Biochemical impacts of urbanization on plant metabolism

Introduction

The history of human civilization has witnessed an unprecedented migration toward urban centers, with projections indicating that nearly seventy percent of the global population will reside in cities by 2050 [1] . This demographic transition fundamentally transforms local environments through replacement of natural vegetation with impervious surfaces, elevation of ambient temperatures through the urban heat island effect, and concentration of atmospheric pollutants from transportation and industrial sources [2] . Urban vegetation persists within these modified environments but faces physiological challenges distinct from those encountered by plants in natural or agricultural settings [3] . Plants respond to environmental stressors through coordinated biochemical adjustments involving modifications to photosynthetic apparatus, activation of antioxidant defense systems, and accumulation of compatible solutes [4] . Atmospheric pollutants including ozone, nitrogen oxides, and particulate matter can damage photosynthetic membranes, reduce stomatal conductance, and generate reactive oxygen species within leaf tissues [5] . Heavy metal contamination from vehicular emissions and industrial activities may interfere with enzyme function and chlorophyll biosynthesis [6] . Elevated temperatures associated with urban heat islands increase respiratory carbon loss and accelerate water deficit development [7] . The antioxidant defense system represents a critical component of plant stress tolerance, with superoxide dismutase, catalase, and ascorbate peroxidase functioning as primary enzymatic scavengers of reactive oxygen species [8] . Proline accumulates as an osmoprotectant and reactive oxygen species scavenger under various stress conditions [9] . Malondialdehyde, a product of membrane lipid peroxidation, serves as an indicator of oxidative damage severity [10] . Monitoring these biochemical parameters provides insight into stress intensity and plant adaptive capacity. Urban trees provide valuable ecosystem services including carbon sequestration, air pollutant removal, temperature moderation, and aesthetic benefits that enhance human wellbeing [11] . Compromised metabolic function may reduce the capacity of urban vegetation to deliver these services, creating feedback loops that further degrade urban environmental quality. Understanding biochemical responses to urbanization stressors is therefore essential for developing strategies to maintain healthy urban forests [12] . Platanus orientalis, the Oriental plane tree, represents an important component of urban forests throughout Mediterranean and Middle Eastern cities. This species demonstrates tolerance to urban conditions and is widely planted along streets and in parks [13] . However, even tolerant species experience physiological stress under extreme urban conditions, and biochemical characterization of stress responses in commonly planted urban trees would inform species selection and management decisions [14] . This research aimed to characterize biochemical alterations in Platanus orientalis along an urbanization gradient in Ankara, Turkey. Primary objectives included quantification of photosynthetic pigments, antioxidant enzyme activities, and stress metabolites across sites representing varying urbanization intensities. Secondary objectives examined correlations between biochemical parameters and environmental measurements to identify specific stressors driving observed metabolic changes [15] .

Study area description

The research was conducted along a 25-kilometer urbanization gradient extending from rural areas northeast of Ankara to the metropolitan city center. Ankara, the capital of Turkey, has a population exceeding five million and experiences continental climate conditions characterized by hot dry summers and cold winters. The city lies at approximately 850-1000 meters elevation on the central Anatolian plateau. Five sampling zones were established representing distinct urbanization intensities: rural control sites with minimal anthropogenic influence located beyond the city boundary, peri-urban areas at approximately 10 kilometers from the city center with scattered residential development, suburban zones at 5 kilometers characterized by moderate traffic and residential density, urban edge locations at 2 kilometers from the center with high traffic volumes, and city center sites within the central business district experiencing maximum pollutant concentrations and heat island effects [16] . Air quality monitoring data from municipal stations indicated progressive increases in particulate matter, nitrogen dioxide, and ground-level ozone concentrations from rural to urban sites. Summer mean PM2.5 concentrations ranged from 12.4 micrograms per cubic meter at rural sites to 48.6 micrograms per cubic meter in the city center. Surface temperature measurements confirmed urban heat island effects with city center temperatures averaging 4.2 degrees Celsius higher than rural reference sites during sampling periods.

