013) reported significantly higher H₂O₂ localization in the leaves of two varieties of Phaseolus vulgaris L. grown in EO₃ conditions in comparison to AO₃ conditions. The high H₂O₂ localization in the leaves is because of the excessive ROS generated due to severe oxidative stress which happens due to elevated O₃ fumigation. With an increased dose of nitrogen treatments, there was a significant degree of reduction in the amount of H₂O₂ localization in the leaves of both varieties ( Figure 4 ). It was reported that H₂O_{2 was} produced in the leaves of Cymopsis tetragonoloba L. Taub. (Cluster bean) was significantly decreased with increasing nitrogen amendment doses (Gupta and Tiwari, 2020). It is suggested that the surplus nitrogen available to the plant gets allocated to improve the antioxidative potential of the plants which results in improved scavenging of the H₂O₂ (Podda et al., 2019). It is noted that N2 treatment was found to be sufficient in the management of O₃ stress under both O₃ exposure conditions.

Malondialdehyde is an intermediate product that is exclusively produced during the process of membrane lipid peroxidation, so measuring the MDA content gives an approximation of the degree of lipid peroxidation. Biochemical analysis of lipid peroxidation showed higher MDA content in the EO₃ conditions in both varieties of Dolichos lablab L. as compared to AO₃ at both developmental stages ( Figures 5 , 6 ). The findings suggest that greater O₃ influx under EO₃ conditions resulted in significant membrane damage in both kinds of Indian beans at both development phases. A 90.2% increase in MDA contents in Vigna mungo L. varieties was reported on exposure to EO₃ conditions as compared to AO₃ conditions (Dhevagi et al., 2021). Since lipid peroxidation is an important marker of O₃ stress tolerance (Iglesias et al., 2006), Kashi Sheetal which showed a lesser degree of membrane lipid peroxidation under EO₃ conditions as compared to Kashi Harittima, was considered to be more tolerant of the two experimental varieties. The level of MDA content in the leaves of both varieties of Dolichos lablab L. was significantly reduced upon nitrogen treatments at both the O₃ concentrations. Similar findings were also reported by Pandey et al. (2018) in which MDA content in the leaves of Triticum aestivum L. was significantly decreased with increased nitrogen amendments. Podda et al. (2019) also found that nitrogen treatment had a beneficial impact on O₃-exposed plants, seeing a substantial decrease in MDA and improved membrane stability after nitrogen treatment at both AO₃ and EO₃ conditions. It was observed that Kashi Sheetal produced a lesser amount of MDA in comparison to the Kashi Harittima, at all the nitrogen treatments during both developmental stages, indicating Kashi Harittima to be was less efficient in scavenging the O₃-induced ROS leading to higher membrane disintegration than the Kashi Sheetal upon nitrogen treatments under both O₃ exposure conditions. This observation points towards the higher sensitivity of the Kashi Harittima towards ozone stress. Furthermore, there was a negligible difference in the response between the N2 and N3 treatments for both varieties upon both O₃ exposure conditions, which specifies the sufficiency of N2 treatment in alleviating O₃ stress in the Indian bean. Pearson’s correlation coefficient test has shown a significant negative correlation of MDA contents with the enzymatic activities, suggesting a reduction in membrane lipid peroxidation of O₃-stressed plants upon N amendments ( Figure 7 ).

