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Individual and combined effects of fluoranthene, phenanthrene, mannitol and sulfuric acid on marigold (Calendula officinalis)
Ecotoxicology and Environmental Safety 148 (2018) 834–841
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Individual and combined effects of fluoranthene, phenanthrene, mannitol and sulfuric acid on marigold (Calendula officinalis) Wahdatullah Khpalwaka,b, Sherif M. Abdel-dayema,c, Hiroshi Sakugawaa,
T
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a
Graduate School of Biosphere Science, Hiroshima University, 1-7-1 Kagamiyama, Higashi-Hiroshima 739-8521, Japan Department of Plant Protection, Faculty of Agriculture, Nangarhar University, Nangarhar, Afghanistan c Department of Pesticide Chemistry, Faculty of Agriculture, Kafrelsheikh University, Kafrelsheikh, Egypt b
A R T I C L E I N F O
A B S T R A C T
Keywords: Antioxidant enzyme activities Fluoranthene Marigold Oxidative stress Reactive oxygen species
A study was conducted to characterize marigold stress response to polycyclic aromatic hydrocarbons (PAHs) (oxidative stress inducers) with and without sulfuric acid (S.Acid; pH 3) (acid-stress inducer), and to evaluate reactive oxygen species (ROS) scavenging activity of mannitol (Mann). Marigold (Calendula officinalis) seedlings were grown in a greenhouse and fumigated with fluoranthene (FLU), phenanthrene (PHE), Mann, and S.Acid individually and in various combinations for 40 days. Various physiological and biochemical parameters among others were analyzed using standard methods. The results revealed that fumigation of FLU induced oxidative stress to the plants via ROS generation leading to negative effects on photosynthesis at near saturating irradiance (Amax), stomatal conductance (Gs), internal carbon dioxide concentration (Ci), leaf water relations and chlorophyll pigments. Significant per cent inhibition of Amax (54%), Gs (86%) and Ci (32%), as well as per cent reductions in chlorophyll a (Chl.a) (33%), Chl.b (34%), and total chlorophyll (Tot. Chl) (48%) contents were recorded in FLU fumigated treatment in comparison to control. Combination of Mann with FLU scavenged the generated ROS and substantially lowered the oxidative stress on the plants hence all the measured parameters were not significantly different from control. PHE fumigation had varied effects on marigold plants and was not as deleterious as FLU. Combined fumigation of S.Acid with both the PAHs had significant negative effect on leaf water relations, and positive effect on fresh and turgid weight of the plants but had no effect on the other measured parameters. The lowest proline contents and highest catalase and ascorbate peroxidase activities in FLU fumigated plants further confirmed that oxidative stress was imposed via the generation of ROS. From the results, it is evident that Mann could be an efficient scavenger of ROS-generated by FLU in the marigold plants. We recommend Mann to be widely used for the protection of higher plants from FLU-generated stress in the urban areas.
1. Introduction Rapid urbanization, industrialization, and an increasing global population in the last few decades have resulted in severely polluted environments. The main air pollutants include gases, suspended particles, ionizing radiation, and noise. Polycyclic aromatic hydrocarbons (PAHs) are among the particulate forms, and are universal organic pollutants emitted into the environment either naturally or through anthropogenic activities. Anthropogenic sources of PAHs include fuel combustion, coke production, bush burning, refining, engine exhausts, and cigarette smoke (Poor et al., 2004). Plant leaves and needles are good sinks of atmospheric PAHs. The complexity of plant organs and the route through which PAHs are transported, stored, or processed influence their environmental fate, and are crucial for the annual cycling of these
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pollutants (Wild et al., 2005). The effects of PAHs on plants range from acute (short period) to chronic (long period) toxicities. The PAHs may enter plants through stomata or the roots. However, the uptake of these lipophilic organic pollutants by plants is primarily via diffusion of gas from the air through the waxy layer of leaves (Keymeulen et al., 2001). The mechanisms underlying PAH toxicity involve the inhibition of biological pathways, including those related to photosynthesis and mitochondrial electron transport. Photosynthesis is a very sensitive indicator of plant stress, and analyses of related activities enable detection of stress-induced changes that can be mitigated if detected early enough. Responses to environmental stresses affect plants in various ways including growth, physiology, and molecular biology. A major plant pathway that leads to stress tolerance involves signal transduction, gene
Corresponding author. E-mail address: [email protected] (H. Sakugawa).
https://doi.org/10.1016/j.ecoenv.2017.11.065 Received 26 July 2017; Received in revised form 9 November 2017; Accepted 24 November 2017 Available online 01 December 2017 0147-6513/ © 2017 Elsevier Inc. All rights reserved.
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or vegetation being “natural air sampler” for the accumulation of PAHs (Oguntimehin et al., 2007, 2010) also justifies the selection of marigold as a model plant for the current study. The main objective of the present study was to investigate the additive, antagonistic, or synergistic effects of two PAHs in both neutral pH and acidic pH by combining with S.Acid (pH 3). Specifically the scavenging activity of Mann was also evaluated through foliar application on marigold plants. Furthermore, we analyzed the eco-physiological characteristics of plants, including net photosynthesis rate at near saturated irradiance (Amax), stomatal conductance (Gs), and intercellular CO2 concentration (Ci). Additionally, we evaluated physiological parameters like chlorophyll a (Chl.a), chlorophyll b (Chl.b), and total chlorophyll (Tot. Chl) contents as well as leaf fresh weight (FW), turgid weight (TW), and dry weight (DW), relative water content (RWC%), and leaf water deficit (LWD%). Biochemical parameters, including CAT and APX activities and proline content, were also investigated.
expression, protein activation, protection, and repair. The joint efforts of physiologists and molecular biologists are therefore required to generate plants that can tolerate changes in the global environment (Rao et al., 2006). Fluoranthene (FLU) and phenanthrene (PHE) are among the United States Environmental Protection Agency (USEPA) priority PAHs with four- and three-rings respectively. FLU is the typical example of PAH highly abundant in many countries including Japan and is mainly emitted in automobile exhaust (Poor et al., 2004). PHE is also widely distributed in the environment which is transferred on the leaf surfaces through atmospheric deposition and mainly accumulates in the lipid layer of plant membranes (Simonich and Hites, 1994). The two PAHs were selected as model compounds because they are ubiquitous in the environment and their accumulation in plants is associated with diverse problems. Additionally, plants are highly sensitive to their presence and/or accumulation. The toxicity of PAHs such as FLU and PHE in plants leads to production of reactive oxygen species (ROS) (Oguntimehin et al., 2007). The damage caused by the generated ROS mostly depends on the type and concentration of PAHs, intensity and duration of the exposure, plant developmental stage, and plant species. The reaction centers of photosystems I and II in chloroplast thylakoids are the major sites of ROS formation. Excessive ROS production induces abiotic and oxidative stresses in plants. Elevated levels of non-metabolized cellular H2O2 can even damage cellular lipids and proteins as well as inactivate key cellular functions (Gill and Tuteja, 2010). Stressinducing H2O2 is normally metabolized and maintained at concentrations necessary for plant growth and development by catalase (CAT) and ascorbate peroxidase (APX) (Gill and Tuteja, 2010). Catalase activity is usually observed in peroxisomes, and this enzyme is activated by relatively high H2O2 concentrations. APX can detoxify even very low concentrations of H2O2 by using ascorbate as the main substrate, and it is active primarily in chloroplasts, the cytosol, mitochondria, and peroxisomes (Gechev et al., 2006). PAHs may act as stressors on the plants both individually or simultaneously with the acid-deposition. Acid deposition or acid rain is natural precipitation of sulfuric or nitric acid in the forms of rain, snow, fog or dusts. Two major sources of acid rain includes natural such as volcanoes and anthropogenic such as burning of coal and fossil fuels, power generation, and exhaust from vehicles and industries. Exposure to high acidity can kill freshwater fish, adversely affect soil chemistry and nutrient availability, inhibit plant growth and physiology, and increase the susceptibility of plants to pests and diseases (Casiday and Frey, 1998; Gheorghe and Ion, 2011). Oguntimehin et al. (2012) in a study of PAHs alone and in combination with the acid mist reported aggravation of the negative effects of PAHs on the plants by acid mist. Mannitol (Mann) is naturally produced in some plants and is a known and efficient scavenger of ROS generated by cell-free oxidantgenerating systems (Upham and Jahnke, 1986). Oguntimehin et al. (2012) reported that Mann has the ability to mitigate the negative effects of acid mist and Fluoranthene in the Japanese red pine. They concluded that Mann was able to reduce the activities of ROS both inside and on the foliar part of the plants. They further recommended Mann to be used in the protection of pine trees and other plants from the air pollutants. Marigold (Calendula officinalis L.), an annual or biennial plant that produces yellow or orange flower heads, belongs to the Asteraceae family. Its leaves and flowers have been valued for their pharmaceutical and cosmetic properties. Possible uses for marigold contents as alternatives to volatile organic compounds in paints have recently been explored by the paint and coating industry. The medicinal and industrial importance of marigold, its capability of absorbing the highest concentration of PAHs from the environment (Nicoleta-Adela HIRISCSU, doctoral thesis, 2015), relatively faster growth and short life cycle were some of the important characteristics considered for its selection in the current study. Moreover, some similar characteristics of marigold with Sunpatiens (Impatiens spp.) as a good bio-indicator and/
