Micro-morphological adaptations and tolerance assessment of selected vascular plant species exposed to vehicular exhausts

The vehicular exhausts on roadsides negatively affect living organisms and disturb their environment. These negative impacts of vehicular exhausts on plants demand a scientific investigation of roadside plants in Abbottabad, Pakistan. So, this observational and experimental study was conducted to evaluate the effect of vehicular exhausts on morphology, epidermal structure and biochemical characteristics of leaves of plants near roadsides and compared with the non-polluted site. The quantitative morphological parameters: leaf area, length of petiole and vein-to-vein difference along with anatomical attributes such as stomata and epidermal cells were recorded. Compared to non-polluted areas, the chlorophyll content of plant leaves from polluted areas was higher. The results indicate that vehicular exhaust negatively affects leaf morphology and increases the Stomatal number at adaxial (front surface) of leaves. The air pollution tolerance index evaluated Melia azedarach and Salix angustifolia as tolerant tree species and Rumex dentatus, Amaranthus viridis and Sonchus hydrophilus as tolerant herbaceous species. In order to survive in polluted environments, this study demonstrated that plants go through adaptive processes.


Introduction
The fast development in industrialization and urbanization contributes to pollute the air (Karmakar et al 2016) by raising the demand for the expansion of roads and vehicles. The expansion of the human population and their needs in cities results in environmental pollution, the most significant among which is air quality generated by vehicular exhausts. Automobile exhaust are a major cause of urban air pollution, influencing human health and vegetation. Afforestation or greenbelt development is a well-known and successful method of pollution abatement worldwide (Saxena Kulshrestha 2016 andGhosh 2013). Plants can assist to reduce level of atmospheric pollution (Abubakar et al 2022).
The tolerance of the plants towards air pollution is affected by diminishing their morphology, biochemistry, anatomy and physiology (Singh et al 2017, Sharma et al 2018. Various research reports sensitive plants were affected by air pollution altering their anatomical, morphological, and physiological and biochemical parameters (Reig-Armiñana et al 2004, Da Silva et al 2005. Exposure to exhaust emissions or pollution can significantly affect the morphological, physiological and biochemical attributes of leafy vegetables (Kohan et al 2021). On the contrary, tolerant plants can tolerate pollutants by regulating biochemical and physiological parameters (Allen et al 1987). Besides the leaf is involved in the gaseous exchange, photosynthetic and transpiration activities, it is considered to be the most sensitive part of the plants towards air pollution. Therefore, the leaves can act as a tool to assess air pollution (Leghari and Zaidi 2013). The alteration by vehicular exhausts occurs in morphology, epidermal anatomy and tolerance level of the of leaf of plants (Shakeel et al 2022). The number of leaves grows but their size reduces substantially at polluted areas compared to plants at controlled sites, possibly to overcome for stress conditions. Similarly, there was decrease in length of petiole, difference in leaf veins, length and width of leaf were also reported. (Dineva 2004, Tiwari et al 2006, Babapour et al 2014). The epidermal cell and stomata were reported to increase in number in the plants growing near roadsides (Achille et al 2015). While others illustrated that air pollution affects the conductance of stomata (Verma, Singh 2006). Therefore, leaves act as an indicator for monitoring the influence of vehicular exhaust.
The air pollution tolerance index formulated by Singh and Rao 1983 was evolved from the biochemical activities of leaves of the plants including pH, relative weight content (RWC), chlorophyll content and ascorbic acid (Molnár et al 2020and Manjunath and Reddy 2019and Ogunkunle et al 2019, and Lohe et al 2015. The air pollution tolerance index (APTI) identify the tolerant plants serve as a sink of pollutants by adapting and showing tolerance to air pollution while other plants show sensitivity by diverging biochemical characteristics of leaves (Bora andJoshi 2014 andSingh et al 1991). In such conditions, different plants growing along roadsides can be used to mitigate air pollutants. However, the mitigation potential of air pollution on plants can also be monitored to find out the influence on micro-morphology and biochemical characteristics for broader evaluation and vegetation of green belt along roadsides. This raises the question that 'Which plants growing along the roadside is more tolerant and less affected by vehicular exhaust'. For this purpose, this study was designed to evaluate the plants adaptation and tolerance along roadsides.
This study focused on the comparative morpho-anatomical and biochemical responses of the leaves of plants at the polluted (roadsides) and controlled areas (away from the city). For this purpose, a total of 06 plants including 2 trees and 04 herbs were investigated. The goal of this analysis was to compare the morphoanatomical and APTI of the selected plants and compared them with other researches in literature.