The presence of an efficient antioxidant defense mechanism allows the higher plants to perceive and decipher ROS signals into necessary cellular responses (Yadav et al., 2019). In the current experiment, it was observed that the endogenous level of antioxidants was higher in Kashi Sheetal as compared to Kashi Harittima, at ambient O₃ exposure, suggesting high tolerance of O₃ stress in Kashi Sheetal. All the antioxidative enzymes showed higher activity under EO₃ conditions as compared to AO₃ conditions and this applies to both varieties at all developmental stages. Similar findings have also been reported by Pandey et al. (2018) in which the activities of antioxidative enzymes increased under EO₃ conditions in two cultivars of Triticum aestivum L. It has been proved that the sustenance and regeneration of the antioxidative enzymatic pool indicate a strengthened defense response capable of O₃ detoxification (Caregnato et al., 2013). In the present study, N amendments enhanced the antioxidant activities in both varieties, thereby proving its potential in the management of O₃ injury. It was observed that the antioxidative system of O₃ sensitive variety Kashi Harittima responded more positively at elevated O₃, compared to ambient O₃. At elevated O₃, Kashi Harittima was capable of sustaining higher increments in the enzyme activities at both developmental stages, which evidently proves that N amendments were more favorable in encountering O₃ stress in this variety, as compared to Kashi Sheetal. As per the PCA analysis, it was observed that Kashi Harittima, as the sensitive variety, demonstrated a stronger association of enzymatic anti-oxidants in component 1 as compared with the Kashi Sheetal ( Figure 8 ). In the ascorbate-glutathione pathway, APX is the essential enzyme known for scavenging H₂O₂ by using ascorbic acid as an electron donor, whereas GR catalyzes glutathione reduction with ascorbate regeneration in plants (Ashraf, 2009). In the present experiment, N amendments intensified the role of APX and GR in inducing the plant’s defense system in both varieties. The higher APX and GR activity can be owed to the greater H₂O₂ level in EO₃ conditions than in AO₃, thereby ensuring the scavenging of the H₂O₂ in order to curtail O₃ stress. Under stressful circumstances, Almeselmani et al. (2006) also observed an increment in APX and GR activity in the tolerant wheat cultivars HD77 and HD2817. The lower levels of H₂O₂ in Kashi Harittima, upon N amendments at elevated O₃ treatment, can be explained by higher increments in CAT activity. Upon N amendments, whereas, Kashi Harittima was able to uphold the activities of SOD and CAT, in elevated O₃-treated plants, Kashi Sheetal showed lesser increments in the response of these two enzymes, during the reproductive stage. It is to be noted that with a gradual increase in the dose of nitrogen treatments, there was a significant surge in the activities of the antioxidative enzymes in both varieties of Dolichos lablab L. at all growth stages. But the degree of increment of the activities of enzymes was insignificant between N2 and N3 treatments, suggesting the adequacy of N2 treatment in the management of O₃ stress.

O₃ exposure is known to adversely affect the physiological parameters of plants, rate of photosynthesis, and stomatal conductance being directly affected (Singh et al., 2014; Tetteh et al., 2016; Dhevagi et al., 2021). This reduced photosynthetic rate caused by elevated ozone may be related to the degradation of the chloroplast structure, which prevents the production of chlorophyll under these conditions (Biswas and Jiang, 2011). In the present study, N supplementation resulted in the improvement of these two parameters in both the varieties of Dolichos lablab, at both O₃ exposure conditions, further justifying the potential role of N amendments in depreciating O₃ stress. This is further proved by significant correlation coefficient values of different physiological parameters of O₃-exposed plants upon N amendments ( Tables 2 , 3 ). Increased g_s upon N amendments have been reported in wheat (Wall et al., 2000), larch (Mao et al., 2014), cluster bean (Gupta and Tiwari, 2020), etc. A decrease in g_s along with an increased A upon N amendments and O₃ exposure in poplar has been reported by Zhang et al. (2018). A comparison of the physiological response of both varieties revealed that upon N supplementation, Kashi Harittima showed higher increments in A and g_s , in comparison to Kashi Sheetal at all ages under both AO₃ and EO₃ conditions. Since the O₃-induced depreciation of cellular performance largely depends upon O₃ flux, which is directly controlled by stomatal movement (Yadav et al., 2019), increased stomatal conductance in O₃-exposed plants of Indian bean in the present study, does not account for the reported improvement in the plant’s performance upon N amendments. It has been reported that the application of a higher N dose leads to an increased influx of O₃, which may lead to the depletion of the antioxidant pool (Harmens et al., 2017; Marzuoli et al., 2018). Non-significant variations in g_s due to O₃ x variety and O₃ x treatment interactions ( Table 1 ) further prove that g_s does not play any substantial role in the ameliorative effect of N amendments in the experimental plants. These observations lead to the conclusion that the upturn in the rate of photosynthesis upon N amendments in O₃-exposed plants can be attributed to the enhanced activities of the enzymatic antioxidant pool, thus proving our hypothesis. Increased enzymatic antioxidants upon N implementation, ensure the scavenging of O₃-induced oxidative entities, thus rendering protection to photosynthetic machinery in the chloroplasts.