2. Materials and methods 2.1. Plant materials and growth conditions Marigold seedlings, pots (25 × 25 cm deep), and commercial soil (Humus) (Iris Ohyama Co. Ltd., Sendai, Japan) were purchased from a nursery at NAFCO (Higashihiroshima, Japan) on October 9, 2015. The pots, with a 15 × 15 cm mesh placed at the bottom, were filled with 10 L soil (i.e., approximately 80% full). One seedling was transferred per each pot, watered thoroughly and kept in a greenhouse (metalframed shelter) at Hiroshima University in Higashihiroshima, Japan. The shelter was well-ventilated, 8 m × 12 m semi-cylindrical structure covered with a 0.06-mm thick Tefzel® film transparent to visible and UV-A light (DuPont, Wilmington, DE, USA) to prevent rain and dew droplets from directly contacting the seedlings. Seedlings were grown normally for approximately 3 months. During this period, they were manually irrigated two or three times per week, depending on the needs of the plants. To avoid being exposed to cold conditions, plants were kept in open-top chambers from early December 2015 to early April 2016. The open-top chambers were covered with the F-CLEAN® transparent ethylene-tetrafluoroethylene copolymer film (Asahi Glass Green-Tech Co. Ltd., Japan), which allowed more than 95% of the sunlight to pass through. A similar experimental design was used in previous studies by Oguntimehin et al. (2007) and Nakatani et al. (2007). Before fumigating the plants, 15 g NPK+Mg granular fertilizer was applied to each pot and repeated after 2 weeks. During these 5 months in the chambers, the pots were rotated monthly to avoid any kind of bias due to their position. 2.2. Treatment preparation and fumigation Plants were fumigated with the following 12 treatments: FLU, PHE, S.Acid, Mann, FLU+Mann, FLU+S.Acid, FLU+Mann+S.Acid, PHE +Mann, PHE+S.Acid, PHE+Mann+S.Acid, Mann+S.Acid, and MilliQ water (control). All treatments were replicated four times i.e., each treatment contained four healthy marigold seedlings. The seedlings were fumigated for 40 days from February to April 2016. Stock solutions of 1 mM FLU (98%, Sigma Aldrich, USA) and PHE (99%, Wako pure chem. Ind. Osaka, Japan) were prepared by dissolving 202.26 mg and 178.23 mg in 1 L 50% aqueous acetone (Sigma Aldrich, USA) and Milli-Q water (Millipore Co., Japan), respectively. The solutions were diluted to 10 µM with Milli-Q water for the fumigation treatments. The final FLU and PHE concentrations were comparable to the lowest concentrations used in previous study by Oguntimehin et al. (2007). The 0.5% acetone in the final FLU solution was previously determined to have no effect on any of the analyzed parameters (Oguntimehin et al., 2007). The 200 mM stock Mann (99%, Nacalai Tesque Inc. Kyoto, Japan) 835
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25 mM potassium phosphate buffer, 0.25 mM ascorbic acid, 0.1 mM EDTA, 0.1 mM fresh H2O2, and 5% (v/v) crude enzyme extract. The oxidation of ascorbate was measured at 290 nm using UV–Vis spectrophotometer. The extinction coefficient used for the calculation was 2.8 mM−1 cm−1, and one unit of APX was determined as the enzyme amount required to oxidize 1 mmol ascorbate in 1 min.
solution was prepared by adding 36.4344 g Mann in 1 L Milli-Q water. The solution was then diluted to 1 mM for the fumigation. Additionally, the 0.1 M stock S.Acid (Nacalai Tesque Inc. Kyoto, Japan) solution was prepared by adding 1.06 mL acid to 150 mL Milli-Q water, and topped up to 200 mL. The stock solution was diluted in Milli-Q water and the pH was adjusted to 3 as measured by the D-51 pH meter (Horiba, Japan) prior to fumigation of seedlings. The solutions were applied to the foliar parts of marigold seedlings using an electronic sprayer with a nozzle (BS-4000, Fujiwara Sangyo, Miki, Japan) from Monday through Friday from 9:00–11:00 a.m. Each seedling received an average of 25 mL solution per day. For all combination treatments, PAHs were pre-applied and allowed to dry for approximately 30 min before the application of S. Acid and Mann.
2.7. Measurement of proline content Proline content was measured using a modified version of the method described by Bates et al. (1973). Frozen leaf tissues were homogenized in liquid nitrogen using a pestle and mortar. The ground samples were suspended in 10 mL 3% sulfosalicylic acid and then centrifuged twice for 5 min each at 10,000×g in 4 °C. The supernatants were collected, and 2-mL aliquots of the extracts were mixed with 2 mL ninhydrin reagent containing glacial acetic acid. Samples were incubated for 1 h in a water bath set at 100 °C, after which they were cooled in an ice bath and then treated with 4 mL toluene (Cica-Reagent, Kanto Chemical Co. Inc., Tokyo, Japan). The solutions were mixed vigorously for approximately 20 s to ensure that the chromophore was in the toluene layer. The absorbance of the chromophore was measured at 520 nm.
2.3. Analysis of photosynthetic activity and gas exchange The Amax, Gs, and Ci were measured with the LI-6400 open-flow infrared gas analyzer (LI-COR Inc., Lincoln, NE, USA) at the end of the fumigation period. Fresh leaves exposed to sunlight were randomly selected for the measurements. The leaves were kept in the leaf chamber in such a way that the entire 2 × 3 cm2 internal area of the chamber was covered with the leaf. The measurements were completed from 10:00 a.m. to 1:00 p.m. on sunny days with a saturating irradiance of 1500 µmol m−2 s−1 (i.e., photosynthetic photon flux density). Leaf temperatures were 20 ± 2 °C, while the leaf-to-air vapor pressure deficit ranged from 0.80 to 1.32 kPa, and the leaf-to-air CO2 concentration was fixed at 370 µmol CO2 mol−1 at 500 µmol s−1.
2.8. Statistical analysis Data were analyzed with the SPSS program (PASW Statistics 18. Ink). The results for the biochemical parameters were based on the average values for three replicates, whereas the data for all other parameters were based on the average values for four replicates. The data are presented as the mean ± standard error. Significant differences among the groups were calculated by a one-way analysis of variance after verifying the homogeneity and normality of the variance (p < 0.05). The means were compared using Duncan's multiple range test (p < 0.05). The relationships among some parameters were evaluated based on Pearson's correlation coefficient (r; p < 0.05 and 0.01).
2.4. Leaf water relations The RWC% was measured as described by Weatherly (1950). Briefly, fresh mature leaves cut into 1-cm2 disks were immediately weighed to determine FW. They were then incubated in Milli-Q water for approximately 24 h under fluorescent light to absorb water, after which excess water was removed by placing the leaf samples on tissue paper. The leaf disks were then weighed to determine TW, and then incubated in a DKN602 drying oven (Yamato Scientific Co. Ltd., Japan) at 70 °C for 72 h to obtain the DW. The RWC% was calculated with the following formula:
3. Results and discussion 3.1. Effect of fumigation on gas exchange parameters
RWC % = (FW − DW )/(TW − DW ) × 100
A summary of results on the effects of fumigation on gas exchange parameters is given in Fig. 1 (A, B and C). From the results, it is evident that the PAHs had different effects on the fumigated marigold plants. Although FLU and PHE treatments negatively affected all the measured gas exchange parameters (i.e., Amax, Gs, and Ci) (Fig. 1), the effects of FLU were significantly different and greater than those of PHE. Highest per cent inhibition of Amax (54%), Gs (86%) and Ci (32%) were recorded in FLU treatment compared to control. The elevated Amax, Gs and Ci values of PHE treatment were however not significantly different (p < 0.05) from the control. The addition of Mann to PAHs treatments had a considerable mitigating effect on FLU, recording 43, 0.6 and 287 compared to control values 38, 0.9 and 247 for Amax, Gs and Ci respectively and were no more statistically different (Fig. 1 A, B, and C). In contrast, the addition of Mann to the PHE treatment had no significant effect and was on par with both PHE-alone and control. Additionally, the Amax, Gs, and Ci values appeared to decrease slightly in response to S.Acid alone or in combination with PAHs or Mann, but the values were not significantly different from the control measurements and the differences were almost negligible. The decrease in Amax, Gs and Ci could be attributed to ROS generation in the cells because of PAH fumigation. Pell et al. (1997) also reported reduction in CO2 assimilation due to ROS accumulation. The stomatal conductance (Gs) decline rapidly in the presence of air pollutants in the plants and thus may lead to stomatal closure. The stomatal closure is therefore considered a good indicator and a valid mechanism of combating environmental stress in the plants (Lefohn,
The LWD% was calculated according to the method described by Kalapos (1994) using the following equation: LWD% = 100 − RWC% 2.5. Chlorophyll concentration Fresh leaves were cut into 1-cm2 disks for a subsequent analysis of chlorophyll contents (i.e., Chl.a, Chl.b, and Tot. Chl). The pigments were extracted with 5 mL NN-dimethylformamide and the concentration was determined by the method of Porra et al. (1989). 2.6. Antioxidant enzymes activities The protein concentrations of leaf enzyme extracts were measured using a protein assay kit (Nacalai Tesque Inc.), based on the Bradford (1976) method. Bovine serum albumin was used as the standard and the enzyme activities were calculated using the protein concentration. The CAT (EC 1.11.1.6) and APX (EC 1.11.1.11) activities were analyzed according to the method developed by Takagi and Yamada (2013). For the CAT activity measurement, the 1-mL assay mixture consisted of 50 mM potassium phosphate buffer (pH 7.0), 10 mM fresh H2O2, and 5% (v/v) crude enzyme extract. Decreases in H2O2 content were monitored at 240 nm, and CAT activity was calculated as mmol H2O2 consumed per minute. The extinction coefficient used for the calculation was 0.04 mM−1 cm−1. The APX activity was measured in a 1-mL assay mixture containing 836
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Fig. 1. Gas exchange parameters (A) Net photosynthesis rate at near saturated irradiance (Amax), (B) Stomatal conductance (Gs) and (C) Intercellular CO2 concentration (Ci). Data are presented as the average value for four marigold seedlings. Identical superscript alphabetical letters indicate the same homogenous groups; different alphabetical letters indicate significant differences at p < 0.05 (Duncan's multiple range test) within each histogram panel.