Study area
Abbottabad 'the city of Pines' lies in Khyber Pakhtunkhwa, Northern Pakistan at 34°92′N latitude and 73°13′E longitude at an altitude of 1,256 m (4,121 ft.) and is a tourist and economic trade Centre. But due to the Karakoram Highway linking Pakistan with China transected through the mid of the city and tourists activities, the traffic increases which in turn affect plants [ figure 1]. The plant samples are collected in the month of August and September in which the day time is about 13 to 14 h. The temperature recorded during the month of August and September was 19°C. While the average rainfall was recorded 989 mm respectively.
To evaluate the impact of vehicular exhaust, the plants from the polluted sites were collected in triplicate growing adjacent to roads of a city in an ice cooler. Similarly, the similar plants in three replications were collected in an ice cooler from the unpolluted sites of the city for comparative analyses.

Micro-morphology
The morphological parameters i.e. leaf area, difference in vein to vein of leaf, and length of petiole were measured and compared. While the epidermal anatomy of the leaves was investigated by a method of (Horanic and Gardner 1967). In this method, a transparent nail paint was gently placed on the surface of the leaves. After desiccation of nail paint, it was gently removed from the superficial layer of leaves and the imprints of the stomata and epidermis appear on nail paint were examined at a 400 X magnification under a microscope IRMECO model IM-800B.

Stomatal index, number of stomata and epidermal cells
The epidermal cell numbers and stomata numbers were investigated by light microscope. While the stomatal index (S.I) of plants were computed (Salisbury et al 1927, Salisbury 1932).
= + E S S.I 100S E = epidermal cell numbers; S = stomata numbers 2.4. Biochemical characterization of plant leaves and apti formulation A Spectrometer (Spectonic 20 model) was used for total chlorophyll measurement by using a method of (Arnon 1949). The sample leaves were grinded with pestle and mortar, 05 grams of extract was reacted with acetone. After filtration, samples absorbance and transmittance were measured. Similarly, Ascorbic acid was measured by using a protocol of (Bajaj and Kaur 1981). For pH analysis, a homogenate of 5 grams of leaves in a distilled water was formed and measured with a portable pH meter KOX206369401 model. The RWC was evaluated as by (Singh 1977): a. Fresh Weight (FW) taken immediately after collection.
b. Turgid weight (TW) 10 grams of fresh weight immersing overnight in water.
c. Dried Weight (DW) After drying of leaves in an oven for 24 h at 70°C.
The APTI was measured as suggested by (Singh and Rao 1983).
A T P R 10 A = Ascorbic acid, T = Total Chlorophyll, p = pH, R = Relative Weight Content

Statistical analyses
The data collected from the replicates of plant species were analyzed statistically. The Two-way Anova and Least significance difference (LSD) were performed to analyzed the data comparatively by using MS-Excel and Statistix 8.1. The data presented in the tables and figures showed means ± standard deviation and alphabets showed different classes. Similarly, Pearson Correlation among different physiological parameters was evaluated to test the tolerance and sensitivity of the plant species.

Results and discussion
The quantitative morphological characters of the leaves of Melia azedarach, Salix babylonica, Parthenium hysterophorus, Rumex dentatus, Amaranthus viridis and Sonchus hydrophilus at both polluted and controlled sites have been comparatively observed and measured.

