In the current study, higher stomatal density and leaf damage were recorded in formaldehyde exposed plants compared to the respective controls. The increase in stomatal density has also been reported in other indoor plants against formaldehyde application (Kim et al. 2013). Plants close their stomata to limit the entry of formaldehyde and tolerate its stress (Kvesitadze et al. 2006; Kim et al. 2013). Similarly, leaf damage has also been well documented in various plants under formaldehyde stress (Kim et al. 2013; Nian et al. 2013; Li et al. 2021). VOCs are known to impart deleterious effects on the basic physiological processes of plants along with damage to cell ultrastructures (Yoo et al. 2006).
The physiological processes of C. comosum were significantly affected by formaldehyde application in the current study. The present study reports a decline in photosynthetic rate after formaldehyde application. Various studies have also reported this decline in different plants under VOCs and ozone stress (Phothi et al. 2016; Hörmann et al. 2018). VOCs are reported to cause damage to PS II quantum yield in light reactions of photosynthesis. Decreased absorption of photons in PS II reduces the formation of NADPH + H+ and ATP which may reduce CO2 fixation, hence leading to reduced photosynthesis (Yoo et al. 2006; Hörmann et al. 2018). Subjection to a higher dose of formaldehyde led to an increased transpiration rate and stomatal conductance in the present study. Gong et al. (2019) has also reported higher stomatal conductance in Epipremnum aureum against benzene stress. A higher transpiration rate reflects higher stomatal conductivity, which is essential for the removal of VOCs by plants (Jin et al. 2013). Whereas the transpiration rate and stomatal conductance significantly declined against low and moderate doses of formaldehyde in the current study. These findings are also supported by previous studies conducted on indoor plants (Yoo et al. 2006; Gong et al. 2019). VOCs including formaldehyde are reported to impart deleterious effects on the basic physiological processes of plants, and damage to cell ultrastructure (Yoo et al. 2006) and reducing the stomatal conductance due to the elasticity of damaged guard cells (Paoletti and Grulke, 2005). A significant rise in the intercellular CO2 level was observed in the current study against formaldehyde doses. This finding is also supported by the research of Yoo et al. (2006) conducted on Spathiphyllum wallisii and Hedera helix under benzene and toluene stress. C. comosum metabolizes formaldehyde by hydrolyzing it into formate. Subsequently, the generated formate is then oxidized to CO2 enzymatically by formate dehydrogenase (Wang et al. 2018). This oxidation could be a possible reason for elevated levels of intercellular CO2 in plants under formaldehyde stress. Furthermore, acidic pH of the control plants shifted to basic after formaldehyde application in current study. Bharti et al. (2018) have also documented a pH shift to basicity in various plants against air pollution stress. Basic leaf pH is known to assist the tolerance capability of plants against air pollutants by enhancing the conversion rate of hexose sugar to vitamin C (Escobedo et al. 2008, Joshi and Bora, 2011). A change in pH is crucial for various enzymatic and physiological responses of plants under stress conditions (Escobedo et al. 2008).
In order to avoid the damage and maintain the balance of oxidation in the leaves, an enzymatic antioxidant system was activated in the current study. Significantly higher SOD, CAT, GPX activity and T-AOC were recorded in the current study. In support of our findings, various reports have documented higher SOD activity (Liang et al. 2019; Khan et al. 2023), CAT activity (Zhao et al. 2019; Li et al. 2021; Khan et al. 2023), GPX activity (Li et al. 2021; Khan et al. 2023)d AOC activity (Li et al. 2019; Khan et al. 2023) in various indoor plants against formaldehyde stress. SOD is an enzymatic antioxidant that catalyzes the elimination of the superoxide anion by dismutating it into O2 and H2O2, eventually eliminating the chances of OH• formation (Mittler, 2002). In the same way, catalase is reported to catalyzing the dismutation of H2O2 into H2O and O2 (Mittler 2002). H2O2 can cross membranes and cause oxidative damage by resulting in 50% reduced activity of various enzymes and programmed cell death (Dat et al. 2000; Bienert et al. 2007). However, the CAT activity did not increase against the highest dose of formaldehyde in current study. The consistent trend of decreasing CAT activity under higher doses of formaldehyde is well documented in various studies (Xue et al. 2008; Linghu et al. 2011; Li et al. 2019). This decline is due to the interactions of formaldehyde with cell’s thiol-disulphide status and the redundancy of reductive H2O2-metabolizing pathways (Mhamdi et al. 2010). Moreover, GPX is also a prominent antioxidant enzyme known to eliminate excessive H2O2 which thus aids the plant to withstand stressful environment (Asada 1999). Higher T-AOC is reported to aid plants in coping up various stresses (Li et al. 2019). TAOs are stable molecules known to donate an electron to a rampaging free radical and neutralize it. This reduces the potential for free radicals to cause cell damage (Lobo et al. 2010).