It was also evident from our study that Mann is an effective scavenger and was able to scavenge the OH radicals generated by PAHs, especially FLU. The current findings on Mann are in accordance with those of Oguntimehin and Sakugawa (2009). Furthermore, we confirm the recommendation by Oguntimehin et al. (2012) to widely use Mann for protection of higher plants from air pollutants.
1991). Despite the existing controversy on whether Amax decreases because of stomatal closure or non-stomatal factors, the current study showed that, the PAHs decreased the Amax (Fig. 1A), Gs (Fig. 1B), and Ci (Fig. 1C). The observed phenomenon could be attributed to stomatal closure, and this is supported by the strong positive correlation between Amax vs Gs, Amax vs Ci and Gs vs Ci as (r = 0.7, 0.6, 0.6 at p < 0.01) obtained in the current study. Kreslavski et al. (2014) reported that in the plants the PAHs especially FLU and PHE accumulates in the thylakoid membranes and leads to alteration of the thylakoids as well as development of oxidative stress that are detrimental to photosynthetic activities and other physiological processes. The results on gas exchange parameters in the current study are consistent with that of Oguntimehin et al. (2007) in which Japanese red pine seedlings were fumigated with FLU and PHE. Our results are also in line with a study by Tomar and Jajoo (2014) in which the phytotoxicity of FLU in wheat plants and the inhibiting effects of FLU on photosynthesis were attributed to the inhibition in the process of CO2 fixation. However the effect of S.Acid alone or in combination with other fumigants contradicted an earlier study that concluded that the presence of FLU exacerbated the adverse effects of an acid treatment (Oguntimehin et al., 2012). This discrepancy is probably because, in the current study each plant received a total of 0.44 × 10−3 mol plant−1 S.Acid during the entire exposure experiment which was three fold lower (1.5 × 10−3 mol plant−1) than those of Oguntimehin et al. (2012). In addition, the plant species used in the current study were different from the previous studies, hence the discrepancies.
3.2. Effect of fumigation on FW, TW, DW, RWC%, and LWD% Results in Table 1 revealed that PHE+S.Acid treatment recorded the highest values (0.11) and (0.15) in comparison to the PHE treatment (0.06) and (0.1) for FW and TW respectively. There were no significant differences (p < 0.05) in any of the treatments except in the FW of PHE+S.Acid in comparison to Control. FW value (0.1) in PHE +S.Acid was the highest among all the other treatments and was significantly different (p < 0.05) from both the Control and PHE treatments. In addition, TW in PHE+S.Acid was the highest and significantly different from PHE treatment but not Control. Generally, the current study revealed that the PAHs had no substantial effects on the FW, TW and DW. However, highest values of FW and TW in PHE+S.Acid treatment could be due to the following reasons; S.Acid at relatively low concentrations might be either detoxified or used in a reductive sulfur cycle to form amino acids or may increase the availability of iron and phosphorus, or improve water penetration in the soils (Ryan et al., 1973; Yahia et al., 1975), whereas PHE is low molecular weight, highly volatile and unstable PAH in comparison to FLU which perhaps pose 837
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chlorophyll content in both FLU and PHE treatments were not significantly different (p < 0.05) from the control values. Nevertheless, Chlorophyll contents except Chl.b in FLU and PHE treatments were significantly different (p < 0.05) from each other. An earlier study by Oguntimehin et al. (2010) confirmed that PAHs can alter chlorophyll pigments in the plants and the reduction in chlorophyll pigments was recognized as an indicator of stress, which was further attributed to the reduction in the chlorophyll pigment capacity of the plants. This finding is consistent with the observations of Oguntimehin et al. (2007) which reported the negative effects of FLU and PHE on certain parameters including total chlorophyll contents in Japanese red pine seedlings, with FLU having greater effect than PHE. Tomar and Jajoo (2015) also reported a decrease in chlorophyll content due to FLU fumigations. In the current study, fumigation of plants with PHE positively affected chlorophyll contents, with the abundance of both Chl.a and Tot. Chl increasing by 32%, and the Chl.b content increasing by 24%. A combined treatment of Mann and PHE caused the chlorophyll level to decline, with Chl.a, Chl.b, and Tot. Chl contents decreasing by 13%, 20%, and 13%, respectively, however, they were not significantly different from control (Table 2). The possible explanation for the effect of PHE on chlorophyll content in the current study could be; PHE is highly volatile in nature, having high vapor pressure (1.21 × 10−4 mm Hg at 25 °C) and easily degradable while FLU has low vapor pressure (9.22 × 10−6 mm Hg at 25 °C) and less degradable (Shiu and Mackay, 1997; Wasik et al., 1983). A part of it, might be evaporated immediately after the fumigation and part of it might have been degraded as reported by Min and Xie (2006) which in both the cases it ultimately results in lower concentration of PHE in the plants which thus results in the synthesis of chlorophyll pigments as reported by Shen et al. (2016). PAHs effect on plants is concentration-dependent. Under lower dose, plant growth might be enhanced because PAHs could be stressors or “hormone” (Laughlin et al., 1982). Apart from that, the short duration of the current study might also be recognized as a limiting factor for evaluating the effect of FLU and the scavenging effect of Mann with special reference to the chlorophyll pigments. But assuming the longer exposure of some biennial or perineal crops, this effect may get clearer and easily visible. Hence, the findings of this study imply that FLU induced the production of free hydroxyl radicals, and the resulting oxidative stress likely had an inhibitory effect on photosystem II as well as on overall chlorophyll pigments producing system (Eisenberg and Cunningham, 1985; ElAlawi et al., 2002). The addition of Mann to the FLU treatment prevented generation of ROS and restricted the ability of FLU to induce oxidative stress.
Table 1 Fresh weight, turgid weight and dry weight of cm2 leaf (g)*. Treatments/ Parameters*
Fresh.W
Turgid.W
Dry.W
Control Mann S.Acid Mann+S.Acid FLU FLU+Mann FLU+S.Acid FLU+Mann+S.Acid PHE PHE+Mann PHE+S.Acid PHE+Mann+S.Acid
0.05 ± 0.01 cde 0.07 ± 0.02bcde 0.08 ± 0.01abc 0.05 ± 0.003de 0.05 ± 0.01de 0.04 ± 0.003e 0.04 ± 0.004e 0.08 ± 0.01abcd 0.06 ± 0.01bcde 0.09 ± 0.01ab 0.1 ± 0.02a 0.04 ± 0.01e
0.12 ± 0.01abcd 0.09 ± 0.02bcd 0.13 ± 0.02ab 0.1 ± 0.01bcd 0.09 ± 0.02bcd 0.07 ± 0.02d 0.08 ± 0.01cd 0.13 ± 0.004abc 0.1 ± 0.01bcd 0.12 ± 0.01abcd 0.15 ± 0.03a 0.09 ± 0.03bcd
0.008 ± 0.0003abc 0.007 ± 0.001c 0.01 ± 0.001ab 0.007 ± 0.0003bc 0.008 ± 0.001abc 0.006 ± 0.0002c 0.006 ± 0.001c 0.011 ± 0.001a 0.009 ± 0.001abc 0.008 ± 0.001abc 0.011 ± 0.002a 0.008 ± 0.002abc
* Data are presented as the average value for three marigold plants ± standard error. Identical superscript alphabetical letters indicate the same homogenous groups; different alphabetical letters indicate significant differences at p < 0.05 (Duncan's multiple range test).
lesser stress to the plants. Results on RWC% and LWD% (Fig. 2) showed that generally, RWC% and LWD% were lowered and increased respectively as a result of fumigations. Despite this phenomenon predominant, the effects were more pronounced with S.Acid treatments. Significantly lower RWC% and higher LWD% in both PHE and PHE +S.Acid treatments were recorded in comparison to control (Fig. 2). Sulfuric acid may acidify cell wall and may harm its stability resulting in a decline in the leaf water content (Anderson, 1972). Current results are in line with that of Gadallah and Sayed (2001) who studied the impact of Kinetin on water relations in sorghum plants at different levels of acidity and reported that plants at pH 3 maintained lower water content than that of neutral pH. In brief, lower pH (pH 3) had greater negative effects on plant leaf water relations in PHE treatment compared to all the other treatments in the current study. 3.3. Effect of fumigation on chlorophyll concentration There were significant differences in the chlorophyll contents (Chl.a, Chl.b, and Tot. Chl.) depending on the fumigation treatments (Table 2). Plants fumigated with FLU recorded the lowest Chl.a, Chl.b, and Tot. Chl contents (36, 11, 48) compared to control (53, 17, 71), which were 33%, 34%, and 32% lower than the control values, respectively. The PHE-treated plants recorded the highest Chl.a, Chl.b, and Tot. Chl contents (70, 21, 93), which were 32%, 24%, and 32% higher than the control measurements respectively. However,
Fig. 2. Relative water content % and leaf water deficit %. Data are presented as the average value for four marigold seedlings. Identical superscript alphabetical letters indicate the same homogenous groups; different alphabetical letters indicate significant differences at p < 0.05 (Duncan's multiple range test) within each histogram panel.