Morphological responses of plant species towards vehicular exhausts
The studies revealed that the leaf area and growth of the plant species at the polluted areas found to decline (John et al 2006, Iqbal et al 2010, Areington et al 2015. This study as in figure 2 showed the adverse effect of road pollutants on plant species present around the roadsides. It is further assumed that the decline in leaf area will also affect photosynthetic activity. The maximum leaf area Rumex dentatus (951.3 ± 248.9 sq mm) and minimum Salix babylonica (123 ± 29.8 sq mm) at the controlled site while Rumex dentatus (898 ± 147.9 sq mm) is maximum and Amaranthus viridis (102 ± 22.6 sq mm) is minimum at the polluted site. Jochner et al 2015 stated that the leaves adopted a defense mechanism by reducing their area and minimized the exposure of the pollutants. The comparison of plants examined that the petiole length of the plant species exhibits reduction at the polluted areas. Petiole length is an essential factor as the average leaves of plants at the contaminated areas were found less in number than the leaves observed in plants at the controlled site. The plant species Sonchus hydrophilus was found sessile during the study. Similar results of reduced petiole length of the plant species at the polluted areas were reported by workers (Stevovi et al 2010 and Leghari and Zaidi 2013). The maximum petiole length was found in Rumex dentatus (12.1 ± 6.96 cm to 12.2 ± 3.2 cm) and minimum in Salix babylonica and Melia azedarach (1.10 ± 0.46 cm to 1.3 ± 0.7 cm) at the polluted and controlled sites, respectively, apart from sessile leaves in Sonchus hydrophilus at both polluted and controlled areas. The comparative means of the petiole length of all the studied plants showed (p > 0.05) from one another (figure 2) at both polluted and controlled area The study conjointly revealed that the leaves gathered from polluted areas showed a reduced vein-to-vein difference compared to controlled environmental conditions as presented in figure 2. The maximum, the more considerable vein-to-vein length was found in Rumex dentatus (1.10 ± 0.32 cm) while Sonchus hydrophilus (1.80 ± 0.36) and minimum in Sonchus hydrophilus (0.1 ± 0.04 cm) and Salix babylonica (0.4 ± 0.03 cm) at the polluted and controlled sites respectively. The studies Shakeel et al 2022) confirmed similar results of decrease in vein-to-vein difference in leaves.

Anatomical adaptations of plant species towards vehicular exhausts
The width and length of the subsidiary cells were also measured to investigate the complete micromorphology of the stomatal apparatus. The stomatal length and width reduces significantly in the plant species present near road sides as compared to remote areas plant species (Allahnouri et al 2018). All the studied plant species confirmed that the subsidiary cells length was found more at both abaxial and adaxial surfaces of leaves of polluted areas. While the subsidiary cells width decreased in leaves collected from the polluted areas as shown in tables 1 and 2 when compared to the plants of the controlled area. However, the dust on leaves at the polluted areas affect the anatomy of leaves, as shown in figures 4(A) and (B). Like most living organisms, it was assumed that plants also modify themselves for their survival by reducing morphology and increasing anatomical characters for their survival in the polluted environment.
The opening and closing of guard cells are important in the regulation of O 2 /CO 2 and water. The width of guard cells at the adaxial side was estimated higher in the studied plants at the polluted sites except for Parthenium  hysterophorus, Rumex dentatus, and Sonchus hysterophorus. As subsidiary cells, the guard cells at the abaxial surface were found higher in length in leaves of all plants and the width showed increased at the abxial sufaces of leaves of Amaranthus viridis, Salix babylonica, and Melia azedarach at polluted areas as presented in figure 4   plants at the controlled areas (table 3). In this case, the herb Amaranthus viridis and a shrub Parthenium hysterophorus show a slightly more Stomatal number at the abaxial surface of polluted areas leaves of plants (Rai and Kulshreshtha 2006) stated that the numbers of stomata increased at the polluted areas. Thus, plants are helpful in resisting air pollutants.
A comparison of leaves epidermal cells number on adaxial surfaces showed reduction at the polluted areas except for a species, Amaranthus viridis with a lower epidermal cells number at the polluted areas (table 4). The abaxial surface showed approximately similar epidermal cell results except for Amaranthus viridis with slight increasing variation at the polluted areas and Salix babylonica at the controlled areas (table 3)