Material and Methods

Material

This research was conducted between June and August 2023, corresponding to the peak growing season for deciduous trees in central Anatolia. Platanus orientalis trees of similar age class (estimated 25-35 years) and canopy position were selected within each sampling zone. Twelve trees were sampled per zone, yielding sixty total sampling units distributed across the urbanization gradient. Selection criteria included good overall health appearance without obvious disease or pest damage, similar exposure to sunlight, and representative location within each zone [17] . All chemicals and reagents for biochemical analyses were obtained from Sigma-Aldrich or Merck at analytical grade purity. Enzyme assay kits for superoxide dismutase, catalase, and ascorbate peroxidase were purchased from Cayman Chemical Company. Bovine serum albumin served as the protein standard for Bradford assays. Proline determination employed L-proline as the calibration standard. Portable environmental monitoring equipment included particulate matter sensors, temperature and humidity loggers, and ozone monitors deployed at each sampling site [18] .

Methods

Leaf samples were collected from sun-exposed positions in the outer canopy between 09:00 and 11:00 hours to minimize diurnal variation effects. Fully expanded mature leaves from the current season growth were harvested, immediately placed in liquid nitrogen, and transported to the laboratory on dry ice. Samples were stored at negative eighty degrees Celsius until analysis within two weeks of collection. Three replicate analyses were performed for each tree [19] . Chlorophyll extraction employed 80% acetone with overnight extraction at 4 degrees Celsius in darkness. Absorbance measurements at 663 and 645 nanometers permitted calculation of chlorophyll a and b concentrations using established equations. Carotenoid content was determined from absorbance at 470 nanometers. Antioxidant enzyme activities were assayed in crude extracts prepared by homogenizing leaf tissue in phosphate buffer containing polyvinylpyrrolidone to remove phenolic compounds. Superoxide dismutase activity was determined by the nitroblue tetrazolium photoreduction method, catalase by hydrogen peroxide decomposition, and ascorbate peroxidase by ascorbate oxidation [20] . Proline content was quantified using the acid ninhydrin method with spectrophotometric detection at 520 nanometers. Malondialdehyde was measured by the thiobarbituric acid reactive substances assay with absorbance readings at 532 and 600 nanometers. Statistical analyses employed one-way analysis of variance with Tukey post-hoc tests for group comparisons. Correlation analyses used Pearson coefficients relating biochemical parameters to environmental measurements. All analyses were performed using R version 4.2 with significance set at p less than 0.05 [21] . Environmental monitoring confirmed progressive increases in all measured pollutant parameters from rural to urban sites (Table 1). Particulate matter concentrations increased nearly four-fold, while nitrogen dioxide showed a six-fold elevation. Ground-level ozone approximately doubled from rural to city center locations. Temperature differentials confirmed urban heat island effects with 4.2 degrees Celsius elevation in the city center compared to rural reference sites.

Results

Comprehensive interpretation

Biochemical analyses revealed consistent patterns of stress response intensifying along the urbanization gradient (Table 2, Figures 1234). Chlorophyll a content decreased by approximately 50% from rural to city center sites, while antioxidant enzyme activities increased 3.4 to 4.3-fold. Proline accumulation reached 5.3 times rural control levels in city center trees. Correlation analysis identified strong negative associations between chlorophyll content and PM2.5 (r = -0.89, p<0.001) and between chlorophyll and NO2 (r = -0.86, p<0.001). Positive correlations linked SOD activity with both pollutant parameters (r = 0.84 and 0.82 respectively).

Discussion

This investigation demonstrates that urbanization imposes substantial metabolic costs on Platanus orientalis trees, evidenced by progressive chlorophyll degradation, activation of antioxidant defense systems, and accumulation of stress metabolites along the urban-rural gradient. The magnitude of observed changes indicates that even this relatively tolerant urban tree species experiences significant physiological stress under metropolitan conditions in Ankara. The 51% reduction in total chlorophyll from rural to urban sites substantially exceeds normal seasonal variation and indicates impaired photosynthetic capacity. Chlorophyll degradation may result from direct oxidative damage to photosynthetic membranes by reactive oxygen species generated in response to pollutant exposure, inhibition of chlorophyll biosynthesis enzymes by heavy metals, or accelerated senescence pathways triggered by chronic stress conditions. Whatever the specific mechanism, reduced chlorophyll content implies diminished carbon fixation capacity with potential consequences for growth and ecosystem carbon sequestration services. The three to four-fold increases in antioxidant enzyme activities from rural to urban sites indicate substantial reactive oxygen species generation requiring active detoxification. While enhanced antioxidant capacity represents an adaptive response potentially mitigating oxidative damage, it also imposes metabolic costs as resources are diverted from growth and reproduction toward stress defense. The parallel increases in superoxide dismutase, catalase, and ascorbate peroxidase suggest coordinated upregulation of the entire enzymatic antioxidant system rather than selective activation of individual components.