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Table 2 Chlorophyll contents (µg/ cm2 leaf) and reduction percentage. Treatments/Parameters*
Chl.a
Chl.a % reduction
Chl.b
Chl.b % reduction
Tot. Chl
Tot. Chl % reduction
Control Mann S.Acid Mann+S.Acid FLU FLU+Mann FLU+S.Acid FLU+Mann+S.Acid PHE PHE+Mann PHE+S.Acid PHE+Mann+S.Acid
53 ± 4abcd 61 ± 1abc 49 ± 6bcd 52 ± 9abcd 36 ± 3d 49 ± 3bcd 43 ± 3 cd 63 ± 7ab 70 ± 5a 46 ± 4bcd 59 ± 8abc 49 ± 8bcd
0.01 −14 8 2 33 8 18 −19 −32 13 −12 7
17 ± 2a 19 ± 1a 16 ± 2a 23 ± 8a 11 ± 1a 18 ± 2a 17 ± 1a 22 ± 5a 21 ± 4a 13 ± 3a 18 ± 5a 12 ± 2a
0 −17 1.6 −36 34 −7.8 −2.8 −32 −24 20 −7.8 27
71 ± 6abc 80 ± 2abc 65 ± 22c 70 ± 12abc 48 ± 3c 65 ± 4abc 59 ± 3bc 82 ± 8ab 93 ± 6a 62 ± 5abc 80 ± 11abc 66 ± 11abc
0 −13 8 2 32 8 17 −15 −32 13 −12 7
* Data are presented as the mean value for three marigold plants ± standard error. Identical superscript alphabetical letters indicate the same homogenous groups; different alphabetical letters indicate significant differences at p < 0.05 (Duncan's multiple range test).
Plants fumigated with FLU exhibited increased CAT and APX activities, while FLU+Mann and control treatments did not significantly influenced the activities of those enzymes (Table 3). In addition, CAT activities in both FLU+Mann and FLU+S.Acid treatments were significantly lower form FLU treatment but not from the control. This suggests that CAT is very sensitive enzyme and react rapidly to the presence of oxidative stress, mainly H2O2. Takagi and Yamada (2013) reported that CAT was the most efficient enzyme among SOD, APX and GR in overcoming H2O2 stress in chenopodiaceous halophytes. The enzymatic activities induced by PHE were not as high as those induced by FLU, suggesting PHE was not as deleterious to the plants as FLU. This is probably because of the difference in molecular weight, vapor pressure, volatility and some other characteristics of FLU and PHE, as discussed earlier in details (3.3. section). The accumulation of ROS in response to FLU increases enzymatic activities, the extent of which is usually dependent on the oxidative stress imposed by FLU as well as the presence of free radicals (Smirnoff, 1993). Plants exposed to oxidative stresses, especially H2O2, exhibit upregulated CAT activity to scavenge the overproduced H2O2 and stabilize the cellular ROS levels (Jannat et al., 2012). Highest APX activity in FLU treatment compared to all the other treatments in the current study (Table 3) suggests that APX is not as sensitive to the oxidative stress as CAT and was only increasing in the highly stressed condition such as in FLU treatment but were not reacting in the mild/ lower stressed conditions. The lower CAT and APX activities in control (Table 3) were likely the result of a lack of stress. However, lower CAT and APX activities in FLU+Mann-treated plants suggest that the ROS generated by FLU was quickly quenched by Mann (i.e., no oxidative stress) but yet the plants needed some time to get relief and overcome the stressed conditions successfully. The findings from the current study on CAT activity contradicts that of Tomar and Jajoo (2015) which reported lowered CAT activity in the wheat plants after oxidative stress by FLU. This difference might be due to the difference in plant species as well as different environmental and study conditions.
3.4. Effect of fumigation on antioxidant enzyme activities The highest proline contents (0.07) were recorded for the control plants and were significantly different (p < 0.05) from all the other treatments (Table 3). Only slight variations were observed among the other treatments, but the differences were not significant. Proline is always present in plants, because it is vital for developing plants (e.g., from transition to the seed development stage). High proline content in the plants is preferable to several species of insects. Plants with higher level of proline are usually used as trap crops in the insect management. Marigold is reported to be a good trap crop in management of several species of insects (Srinivasan et al., 2008) which thus justifies the higher level of proline in control plants. Proline is catabolized immediately after plants recovery from any kind of stress. Findings from the current study on proline contents are consistent with those of Khalil et al. (2016). Previous investigations confirmed that proline content increases as an adaptive plant response. Immediately after plants overcome stress conditions, the accumulated proline is rapidly oxidized to glutamate. The lowest proline contents following the FLU and PHE treatments were due to the downregulation of proline contents after the plant antioxidant system responded to the stress condition. Proline is an ROS scavenger that can maintain cell membrane permeability as well as increase enzyme activities and stabilize protein structures (Yuyan et al., 2013). The rapid proline catabolism in plants recovering from stress conditions helps to induce some metabolic pathways, including those affecting the restoration of chloroplast activities (Vankova et al., 2012).
Table 3 Changes to antioxidant enzyme activities. Treatments/ Parameters*
Proline (µmol g−1 FW)
Catalase (U. mg protein−1 min−1)
APX (U. mg protein−1 min−1)
Control Mann S.Acid Mann+S.Acid FLU FLU+Mann FLU+S.Acid FLU+Mann +S.Acid PHE PHE+Mann PHE+S.Acid PHE+Mann +S.Acid
0.07 ± 0.003a 0.02 ± 0.001b 0.01 ± 0.002b 0.02 ± 0.001b 0.02 ± 0.002b 0.02 ± 0.003b 0.02 ± 0.001b 0.02 ± 0.002b
35 ± 2bcd 5 ± 1d 37 ± 5b 11 ± 2bcd 240 ± 12a 20 ± 1bc 38 ± 15bcd 14 ± 2 cd
0.4 ± 0.03b 0.1 ± 0.02b 0.4 ± 0.1b 0.1 ± 0.03b 4.2 ± 0.9a 0.3 ± 0.01b 0.3 ± 0.1b 0.2 ± 0.04b
0.01 ± 0.002b 0.02 ± 0.005b 0.02 ± 0.003b 0.01 ± 0.001b
18 ± 5bcd 30 ± 14b 26 ± 3bc 16 ± 1bcd
0.2 ± 0.1b 0.3 ± 0.1b 0.2 ± 0.03b 0.2 ± 0.03b
4. Conclusions This study evaluated the individual and combined effects of fluoranthene, phenanthrene, mannitol and sulfuric acid on marigold (Calendula officinalis). The results revealed that FLU and PHE fumigations are detrimental to some ecophysiological and biochemical parameters of marigold plants. The PAHs, in particular FLU induced significant oxidative stress in terms of reduction in gas exchange parameters and chlorophyll pigments, imbalance in leaf water relations, and increase in antioxidant enzymes activities via generation of ROS. The oxidative stress was not induced when Mann (an ROS-scavenger) was combined with FLU, and this implies that the ROS generated was quenched by Mann. In contrast, PHE-fumigation had varied effects on
* Data are presented as the mean value for three marigold plants ± standard error. Identical superscript alphabetical letters indicate the same homogenous groups; different alphabetical letters indicate significant differences at p < 0.05 (Duncan's multiple range test).
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the physiological and biochemical parameters of marigold plants and was not as deleterious as FLU. Furthermore, combination of S.Acid (pH 3) with both FLU and PHE had varied effect on the measured parameters including the deleterious effect on leaf water relations and beneficial in the FW and TW of the plants. This is most likely because the S.Acid concentration used in this study was not high enough and served as a source of sulfur, required for normal seedling development. Moreover, antioxidant enzymes activities enhanced to ROS accumulation especially due to FLU fumigation and such activities were not high enough when Mann was combined with FLU. Generally, the FLU fumigation induced severe oxidative stress to marigold plants by generating ROS which was well represented by alteration of gas exchange parameters, lowered chlorophyll pigments and enhanced antioxidant enzymes activities, and such changes were not observed when ROS was quenched by the addition of Mann. The effect of PHE was not as deleterious as FLU on marigold plants and will require further investigations in both neutral and acidic pH. Mannitol seemed to be more effective against high molecular weight PAH (having low volatility) than low molecular weight PAH (highly volatile) such as FLU and PHE respectively. In light of the current study, Mann is recommended to be widely used for the protection of higher plants from high molecular weight PAHs-stress in the urban areas.
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Acknowledgments The authors thank Dr. Hayam Ibrahim Attia Elsawy for her technical assistance, Dr. Kazuhiko Takeda and Mr. Hoseki Ueno for assistance throughout the experiment and Mr. Russel Chidya for his inputs in the revision of manuscript. Funding This work was partly supported by Project for the Promotion and Enhancement of the Afghan Capacity for Effective Development (PEACE) from Japan International Cooperation Agency (JICA). Conflicts of interest None. References Anderson, W.P., 1972. Ion transport in the cells of higher plant tissues. Annu. Rev. Plant Physiol. 23, 51–72. Bates, L.S., Waldren, R.P., Teare, I.D., 1973. Rapid determination of free proline for water stress studies. Plant Soil. 39, 205–207. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Casiday, R., Frey, R., 1998. Acid rain. Inorganic Reactions Experiment Tutorial. Department of Chemistry, Washington University. Retrieved February 2012. 〈http:// www.chemistry.wustl.edu/wedudev/LabTutorials/Water/FreshWater/acidrain. html〉. Eisenberg, W.C., Cunningham, D.L.B., 1985. Analysis of polycyclic aromatic hydrocarbons in diesel emissions using high performance liquid chromatography: a method development study. In: Cook, M., Dennis, A.J. (Eds.), Polycyclic Aromatic Hydrocarbons: Mechanisms Methods, and Metabolism. Columbus, Battelle Press, OH, pp. 79–393. El-Alawi, Y.S., McConkey, B.J., Dixon, D.G., Greenberg, B.M., 2002. Measurement of short and long-term toxicity of polycyclic aromatic hydrocarbons using luminescent bacteria. Ecotoxicol. Environ. Saf. 51, 12–21. Gadallah, M.A.A., Sayed, S.A., 2001. The impact of Kinetin application on water relations, leaf osmotic potential and soluble carbon and nitrogen compound contents in Sorghum bicolor plants growing at varying levels of soil acidity. Pak. J. Biol. Sci. 4, 10–16. http://dx.doi.org/10.3923/pjbs.2001.10.16. Gechev, T.S., Van Breusegem, F., Stone, J.M., Denev, I., Laloi, C., 2006. Reactive oxygen species as signals that modulate plant stress responses and programmed cell death. Bio Essays 28, 1091–1101. Gheorghe, I.F., Ion, B., 2011. The effect of air pollutants on vegetation and the role of vegetation in reducing atmospheric pollution. pp. 241–280. Available online; 〈http://cdn.intechopen.com/pdfs-wm/18642.pdf〉. Gill, S.S., Tuteja, N., 2010. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol. Biochem. 48, 909–930.