Current study reports a significant rise in TPC and TFC against formaldehyde application after tested durations. These findings are in alignment with the findings of Khan et al. (2023). Phenolic contents are a prominent non-enzymatic antioxidant (Samec et al. 2021) that aids plants by donating electrons to guaiacol-type peroxidases for the catabolism of H2O2 formed under harsh environmental conditions (Sakihama et al. 2002). Moreover, formaldehyde is shown to enhance the expression of enzymes that are involved in phenolic transformations, such as peroxidase (Peng et al. 2022). Whereas, flavonoids are reported to scavenge singlet oxygen and to reduce the damages to the outer envelope of the chloroplastic membrane (Agati et al. 2012).
Increased proline, total protein and MDA content was recorded in C. comosum plants against formaldehyde application in the current study. Various researchers have reported higher proline content (Wang et al. 2020; Markazi et al. 2022; Khan et al. 2023), total protein content (Li et al., 2019) and MDA content (Li et al. 2019; Wang et al. 2020; Qiao et al. 2023) in various indoor plants against formaldehyde stress. In stressful conditions, proline works as an osmo-protectant, maintains redox balance inside the plant cell and stabilizes cellular structures and enzymes (Meena et al. 2019). It quenches ROS including singlet oxygen, superoxide and hydroxyl radicals (Rehman et al. 2021). MDA is a byproduct of lipid peroxidation of polyunsaturated fatty acids caused by hydroxyl radicals, which is induced under formaldehyde stress in plants (Li et al. 2019). However, after subsequent increase, a reduction in MDA content at extended time durations was observed in the current study. This reduction is possibly associated with the decrease in formaldehyde level being metabolized by C. comosum (Li et al. 2021; Wu et al. 2022). Under abiotic stress conditions, ROS accumulation occurs in plants. This leads to the requirement of new protein synthesis to defend against ROS and replace existing damaged proteins (Qazi et al. 2019; Salih et al. 2020).
Formaldehyde application also significantly escalated the chlorophyll and carotenoid content in this study. This follows the pattern of previous studies conducted on Pisum sativum and C. comosum against formaldehyde stress (Erofeeva, 2018; Paresh et al. 2018; Khan et al. 2023). Under paradoxical effects, the toxic impact of higher formaldehyde level is reduced, leading to increased chlorophyll content in plants (Erofeeva, 2018). Additionally, a higher carotenoid content recorded in the current study could be a possible reason of an inclined chlorophyll content against formaldehyde. Carotenoids act as an antioxidants to protect chlorophyll and photosynthetic apparatus by quenching of 3Chl* and 1O2 by excitation transfer mechanism followed by thermal energy dissipation (Triantaphylidès & Havaux, 2009; Yuan et al. 2015).
Elevated TSS content in C. comosum against formaldehyde application is recorded in the current research, which is supported by the report of Skłodowska et al. (2023), conducted on C. comosum against formaldehyde stress. Formaldehyde is known to be oxidized and utilized in the Calvin cycle after being absorbed by plant leaves (Salonen et al. 2009; Kim et al. 2010). This could be a possible reason for inclined TSS content recorded in this study.
PCA indicated that SOD, CAT, GPX, MDA, TPC, TFC, TAO, carotenoids, TSS and intercellular CO2 contributed the most to the total variance and can be considered biochemical markers for formaldehyde stress in C. comosum. Different researchers have documented these parameters individually to respond significantly against formaldehyde stress (Yoo et al. 2006; Liang et al. 2019; Li et al. 2019; Zhao et al. 2019; Wang et al. 2020; Li et al. 2021; Khan et al. 2023; Qiao et al. 2023; Skłodowska et al. 2023). However, according to our extensive literature review, no research study has documented these parameters as biomarkers in C. comosum against different levels of formaldehyde for a wide range durations.