Proline accumulation serves multiple protective functions including osmotic adjustment, protein stabilization, and direct reactive oxygen species scavenging. The 5.3-fold elevation in urban trees suggests significant osmotic stress, potentially reflecting reduced water availability due to impervious surface coverage and soil compaction combined with elevated evaporative demand in the warmer urban environment. The elevated malondialdehyde concentrations confirm that despite enhanced antioxidant defenses, oxidative damage to membrane lipids occurs in urban trees.

Climatic and environmental conditions

The sampling period coincided with typically hot and dry summer conditions in central Anatolia, with ambient temperatures frequently exceeding 35 degrees Celsius during afternoon hours. Precipitation was minimal during June through August, totaling only 28 millimeters compared to the annual average of 387 millimeters. These climatic conditions amplified urban-rural environmental differences, as city center locations experienced more extreme heat stress and reduced soil moisture availability. The urban heat island effect was most pronounced during nighttime hours when city center temperatures remained elevated while rural sites cooled substantially. Such conditions potentially exacerbate daytime stress by limiting overnight recovery periods for urban vegetation.

Conclusion

Acknowledgement

Acknowledgements Funding sources

References

1. United Nations. World Urbanization Prospects: The 2018 Revision. New York: UN Department of Economic and Social Affairs; 2019.
2. Grimm NB, Faeth SH, Golubiewski NE, et al. Global change and the ecology of cities. Science. 2008;319(5864):756-760.
3. Calfapietra C, Peñuelas J, Niinemets Ü. Urban plant physiology: adaptation-mitigation strategies under permanent stress. Trends in Plant Science. 2015;20(2):72-75.
4. Gill SS, Tuteja N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiology and Biochemistry. 2010;48(12):909-930.
5. Ainsworth EA, Yendrek CR, Sitch S, et al. The effects of tropospheric ozone on net primary productivity and implications for climate change. Annual Review of Plant Biology. 2012;63:637-661.
6. Nagajyoti PC, Lee KD, Sreekanth TVM. Heavy metals, occurrence and toxicity for plants: a review. Environmental Chemistry Letters. 2010;8(3):199-216.
7. Oke TR. The energetic basis of the urban heat island. Quarterly Journal of the Royal Meteorological Society. 1982;108(455):1-24.
8. Apel K, Hirt H. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annual Review of Plant Biology. 2004;55:373-399.
9. Szabados L, Savouré A. Proline: a multifunctional amino acid. Trends in Plant Science. 2010;15(2):89-97.
10. Hodges DM, DeLong JM, Forney CF, Prange RK. Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues. Planta. 1999;207(4):604-611.
11. Nowak DJ, Crane DE, Stevens JC. Air pollution removal by urban trees and shrubs in the United States. Urban Forestry and Urban Greening. 2006;4(3-4):115123.
12. Escobedo FJ, Kroeger T, Wagner JE. Urban forests and pollution mitigation: analyzing ecosystem services and disservices. Environmental Pollution. 2011;159(89):2078-2087.
13. Ferrini F, Bussotti F, Tattini M, Fini A. Trees in the urban environment: response mechanisms and benefits for the ecosystem. Biologia Plantarum. 2014;58(3):399412.
14. Sjöman H, Hirons AD, Bassuk NL. Urban forest resilience through tree selection-Variation in drought tolerance in Acer. Urban Forestry and Urban Greening. 2015;14(4):858-865.
15. Przybysz A, Saebø A, Hanslin HM, Gawroński SW. Accumulation of particulate matter and trace elements on vegetation as affected by pollution level, rainfall and the passage of time. Science of the Total Environment. 2014;481:360-369.
16. Turkish Statistical Institute. Population and Housing Census 2021. Ankara: TUIK; 2022.
17. Petrova S, Yurukova L, Velcheva I. Possibilities of using deciduous tree species in biomonitoring of heavy metals in urban areas. Atmospheric Environment. 2014;89:750-757.
18. Lichtenthaler HK. Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods in Enzymology. 1987;148:350-382.
19. Beauchamp C, Fridovich I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Analytical Biochemistry. 1971;44(1):276-287.
20. Bates LS, Waldren RP, Teare ID. Rapid determination of free proline for water-stress studies. Plant and Soil. 1973;39(1):205-207.
21. R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. 2022.