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water penetration into dry calcareous and sodic soils. Soil Sci. Sot. Am. Proc. Yuyan, An, Zhang, M., Liu, G., Han, R., Liang, Z., 2013. Proline accumulation in leaves of Periploca sepium via both biosynthesis up-regulation and transport during recovery from severe drought. PLoS ONE 8 (7), e69942. http://dx.doi.org/10.1371/journal. pone.0069942.
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Individual and combined effects of fluoranthene, phenanthrene, mannitol and sulfuric acid on marigold (Calendula officinalis) Wahdatullah Khpalwaka,b, Sherif M. Abdel-dayema,c, Hiroshi Sakugawaa,
T
⁎
a
Graduate School of Biosphere Science, Hiroshima University, 1-7-1 Kagamiyama, Higashi-Hiroshima 739-8521, Japan Department of Plant Protection, Faculty of Agriculture, Nangarhar University, Nangarhar, Afghanistan c Department of Pesticide Chemistry, Faculty of Agriculture, Kafrelsheikh University, Kafrelsheikh, Egypt b
A R T I C L E I N F O
A B S T R A C T
Keywords: Antioxidant enzyme activities Fluoranthene Marigold Oxidative stress Reactive oxygen species
A study was conducted to characterize marigold stress response to polycyclic aromatic hydrocarbons (PAHs) (oxidative stress inducers) with and without sulfuric acid (S.Acid; pH 3) (acid-stress inducer), and to evaluate reactive oxygen species (ROS) scavenging activity of mannitol (Mann). Marigold (Calendula officinalis) seedlings were grown in a greenhouse and fumigated with fluoranthene (FLU), phenanthrene (PHE), Mann, and S.Acid individually and in various combinations for 40 days. Various physiological and biochemical parameters among others were analyzed using standard methods. The results revealed that fumigation of FLU induced oxidative stress to the plants via ROS generation leading to negative effects on photosynthesis at near saturating irradiance (Amax), stomatal conductance (Gs), internal carbon dioxide concentration (Ci), leaf water relations and chlorophyll pigments. Significant per cent inhibition of Amax (54%), Gs (86%) and Ci (32%), as well as per cent reductions in chlorophyll a (Chl.a) (33%), Chl.b (34%), and total chlorophyll (Tot. Chl) (48%) contents were recorded in FLU fumigated treatment in comparison to control. Combination of Mann with FLU scavenged the generated ROS and substantially lowered the oxidative stress on the plants hence all the measured parameters were not significantly different from control. PHE fumigation had varied effects on marigold plants and was not as deleterious as FLU. Combined fumigation of S.Acid with both the PAHs had significant negative effect on leaf water relations, and positive effect on fresh and turgid weight of the plants but had no effect on the other measured parameters. The lowest proline contents and highest catalase and ascorbate peroxidase activities in FLU fumigated plants further confirmed that oxidative stress was imposed via the generation of ROS. From the results, it is evident that Mann could be an efficient scavenger of ROS-generated by FLU in the marigold plants. We recommend Mann to be widely used for the protection of higher plants from FLU-generated stress in the urban areas.
1. Introduction Rapid urbanization, industrialization, and an increasing global population in the last few decades have resulted in severely polluted environments. The main air pollutants include gases, suspended particles, ionizing radiation, and noise. Polycyclic aromatic hydrocarbons (PAHs) are among the particulate forms, and are universal organic pollutants emitted into the environment either naturally or through anthropogenic activities. Anthropogenic sources of PAHs include fuel combustion, coke production, bush burning, refining, engine exhausts, and cigarette smoke (Poor et al., 2004). Plant leaves and needles are good sinks of atmospheric PAHs. The complexity of plant organs and the route through which PAHs are transported, stored, or processed influence their environmental fate, and are crucial for the annual cycling of these
⁎
pollutants (Wild et al., 2005). The effects of PAHs on plants range from acute (short period) to chronic (long period) toxicities. The PAHs may enter plants through stomata or the roots. However, the uptake of these lipophilic organic pollutants by plants is primarily via diffusion of gas from the air through the waxy layer of leaves (Keymeulen et al., 2001). The mechanisms underlying PAH toxicity involve the inhibition of biological pathways, including those related to photosynthesis and mitochondrial electron transport. Photosynthesis is a very sensitive indicator of plant stress, and analyses of related activities enable detection of stress-induced changes that can be mitigated if detected early enough. Responses to environmental stresses affect plants in various ways including growth, physiology, and molecular biology. A major plant pathway that leads to stress tolerance involves signal transduction, gene
Corresponding author. E-mail address: [email protected] (H. Sakugawa).
https://doi.org/10.1016/j.ecoenv.2017.11.065 Received 26 July 2017; Received in revised form 9 November 2017; Accepted 24 November 2017 Available online 01 December 2017 0147-6513/ © 2017 Elsevier Inc. All rights reserved.
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or vegetation being “natural air sampler” for the accumulation of PAHs (Oguntimehin et al., 2007, 2010) also justifies the selection of marigold as a model plant for the current study. The main objective of the present study was to investigate the additive, antagonistic, or synergistic effects of two PAHs in both neutral pH and acidic pH by combining with S.Acid (pH 3). Specifically the scavenging activity of Mann was also evaluated through foliar application on marigold plants. Furthermore, we analyzed the eco-physiological characteristics of plants, including net photosynthesis rate at near saturated irradiance (Amax), stomatal conductance (Gs), and intercellular CO2 concentration (Ci). Additionally, we evaluated physiological parameters like chlorophyll a (Chl.a), chlorophyll b (Chl.b), and total chlorophyll (Tot. Chl) contents as well as leaf fresh weight (FW), turgid weight (TW), and dry weight (DW), relative water content (RWC%), and leaf water deficit (LWD%). Biochemical parameters, including CAT and APX activities and proline content, were also investigated.