The analysis of yield parameters showed a significant reduction in the EO₃ conditions in comparison to AO₃ conditions which accounts for both varieties of Dolichos lablab L. at all ages of growth ( Table 4 ). An earlier study found that the yield of selected varieties of wheat (HUW 234 and 468, HD 3086 and 3118) and black gram (CO6 and VBN 1-8) were markedly reduced under EO₃ as compared to AO₃ (Yadav et al., 2019; Dhevagi et al., 2021). Both varieties of the Indian bean exhibited a significant increase in the weight of grains plant^-1 upon all doses of N amendments (N1, N2, and N3). However, the difference in the magnitude of the increase in yield of N2 and N3 was inconsequential for both varieties indicating the sufficiency of N2 treatment in ameliorating oxidative conditions. Interestingly, the O₃-sensitive Kashi Harittima variety showed a higher percent increment in the number/weight of seeds plant^-1 upon N amendments as compared to Kashi Sheetal ( Table 4 ). Higher enhancement in the antioxidative enzyme activity and greater increments in the rate of photosynthesis explain the higher yield improvements in Kashi Harittima. A previous experiment also reported similar findings in which an O₃-sensitive wheat cultivar showed a higher percent increment in response to N amendments as compared to an O₃-tolerant wheat cultivar under both AO₃ and EO₃ conditions (Pandey et al., 2018). The above results prove our first hypothesis that nitrogen treatments assist in strengthening the antioxidative enzyme pool for defense against oxidative stress conditions. In the present study, the analysis of H₂O₂ localization, antioxidative enzymes, physiological traits, and yield revealed that the Kashi Sheetal variety outperformed the Kashi Harittima variety in every aspect proving the second hypothesis that the differential response of the varieties to varied O₃ conditions helps in determining their sensitivity to O₃ stress. The more positive response of the O₃-sensitive varieties upon N exposure provides a promising feature and can be used for promoting the farming of O₃-sensitive varieties, thereby boosting agricultural production.

This experiment justifies the use of N amendments as an effective measure for the management of O₃ injury. The higher endogenous levels of H₂O₂ accumulation and enzymatic antioxidant contents upon O₃ exposure have established Kashi Sheetal to be O₃ tolerant. The interaction between N amendments and O₃ exposure had a more positive effect on O₃-sensitive Kashi Harittima, as compared to Kashi Sheetal. Since the increased stomatal conductance upon N fertilization does not restrict the entry of O₃ in plants, the higher photosynthetic rate, and subsequently yield were maintained by a stimulated enzymatic antioxidative response in plants at both O₃ exposure conditions. The enzymatic response showed significant variations due to ozone and treatment and ozone and variety interactions, whereas the variations of stomatal conductance were insignificant, proving our theory. A more positive response of Kashi Harittima to N supplementation at both O₃ exposure conditions can be associated with the sustenance of its higher enzymatic response at the reproductive stage as well, which was more prominent at the EO₃ condition. The current study proved that N2 treatment (1.5 times recommended dose) was sufficient to partially ameliorate O₃ stress and that larger nitrogen doses may not be more successful in doing so because they did not confer any further perks on the plant’s growth and development. However, more experiments are required to establish a dose-response relationship between N fertilization and O₃ exposure doses.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

Conceptualization, writing-original draft, AS. Data curation, PM. Software, AM. Conceptualization, supervision, ST. All authors contributed to the article and approved the submitted version.