The Effect of Gasoline Exhaust Pollution on Some Anatomical, Physiological and Morphological Characteristics of Spinach

Introduction
Depending on the fuel type, the main vehicle exhaust emissions are nitrogen oxides (NOx), carbon oxides, sulfur oxides (SOx), carbon particles, heavy metals, water vapor and hydrocarbons including aldehydes, single hydrocarbons and polyaromatic hydrocarbons, alcohols, olefins, alkyl nitriles that along with secondary pollutants such as ozone, have negative effects on plant health. Agricultural lands which are adjacent to urban areas, are increasingly vulnerable to vehicle pollution.
The plants suffer a lot from the pollution caused by exhaust of vehicles because they can‌ not move away from the source of contamination.
Plants exposed to exhaust of vehicles, show many disruptions in their general appearance, which are referred to as "visible injuries". In fact, these visible damages to the plants reflect the physiological changes that come with the contaminants. These physiological changes may be considered as "hidden injuries".
There is concern about the compounds of contaminants, because there is an evidence that the plant response to multi-pollutant mixtures may be different from plant response to a pollutant. For example, one hour exposure to combination of SO2 gases with concentration of 1310 micrograms per cubic meter and NO2 with concentration of 940 micrograms per cubic meter resulted in low leaf damage, while none of the gases do not cause damage alone in this concentrations.
There are many reports on the effects of pollutants on the anatomy, physiology and morphology of different plant species.
The importance of stomata in plant protection against air pollutants has been investigated and it has been shown that the closure of stomata helps to protect plants from pollution damage.
Vehicle contaminants affect photosynthesis and cause oxidative stress in plants.
Air pollution caused a significant increase in membrane peroxidation in Lavandula officinalis Chaix. and Ligustrum vulgare L. planted in the margin of the streets of polluted and cleaned areas of Tehran.
The activity of Antioxidants enzymes (catalase, peroxidase and ascorbate peroxidase) increased in different levels in contaminated areas compared to clean areas.
There are not enough researches on the effect of gasoline exhaust pollution on leafy vegetables, especially spinach.
According to the considerable amount of cultivated spinach, especially in Iran are in outdoors and on the side of the roads or contaminated urban areas, therefore, the effects of the gasoline exhaust pollutants on some anatomical, physiological and morphological characteristics of spinach (spinacia oleraceae var virofly) was investigated.

Material and method
The experiment was conducted in factorial test based on a completely randomized design with three replications in greenhouse of Isfahan University of Technology.
12 small chambers (length: 100 cm, width 70 cm and height: 70 cm) and an exhaust generator for transferring the contaminants to the chambers were constructed.
VARIOPLUS MRU device was used to analyze the output gases and determine the concentration of contaminants from burning gasoline.
The seeds of Spinacia oleraceae var. viroflay were directly cultured in pots No. 4 which were filled with sand and soil mixtures of 1:2.
When the spinach seedlings reached to 4-leafy stage, transferred to the chambers and exposed separately to the gasoline exhaust pollution for three hours in three different exposure times (10, 20 and 30 days) and after the exposure time, their anatomical, physiological and morphological traits of plants were measured and compared with control plants.
The relative water content was measured by the method of Ritchie et al. (1990). Electrolyte leakage was measured by Lutts et al. (1995).
The total chlorophyll content and carotenoid content of leaves were extracted by 100% acetone solvent method. The absorbance of light at 661.6 and 168.8 for total chlorophyll and 470 nm for carotenoids were read.
The chlorophyll fluorescence concentration was measured by a chlorophyll meter machine (OS-30P model manufactured by Opti-science, UK) and the Fv/Fm ratio (photochemical effect of photosystem 2) was reported.
Factors related to leaf gas exchange were measured by a portable photosynthesis measurement (LCi model manufactured by ADC Bioscientific Ltd, UK). The amount of proline was measured using the Bates method (1973).
Catalase activity was measured using modified Aebi method (1984) at 240 nm wavelength. Ascorbate peroxidase activity was estimated by modified Nakano and Asada (1981) by absorbance reduction at 290 nm. The activity of the guaiacol peroxidase enzyme was determined by the Chance and Maehli method (1955) with a slight change.
Anatomical traits were observed by optical microscope and measured through the Edn-2 software.
Statistical analysis of data of all traits were performed using statistical software Statistix 8 and comparison of meanings performed based on minimum significant difference test (LSD) at 5% probability level.
Discussion of Results
Anatomical, physiological and morphological characteristics of spinach plants affected by gasoline exhaust pollution in every three exposure time.
Exposure to contaminants for 10 days, significantly increased total chlorophyll content, CO2 intracellular concentration, stomatal conductance and net photosynthetic rate compared to controls.
No reduction was seen in all anatomical, physiological and morphological characteristics of spinach plants exposured to contaminants for 10 days compared to the controls.
The number of stomata, proline content and antioxidant enzymes activity including catalase, ascorbate peroxidase and guaiacol peroxidase in plants exposed to contaminants for 20 days showed significant increase compared to the control plants.
Dry weight, relative water content of leaves and stomatal conductance of plants exposed to contaminants for 20 days showed a significant decrease compared to the control plants.
Plants exposed to the pollutants for 30 days, showed significant increases in antioxidant enzymes activity such as catalase, ascorbate peroxidase and guaiacol peroxidase, proline content, electrolyte leakage and the number of stomata per unit compared to controls. But total chlorophyll content, carotenoid, photosynthesis rate, stomatal conductance, chlorophyll fluorescence, relative water content of leaves, fresh weight, dry weight and length of stomata significantly decreased compared to control plants.
Stomata pores in spinach plants that were exposed to the pollutants for 30 days shrinked due to closure of stomata by the pollutants as against other exposure days and control plants.
Significant differences in the xylem and phloem of plants that had the highest and the lowest exposure time to the pollutants were not observed.
Conclusion
Exposure to contaminants for 10 days, significantly increased total chlorophyll content and leaf gas-exchange parameters that are most likely because of the contents in contaminants and also the role of CO2 in photosynthesis process and the presence of nitrogen and sulfur in NO2 and SO2 gases.
Between the three time of exposure in this study, the most trait changes were observed in plants exposed to pollutants for 30 days.
Significant increase in antioxidant enzymes, proline, ion leakage and number of stomata was observed in spinach plants exposed to pollutants for 30 days compared to control plants. The highest increase in this interval was related to the activity of the guaiacol peroxidase enzyme and then the amount of proline.
Among the anatomical traits, the highest change was shown in the number of stoma (62.20%) of plants exposed to pollutants for 30 days compared to controls.
Significant decrease in total chlorophyll, carotenoid, photosynthesis rate, stomatal conductance, chlorophyll fluorescence, relative water content of leaves, fresh weight, dry weight and length of stomata in spinach plants exposured for 30 days was observed compared to control plants.
The highest decrease in plants exposed to pollutants for 30 days compared to controls was observed in stomatal conductance (39.21%) and the lowest reduction was observed in the amount of chlorophyll fluorescence (2.56%).
The results showed that the exposure of spinach plants to pollutants, especially 30-days exposure, caused morphological, physiological and anatomical reactions due to contaminants that could be considered these reactions as the adaptation of these plants to the stress of contamination, in order to adapt or to deal with stress conditions and thus to survive the plant under pollution stress conditions.