expression, protein activation, protection, and repair. The joint efforts of physiologists and molecular biologists are therefore required to generate plants that can tolerate changes in the global environment (Rao et al., 2006). Fluoranthene (FLU) and phenanthrene (PHE) are among the United States Environmental Protection Agency (USEPA) priority PAHs with four- and three-rings respectively. FLU is the typical example of PAH highly abundant in many countries including Japan and is mainly emitted in automobile exhaust (Poor et al., 2004). PHE is also widely distributed in the environment which is transferred on the leaf surfaces through atmospheric deposition and mainly accumulates in the lipid layer of plant membranes (Simonich and Hites, 1994). The two PAHs were selected as model compounds because they are ubiquitous in the environment and their accumulation in plants is associated with diverse problems. Additionally, plants are highly sensitive to their presence and/or accumulation. The toxicity of PAHs such as FLU and PHE in plants leads to production of reactive oxygen species (ROS) (Oguntimehin et al., 2007). The damage caused by the generated ROS mostly depends on the type and concentration of PAHs, intensity and duration of the exposure, plant developmental stage, and plant species. The reaction centers of photosystems I and II in chloroplast thylakoids are the major sites of ROS formation. Excessive ROS production induces abiotic and oxidative stresses in plants. Elevated levels of non-metabolized cellular H2O2 can even damage cellular lipids and proteins as well as inactivate key cellular functions (Gill and Tuteja, 2010). Stressinducing H2O2 is normally metabolized and maintained at concentrations necessary for plant growth and development by catalase (CAT) and ascorbate peroxidase (APX) (Gill and Tuteja, 2010). Catalase activity is usually observed in peroxisomes, and this enzyme is activated by relatively high H2O2 concentrations. APX can detoxify even very low concentrations of H2O2 by using ascorbate as the main substrate, and it is active primarily in chloroplasts, the cytosol, mitochondria, and peroxisomes (Gechev et al., 2006). PAHs may act as stressors on the plants both individually or simultaneously with the acid-deposition. Acid deposition or acid rain is natural precipitation of sulfuric or nitric acid in the forms of rain, snow, fog or dusts. Two major sources of acid rain includes natural such as volcanoes and anthropogenic such as burning of coal and fossil fuels, power generation, and exhaust from vehicles and industries. Exposure to high acidity can kill freshwater fish, adversely affect soil chemistry and nutrient availability, inhibit plant growth and physiology, and increase the susceptibility of plants to pests and diseases (Casiday and Frey, 1998; Gheorghe and Ion, 2011). Oguntimehin et al. (2012) in a study of PAHs alone and in combination with the acid mist reported aggravation of the negative effects of PAHs on the plants by acid mist. Mannitol (Mann) is naturally produced in some plants and is a known and efficient scavenger of ROS generated by cell-free oxidantgenerating systems (Upham and Jahnke, 1986). Oguntimehin et al. (2012) reported that Mann has the ability to mitigate the negative effects of acid mist and Fluoranthene in the Japanese red pine. They concluded that Mann was able to reduce the activities of ROS both inside and on the foliar part of the plants. They further recommended Mann to be used in the protection of pine trees and other plants from the air pollutants. Marigold (Calendula officinalis L.), an annual or biennial plant that produces yellow or orange flower heads, belongs to the Asteraceae family. Its leaves and flowers have been valued for their pharmaceutical and cosmetic properties. Possible uses for marigold contents as alternatives to volatile organic compounds in paints have recently been explored by the paint and coating industry. The medicinal and industrial importance of marigold, its capability of absorbing the highest concentration of PAHs from the environment (Nicoleta-Adela HIRISCSU, doctoral thesis, 2015), relatively faster growth and short life cycle were some of the important characteristics considered for its selection in the current study. Moreover, some similar characteristics of marigold with Sunpatiens (Impatiens spp.) as a good bio-indicator and/
2. Materials and methods 2.1. Plant materials and growth conditions Marigold seedlings, pots (25 × 25 cm deep), and commercial soil (Humus) (Iris Ohyama Co. Ltd., Sendai, Japan) were purchased from a nursery at NAFCO (Higashihiroshima, Japan) on October 9, 2015. The pots, with a 15 × 15 cm mesh placed at the bottom, were filled with 10 L soil (i.e., approximately 80% full). One seedling was transferred per each pot, watered thoroughly and kept in a greenhouse (metalframed shelter) at Hiroshima University in Higashihiroshima, Japan. The shelter was well-ventilated, 8 m × 12 m semi-cylindrical structure covered with a 0.06-mm thick Tefzel® film transparent to visible and UV-A light (DuPont, Wilmington, DE, USA) to prevent rain and dew droplets from directly contacting the seedlings. Seedlings were grown normally for approximately 3 months. During this period, they were manually irrigated two or three times per week, depending on the needs of the plants. To avoid being exposed to cold conditions, plants were kept in open-top chambers from early December 2015 to early April 2016. The open-top chambers were covered with the F-CLEAN® transparent ethylene-tetrafluoroethylene copolymer film (Asahi Glass Green-Tech Co. Ltd., Japan), which allowed more than 95% of the sunlight to pass through. A similar experimental design was used in previous studies by Oguntimehin et al. (2007) and Nakatani et al. (2007). Before fumigating the plants, 15 g NPK+Mg granular fertilizer was applied to each pot and repeated after 2 weeks. During these 5 months in the chambers, the pots were rotated monthly to avoid any kind of bias due to their position. 2.2. Treatment preparation and fumigation Plants were fumigated with the following 12 treatments: FLU, PHE, S.Acid, Mann, FLU+Mann, FLU+S.Acid, FLU+Mann+S.Acid, PHE +Mann, PHE+S.Acid, PHE+Mann+S.Acid, Mann+S.Acid, and MilliQ water (control). All treatments were replicated four times i.e., each treatment contained four healthy marigold seedlings. The seedlings were fumigated for 40 days from February to April 2016. Stock solutions of 1 mM FLU (98%, Sigma Aldrich, USA) and PHE (99%, Wako pure chem. Ind. Osaka, Japan) were prepared by dissolving 202.26 mg and 178.23 mg in 1 L 50% aqueous acetone (Sigma Aldrich, USA) and Milli-Q water (Millipore Co., Japan), respectively. The solutions were diluted to 10 µM with Milli-Q water for the fumigation treatments. The final FLU and PHE concentrations were comparable to the lowest concentrations used in previous study by Oguntimehin et al. (2007). The 0.5% acetone in the final FLU solution was previously determined to have no effect on any of the analyzed parameters (Oguntimehin et al., 2007). The 200 mM stock Mann (99%, Nacalai Tesque Inc. Kyoto, Japan) 835
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25 mM potassium phosphate buffer, 0.25 mM ascorbic acid, 0.1 mM EDTA, 0.1 mM fresh H2O2, and 5% (v/v) crude enzyme extract. The oxidation of ascorbate was measured at 290 nm using UV–Vis spectrophotometer. The extinction coefficient used for the calculation was 2.8 mM−1 cm−1, and one unit of APX was determined as the enzyme amount required to oxidize 1 mmol ascorbate in 1 min.
solution was prepared by adding 36.4344 g Mann in 1 L Milli-Q water. The solution was then diluted to 1 mM for the fumigation. Additionally, the 0.1 M stock S.Acid (Nacalai Tesque Inc. Kyoto, Japan) solution was prepared by adding 1.06 mL acid to 150 mL Milli-Q water, and topped up to 200 mL. The stock solution was diluted in Milli-Q water and the pH was adjusted to 3 as measured by the D-51 pH meter (Horiba, Japan) prior to fumigation of seedlings. The solutions were applied to the foliar parts of marigold seedlings using an electronic sprayer with a nozzle (BS-4000, Fujiwara Sangyo, Miki, Japan) from Monday through Friday from 9:00–11:00 a.m. Each seedling received an average of 25 mL solution per day. For all combination treatments, PAHs were pre-applied and allowed to dry for approximately 30 min before the application of S. Acid and Mann.
2.7. Measurement of proline content Proline content was measured using a modified version of the method described by Bates et al. (1973). Frozen leaf tissues were homogenized in liquid nitrogen using a pestle and mortar. The ground samples were suspended in 10 mL 3% sulfosalicylic acid and then centrifuged twice for 5 min each at 10,000×g in 4 °C. The supernatants were collected, and 2-mL aliquots of the extracts were mixed with 2 mL ninhydrin reagent containing glacial acetic acid. Samples were incubated for 1 h in a water bath set at 100 °C, after which they were cooled in an ice bath and then treated with 4 mL toluene (Cica-Reagent, Kanto Chemical Co. Inc., Tokyo, Japan). The solutions were mixed vigorously for approximately 20 s to ensure that the chromophore was in the toluene layer. The absorbance of the chromophore was measured at 520 nm.
2.3. Analysis of photosynthetic activity and gas exchange The Amax, Gs, and Ci were measured with the LI-6400 open-flow infrared gas analyzer (LI-COR Inc., Lincoln, NE, USA) at the end of the fumigation period. Fresh leaves exposed to sunlight were randomly selected for the measurements. The leaves were kept in the leaf chamber in such a way that the entire 2 × 3 cm2 internal area of the chamber was covered with the leaf. The measurements were completed from 10:00 a.m. to 1:00 p.m. on sunny days with a saturating irradiance of 1500 µmol m−2 s−1 (i.e., photosynthetic photon flux density). Leaf temperatures were 20 ± 2 °C, while the leaf-to-air vapor pressure deficit ranged from 0.80 to 1.32 kPa, and the leaf-to-air CO2 concentration was fixed at 370 µmol CO2 mol−1 at 500 µmol s−1.
2.8. Statistical analysis Data were analyzed with the SPSS program (PASW Statistics 18. Ink). The results for the biochemical parameters were based on the average values for three replicates, whereas the data for all other parameters were based on the average values for four replicates. The data are presented as the mean ± standard error. Significant differences among the groups were calculated by a one-way analysis of variance after verifying the homogeneity and normality of the variance (p < 0.05). The means were compared using Duncan's multiple range test (p < 0.05). The relationships among some parameters were evaluated based on Pearson's correlation coefficient (r; p < 0.05 and 0.01).
2.4. Leaf water relations The RWC% was measured as described by Weatherly (1950). Briefly, fresh mature leaves cut into 1-cm2 disks were immediately weighed to determine FW. They were then incubated in Milli-Q water for approximately 24 h under fluorescent light to absorb water, after which excess water was removed by placing the leaf samples on tissue paper. The leaf disks were then weighed to determine TW, and then incubated in a DKN602 drying oven (Yamato Scientific Co. Ltd., Japan) at 70 °C for 72 h to obtain the DW. The RWC% was calculated with the following formula:
3. Results and discussion 3.1. Effect of fumigation on gas exchange parameters
RWC % = (FW − DW )/(TW − DW ) × 100
A summary of results on the effects of fumigation on gas exchange parameters is given in Fig. 1 (A, B and C). From the results, it is evident that the PAHs had different effects on the fumigated marigold plants. Although FLU and PHE treatments negatively affected all the measured gas exchange parameters (i.e., Amax, Gs, and Ci) (Fig. 1), the effects of FLU were significantly different and greater than those of PHE. Highest per cent inhibition of Amax (54%), Gs (86%) and Ci (32%) were recorded in FLU treatment compared to control. The elevated Amax, Gs and Ci values of PHE treatment were however not significantly different (p < 0.05) from the control. The addition of Mann to PAHs treatments had a considerable mitigating effect on FLU, recording 43, 0.6 and 287 compared to control values 38, 0.9 and 247 for Amax, Gs and Ci respectively and were no more statistically different (Fig. 1 A, B, and C). In contrast, the addition of Mann to the PHE treatment had no significant effect and was on par with both PHE-alone and control. Additionally, the Amax, Gs, and Ci values appeared to decrease slightly in response to S.Acid alone or in combination with PAHs or Mann, but the values were not significantly different from the control measurements and the differences were almost negligible. The decrease in Amax, Gs and Ci could be attributed to ROS generation in the cells because of PAH fumigation. Pell et al. (1997) also reported reduction in CO2 assimilation due to ROS accumulation. The stomatal conductance (Gs) decline rapidly in the presence of air pollutants in the plants and thus may lead to stomatal closure. The stomatal closure is therefore considered a good indicator and a valid mechanism of combating environmental stress in the plants (Lefohn,
The LWD% was calculated according to the method described by Kalapos (1994) using the following equation: LWD% = 100 − RWC% 2.5. Chlorophyll concentration Fresh leaves were cut into 1-cm2 disks for a subsequent analysis of chlorophyll contents (i.e., Chl.a, Chl.b, and Tot. Chl). The pigments were extracted with 5 mL NN-dimethylformamide and the concentration was determined by the method of Porra et al. (1989). 2.6. Antioxidant enzymes activities The protein concentrations of leaf enzyme extracts were measured using a protein assay kit (Nacalai Tesque Inc.), based on the Bradford (1976) method. Bovine serum albumin was used as the standard and the enzyme activities were calculated using the protein concentration. The CAT (EC 1.11.1.6) and APX (EC 1.11.1.11) activities were analyzed according to the method developed by Takagi and Yamada (2013). For the CAT activity measurement, the 1-mL assay mixture consisted of 50 mM potassium phosphate buffer (pH 7.0), 10 mM fresh H2O2, and 5% (v/v) crude enzyme extract. Decreases in H2O2 content were monitored at 240 nm, and CAT activity was calculated as mmol H2O2 consumed per minute. The extinction coefficient used for the calculation was 0.04 mM−1 cm−1. The APX activity was measured in a 1-mL assay mixture containing 836
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Fig. 1. Gas exchange parameters (A) Net photosynthesis rate at near saturated irradiance (Amax), (B) Stomatal conductance (Gs) and (C) Intercellular CO2 concentration (Ci). Data are presented as the average value for four marigold seedlings. Identical superscript alphabetical letters indicate the same homogenous groups; different alphabetical letters indicate significant differences at p < 0.05 (Duncan's multiple range test) within each histogram panel.