Impact of vehiular echausts on air pollution tolerance index and associative attributes
The increase in acidity enhances the sensitivity of stomata against air pollution. The pH alteration effect the transpiration rate and respiration in plants (Khanoranga and Khalid S 2019). The plants at the polluted areas exhibited more acidic pH due to acidic gaseous pollutants on roads caused by vehicles (Pawar et al 2010). An acidic leaf pH was observed in the plants exposed to SO x , NO x and CO 2 . The acidic pH present in plants at the polluted areas is slightly lower (p > 0.05) which do not cause physiological disturbance in plants to a great extent. As stated previously during this study, the Stomatal number were higher in plants at the polluted areas which may also help to decline the toxic effect of air pollutants by gaseous exchange through stomata. The pH difference between both areas plants is marginal similar with Prajapati and Tripathi 2008 which investigated minimal effect on chlorophyll and Ascorbic acid content and evaluated as tolerant.
The growth, development, biomass enrichment particularly photosynthesis content are dependent on chlorophyll production. During this study, chlorophyll was found to be greater in plants at the polluted areas. It is a sequence of facts that the increased Stomatal number at the polluted areas may be evident for more gaseous exchange suitable for photosynthetic activity. The higher content of chlorophyll help plants to tolerate pollutant condition by increasing food production. The total chlorophyll varies in different plants (Karmakar and Padhy 2019, Kaur and Nagpal 2017, Banerjee et al 2019, Prajapati and Tripathi 2008and Shakeel et al 2022. During the study, Amaranthus viridis is the only plants at the polluted areas with less chlorophyll content than the controlled areas. Moreover, it was found in Parthenium hysterophorus while minimum in Melia azedarach at the polluted areas. The degradation in the photosynthetic pigment provide bio indication of air pollutants on plants, the chlorophyll content decline shows sensitivity (Woo and Je 2006) while higher chlorophyll level of the plants shows tolerance (Singh and Verma 2007). Moreover, this study hypothesizes that the increasing Stomatal number enhance the total chlorophyll in the plants.
Ascorbic acid acts as a reducing agent for carbon fixation in photosynthesis ( The RWC of the examined plants was highest for Parthenium hysterophorus and lowest for Amaranthus viridis. The plants Salix babylonica and Parthenium hysterophorus have been reported to have an RWC greater than 30%. These plants with an elevated RWC help remove pollutants. An increase in RWCs increases stomatal activity, which in turn increases photosynthesis. The highest percentage of RWC has been reported to be consistent with the results at contaminated areas (Kanwar et al 2016and Tak and Kakde 2017and Patel and Kousar 2011. The higher RWC on the polluted areas helps plants cope with the dry conditions (Tyree et al 1991). As RWC increases, plants become more tolerant to air pollution (Singh et al 1991) which increases cell permeability (Oleinikova 1969) and provides tolerance to pollutants (Achakzai et al 2017).
APTI was estimated to be highest in Salix babylonica and lowest in Parthenium hysterophorus at the contaminated areas. The results of the present study showed the order of APTI in the plants Salix babylonica (22.5 ± 1.7)> Amaranthus viridis (20.8 ± 3.55)> Sonchus hydrophilus (20.4 ± 0.82)> Melia azedarach (16.7 ± . The linear regression plot between biochemical parameters (ascorbic acid, total chlorophyll, pH and RWC) and APTI has shown a strong correlation between APTI and ascorbic acid (R 2 = 0.8218) and a weak correlation between total chlorophyll content (R 2 = 0.1232). , pH (R 2 = 0.0119) and RWC (R 2 = 0.1208) (figure 5). Therefore, it has indicated that ascorbic acid is the main factor in combating air pollution similar to results of Banerjee et al 2021,

Conclusion
The morphological, anatomical and biochemical activities of the sensitive part of the plants i.e. leaf is important for the assessment of vehicular exhausts impacts on the plants. Therefore, the research was carried out to monitor the effect of vehicular emissions on morphology, anatomy and biochemical activities of leaves of plant species growing along road sides of Abbottabad and compared with the controlled sites. This study disclosed that the air pollution caused by vehicular exhaust negatively affect morphological characteristics of plants. Similarly, the epidermal anatomy of leaves revealed that the adaxial (frontal) surfaces were affected more than abaxial (back) surfaces by the vehicular exhausts. The APTI is an important cost effective method for the determination, control and improvement of air pollution in cities. Similarly, Salix babylonica (Tree), Amaranthus viridis (Herb), Sonchus hydrophilus (Herb) and Melia azedarach (Tree) have been recommended for planting green belts as tolerant plants. Furthermore, there is a requirement for further research in order to determine genetic aberrations and biochemical activities of plants as a result of exposure to a particular pollutant.

Data availability statement
All data that support the findings of this study are included within the article (and any supplementary files).