It was also evident from our study that Mann is an effective scavenger and was able to scavenge the OH radicals generated by PAHs, especially FLU. The current findings on Mann are in accordance with those of Oguntimehin and Sakugawa (2009). Furthermore, we confirm the recommendation by Oguntimehin et al. (2012) to widely use Mann for protection of higher plants from air pollutants.
1991). Despite the existing controversy on whether Amax decreases because of stomatal closure or non-stomatal factors, the current study showed that, the PAHs decreased the Amax (Fig. 1A), Gs (Fig. 1B), and Ci (Fig. 1C). The observed phenomenon could be attributed to stomatal closure, and this is supported by the strong positive correlation between Amax vs Gs, Amax vs Ci and Gs vs Ci as (r = 0.7, 0.6, 0.6 at p < 0.01) obtained in the current study. Kreslavski et al. (2014) reported that in the plants the PAHs especially FLU and PHE accumulates in the thylakoid membranes and leads to alteration of the thylakoids as well as development of oxidative stress that are detrimental to photosynthetic activities and other physiological processes. The results on gas exchange parameters in the current study are consistent with that of Oguntimehin et al. (2007) in which Japanese red pine seedlings were fumigated with FLU and PHE. Our results are also in line with a study by Tomar and Jajoo (2014) in which the phytotoxicity of FLU in wheat plants and the inhibiting effects of FLU on photosynthesis were attributed to the inhibition in the process of CO2 fixation. However the effect of S.Acid alone or in combination with other fumigants contradicted an earlier study that concluded that the presence of FLU exacerbated the adverse effects of an acid treatment (Oguntimehin et al., 2012). This discrepancy is probably because, in the current study each plant received a total of 0.44 × 10−3 mol plant−1 S.Acid during the entire exposure experiment which was three fold lower (1.5 × 10−3 mol plant−1) than those of Oguntimehin et al. (2012). In addition, the plant species used in the current study were different from the previous studies, hence the discrepancies.
3.2. Effect of fumigation on FW, TW, DW, RWC%, and LWD% Results in Table 1 revealed that PHE+S.Acid treatment recorded the highest values (0.11) and (0.15) in comparison to the PHE treatment (0.06) and (0.1) for FW and TW respectively. There were no significant differences (p < 0.05) in any of the treatments except in the FW of PHE+S.Acid in comparison to Control. FW value (0.1) in PHE +S.Acid was the highest among all the other treatments and was significantly different (p < 0.05) from both the Control and PHE treatments. In addition, TW in PHE+S.Acid was the highest and significantly different from PHE treatment but not Control. Generally, the current study revealed that the PAHs had no substantial effects on the FW, TW and DW. However, highest values of FW and TW in PHE+S.Acid treatment could be due to the following reasons; S.Acid at relatively low concentrations might be either detoxified or used in a reductive sulfur cycle to form amino acids or may increase the availability of iron and phosphorus, or improve water penetration in the soils (Ryan et al., 1973; Yahia et al., 1975), whereas PHE is low molecular weight, highly volatile and unstable PAH in comparison to FLU which perhaps pose 837
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chlorophyll content in both FLU and PHE treatments were not significantly different (p < 0.05) from the control values. Nevertheless, Chlorophyll contents except Chl.b in FLU and PHE treatments were significantly different (p < 0.05) from each other. An earlier study by Oguntimehin et al. (2010) confirmed that PAHs can alter chlorophyll pigments in the plants and the reduction in chlorophyll pigments was recognized as an indicator of stress, which was further attributed to the reduction in the chlorophyll pigment capacity of the plants. This finding is consistent with the observations of Oguntimehin et al. (2007) which reported the negative effects of FLU and PHE on certain parameters including total chlorophyll contents in Japanese red pine seedlings, with FLU having greater effect than PHE. Tomar and Jajoo (2015) also reported a decrease in chlorophyll content due to FLU fumigations. In the current study, fumigation of plants with PHE positively affected chlorophyll contents, with the abundance of both Chl.a and Tot. Chl increasing by 32%, and the Chl.b content increasing by 24%. A combined treatment of Mann and PHE caused the chlorophyll level to decline, with Chl.a, Chl.b, and Tot. Chl contents decreasing by 13%, 20%, and 13%, respectively, however, they were not significantly different from control (Table 2). The possible explanation for the effect of PHE on chlorophyll content in the current study could be; PHE is highly volatile in nature, having high vapor pressure (1.21 × 10−4 mm Hg at 25 °C) and easily degradable while FLU has low vapor pressure (9.22 × 10−6 mm Hg at 25 °C) and less degradable (Shiu and Mackay, 1997; Wasik et al., 1983). A part of it, might be evaporated immediately after the fumigation and part of it might have been degraded as reported by Min and Xie (2006) which in both the cases it ultimately results in lower concentration of PHE in the plants which thus results in the synthesis of chlorophyll pigments as reported by Shen et al. (2016). PAHs effect on plants is concentration-dependent. Under lower dose, plant growth might be enhanced because PAHs could be stressors or “hormone” (Laughlin et al., 1982). Apart from that, the short duration of the current study might also be recognized as a limiting factor for evaluating the effect of FLU and the scavenging effect of Mann with special reference to the chlorophyll pigments. But assuming the longer exposure of some biennial or perineal crops, this effect may get clearer and easily visible. Hence, the findings of this study imply that FLU induced the production of free hydroxyl radicals, and the resulting oxidative stress likely had an inhibitory effect on photosystem II as well as on overall chlorophyll pigments producing system (Eisenberg and Cunningham, 1985; ElAlawi et al., 2002). The addition of Mann to the FLU treatment prevented generation of ROS and restricted the ability of FLU to induce oxidative stress.
Table 1 Fresh weight, turgid weight and dry weight of cm2 leaf (g)*. Treatments/ Parameters*
Fresh.W
Turgid.W
Dry.W
Control Mann S.Acid Mann+S.Acid FLU FLU+Mann FLU+S.Acid FLU+Mann+S.Acid PHE PHE+Mann PHE+S.Acid PHE+Mann+S.Acid
0.05 ± 0.01 cde 0.07 ± 0.02bcde 0.08 ± 0.01abc 0.05 ± 0.003de 0.05 ± 0.01de 0.04 ± 0.003e 0.04 ± 0.004e 0.08 ± 0.01abcd 0.06 ± 0.01bcde 0.09 ± 0.01ab 0.1 ± 0.02a 0.04 ± 0.01e
0.12 ± 0.01abcd 0.09 ± 0.02bcd 0.13 ± 0.02ab 0.1 ± 0.01bcd 0.09 ± 0.02bcd 0.07 ± 0.02d 0.08 ± 0.01cd 0.13 ± 0.004abc 0.1 ± 0.01bcd 0.12 ± 0.01abcd 0.15 ± 0.03a 0.09 ± 0.03bcd
0.008 ± 0.0003abc 0.007 ± 0.001c 0.01 ± 0.001ab 0.007 ± 0.0003bc 0.008 ± 0.001abc 0.006 ± 0.0002c 0.006 ± 0.001c 0.011 ± 0.001a 0.009 ± 0.001abc 0.008 ± 0.001abc 0.011 ± 0.002a 0.008 ± 0.002abc
* Data are presented as the average value for three marigold plants ± standard error. Identical superscript alphabetical letters indicate the same homogenous groups; different alphabetical letters indicate significant differences at p < 0.05 (Duncan's multiple range test).
lesser stress to the plants. Results on RWC% and LWD% (Fig. 2) showed that generally, RWC% and LWD% were lowered and increased respectively as a result of fumigations. Despite this phenomenon predominant, the effects were more pronounced with S.Acid treatments. Significantly lower RWC% and higher LWD% in both PHE and PHE +S.Acid treatments were recorded in comparison to control (Fig. 2). Sulfuric acid may acidify cell wall and may harm its stability resulting in a decline in the leaf water content (Anderson, 1972). Current results are in line with that of Gadallah and Sayed (2001) who studied the impact of Kinetin on water relations in sorghum plants at different levels of acidity and reported that plants at pH 3 maintained lower water content than that of neutral pH. In brief, lower pH (pH 3) had greater negative effects on plant leaf water relations in PHE treatment compared to all the other treatments in the current study. 3.3. Effect of fumigation on chlorophyll concentration There were significant differences in the chlorophyll contents (Chl.a, Chl.b, and Tot. Chl.) depending on the fumigation treatments (Table 2). Plants fumigated with FLU recorded the lowest Chl.a, Chl.b, and Tot. Chl contents (36, 11, 48) compared to control (53, 17, 71), which were 33%, 34%, and 32% lower than the control values, respectively. The PHE-treated plants recorded the highest Chl.a, Chl.b, and Tot. Chl contents (70, 21, 93), which were 32%, 24%, and 32% higher than the control measurements respectively. However,
Fig. 2. Relative water content % and leaf water deficit %. Data are presented as the average value for four marigold seedlings. Identical superscript alphabetical letters indicate the same homogenous groups; different alphabetical letters indicate significant differences at p < 0.05 (Duncan's multiple range test) within each histogram panel.
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Table 2 Chlorophyll contents (µg/ cm2 leaf) and reduction percentage. Treatments/Parameters*
Chl.a
Chl.a % reduction
Chl.b
Chl.b % reduction
Tot. Chl
Tot. Chl % reduction
Control Mann S.Acid Mann+S.Acid FLU FLU+Mann FLU+S.Acid FLU+Mann+S.Acid PHE PHE+Mann PHE+S.Acid PHE+Mann+S.Acid
53 ± 4abcd 61 ± 1abc 49 ± 6bcd 52 ± 9abcd 36 ± 3d 49 ± 3bcd 43 ± 3 cd 63 ± 7ab 70 ± 5a 46 ± 4bcd 59 ± 8abc 49 ± 8bcd
0.01 −14 8 2 33 8 18 −19 −32 13 −12 7
17 ± 2a 19 ± 1a 16 ± 2a 23 ± 8a 11 ± 1a 18 ± 2a 17 ± 1a 22 ± 5a 21 ± 4a 13 ± 3a 18 ± 5a 12 ± 2a
0 −17 1.6 −36 34 −7.8 −2.8 −32 −24 20 −7.8 27
71 ± 6abc 80 ± 2abc 65 ± 22c 70 ± 12abc 48 ± 3c 65 ± 4abc 59 ± 3bc 82 ± 8ab 93 ± 6a 62 ± 5abc 80 ± 11abc 66 ± 11abc
0 −13 8 2 32 8 17 −15 −32 13 −12 7
* Data are presented as the mean value for three marigold plants ± standard error. Identical superscript alphabetical letters indicate the same homogenous groups; different alphabetical letters indicate significant differences at p < 0.05 (Duncan's multiple range test).
Plants fumigated with FLU exhibited increased CAT and APX activities, while FLU+Mann and control treatments did not significantly influenced the activities of those enzymes (Table 3). In addition, CAT activities in both FLU+Mann and FLU+S.Acid treatments were significantly lower form FLU treatment but not from the control. This suggests that CAT is very sensitive enzyme and react rapidly to the presence of oxidative stress, mainly H2O2. Takagi and Yamada (2013) reported that CAT was the most efficient enzyme among SOD, APX and GR in overcoming H2O2 stress in chenopodiaceous halophytes. The enzymatic activities induced by PHE were not as high as those induced by FLU, suggesting PHE was not as deleterious to the plants as FLU. This is probably because of the difference in molecular weight, vapor pressure, volatility and some other characteristics of FLU and PHE, as discussed earlier in details (3.3. section). The accumulation of ROS in response to FLU increases enzymatic activities, the extent of which is usually dependent on the oxidative stress imposed by FLU as well as the presence of free radicals (Smirnoff, 1993). Plants exposed to oxidative stresses, especially H2O2, exhibit upregulated CAT activity to scavenge the overproduced H2O2 and stabilize the cellular ROS levels (Jannat et al., 2012). Highest APX activity in FLU treatment compared to all the other treatments in the current study (Table 3) suggests that APX is not as sensitive to the oxidative stress as CAT and was only increasing in the highly stressed condition such as in FLU treatment but were not reacting in the mild/ lower stressed conditions. The lower CAT and APX activities in control (Table 3) were likely the result of a lack of stress. However, lower CAT and APX activities in FLU+Mann-treated plants suggest that the ROS generated by FLU was quickly quenched by Mann (i.e., no oxidative stress) but yet the plants needed some time to get relief and overcome the stressed conditions successfully. The findings from the current study on CAT activity contradicts that of Tomar and Jajoo (2015) which reported lowered CAT activity in the wheat plants after oxidative stress by FLU. This difference might be due to the difference in plant species as well as different environmental and study conditions.
3.4. Effect of fumigation on antioxidant enzyme activities The highest proline contents (0.07) were recorded for the control plants and were significantly different (p < 0.05) from all the other treatments (Table 3). Only slight variations were observed among the other treatments, but the differences were not significant. Proline is always present in plants, because it is vital for developing plants (e.g., from transition to the seed development stage). High proline content in the plants is preferable to several species of insects. Plants with higher level of proline are usually used as trap crops in the insect management. Marigold is reported to be a good trap crop in management of several species of insects (Srinivasan et al., 2008) which thus justifies the higher level of proline in control plants. Proline is catabolized immediately after plants recovery from any kind of stress. Findings from the current study on proline contents are consistent with those of Khalil et al. (2016). Previous investigations confirmed that proline content increases as an adaptive plant response. Immediately after plants overcome stress conditions, the accumulated proline is rapidly oxidized to glutamate. The lowest proline contents following the FLU and PHE treatments were due to the downregulation of proline contents after the plant antioxidant system responded to the stress condition. Proline is an ROS scavenger that can maintain cell membrane permeability as well as increase enzyme activities and stabilize protein structures (Yuyan et al., 2013). The rapid proline catabolism in plants recovering from stress conditions helps to induce some metabolic pathways, including those affecting the restoration of chloroplast activities (Vankova et al., 2012).
Table 3 Changes to antioxidant enzyme activities. Treatments/ Parameters*
Proline (µmol g−1 FW)
Catalase (U. mg protein−1 min−1)
APX (U. mg protein−1 min−1)
Control Mann S.Acid Mann+S.Acid FLU FLU+Mann FLU+S.Acid FLU+Mann +S.Acid PHE PHE+Mann PHE+S.Acid PHE+Mann +S.Acid
0.07 ± 0.003a 0.02 ± 0.001b 0.01 ± 0.002b 0.02 ± 0.001b 0.02 ± 0.002b 0.02 ± 0.003b 0.02 ± 0.001b 0.02 ± 0.002b
35 ± 2bcd 5 ± 1d 37 ± 5b 11 ± 2bcd 240 ± 12a 20 ± 1bc 38 ± 15bcd 14 ± 2 cd
0.4 ± 0.03b 0.1 ± 0.02b 0.4 ± 0.1b 0.1 ± 0.03b 4.2 ± 0.9a 0.3 ± 0.01b 0.3 ± 0.1b 0.2 ± 0.04b
0.01 ± 0.002b 0.02 ± 0.005b 0.02 ± 0.003b 0.01 ± 0.001b
18 ± 5bcd 30 ± 14b 26 ± 3bc 16 ± 1bcd
0.2 ± 0.1b 0.3 ± 0.1b 0.2 ± 0.03b 0.2 ± 0.03b
4. Conclusions This study evaluated the individual and combined effects of fluoranthene, phenanthrene, mannitol and sulfuric acid on marigold (Calendula officinalis). The results revealed that FLU and PHE fumigations are detrimental to some ecophysiological and biochemical parameters of marigold plants. The PAHs, in particular FLU induced significant oxidative stress in terms of reduction in gas exchange parameters and chlorophyll pigments, imbalance in leaf water relations, and increase in antioxidant enzymes activities via generation of ROS. The oxidative stress was not induced when Mann (an ROS-scavenger) was combined with FLU, and this implies that the ROS generated was quenched by Mann. In contrast, PHE-fumigation had varied effects on
* Data are presented as the mean value for three marigold plants ± standard error. Identical superscript alphabetical letters indicate the same homogenous groups; different alphabetical letters indicate significant differences at p < 0.05 (Duncan's multiple range test).
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the physiological and biochemical parameters of marigold plants and was not as deleterious as FLU. Furthermore, combination of S.Acid (pH 3) with both FLU and PHE had varied effect on the measured parameters including the deleterious effect on leaf water relations and beneficial in the FW and TW of the plants. This is most likely because the S.Acid concentration used in this study was not high enough and served as a source of sulfur, required for normal seedling development. Moreover, antioxidant enzymes activities enhanced to ROS accumulation especially due to FLU fumigation and such activities were not high enough when Mann was combined with FLU. Generally, the FLU fumigation induced severe oxidative stress to marigold plants by generating ROS which was well represented by alteration of gas exchange parameters, lowered chlorophyll pigments and enhanced antioxidant enzymes activities, and such changes were not observed when ROS was quenched by the addition of Mann. The effect of PHE was not as deleterious as FLU on marigold plants and will require further investigations in both neutral and acidic pH. Mannitol seemed to be more effective against high molecular weight PAH (having low volatility) than low molecular weight PAH (highly volatile) such as FLU and PHE respectively. In light of the current study, Mann is recommended to be widely used for the protection of higher plants from high molecular weight PAHs-stress in the urban areas.
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